Exploring natural and pharmocological products for the ...

University of Veterinary Medicine Hannover

Exploring natural and pharmocological products for the

ability to boost neutrophils against bacterial infections

Thesis

Submitted in partial fulfilment of the requirements for the degree

-Doctor of Veterinary Medicine-

Doctor medicinae veterinariae

(Dr med vet)

by

Natalja Jerjomiceva

Latvia

Hannover 2013

Academic supervision Prof Dr Hassan Y Naim

Department of Physiological Chemistry


Dr Maren von Koumlckritz-Blickwede



Referee Apl- Prof Dr Stefan Schwarz

Institute of Farm Animal Genetics

Friedrich-Loeffler-Institut (FLI) Neustadt-Mariensee

Day of the oral examination 17052013

This work was supported by a grant from Akademie fuumlr Tiergesundheit (AfT) Bonn

Germany

To my parents

Contents

Chapter 1 General introduction 7

11 Antibiotic resistant bacterial infections 10

12 Drug discovery from the plant extract 12

13 Use of plants with immunomodulatory activity in folklore

medicine 17

14 The UAH natural products drug discovery group 26

15 Guarea kunthiana 28

16 Fluoroquinolones 31

17 Enrofloxacin 33

18 Goal 37

Chapter 2 Materials and methods 45

21 Plant material 47

22 Drugs 47

23 Bacterial strains 47

24 Preparation of plant extract 47

25 Effect of Guarea kunthiana on growth of bacteria 48

26 Isolation and preparation of human neutrophils 48

27 Isolation and preparation of bovine neutrophils 49

28 Neutrophil killing assay 49

29 Determination of phagocytotic uptake 50

210 NET visualization and quantification 50

211 Degranulation 51

212 Oxidative burst 51

213 Effect of nocodazole and cytochalasin D on the Guarea

kunthiana or enrofloxacin-mediated NET formation 51

214 Western blotting analysis of PAD-4 expression 52

215 Livedead viabilitycytotoxicity assay 52

216 Measurement of membrane integritycell death 53

217 Statistical analysis 53

Chapter 3 Guarea kunthiana bark extract enhance the antimicrobial

activities of human and bovine neutrophils 57

Chapter 4 Enrofloxacin enhances the formation of neutrophil

extracellular traps in bovine granulocytes 77

Chapter 5 General discussion and future outlook 91

Chapter 6 Summary 105

Chapter 7 Zusammenfassung 109

Appendix - List of figures and tables

- Abstracts

- Acknowledgements

- Erklaumlrung

- Curriculum Vita

- List of abbreviations

Chapter 1

General introduction

General introduction Chapter 1

9

Entering into its 7th decade the era of antimicrobial therapy has greatly reduced

the morbidity and mortality of infectious diseases However the emergence of resistant

microorganisms has now reached epidemic proportions and poses great challenges to

human and veterinary medicine For essentially every antibiotic ever developed

resistance has developed in one or another potential pathogen Worrisome trends are

particularly evident in several important Gram-positive bacterial species including

Staphylococcus (S) aureus which are increasingly unresponsive to first-line antibiotic

therapies Recently new strains of methicillin-resistant S aureus (MRSA) have

emerged from the animal kingdom and can be transmitted to humans and cause severe

zoonotic infections Innovative approaches to broad-spectrum antibacterial treatment

are urgently needed An alternative approach for the treatment of difficult infections

such as those involving antimicrobial resistance or compromised host immunity could

be the pharmacological enhancement of the antimicrobial capabilities of phagocytes

Pharmacological agents that boost the host immune system could conceivably be used

alongside conventional antibiotic treatment for successful therapy of the infection The

goal of my study at the Department of Physiological Chemistry of the University of

Veterinary Medicine Hannover was to search for (1) novel natural products and (2)

known pharmaceutical products with the ability to boost the antimicrobial functions of

neutrophils as the first line of defence against bacterial infections

Figure 1-1 Neutrophil engulfing bacteria (wwwchronicprostatitiscomimagesneutrophiljpg)


10

11 Antibiotic resistant bacterial infections

The ―golden age of antibiotics began with the production of penicillin in 1941

when this compound discovered by Alexander Fleming in 1928 was finally mass-

produced and first made available for limited clinical trials (Soares et al 2012) Around

1946 penicillin became generally available for treatment of bacterial infections

especially those caused by staphylococci and streptococci Initially the antibiotic was

effective against all sorts of infections caused by these two Gram-positive bacteria

Resistance to penicillin in some strains of staphylococci was recognized almost

immediately (Todar 2002)

Since the late 1940s and early 1950s streptomycin chloramphenicol and

tetracycline have been widely used in antibiotic chemotherapy These antibiotics were

effective against the full array of bacterial pathogens including Gram-positive and Gram-

negative bacteria intracellular parasites and the tuberculosis bacillus

By the 1960s it became apparent that some bacterial pathogens were

developing resistance to antibiotic-after-antibiotic at a rate faster than new antibiotics

could be brought to market The most important pathogens to emerge in multiple drug

resistant forms so far have been Mycobacterium tuberculosis and Staphylococcus

aureus (Todar 2002)

Serious infections caused by bacteria that have become resistant to commonly

used antibiotics have become a major global healthcare problem in the 21st century

(Alanis 2005) In the past 60 years antibiotics have been critical in the fight against

infectious disease caused by bacteria and other microbes However disease-causing

microbes that have become resistant to antibiotic drug therapy are an increasing public

health problem Wound infections gonorrhea tuberculosis pneumonia septicemia and

childhood ear infections are just a few of the diseases that have become hard to treat

with antibiotics One part of the problem is that bacteria and other microbes that cause

infections are remarkably resilient and have developed several ways to resist antibiotics

and other antimicrobial drugs Another part of the problem is due to increasing use and

misuse of existing antibiotics in human and veterinary medicine and in agriculture

Nowadays about 70 percent of the bacteria that cause infections in hospitals are

resistant to at least one of the drugs most commonly used for treatment Some


11

organisms are resistant to all approved antibiotics and can only be treated with

experimental and potentially toxic drugs (Todar 2002) Worrisome trends are

particularly evident in several important Gram-positive and Gram-negative bacterial

species specifically methicillin-resistant Staphylococcus aureus (MRSA) vancomycin-

resistant enterococci (VRE) and floroquinolone-resistant Pseudomonas aeruginosa

(FQRP) which are increasingly unresponsive to first-line antibiotic therapy

Figure 1-2 Development of multidrug-resistant (MDR) pathogens Number of MDR pathogens per 1000 patient days split into methicillin-resistant Staphylococcus aureus (MRSA) vancomycin-resistant enterococci (VRE) imipenem-resistant Acinetobacter baumannii (Imi R Aci) and group 3 cephalosporinresistant-resistant Klebsiella pneumoni (G3C R Kleb) and group 3 cephalosporin-resistant Escherichia coli (G3C R Eco) (Mattner et al 2012)

Obviously if a bacterial pathogen is able to develop or acquire resistance to an

antibiotic then that substance becomes useless in the treatment of the infectious

disease caused by that pathogen So as pathogens develop resistance there is the

immediate need to find new (different) antibiotics to fill the place of the old ones in

treatment regimes


12

12 Drug discovery from the plant extract

Researchers have proved that plant extracts can be used to cure many health

ailments Figure 1-3 shows that 37 of all drugs developed in the time frame from 1981

to 2006 originate from natural products This indicates that natural products continue to

play a highly significant role in the drug discovery and development process

Figure 1-3 Natural products as sources of new drugs over the last 25 years (Newman and Cragg 2007) ―B Biological usually a large (gt45 residues) peptide or protein either isolated from an organismcell line or produced by biotechnological means in a surrogate host ―N Natural product ―ND Derived from a natural product and is usually a semisynthetic modification ―S Totally synthetic drug often found by random screeningmodification of an existing agent ―S Made by total synthesis but the pharmacophore iswas from a natural product NM Natural product mimic ―V Vaccine

Today there are at least 120 distinct chemical substances derived from plants

that are considered as important drugs and that are currently in use in one or more

countries in the world These chemical substances are shown in the Table 1-1 (Taylor

2000) It is estimated that 60 of antitumor and antiinfection drugs already on the

market or under clinical trial derive from a natural source Natural compounds can be

lead compounds allowing the design and rational planning of new drugs biomimetic

30

10

4

104

14

5

23

S SNM S SNM V B N ND


13

synthesis development and the discovery of new therapeutic properties not yet

attributed to known compounds (Rates 2000)

For example many years ago a plant chemical was discovered in a tropical

plant Cephaelis ipecacuanha and the chemical was named emetine A drug was

developed from this plant chemical called Ipecac which was used for many years to

induce vomiting mostly if someone accidently swallowed a poisonous or harmful

substance Another example is the plant chemical named taxol which derives from

Taxus brevifolia (Table 1-1) A pharmaceutical company copied this chemical and

patented a drug named Paclitaxeltrade which is used against various types of tumors

today

Cynarin is a plant chemical found in the common artichoke (Cynara scolymus) A

cynarin drug is sold for liver problems and hypertension which simply consists of this

one chemical extracted from the artichoke plant or a plant extract which has been

standardized to contain a specific milligram amount of this one chemical Some of the

drugchemicals shown below are still sold as plant based drugs requiring the processing

of the actual plant material Others have been chemically copied or synthesized by

laboratories and no plant materials are used in the manufacture of the drug A good

example of this is the plant chemical quinine which was discovered in a rainforest tree

(Cinchona ledgeriana) over 100 years ago For many years the quinine chemical was

extracted from the bark of this tree and processed into pills to treat malaria Then a

scientist was able to synthesize or copy this plant alkaloid into a chemical drug without

using the original tree bark for manufacturing the drug Today all quinine drugs sold are

manufactured chemically without the use of any tree bark However another chemical

in the tree called quinidine which was found to be useful for various heart conditions

could not be completely copied in the laboratory and the tree bark is still harvested and

used to extract this plant chemical from it (Taylor 2000)

Table 1-1 Chemical substances derived from plants (Taylor 2000)

DrugChemicals ActionClinical Use Plant Source

Acetyldigoxin Cardiotonic Digitalis lanata

Adoniside Cardiotonic Adonis vernalis

Aescin Antiinflammatory Aesculus hippocastanum


14

Aesculetin Antidysentery Frazinus rhychophylla

Agrimopho Antihelmintic Agrimonia supatoria

Ajmalicine Circulatory disoders Rauvolfia sepentina

Allantoin Vulnerary Several plants

Allyl isothiocyanate Rubefacient Brassica nigra

Anabesine Skeletal muscle relaxant Anbasis sphylla

Andrographolide Baccillary dysentery Andrographis paniculata

Anisodamine Anticholinergic Anisodus tanguticus

Anisodine Anticholinergic Anisodus tanguticus

Arecoline Anthelmintic Areca catechu

Asiaticoside Vulnerary Centella asiatica

Atropine Anticholinergic Atropa belladonna

Benzyl benzoate Scabicide Several plants

Berberine Bacillary dysentery Berberis vulgaris

Borneol Antitussive Ardisia japonica

Bromelain Anticancerous Betula alba

Borneol Antipyretic analgesic antiinflamatory

Several plants

Bromelain Antiinflammatory proteolytic Ananas comosus

Caffeine CNS stimulant Camellia sinensis

Camphor Rubefacient Cinnamomum camphora

Camptothecin Anticancerous Camptotheca acuminata

(+)-Catetchin Haemostatic Potentilla fragarioides

Chymopapain Proteolytic mucolytic Carica papaya

Cissampeline Skeletal muscle relaxant Cissampelos pareira

Cocaine Local anaesthetic Erythroxylum coca

Codeine Analgesic antitussive Papaver somniferum

Colchiceine Antitumor agent Colchicum autumnale

Colchicine Antitumor agent antigout Colchicum autumnale

Convallatoxin Cardiotonic Convallaria majalis

Curcumin Choleretic Curcuma longa

Cynarin Choleretic Cynara scolymus

Danthron Laxative Cassia species

Demecolcine Antitumor agent Colchicum autumnale

Deserpidine Antihypertensive tranquillizer Rauvolfia canescens

Deslanoside Cardiotonic Digitalis lanata

LndashDope Antiparkinsonism Mucuna sp


15

Digitalin Cardiotonic Digitalis purpurea

Digitoxin Cardiotonic Digitalis purpurea

Digoxin Cardiotonic Digitalis purpurea

Emetine Amoebicide emetic Cephaelis ipecacuanha

Ephedrine Antihistamine Ephedra sinica

Etoposide Antitumor agent Podophyllum peltatum

Galanthamine Cholinesterase inhibitor Lycoris squamigera

Gitalin Cardiotonic Digitalis purpurea

Glaucarubin Amoebicide Simarouba glauca

Glaucine Antitussive Glaucium flavum

Glasiovine Antidepressant Octea glaziovii

Glycyrrhizin Sweetener Addisonrsquos disease Glycyrrhiza glabra

Gossypol Male contraceptive Gossypium species

Hemsleyadin Bacillary dysentery Hemseya amabilis

Hesperidin Capillary fragility Citrus species

Hydrastine Hemostatic astringent Hydrastis canadensis

Hyoscyamine Anticholinergic Hyoscyamus niger

Irinotecan Anticancer antitumor agent Camptotheca acuminata

Kaibic acud Ascaricide Digenea simplex

Kawain Tranquillizer Piper methysticum

Kheltin Bronchodilator Ammi visaga

Lanatosides A B C Cardiotonic Digitalis lanata

Lapachol Anticancer antitumor Tabebuia sp

a-Lobeline Smoking deterrant respiratory stimulant

Lobelia inflata

Menthol Rubefacient Mentha species

Methyl salicylate Rubefacient Gaultheria procumbens

Monocrotaline Antitumor agent (topical) Crotalaria sessiliflora

Morphine Analgesic Papaver somniferum

Neoandrograpolide Dysentery Andrographis paniculata

Nicotine Insecticide Nicotiana tabacum

Nordihydroguaiaretic acid Antioxidant Larrea divaricata

Noscapine Antitussive Papaver somniferum

Ouabain Cardiotonic Strophanthus gratus

Pachycarpine Oxytocic Sophora pschycarpa

Palmatine Antipyretic detoxicant Coptis japonica

Papain Proteolytic mucolytic Carica papaya


16

Papavarine Smooth muscle relaxant Papaver somniferum

Phyllodulcin Sweetner Hydeangea macrophylla

Physostigmine Cholinesterase inhibitor Psysostigma venenosum

Picrotoxin Analeptic Anamirta cocculuc

Pilocarpine Parasympathomimetic Pilocarpus jaborandi

Pinitol Expectorant Several plants

Podolphyllotoxin Antitumor anticancer agent Podophyllum peltatum

Protoveratrine A B Antihypertensives Veratrum album

Pseudoephredine Sympathomimetic Ephedra sinica

Pseudoephedrine nor- Sympathomimetic Ephedra sinica

Quinidine Antiarrhythmic Cinchona ledgeriana

Quinine Antimalarial antipyretic Cinchona ledgeriana

Qulsqualic acid Antihelmintic Quisqualis indica

Rescinnamine Antihypertensive tranquillizer Rauvolfia serpentina

Reserpine Antihypertensive tranquillizer Rauvolfia serpentina

Rhomitoxin Antihypertensive tranquillizer Rauvolfia molle

Rorifone Antitussive Rorippa indica

Rotenone Piscicide insecticide Lonchocarpus nicou

Rotundine Analagesic sedative tranquillizer Stephania sinica

Rutin Capillary fragility Citrus species

Salicin Analgesic Salix alba

Sanguinarine Dental plaque inhibitor Sanguinaria canadensis

Santonin Ascaride Artemisia maritma

Scillarin A Cardiotonic Urginea maritima

Scopolamine Setative Datura species

Sennosides A B Laxative Cassia species

Silymarin Antihepatotoxic Silybum marianum

Sparteine Oxytocic Cytisus scoparius

Stevioside Sweetner Stevia rebaudiana

Strychnine CNS stimulant Strychos nux-vomica

Taxol Antitumor agent Taxus brevifolia

Teniposide Antitumor agent Taxus brevifolia

A-Tetrahydrocannabinol (THC) Antiemetic decrease occular tension

Podophyllum peltatum

Tetrahydropaltine Analgesic sedative tranquillizer Cannabis sativa

Tetrahydropalatine Analgesic sedative tranquillizer Corydalis ambigua

Tetrandrine Antihypertensive Stephania tetrandra


17

Theobromine Diuretic vasodilator Theobroma cacao

Theophylline Diuretic bronchodilator Theobroma cacao and others

Thymol Antifungal (topical) Thymus vulgaris

Topotecan Antitumor anticancer agent Camptotheca acuminata

Trichosanthin Abortifacient Trichosanthes kirilowii

Tubocurarine Skeletal muscle relaxant Chondodendron tomentosum

Valapotriates Sedative Valeriana officinalis

Vasicine Cerebral stimulant Vinca minor

Vinblastine Antitumor antileukemic agent Catharanthus roseus

Vincristine Antitumor antileukemic agent Cataranthus roseus

Yohimbine Aphrodisiac Pausinystalia yohimbe

Yuanhuacine Abortifacient Daphne genkwa

13 Use of plants with immunomodulatory activity in folklore medicine

For thousands of years natural products have played an important role

throughout the world in treating and preventing human diseases Extracts from plants

have known to possess many beneficial qualities to augment the overall health of

human beings The use of plant species for treatment of various human ailments has

been mentioned in Ayurveda and in other traditional medicine sources Some of these

drugs are believed to enhance the natural resistance of body infections Thus there are

many plants having immunomodulatory activities

Immunomodulation is the regulation and modulation of immunity either by

enhancing or by reducing the immune response In other words immunomodulation

involves a change in the human bodys immune system caused by agents that activate

or suppress the function if immune cells If modulation of the immune system results in

an enhancement of the immune reaction it is known as immunostimulation The are two

main categories of immunostimulators (1) specific immunostimulators that provide

antigenic specificity in immune response such as vaccines or any antigen and (2) non-

specific immunostimulators that act irrespective of antigenic specificity (Kumar et al

2011)


18

Examples of plants with immunomodulatory activity in folklore medicine

Modulation of immune response to alleviate diseases has been of interest since

a long time ago Some of the plants which have shown reported immunomodulatory

activity are described below and in Table 1-2

The medicinal usage of garlic (Allium sativum) has been known for centuries In

the last decades garlic has been reported to display antibiotic activities including

antifungal and antibacterial activities It is also reported to have hypolipidemic

antiartherosclerosic anticancerogenic and antimutagenic activities It has been widely

used in folk medicine since ancient times in India Egypt Greece Rome and China for

a variety of diseases including abdominal pain (intestinal ulcer) parasitic infections

insect and snakes bites hemorrhoid rheumatism and other ailments Scientific studies

have demonstrated that garlic enhances natural killer (NK) activity T-lymphocyte

proliferation delayed type hypersensitivity (DTH) and humoral activity against sheep red

blood cells (SRBC) (Ghazanfari et al 2002)

Anacyclus pyrethrum (L) Link (family Compositae) and Alpinia galanga (L)

Willd (family Zingaberaceae) are widely used in folk remedies for several diseases The

roots of Anacyclus pyrethrum are used to stimulate salivary glands to cure chronic

catarrh of the head and nostrils and to clear the brain by exciting a free flow of nasal

mucous tears and by stimulating the blood flow to the tissues The rhizomes of Alpinia

galanga treat problems associated with the digestive system and relieve bronchitis

measles rubella and cholera Traditional medicine in Algeria has for centuries used the

roots of Anacyclus pyrethrum and Alpina galanga for the treatment of respiratory

infections A recent study demonstrated that the polysaccharide extracts of Anacyclus

pyrethrum and Alpinia galanga markedly enhanced the lymphocyte proliferation in vivo

(Bendjeddou et al 2003)

Anoectochilus formosanus Hayata from genus Anoectochilus (Orchidaceae) is

distributed from India the Himalayas Southeast Asia and Indonesia to New Caledonia

and Hawai It has been used by local people to cure snake bites Anoectochilus spp are

nowadays known as expensive folk medicinal plants used to treat cancer hypertension

diabetes mellitus hepatitis tuberculosis consumption and nephritis in Taiwan A

formosanus Hyata has been called ―King Medicine because of its diverse


19

pharmacological effects including antiinflammatory hepatoprotective activities and

antioxidant activities It has been reported that the methanol extract of A formosanus

exhibits antilipid peroxidation antisuperoxide formation and free-radical-scavenging

activity Furthermore it was shown that the aqueous extract of A formosanus might

completely prevent oxidative stress The aqueous extract of A formosanus could

enhance the viability and phagocytosis of macrophages indicating that it might not only

be used as a hepatoprotective drug but also as a prominent immunomodulating agent

(Tseng et al 2005)

Argyreia speciosa Sweet (Convolvulaceae) is a woody climber found throughout

India The roots of this plant have been regarded as alterative and tonic and are said to

be useful in rheumatism and diseases of the nervous system The leaves are used by

natives as a local stimulant and rubefacient in skin diseases Previous studies have

shown the plant seed oil to possess antibacterial and antifungal properties

Phytochemical screening of the plant has shown the presence of lipids flavonoids

triterpenes and phenylpropanoids The ethanolic extract of the roots of A speciosa was

found to stimulate the cellular and humoral immune system (Gokhale et al 2002)

Caesalpinia bonducella Fleming (Caesalpiniaceae) is a plant well known for its

medicinal value in Indian Ayurveda It is reported that this plant has multiple therapeutic

properties like antipyuretic antidiuretic anthelmintic and antibacterial antianaphylactic

and antidiarrheal antiviral antiasthmatic antiamoebic and antiestrogenic Futhermore

it has also been revealed that Caesalpinia bonducella has been traditionally used for the

treatment of tumor inflammation and liver disoders Besides the aqueous solution of

the outer shell of the seed of Caesalpinia bonducella has also been used traditionally by

the tribial people of Andaman and Nicober Islands for the relief of the symptoms of

diabetes mellitus Ethanolic seed extract of Caesalpinia bonducella seeds contain

alkaloid saponins terpenoids phenolics flavonoids and polysaccharides as essential

phytochemicasl A recent study demonstrated that ethanolic extract derived from

Caesalphinia seeds may stimulate both cellular and humoral immune responses and

can be served as an effective immunomodulatory candidate (Shukla et al 2009)

Tea from the plant Camellia sinensis L is one of the most popular beverages

consumed worldwide in its green black or prolong form It contains many compounds


20

such as polyphenols polysaccharides amino acids vitamins etc and it reduces the

risk of a variety of diseases The crude tea leaf extract contains high amount of

catechins which are the main polyphenols in green tea extracts Catechin-

polysaccharide complexes have been shown to be involved in the immunomodulating

activity of tea extracts (Monobe et al 2008)

Capparis zeylanica L (family Capparidaceae) commonly known as Indian

caper is a climbing shrub found throughout India and has been used as a Rasayana

plants particularly recommended for the treatment of immune disorders The various

species of genus Capparis are useful in the treatment of cough asthma inflammation

fevers Cholera and also useful as poultice in gout and rheumatism The study shows

that Capparis zeylanica is able to evoke a significant increase in percent neutrophils

that may help in increasing immunity of body against microbial infections The

administration of Capparis zeylanica significantly ameliorated the total white blood cell

(WBC) count red blood cell (RBC) count hemoglobin and platelets count and also

restored the myelosuppressive effects induced by cyclophosphamide The

investigations made by Ghule et al (2006) suggest that Capparis zeylanica is a potent

immunostimulatory agent stimulating both the specific and non-specific immune

mechanisms (Ghule et al 2006)

Cedrela lilloi and Trichilia elegans belonging to the Meliaceae family grow in

Argentina In traditional medicine they are used to treat fever malaria and rheumatisms

Both extracts showed a strong anticomplementary activity and inhibited the

phagocytosis of opsonized sheep erythrocytes and the activation of the oxidative

metabolism by opsonized zymosan on peritoneal macrophages The in vitro proliferation

of spleen T-lymphocytes was also impaired (Nores et al 1997)

Clausena excavata Burm f a wild shrub of the Rutaceae family is widely

distributed in south Asia It has been used in folk medicines for the treatment of cancer

and several disorders in the east of Thailand Its leaves and stems are also used for

treatment of colic cough headache rhinitis sore wounds yaws and detoxification The

main constituents of this plant have been revealed to be carbazole alkaloids and

coumarins The study by Manosroi et al (2003) demonstrated that aqueous acetone


21

and folklore extracts of Clausena excavata stimulated phagocytotic activity on

lysosomal enzymes (Manosroi et al 2003)

Hawthorn extract is among the most popular herbal remedies in European

countries as well as in the United States Preparations marketed as prescription or over-

the-counter medicines usually contain extracts derived from Crataegus laevigata and

other Crataegus spp Hawthorn extract is advocated as an oral treatment option for

chronic heart failure because of its cardiotonic and cardioprotective properties The

main finding of the study made by Dalli et al (2008) is that dry extract of leaves and

flowers of C laevigata inhibits a number of functional outputs of activated human

neutrophils oxidative burst elastase release chemotaxis and phagocytosis which are

potentially relevant to the pathophysiology of chronic cardiac failure (Dalli et al 2008)

Curculigo orchioides Gaerten is a small herb found in India Its tuberous roots or

rhizomes are used as an alterative demulscent diuretic restorative agent and for the

treatment of jaundice It is also the component of several Ayurvedic tonics In China it is

being used for the treatment of decline in strength The root powder is said to stop

bleeding and heal wounds Methanolic extracts of the roots have been shown to

enhance phagocytic activity of macrophages (Lakshmi et al 2003)

Curcuma xanthorrhiza Roxb has traditionally been used in Indonesia as a

traditional medicine in the treatment of various diseases This plant possesses a variety

of biological activities including antiinflammatory anticarcinogenic wound-healing and

serum cholesterol-lowering effects The study by Kim et al 2007 shows that crude

polysaccharide extract (CPE) from this plant induces macrophage activation and

immunostimulating activity through the NF-kB signalling pathway (Kim et al 2007)

The discovery and identification of new antitumor drugs which can potentiate the

immune function has become an important goal of research in immunopharmacology

and oncotherapy Lycium barbarum plays multiple roles in pharmacological and

biological functions in well-known Chinese traditional medicine L barbarum

polysaccharidendashprotein complex (LBP) is its important bioactive component It was

reported that the crude LBP could significantly suppress the growth of malignant tumor

in vivo The study by Gan et al 2004 shows that LPB of those plants can significantly

inhibit the growth of mouse transplantable sarcoma It also could markedly increase


22

macrophage phagocytosis antibody secretion by spleen cells and spleen lymphocyte

proliferation It also increased the IL-2 production and decreased lipid peroxidation (Gan

et al 2004)

Pfaffia paniculata (Brazilian ginseng) is a plant of Brazilian origin It has been

indicated as a tonic aphrodisiac analgesic and for antidiabetic purposes Antineoplastic

properties are also attributed to the roots of this plant Furthermore it has also been

shown to present analgesic and antiinflammatory effects The main isolated

components of P paniculata are stigmasterol sitosterol and their glycosides allantoin

pfaffic acid and the saponins termed pfaffosides A B C D E and F The study by

Pinello et al 2005 demonstrated that the methanolic extract of Pfaffia paniculata

increases the spreading index of macrophages (Pinello et al 2005)

Pouteria cambodiana (Pierre ex Dubard) Baehni is a plant of the Sapotaceae

family widely distributed in Asia The decoction of its bark has been orally taken daily

by breast feeding mothers for lactation promotion in Thailand Other parts of this plant

have been used in folklore medicines for the treatment of nausea vomiting fever and

back pain Bark decoction are used as antipyretic in Mexico as well as to treat skin

eruptions in Cuba The study by Manosroi et al 2005 showed that extracts from

Pouteria cambodiana (Pierre ex Dubard) Baehni present in vitro immunomodulatory

activity due to macrophage phagocytotic activity on lysosomal enzyme and splenocyte

proliferation These effects explain at least in part the Thai folklore application of this

plant in the treatment of fever and skin eruption (Manosroi et al 2005)

Tephrosia purpurea L (Lenguminosae) belongs to the Ayurvedic system of

medicine The whole plant has been used to cure tumours ulcers leprosy allergic and

inflammatory conditions such as rheumatism asthma and bronchitis Phytochemical

investigations on T purpurea have revealed the presence of glycosides rotenoids

isoflavones flavones chalcones flavanols and sterols The study made by Damre et

al 2003 demonstrated that oral administration of flavonoid fraction of Tephrosia

purpurea (FFTP) significantly inhibited sheep RBCs-induced delayed type

hypersensitivity reactions (Damre et al 2003)

The table below summarizes the immunomodulatory plant effects described

above


23

Table 1-2 Known plants with immunodulatory activity

Name Distribution Activity Folklore treatment Effect References

Allium sativum

(Amaryllidaceae) Asia Africa

Europe

Immunomodulatory antibiotic antifungal

antibacterial hypolipedemic

antiartherosclerosis anticarcinogenesis

Pain (intestinal ulcer) parasitic infection insect and snake bites hemorrhoid

rheumatism and other ailments

Enhances DTH NK activity and T-lymphocyte

prolifeation

Ghazanfari et al 2002

Anacyclus pyrethrum (L)

(Compositae) Alpinia galanga (L)

Willd (Zingaberaceae)

Algeria Immunostimulating

Anacyclus pyrethrum stimulates salivary glands to cure chronic catarrh

of the head and nostrils Alpinia galanga problems with the digestive system relieve bronchitis

measles rubella and cholera Citrullus colocynthis cancer breast inflammation adenopathy anemia fever ulcers bronchitis diabetes

Stimulate the immune response by acting on macrophages from the

systemic immune compartment

Bendjeddou et al 2003

Anoectochlus formosanus

Hayata (Orchidaceae)

Taiwan

Antitumor immunostimulating antiinflammation hepatoprotective

antioxidant

Cancer hypertension diabetes mellitus hepatitis tuberculosis consumption and

nephritis

Enhance the viability and phagocytosis of peritoneal

macrophages

Tseng et al 2005


24

Argyreia speciosa (Convolvulaceae)

India Immunomodulatory

Ayurveda medicine rheumatism and disease of nervous system skin

diseases Plant seed oil antibacterial and anti-fungal activity

Stimulate cellular and humoral immunity Effects phagocytic function of the

cells of the reticuloendothelial

system

Gokhale et al 2002

Caesalpinia bonducella FLEMINS

(Caesalpiniaceae)

India

Myanmar and Sri Lanka

Immunomodulatory antipyuretic antidiuretic

anthelmintic antibacterial

antianaphylactic antidiarrheal antiviral

antiasthmatic antiamoebic and antiestrogenic

Tumor inflammation liver disorders

diabetes mellitus

Stimulate cellular and humoral immune

responses increase the amount of neutrophils

ameliorate the total WBCs RBCs

haemoglobin and platelets count

Shukla et al 2009

Capparis zeylanica Linn

(Capparidaceae) India Immunostimulatory Immune disoders

Enhances the phagocytic function ameliorated the

total WBCs RBCs haemoglobin and platelets

count

Ghule et al 2006

Cedrela lilloi and Trichilia elegans

(Meliaceae) Argentina Immunomodulatory Fever malaria rheumatism

Inhibit T-lymphocyte proliferation phagocytic capability and oxidative

metabolism of peripheral blood monocytes and

polymorphonuclear cells

Nores et al 1997

Clausena excavate Burm f (Rutaceae)

South Asia Immunomodulatory Cancer and several disorders colic

cough headache rhinitis sore wounds

Inhibit phagocytic activity

of macrophages splenocyte proliferation

Manosroi et al 2003


25

Curculigo orchioides

(Amaryllidacea)

India in the sub-tropical Himalayas

Immunostimulatory

Alterative demulscent diuretic restorative and for treatment of jaundice The root powder is said to stop bleeding

and heal wounds

Increase macrophage phagocytosis activity

Lakshmi et al 2003

Curcuma

xanthorrhiza (Zingiberaceae)

Indonesia Antiinflammatory anticarcinogenic

immunostimmulating

Wound healing cholesterol-lowering effect

Phagocytosis activity of macrophages lysosomal

enzyme activity splenocyte proliferation

Kim et al 2007

Lycium barbarum (Solanaceae)

China Immunomodulating

Against human pathogenic bacteria and fungi

Increase macrophages phagocytosis lymphocyte

proliferation IL-2 expression decrease of

lipid peroxidation

Gan et al 2004

Pfaffia paniculata (Amaranthaceae)

Brazil

Anticancer analgesic antiinflammmatory

Tonic aphrodisiac and analgesic purposes for diabetes mellitus

treatment

Increase of macrophage spreading and phagocytosis

Pinello et at 2005

Pouteria

cambodiana (Sapotaceae)

Brazil Immunomodulatory

Lactation promotion treatment of nausea vomiting fever back pain

Antipyretic in Mexico skin eruptions in Cuba

Enhance macrophage phagocytosis

degranulation splenocyte proliferation lysosomal enzyme activity inhibit

oxidative burst

Manosroi et al 2005

Tephrosia purpurea

(Leguminosae)

India Immunomodulatory To cure tumors ulcers leprosy allergic

rheumatism asthma and bronchitis

Significantly inhibited sheep red blood cells-induced delayed type

hypersensitivity reaction

Damre et al 2003


26

In earlier times all drugs and medicinal agents were derived from natural

substances and most of these remedies were obtained from higher plants Today

many new chemotherapeutic agents are synthetically derived based on rational drug

design The study of natural products has advantages over synthetic drug design since

they may lead to the discovery of new structural features with novel biological activities

Not only do higher plants continue to serve as important sources of new drugs but

phytochemicals derived from them are also extremely useful as lead structures for

synthetic modification and optimization of bioactivity The starting materials for about

one-half of the medicines we use today derive from natural sources Virtually every

pharmacological class of drugs includes a natural product prototype The future of

higher plants as sources of medicinal agents for use in investigation prevention and

treatment of diseases is very promising

Immunomodulation using medicinal plants can provide an alternative to

conventional chemotherapy for a variety of diseases especially when host defense

mechanism has to be activated under the conditions of impaired immune response or

when a selective immunosuppression is desired in situations like autoimmune disorders

There is great potential for the discovery of new specific immunomodulators which

mimic or antagonize the biological effects of cytokines and interleukins Natural

remedies should be revisited as important sources of novel ligands capable of targeting

specific cellular receptors (Alamgir and Uddin 2010)

14 The UAH natural products drug discovery group

The Natural Products Drug Discovery Group at the University of Alabama in

Huntsville (UAH) is interested in tropical rainforest plants as sources of new drugs This

group has been looking for new anticancer drugs new antibacterial agents and new

antiviral drugs since 2000 They have recorded a hit rate of over 30 (a hit being the

discovery of a plant that appears to have interesting biological properties) Extracts that

show potential medicinal utility are subjected to bioactivity-directed fractionation and

isolation The structures of the active components are then elucidated using

spectroscopic and crystallographic techniques


27

With funds from the National Institutes of Health members of the group are using

their experience in plant biochemistry and ecology to find and harvest medicines from

tropical rainforest plants The Natural Products Drug Discovery Group has been

studying tropical plants for over ten years - rainforest plants from Costa Rica as well as

tropical Australia The Group currently has field laboratories located in Monteverde

Costa Rica and Paluma North Queensland Australia (Setzer 2011)

From Monteverde Costa Rica they have already collected 165 species

representing 61 families Crude plant extracts were tested for in vitro bactericidal and

fungicidal activity as well as cytotoxic and antiherpes activity 123 extracts exhibited

notable cytotoxicity 62 showed antibacterial activity 4 showed antifungal activity and 8

showed promising antiviral activity (Setzer et al 2003)

Here are some representative publications of the UAH Natural Products Drug

Discovery Group about the plants from Monteverde Costa Rica

Hassanzadeh SL Tuten JA Vogler B Setzer WN (2010) The chemical

composition and antimicrobial activity of the leaf oil of Cupressus lusitanica from

Monteverde Costa Rica Pharmacognosy Research 2 19-21

Cupressus lusitanica has been planted in Monteverde as a windbreak to protect

dairy cows from harsh winds The leaves of this plant are used to cure some skin

diseases caused by dermatophytes and have also been used to ward off insects from

stored grain In Costa Rica a drink made by steeping a branch in alcohol is taken to

alleviate coughs and cold symptoms In this report the authors present the leaf

essential oil composition and antimicrobial activity of C lusitanica from Monteverde

Costa Rica

Setzer WN Vogler B Schmidt JM Petty JL Haber WA (2005) Isolation of

cupanioside a novel cytotoxic and antibacterial long-chain fatty alcohol

glycoside from the bark of Cupania glabra Planta Medica 71 686-688

The crude dichloromethane extract from the stem bark of Cupania glabra

(Sapindaceae) showed in-vitro cytotoxic activity against Hep G2 MDA-MB-231 Hs

578T MCF-7 and PC-3 cells and antibacterial activity against Bacillus cereus

Staphylococcus aureus and Escherichia coli Bioactivity-directed fractionation led to


28

isolation of the novel 1-O-[234-tri-O-acetyl-a-L-rhamnopyranosyl-(1reg2)-b-D-

glucopyranosyl] hexadecanol (cupanioside) as the cytotoxic agent The authors

elucidated its structure by analysis of 2D-NMR spectra

Setzer MC Moriarity DM Lawton RO Setzer WN Gentry GA Haber WA

(2003) The phytomedicinal potential of tropical cloudforest plants from

Monteverde Costa Rica Revista Biologica Tropical 51 647-674

A pharmacological survey of plants from Monteverde Costa Rica including 165

species representing 61 families has been carried out The authors tested this crude

plant extracts for in vitro bactericidal and fungicidal activity as well as cytotoxic and anti-

herpes activity Of these 123 extracts exhibited notable cytotoxicity 62 showed

antibacterial activity 4 showed antifungal activity and 8 showed promising antiviral

activity These results underscore the phytomedicinal potential of Neotropical cloud

forests

Preliminary experiments performed by my supervisor Dr Maren von Koumlckritz-

Blickwede revealed that an acetone extract of the bark from Guarea kunthiana

(Meliaceae) is able to boost the phagocytosis of FITC-labelled S aureus bioparticles

However those results were only performed once (n = 1) and therefore needed to be

repeated and validated Based on those preliminary results I focussed on this plant

extract in my doctoral thesis

15 Guarea kunthiana

Guarea kunthiana from the plant family Meliaceae was found at the Monteverde

Cloud Forest Reserve (103483 N 847633 W 1530 m above sea level) and a voucher

specimen (Haber 3799) has been deposited in the Missouri Botanical Garden

Herbarium

The plant family Meliaceae has been a rich source of biosynthetically modified

triterpenes that have useful biological activity eg insect antifeedants Their structural

complexity and diversity attracted the attention of the chemists (Tinto et al 1991)


29

G kunthiana commonly known as cocora (Ecuador) or jatuauba (Brasilia) is

widely distributed in South and Central America It is found in lowland as well as

mountain tropical moist forest and swamps

G kunthiana is used in folk remedies for several diseases The juice of the

crushed bark of this tree is used in Ecuador in the treatment of malaria (DeFilipps et al

2004) It is also used to treat post-natal depression reputed to be good for the

treatment of asthma and stomachache (Coelho et al 2006) The topical application of

aqueous infusion made from the leaf of this plant is particularly recommended for

healing of wounds as a disinfectant (Tene et al 2007) In Brazilia this plant was

traditionally used in medicine as an antiinflammatory agent (de Mesquita et al 2005)

G kunthiana shows also activity against different protozoan infections

Crude extracts of G kunthiana were tested against leishmaniasis Results

showed an inhibition greater than 50 at a concentration of 15 microgmL and an inhibitory

concentration (IC) 50 value ranging from 5 to 10 microgmL for the hexanic extracts of G

kunthiana root against promastigote forms of Leishmania donovani (de Mesquita et al

2009)

Chagas disease also known as American Trypanosomiasis is caused by the

flagellated protozoan Trypanosoma cruzi Chagas and its transmission to vertebrate

hosts is carried out by haematophagous insects from the Triatominae subfamily

through feces contamination via mucosa or skin wounds Pyrethroids have been the

main insecticides used against these insects However some populations of insects

have shown significant levels of resistance to several pyrethroids indicating the need of

new insecticides for the control of triatomines (Coelho et al 2006) Root or stem

extracts of G kunthiana have been discussed as alternative drug against the insects

Table 1-3 shows the effect of crude extract of G kunthiana (root ethanol stem hexane

and ethanol) on the mortality of Rhodnius milesi a member of the family Triatominae


30

Table 1-3 Mortality rate of R milesi when topically applied with extract from G kunthiana (n = 20) (Coelho et al 2006)

Species

Plant

part

used

Solvent

Mortality rate ()

24 h 48 h 72 h 7 days 14 days 21 days 28 days

G kunthiana

Root Hexane 0 5 5 5 5 5 5

Ethanol 0 0 5 10 15 20 25

Stem

Hexane 25 30 35 40 40 40 40

Ethanol 10 15 15 20 20 20 20

Leaves Ethanol 5 5 5 5 5 5 5

For the extract application on tripanosomes 50 microg of the extract were resolved in

acetone or ethanol Triatomines were observed over a 28-day period Hexanic and

ethanolic extracts of G kunthiana caused mortality between 5 and 40 of R milesi in

comparison with the controls which showed no insect mortality However the hexanic

stem extract of G kunthiana presented the most rapid activity since it killed 25 of the

bugs 24 h after the application and 40 after seven days of the experiment (Coehlo et

al 2006)

The main components isolated from the G kunthiana were ecuadorin a novel

tetranortrirerpenoid (Tinto et al 1991) and limonoids (Espindola 2006) thus

accounting for a wide variety of therapeutic effects of G kunthiana such as antibacterial

and antimalarial functions

Different parts of G kunthiana leaves (hexane ethanol) fruits (hexane ethanol)

stem (wood + bark) (hexane dichloromethane ethanol) root (wood + bark) (ethanol)

were tested for cytotoxic activity against human cancer tumor cells but results were not

significant (de Mesquita et al 2005)

All these studies show that extracts from G kunthiana might be promising

candidates as immunomodulatory agents So far nothing is known about the effect of G

kunthiana extracts on neutrophil functions as the first line of defence against bacterial

infections


31

16 Fluoroquinolones

General structure

Quinolones are entirely synthetic antibacterial drugs with the first representative

(nalidixic acid) having been synthesized in 1962 Their structures are based on a 4-oxo-

14-dihydroquinolone skeleton (Labro 2000) They have evolved from agents used

solely for the treatment of urinary tract infections to molecules with potent activity

against a wide spectrum of significant bacterial pathogens with resultant broad clinical

utility This evolutionary pattern has arisen through the development of new core and

side-chain structures with associated improvements in activity pharmacokinetics and

tolerability and through the selection of molecules that remain useful and well tolerated

(Ball 2000)

All quinolones with antibacterial activity have a 4-quinolone nucleus with a

nitrogen atom at position 1 a carboxyl group at position 3 and a ketone at position 4

The structure of the quinolones has developed along two parallel pathways the

naphthyridones in which a carbon atom is substituted for nitrogen at position 8 of the

naphthyridine nucleus As mentioned above the first member of the quinolone class of

antibiotics was nalidixic acid which has limited antibacterial activity against Gram-

positive bacteria (Owens et al 2000) Since then thousand of compounds have been

synthesized of which the 6-fluorinated molecules (fluoroquinolones) represent a

breakthrough in 4-quinolone research (Labro 2000)

Recently interest in the quinolone antibiotics has intensified following the

publication of clinical and pre-clinical data confirming their potential for use in treatment

of tuberculosis (TB) (Duncan and Barry 2004) Data from quinolone studies suggest

that these drugs may be used to shorten the duration of chemotherapy In the

development of fluoroquinolone containing third-line regimens moxifloxacin was found

to be superior to ofloxacin or levofloxacin when killing of bacteria were achieved in nine

months (Veziris et al 2003 Duncan and Barry 2004)

All fluoroquinolones have a fluorine substitution at the 6-position which confers

greater antibacterial potency and a broader spectrum of activity (Owens et al 2000)

than that of nalidixic acid and other nonfluorinated quinolones (eg cinoxacin oxilinic


32

acid) Addition of the fluorine and piperazine moiety at positions 6 and 7 respectively

substitution of carbon for nitrogen at position 8 and modification of the side chain at

position 1 yielded the second generation agents ciprofloxacin and ofloxacin Other

modifications to the fluoroquinolone structure yielded third generation agents (eg

levofloxacin sparfloxacin grepafloxacin) with an improved antibacterial spectrum of

activity greater potency and an extended half-life Recently a fourth generation of

quinolones (eg gatifloxacin moxifloxacin trovafloxacin) with expanded coverage

against anaerobes has been developed (Ball 2000 Owens et al 2000) The methoxy

group at position 8 of moxifloxacin and gatifloxavin theoretically may confer enhanced

activity against resistant Gram-positive bacteria and reduce development of resistance

(Owens et al 2000)

Distribution

Fluoroquinolones achieve tissue concentrations that are at least as high as

achievable plasma concentrations with the exception of the central nervous system and

the eye This is true in many species including cattle Interestingly fluoroquinolones are

rapidly accumulated in macrophages and neutrophils achieving an intracellular

concentration four to eight times higher than the extracellular concentration In vivo the

concentration of fluoroquinolones in alveolar macrophages (Ams) was 14-18 times

higher than in serum (Schoevers et al 1999) A high concentration in leukocytes may

explain the higher fluoroquinolone concentration in infected tissue compared to healthy

tissue (The United States Pharmacopeial Convention 2007)

Effects of quinolones on bacteria

The quinolones are bactericidal agents These agents rapidly inhibit DNA

synthesis Inhibition of DNA gyrasestopoisomerases prevents supercoiling of the

chromosomal DNA Only in a supercoiled state the chromosomal DNA fits into the

bacterial cell If the chromosomal DNA is in a relaxed state the corresponding bacterial

cell will burst (Wolfson and Hooper 1989)


33

17 Enrofloxacin

Fluoroquinolones are gaining widespread acceptance in veterinary medicine

because of their wide spectrum of activity and favorable pharmacokinetic behavior

They generally present very good activities against a broad spectrum of aerobic

bacteria including Pasteurella spp and against Mycoplasma Generally

fluoroquinolones are characterized by excellent tissue penetration high bioavailabilities

and long terminal half-life (de Lucas et al 2008) Enrofloxacin is a synthetic

chemotherapeutic agent from the class of the fluoroquinolone carboxylic acid derivatives

It was developed in 1983 and was the first fluoroquinolone approved for use in animals

Enrofloxacin was first synthesized after a series of chemical modifications of nalidixic

acid The antibacterial properties and absorption of the molecule were increased and

their adverse effects were reduced (Ellakany et al 2007)

Enrofloxacin presents 14-dihydro-1-cyclopropyl-7-(4-ethyl-1-piperazinyl)-6-

fluoro-oxo-3-quinolo 3-quinoline carboxylic acid The molecular formula is

C19H22FN3O3 A crystalline active substance with faint yellow color that was obtained to

develop in high purity is hardly soluble in water at pH 7 But as the molecule contains

acidic and basic groups it is easily dissolved at both alkaline and acidic pH

(Babaahmady and Khosravi 2011)

Enrofloxacin is a fluoroquinolone exclusively developed for companion and farm

animals including cattle Like other fluoroquinolones enrofloxacin exhibits a broad

spectrum of antibacterial activity Enrofloxacin has demonstrated a significant post-

antibiotic effect for both Gram-negative and Gram-positive bacteria and is active in both

stationary and growth phases of bacterial replication It is effective against

Pseudomonas Klebsiella Escherichia Enterobacter Campylobacter Shigella

Salmonella Aeromonas Haemophilus Proteus Yersinia Serratia Vibrio Brucella

Chlamydia trachomatis Staphylococcus (including penicillinase-producing and

methicillin-resistant strains) Mycoplasma Mycobacterium (Babaahmady and Khosravi

2011)

Enrofloxacin has a similar spectrum of activity as ciprofloxacin but it has been

shown that enrofloxacin has a better bioavailability With the exception of cerebral-

spinal fluid enrofloxacin attains therapeutic levels in most body tissues (see Figure 1-


34

4) so it has been formulated as the antibiotic of choice for the treatment of difficult

infections particularly those that need long-term antibiotics like osteomyelitis sinusitis

infections otitis difficult soft-tissue infections peritonitis and pleuritis or pneumonia

Animals with impaired kidney or liver function may need extra monitoring and dose

adjustments to prevent excess drug accumulation because enrofloxacin is eliminated

by both renal and hepatic metabolism (Babaahmady and Khosravi 2011)

The bactericidal activity of enrofloxacin is concentration-dependent with

susceptible bacterial cell death occurring within 20ndash30 minutes of exposure

Figure 1-4 Mean concentrations of enrofloxacin in plasma subcutaneous and intramuscular interstitial fluid (ISF) and pleural fluid in calves after a subcutaneous injection of 125 mgkg Concentration (microgmL) shown on a semilogarithmic axic (Dosogne et al 2002)

In cattle enrofloxacin is usually used for the treatment of bovine respiratory

disease complex caused by Mannheimia haemolytica Pasteurella multocida and

Histophilus somni (formerly known as Haemophilus somnus) (Davis et al 2007) and

also to treat digestive diseases (de Lucas et al 2008)

The most common bacterial infection in dairy cows is mastitis which is

economically the most costly disease in the dairy industry Escherichia coli is one of the

most common causes of mastitis in dairy cattle It is known that the incidence of acute

coliform mastitis is highest between parturition and peak lactation 40 of all cases of

Escherichia coli mastitis occur during the first 4 weeks after calving (Hoeben et al

2000) Acute coliform mastitis is generally treated with antibiotics amongst other drugs


35

Unfortunately treatment is often initiated too late as the inflammatory cascade has

already started by the time the farmer observes signs of mastitis

Enrofloxacin treatment reduces bacterial growth within the mammary gland

(Monfardini et al 1999) and accelerates recovery of milk production during E coli

mastitis (Hoeben et al 1999) Therefore enrofloxacin is often chosen as the drug of

choice for the treatment of mastitis

Figure 1-5 Growth of E coli (CFU ml milk) in milk of challenged quarters during induced E coli mastitis Control (white symbol) and enrofloxacin treated (black symbol) early post partum dairy cows Significant differences between the control and the enrofloxacin-treated group at each time point if they occur are indicated with asteriks (Dosogne et al 2002)

However efficient removal of invading bacteria requires both activity of the

antibiotic against the bacteria and an optimal activity of the immune system of the

animal (Hoeben et al 2000) Interestingly enrofloxacin has been shown to stimulate

the release of oxidative burst in bovine neutrophils (Figure 1-6) thereby improving the

antimicrobial capabilities of neutrophils against E coli Luminol-enhanced phorbol-12-

myristate-13-acetate (PMA)-stimulated chemiluminescence was used to measure the

respiratory burst activity of the isolated polymorphonuclear leukocytes Increasing doses

of enrofloxacin enhanced chemiluminescence (Hoeben et al 1999)


36

Figure 1-6 Influence of different doses of antibiotics on chemiluminescence in a cell-free system Chemiluminescence index = (AUC of the antibiotic-incubated cells) (AUC of the vehicle treated cells) 100 danofloxacin Na

+-ceftiofur penicillin spiramycin enrofloxacin erythromycin X

oxytetracycline chloramphenicol (Hoeben et al 1999)

But although enrofloxacin increased oxidative burst it did not increase

phagocytosis of E coli (Figure 1-7) (Hoeben et al 1999) However the detailed

mechanisms behind this phenomenon still remined to be determined

Figure 1-7 Influence of different doses of antibiotics on myeloperoxidase activity measured in terms of oxidation of ortho-dianisidine danofloxacin Na

+- ceftiofur penicillin spiramycin

enrofloxacin erythromycin X oxytetracycline+ sulphadiazine chloramphenicol


37

18 Goal

As already mentioned in the introduction the efficient removal of invading

bacteria requires both activity of the antibiotic against the bacteria and an optimal

activity of the immune system (Hoeben et al 2000) The immune system protects the

body from microbes that invade and harm the host Neutrophils which make up about

60 percent of all white blood cells are the largest cellular component of the immune

system They are the first immune cells recruited from the bloodstream to a site of

inflammation Neutrophils mature in the bone marrow and when terminally

differentiated they are released into the blood stream where they have a short life span

of only a few hours (Brinkmann and Zychlinsky 2007)

Neutrophils are essential for host defense against many bacterial and fungal

agents To fulfill this function neutrophils have a large antimicrobial arsenal at their

disposal When neutrophils are activated in response to inflammatory stimuli the major

events that occur include chemotaxis phagocytosis secretion of enzymes

(degranulation) and the oxidative bursts of metabolism (Brown and Roth 1990) In

2004 Brinkmann and colleagues described a striking new observation that activation of

neutrophils causes the release of web-like structures of DNA The formation of

neutrophil extracellular traps (NETs) has been recognized as a novel and important

mechanism of the host innate immune response against infections (reviewed by von

Kockritz-Blickwede and Nizet 2009)Neu

An alternative approach for the treatment of difficult infections such as those

involving antimicrobial resistance or compromised host immunity could be the

pharmacological enhancement of the antimicrobial capabilities of neutrophils

The overall goal of this study is to search for (1) novel natural products (Chapter

3) and (2) known pharmacological products (Chapter 4) with the ability to boost the host

immune defense against bacterial infections (3) to test how these products can modify

the main functions of neutrophils phagocytosis degranulation and formation of

neutrophil extracellular traps (Chapter 3 and 4)

Based on preliminary data obtained by my supervisor Dr Maren von Koumlckritz-

Blickwede I characterized the effect of bark extracts of G kunthinia on human and

bovine neutrophil functions Furthermore the biochemical mechanisms behind the


38

effect were studied (Chapter 3) In the fourth chapter I investigated the effect of

enrofloxacin on bovine neutrophil functions

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Mooto BS Jativa C Tinto WF Reynolds WF and McLean S (1991) Ecuadorin a novel tetranotriterpenoid of Guarea kunthiana structure elucidation by 2-D NMR spectroscopy Canadian Journal of Chemistry 70 1260-1264

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Newman DJ and Cragg GM (2007) Natural products as sources of new drugs over the last 25 years Journal of Natural Products 70 461-477

Nores MM Courreges MC Benencia F Couombie FC (1997) Immunomodulatory activities of Cedrela lilloi and Trichilia elegans aqueous leaf extracts Journal of Ethnopharmacology 55 99-106

Owen RC and Ambrose PG (2005) Antimicrobial safety focus on fluororquinolones Clinical Infectious Diseases 41 144-157

Papayannopoulos V and Zychlinsky A (2009) NETs a new strategy for using old weapons Cell Press 30 513-520

Pinello KC Fonseca Ede S Akisue G Silva AP Salgado Oloris SC Sakai M Matsuzaki P Nagamine MK Palermo Neto J Dagli ML (2006) Effects of Pfaffia paniculata (Brazilian ginseng) extract on macrophage activity Life Sciences 78 1287-1292

Rates SMK (2001) Plants as source of drugs Toxicon 39 603-613

Sarkozy G (2001) Quinolones a class of antimicrobial agents Veterinary Medicine-Czech 46 257-274

Scharlach A Wagner D Dreesman J Pulz M (2011) Antimicrobial resistance monitoring in Lower Saxony (ARMIN) first trends for MRSA ESBL-producing Escherichia coli and VRE from 2006 to 2010 Gesundheitswesen 73 744-747

Schoevers EJ van Leengoed LAMG Verheijden JHM and Niewold TA (1999) Effects of enrofloxacin on porcine phagocytic function Antimicrobial Agents and Chemotherapy 43 2138-2143

Segal AW (2005) How neutrophils kill microbes Annual Review of Immunology 23 197-223


42

Setzer WN (2011) Drugs from the cloudforest the search for new medicines from Monteverde Costa Rica Natural Product Communications 6 1549-1548

Setzer WN Vogler B Schmidt JM Petty JL Haber WA (2005) Isolation of cupanioside a novel cytotoxic and antibacterial long-chain fatty alcohol glycoside from the bark of Cupania glabra Planta Medica 71 686-688

Setzer MC Moriarity DM Lawton RO Setzer WN Gentry GA Haber WA (2003) Phytomedicinal potential of tropical cloudforest plants from Monteverde Costa Rica Revista Biologica Tropical 51 647-674

Soares GMS Figueiredo LC Faveri M Cortelli SC Duarete PM Feres M (2012) Mechanisms of action of systemic antibiotics used in periodontal treatment and mechanisms of bacterial resistance to these drugs Journal of ApplIied Oral Science 20 295-309

Szmolka A Anjum MF La Ragione RM Kaszanyitzky EJ Nagy B (2012) Microarray based comparative genotyping of gentamicin resistant Escherichia coli strains from food animals and humans Veterinary Microbiology 156 110-118

Taylor L (2000) Plant based drugs and medicines httprainforest-databasecomplantsplantdrugshtm

Tene V Malagon O Finzi PV Vidari G Armijos C Zaragoza T (2006) An ethnobotanical survey of medicinal plants used in Loja and Zamora-Chinchipe Ecuador Jounal of Ethnopharmacology 111 63-81

Tinto WF Reynolds WF and McLean S (1991) Ecuadorin a novel tetranortriterpenoid of Gurea kunthiana structure elucidation by 2-D NMR spectroscopy Canadian Journal of Chemistry 70 1260-

Todar K (2002) Todars Online Textbook of Bacteriology httptextbookofbacteriologynet 1-580

Tseng C-C Shang H-F Wang L-F Su B Hsu C-C Kao H-Y Cheng K-Y (2006) Antitumor and immunostimulating effects of Anoedtochilus formosanus Hayata Phytomedicine 13 366-370

Urban F Lourido S and Zychlinsky A (2006) How do microbes evade neutrophil killing Cellular Microbiology 8 1687-1696

Veziris N Truffot-Pernot C Aubry A Jarlier V and Lounis N (2003) Fluoroquinolone-containing third-line regimen against Mycobacterium tuberculosis in vivo Antimicrobial Agents and Chemotherapy 47 3117-3122

von Koumlckritz-Blickwede M Nizet V (2009) Innate immunity turned inside-out antimicrobial defence by phagocyte extracellular traps Journal of Molecular Medicine 87 775-783


43

Wolfson JS and Hooper DC (1989) Fluoroquinolone antimicrobial agents Clinical Microbiology Reviews 2 378-424

Wright GD (2010) Antibiotic resistance where does it come from and what can we do about it Journal of Biology 8 1-6

Chapter 2

Materials and methods

Materials and Methods Chapter 2

47

21 Plant material

Guarea kunthiana (G kunthiana) was collected in May 2003 from the

Monteverde Cloud Forest Reserve Costa Rica (10 171 N 84 471 W 1570 m

about sea level) The tree was identified by William A Haber and a voucher


Herbarium

22 Drugs

Enrofloxacin (Bayer Animal Health GmbH) was dissolved in 01 M

natriumhydroxid (NaOH) at a concentration of 10 mgmL per stock For all

experiments a final concentration of 10 microgmL enrofloxacin was used

23 Bacterial strains

Escherichia coli K-12 and Staphylococcus aureus Newman were used in this

study E coli K-12 was grown in LB medium at 37degC with shaking Fresh overnight

cultures were diluted 1100 in LB and then grown to logarithmic growth phase (OD600

= 05) Bacterial suspensions were used directly for the following experiments by

diluting the bacteria in respective cell culture media to the desired concentration

aiming a multiplicity of infection (MOI) of 21 colony forming units (cfu) per cell

S aureus Newman was grown in Brain-Heart Infusion (BHI) medium at 37degC

with shaking Fresh overnight cultures were diluted 1100 in BHI and then grown to

logarithmic growth phase (OD600 = 05) After centrifugation at 3000 g for 10 min the

pellet was resuspended in sterile phosphate-buffered saline (PBS) to reach the

desired bacterial concentration of MOI 2

24 Preparation of plant extract

The bark was chopped and air-dried 4559 g dried bark was extracted with

refluxing acetone using a Soxhlet extractor for 4 hours The solvent was evaporated

to give 43 g crude extract For further experiments the crude extract was dissolved

in dimethyl sulfoxide (DMSO) 10 mgmL per stock and tested with a final

concentration of 1 microgmL


48

25 Effect of Guarea kunthiana on growth of bacteria

For growth curve analysis midlog phase of bacterial cultures were diluted

1100 in respective medium in the presence or absence of 1 microgmL G kunthiana bark

extract or vehicle control (DMSO) incubated at 37degC + 5 CO2 and bacterial density

(optical density at 600 nm) was measured hourly

26 Isolation and preparation of human neutrophils

Human neutrophils were isolated from healthy donors by using the

PolymorphPrep system (Axis-Shield) as previously described (von Koumlckritz-

Blickwede et al 2010) Briefly neutrophils were separated at the gradient interface

washed with H2O to lyse erythrocytes and resuspended in RPMI (Roswell Park

Memorial Institute medium) containing 2 nuclease-free fetal calf serum (FCS)

which was heat-inactivated at 70degC as described by von Koumlckritz-Blickwede et al

(2009) Neutrophil viability was checked by Trypan dye exclusion test using

hemocytometer and the count was adjust to 2 106 cellmL Neutrophils were treated

with G kunthiana extract or respective vehicle control for 2 h at 37degC in 5 CO2

Figure 2-1 Density gradient before and after centrifugation of PolymorphPrep with human blood


49

27 Isolation and preparation of bovine neutrophils

Bovine neutrophils were isolated from fresh blood of healthy animals by

density gradient centrifugation using Biocoll Therefore 20 mL of blood were layered

on top of 15 mL Biocoll (Biochrom) and centrifuged at 1100 g for 30 min at 10degC

(without brake) The supernatant including mononuclear cells and plasma was

discarded The resulting cell pellet including erythrocytes and granulocytes was

harvested Cells were washed with H2O to lyse erytrocytes and were resuspended in

RPMI containing 2 nuclease-free (heat-inactivated at 70degC) fetal calf serum (FCS)

Cell viability was checked by Trypan blue dye exclusion test using hemocytometer

and the cell suspension was adjusted to 2 106 cellsmL Neutrophils were treated

with G kunthiana extract for 5 h or with enrofloxacin (10 microgmL) for 2 h at 37degC in 5

CO2

Figure 2-2 Density gradient before and after centrifugation of Biocoll with bovine blood

28 Neutrophil killing assay

Neutrophils were plated in nontreated tissue culture plates at a concentration

of 5 105 cells250 microL The cells were treated with 1 microgmL G kunthiana bark extract

or with 25 nM phorbol 12-myristate 13-acetate (PMA) and incubated for 2 h (human

neutrophils) or 5 h (bovine neutrophils) at 37degC in 5 CO2 To block phagocytosis


50

neutrophils were additionally treated with 10 microgmL cytochalasin D for 15 min After

incubation the neutrophils were infected with bacteria (E coli K-12 or S aureus

Newman) at a MOI of 2 The plates were centrifuged at 370 g for 5 min and

incubated for 30 min at 37degC in 5 CO2 Serial dilutions in sterile PBS were plated on

agar plates for enumeration of surviving bacteria The percentage of surviving

bacteria was calculated in comparison to bacterial growth control grown under the

same conditions in the absence of cells

29 Determination of phagocytotic uptake

Neutrophils (5 105 cells250 microL) were treated with G kunthiana acetone bark

extract (human neutrophils for 2 h bovine neutrophils for 5 h) or with enrofloxacin for

2 h at 37degC in 5 CO2 Then 10 microL heat-killed fluorescein isothiocyanate (FITC)-

labeled Escherichia coli (K-12 strain Sigma) and 5 microl Staphylococcus aureus (Wood

strain Sigma) were incubated with neutrophils for 30 min at 37degC in 5 CO2 The

cells were washed with PBS and centrifuged at 370 g to remove non-phagocytosed

bacteria FITC-fluorescence was measured by Beckman Coulter EPICS XL Flow

Cytometer and recorded on a logarithmic scale from 1 to 5000 Mean fluorescence

intensity represents the mean percent number of FITC-labeled E coli and S aureus

attached or ingested per neutrophil

210 NET visualization and quantification

Neutrophils (1 106 cells500 microL) were seeded on poly-L-lysine-coated cover

slides and treated with G kunthiana (human neutrophils for 2 h bovine neutrophils

for 5 h) or with enrofloxacin for 2 h at 37degC in 5 CO2 After incubation cells were

fixed with 4 paraformaldehyde washed with PBS and blocked with 2 bovine

serum albumin (Sigma) in PBS + 02 Triton X-100 for 45 min at room temperature

To visualize NETs the slides were incubated overnight at +4degC with antibodies

against histone H2A-H2B-DNA complex (mouse monoclonal anti-H2A-H2B-DNA

PL2ndash6 05 microgmL Losman et al 1992) After incubation the slides were washed 3

times with PBS and incubated for 45 min at room temperature with secondary

antibodies Alexa fluor 488 rabbit anti-mouse IgG (1500 Invitrogen) After washing


51

the slides were mounted on glass slides using Prolong Gold with 46-diamidino-2-

phenylindole (DAPI) (Invitrogen) Washing steps were conducted with PBS and the

antibodies were diluted in 2 BSA-PBS + 02 Triton X-100 Images were recorded

using a Leica TCS SP5 confocal microscope with a HCX PL APO 40 075-125 oil

immersion objective Settings were adjusted with control preparations using an

isotype control antibody The total amount of neutrophils and the amount of

neutrophils releasing NETs per field of view were counted in 5 individual images per

sample

211 Degranulation

Neutrophils (2 106 cellsmL) were treated with G kunthiana extract (human

neutrophils for 2 h bovine neutrophils for 5 h) or with enrofloxacin for 2h at 37degC in

5 CO2 Degranulation of neutrophils after treatment with G kunthiana or

enrofloxacin was measured by the Beckman Coulter EPICS XL Flow Cytometer via

measurement of the cell size (sideward scatter SSC)

212 Oxidative burst

Oxidative burst was determined by change in fluorescence resulting from

oxidation of the fluorescent probe 2rsquo7rsquo-dichlorofluorescein Briefly 5 105 cells250

microL were treated with G kunthiana (human neutrophils for 2 h bovine neutrophils for

5 h) or with enrofloxacin for 2 h at 37degC in 5 CO2 After incubation cells were then

incubated with fluorescent dye 27-dichlorofluorescein (DCF 10 microM) for 30 min at

37degC in 5 CO2 The cells were washed with PBS The oxidative burst was analyzed

by respective fluorescent probes using Beckman Coulter EPICS XL Flow Cytometer

213 Effect of nocodazole and cytochalasin D on the Guarea kunthiana or

enrofloxacin-mediated NET formation

Neutrophils (1 105 cellsmL) were treated with G kunthiana or enrofloxacin

in the presence or absence of nocodazole (10 microM Sigma) or cytochalasin D (10 microM

Sigma) for 2 h at 37degC and 5 CO2 Images were recorded using a Leica TCS SP5


52

Confocal Microscope The total amount of neutrophils and the amount of neutrophils

releasing NETs per field of view were counted in 5 individual images per sample

214 Western blotting analysis of PAD-4 expression

A total amount of 2 107 cells were incubated in the presence of 10 microgmL

enrofloxacin or vehicle control for 2 h at 37degC then centrifuged for 7 min at 140 g

The cell pellet was resuspended in 200 microL lysis buffer with proteinase inhibitors and

incubated for 1 h at 4degC under constant agitation Then the samples were centrifuged

for 15 min at 13000 g and 4degC Equal protein amounts from each sample

(enrofloxacin-treated and vehicle control cells) were denaturated in boiling Laemmli

buffer + 001 DTT for 5 min Samples were separated in 10 SDS-PAGE After

electrophoresis proteins were transferred to PVDF membranesThe membranes

were blocked for 1 h at room temperature in Tris-buffered saline + 01 Tween 20

(TBS) (supplemented with 5 fat free dried milk and 3 bovine serum albumin) and

then incubated with primary antibodies polyclonal rabbit anti-PAD-4 (12000 (Wang et

al 2004)) and monoclonal mouse anti-β-Actin (110000 Santa Cruz) diluted in TBS

(supplemented with 2 fat free dried milk) overnight at 4degC under agitation After

washing in TBS blots were incubated for 1 h at room temperature with the respective

secondary anti-rabbit (15000) for PAD-4 and anti-mouse (15000) for β-Actin

antibodies diluted in TBS with 5 fat free dried milk Membranes were washed in

TBS and followed by development with Super Signal West Femto Chemiluminescent

Substrate reagents (Pierce Thermo Scientific)

215 Livedead viabilitycytotoxicity assay

Bovine neutrophils (1 106 cells500 microL) were plated in 24 well plates and

treated with enrofloxacin for 2 h at 37degC in 5 CO2 After incubation cells were

washed with PBS to reduce unspecific background staining and stained for 30 min at

room temperature in the dark with 150 microL LiveDead ViabilityCytotoxicity Assay kit

for mammalian cells (Invitrogen) Images were recorded using a Leica TCS SP5

Confocal Microscope as described above


53

216 Measurement of membrane integritycell death

Neutrophils were isolated and treated as described above Then the cells were

centrifuged at 370 g for 5 min the supernatant was harvested and release of lactate-

dehydrogenase (LDH) as a marker for cell membrane integritycell death was

quantified using the CytotoxONE Reagent (Promega) as recommended by the

manufacturer Total cell LDH levels (100) were determined using cells lysed with

02 Triton X-100

217 Statistical analysis

Data were analyzed using Excel 2003 (Microsoft) and GraphPad Prism 50

(GraphPad Software) All experiments were performed at least three independent

times Differences between the two groups were analyzed by using a paired one-

tailed Studentrsquos t-test The significance is indicated as p lt 005 p lt 0005

References

Brinkmann V Reichard U Goosmann C Fauler B Uhlemann Y Weiss DS Weinrauch Y Zychlinsky A (2004) Neutrophil extracellular traps kill bacteria Science 3031532-1535

Boothe DM (1994) Enrofloxacin revisited Veterinary Medicine 8 744-753

Chow OA von Koumlckritz-Blickwede M Bright AT Hensler ME Zinkernagel AS Cogen AL Gallo RL Wang Y Glass CK Nizet V (2010) Statins enhance formation of phagocyte extracellular traps Cell Host amp Microbe 8 445-454


Fuch TA Abed U Goosmann C Hurwitz R Schulze I Wahn V Weinrauch Y Brinkmann V and Zychlinsky A (2007) Novel cell death program leads to neutrophil extracellular traps The Journal of Cell Biology 176 231-241

Hoeben D Dosogne H Heyneman R Burvenich C (1997) Effect of antibiotics on the phagocytotic and respiratory burst activity of bovine granulocytes European Journal of Pharmacology 332 289-297

Kabelitz D and Kaufmann SHE (2010) Immunology of infections Methods in Microbiology 37 139-160


54

Kaufmann SH and Kabelitz D (2010) Immunology of infection immunology Academic Press 139-160

Koul O and Walia S (2009) Comparing impacts of plant extracts and pure allelochemicals and implications for pest control Perspectives in Agriculture Veterinary Science Nutrition and Natural Resources 4 29-30 Lacy P (2006) Mechanism of degranulation in neutrophils Allergy Asthma and Clinical Immunology 2 1-11

Lindner R Naim HY (2009) Domains in biological membranes Experimental Cell Research 315 2871-2878

Losman MJ Fasy TM Novick KE Monestier M (1992) Monoclonal autoantibodies to subnucleosomes from a MRLMp(-)++ mouse Oligoclonality of the antibody response and recognition of a determinant composed of histones H2A H2B and DNA Journal of Immunology 148 1561-1569

Menegazzi R Decleva E and Dri P (2012) Killing by neutrophil extracellular traps fact or folklore Blood 119 1214-1216


Neeli I Dwivedi N Khan S Radic M (2009) Regulation of extracellular chromatin release from neutrophils Journal of Innate Immunity 1 194ndash201 Papayannopoulos V and Zychlinsky A (2009) NETs a new strategy for using old weapons Cell Press 30 513-520

Pohlit AM Rezende AR Baldin ELL Lopes NP de Andrade Neto VF (2011) Plant extracts isolated phytochemicals and plant-derived agents which are lethal to arthropod vectors of human tropical diseases - A Review Planta Medica 77 618-630 Urban F Lourido S and Zychlinsky A (2006) How do microbes evade neutrophil kiling Cellular Microbiology 8 1687-1696

von Koumlckritz-Blickwede M and Nizet V (2009) Innate immunity turned inside-out antimicrobial defence by phagocyte extracellular traps Journal of Molecular Medicine 87 775-783

von Koumlckritz-Blickwede M Chow O Ghochani M Nizet V (2010) Visualization and functional evaluation of phagocyte extracellular traps Methods in Microbiology 37 139-160


55

Wang Y Li M Stadler S Correll S Li P Wang D Hayama R Leonelli L Han H Grigoryev SA Allis CD Coonrod SA (2009) Histone hypercitrullination

mediates chromatin decondensation and neutrophil extracellular trap formation Journal of Cell Biology 184 205-213

Wang Y Wysocka J Sayegh J Lee YH Perlin JR Leonelli L Sonbuchner LS McDonald CH Cook RG Dou Y Roeder RG Clarke S Stallcup MR Allis CD Coonrod SA (2004) Human PAD-4 regulates histone arginine methylation

levels via demethylimination Science 306 279-283

Yipp BG Petri B Salina D Jenne CN Scott BN Zbytnuik LD Pittman K Asaduzzaman M Wu K Meijndert HC Malawista SE de Boisfleury CA Zhang K Conly J Kubes P (2012) Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo Nature Medicine 18 1386-1393

Yousefi S Gold JA Andina N Lee JJ Kelly AM Kozlowski E Schmid I Straumann A Reichenbach J Gleich GJ Simon HU (2008) Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense Nature Medicine 14 949-953

Yousefi S Mihalache C Kozlowski E Schmid I Simon HU (2009) Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps Cell Death and Differentiation 16 1438-1444

Chapter 3

Guarea kunthiana bark extract enhance the antimicrobial

activities of human and bovine neutrophils

Natalja Jerjomiceva1 Hisham Seri

12 Ragheda Yaseen

1 William N Setzer

3 Hassan Y Naim

1 Maren

von Koumlckritz-Blickwede1

1Department of Physiological Chemistry University of Veterinary Medicine Hannover Germany

2Department of Clinical Studies College of Veterinary Medicine Sudan University of Science and

Technology and 3Department of Chemistry University of Alabama Huntsville

corresponding authors Dr Maren von Koeckritz-Blickwede and Prof Dr Hassan Naim Department of

Physiological Chemistry University of Veterinary Medicine Hannover Buumlnteweg 17 30559 Hannover

Germany e-mail mkoeckbltiho-hannoverde hassannaimtiho-hannoverde

(Manuscript submitted)

58

The extent of Natalja Jerjomiceva contribution to the article is evaluated according to

the following scale

A has contributed to collaboration (0-33)

B has contributed significantly (34-66)

C has essentially performed this study independently (67-100)

1 Design of the project including design of individual experiments B

2 Performing of the experimental part of the study C

3 Analysis of the experiments C

4 Presentation and disscusion of the study in article form C

G kunthiana enhance the antimicrobial activit ies of neutrophils Chapter 3

59

Abstract

Ethnopharmacological relevance

Guarea kunthiana commonly known as cocora or jatuauba is widely distributed

in South and Central America It is used in folk remedies for several diseases eg

malaria stomachache or for desinfecting and healing of wounds The aim of this study

was to investigate the effect of G kunthiana bark extract on antimicrobial functions of

neutrophils as the first line of defense against infections


Neutrophils were isolated from fresh bovine or human blood of healthy donors by

density gradient centrifugation treated with G kunthiana crude acetone bark extract for

2 h or 5 h and then the antimicrobial activities against Escherichia coli or

Staphylococcus aureus were investigated

Results

G kunthiana bark extract exhibited no direct antimicrobial effect against the

bacteria However neutrophils treated with G kunthiana bark extract showed

significantly induced antimicrobial activity eg phagocytosis against E coli or S

aureus Interestingly using immunofluorescence microscopy we demonstrated that G

kunthiana bark extract induce the formation of a recently discovered innate immune

defense mechanism namely the formation of neutrophil extracellular traps (NETs) This

effect was abolished when treating the cells with nocodazole indicating that cytoskeletal

rearrangements are involved in this process

Conclusions

Our study lead to the conclusion that G kunthiana bark extract might have a

beneficial effect on the host innate immune system by boosting the antimicrobial

capacities of neutrophils as the first line of defense against invading pathogens


60

Introduction

The emergence of resistant microorganisms has now reached epidemic

proportions and poses great challenges to human and veterinary medicine An

alternative approach for the treatment of difficult infections such as those involving

antimicrobial resistance or compromised host immunity could be the pharmacological

enhancement of the antimicrobial capabilities of phagocytes The goal of this study was

to characterize the effect of plant extracts on the antimicrobial functions of neutrophils

as the first line of defense against infections

Guarea kunthiana A Juss commonly known as cocora (Costa Rica Ecuador) or

jatuauba (Brasilia) belongs to the plant family Meliaceae and is widely distributed in

South and Central America It is found in lowland as well as montane tropical moist

forest and swamps In folk remedies it is used for the treatment of several diseases The

juice of the crushed bark of this tree is used in Ecuador for the treatment of malaria

(DeFilipps et al 2004) It is also used to treat post-natal depression reputed to be good

for the treatment of asthma and stomachache (Coelho et al 2006) The topical

application of aqueous infusion made from the leaf of this plant is particulary

recommended for healing of wounds and as disinfectors (Tene et al 2007) In Brasilia

this plant was traditionally used in medicine as an anti-inflammatory agent (de Mesquita

et al 2005)

Crude extracts of G kunthiana showed an inhibition greater than 50 at a

concentration of 15 microgmL and an IC50 value ranging from 5 to 10 microgmL for the hexanic

extracts of G kunthiana root against promastigote forms of L donovani (de Mesquita et

al 2005) The hexanic stem extract of G kunthiana presented a very rapid and efficient

activity against Rhodnius milesi since it killed 25 of the bugs 24 h after the

application and 40 after seven days (Coelho et al 2006 Koul and Walia 2009)

The mechanisms of action how the extracts might act against malaria or other

pathogens as wound healing or as desinfecting agent are still not known Our present

study was undertaken to examine the effect of G kunthiana on the antimicrobial

activities of neutrophils as the first line of defense against infections


61


Plant material The plant was collected in May 2003 from the Monteverde Cloud Forest

Reserve Costa Rica (10 171 N 84 471 W 1570 m above sea level) The tree was

identified by William A Haber and a voucher specimen (Haber 3799) has been

deposited in the Missouri Botanical Garden Herbarium

Preparation of plant extract The bark was chopped and air-dried 4559 g dried bark

was extracted with refluxing acetone using a Soxhlet extractor for 4 hours The solvent

was evaporated to give 43 g crude extract For further experiments the crude extract

was dissolved in dimethyl sulfoxide (DMSO) 10 mgmL per stock and tested with a final


Isolation and preparation of human neutrophils Human neutrophils were isolated

from healthy donors by using the PolymorphPrep system (Axis-Shield) as previously

described (von Koumlckritz-Blickwede et al 2010) Briefly neutrophils were separated at

the gradient interface washed with H2O to lyse erythrocytes and resuspended in RPMI

containing 2 nuclease-free fetal calf serum (FCS) which was heat-inactivated at 70degC

as described by von Koumlckritz-Blickwede et al (2009) Neutrophil viability was checked

by Trypan dye exclusion test using hemocytometer and the count was adjust to 2 106

cellmL Neutrophils were treated with G kunthiana extract or respective vehicle control

for 2 h at 37degC in 5 CO2

Isolation and preparation of bovine neutrophils Bovine neutrophils were isolated

from fresh blood of healthy animals by density gradient centrifugation using Biocoll

Therefore 20 mL of blood were layered on top of 15 mL Biocoll (Biochrom) and

centrifuged at 1100 g for 30 min at 10degC (without brake) The supernatant including

mononuclear cells and plasma was discarded The resulting cell pellet including

erythrocytes and granulocytes was harvested Cells were washed with H2O to lyse

erytrocytes and were resuspended in RPMI containing 2 nuclease-free (heat-

inactivated at 70degC) fetal calf serum (FCS) Cell viability was checked by trypan blue

dye exclusion test using hemocytometer and the cell suspension was adjusted to 2

106 cellsmL Neutrophils were treated with G kunthiana extract or respective vehicle

control (DMSO) for 5 h at 37degC in 5 CO2


62

Bacterial strains Escherichia coli K-12 and Staphylococcus aureus Newman were

used in this study E coli K-12 was grown in LB medium at 37degC with shaking Fresh

overnight cultures were diluted 1100 in LB and then grown to logarithmic growth phase

(OD600 = 05) Bacterial suspensions were used directly for the following experiments by

diluting the bacteria in respective cell culture media to the desired concentration aiming

a multiplicity of infection (MOI) of 21 colony forming units (cfu) per cell

S aureus Newman was grown in Brain-Heart Infusion (BHI) medium at 37degC with

shaking Fresh overnight cultures were diluted 1100 in BHI and then grown to


pellet was resuspended in sterile phosphate-buffered saline (PBS) to reach the desired

bacterial concentration of MOI 2

Effect of Guarea kunthiana on growth of bacteria For growth curve analysis midlog

phase of bacterial cultures were diluted 1100 in respective medium in the presence or

absence of 1 microgmL G kunthiana bark extract or vehicle control (DMSO) incubated at

37degC + 5 CO2 and bacterial density (optical density at 600 nm) was measured hourly

Neutrophil killing assay Neutrophils were plated in nontreated tissue culture plates at

a concentration of 5 105 cells250microL The cells were treated with 1 microgmL G

kunthiana bark extract or with 25 nM phorbol 12-myristate 13-acetate (PMA) and

incubated for 2 h (human neutrophils) or 5 h (bovine neutrophils) at 37degC in 5 CO2 To

block phagocytosis neutrophils were additionally treated with 10 microgmL cytochalasin D

for 15 min After incubation the neutrophils were infected with bacteria (E coli K-12 or

S aureus Newman) at a MOI of 2 The plates were centrifuged at 370 g for 5 min and


agar plates for enumeration of surviving bacteria The percentage of surviving bacteria

was calculated in comparison to bacterial growth control grown under the same

conditions in the absence of cells

Determination of phagocytotic uptake Neutrophils (5 105 cells250 microL) were

treated with G kunthiana acetone bark extract (human neutrophils for 2 h bovine

neutrophils for 5 h) at 37degC in 5 CO2 Then heat-killed fluorescein isothiocyanate

(FITC)-labeled Escherichia coli (K-12 strain Sigma) and Staphylococcus aureus (Wood

strain Sigma) were co-incubated with neutrophils for 30 min at 37degC in 5 CO2


63

Afterwards the cells were washed with PBS to remove unbound bacteria FITC

fluorescence as a marker for phagocytosis was measured by Beckman Coulter EPICS

XL Flow Cytometer and recorded on a logarithmic scale from 1 to 5000 Mean

fluorescence intensity represents the mean percent number of FITC-labeled E coli and

S aureus attached or ingested per neutrophils

NET visualization and quantification Neutrophils (1 106 cells500 microL) were seeded

on poly-L-lysine-coated cover slides and treated with G kunthiana (human neutrophils

for 2 h bovine neutrophils for 5 h) at 37degC in 5 CO2 After incubation cells were fixed

with 4 paraformaldehyde washed with PBS and blocked with 2 bovine serum

albumin (Sigma) in PBS + 02 Triton X-100 for 45 min at room temperature To

visualize NETs the slides were incubated overnight at +4degC with antibodies against

histone H2A-H2B-DNA complex (mouse monoclonal anti-H2A-H2B-DNA PL2ndash6 05

microgmL Losman et al 1992) After incubation the slides were washed 3 times with PBS

and incubated for 45 min at room temperature with secondary antibodies Alexa fluor

488 rabbit anti-mouse IgG (1500 Invitrogen) After washing the slides were mounted

on glass slides using Prolong Gold with 46-diamidino-2-phenylindole (DAPI)

(Invitrogen) Washing steps were conducted with PBS and the antibodies were diluted

in 2 BSA-PBS + 02 Triton X-100 Images were recorded using a Leica TCS SP5

confocal microscope with a HCX PL APO 40 075-125 oil immersion objective

Settings were adjusted with control preparations using an isotype control antibody The

total amount of neutrophils and the amount of neutrophils releasing NETs per field of

view were counted in 5 individual images per sample

Degranulation Neutrophils (2 106 cellsmL) were treated with G kunthiana extract

(human neutrophils for 2 h bovine neutrophils for 5 h) at 37degC in 5 CO2

Degranulation of neutrophils after treatment with G kunthiana was measured by the

Beckman Coulter EPICS XL Flow Cytometer via measurement of the cell size (sideward

scatter SSC)

Oxidative burst Oxidative burst was determined by change in fluorescence resulting

from oxidation of the fluorescent probe 2rsquo7rsquo-dichlorofluorescein Briefly 5 105

cells250 microL were treated with G kunthiana (human neutrophils for 2 h bovine

neutrophils for 5 h) at 37degC in 5 CO2 After incubation cells were then incubated with


64

fluorescent dye 27-dichlorofluorescein (DCF 10 microM) for 30 min at 37degC in 5 CO2

The cells were washed with PBS The oxidative burst was analyzed by respective

fluorescent probes using Beckman Coulter EPICS XL Flow Cytometer

Effect of nocodazole on the Guarea kunthiana-mediated NET formation

Neutrophils (1 106 cells500 microL) were treated with G kunthiana and nocodazole (10

microM Sigma) for 2 h at 37degC in 5 CO2 Images were recorded using a Leica TCS SP5



Statistical analysis Data were analyzed by using a paired one-tailed t-test (GraphPad

Software) Experiments were performed as at least 3 independent experiments Values

less than 005 were considered significant

Results

Effect of Guarea kunthiana on growth of bacteria

To determine if the plant extract has a direct antimicrobial effect against bacteria

G kunthiana was tested for antimicrobial activity against E coli and S aureus as

prototype bacteria G kunthiana extract exhibited no direct antimicrobial effect on the

growth of bacteria E coli K-12 or S aureus Newman (Figure 3-1)

Figure 3-1 Effect of G kunthiana on the growth of (a) E coli K-12 and (b) S aureus Newman Bacteria and plant extract were diluted in medium and incubated at 37

ᵒC Optical density at 600 nm was measured

every 30 min


65

Effect of Guarea kunthiana on the antimicrobial activity of neutrophils

For testing antimicrobial activities of neutrophils after treatment with Guarea

kunthiana neutrophils were isolated from bovine or human blood by density gradient

centrifugation treated with G kunthiana (human neutrophils for 2 h bovine neutrophils

for 5 h) and then co-incubated with Gram-positive S aureus and Gram-negative E coli

for 30 min at 37ᵒC

Figure 3-2 Effect of G kunthiana on the antimicrobial activity of neutrophils Treatment of neutrophils with G kunthiana resulted in significant higher antimicrobial activity against the tested bacteria E coli (a) tested with human neutrophils after 2 h of co-incubation (n = 6) and with (c) bovine neutrophils after 5 h of incubation (n = 7) S aureus (b) with human neutrophils after 2 h of incubation (n = 5) and (d) with bovine neutrophils after 5 h of incubation (n = 5) p lt 005

The percentage of surviving bacteria compared to a growth control was

quantified Interestingly neutrophils treated with G kuntiana showed a distinctly

(

b)


66

enhanced antimicrobial activity against E coli as well as S aureus in human and bovine

neutrophils (Figure 3-2)

Classically neutrophils have been shown to exhibit two major antimicrobial

strategies to kill invading pathogens First the oxidative burst-dependent phagocytosis

which involves the engulfment and subsequent elimination of microbes in specialized

phagolysosome compartments and second oxidative burst-independent

degranulation which releases antimicrobial molecules into the extracellular milieu

(reviewed by von Koumlckritz-Blickwede and Nizet 2009) Recently a novel defense

mechanism of neutrophils was discovered namely the formation of neutrophil

extracellular traps (NETs) (Brinkmann et al 2004) NETs are extracellularly released

fibers that consist of nuclear and mitochondrial DNA with associated histones which are

able to entrap immobilize and kill various bacterial parasitic and fungal pathogens

(reviewed by von Koumlckritz-Blickwede and Nizet 2009)

In the following experiments the effect of G kunthiana on all mentioned

antimicrobial neutrophil strategies was tested phagocytosis degranulation and NET-

formation

Effect of Guarea kunthiana on phagocytotic uptake

First we characterized the effect of G kunthiana bark extract on phagocytic

uptake of S aureus and E coli fluorescent bioparticles

As shown in Figure 3-3 and Figure 3-4 there was a slight but a signifcant

induction of phagocytosis of E coli bioparticles detectable in human neutrophils after

treatment with G kunthiana compared to vehicle control In case of S aureus G

kunthiana only enhanced its uptake in case of bovine neutrophils Cytochalasin D was

used as negative control since it blocks the cytoskeletal rearrangements required for

phagocytosis of bacteria As shown in Figure 3-3 and 3-4 cytochalasin D completely

blocked the intracellular uptake of bacteria


67

Figure 3-3 Effect of G kunthiana on phagocytosis of bacteria in human neutrophils G kunthiana significantly induces uptake of (a) E coli in human neutrophils after 2 h of incubation (n = 4) but it has no effect on (b) S aureus (n = 3) Cytochalasin D was used as a negative control p lt 005

Figure 3-4 Effect of G kunthiana on phagocytosis of bacteria in bovine neutrophils G kunthiana has no effect on uptake of (a) E coli in bovine neutrophils after 5 h of incubation (n = 5) but it significantly induces uptake of (b) S aureus (n = 7) Cytochalasin D was used as a negative control p lt 005

(b)


68

In good correlation to above-mentioned data the phenotype shown in Figure 3-

2 that G kunthiana induces antimicrobial activity of neutrophils is abolished when

treating the cells with cytochalasin D (Figure 3-5) These data indicate that

phagocytosis is involved in the G kunthiana-induced antimicrobial activities of

neutrophils

Figure 3-5 Effect of cytochalasin D on antimicrobial activity of neutrophils (a) Percentage of surviving E coli (n = 3) in human neutrophils after 2 h of incubation and (b) S aureus (n = 5) in bovine neutrophils after 5 h of incubation Cytochalasin D inhibits phagocytosis and abolishes the G kunthiana-induced antimicrobial activity of neutrophils

Effect of Guarea kunthiana on degranulation

Neutrophils are able to release granule-derived mediators or antimicrobial

peptides by degranulation or exocytosis of membrane-bound secretory granules (Lacy

2006) Quantitative measurement of neutrophil degranulation after treatment with G

kunthiana was perfomed using flow cytometry based on quantification of granularity

(sideward-scatter SSC) As shown in Figure 3-6 G kunthiana significantly induces

degranulation in bovine neutrophils but not in human neutrophils PMA was used as

positive control and revealed significant degranulation as expected


69

Figure 3-6 Effect of G kunthiana on degranulation (a) in human neutrophils after 2 h of incubation (n = 4) and (b) in bovine neutrophils after 5 h of incubation (n = 3) Degranulation of neutrophils after treatment with G kunthiana was measured with flow cytometry and analyzed the cell size (sideward scatter) PMA was used as positive control p lt 005

Effect of Guarea kunthiana on NET Formation

Among the strategies that neutrophils use to kill invading microorganisms great

emphasis has recently been placed on the role of the above-mentioned NETs which

are nowadays considered as an important part of the neutrophil response to microbes

(Brinkmann et al 2004) Interestingly using immunofluorescent microscopy with NET-

specific antibodies we could demonstrate that G kunthiana significantly induces the

formation of NETs in both human as well as bovine neutrophils (Figure 3-7) Whereas

only approximately 20 of DMSO-treated control cells showed a spontaneous release

of NETs NET-formation increased up to 50 in the presence of G kunthiana bark

extract


70

Figure 3-7 G kunthiana significantly induces NET-formation (a) in human neutrophils after 2 h of incubation und (b) in bovine neutrophils after 5 h of incubation The total amount of neutrophils and the amount of neutrophils releasing NETs per field of view were counted in 5 individual images per sample The results of 4 independent experiments were analyzed p lt 005

Figure 3-8 Representative fluorescent image of NETs induced after treatment with G kunthiana Boosting of G kunthiana induces NETs formation in human neutrophils after 2 h of incubation NETs were visualized using a double-staining of DAPI to stain DNA (blue) monoclonal mouse anti-H2A-H2B-DNA complex antibody followed by an Alexa 488-rabbit anti-mouse antibody (green)

(

a)


71

Effect of Guarea kunthiana on oxidative burst

Formation of NETs has been characterized as a process that involves formation

of reactive oxygen species (ROS) by NADPH oxidases (Urban et al 2006) To see if

the blocking of NADPH-oxidases affects the G kunthiana-mediated NET-formation

human and bovine neutrophils were additionally incubated with diphenylene iodonium

(DPI) As shown in Figure 3-9 blocking of oxidative burst significantly decrease the G

kunthiana-mediated NET formation indicating that this NET-formation is ROS-

dependent

Figure 3-9 Effect of blocking of oxidative burst on the G kunthiana-mediated NET formation (a) in human neutrophils after 2h of incubation and (b) in bovine neutrophils after 2h of incubation The total amount of neutrophils and the amount of neutrophils releasing NETs per field of view were counted in 5 individual images per sample The results of 4 independent experiments were analyzed p lt 005

To determine if G kunthiana has a general effect on oxidative burst in human

and bovine neutrophils quantitative measurement of ROS was done with the

fluorescent dye 27-dichlorofluorescein using flow cytometry PMA was used as positive

control and revealed significant boosting of ROS formation in the cells However

treatment of neutrophils with G kunthiana significantly inhibited oxidative burst (Figure

3-10)


72

Figure 3-10 Effect of G kunthiana on oxidative burst (a) in human neutrophils after 2 h of incubation (n = 3) and (b) in bovine neutrophils after 5 h of incubation (n = 4) Oxidative burst (relative fluorescent indicated by x-mean value of the respective fluorescence detector) of neutrophils after treatment with G kunthiana was measured with flow cytometry PMA was used as positive control p lt 005

Effect of nocodozole on Guarea kunthiana-mediated NET formation

To test the involvement of microtubules in G kunthiana mediated NET formation

we incubated neutrophils with nocodazole a drug that interferes with tubulin

polymerization into microtubules As shown in Figure 3-11 significantly decreased the

G kunthiana-mediated NET production indicating that microtubules are involved in this

process

Figure 3-11 Effect of nocodazole on G kunthiana-mediated NET formation in bovine neutrophils Bovine neutrophils were treated with G kunthiana and 10 microM Nocodazole (Noc) for 5 h Images were recorded using a confocal microscope The total amount of neutrophils and the amount of neutrophils releasing NETs per field of view were counted in 5 individual images per sample The results of 3 independent experiments were analyzed p lt 005


73

Discussion

In conclusion we demonstrated that treatment of neutrophils with G kunthiana

crude acetone bark extract led to a significant boost of the antimicrobial activities of

bovine and human neutrophils against the Gram-positive bacterium S aureus and

Gram-negative E coli Interestingly G kunthiana significantly boosted various

antimicrobial activities of neutrophils First there is only a slight effect on the classical

neutrophil functions degranulation and phagocytosis Second G kunthiana showed a

significant induction of the formation of NETs The phenotype was clear in neutrophils

derived from human as well as bovine cells indicating that this is not a species-specific

effect

NETs have been originally discovered in 2004 (Brinkmann et al 2004) and have

been shown to consist of extracellularly released DNA with associated histones

proteases and antimicrobial peptides that are able to entrap and kill various microbes

Nowadays there is increasing evidence about the molecular mechanisms associated

with the release of NETs Neeli et al (2009) showed that microtubule depolymerization

by nocodazole prevented the LPS-induced NET-formation in human neutrophils (Neeli

et al 2009) In good accordance to these data we also observed that treatment of

bovine neutrophils with nocodazole prevented G kunthiana extract to induce NET

formation in the cells (Figure 3-11)

Furthermore we showed that NADPH oxidases are involved in the G kunthiana

ndashinduced NET-formation When we blocked neutrophil ROS production using the

NADPH oxidase inhibitor DPI the level of NET generation was clearly reduced

Combined with the observation that G kunthiana treatment reduced overall ROS

production in the neutrophils the evidence suggests that G kunthiana may predispose

cells to enter the NET-formation pathway in response to a lower threshold level of ROS

signal This phenotype is similar to a phenotype of NET-formation which was previously

shown for statins the pharmacological inhibitors of the 3-hydroxy 3-methylglutaryl

coenzyme A (HMG-CoA) reductase the rate-limiting enzyme in cholesterol biosynthesis

(Chow et al 2010) These data by Chow et al revealed a beneficial effect of statins on

S aureus clearance using in vivo ex vivo and in vitro models of phagocyte function


74

However the in vivo effect of G kunthiana on neutrophil functions remains to be

determined

In conclusion we can say that the bark from G kunthiana induces various

functional outputs of activated human and bovine neutrophils Future work will focus on

the characterizatoon of the bark extract components involved in immune boosting

functions The main components of the G kunthiana that have already been isolated

are terpenoids (Tinto at al 1991 Garcez et al 2004) and limonoids (Laila Espindola

2006) In general the Meliaceae has been a rich source of biosynthetically modified

triterpenes that have useful biological activity some are insect antifeedants and some

are cytotoxic and may be useful medicinally (Taylor 1984) Their structural complexity

and diversity attracts the attention of the chemist

Finally this project might help to identify new therapeutic targets based on

natural products which can be further developed as new therapeutic treatment

strategies against bacterial infections in human as well as animals In recent years the

emergence of numerous antibiotic resistant bacterial pathogens has led to an urgent

need for new antibacterial agents Augmentation of the host response by

immunomodulators is an alternative to the use of antibiotics in the prevention andor

treatment of infections caused by antibiotic-resistant bacteria (Ahn et al 2006)

Acknowledgements

This work was supported by a grant from Akademie fuumlr Tiergesundheit (AtF)

WNS is grateful to the Monteverde Cloud Forest Preserve and the Tropical

Science Center for granting permission to collect plant materials under a cooperative

rights agreement and to the Commission for the Development of Biodiversity of Costa

Ricarsquos Ministry of the Environment Energy and Telecommunications for Research

Permit R-001-2006-OT-CONAGEBIO


75

References

Ahn J-Y Choi I-S Shim J-Y Yun E-K Yun Y-S Jeong G and Song J-Y (2006) The immunomodulator gingsan induces resistance to experimental sepsis by inhibiting Toll-like receptormediated inflammatory signal European Journal of Immunology 36 37-45


Coelho AA de Paula JE and Espindola LS (2006) Insecticidal activity of Cerrado plant extracts on Rhodnius milesi Carcavallo Rocha Calvao and Jurberg (Hemiptera Reduviidae) under Laboratory Conditions Neutrophical Entmology 35 133-138


de Mesquita ML Paula JE Pessoa C de Moraes MO Costa-Lotufo LV Grougnet R Michel S Tillequin F Erspindola LS (2009) Cytotoxic activity of Brazilian Cerrado plants used in traditional medicien against cancer cell lines Journal of Ethnopharmacology 123 439-445

de Mesquita ML Desrivot J Bories C Fournet A de Pauna JE Grellier P Espindola LS (2005) Antileishmanial and trypanocidal activity of Brazilian Cerrado plants Memorias do Instituto Oswaldo Cruz 7 783-787 Espindola LS (2006) Limonoide de Guarea kunthiana com potencial leishmanicida 1-77

Fuch TA Abed U Goosmann C Hurwitz R Schulze I Wahn V Weinrauch Y Brinkmann V and Zychlinsky A (2007) Novel cell death program leads to neutrophil extracellular traps The Journal of Cell Biology 176 231-241 Garcez FR Garcez WS da Silva AFG de Caacutessia Bazzo R and Ubirazilda MR (2004) Terpenoid constituents from leaves of Guarea kunthiana Journal of Brazilian Chemical Society 5 767-772

Lacy P (2006) Mechanism of degranulation in neutrophils Allergy Asthma and Clinical Immunology 2 1-11


Neeli I Dwivedi N Khan S Radic M (2009) Regulation of extracellular chromatin release from neutrophils Journal of Innate Immunity 1 194ndash201


76

Kabelitz D and Kaufmann SHE (2010) Immunology of infections Methods in Microbiology 37 139-160 Kaufmann SH and Kabelitz D (2010) Immunology of infection immunology Academic Press 139-160

Koul O and Walia S (2009) Comparing impacts of plant extracts and pure allelochemicals and implications for pest control Perspectives in Agriculture Veterinary Science Nutrition and Natural Resources 4 29-30

Losman MJ Fasy TM Novick KE (1992) Monestier M Monoclonal autoantibodies to subnucleosomes from a MRLMp (-)++ mouse Oligoclonality of the antibody response and recognition of a determinant composed of histones H2A H2B and DNA Journal of Immunology 148 1561-1569




Pohlit AM Rezende AR Baldin ELL Lopes NP de Andrade Neto VF (2011) Plant extracts isolated phytochemicals and plant-derived agents which are lethal to arthropod vectors of human tropical diseases - A Review Planta Medica 77 618-630

Tene V Malagon O Finzi PV Vidari G Armijos C Zaragoza T (2007) An ethnobotanical survey of medicinal plants used in Loja and Zamora-Chinchipe Ecuador Journal of Ethnopharmacology 1 63-81

Urban F Lourido S and Zychlinsky A (2006) How do microbes evade neutrophil kiling Cellular Microbiology 8 1687-1696


von Koumlckritz-Blickwede M Chow O Ghochani M Nizet V (2010) Visualization and functional evaluation of phagocyte extracellular traps In Methods in Microbiology (Volume 37) Immunology of Infection Immunology 3rd edition (Kaufmann SH and Kabelitz D) Academic Press 139-160

Chapter 4

Enrofloxacin enhances the formation of neutrophil extracellular traps

in bovine granulocytes


12 Lena Voumlllger

1 Hassan Y Naim

1 Maren von Koumlckritz-Blickwede

1



Technology





78


the following scale








Enrofloxacin induces the formation of NETs in bovine neutrophils Chapter 4

79

Abstract

Several antibiotics are known for their ability to accumulate in neutrophils as the

first line of defence against pathogens This manuscript demonstrates for the first time

that an antibiotic namely the fluoroquinolone enrofloxacin enhances the formation of

bovine neutrophil extracellular traps (NETs) in a reactive oxygen species-and

cytoskeletal-dependent manner


80

Introduction

Intramammary infections as a major cause of mastitis in dairy cows have recently

received a lot of attention because of their major economic impact (Barlow 2011) The

fluoroquinolone enrofloxacin is authorized for lactating cattle and is one of the few

antimicrobial drugs that have been recommended for the treatment of S aureus and E

coli-induced mastitis due to its favorable pharmacokinetic and pharmacodynamic

properties Like other fluoroquinolones enrofloxacin exhibits a broad spectrum of

antibacterial activity against both Gram-positive and Gram-negative bacteria in

diseased animals (Boothe 1994) The bactericidal activity of enrofloxacin is

concentration-dependent and is caused by inhibition of the bacterial DNA gyrase (a type-

II topoisomerase) thereby preventing DNA supercoiling and DNA synthesis Interestingly

enrofloxacin has been shown to stimulate the oxidative burst of bovine granulocytes in

vitro at a concentration of 10 microgml (Hoeben et al 1997) which reflects a physiological

relevant concentration received in infected tissue upon treatment with enrofloxacin (Ziv

et al 1973) However it is completely unclear if enrofloxacin modulates additional

antimicrobial granulocyte functions

Granulocytes play a key role in the early innate immune defence against bacterial

infections during mastitis in dairy cows (Burvenich et al 1994) Until a few years ago

granulocytes eg neutrophils were thought to employ essentially two major antimicrobial

strategies to entrap and kill invading pathogens First the oxidative burst-dependent

phagocytosis which involves the engulfment and subsequent elimination of microbes in

specialized phagolysosome compartments and second oxidative burst-independent


(reviewed by von Koumlckritz-Blickwede and Nizet 2009) Recently a third strategy was

discovered namely the formation of neutrophil extracellular traps (NETs) (Brinkmann et

al 2004) NETs consist of nuclear and mitochondrial DNA with associated histones and

have recently been identified as a novel extracellular host innate immune defence

mechanism of granulocytes by mediating extracellular entrapment and subsequent

immobilization of invading pathogens (reviewed by von Koumlckritz-Blickwede and Nizet

2009)


81

The goal of this study was to investigate the effect of enrofloxacin on bovine

granulocyte functions with special focus on NET-formation and to characterize the

underlying biochemical mechanisms of the observed phenotypes


Drugs Enrofloxacin (Bayer Animal Health GmbH) was dissolved in 01 M

natriumhydroxid (NaOH) at a concentration of 10 mgmL per stock For all experiments

a final concentration of 10 microgmL enrofloxacin was used

Isolation and preparation of bovine granulocytes Bovine granulocytes were isolated

from fresh blood of healthy animals by density gradient centrifugation Therefore 20 mL

of blood were layered on top of 15 mL Biocoll (Biochrom) and centrifuged at 1100 g for

30 min at 10degC (without brake) The supernatant including mononuclear cells and

plasma was discarded The resulting cell pellet including erythrocytes and granulocytes

was harvested Cells were washed with H2O to lyse erytrocytes and were resuspended

in RPMI containing 2 nuclease-free (heat-inactivated at 70degC) fetal calf serum (FCS)

Granulocyte viabilitywas checked by trypan blue dye exclusion test using

haemocytometer and the cell suspension was adjusted to 2 106 cellsmL

Granulocytes were treated with enrofloxacin (10 microgmL) or respective vehicle control

(NaOH) for 2 h at 37degC in 5 CO2

Determination of phagocytic bacterial uptake Bovine granulocytes (2 106

cellsmL) were treated with enrofloxacin for 2 h at 37degC in 5 CO2 Then 10 microL heat-

killed fluorescein isothiocyanate (FITC)-labeled Escherichia coli (K-12 strain Sigma)

and 5 microl Staphylococcus aureus (Wood strain Sigma) were incubated with neutrophils

for 30 min at 37degC in 5 CO2 The cells were washed with PBS and centrifuged at 370

g to remove non-phagocytosed bacteria FITC-fluorescence was measured by Beckman

Coulter EPICS XL Flow Cytometer and recorded on a logarithmic scale from 1 to 5 000

Mean fluorescence intensity represents the mean percent number of FITC-labeled E

coli and S aureus attached or ingested per neutrophil

NET visualization and quantification Bovine neutrophils (1 106 cells500 microL) were

seeded on poly-L-lysine-coated cover slides and treated with enrofloxacin for 2 h at

37degC in 5 CO2 After incubation cells were fixed with 4 paraformaldehyde washed


82

with PBS and blocked with 2 bovine serum albumin (Sigma) in PBS + 02 TritonX-

100 for 45 min at room temperature To visualize NETs the slides were incubated

overnight at 4degC with mouse monoclonal anti-H2A-H2B-DNA antibodies (PL2ndash6

11000) (Losman et al 1992) After incubation the slides were washed 3 times with

PBS and incubated for 45 min at room temperature with secondary antibodies Alexa

fluor 488 goat anti-mouse IgG (1500 Invitrogen) After washing the slides were

mounted on glass slides using ProlongGoldreg antifade with DAPI (Invitrogen) Washing

steps were conducted with PBS and the antibodies were diluted in 2 BSA-PBS +

02 Triton X-100 After mounting slides were analyzed by confocal fluorescence

microscopy using a Leica TCS SP5 confocal microscope with a HCX PL APO 40 x 075-

125 oil immersion objective Settings were adjusted with control preparations using an

isotype control antibody For each preparation five randomly selected images were

acquired and used for quantification of NET-producing cells Data were expressed as

percentages of NET-forming cells in relation to the total number of cells or as area

covered with NETs The mean value derived from n = 5 images for each condition per

experiment was used for statistical analysis

Degranulation Bovine granulocytes (2 106 cellsmL) were treated with enrofloxacin

for 2 h at 37degC in 5 CO2 Degranulation of granulocytes after treatment with antibiotic

compared to vehicle control was measured by Beckman Coulter EPICS XL Flow

Cytometer using the sideward scatter (SSC)


from oxidation of the fluorescent probe 27-dichlorofluorescein (DCF) in response to

reactive oxygen species (ROS) Briefly 5 105 cells250 microL were treated with

enrofloxacin for 2 h at 37degC in 5 CO2 After incubation cells were then incubated with

10 microM DCF for 30 min at 37degC in 5 CO2 The cells were washed with PBS by

centrifugation The relative oxidative burst was analyzed using the fluorescence

detector FL-1 of a Beckman Coulter EPICS XL Flow Cytometer

Effect of nocodazole and cytochalasin D on the enrofloxacin-mediated NET

formation Bovine neutrophils (1 105 cellsmL) were treated with enrofloxacin or

NaOH vehicle control in the presence or absence of nocodazole (10 microM Sigma) or


83

cytochalasin D (10 microM Sigma) for 2 h at 37degC and 5 CO2 Microscopic quantification

of NETs was performed as described above

Western blotting analysis of PAD-4 expression A total amount of 2 107 cells were

incubated in the presence of 10 microgmL enrofloxacin or vehicle control for 2 h at 37degC

then centrifuged for 7 min at 140 g The cell pellet was resuspended in 200 microL lysis

buffer with proteinase inhibitors and incubated for 1 h at 4degC under constant agitation

Then the samples were centrifuged for 15 min at 13000 g and 4degC Equal protein

amounts from each sample (enrofloxacin-treated and vehicle control cells) were

denaturated in boiling Laemmli buffer + 001 DTT for 5 min Samples were separated

in 10 SDS-PAGE After electrophoresis proteins were transferred to PVDF

membranesThe membranes were blocked for 1h at room temperature in Tris-buffered

saline + 01 Tween 20 (TBS) (supplemented with 5 fat free dried milk and 3

bovine serum albumin) and then incubated with primary antibodies polyclonal rabbit

anti-PAD-4 (12000 (Wang et al 2004)) and monoclonal mouse anti-β-Actin (110000

Santa Cruz) diluted in TBS (supplemented with 2 fat free dried milk) overnight at 4degC

under agitation After washing in TBS blots were incubated for 1h at room temperature

with the respective secondary anti-rabbit (15000) for PAD-4 and anti-mouse (15000)

for β-Actin antibodies diluted in TBS with 5 fat free dried milk Membranes were

washed in TBS and followed by development with Super Signal West Femto

Chemiluminescent Substrate reagents (Pierce Thermo Scientific)

Livedead viabilitycytotoxicity assay Bovine neutrophils (1 106 cells500 microL) were

plated in 24 well plates and treated with enrofloxacin for 2 h at 37degC in 5 CO2 After

incubation cells were washed with PBS to reduce unspecific background staining and

stained for 30 min at room temperature in the dark with 150 microL LiveDead

ViabilityCytotoxicity Assay kit for mammalian cells (Invitrogen) Images were recorded

using a Leica TCS SP5 Confocal Microscope as described above

Measurement of membrane integritycell death Neutrophils were isolated and

treated as described above Then the cells were centrifuged at 370 g for 5 min the

supernatant was harvested and release of lactate-dehydrogenase (LDH) as a marker

for cell membrane integritycell death was quantified using the CytotoxONE Reagent


84

(Promega) as recommended by the manufacturer Total cell LDH levels (100) were

determined using cells lysed with 02 Triton X-100

Statistical analysis Data were analyzed using Excel 2003 (Microsoft) and GraphPad

Prism 50 (GraphPad Software) All experiments were performed at least three

independent times Differences between the two groups were analyzed by using a

paired one-tailed Studentrsquos t-test The significance is indicated as p lt 005 p lt 0005

and p lt 0001

Results and Discussion

Effect of enrofloxacinon antimicrobial defence strategies of bovine granulocytes

Primary blood-derived granulocytes were isolated from fresh blood of healthy cows by

density gradient centrifugation After treatment of the cells with 10 microgmL enrofloxacin or

vehicle control phagocytosis degranulation oxidative burst and NET-formation were

analyzed Quantitative measurement of neutrophil degranulation after treatment with

enrofloxacin was performed using flow cytometry based on quantification of cell

granularity using the sideward-scatter (SSC) The results in Figure 4-1 a show that

enrofloxacin has no effect on granularity of the cells PMA was used as positive control

and showed significant degranulation of the treated cells

In a next step neutrophils were incubated in the presence of fluorescent labeled

E coli or S aureus bioparticles In accordance with a previous publication by Hoeben et

al 1997 enrofloxacin treatment of the cells did not alter the phagocytosis of E coli

bioparticles (Figure 4-1 bi) However there was significant uptake of S aureus

bioparticles detectable after treatment with enrofloxacin compared to vehicle control

(Figure 3-1 bii) These data indicate that enrofloxacin modulates oxidative burst-

dependent defence strategies of granulocytes eg phagocytosis of S aureus


85

Oxidative burstDegranulation

Phagocytosis

E coli S aureus

a c

bi bii

CtrEnro

25nM P

MA

0

50

100

150

200

400

600

800

Rel

ativ

e o

xid

ativ

e b

urs

t

[x-M

ean

of F

L-1

]

Ctr

Enro

25nM P

MA

0

200

400

600

n s

Rel

ativ

e d

egra

nu

latio

n [S

SC

val

ue]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

20

25

n s

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

NET-formation

di dii

diii

Ctr

Ctr + D

PI

Enro

Enro +

DPI

0

10

20

30

NE

T-r

elea

sin

g c

ells

[]

Figure 4-1 Effect of enrofloxacinon antimicrobial defence strategies of bovine granulocytes Granulocytes were isolated by density gradient centrifugation and treated with 10 microgmL enrofloxacin for 2 h (a) Relative oxidative burst measured by flow cytometry using DCF as fluorescent probe (b) Relative degranulation measured by flow cytometry (c) Neutrophil phagocytosis of FITC-labeled bacteria (i) E coli and (ii) S aureus PMA was used as a positive control and the phagocytosis-blocking agent cytochalasin D was used as negative control (d) Formation of NETs visualized by immunofluorescence microscopy using an antibody against histone-DNA-complexes (green) and DAPI to stain DNA (blue) (di) and (dii) are showing representative images of the quantification depicted in (diii) The results of minimum 3 independent experiments were analyzed using a paired one-tailed t-test (p lt 005)


86

Thus to confirm that enrofloxacin alters the oxidative burst flow cytometry

studies were performed using the cell permeant fluorogenic probe 27-

dichlorofluorescein Again PMA was used as positive control to exclude technical

problems As shown in Figure 4-1c enrofloxacin significantly induced production of

ROS This finding goes in line with the publication from Hoeben et al who demonstrated

increased chemiluminescense of bovine granulocytes (Hoeben et al 1997)

Finally the formation of NETs was quantified using immunofluorescent

microscopy with histone (H1 and H2)-DNA-specific antibodies Interestingly we

demonstrated that enrofloxacin significantly induces the formation of NETs in bovine

granulocytes after 2 h of treatment with enrofloxacin (Figure 4-1 d) To see if blocking

of oxidative burst alters the enrofloxacin-dependent NET-induction bovine granulocytes

were incubated with DPI to block the NADPH-dependent formation of ROS As shown in

Figure 4-1 diii blocking of oxidative burst significantly decreased the enrofloxacin-

induced NET formation indicating that the phenomenon is ROS-dependent

Biochemical mechanisms associated with enrofloxacin-induced formation of

NETs Initially when NETs were discovered it was believed that cells die during an

active process of release of intracellular DNA and histones a process called NETosis

(Fuchs et al 2007) Recently it has been shown that NETosis involves viable cells eg

eosinophils or neutrophils that do not undergo lysis (Yousefi et al 2008 Yoursefi et al

2009) and retain the ability to multitask (Yipp et al 2012) Immunofluorescence

microscopic investigations shown in Figure 4-2 ai revealed that viable as well as dead

cells release extracellular DNA and may contribute to the enrofloxacin-mediated NET-

induction To quantify if enrofloxacin has a significant cytotoxic effect on the bovine

neutrophils we measured the membrane integrity of the cells by quantifying LDH

release of neutrophils after treatment with enrofloxacin (Figure 4-2 aii) Importantly we

found that enrofloxacin does not induce loss of cell membrane integrity and thus does

not impair viability of the cells


87

Cell death

Red = extracellular DNA (dead cell)

green = calcein activity (living cell)

NaO

HEnr

o

0

5

10

15 n s

LD

H r

ele

as

e in

[Co

mp

are

d t

o t

ota

l c

ell ly

sis

]

Enr

o

Enr

o +

Cyt

D

Ctr +

Cyt

D

0

5

10

15

20

25

ns

NE

T-r

ele

as

ing

ce

lls

[

]

NaOH

74 kDa

PAD4 M Enro

Ctr

Enr

o

0

50

100

150

200

rela

tiv

e P

AD

-4-p

rote

in e

xp

res

sio

n in

[co

mp

are

d t

o

-ac

tin

ex

pre

ss

ion

]

PAD-4-expression Role of cytoskeleton in

NET formation

ai

aii

bi

bii

ci

cii

Enr

o

Enr

o +

Noc

Ctr +

Noc

0

10

20

30

40

n s

NE

T-r

ele

as

ing

ce

lls

[

]

74 kDa

50 kDa

33 kDa

M Enro Ctr

PAD-4

β-actin

Figure 4-2 Biochemical mechanisms associated with enrofloxacin-induced formation of NETs Bovine

granulocytes were treated with 10 microgmL enrofloxacin for 2 h (ai) Viability of cells Representative fluorescent image of granulocytes stained with livedead viabilitycytotoxicity kit for mammalian cells (Cytoplasm of viable cells is stained in green and dead cells and extracellular DNA are stained in red) (aii) Membrane integritycell viability was measured by quantifying the release of LDH (b) Effect of enrofloxacin on PAD-4 protein expression as analyzed by Western Blot (bi) is showing a representative blot (bii) is indicating the densitometric quantification of PAD-4 band intensities compared to β-actin signal from 5 independent experiments (c) Effect of nocodazole and cytochalasin D on enrofloxacin-mediated NET formation in bovine neutrophils The results of minimum 3 independent experiments were analyzed using a paired one-tailed t-test

The histone hypercitrullination catalyzed by peptidylarginine deiminase 4 (PAD-4)

has been shown to be involved in the chromatin decondensation during formation of

NETs (Neeli et al 2009 Wang et al 2009) In good correlation to these data we

observed that enrofloxacin significantly induced the relative PAD-4 protein expression in

bovine granulocytes (Figure 4-2 b) To additionally test the involvement of microtubules


88

and actin filaments in enrofloxacin mediated NET formation we incubated neutrophils

with nocodazole a drug that interferes with tubulin polymerization into microtubules or

cytochalasin D a drug that disrupts the polymerization of actin filaments Both

treatments significantly decreased the NET production in bovine granulocytes after 2 h

of incubation (Figure 4-2 c) Thus our data indicate that enrofloxacin-mediated NET

formation requires functional tubulin and actin filaments similar as previously shown for

chromatin release by neutrophils in response to LPS (Neeli et al 2009)

Conclusions

In summary this is the first report which shows that an antibiotic chemotherapy

modulates the ROS-dependent formation of NETs as novel innate immune function of

granulocytes facilitating entrapment and subsequent immobilization of bacteria and

bacterial toxins Since the use of a bactericidal antibiotic could result in enhanced

release of endotoxins eg LPS from the Gram-negative bacterial cell membrane

antibiotic treatment is often asociated with massive plasma tumor necrosis factor-α

(TNF-α) concentrations and high inflammation However it is known that enrofloxacin

treatment of dairy cows with acute E coli mastitis is not associated with a significant

increase of LPS level in plasma (Dosogne et al 2002) Based on our findings it may be

hypothesized that enrofloxacin-mediated NET-induction might facilitate immobilization of

E coli and its released toxins and thereby diminishes high inflammatory reactions

during bacteriostatic antibiotic treatment

Overall the observed effects of enrofloxacin on bovine granulocyte functions

might be of importance during treatment of infectious diseases in normal and

immunocompromised animals In general the ability of an antibiotic chemotherapy to

induce NET-formation could substantially influence the management of an infection

Acknowledgements

We wish to thank Friederike Reuner for excellent tecnical assistance and

Yanming Wang for providing the PAD-4 antibody


89

Funding

This work was supported by a grant from the Akademie fuumlr Tiergesundheit (AfT)

References

Barlow J (2011) Mastitis therapy and antimicrobial susceptibility a multispecies review with a focus on antibiotic treatment of mastitis in dairy cattle Journal of Mammary Gland

Biology and Neoplasia 16 383-407



Burvenich C Paape MJ Hill AW Guidry AJ Miller RH Heyneman R Kremer WDJ Brand A (1994) Role of the neutrophil leukocyte in the local and systemic reactions during experimentally induced E coli mastitis in cows immediately after calving Veterinary Quarterly 16 45-50

Dosogne H Meyer E Sturk A van Loon J Massart-Leeumln AM Burvenich C (2002) Effect of enrofloxacin treatment on plasma endotoxin during bovine Escherichia coli mastitis Journal of Inflammatory Research 51 201ndash205

Fuchs TA Abed U Goosmann C Hurwitz R Schulze I Wahn V Weinrauch Y Brinkmann V Zychlinsky A (2007) Novel cell death program leads to neutrophil extracellular traps Journal of Cell Biology 176 231-41 Hoeben D Dosogne H Heyneman R Burvenich C (1997) Effect of antibiotics on the phagocytotic and respiratory burst activity of bovine granulocytes European Journal of Pharmacology 332 289-297





90

Wang Y Li M Stadler S Correll S Li P Wang D Hayama R Leonelli L Han H Grigoryev SA Allis CD Coonrod SA (2009) Histone hypercitrullination mediates

chromatin decondensation and neutrophil extracellular trap formation Journal of Cell Biology 184 205-213

Wang Y Wysocka J Sayegh J Lee YH Perlin JR Leonelli L Sonbuchner LS McDonald CH Cook RG Dou Y Roeder RG Clarke S Stallcup MR Allis CD Coonrod SA (2004) Human PAD-4 regulates histone arginine methylation levels

via demethylimination Science 306 279-283

Yipp BG Petri B Salina D Jenne CN Scott BN Zbytnuik LD Pittman K Asaduzzaman M Wu K Meijndert HC Malawista SE de Boisfleury Chevance A Zhang K Conly J Kubes P (2012) Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo Nature Medicine 18 1386-1393


Yousefi S Mihalache C Kozlowski E Schmid I Simon HU (2009) Viable neutrophils release mitochnondrial DNA to form neutrophil extracellular traps Cell Death and Differentiation 16 1438-1444

Ziv G Gordin S Bachar G Bernstein S (1973) Concentration and persistence of antibiotics in milk following intramammary infusion in cows Refuah Veterinarith 30 85-100

Chapter 5

General discussion and future outlook

General discussion and future outlook Chapter 5

93

Discussion

Infections caused by antibiotic resistant bacteria represent a significant burden to

healthcare systems worldwide today Resistance among commonly encountered

pathogens has emerged as a consequence of the selective pressures of antimicrobial

use The increased mortality and morbidity rates associated with infection by antibiotic-

resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and

vancomycin-resistant enterococci (VRE) have become a common problem and threat

to public health

The aim of the present study was to test natural and pharmacological products

for their ability to enhance the antimicrobial capabilities of the immune system

The immune system is one of naturersquos more fascinating creations that protects

the body against infectious organisms and other invaders If bacteria can develop

resistance to antibiotics the immune system can evolve not only a memory of specific

antibodies to the current infection and any similar to it but also the ability to fight more

effectively the next time it is challenged or attacked If antibiotics fail the immune

system will help us fight against antibotic resistant bacteria

The immune system is made up of a network of cells tissues and organs that

work together to protect the body Neutrophils constitute the dominant cell population in

the circulation that mediates the earliest innate immune responses to infections There

is a dramatic increase in mortality from infection in people with quantitative or qualitative

neutrophil defects thus providing clinical confirmation on the important role of

neutrophils in maintaining good health Thereby this study was focusing on neutrophils

as the first line of defence against invading pathogens

Beginning with a landmark study by Brinkmann et al (2004) the fundamental

conception of how and where neutrophils kill pathogenic microbes has been altered in a

most fascinating and provocative way In this study the formation of neutrophil

extracellular traps (NETs) has been recognized as a novel and important mechanism of

the host innate immune response against infections

NETs are DNA-based net-like fibers that mediate an antimicrobial function

outside the cell These structures bind microorganisms prevent their spreading and

ensure a high local concentration of antimicrobial agents capable of inhibiting or killing


94

the invading pathogens extracellularly (Kabelitz and Kaufmann 2010) Stimulation of

neutrophils results in the activation of NADPH oxidases and the formation of reactive

oxygen species (ROS) ROS signalling is required for the novel cell death pathway of

NETosis which is characterized by the disruption of the nuclear membrane chromatin

decondensation and the mixing of nuclear contents with cytoplasmic and granular

proteins As a final step nuclear and granular components are released by the dead cell

generating the extracellular NETs (reviewed by von Koumlckritz-Blickwede M and Nizet

V 2009)

A variety of different proinflammatory stimuli have been shown to activate

formation of NETs including hydrogen peroxide (H2O) bacterial lipopolysaccharide

(LPS) the mitogen phorbol myristate acetate (PMA) and the CXC family chemokine

interleukin 8 (IL-8) (Brinkmann et al 2004) Bactericidal activity of NET-associated

histones has been proven against Mycobacterium tuberculosis Escherichia coli

Shigella flexneri Salmonella enterica Staphylococcus aureus Streptococcus

pyogenes and Bacillus anthracis (Kawasaki et al 2008) Moreover NETs are effective

against a variety of different hyphae or yeast forms of Candida albicans (Urban et al

2006) and the protozoan parasite Leishmania amazonensis (Guimaraes-Costa et al

2009)

But importantly NETs are not effective against all bacteria and infections Some

microbes have a mechanism to avoid entrapment or killing by NETs Certain leading

bacterial pathogens have evolved mechanisms to avoid NET-based immune clearance

either through NET degradation resistance to the intrinsic antimicrobial effectors with

NETs or the suppression of NET production For example H influenzae is resistant

against NET-killing by surface lipooligosaccharides that allow the organism to survive

within NETs in the middle ear cavity (Hong et al 2009) S pneumoniae expression of

polysaccharide capsule a classical virulence factor of the pathogen significantly

reduced the trapping of the bacterium within NETs providing another potential

mechanistic contribution of capsule to disease progression Streptococcus agalactiae

suppress the NETs by sialic acid engagement of Siglec receptors and consequent

inhibitory signaling (Carlin et al 2009)


95

However recent research showed that boosting of NETs may improve the

antimicrobial capabilities of neutrophils and thereby improve the outcome of an MRSA

infection in mice This study shows that in response to the main bacterial pathogen S

aureus statins were able to enhance formation of NETs and promote bacterial killing

(Ohn et al 2010) These data lead to the suggestion that NETs might serve as a novel

therapeutic target against S aureus infections Interestingly during my doctoral thesis

it was discovered that the bark extract of G kunthiana and the antibiotic enrofloxacin

are able to boost the formation of NETs

G kunthiana is widely used in folk remedies for the treatment of malaria

(DeFilipps et al 2004) post-natal depression asthma stomachache (Coelho et al

2006) and as an antiinflammatory agent (de Mesquita et al 2005)

In this study it was found that acetone extracts of G kunthiana exerted in vitro

immunomodulatory activities in human and bovine neutrophils It was demonstrated that

G kunthiana significantly inhibits the growth of E coli and S aureus (Figure 3-2) and

induces the entrapment of these bacteria when labelled with FITC (Figure 3-3) and

Figure 3-4) Besides this the study was also able to show that G kunthiana

significantly enhances NET release in bovine and human neutrophils (Figure 3-7)

Interestingly after treatment of bovine neutrophils with cytochalasin D or nocodazole

the amounts of NETs produced were significantly decreased (Figure 3-11) indicating

that actin and tubulin are involved in GUKUBA-mediated NET release Finally it was

found that G kunthiana decrease the release of reactive oxygen species (Figure 3-10)

thus explaining the use of G kunthiana in folk remedies against inflammation

Overall the results show that G kunthiana treatment of human and bovine

neutrophils is associated with increased phagocytosis formation of NETs and reduction

of oxidative burst which are the key effectors of bacterial killing and reduction of

inflammation Work focussing on the biochemical characterization of the mechansims

behind this phenomenon is currently being performed

Nowadays there is increasing evidence that certain antibiotics might have

immunomodulatory functions The immunomodulatory effects of antibiotics include

alteration of phagocytosis chemotaxis endotoxin release cytokine production and

hematopoietic recovery after imunosuppression Moreover some antibiotics can affect


96

the life-span of immune and inflammatory cells through the induction or inhibition of

apoptosis (Choi et al 2003) Such properties may have clinical significance for the

modulation of immune response of patients especially those who are immunodeficient

and those with microbial infections that have harmful inflamatory effects (eg septic

shock) Thus antibiotics may play a dual role in infections by having both direct

antimicrobial effects as well as indirect effects which can be eitheir beneficial or

detrimental to host response (Araujo et al 2002) Among the various classes of

antibiotics it is known that fluoroquinolones are able to exert immunomodulatory effects

(Ono et al 2000)

Many antimicrobials that are able to cross cell membranes of phagocytes have

some interaction with their host cells (Acoin 1996) For fluoroquinolones it was

successfully demonstrated under in vitro conditions that there are no detrimental effects

on viability phagocytosis or chemotaxis of neutrophils In contrast these drugs have

been shown to exhibit a synergistic effect with the major killing mechanisms used by

phagocytic cells namely oxidative damage of the pathogens due to superoxide

production (respiratory burst) (Acoin 1996) Fluoroquinolones additionally have been

demonstrated to synergistically utilize oxygen-dependent killing mechanisms used by

phagocytes to enhance their intracellular killing ability (Acoin 1996) It is known that

quinolone interaction with topoisomerase stimulates the oxidation of NADPH through

the electron transport chain which is dependent on the tricarboxylic acid cycle

Hyperactivation of the electron transport chain stimulates superoxide formation

Superoxide damages Fe-S clusters making ferrous iron available for oxidation by the

Fenton reaction The Fenton reaction then leads to the formation of hydroxyl radicals

which damage DNA lipids and proteins This contributes to antibiotic-induced cell death

(Kohanski et al 2010) These effects of fluoroquinolones on polymorph nuclear cells

as important parts of the immune system result in more effective phagocytosis and

killing of pathogens at the site of infection

Upon chemotactic stimulation mobile phagocytes accumulate at the site of

infection in large numbers Cells loaded with high concentrations of active drug seem to

be a reasonable vehicle for delivering fluoroquinolones directly to the infected tissues

(Acoin 1996) In a drug-free environment these drugs rapidly efflux from the


97

phagocytes and act directly against pathogens Phagocytes therefore were proposed

to act as the drug delivery device for fluoroquinolones to the site of infection (Boothe

1997)

In this study an immune boosting effect of enrofloxacin on bovine neutrophil

function was demonstrated Enrofloxacin was able to induce the phagocytosis of

fluorescently labelled S aureus but it has no effect on the FITC labelled E coli (Figure

4-1 b) which is in agreement with the findings of Hoeben et al (1997)

The most important finding that was observed was that enrofloxacin-treated

bovine neutrophils are able to induce the release of NETs (Figure 4-1 d)

As it was described before NET-formation is dependent on ROS production by

the multienzyme complex NADPH oxidase Using flow cytometry it was found that

enrofloxacin was able to induce the release of oxidative burst (Figure 4-1 c) In

accordance with those data when blocking neutrophil ROS production using the

NADPH oxidase inhibitor diphenylene iodonium (DPI) the level of NET generation was

clearly reduced (Figure 4-1 diii)

Furthermore when neutrophils were incubated with nocodazole a drug that

interferes with tubulin polymerization into microtubules or cytochalasin D a drug that

disrupts the polymerization of actin filaments the release of NETs were significantly

decreased compared to the controls (Figure 4-2 ci and cii) These data indicate that

actin and tubulin are in the enrofloxacin-mediated NET release similar as G kunthiana

Another important event in NET-formation and chromatin decondensation is

histone hypercitrullination a reaction catalyzed by peptidyl arginine deiminase 4 (PAD-

4) in which histone arginines are converted to citrullines by deimination In this study it

was demonstrated that enrofloxacin significantly induces the release of PAD-4 (Figure

4-2 b)

All together this study shows that treatment with G kunthiana and enrofloxacin

both increased the formation of NETs a novel host innate immune defence against

bacteria This data correlates with recent research about the effect of the feed additive

β-glucan on the degradation of NETs by the important fish pathogen Aeromonas

hydrophila The prementioned study shows that treatment of cells with β-glucan

significantly protects the NETs against bacterial degradation (Brogden et al 2012)


98

It is important to mention that the formation of ETs by phagocytic cells has been

shown to not only exert antibacterial effects but also to provoke inflammation There is

abounding evidence that NETs are also generated upon non-infectious stimuli in various

clinical settings In acute or chronic inflammatory disorders aberrantly enhanced NET

formation andor decreased NET degranulation seems to correlate with disease

outcome (Loumlgters et al 2009) In a host setting however there are pathways available

to limit these aberrations like destroying NETs by endogenous nucleases eg DNase

There is a delicate balance that should be maintained in order to allow NETs to perform

their duty yet disrupt them after they have fulfilled their antimicrobial activity upon

infection In cases of MRSA infections this improves the outcome of an infection and

thus ―NETs might be used as a possible therapeutic target (Behrens et al 2010 and

Ohn et al 2010)

Recently a very important publication showed that neutrophils do not always die

during NET-formation and can still undergo phagocytosis (Yipp et al 2012) These data

go in line with this studies where cell death is not always associated with NET-

formation Thus the boosting of NETs against certain infections eg MRSA in living

neutrophils can have high potentional value and its applications might be expanded

when combined with antibiotics

Future outlook

This study investigated the effect of G kunthiana and enrofloxacin on the

antimicrobial activity of neutrophils against E coli K-12 and S aureus Newman Both of

these bacteria are antibiotic susceptible prototype bacteria Future work needs to be

done with antibiotic resistant bacteria to see if neutrophils after G kunthiana and

enrofloxacin treatment can be boosted as well against antibiotic resistant bacteria eg

MRSA

Further studies may include looking at the biochemical mechanisms involved in

the process of G kunthiana and enrofloxacin-mediated NETs formation Two

dimensional gel electrophoresis and MALDI-TOF will be used to identify proteins

differentially expressed after exposure of neutrophils to G kunthiana and enrofloxacin

Verification of differential expression will also be conducted by RT-PCR


99

To elucidate the immunomodulatory mechanisms of G kunthiana and

enrofloxacin on neutrophils the following parameters for biological activity still need to

be measured 1) release of cytokines such as TNF-α or IL-6 that can be quantified by

commercially available standard ELISA-techniques 2) release and production of

antimicrobial peptidesproteins that can be analyzed and quantified in supernatant of

cells or total cell extracts respectively by 2D-gelelectrophoresis Western-Blot and

subsequent immune-staining using commercially available antibodies against selected

antimicrobial peptides

As in this study a total bark extract of G kunthiana was used future studies

should focus on the identification of the active compound in this crude bark extract

Finally the described effects should be studied in vivo eg using a mouse model of

infection All these studies will help to find an alternative approach for the treatment of

difficult infections such as those involving antimicrobial resistance or compromised host

immunity

References

Acoin DP (1996) Intracellular-intraphagocytic dynamics of fluoroquinolone antibiotics a comparative review Compendium on Continuing Education for the Practicing Veterinarian 18 9-13

Araujo FG Slifer TL and Remington JS (2002) Effect of moxifloxacin on secretion of cytokines by human monocytes stimulated with lypopolysaccharide Clinical Microbiology and Infection 8 26-30

Berends ETM Horswill AR Haste NM Monestier M Nizet V von Koumlckritz-Blickwede M (2010) Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps Journal of Innate Immunity 2 576-586

Boothe DM (1997) Principles of drug selection for respiratory infections in cats Compendium on Continuing Education for the Practicing Veterinarian 19 5-15

Brinkmann V Reichard U Goosmann C Fauler B Uhlemann Y Weiss DS Weinrauch Y Zychlinsky A (2004) Neutrophil extracellular traps kill bacteria Science 303 1532ndash1535

Brinkmann V and Zychlinsky A (2007) Beneficial suicide why neutrophils die to make NETs Nature Reviews Microbiolology 5 577-582


100

Brogden G von Koumlckritz-Blickwede M Adamek M Reuner F Jung-Schroers V Naim HY Steinhagen D (2012) β-Glucan protects neutrophil extracellular traps against degradation by Aeromonas hydrophila in carp (Cyprinus carpio) Fish Shellfish Immunology 33 1060-1064

Burvenich C Paape MJ Hill AW Guidry AJ Miller RH Heyneman R Kremer WDJ and Brand A (1994) Role of the neutrophil leukocyte in the local and systemic reactions during experimentally induced Ecoli mastitis in cows immediately after calving Veterinary Quarterly 16 45-50

Carlin AF Uchiyama S Chang YC Lewis AL Nizet V Varki A (2009) Molecular mimicry of host sialylated glycans allows a bacterial pathogen to engage neutrophil Siglec-9 and dampen the innate immune response Blood 113 3333ndash3336

Choi J-H Song M-J Kim S-H Choi S-M Lee D-G Yoo J-H and Shin W-S(2003) Effect of moxifloxacin on production of proinflammatory cytokines from human peripheral blood mononuclear cells Antimicrobial Agents and Chemotherapy 47 3704-3707

Chow OA von Koumlckritz-Blickwede M Bright AT Henaler ME Zinkernagel AS Cogen AL Gallo RL Moneetler M Wang Y Glass CK and Nizet V (2010) Statins enhance formation of phagocyte extracellular traps Cell Host amp Microbe 8 445-454

Coelho AAM de Paula JE and Espindola LS (2006) Insecticidal activity of Cerrado plant extracts on Rhodnius milesi Carcavallo Rocha Calvao and Jurberg (Hemiptera Reduviidae) under laboratory conditions Neutrophical Entomology 35 133-138

Davis JL Foster DM Papich MG (2007) Pharmacokinetics and tissue distribution of enrofloxacin and its active metabolite ciprofloxacin in calves Journal of Veterinary Pharmacology and Therapeutics 30 564ndash571

de Mesquita ML Desrivot J Bories C Fournet A de Pauna JE Grellier P Espindola LS (2005) Antileishmanial and trypanocidal activity of Brazilian Cerrado plants Memorias do Instituto Oswaldo Cruz 7 783-787




101

Dosogne H Meyer E Sturk A van Loon J Massart-Leeumln AM and Burvenich C (2002) Effect of enrofloxacin treatment on plasma endotoxin during bovine Escherichia coli mastitis Inflammation Research 51 201ndash205

Espindola LS (2006) Limonoide de Guarea kunthiana com potencial leishmanicida 1-77

Garcez FR Garceza WS da Silvaa AFG de Caacutessia Bazzoa R and Ubirazilda MR (2004) Terpenoid constituents from leaves of Guarea kunthiana Journal of Brazilian Chemical Society 5 767-772

Guimaratildees-Costa AB Nascimento M T Froment GS Soares R P Morgado F N Conceiccedilatildeo-Silva F Saraiva EM (2009) Leishmania amazanensis promastigotes induce and are killed by neutrophil extracellular traps Proceedings of the National Academy of Sciences of the United States of America 106 6748ndash6753

Hoeben D Monfardini E Burvenich C and Joumlrn H (2000) Treatment of acute Escherichia coli mastitis in cows with enrfloxacin effect on clinical signs and chemiluminescence of circulating neutrophils Journal of Dairy Research 67 485-502


Hong W Juneau RA Pang B Swords WE (2009) Survival of bacterial biofilms within neutrophil extracellular traps promotes nontypeable Haemophilus influenzae persistence in the chinchilla model for otitis media Journal of Innate Immunity 1 215ndash224

Idowu OR Peggins JO Cullison R von Bredow J (2010) Comparative pharmacokinetics of enrofloxacin and ciprofloxacin in lactatingdairy cows and beef steers following intravenous administration of enrofloxacin Research in Veterinary Science 89 30ndash235

Kabelitz D Kaufmann SHE (2010) Immunology of infections Methods in Microbiology 37 139-160

Kaplan MJ Radic M (2012) Neutrophil extracellular traps double-edged swords of innate immunity Journal of Immunology 189 2689-2695

Kawasaki H Iwamuro S (2008) Potential roles of histones in host defense as antimicrobial agents Infectious DisordersmdashDrug Targets 8 195ndash205

Kohanski MA Dwyer DJ Collins JJ (2010) How antibiotics kill bacteria from targets to network Nature Reviews Microbiology 8 423-445

Koul O and Walia S (2009) Comparing impacts of plant extracts and pure allelochemicalc and implications for pest control 4 29-30


102

Loumlgters T Margraf S Altrichter J Cinatl J Mitzner S Windolf J Scholz M (2009) The clinical value of neutrophil extracellular traps Medical Microbiology and Immunology 198 211-219

Monfardini E Burvenich C Massart-LeeEgraven AM Smits E Paape MJ (1999) Effect of antibiotic induced bacterial clearance in the udder on L-selectin shedding of blood neutrophils in cows with Escherichia coli mastitis Veterinary Immunology and Immunopathology 67 373-384

Mooto BS Jativa C Tinto WF Reynolds WF and McLean S (1991) Ecuadorin a novel tetranotriterpenoid of Guarea kunthiana structure elucidation by 2-D NMR spectroscopy Candian Journal of Chemistry 70 1260-1264


Ohn AC von Koumlckritz-Blickwede M Bright AT Hensler ME Zinkernagel AS Cogen AL Gallo RL Monestier M Wang Y Glass CK Nizet V (2010) Statins enhance formation of phagocyte extracellular traps Cell Host amp Microbe 8 445-454

Ono Y Ohmoto Y Ono K Sakata Y Murata K (2000) Effect of grepafloxacin on cytokine production in vitro Journal of Antimicrobial Chemotherapy 46 91-94


Petersson-Wolfe CS Mullarky IK Jones GM (1997) Staphylococcus aureus mastitis cause detection and control European Journal of Pharmacology 322 289ndash297

Pohlit AM Rezende AR Baldin ELL Lopes NP de Andrade Neto VF (2011) Plant extracts isolated phytochemicals and plant-derived agents which are lethal to anthropod vectors of human tropical diseases-a review Planta Medica 77 618-630

Schoevers EJ van Leengoed LANG Verheijden JHM and Niewold TA (1999) Effects of enrofloxacin on pocine phagocytic function Antimicrobial Agents and Chemotherapy 43 2138-2143



Urban F Reichard U Brinkmann V Zychlinsky A (2006) Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms Cell Microbiology 8 668ndash676


103

von Koumlckritz-Blickwede Maren and Nizet V (2009) Innate immunity turned inside-out antimicrobial defense by phagocyte extracellular traps Journal of Molecular Medicine 87 775-783

Wang X Zhao X Malik M Drlica K (2010) Contribution of reactive oxygen species to pathways of quinolone-mediated bacterial cell death Journal of Antimicrobial Chemotherapy 65 520-524

Wellnitz O Bruckmaier RM (2012) The innate immune response of the bovine mammary gland to bacterial infection Veterinary Journal 192 148-152

Yipp BG Petri B Salina D Jenne CN Scott BNV Zbytnuik LD Pittman K Asaduzzaman M Wu K Meijndert HC Malawista SE Chevance AB Zhang K Conly J Kubes P (2012) NETs are essential for limiting acute Saureus dissemination Nature Medicine 18 1386-1393

Chapter 6

Summary

Summary Chapter 6

107

Summary

Natalja Jerjomiceva

Exploring natural and pharmocological products for the ability to boost

neutrophils against bacterial infections

Since the middle of the 20th century major advances in antibacterial drug

development and other means of infection control helped turn the tide in the favor of

humans Regarding bacterial infections the situation dramatically improved when

penicillin became available for use in the early 1940s However the euphoria over the

potential conquest of infectious diseases was short lived Almost as soon as

antibacterial drugs were deployed bacteria responded by manifesting various forms of

resistance As antimicrobial usage increased so did the level and complexity of the

resistance mechanisms exhibited by bacterial pathogens The struggle to gain the upper

hand against infections continues to this day however the number of scientists working

on antibacterial agents is decreasing and bacteria are still evolving ever more

sophisticated mechanisms of resistance



pharmacological enhancement of the antimicrobial capabilities of phagocytes


alongside conventional antibiotic treatments for successful therapy of the infection

In this work the effect of a novel plant extract Guarea kunthiana (Chapter 3) and

a well-known antibiotic enrofloxacin (Chapter 4) on neutrophil functions was studied by

investigting their ability to stimulate the host immune defence against bacterial

infections

This study identified that G kunthiana is able to boost the antimicrobial activities

of bovine and human neutrophils Interestingly G kunthiana exhibited no direct

antimicrobial effect on the bacteria but neutrophils treated with G kunthiana showed

significantly induced growth inhibition of E coli and S aureus This effect was abolished

when treating the cells with cytochalasin D indicating that phagocytosis is involved in

Summary Chapter 6

108

this process We were also able to demonstrate that G kunthiana induces the formation

of antimicrobial neutrophil extracellular traps (NETs)

After treating bovine neutrophils with enrofloxacin it was found that they exhibited

increased levels of oxidative burst Furthemore there was a significant effect of

enrofloxacin on intracellular uptake of FITC-labeled S aureus detectable Interestingly

the treatment of neutrophils with enrofloxacin resulted in the formation of neutrophil

extracellular trap (NETs) But after treating the cells with cytochalasin D and

nocodozole the enrofloxacin-mediated NET-induction was abolished indicating that

actin or tubulin polymerization is involved in th enrofloxacin-mediated induction of NETs

The present investigations suggest that G kunthiana and enrofloxacin may stimulate

cellular immune responses and thereby might improve the outcome of an infection

Chapter 7

Zusammenfassung

Zusammenfassung Chapter 7

111

Zusammenfassung

Natalja Jerjomiceva

Wirkung von Naturprodukten und pharmakologischen Substanzen auf die

Faumlhigkeit Neutrophile gegen bakterielle Infektionen zu stimulieren

Seit etwa der Mitte des 20 Jahrhunderts haben groszlige Fortschritte in der

Entwicklung von antimikrobiellen Medikamenten und anderen Mitteln der

Infektionskontrolle das Blatt bezuumlglich bakterieller Infektionen zugunsten der Menschen

gewendet Seitdem Penicillin in den 1940er Jahren entwickelt wurde hat sich die

Situation drastisch verbessert Jedoch war die Euphorie uumlber den potenziellen Sieg

uumlber Infektionskrankheiten nur kurzlebig Fast genauso schnell wie antibakterielle

Medikamente eingesetzt wurden entwickelten Bakterien Resistenzen gegen diese

Antibiotika Aus diesem Grund dauert der Kampf gegen Infektionen bis heute an zumal

sich die Entwicklung neuer antibakterieller Medikamente verringert

Ein alternatives Konzept fuumlr die Behandlung von Infektionen welche durch

Antibiotikaresistenzen oder Wirtsimmunitaumlt erschwert werden koumlnnte die

pharmakologische Steigerung der antimikrobiellen Faumlhigkeiten von Phagozyten sein

Pharmakologische Wirkstoffe die das Immunsystem des Wirts staumlrken koumlnnen neben

einer konventionellen Antibiotika-Behandlung fuumlr eine erfolgreiche Therapie von

Infektionen verwendet werden

In dieser Arbeit wurde die Wirkung des neuartigen Pflanzenextrakts Guarea

kuthiana (Kapitel 3) sowie des bekannten Antibiotikums Enrofloxacin (Kapitel 4) auf die

Funktionen von Neutrophilen untersucht Dabei wurde die Faumlhigkeit dieser beiden

Wirkstoffe die Immunantwort des Wirts gegen bakterielle Infektionen zu stimulieren

analysiert

Es konnte gezeigt werden dass G kunthiana die antimikrobiellen Aktivitaumlten von

bovinen und humanen Neutrophilen steigern kann Interessanterweise zeigt G

kunthiana keine direkte antimikrobielle Wirkung auf die Bakterien jedoch ist nach der

Behandlung von Neutrophilen mit G kunthiana eine signifikante Wachstumshemmung

von E coli und S aureus zu beobachten Dieser Effekt wurde durch die Behandlung der

Zellen mit Cytochalasin D aufgehoben was zeigt dass Phagozytose an diesem


112

Prozess beteiligt ist Es konnte auch demonstriert werden dass G kunthiana die

Bildung von antimikrobiellen neutrophilen extrazellulaumlren Fallen (NETs) induziert

In dieser Arbeit konnte zudem gezeigt werden dass eine Behandlung der

Neutrophilen mit Enrofloxacin zu einer erhoumlhten Bildung von Sauerstoffderivaten fuumlhrt

Auszligerdem ist eine signifikante Auswirkung auf die intrazellulaumlre Aufnahme von FITC-

markierten S aureus in Enrofloxacin behandelten Zellen zu erkennen

Interessanterweise induziert die Behandlung von Neutrophilen mit Enrofloxacin

ebenfalls die Bildung von NETs Jedoch wurde nach der Behandlung der Zellen mit

Cytochalasin D oder Nocodazole die Enrofloxacin-vermittelte NETs-Induktion

aufgehoben was darauf hinweist dass die Polymerisation von Aktin und Tubulin an der

durch Enrofloxacin-vermittelte Induktion der neutrophilen extrazellulaumlren Fallen beteiligt

ist

Die vorliegende Studie zeigt dass das Pflanzenextrakt G kunthiana und das

Antibiotikum Enrofloxacin die zellulaumlre Immunantwort stimulieren koumlnnen und somit den

Infektionsverlauf protektiv beeinflussen koumlnnen

Appendix

Appendix

115

List of figures and tables

Figure 1-1 Neutrophil engulfing bacteria 9

Figure 1-2 Development of multidrug-resistant (MDR) pathogens 11

Figure 1-3 Natural products as sources of new drugs over the last 25 years 12

Figure 1-4 Mean concentrations of enrofloxacin in plasma subcutaneous

and intramuscular interstitial fluid and pleural fluid in calves after

subcutaneous injection of 125 mgkg 34

Figure 1-5 Growth of E coli in milk of challenged quarters during induced E

coli mastitis 35

Figure 1-6 Influence of different doses of antibiotics on chemiluminescence

in a cell-free system 36

Figure 1-7 Influence of different doses of antibiotics on myeloperoxidase

activity measured interms of oxidation of ortho-dianiside 36

Figure 2-1 Density gradient before and after centrifugation of

PolymorphPrep with human blood 48

Figure 2-2 Density gradient before and after centrifugation of Biocoll with

bovine blood 49

Figure 3-1 Effect of G kunthiana on the growth of E coli K-12 and S

aureus Newman 64

Figure 3-2 Effect of G kunthiana on the antimicrobial activity of neutrophils 65

Figure 3-3 Effect of G kunthiana on phagocytosis of bacteria in human

neutrophils 67

Figure 3-4 Effect of G kunthiana on phagocytosis of bacteria in bovine

neutrophils 67

Figure 3-5 Effect of cytochalasin D on antimicrobial activity of neutrophils 68

Figure 3-6 Effect of G kunthiana on degranulation in human and bovine

neutrophils 69

Figure 3-7 G kunthiana significantly induces NET-formation in human and

bovine neutrophils 70

Appendix

116

Figure 3-8 Representative fluorescent image of NETs induced after

treatment with G kunthiana 70

Figure 3-9 Effect of blocking of oxidative burst on the G kunthiana-

mediated NET formation 71

Figure 3-10 Effect of G kunthiana on oxidative burst 72

Figure 3-11 Effect of nocodazole on G kunthiana-mediated NET formation in


Figure 4-1 Effect of enrofloxacin on antimicrobial defence strategies of

bovine granulocytes 85

Figure 4-2 Biochemical mechanisms associated with enrofloxacin-induced

formation of NETs 87

Table 1-1 Chemical substances derived from plants 13

Table 1-2 Known plants with immunodulatory activity 23

Table 1-3 Mortality rate of R milesi when topically applied with extract from

G kunthiana 30

Abstracts

Effect of enrofloxacin on antimicrobial activities of bovine neutrophil functions

Natalja Jerjomiceva Hisham Seri Martin Heine Hassan Y Naim Maren von Koumlckritz-Blickwede

Institut fuumlr Physiologische Chemie Tieraumlrztliche Hochschule Hannover

Several classes of drugs such as fluoroquinolones are actively accumulated in

neutrophils that mediate the first line of defence against invading pathogens There is

increasing evidence that some of those antibiotics are able to additionally modulate the

function of the immune cell A better understanding of these antibiotic-mediated

changes might allow a more rational application of the respective antibiotics with regard

to successful therapy of infections The goal of this study is to investigate the

mechanism of enrofloxacin-mediated modulation of bovine neutrophil functions


animals including cattle

Bovine neutrophils were isolated from fresh blood by density gradient

centrifugation and were characterized by flow cytometry After treatment of the cells with

enrofloxacin they exhibited increased oxidative burst as measured by flow cytometry

utilizing dichlorofluorescein as a marker Furthermore there was a significant effect of

enrofloxacin on intracellular uptake of FITC labelled Staphylococcus aureus

Interestingly using immunofluorescence we were able to show that treatment of

neutrophils with enrofloxacin resulted in the formation of neutrophil extracellular traps

(NETs) NETs have recently been identified as a novel host innate immune defence

mechanisms of granulocytes They consist of nuclear and mitochondrial DNA with

associated histones and antimicrobial peptides and are able to entrap and kill invading

pathogens When treating the cells with cytochalasin D the enrofloxacin-mediated NET-

induction was abolished indicating that the actin polymerization is involved in this

process Current work is focussing on the biochemical mechanisms involved in the

enrofloxacin-mediated induction of NETs

In summary the observed effects of enrofloxacin on bovine neutrophil functions


immunocompromised animals and thus could substantially influence the management

of infections

16-18 February 2012 20 Tagung der Fachgruppe Physiologie und Biochemie der

Deutschen Veterinaumlrmedizinischen Gesellschaft Munich Germany

Pharmacological enhancement of the antimicrobial capabilities of bovine and

human neutrophils using Guarea kunthiana extract


1 William N Setzer

2 Hassan Y Naim

1 Maren von Koumlckritz-

Blickwede1

1Institut fuumlr Physiologische Chemie Tieraumlrztliche Hochschule Hannover und

2Department of

Chemistry University of Alabama Huntsville


proportions and poses great challenges to the human and veterinary medicine An



enhancement of the antimicrobial capabilities of phagocytes Pharmacological agents

which boost the host immune system could conceivably be used alongside conventional

antibiotic treatment for successful therapy of the infection The goal of this study is to

search for novel natural products with the ability to boost the host immune defence

against bacterial infections

By screening a library of plant extracts collected from the Monteverde Cloud

Forest Reserve Costa Rica we identified an extract of the plant Guarea kunthiana

(GUKUBA) to be able to boost the antimicrobial activities of bovine and human

neutrophils Neutrophils were isolated from bovine or human blood by density gradient

centrifugation treated with GUKUBA for 2 or 5 h and then the antimicrobial activity

against Escherichia coli or Staphylococcus aureus was investigated Interestingly

GUKUBA extracts exhibited no direct antimicrobial effect on the bacteria However

neutrophils treated with GUKUBA showed significantly induced growth inhibition of E

coli as well as S aureus This effect was abolished when treating the cells with

cytochalasin D indicating that phagocytosis in involved in this process Furthermore

using immunofluorescence microscopy we could demonstrate that GUKUBA induces

the formation of antimicrobial neutrophil extracellular traps (NETs) which are able to

entrap and kill the bacteria Current work is focussing on the biochemical

characterization of the mechanisms behind this phenomenon

Finally this project might identify new therapeutic targets based on natural

products which can be further developed as new therapeutic treatment strategies


We are grateful to the Monteverde Cloud Forest Preserve and the Tropical

Science Center for granting us permission to collect plant materials under a cooperative




30 September ndash 3 October 2012 64 Jahrestagung der Deutschen Gesellschaft fuumlr

Hygiene und Mikrobiologie Hamburg Germany

9 July 2012 Center for Infection Medicine University of Veteriary Medicine Hannover

Germany



Acknowledgements

I would like to express my gratitude to the Director of the Department of

Physiological Chemistry Prof Dr Hassan Naim for the great opportunity to

work in his lab for his advices and unsurpassed knowledge in Biochemistry

I am sincerely and heartily grateful to my supervisor Dr Maren von Koumlckritz-

Blickwede for the support guidance and patience she showed me throughout

my dissertation I am sure it would have not been possible without her help

Maren I could not have wished a better supervisor

I am grateful to all my colleagues Marc Behrendt Sonja Schmidt Gabriele

Wetzel Heike Kanapin Stephanie Geveke Graham Brogden Eva

Toennings Birthe Gericke Petra Luumlthje Mahdi Amiri Aleksander Prokscha

Stefanie Blodkamp Nathalie Zeitouni Ragheda Yaseen Lena Diekmann

Sandra Pfeifer who mainted a pleasant atmosphere and have been close to

me everyday being nice colleagues

I thank Martin Heine for his high qualified help in many problems with

laboratory equipment and PC

I wish to thank Friederike Reuner who gladly helped me very much anytime

when needed

I owe sincere and earnest thankfulness to Katia Maalouf Ariane Neumann

and Lena Voumlllger for their good edvices kindness friendship and support

throughout my project

I would like to show my gratitude to Hisham Seri who kindly helped me with

the practical part of my project

I would like to thank Prof William Setzer for providing me the plant extract

and whom sadly I never met in person

I thank Dr Marc Monestier for providing the anti-histone-DNA-antibody

I am truly indebted and thankful to Maritta Ledwoch for her invaluable help

Besides I would like to thank my friends Yidan Huang Rindrahatsarana

Ramanankirahina Lilja Ivanova Karina Petjukevica and Margarita Petrenko

that boosted me morally all the time

I would like to thank my parents and my boyfriend Artis for their love

unequivocal support and encouragement throughout my study

During my thesis I received financial support from the Akademie fuumlr

Tiergesundheit (AfT) Bonn Germany

For any errors or inadequacies that may remain in this work of course the

responsibility is entirely my own

Erklaumlrung

Hiermit erklaumlre ich dass ich die Dissertation bdquoExploring natural and

pharmocological products for the ability to boost neutrophils against bacterial

infectionsrdquo selbstaumlndig verfaβt habe

Ich habe keine entgeltliche Hilfe von Vermittlungs-bzw Beratungsdiensten

(Promotionsberater oder anderer Personen) in Anspruch genommen

Niemand hat von mir unmittelbar oder mittelbar entgeltliche Leistungen fuumlr

Arbeit erhalten die im Zusammenhang mit dem Inhalt der vorgelegten

Dissertation stehen

Ich habe die Dissertation an folgenden Institutionen angefertigt

Institut fuumlr Physiologische Chemie Stiftung Tieraumlrztliche Hochschule

Hannover

Die Dissertation wurde bisher nicht fuumlr eine Pruumlfung oder Promotion oder fuumlr

einen aumlhnlichen Zweck zur Beurteilung eingereicht

Ich versichere dass ich die vorstehenden Angaben nach bestem Wissen

vollstaumlndig und der Wahrheit entsprechend gemacht habe

Hannover den 300413

Natalja Jerjomiceva

Curriculum Vitae

Name Natalja Jerjomiceva

Date and place of birth 24071984 Riga Latvia

Education since Januar 2013 Internship in the Small Animal

Clinic at the University of Veterinary Medicine

Hannover Germany

since September 2010 Doctoral studies at the


Germany

2009 - 2010 Veterinary surgeon in the Veterinary

Clinic ―BIOVET and ―TERION Riga Latvia

September 2009 ndash October 2009 Practical training

at the Department of Physiological Chemistry


Germany

March 2009 ndash Mai 2009 Practical training at the

University of Veterinary Medicine Vienna (Austria)

as part of the ERASMUS exchange program

September 2003 ndash Juny 2009 Faculty of

Veterinary Medicine Jelgava Latvia

Publications as poster abstracts

30 September ndash 3 October 2012 64 Jahrestagung

der Deutschen Gesellschaft fuumlr Hygiene und

Mikrobiologie Hamburg Germany Natural

products that boost the host immune defence


9 July 2012 Center for Infection Medicine

University of Veteriary Medicine Hannover

Germany Pharmacological of the antimicrobial

capabilities of bovine and human neutrophils

using Guarea kunthiana extract

16-18 February 2012 20 Tagung der Fachgruppe

Physiologie und Biochemie der Deutschen

Veterinaumlrmedizinischen Gesellschaft Munich

Germany Pharmacological enhancement of the

antimicrobial capabilities of bovine and human

neutrophils using Guarea kunthiana extract

List of abbreviations

BHI brain-heart infusion

Burm f Nicolaas Laurens Burman

CPE crude polysaccharide extract

CytD cytochalasin D

E coli Escherichia coli

EDTA ethylene-diamine-tetraacetic acid

ELISA enzyme-linked immunosorbent assay

FACS fluorescence-activated cell sorting

FCS fetal calf serum

Fe iron

FITC fluorescein isothiocyanate

FQRP floroquinolone-resistant Pseudomonas aeruginosa

g gravitational acceleration

G3C R Eco group 3 cephalosporin-resistant Escherichia coli

G3C R Kleb group 3 cephalosporinresistant-resistant Klebsiella pneumoni

G kunthiana Guarhea kunthuiana

DAPI 46-diamidino-2-phenylindole

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

DPI diphenylene iodonium

DTH delayed type hypersensitivity

IC inhibitory concentration

IgG immunoglobulin G

IL-2 Interleukin-2

Imi R Aci imipenem-resistant Acinetobacter baumannii

ISF Interstitial fluid

kDa kilo Dalton

LB Lysogeny broth

Link Johann Heinrich Friedrich Link

Linn Carl Linnaeus

LPS lipopolysaccharide

microl microliter

M molar

MALDI-TOF matrix-assisted laser desorptionionization- time-of-flight mass spectrometer

MDR development of multidrug-resistant

mg milligram

min minute

ml millilitre

mm millimeter

mM millimolar

MOI multiplicity of infection

MRSA methicillin-resistant Staphylococcus aureus

NaOH sodium xydroxide

NET neutrophyl extracellular trap

NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells

NK natural killer

Noc nocodazole

OD optical density

PAGE polyacrylamide gel electrophoresis

PAD4 peptidyl arginine deiminase 4

PMA phorbol myristate acetate

PMN polymorphonuclear leukocytes

RBC red blood cell

ROS reactive oxygen species

RPMI Roswell Park Memorial Institute medium

RT-PCR reverse transcription polymerase chain reaction

SDS sodium dodecyl sulphate

TBS tris-buffered saline

THB Todd-Hewitt broth

TNF-α tumor necrosis factor-alpha

U unit

S sulfur

S aureus Staphylococcus aureus

SSC sideward-scatter

VRE vancomycin-resistant enterococci

WBS white blood cell

Willd Carl Ludwig Willdenow

Academic supervision Prof Dr Hassan Y Naim



Dr Maren von Koumlckritz-Blickwede



Referee Apl- Prof Dr Stefan Schwarz

Institute of Farm Animal Genetics

Friedrich-Loeffler-Institut (FLI) Neustadt-Mariensee

Day of the oral examination 17052013

This work was supported by a grant from Akademie fuumlr Tiergesundheit (AfT) Bonn

Germany

To my parents

Contents





medicine 17




17 Enrofloxacin 33

18 Goal 37



22 Drugs 47

























- Abstracts

- Acknowledgements

- Erklaumlrung

- Curriculum Vita


Chapter 1



9






















10


















aureus (Todar 2002)















11












treatment regimes


12













30

10

4

104

14

5

23



13










today























14


















Several plants





















15

























Lobelia inflata














16







































17





























2011)


18

































19








(Tseng et al 2005)

























20
































21

































22



et al 2004)




























above


23



Allium sativum


Europe







prolifeation














Taiwan


antioxidant


nephritis


macrophages

Tseng et al 2005


24







system

Gokhale et al 2002


(Caesalpiniaceae)

India







diabetes mellitus





Shukla et al 2009





count

Ghule et al 2006






Nores et al 1997






Manosroi et al 2003


25


(Amaryllidacea)


Immunostimulatory


and heal wounds


Lakshmi et al 2003

Curcuma



immunostimmulating




Kim et al 2007






lipid peroxidation

Gan et al 2004


Brazil



treatment


Pinello et at 2005

Pouteria







oxidative burst

Manosroi et al 2005

Tephrosia purpurea

(Leguminosae)





Damre et al 2003


26































27























Costa Rica









28













forests







15 Guarea kunthiana




Herbarium





29
















2009)












30


Species

Plant

part

used

Solvent

Mortality rate ()


G kunthiana


Ethanol 0 0 5 10 15 20 25

Stem

Hexane 25 30 35 40 40 40 40

Ethanol 10 15 15 20 20 20 20








al 2006)












infections


31

16 Fluoroquinolones

General structure









(Ball 2000)





















32











(Owens et al 2000)

Distribution

















33

17 Enrofloxacin



























2011)





34





















35

















36











37

18 Goal
































38



References














39














40














41
















42















43



Chapter 2



47

21 Plant material





Herbarium

22 Drugs























48


















49












CO2








50































51








sample

211 Degranulation






212 Oxidative burst














52






























53







02 Triton X-100






References









54












55








Chapter 3




12 Ragheda Yaseen

1 William N Setzer

3 Hassan Y Naim

1 Maren









58


the following scale









59

Abstract












Results









Conclusions





60

Introduction


















et al 2005)












61
































62

































63



























scatter SSC)






64












Results








every 30 min


65










(

b)


66
















formation












67



(b)


68





neutrophils











69











extract


70



(

a)


71








dependent







3-10)


72







process



73

Discussion









effect






















74


determined

















Acknowledgements








75

References












76












Chapter 4




12 Lena Voumlllger

1 Hassan Y Naim


1



Technology





78


the following scale









79

Abstract







80

Introduction





























2009)


81
































82
































83
































84







and p lt 0001



















85


Phagocytosis

E coli S aureus

a c

bi bii

CtrEnro

25nM P

MA

0

50

100

150

200

400

600

800

Rel

ativ

e o

xid

ativ

e b

urs

t

[x-M

ean

of F

L-1

]

Ctr

Enro

25nM P

MA

0

200

400

600

n s

Rel

ativ

e d

egra

nu

latio

n [S

SC

val

ue]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

20

25

n s

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

NET-formation

di dii

diii

Ctr

Ctr + D

PI

Enro

Enro +

DPI

0

10

20

30

NE

T-r

elea

sin

g c

ells

[]



86





























87

Cell death



NaO

HEnr

o

0

5

10

15 n s

LD

H r

ele

as

e in

[Co

mp

are

d t

o t

ota

l c

ell ly

sis

]

Enr

o

Enr

o +

Cyt

D

Ctr +

Cyt

D

0

5

10

15

20

25

ns

NE

T-r

ele

as

ing

ce

lls

[

]

NaOH

74 kDa

PAD4 M Enro

Ctr

Enr

o

0

50

100

150

200

rela

tiv

e P

AD

-4-p

rote

in e

xp

res

sio

n in

[co

mp

are

d t

o

-ac

tin

ex

pre

ss

ion

]


NET formation

ai

aii

bi

bii

ci

cii

Enr

o

Enr

o +

Noc

Ctr +

Noc

0

10

20

30

40

n s

NE

T-r

ele

as

ing

ce

lls

[

]

74 kDa

50 kDa

33 kDa

M Enro Ctr

PAD-4

β-actin









88








Conclusions

















Acknowledgements




89

Funding


References












90









Chapter 5



93

Discussion







to public health

























94








V 2009)










2009)














95

































96









(Ono et al 2000)
























97



1997)






















4-2 b)








98












Ohn et al 2010)







Future outlook






MRSA







99














immunity

References








100












101















102















103





Chapter 6

Summary

Summary Chapter 6

107

Summary

Natalja Jerjomiceva






















infections






Summary Chapter 6

108












Chapter 7

Zusammenfassung


111

Zusammenfassung

Natalja Jerjomiceva






















analysiert








112












ist




Appendix

Appendix

115









coli mastitis 35








bovine blood 49


aureus Newman 64



neutrophils 67


neutrophils 67



neutrophils 69



Appendix

116















G kunthiana 30

Abstracts






























of infections






1 William N Setzer

2 Hassan Y Naim


Blickwede1


2Department of




































Germany



Acknowledgements

















when needed



















Erklaumlrung








Dissertation stehen



Hannover





Hannover den 300413

Natalja Jerjomiceva

Curriculum Vitae





Hannover Germany



Germany






Germany



























CytD cytochalasin D






Fe iron














IL-2 Interleukin-2



kDa kilo Dalton

LB Lysogeny broth


Linn Carl Linnaeus


microl microliter

M molar



mg milligram

min minute

ml millilitre

mm millimeter

mM millimolar






NK natural killer

Noc nocodazole

OD optical density





RBC red blood cell








U unit

S sulfur






To my parents

Contents





medicine 17




17 Enrofloxacin 33

18 Goal 37



22 Drugs 47

























- Abstracts

- Acknowledgements

- Erklaumlrung

- Curriculum Vita


Chapter 1



9






















10


















aureus (Todar 2002)















11












treatment regimes


12













30

10

4

104

14

5

23



13










today























14


















Several plants





















15

























Lobelia inflata














16







































17





























2011)


18

































19








(Tseng et al 2005)

























20
































21

































22



et al 2004)




























above


23



Allium sativum


Europe







prolifeation














Taiwan


antioxidant


nephritis


macrophages

Tseng et al 2005


24







system

Gokhale et al 2002


(Caesalpiniaceae)

India







diabetes mellitus





Shukla et al 2009





count

Ghule et al 2006






Nores et al 1997






Manosroi et al 2003


25


(Amaryllidacea)


Immunostimulatory


and heal wounds


Lakshmi et al 2003

Curcuma



immunostimmulating




Kim et al 2007






lipid peroxidation

Gan et al 2004


Brazil



treatment


Pinello et at 2005

Pouteria







oxidative burst

Manosroi et al 2005

Tephrosia purpurea

(Leguminosae)





Damre et al 2003


26































27























Costa Rica









28













forests







15 Guarea kunthiana




Herbarium





29
















2009)












30


Species

Plant

part

used

Solvent

Mortality rate ()


G kunthiana


Ethanol 0 0 5 10 15 20 25

Stem

Hexane 25 30 35 40 40 40 40

Ethanol 10 15 15 20 20 20 20








al 2006)












infections


31

16 Fluoroquinolones

General structure









(Ball 2000)





















32











(Owens et al 2000)

Distribution

















33

17 Enrofloxacin



























2011)





34





















35

















36











37

18 Goal
































38



References














39














40














41
















42















43



Chapter 2



47

21 Plant material





Herbarium

22 Drugs























48


















49












CO2








50































51








sample

211 Degranulation






212 Oxidative burst














52






























53







02 Triton X-100






References









54












55








Chapter 3




12 Ragheda Yaseen

1 William N Setzer

3 Hassan Y Naim

1 Maren









58


the following scale









59

Abstract












Results









Conclusions





60

Introduction


















et al 2005)












61
































62

































63



























scatter SSC)






64












Results








every 30 min


65










(

b)


66
















formation












67



(b)


68





neutrophils











69











extract


70



(

a)


71








dependent







3-10)


72







process



73

Discussion









effect






















74


determined

















Acknowledgements








75

References












76












Chapter 4




12 Lena Voumlllger

1 Hassan Y Naim


1



Technology





78


the following scale









79

Abstract







80

Introduction





























2009)


81
































82
































83
































84







and p lt 0001



















85


Phagocytosis

E coli S aureus

a c

bi bii

CtrEnro

25nM P

MA

0

50

100

150

200

400

600

800

Rel

ativ

e o

xid

ativ

e b

urs

t

[x-M

ean

of F

L-1

]

Ctr

Enro

25nM P

MA

0

200

400

600

n s

Rel

ativ

e d

egra

nu

latio

n [S

SC

val

ue]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

20

25

n s

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

NET-formation

di dii

diii

Ctr

Ctr + D

PI

Enro

Enro +

DPI

0

10

20

30

NE

T-r

elea

sin

g c

ells

[]



86





























87

Cell death



NaO

HEnr

o

0

5

10

15 n s

LD

H r

ele

as

e in

[Co

mp

are

d t

o t

ota

l c

ell ly

sis

]

Enr

o

Enr

o +

Cyt

D

Ctr +

Cyt

D

0

5

10

15

20

25

ns

NE

T-r

ele

as

ing

ce

lls

[

]

NaOH

74 kDa

PAD4 M Enro

Ctr

Enr

o

0

50

100

150

200

rela

tiv

e P

AD

-4-p

rote

in e

xp

res

sio

n in

[co

mp

are

d t

o

-ac

tin

ex

pre

ss

ion

]


NET formation

ai

aii

bi

bii

ci

cii

Enr

o

Enr

o +

Noc

Ctr +

Noc

0

10

20

30

40

n s

NE

T-r

ele

as

ing

ce

lls

[

]

74 kDa

50 kDa

33 kDa

M Enro Ctr

PAD-4

β-actin









88








Conclusions

















Acknowledgements




89

Funding


References












90









Chapter 5



93

Discussion







to public health

























94








V 2009)










2009)














95

































96









(Ono et al 2000)
























97



1997)






















4-2 b)








98












Ohn et al 2010)







Future outlook






MRSA







99














immunity

References








100












101















102















103





Chapter 6

Summary

Summary Chapter 6

107

Summary

Natalja Jerjomiceva






















infections






Summary Chapter 6

108












Chapter 7

Zusammenfassung


111

Zusammenfassung

Natalja Jerjomiceva






















analysiert








112












ist




Appendix

Appendix

115









coli mastitis 35








bovine blood 49


aureus Newman 64



neutrophils 67


neutrophils 67



neutrophils 69



Appendix

116















G kunthiana 30

Abstracts






























of infections






1 William N Setzer

2 Hassan Y Naim


Blickwede1


2Department of




































Germany



Acknowledgements

















when needed



















Erklaumlrung








Dissertation stehen



Hannover





Hannover den 300413

Natalja Jerjomiceva

Curriculum Vitae





Hannover Germany



Germany






Germany



























CytD cytochalasin D






Fe iron














IL-2 Interleukin-2



kDa kilo Dalton

LB Lysogeny broth


Linn Carl Linnaeus


microl microliter

M molar



mg milligram

min minute

ml millilitre

mm millimeter

mM millimolar






NK natural killer

Noc nocodazole

OD optical density





RBC red blood cell








U unit

S sulfur






Contents





medicine 17




17 Enrofloxacin 33

18 Goal 37



22 Drugs 47

























- Abstracts

- Acknowledgements

- Erklaumlrung

- Curriculum Vita


Chapter 1



9






















10


















aureus (Todar 2002)















11












treatment regimes


12













30

10

4

104

14

5

23



13










today























14


















Several plants





















15

























Lobelia inflata














16







































17





























2011)


18

































19








(Tseng et al 2005)

























20
































21

































22



et al 2004)




























above


23



Allium sativum


Europe







prolifeation














Taiwan


antioxidant


nephritis


macrophages

Tseng et al 2005


24







system

Gokhale et al 2002


(Caesalpiniaceae)

India







diabetes mellitus





Shukla et al 2009





count

Ghule et al 2006






Nores et al 1997






Manosroi et al 2003


25


(Amaryllidacea)


Immunostimulatory


and heal wounds


Lakshmi et al 2003

Curcuma



immunostimmulating




Kim et al 2007






lipid peroxidation

Gan et al 2004


Brazil



treatment


Pinello et at 2005

Pouteria







oxidative burst

Manosroi et al 2005

Tephrosia purpurea

(Leguminosae)





Damre et al 2003


26































27























Costa Rica









28













forests







15 Guarea kunthiana




Herbarium





29
















2009)












30


Species

Plant

part

used

Solvent

Mortality rate ()


G kunthiana


Ethanol 0 0 5 10 15 20 25

Stem

Hexane 25 30 35 40 40 40 40

Ethanol 10 15 15 20 20 20 20








al 2006)












infections


31

16 Fluoroquinolones

General structure









(Ball 2000)





















32











(Owens et al 2000)

Distribution

















33

17 Enrofloxacin



























2011)





34





















35

















36











37

18 Goal
































38



References














39














40














41
















42















43



Chapter 2



47

21 Plant material





Herbarium

22 Drugs























48


















49












CO2








50































51








sample

211 Degranulation






212 Oxidative burst














52






























53







02 Triton X-100






References









54












55








Chapter 3




12 Ragheda Yaseen

1 William N Setzer

3 Hassan Y Naim

1 Maren









58


the following scale









59

Abstract












Results









Conclusions





60

Introduction


















et al 2005)












61
































62

































63



























scatter SSC)






64












Results








every 30 min


65










(

b)


66
















formation












67



(b)


68





neutrophils











69











extract


70



(

a)


71








dependent







3-10)


72







process



73

Discussion









effect






















74


determined

















Acknowledgements








75

References












76












Chapter 4




12 Lena Voumlllger

1 Hassan Y Naim


1



Technology





78


the following scale









79

Abstract







80

Introduction





























2009)


81
































82
































83
































84







and p lt 0001



















85


Phagocytosis

E coli S aureus

a c

bi bii

CtrEnro

25nM P

MA

0

50

100

150

200

400

600

800

Rel

ativ

e o

xid

ativ

e b

urs

t

[x-M

ean

of F

L-1

]

Ctr

Enro

25nM P

MA

0

200

400

600

n s

Rel

ativ

e d

egra

nu

latio

n [S

SC

val

ue]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

20

25

n s

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

NET-formation

di dii

diii

Ctr

Ctr + D

PI

Enro

Enro +

DPI

0

10

20

30

NE

T-r

elea

sin

g c

ells

[]



86





























87

Cell death



NaO

HEnr

o

0

5

10

15 n s

LD

H r

ele

as

e in

[Co

mp

are

d t

o t

ota

l c

ell ly

sis

]

Enr

o

Enr

o +

Cyt

D

Ctr +

Cyt

D

0

5

10

15

20

25

ns

NE

T-r

ele

as

ing

ce

lls

[

]

NaOH

74 kDa

PAD4 M Enro

Ctr

Enr

o

0

50

100

150

200

rela

tiv

e P

AD

-4-p

rote

in e

xp

res

sio

n in

[co

mp

are

d t

o

-ac

tin

ex

pre

ss

ion

]


NET formation

ai

aii

bi

bii

ci

cii

Enr

o

Enr

o +

Noc

Ctr +

Noc

0

10

20

30

40

n s

NE

T-r

ele

as

ing

ce

lls

[

]

74 kDa

50 kDa

33 kDa

M Enro Ctr

PAD-4

β-actin









88








Conclusions

















Acknowledgements




89

Funding


References












90









Chapter 5



93

Discussion







to public health

























94








V 2009)










2009)














95

































96









(Ono et al 2000)
























97



1997)






















4-2 b)








98












Ohn et al 2010)







Future outlook






MRSA







99














immunity

References








100












101















102















103





Chapter 6

Summary

Summary Chapter 6

107

Summary

Natalja Jerjomiceva






















infections






Summary Chapter 6

108












Chapter 7

Zusammenfassung


111

Zusammenfassung

Natalja Jerjomiceva






















analysiert








112












ist




Appendix

Appendix

115









coli mastitis 35








bovine blood 49


aureus Newman 64



neutrophils 67


neutrophils 67



neutrophils 69



Appendix

116















G kunthiana 30

Abstracts






























of infections






1 William N Setzer

2 Hassan Y Naim


Blickwede1


2Department of




































Germany



Acknowledgements

















when needed



















Erklaumlrung








Dissertation stehen



Hannover





Hannover den 300413

Natalja Jerjomiceva

Curriculum Vitae





Hannover Germany



Germany






Germany



























CytD cytochalasin D






Fe iron














IL-2 Interleukin-2



kDa kilo Dalton

LB Lysogeny broth


Linn Carl Linnaeus


microl microliter

M molar



mg milligram

min minute

ml millilitre

mm millimeter

mM millimolar






NK natural killer

Noc nocodazole

OD optical density





RBC red blood cell








U unit

S sulfur















- Abstracts

- Acknowledgements

- Erklaumlrung

- Curriculum Vita


Chapter 1



9






















10


















aureus (Todar 2002)















11












treatment regimes


12













30

10

4

104

14

5

23



13










today























14


















Several plants





















15

























Lobelia inflata














16







































17





























2011)


18

































19








(Tseng et al 2005)

























20
































21

































22



et al 2004)




























above


23



Allium sativum


Europe







prolifeation














Taiwan


antioxidant


nephritis


macrophages

Tseng et al 2005


24







system

Gokhale et al 2002


(Caesalpiniaceae)

India







diabetes mellitus





Shukla et al 2009





count

Ghule et al 2006






Nores et al 1997






Manosroi et al 2003


25


(Amaryllidacea)


Immunostimulatory


and heal wounds


Lakshmi et al 2003

Curcuma



immunostimmulating




Kim et al 2007






lipid peroxidation

Gan et al 2004


Brazil



treatment


Pinello et at 2005

Pouteria







oxidative burst

Manosroi et al 2005

Tephrosia purpurea

(Leguminosae)





Damre et al 2003


26































27























Costa Rica









28













forests







15 Guarea kunthiana




Herbarium





29
















2009)












30


Species

Plant

part

used

Solvent

Mortality rate ()


G kunthiana


Ethanol 0 0 5 10 15 20 25

Stem

Hexane 25 30 35 40 40 40 40

Ethanol 10 15 15 20 20 20 20








al 2006)












infections


31

16 Fluoroquinolones

General structure









(Ball 2000)





















32











(Owens et al 2000)

Distribution

















33

17 Enrofloxacin



























2011)





34





















35

















36











37

18 Goal
































38



References














39














40














41
















42















43



Chapter 2



47

21 Plant material





Herbarium

22 Drugs























48


















49












CO2








50































51








sample

211 Degranulation






212 Oxidative burst














52






























53







02 Triton X-100






References









54












55








Chapter 3




12 Ragheda Yaseen

1 William N Setzer

3 Hassan Y Naim

1 Maren









58


the following scale









59

Abstract












Results









Conclusions





60

Introduction


















et al 2005)












61
































62

































63



























scatter SSC)






64












Results








every 30 min


65










(

b)


66
















formation












67



(b)


68





neutrophils











69











extract


70



(

a)


71








dependent







3-10)


72







process



73

Discussion









effect






















74


determined

















Acknowledgements








75

References












76












Chapter 4




12 Lena Voumlllger

1 Hassan Y Naim


1



Technology





78


the following scale









79

Abstract







80

Introduction





























2009)


81
































82
































83
































84







and p lt 0001



















85


Phagocytosis

E coli S aureus

a c

bi bii

CtrEnro

25nM P

MA

0

50

100

150

200

400

600

800

Rel

ativ

e o

xid

ativ

e b

urs

t

[x-M

ean

of F

L-1

]

Ctr

Enro

25nM P

MA

0

200

400

600

n s

Rel

ativ

e d

egra

nu

latio

n [S

SC

val

ue]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

20

25

n s

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

NET-formation

di dii

diii

Ctr

Ctr + D

PI

Enro

Enro +

DPI

0

10

20

30

NE

T-r

elea

sin

g c

ells

[]



86





























87

Cell death



NaO

HEnr

o

0

5

10

15 n s

LD

H r

ele

as

e in

[Co

mp

are

d t

o t

ota

l c

ell ly

sis

]

Enr

o

Enr

o +

Cyt

D

Ctr +

Cyt

D

0

5

10

15

20

25

ns

NE

T-r

ele

as

ing

ce

lls

[

]

NaOH

74 kDa

PAD4 M Enro

Ctr

Enr

o

0

50

100

150

200

rela

tiv

e P

AD

-4-p

rote

in e

xp

res

sio

n in

[co

mp

are

d t

o

-ac

tin

ex

pre

ss

ion

]


NET formation

ai

aii

bi

bii

ci

cii

Enr

o

Enr

o +

Noc

Ctr +

Noc

0

10

20

30

40

n s

NE

T-r

ele

as

ing

ce

lls

[

]

74 kDa

50 kDa

33 kDa

M Enro Ctr

PAD-4

β-actin









88








Conclusions

















Acknowledgements




89

Funding


References












90









Chapter 5



93

Discussion







to public health

























94








V 2009)










2009)














95

































96









(Ono et al 2000)
























97



1997)






















4-2 b)








98












Ohn et al 2010)







Future outlook






MRSA







99














immunity

References








100












101















102















103





Chapter 6

Summary

Summary Chapter 6

107

Summary

Natalja Jerjomiceva






















infections






Summary Chapter 6

108












Chapter 7

Zusammenfassung


111

Zusammenfassung

Natalja Jerjomiceva






















analysiert








112












ist




Appendix

Appendix

115









coli mastitis 35








bovine blood 49


aureus Newman 64



neutrophils 67


neutrophils 67



neutrophils 69



Appendix

116















G kunthiana 30

Abstracts






























of infections






1 William N Setzer

2 Hassan Y Naim


Blickwede1


2Department of




































Germany



Acknowledgements

















when needed



















Erklaumlrung








Dissertation stehen



Hannover





Hannover den 300413

Natalja Jerjomiceva

Curriculum Vitae





Hannover Germany



Germany






Germany



























CytD cytochalasin D






Fe iron














IL-2 Interleukin-2



kDa kilo Dalton

LB Lysogeny broth


Linn Carl Linnaeus


microl microliter

M molar



mg milligram

min minute

ml millilitre

mm millimeter

mM millimolar






NK natural killer

Noc nocodazole

OD optical density





RBC red blood cell








U unit

S sulfur






Chapter 1



9






















10


















aureus (Todar 2002)















11












treatment regimes


12













30

10

4

104

14

5

23



13










today























14


















Several plants





















15

























Lobelia inflata














16







































17





























2011)


18

































19








(Tseng et al 2005)

























20
































21

































22



et al 2004)




























above


23



Allium sativum


Europe







prolifeation














Taiwan


antioxidant


nephritis


macrophages

Tseng et al 2005


24







system

Gokhale et al 2002


(Caesalpiniaceae)

India







diabetes mellitus





Shukla et al 2009





count

Ghule et al 2006






Nores et al 1997






Manosroi et al 2003


25


(Amaryllidacea)


Immunostimulatory


and heal wounds


Lakshmi et al 2003

Curcuma



immunostimmulating




Kim et al 2007






lipid peroxidation

Gan et al 2004


Brazil



treatment


Pinello et at 2005

Pouteria







oxidative burst

Manosroi et al 2005

Tephrosia purpurea

(Leguminosae)





Damre et al 2003


26































27























Costa Rica









28













forests







15 Guarea kunthiana




Herbarium





29
















2009)












30


Species

Plant

part

used

Solvent

Mortality rate ()


G kunthiana


Ethanol 0 0 5 10 15 20 25

Stem

Hexane 25 30 35 40 40 40 40

Ethanol 10 15 15 20 20 20 20








al 2006)












infections


31

16 Fluoroquinolones

General structure









(Ball 2000)





















32











(Owens et al 2000)

Distribution

















33

17 Enrofloxacin



























2011)





34





















35

















36











37

18 Goal
































38



References














39














40














41
















42















43



Chapter 2



47

21 Plant material





Herbarium

22 Drugs























48


















49












CO2








50































51








sample

211 Degranulation






212 Oxidative burst














52






























53







02 Triton X-100






References









54












55








Chapter 3




12 Ragheda Yaseen

1 William N Setzer

3 Hassan Y Naim

1 Maren









58


the following scale









59

Abstract












Results









Conclusions





60

Introduction


















et al 2005)












61
































62

































63



























scatter SSC)






64












Results








every 30 min


65










(

b)


66
















formation












67



(b)


68





neutrophils











69











extract


70



(

a)


71








dependent







3-10)


72







process



73

Discussion









effect






















74


determined

















Acknowledgements








75

References












76












Chapter 4




12 Lena Voumlllger

1 Hassan Y Naim


1



Technology





78


the following scale









79

Abstract







80

Introduction





























2009)


81
































82
































83
































84







and p lt 0001



















85


Phagocytosis

E coli S aureus

a c

bi bii

CtrEnro

25nM P

MA

0

50

100

150

200

400

600

800

Rel

ativ

e o

xid

ativ

e b

urs

t

[x-M

ean

of F

L-1

]

Ctr

Enro

25nM P

MA

0

200

400

600

n s

Rel

ativ

e d

egra

nu

latio

n [S

SC

val

ue]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

20

25

n s

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

NET-formation

di dii

diii

Ctr

Ctr + D

PI

Enro

Enro +

DPI

0

10

20

30

NE

T-r

elea

sin

g c

ells

[]



86





























87

Cell death



NaO

HEnr

o

0

5

10

15 n s

LD

H r

ele

as

e in

[Co

mp

are

d t

o t

ota

l c

ell ly

sis

]

Enr

o

Enr

o +

Cyt

D

Ctr +

Cyt

D

0

5

10

15

20

25

ns

NE

T-r

ele

as

ing

ce

lls

[

]

NaOH

74 kDa

PAD4 M Enro

Ctr

Enr

o

0

50

100

150

200

rela

tiv

e P

AD

-4-p

rote

in e

xp

res

sio

n in

[co

mp

are

d t

o

-ac

tin

ex

pre

ss

ion

]


NET formation

ai

aii

bi

bii

ci

cii

Enr

o

Enr

o +

Noc

Ctr +

Noc

0

10

20

30

40

n s

NE

T-r

ele

as

ing

ce

lls

[

]

74 kDa

50 kDa

33 kDa

M Enro Ctr

PAD-4

β-actin









88








Conclusions

















Acknowledgements




89

Funding


References












90









Chapter 5



93

Discussion







to public health

























94








V 2009)










2009)














95

































96









(Ono et al 2000)
























97



1997)






















4-2 b)








98












Ohn et al 2010)







Future outlook






MRSA







99














immunity

References








100












101















102















103





Chapter 6

Summary

Summary Chapter 6

107

Summary

Natalja Jerjomiceva






















infections






Summary Chapter 6

108












Chapter 7

Zusammenfassung


111

Zusammenfassung

Natalja Jerjomiceva






















analysiert








112












ist




Appendix

Appendix

115









coli mastitis 35








bovine blood 49


aureus Newman 64



neutrophils 67


neutrophils 67



neutrophils 69



Appendix

116















G kunthiana 30

Abstracts






























of infections






1 William N Setzer

2 Hassan Y Naim


Blickwede1


2Department of




































Germany



Acknowledgements

















when needed



















Erklaumlrung








Dissertation stehen



Hannover





Hannover den 300413

Natalja Jerjomiceva

Curriculum Vitae





Hannover Germany



Germany






Germany



























CytD cytochalasin D






Fe iron














IL-2 Interleukin-2



kDa kilo Dalton

LB Lysogeny broth


Linn Carl Linnaeus


microl microliter

M molar



mg milligram

min minute

ml millilitre

mm millimeter

mM millimolar






NK natural killer

Noc nocodazole

OD optical density





RBC red blood cell








U unit

S sulfur







9






















10


















aureus (Todar 2002)















11












treatment regimes


12













30

10

4

104

14

5

23



13










today























14


















Several plants





















15

























Lobelia inflata














16







































17





























2011)


18

































19








(Tseng et al 2005)

























20
































21

































22



et al 2004)




























above


23



Allium sativum


Europe







prolifeation














Taiwan


antioxidant


nephritis


macrophages

Tseng et al 2005


24







system

Gokhale et al 2002


(Caesalpiniaceae)

India







diabetes mellitus





Shukla et al 2009





count

Ghule et al 2006






Nores et al 1997






Manosroi et al 2003


25


(Amaryllidacea)


Immunostimulatory


and heal wounds


Lakshmi et al 2003

Curcuma



immunostimmulating




Kim et al 2007






lipid peroxidation

Gan et al 2004


Brazil



treatment


Pinello et at 2005

Pouteria







oxidative burst

Manosroi et al 2005

Tephrosia purpurea

(Leguminosae)





Damre et al 2003


26































27























Costa Rica









28













forests







15 Guarea kunthiana




Herbarium





29
















2009)












30


Species

Plant

part

used

Solvent

Mortality rate ()


G kunthiana


Ethanol 0 0 5 10 15 20 25

Stem

Hexane 25 30 35 40 40 40 40

Ethanol 10 15 15 20 20 20 20








al 2006)












infections


31

16 Fluoroquinolones

General structure









(Ball 2000)





















32











(Owens et al 2000)

Distribution

















33

17 Enrofloxacin



























2011)





34





















35

















36











37

18 Goal
































38



References














39














40














41
















42















43



Chapter 2



47

21 Plant material





Herbarium

22 Drugs























48


















49












CO2








50































51








sample

211 Degranulation






212 Oxidative burst














52






























53







02 Triton X-100






References









54












55








Chapter 3




12 Ragheda Yaseen

1 William N Setzer

3 Hassan Y Naim

1 Maren









58


the following scale









59

Abstract












Results









Conclusions





60

Introduction


















et al 2005)












61
































62

































63



























scatter SSC)






64












Results








every 30 min


65










(

b)


66
















formation












67



(b)


68





neutrophils











69











extract


70



(

a)


71








dependent







3-10)


72







process



73

Discussion









effect






















74


determined

















Acknowledgements








75

References












76












Chapter 4




12 Lena Voumlllger

1 Hassan Y Naim


1



Technology





78


the following scale









79

Abstract







80

Introduction





























2009)


81
































82
































83
































84







and p lt 0001



















85


Phagocytosis

E coli S aureus

a c

bi bii

CtrEnro

25nM P

MA

0

50

100

150

200

400

600

800

Rel

ativ

e o

xid

ativ

e b

urs

t

[x-M

ean

of F

L-1

]

Ctr

Enro

25nM P

MA

0

200

400

600

n s

Rel

ativ

e d

egra

nu

latio

n [S

SC

val

ue]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

20

25

n s

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

NET-formation

di dii

diii

Ctr

Ctr + D

PI

Enro

Enro +

DPI

0

10

20

30

NE

T-r

elea

sin

g c

ells

[]



86





























87

Cell death



NaO

HEnr

o

0

5

10

15 n s

LD

H r

ele

as

e in

[Co

mp

are

d t

o t

ota

l c

ell ly

sis

]

Enr

o

Enr

o +

Cyt

D

Ctr +

Cyt

D

0

5

10

15

20

25

ns

NE

T-r

ele

as

ing

ce

lls

[

]

NaOH

74 kDa

PAD4 M Enro

Ctr

Enr

o

0

50

100

150

200

rela

tiv

e P

AD

-4-p

rote

in e

xp

res

sio

n in

[co

mp

are

d t

o

-ac

tin

ex

pre

ss

ion

]


NET formation

ai

aii

bi

bii

ci

cii

Enr

o

Enr

o +

Noc

Ctr +

Noc

0

10

20

30

40

n s

NE

T-r

ele

as

ing

ce

lls

[

]

74 kDa

50 kDa

33 kDa

M Enro Ctr

PAD-4

β-actin









88








Conclusions

















Acknowledgements




89

Funding


References












90









Chapter 5



93

Discussion







to public health

























94








V 2009)










2009)














95

































96









(Ono et al 2000)
























97



1997)






















4-2 b)








98












Ohn et al 2010)







Future outlook






MRSA







99














immunity

References








100












101















102















103





Chapter 6

Summary

Summary Chapter 6

107

Summary

Natalja Jerjomiceva






















infections






Summary Chapter 6

108












Chapter 7

Zusammenfassung


111

Zusammenfassung

Natalja Jerjomiceva






















analysiert








112












ist




Appendix

Appendix

115









coli mastitis 35








bovine blood 49


aureus Newman 64



neutrophils 67


neutrophils 67



neutrophils 69



Appendix

116















G kunthiana 30

Abstracts






























of infections






1 William N Setzer

2 Hassan Y Naim


Blickwede1


2Department of




































Germany



Acknowledgements

















when needed



















Erklaumlrung








Dissertation stehen



Hannover





Hannover den 300413

Natalja Jerjomiceva

Curriculum Vitae





Hannover Germany



Germany






Germany



























CytD cytochalasin D






Fe iron














IL-2 Interleukin-2



kDa kilo Dalton

LB Lysogeny broth


Linn Carl Linnaeus


microl microliter

M molar



mg milligram

min minute

ml millilitre

mm millimeter

mM millimolar






NK natural killer

Noc nocodazole

OD optical density





RBC red blood cell








U unit

S sulfur







10


















aureus (Todar 2002)















11












treatment regimes


12













30

10

4

104

14

5

23



13










today























14


















Several plants





















15

























Lobelia inflata














16







































17





























2011)


18

































19








(Tseng et al 2005)

























20
































21

































22



et al 2004)




























above


23



Allium sativum


Europe







prolifeation














Taiwan


antioxidant


nephritis


macrophages

Tseng et al 2005


24







system

Gokhale et al 2002


(Caesalpiniaceae)

India







diabetes mellitus





Shukla et al 2009





count

Ghule et al 2006






Nores et al 1997






Manosroi et al 2003


25


(Amaryllidacea)


Immunostimulatory


and heal wounds


Lakshmi et al 2003

Curcuma



immunostimmulating




Kim et al 2007






lipid peroxidation

Gan et al 2004


Brazil



treatment


Pinello et at 2005

Pouteria







oxidative burst

Manosroi et al 2005

Tephrosia purpurea

(Leguminosae)





Damre et al 2003


26































27























Costa Rica









28













forests







15 Guarea kunthiana




Herbarium





29
















2009)












30


Species

Plant

part

used

Solvent

Mortality rate ()


G kunthiana


Ethanol 0 0 5 10 15 20 25

Stem

Hexane 25 30 35 40 40 40 40

Ethanol 10 15 15 20 20 20 20








al 2006)












infections


31

16 Fluoroquinolones

General structure









(Ball 2000)





















32











(Owens et al 2000)

Distribution

















33

17 Enrofloxacin



























2011)





34





















35

















36











37

18 Goal
































38



References














39














40














41
















42















43



Chapter 2



47

21 Plant material





Herbarium

22 Drugs























48


















49












CO2








50































51








sample

211 Degranulation






212 Oxidative burst














52






























53







02 Triton X-100






References









54












55








Chapter 3




12 Ragheda Yaseen

1 William N Setzer

3 Hassan Y Naim

1 Maren









58


the following scale









59

Abstract












Results









Conclusions





60

Introduction


















et al 2005)












61
































62

































63



























scatter SSC)






64












Results








every 30 min


65










(

b)


66
















formation












67



(b)


68





neutrophils











69











extract


70



(

a)


71








dependent







3-10)


72







process



73

Discussion









effect






















74


determined

















Acknowledgements








75

References












76












Chapter 4




12 Lena Voumlllger

1 Hassan Y Naim


1



Technology





78


the following scale









79

Abstract







80

Introduction





























2009)


81
































82
































83
































84







and p lt 0001



















85


Phagocytosis

E coli S aureus

a c

bi bii

CtrEnro

25nM P

MA

0

50

100

150

200

400

600

800

Rel

ativ

e o

xid

ativ

e b

urs

t

[x-M

ean

of F

L-1

]

Ctr

Enro

25nM P

MA

0

200

400

600

n s

Rel

ativ

e d

egra

nu

latio

n [S

SC

val

ue]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

20

25

n s

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

NET-formation

di dii

diii

Ctr

Ctr + D

PI

Enro

Enro +

DPI

0

10

20

30

NE

T-r

elea

sin

g c

ells

[]



86





























87

Cell death



NaO

HEnr

o

0

5

10

15 n s

LD

H r

ele

as

e in

[Co

mp

are

d t

o t

ota

l c

ell ly

sis

]

Enr

o

Enr

o +

Cyt

D

Ctr +

Cyt

D

0

5

10

15

20

25

ns

NE

T-r

ele

as

ing

ce

lls

[

]

NaOH

74 kDa

PAD4 M Enro

Ctr

Enr

o

0

50

100

150

200

rela

tiv

e P

AD

-4-p

rote

in e

xp

res

sio

n in

[co

mp

are

d t

o

-ac

tin

ex

pre

ss

ion

]


NET formation

ai

aii

bi

bii

ci

cii

Enr

o

Enr

o +

Noc

Ctr +

Noc

0

10

20

30

40

n s

NE

T-r

ele

as

ing

ce

lls

[

]

74 kDa

50 kDa

33 kDa

M Enro Ctr

PAD-4

β-actin









88








Conclusions

















Acknowledgements




89

Funding


References












90









Chapter 5



93

Discussion







to public health

























94








V 2009)










2009)














95

































96









(Ono et al 2000)
























97



1997)






















4-2 b)








98












Ohn et al 2010)







Future outlook






MRSA







99














immunity

References








100












101















102















103





Chapter 6

Summary

Summary Chapter 6

107

Summary

Natalja Jerjomiceva






















infections






Summary Chapter 6

108












Chapter 7

Zusammenfassung


111

Zusammenfassung

Natalja Jerjomiceva






















analysiert








112












ist




Appendix

Appendix

115









coli mastitis 35








bovine blood 49


aureus Newman 64



neutrophils 67


neutrophils 67



neutrophils 69



Appendix

116















G kunthiana 30

Abstracts






























of infections






1 William N Setzer

2 Hassan Y Naim


Blickwede1


2Department of




































Germany



Acknowledgements

















when needed



















Erklaumlrung








Dissertation stehen



Hannover





Hannover den 300413

Natalja Jerjomiceva

Curriculum Vitae





Hannover Germany



Germany






Germany



























CytD cytochalasin D






Fe iron














IL-2 Interleukin-2



kDa kilo Dalton

LB Lysogeny broth


Linn Carl Linnaeus


microl microliter

M molar



mg milligram

min minute

ml millilitre

mm millimeter

mM millimolar






NK natural killer

Noc nocodazole

OD optical density





RBC red blood cell








U unit

S sulfur







11












treatment regimes


12













30

10

4

104

14

5

23



13










today























14


















Several plants





















15

























Lobelia inflata














16







































17





























2011)


18

































19








(Tseng et al 2005)

























20
































21

































22



et al 2004)




























above


23



Allium sativum


Europe







prolifeation














Taiwan


antioxidant


nephritis


macrophages

Tseng et al 2005


24







system

Gokhale et al 2002


(Caesalpiniaceae)

India







diabetes mellitus





Shukla et al 2009





count

Ghule et al 2006






Nores et al 1997






Manosroi et al 2003


25


(Amaryllidacea)


Immunostimulatory


and heal wounds


Lakshmi et al 2003

Curcuma



immunostimmulating




Kim et al 2007






lipid peroxidation

Gan et al 2004


Brazil



treatment


Pinello et at 2005

Pouteria







oxidative burst

Manosroi et al 2005

Tephrosia purpurea

(Leguminosae)





Damre et al 2003


26































27























Costa Rica









28













forests







15 Guarea kunthiana




Herbarium





29
















2009)












30


Species

Plant

part

used

Solvent

Mortality rate ()


G kunthiana


Ethanol 0 0 5 10 15 20 25

Stem

Hexane 25 30 35 40 40 40 40

Ethanol 10 15 15 20 20 20 20








al 2006)












infections


31

16 Fluoroquinolones

General structure









(Ball 2000)





















32











(Owens et al 2000)

Distribution

















33

17 Enrofloxacin



























2011)





34





















35

















36











37

18 Goal
































38



References














39














40














41
















42















43



Chapter 2



47

21 Plant material





Herbarium

22 Drugs























48


















49












CO2








50































51








sample

211 Degranulation






212 Oxidative burst














52






























53







02 Triton X-100






References









54












55








Chapter 3




12 Ragheda Yaseen

1 William N Setzer

3 Hassan Y Naim

1 Maren









58


the following scale









59

Abstract












Results









Conclusions





60

Introduction


















et al 2005)












61
































62

































63



























scatter SSC)






64












Results








every 30 min


65










(

b)


66
















formation












67



(b)


68





neutrophils











69











extract


70



(

a)


71








dependent







3-10)


72







process



73

Discussion









effect






















74


determined

















Acknowledgements








75

References












76












Chapter 4




12 Lena Voumlllger

1 Hassan Y Naim


1



Technology





78


the following scale









79

Abstract







80

Introduction





























2009)


81
































82
































83
































84







and p lt 0001



















85


Phagocytosis

E coli S aureus

a c

bi bii

CtrEnro

25nM P

MA

0

50

100

150

200

400

600

800

Rel

ativ

e o

xid

ativ

e b

urs

t

[x-M

ean

of F

L-1

]

Ctr

Enro

25nM P

MA

0

200

400

600

n s

Rel

ativ

e d

egra

nu

latio

n [S

SC

val

ue]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

20

25

n s

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

NET-formation

di dii

diii

Ctr

Ctr + D

PI

Enro

Enro +

DPI

0

10

20

30

NE

T-r

elea

sin

g c

ells

[]



86





























87

Cell death



NaO

HEnr

o

0

5

10

15 n s

LD

H r

ele

as

e in

[Co

mp

are

d t

o t

ota

l c

ell ly

sis

]

Enr

o

Enr

o +

Cyt

D

Ctr +

Cyt

D

0

5

10

15

20

25

ns

NE

T-r

ele

as

ing

ce

lls

[

]

NaOH

74 kDa

PAD4 M Enro

Ctr

Enr

o

0

50

100

150

200

rela

tiv

e P

AD

-4-p

rote

in e

xp

res

sio

n in

[co

mp

are

d t

o

-ac

tin

ex

pre

ss

ion

]


NET formation

ai

aii

bi

bii

ci

cii

Enr

o

Enr

o +

Noc

Ctr +

Noc

0

10

20

30

40

n s

NE

T-r

ele

as

ing

ce

lls

[

]

74 kDa

50 kDa

33 kDa

M Enro Ctr

PAD-4

β-actin









88








Conclusions

















Acknowledgements




89

Funding


References












90









Chapter 5



93

Discussion







to public health

























94








V 2009)










2009)














95

































96









(Ono et al 2000)
























97



1997)






















4-2 b)








98












Ohn et al 2010)







Future outlook






MRSA







99














immunity

References








100












101















102















103





Chapter 6

Summary

Summary Chapter 6

107

Summary

Natalja Jerjomiceva






















infections






Summary Chapter 6

108












Chapter 7

Zusammenfassung


111

Zusammenfassung

Natalja Jerjomiceva






















analysiert








112












ist




Appendix

Appendix

115









coli mastitis 35








bovine blood 49


aureus Newman 64



neutrophils 67


neutrophils 67



neutrophils 69



Appendix

116















G kunthiana 30

Abstracts






























of infections






1 William N Setzer

2 Hassan Y Naim


Blickwede1


2Department of




































Germany



Acknowledgements

















when needed



















Erklaumlrung








Dissertation stehen



Hannover





Hannover den 300413

Natalja Jerjomiceva

Curriculum Vitae





Hannover Germany



Germany






Germany



























CytD cytochalasin D






Fe iron














IL-2 Interleukin-2



kDa kilo Dalton

LB Lysogeny broth


Linn Carl Linnaeus


microl microliter

M molar



mg milligram

min minute

ml millilitre

mm millimeter

mM millimolar






NK natural killer

Noc nocodazole

OD optical density





RBC red blood cell








U unit

S sulfur







12













30

10

4

104

14

5

23



13










today























14


















Several plants





















15

























Lobelia inflata














16







































17





























2011)


18

































19








(Tseng et al 2005)

























20
































21

































22



et al 2004)




























above


23



Allium sativum


Europe







prolifeation














Taiwan


antioxidant


nephritis


macrophages

Tseng et al 2005


24







system

Gokhale et al 2002


(Caesalpiniaceae)

India







diabetes mellitus





Shukla et al 2009





count

Ghule et al 2006






Nores et al 1997






Manosroi et al 2003


25


(Amaryllidacea)


Immunostimulatory


and heal wounds


Lakshmi et al 2003

Curcuma



immunostimmulating




Kim et al 2007






lipid peroxidation

Gan et al 2004


Brazil



treatment


Pinello et at 2005

Pouteria







oxidative burst

Manosroi et al 2005

Tephrosia purpurea

(Leguminosae)





Damre et al 2003


26































27























Costa Rica









28













forests







15 Guarea kunthiana




Herbarium





29
















2009)












30


Species

Plant

part

used

Solvent

Mortality rate ()


G kunthiana


Ethanol 0 0 5 10 15 20 25

Stem

Hexane 25 30 35 40 40 40 40

Ethanol 10 15 15 20 20 20 20








al 2006)












infections


31

16 Fluoroquinolones

General structure









(Ball 2000)





















32











(Owens et al 2000)

Distribution

















33

17 Enrofloxacin



























2011)





34





















35

















36











37

18 Goal
































38



References














39














40














41
















42















43



Chapter 2



47

21 Plant material





Herbarium

22 Drugs























48


















49












CO2








50































51








sample

211 Degranulation






212 Oxidative burst














52






























53







02 Triton X-100






References









54












55








Chapter 3




12 Ragheda Yaseen

1 William N Setzer

3 Hassan Y Naim

1 Maren









58


the following scale









59

Abstract












Results









Conclusions





60

Introduction


















et al 2005)












61
































62

































63



























scatter SSC)






64












Results








every 30 min


65










(

b)


66
















formation












67



(b)


68





neutrophils











69











extract


70



(

a)


71








dependent







3-10)


72







process



73

Discussion









effect






















74


determined

















Acknowledgements








75

References












76












Chapter 4




12 Lena Voumlllger

1 Hassan Y Naim


1



Technology





78


the following scale









79

Abstract







80

Introduction





























2009)


81
































82
































83
































84







and p lt 0001



















85


Phagocytosis

E coli S aureus

a c

bi bii

CtrEnro

25nM P

MA

0

50

100

150

200

400

600

800

Rel

ativ

e o

xid

ativ

e b

urs

t

[x-M

ean

of F

L-1

]

Ctr

Enro

25nM P

MA

0

200

400

600

n s

Rel

ativ

e d

egra

nu

latio

n [S

SC

val

ue]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

20

25

n s

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

Ctr

Ctr + C

yt DEnro

Enro +

Cyt D

0

5

10

15

Rel

ativ

e p

hag

ocy

tosi

s

[Gx-

Mea

n o

f FL

-1]

NET-formation

di dii

diii

Ctr

Ctr + D

PI

Enro

Enro +

DPI

0

10

20

30

NE

T-r

elea

sin

g c

ells

[]



86





























87

Cell death



NaO

HEnr

o

0

5

10

15 n s

LD

H r

ele

as

e in

[Co

mp

are

d t

o t

ota

l c

ell ly

sis

]

Enr

o

Enr

o +

Cyt

D

Ctr +

Cyt

D

0

5

10

15

20

25

ns

NE

T-r

ele

as

ing

ce

lls

[

]

NaOH

74 kDa

PAD4 M Enro

Ctr

Enr

o

0

50

100

150

200

rela

tiv

e P

AD

-4-p

rote

in e

xp

res

sio

n in

[co

mp

are

d t

o

-ac

tin

ex

pre

ss

ion

]


NET formation

ai

aii

bi

bii

ci

cii

Enr

o

Enr

o +

Noc

Ctr +

Noc

0

10

20

30

40

n s

NE

T-r

ele

as

ing

ce

lls

[

]

74 kDa

50 kDa

33 kDa

M Enro Ctr

PAD-4

β-actin









88








Conclusions

















Acknowledgements




89

Funding


References












90









Chapter 5



93

Discussion







to public health

























94








V 2009)










2009)














95

































96









(Ono et al 2000)
























97



1997)






















4-2 b)








98












Ohn et al 2010)







Future outlook






MRSA







99














immunity

References








100












101















102















103





Chapter 6

Summary

Summary Chapter 6

107

Summary

Natalja Jerjomiceva






















infections






Summary Chapter 6

108












Chapter 7

Zusammenfassung


111

Zusammenfassung

Natalja Jerjomiceva






















analysiert








112












ist




Appendix

Appendix

115









coli mastitis 35








bovine blood 49


aureus Newman 64



neutrophils 67


neutrophils 67



neutrophils 69



Appendix

116















G kunthiana 30

Abstracts






























of infections






1 William N Setzer

2 Hassan Y Naim


Blickwede1


2Department of




































Germany



Acknowledgements

















when needed



















Erklaumlrung








Dissertation stehen



Hannover





Hannover den 300413

Natalja Jerjomiceva

Curriculum Vitae





Hannover Germany



Germany






Germany



























CytD cytochalasin D






Fe iron














IL-2 Interleukin-2



kDa kilo Dalton

LB Lysogeny broth


Linn Carl Linnaeus


microl microliter

M molar



mg milligram

min minute

ml millilitre

mm millimeter

mM millimolar






NK natural killer

Noc nocodazole

OD optical density





RBC red blood cell








U unit

S sulfur






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