The pathogenesis of Leishmania/HIV co-infection: cellular ... · PATHOGENESIS OF Leishmania/HIV...

The pathogenesis of LeishmaniaHIV co-infectioncellular and immunological mechanisms

M OLIVIER R BADAROdagger F J MEDRANODagger and J MORENOsect

Faculty of Medicine McGill University Lyman DuV Medical Building 3775 University StreetMontreal Quebec H3A 2B4 CanadadaggerUniversidade Federal da Bahia Departamento de Medicina Rua Frei Henrique 8 Nazare40050420 Salvador BA BrazilDaggerViral Hepatitis and AIDS Study Group Department of Internal MedicineHospital Universitario Virgen del Roc otilde o Avenida Manuel Siurot sn 41013 Seville SpainsectWHO Collaborating Centre for Leishmaniasis Servicio de Parasitologotilde a Centro Nacionalde Microbiologotilde a Instituto de Salud Carlos III Carretera MajadahondandashPozuelo Km 228220 Majadahonda Madrid Spain

Received and accepted 30 May 2003

The intracellular protozoan parasites of the genus Leishmania have been recognized as opportunistic pathogensin immunosuppressed individuals including those infected with human immunode ciency virus type-1 (HIV-1)Leishmaniasis and AIDS overlap in several sub-tropical and tropical regions around the world including theMediterranean area In 1994 3ndash7 of HIV-1-infected individuals in southern Europe developed visceralleishmaniasis In humans interestingly both HIV-1 and Leishmania interact with invade and multiply withincells of myeloid or lymphoid origin The combined modulation of Leishmania- and HIV-1-related pathogenesisin the co-infected cases is therefore probably a realistic goal In the light of the recent demonstration thatL donovani can up-regulate HIV-1 replication both in monocytoid and lymphoid cells in vitro and in co-infectedindividuals it is clear from the epidemiological data available that Leishmania can probably act as a powerfulco-factor in the pathogenesis of HIV-1 infection In those who are co-infected complex mechanisms involvingcytokine secretion and cellular-signalling events play pivotal roles in the Leishmania-mediated activation andpathogenesis of HIV-1 An overview of the recent ndings concerning this LeishmaniaHIV-1 interaction ispresented here

The aetiological role of the human immuno- recognized as the predominant cell line pro-de ciency virus type-1 (HIV-1) in the patho- ductively infected with HIV in the lymphgenesis of the acquired immunode ciency nodes lungs and central nervous systemsyndrome (AIDS) is rmly established (Meltzer et al 1990) Monocytes and macro-(Popovic et al 1984 Levy 1993) HIV-1 is phages represent an important reservoir forknown to propagate mainly in T-lymphocytes HIV and serve as vehicles that disseminatebecause these cells express the primary the virus throughout the host In the infectedcellular receptor for viral entry into target cells individual HIV-1 may replicate undetectedthe surface molecule CD4 (Dalgleish et al at low levels for prolonged periods with1984 Klatzmann et al 1984 McDougal minimal clinical manifestation An extended1986) However the macrophage has been period of latency the development of various

and repetitive opportunistic infections attri-butable to the state of immunode ciencyReprint requests to M Olivier

E-mail martinoliviermcgillca fax +1 514 398 7052 induced by the infection and the period

DOI 101179000349803225002561

Annals of Tropical Medicine amp Parasitology Vol 97 Supplement No 1 S79ndashS98 (2003)

copy 2003 The Liverpool School of Tropical Medicine

80 OLIVIER ET AL

between time of infection and onset of full- Bernier et al (1995) observed thatL donovani its surface lipophosphoglycanblown AIDS (Melbye et al 1986 Fauci

1988a) are indeed the principal clinical (LPG) and diverse structural componentsof the parasite could each induce viralcharacteristics of HIV-1 infection Co-

infection with certain opportunistic micro- replication in several monocytoid cell lineschronically infected with HIV-1 [Fig 1(a)]organisms including the protozoan parasites

of the genus Leishmania may lead not The LPG molecule a glycoconjugate isone of the major constituents expressed ononly to direct pathogenesis and morbidity

but may also play an important and active the surface of the Leishmania promastigoteWhen a promastigote is inoculated into arole in the progression of HIV-1 infection

toward AIDS Such opportunistic infections human host by an infected sand y it isengulfed into the phagolysosome of a macro-induce in ammatory responses and cellular-

signalling events in their host that could phage where it rapidly diVerentiates intoan amastigote During its engulfment thepromote viral replication (Tremblay et al

1996) Interestingly it is known that certain parasite loses most of its structural LPG atthe surface of the phagocyte and retainsstimuli can activate regulatory elements

located within the long-terminal-repeat (LTR) only the intramembrane component Thiscomponent the phosphatidyl inositol coresequences of HIV-1 and may induce HIV-1

gene expression from latent proviruses thus (core-PI) is solely encountered on the sur-face of the amastigote The LPG surfaceaccelerating viral replication and disease

progression (Fauci 1988a b) It seems likely molecule has been recognized as favouringthe intracellular survival and establishment ofthat cellular activation induced in the course

of infection by opportunistic pathogens the parasite (Turco 1999) The alteration ofsignalling events dependent on the secondcould lead to the activation of the regulatory

elements of HIV-1 messenger protein kinase C (PKC) has beenimplicated in the LPG-mediated protectionAlthough the role of protozoan parasites

as possible co-factors favouring the pro- of the parasite within the macrophage phago-lysosome and the LPG-induced inhibitiongression of HIV-1 infection towards AIDS

still needs much study Tremblay et al of several important macrophage functionssuch as the generation of oxygen radicals(1996) discussed the putative participation

of Leishmania in the pathogenesis of HIV-1 (Handman et al 1986) Among other host-cell dysfunctions induced by Leishmaniainfection Their ideas were based on the

observation that the geographical distributions are inhibiting mechanisms (possibly LPG-independent) that can lead to the alterationof HIV and Leishmania overlap in several

countries Many cases of L infantumHIV-1 of several signalling pathways in which pro-tein tyrosine kinase (PTK) PKC and calciumco-infection have already been recorded

around the Mediterranean basin particularly (Ca2+ ) participate (Olivier et al 1992a b1998)in France Spain and Italy ( Jeannel et al

1989 Fillola et al 1992) There are clini- In a recent Spanish study which is dis-cussed at length below L infantum LPG wascal and epidemiological reports implicating

Leishmania as an opportunistic pathogen found to be not only an excellent activatorof HIV-1 replication within latently infectedin immunosuppressed AIDS patients (Alvar

et al 1987) As leishmanial parasites con- monocytoid cells but also a potent inducerof HIV-1 LTR transcription and viral repli-stitute a major public-health risk through-

out subtropical and tropical areas of the cation in T-cells [Fig 1(b) unpubl obs]In vitro rapid upregulation of HIV-1 LTRworld ( Jeronimo et al 1994 Marty et al

1994 Zijlstra et al 1994) it should come activity was seen following LPG additionpossibly indicating a direct eVect on theas no surprise if LeishmaniaHIV co-infection

emerges in patients living in these regions LTR-dependent gene expression of HIV-1

PATHOGENESIS OF LeishmaniaHIV CO-INFECTION 81

FIG 1 Induction of HIV-1 expression by lipophosphoglycan (LPG) in cells of the monocytoid and lymphoidlineages (a) Induction of HIV-1 expression in monocytoid (U1) cells by Leishmania donovani (one parasiteU1cell) 10 mm LPG mdash with or without antibodies to tumour necrosis factor-a (anti-TNF-a at 2 mgml) 10 mm

phosphoglycan (PG) 10 mm of the phosphatidyl inositol core of LPG (core-PI) or as a positive control 10 nm

phorbol 12-myristate 13-acetate (PMA) (b) Activation of HIV-1 transcription in lymphoid T (1G5) cells by10 mm LPG 10 mm core-PI or as a positive control phytohaemagglutinin (PHA at 3 mgml) (c) Signalling andactivation of the HIV-1 long-terminal repeats in infected lymphoid T (1G5) cells subjected to signalling-inhibitortreatment 1 h prior to stimulation with 10 mm LPG the inhibitors were herbimycin A (HerA at 01 mm) H7(5 mm) HA1004 (HA 5 mm) MDL12330 (MDL 125 mm) the membrane permeant acetomethoxy-ester formof bis-(aminophenoxy)ethane-tetraacetic acid (BAPTAAM 5 mm) and W7 (5 mm) (d) Activation of the HIV-1long-terminal repeats in Jurkat cells that had been transfected with wild-type nuclear factor-kB (NF-kB f ) ormutated constructs (e ) of this factor following stimulation for 24 h with PHA (at 3 mgml) or LPG (at 10 mm)For each experiment the results shown are mean values with se shown as error bars All the results for the testcultures diVered signi cantly from those of the respective control cultures (Plt001) In each culture viral loadwas measured as the level of reverse transcriptase activity (a) or after attaching a luciferase gene to the longterminal repeat of the HIV by measuring luciferase activity (b c and d)

Various intracellular second messengers modulin were also important physiologicaleVectors for the Leishmania-LPG-inducedwhose involvement was crucial to the LPG-

mediated activation of the HIV-1 LTR viral activation Ca2+ is known to be aregulator of several second messengers insequence were also identi ed Both PTK-

and protein-kinase-AcAMP-dependent sig- pivotal transductional mechanisms (Lewisand Cahalan 1995) Such observations werenalling for example were key events in the

LPG-induced activation of HIV-1 LTR not surprising as the Leishmania parasiteand its surface LPG can rapidly modulatetranscription [Fig 1(c)] The divalent cation

Ca2+ and the Ca2+ -binding protein cal- Ca2+ homeostasis and mobilization within

82 OLIVIER ET AL

the phagocytic host cell (Olivier et al 1992a studies have been performed in vitro it isrealistic to believe that during the course ofOlivier 1996 Mans eld and Olivier 2002)

Signalling events often result in increased Leishmania infection in an individual aZictedby HIV-1 free or phagocyte-presented para-gene expression which must involve a tran-

scriptional factor such as nuclear factor-kB site constituents could similarly exacerbateHIV-1 replication in cells of monocytoid or(NF-kB) As expected the LPG-mediated

HIV-1-LTR expression was found to involve lymphoid originResearch by Easterbrook et al (1995)the NF-kB binding region the LTR acti-

vation induced by LPG being completely demonstrated that interactions between LPGand T lymphoid cells lead to the inhibitionabolished in cells transiently transfected

with a mutated NF-kB construct [Fig 1(d) of the HIV-1-induced formation of syncytiaAs the formation of these giant multi-unpubl obs] LPG is capable of inducing

NF-kB translocation to the nucleus (Bernier nucleated cells is an important event in thedevelopment of HIV-1 pathogenesis (Levyet al 1998) The fact that the regulation

of NF-kB involves several protein kinases 1993) some interest in the potential use ofLeishmania LPG to inhibit the progressionsuch as protein kinases A and C PTK and

the Ca2+-dependent phosphatase calcineurin of HIV-1 infection has developed Howeverany potential bene ts from the use of LPG(Frantz et al 1994 Barbeau et al 1997)

helps to explain why so many second- in the design of new therapeutic strategiesagainst HIV-1 have to be carefully balancedmessenger antagonists blocked LPG-induced

HIV-1 LTR activation through NF-kB- against the potential disadvantages (Bernieret al 1995 1998) In addition to its capacitydependent events (Folks et al 1987 Duh

et al 1989 Pomerantz et al 1990) The to activate HIV-1 replication the LPGmolecule can also stimulate the release ofLeishmania LPG molecule is not the only

pathogenic component to activate NF-kB several in ammatory molecules such asprostaglandin E2 (PGE2 Matte et al 2001)mdash bacterial lipopolysaccharide (LPS) and

the lipoarabinomannan of Mycobacterium PGE2 is reported to be secreted during thecourse of both Leishmania (Reiner andtuberculosis are also recognized as powerful

inducers of NF-kB activation (Bernier et al Malemud 1984) and HIV-1 infection (Abelet al 1992 Foley et al 1992) It seems1998 Zhang et al 1995)

Like the LPG itself the core-PI mdash the likely that PGE2 plays a role in the develop-ment of HIV-1-related pathogenesis parti-intramembrane structural component of LPG

that is present on the amastigote surface mdash cularly in those co-infected with Leishmaniasince this prostaglandin is a powerful induceris also a powerful inducer of HIV-1 LTR

activity This is of prime importance consider- of HIV-1 replication (Dumais et al 1998)In summary it has been established thating that it is under the amastigote stage that

leishmanial infection progresses within the L infantum LPG and its core-PI moiety canvia complex biochemical pathways involvinghuman host Repeated units of the Leish-

mania LPG molecule can be detected on the the participation of transcriptional factorssuch as NF-kB activate HIV-1 LTR tran-surface of Leishmania-infected mononuclear

phagocytic cells (Turco 1999) Leishmania- scription in host cells of the monocytoid andlymphoid lineages It is suYcient to exposeinfected macrophages that are involved in cellndash

cell interactions with T-lymphocytes during HIV-1-infected T-cells or phagocytic cellsto LPG or Leishmania-infected mononuclearthe process of antigenic presentation (which

may involve the presentation of diVerent phagocytes to trigger activation of latent pro-virus DNA (Fig 2 unpubl obs) In thosestructural components of the LPG molecule)

might therefore be suYcient to trigger co-infected with Leishmania and HIV-1 theprotozoan parasite seems to be an importantsignalling events in HIV-1-infected CD4+

T-lymphocytes Although most of the relevant co-factor that helps promote the switch from


FIG

2

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HIV

-1ex

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84 OLIVIER ET AL

a state of clinical latency (in terms of the Although humans infected with HIV donot show evidence of a response that is pre-HIV-1 infection) to virus-related disease

The elucidation of the cellular events that dominantly of the Th1 or Th2 type (Graziosiet al 1994) some advances toward a betteroccur during Leishmania infection HIV-1

infection and LeishmaniaHIV-1 co-infection understanding of the immunopathogenesisof this life-threatening infection have beenshould speed the identi cation of speci c

cellular targets and the development of new made since HIV-1 was discovered A pro-found disruption in cell-mediated immunitytherapeutic approaches that could control

the associated diseases is of course central to the immune defectin AIDS but nearly every aspect of the hostrsquosnormal immune response is aVected by HIV

Th1- AND Th2-TYPE RESPONSES infection (Pantaleo et al 1993 1994 Fauci1996)

HIV can interact with T-helper and T-Some of the interactions that occur betweenHIV infection and parasitic diseases can be suppressor lymphocytes monocytes macro-

phages dendritic cells B-cells and microgliaanticipated from the immunology of para-sitic infections in humans and the known cells in the central nervous system (CNS) In

addition several chemokines and RANTESeVects of HIV on the immune system of itshosts The viral infection may lead to the loss (lsquoregulated upon activation normally T-cell

expressed and secreted chemokinersquo) receptorsor non-development of protective immunitypermitting unrestricted proliferation of the are down-regulated during the primary

infection (Fauci 1996 Weissman and Fauciparasites whereas the stimulation by theparasites of host cells that are latently or 1997 Zaitseva et al 1998) The dynamics

of HIV replication and its contribution tochronically infected with the virus may leadto increased expression of HIV or its pro- T-cell and macrophage destruction lead to

the inevitable and irreversible incapacitationtein products (Morrow et al 1989) Leish-maniasis is one of a group of parasitic of the hostrsquos immune system leaving it unable

to limit existing co-infections or to preventdiseases in which the pattern of responseby the T-helper (Th) cells of an immuno- opportunistic microbes from infecting the

host and causing morbidity (Ho et al 1995competent host correlates well with theclinical picture and the severity of the Mellors et al 1996 Perelson et al 1996)

The level of destruction of CD4+ cellsdisease (Reed and Scott 1993) In immuno-competent humans and murine models mdash as quantitatively expressed by counts of

such cells in the peripheral blood mdash appearsresistance to leishmaniasis and the self-limitation of the disease are associated with closely correlated with the risk that an

opportunistic infection will have clinicala Th1 cytokine pro le (Barral-Neto et al1995) Most patients who have localized and manifestations (Mellors et al 1997) Most

patients who have lt100 CD4+ cellsml inself-limited mucocutaneous leishmaniasisfor example have good lymphoproliferative their peripheral blood have had at least one

episode of illness as the result of an oppor-responses to leishmanial antigens and pro-duce large amounts of interleukin-2 (IL-2) tunistic infection (Frank et al 1998) The

pattern and severity of several infectiousand interferon-c (IFN-c) even in the activephase of their disease In contrast suscepti- diseases ( leishmaniasis being one of the best

examples) are markedly worsened by HIVbility to the leishmanial infection and diseaseexacerbation are associated in the immuno- co-infection although curiously the clinical

manifestations of leishmanial infection incompetent with cytokine responses of theTh2 type in which IL-4 IL-5 and large those with other immunosuppressive disease

are generally similar to those seen in theamounts of IL-10 predominate (Pirmez et al1993 Scott 1993) immunocompetent (Badaro et al 1986a b)


Almost all (90) of the approximately 700 IL-6 and IL-10) diVer signi cantly fromthose seen in simple HIV infection (Preisercases of LeishmaniaHIV co-infection reviewed

by the World Health Organization (1997) had et al 1996) When Nigro et al (1999) usedphytohaemagglutinin (PHA) to stimulatefewer than 200 CD4+ cellsml In southern

Europe there are demographic clinical peripheral-blood mononuclear cells (PBMC)from HIV-positives they found that theand epidemiological diVerences between the

immunocompetent who have visceral leish- responses varied depending on whether thecells had come from a patient co-infectedmaniasis (VL) and the HIV-infected cases of

the disease (Desjeux and Alvar 2003) Those with Leishmania or not Although there weremarked increases in the production of bothco-infected with HIV may develop VL

though infected with a Leishmania zymodeme IFN-c and IL-2R if the cells came from apatient without Leishmania infection therethat only causes cutaneous leishmaniasis in

the immunocompetent their leishmaniasis was only a signi cant increase in the pro-duction of IL-2R if the cells came from amay be particularly severe and unresponsive

to treatment and they may have amastigotes case of co-infection Cultured PBMC fromthe cases of LeishmaniaHIV co-infectionin tissues such as the intestine that are never

found infected in the immunocompetent also released greater quantities of IL-4 andIL-10 than the cells from patients who were(Badaro et al 1986a Badaro 1997 Munoz-

Rodrotilde guez et al 1997 Sebastian et al 1997 not infected with Leishmania These obser-vations con rm that co-infection with HIVMoreno-Camacho et al 1998 Vasquez-

Pineiro et al 1998 Wolday et al 1998 exacerbates the Th2-type responses of VLpatients (Preiser et al 1996) In additionDesjeux and Alvar 2003) Those co-infected

with HIV also have unusually high numbers the observation that IL-4 is overproduced inthe co-infected patients supports the hypo-of amastigotes in their reticulo-endothelial

system (Mart otilde nez et al 1993) thesis that HIV infection in association withother Th2-type inducing infections couldExactly how HIV circumvents the hostrsquos

immune defences still baZes investigators represent an indirect feature of Th2 cellexpansion among LeishmaniaHIV co-infectedBoth Leishmania and HIV share the same

target cells The results of in-vitro studies have subjects (Clerici et al 1993 Klein et al 1997Nigro et al 1999) If immunocompetentdemonstrated that L infantum induces the

expression of latent HIV-1 by up-regulating acute cases of VL generally respond well toantileishmanial therapy with resolution of theCCRS and CRXY receptors and GP120

on the surface of CD4+ lymphocytes and active infection and the complete regressionof the signs and symptoms of the diseasemacrophages (Bernier et al 1998) Con-

sequently more virus infects the CD4+ VL patients with HIV co-infection howeverrespond poorly to leishmaniasis treatmentcells which are virtually destroyed by the

L infantum whether the parasites are of a (Russo et al 1996) and prophylactic therapymust be administered frequently to controldermotropic or viscerotropic zymodeme

Thus in those co-infected with HIV severe the active disease This bleak clinical picturesupports the theory that irreversible defectsforms of leishmaniasis can arise irrespective

of the L infantum zymodeme involved in the T-cell-related immune responsetriggered by Th2 cytokine over-expansion( Jimenez et al 1991 1996 Gradoni and

Gramiccia 1994) Prolonged Th2-type acti- are caused by the dual infection (Zumlaand Croft 1992 Nigro et al 1999) Suchvation and increased viral replication have

been documented in patients co-infected defects favour the progression to AIDS inHIV-positive subjectswith Leishmania and HIV despite treatment

of their leishmaniasis (Cacopardo et al 1996) Memory cells also appear to be aVectedin LeishmaniaHIV co-infection The delayed-The Th2-type cytokine pro les in Leishmania

HIV co-infection (with high levels of IL-4 type hypersensitivity reaction (DTH) is the

86 OLIVIER ET AL

hallmark of hypersensitivity in tegumental associated with the Th2 and Th1 pro lesof cytokine production respectively (Reedleishmaniasis (Badaro 1997) but several

HIV-positive cases of mucocutaneous leish- and Scott 1993) Intense production ofTNF-a is also observed in patients duringmaniasis have been found to give a negative

result when tested with the leishmanin antileishmanial infections (Cenini et al 1993)Therefore both VL and HIV-1 infectiongen (Badaro 1997 De Souza et al 1998)

Hopefully use of highly active antiretroviral are associated with a depression in T-cellresponse and similar disturbances in cyto-therapy (HAART) will induce a reversal of

cytokine pro les in HIV-positive patients kine patterns To date only one study hasfocused on the Th1Th2 dynamic of thefrom type-2 to type-1 patterns and enhance

the eYcacy of antileishmanial therapy in VLHIV-1 co-infection (Cacopardo et al1996) increases in the serum concentrationsthose co-infected with Leishmania (Nabors

and Farrell 1996 Kubar et al 1998) of Th2 cytokines and HIV viraemia togetherwith a decrease in the serum concentrationsof IL-2 and IL-12 (ie cytokines associated

PRO-INFLAMMATORY CYTOKINES with the Th1 response) were observed inHIV-positive patients after they developedVL Cacopardo et al (1996) proposed that anAs mentioned above the life-cycle of HIV-1

infection is characterized by a prolonged and irreversible switch from a type-1 to a type-2response occurs during the co-infection Asvariable period of clinical latency usually

with a high viral turnover (Ho et al 1995) the four patients included in this study hadsurprisingly high counts of CD4+ cells atprogressive loss of T-helper-cell-mediated

immunity and depletion of CD4+ T-cells baseline however it is diYcult to extra-polate the results to the majority of HIV-(Clerici et al 1989) The sequential decline

and ablation of cell-mediated immunity infected patients who have lower counts Amore detailed account of the immunologicalresults in the development of opportunistic

diseases The opportunistic pathogens may status induced by the co-infection is stilllackingfurther modify the immunological status of

the host and in uence the outcome of the The serum cytokine concentrations andperipheral T-cell sub-populations of eightHIV-related disease A Th1 response char-

acterized by the production of IFN-c and HIV-1-infected patients have been studiedbefore during and after active VL and com-IL-2 is present during the early stages of

HIV infection the progression to AIDS pared with those of appropriate controls(unpubl obs) In this investigation thebeing associated with a switch to a Th2

response marked by the production of IL-4 HIV-1-infected patients at VL diagnosisshowed signi cantly higher serum concen-and IL-10 (Clerici et al 1993) Tumour

necrosis factor-a (TNF-a) may also play a trations of IFN-c than the matched HIV-positive (but Leishmania-negative) controlspivotal role in HIV-1 infection since it can

enhance the replication of the virus (Mellors and lower serum concentrations of IL-10than the immunocompetent VL controlset al 1991) and high serum concentrations

of TNF-a and TNF-b have been associated (Fig 3) These results show that the Th1Th2 response induced by the co-infectionwith a progression to AIDS (Aukrust et al

1994 Medrano et al 1998a) diVers from that induced by either singleinfection and indicate an unexpected non-Immunologically VL is also accom-

panied by impaired (Leishmania-speci c) synergistic eVect of the two infections onTh1Th2 polarity in vivo In the co-infectedcell-mediated immunity and by a decline in

the number of CD4+ cells (Cenini et al cases there were no relevant changes in theserum concentrations of IFN-c or IL-101993) Susceptibility and resistance to Leish-

mania in experimental models have been during the 6 months of follow-up after the


FIG 4 Changes in the serum concentrations ofinterferon-c (a) and interleukin-10 (b) observed in eightHIV-1-infected patients before and after they werefound to have visceral leishmaniasis (VL) For eachexperiment the results shown are mean values withse shown as error barsFIG 3 The serum concentrations of tumour necrosis

factor-a (a) interferon-c (b) and interleukin-10 (c)observed in cases of Leishmania infantumHIV-1 co-infection (e measured when visceral leishmaniasis more importantly that the serum levels ofwas diagnosed) and as controls individuals who TNF-a in the co-infected cases remainedwere Leishmania-negativeHIV-positive (^) Leishmania- high after recovery from VL (Fig 5) ThirdlypositiveHIV-negative (f ) or Leishmania-negative and

that CD4+ counts in the co-infected casesHIV-negative (_) For each experiment the resultsfell as active VL developed and remainedshown are mean values with se shown as error bars

Values that are signi cantly diVerent from those for low thereafter Fourthly that there was pro-the co-infected cases (Plt005) gressive seroconversion for the p24 HIV-1

antigen throughout the follow-up periodAn increase in serum HIV viraemia was alsodiagnosis of VL (Fig 4) This observation

probably indicates an impairment in the syn- observed in the patients after the onset ofacute VL (Fig 5) As HIV-1 directly infectsthesis of Th1Th2 cytokines mdash an impair-

ment expected during the profound immuno- cells of the immune system and triggers arobust immune response it is an importantsuppression of the advanced stages of HIV-1

infection (Fauci 1996) and persistent source of immune activation(Fauci 1996) linked to TNF-a secretion byThe recent Spanish study had several

other major ndings (unpubl obs) Firstly macrophages and lymphocytes (Aukrust et al1994) The release of pro-in ammatorythat HIV-1-infected patients with sympto-

matic VL have much higher serum concen- cytokines is considered to be involved in themechanism of defence against Leishmaniatrations of TNF-a than Leishmania-negative

HIV-1-positives (or than themselves before and other intracellular pathogens (Barral-Netto et al 1991 Cenini et al 1993 Haugthey developed VL Fig 3) Secondly and

88 OLIVIER ET AL

TNF-a release from monocytes latentlyinfected with HIV-1 and promote viral repli-cation The results of the more recent studyby Medrano et al (1998b) indicate that thiseVect of leishmanial infection on TNF-aproduction may also occur in vivo These ndings have important implications becausestrong activation of the TNF-a system isconsidered to play an important role in theprogression of HIV infection to AIDS Thedeleterious immunological and virologicaleVects of chronic activation of TNF-a couldexplain the dynamics of the CD4+-celldecline the increase in serum RNA viraemiaand the p24 seroconversion observed byMedrano et al (1998b) in the co-infectedpatients during the course of their VL

The data presented here demonstrate thatan aberrant persistent activation of TNF-aproduction with possibly harmful immuno-logical and virological consequences occursin patients with HIV-1 and Leishmaniaco-infection Although it is not possibleto discern clearly the exact causendasheVectrelationship between the parasite and theviral activation the results of several studies(Bernier et al 1995 Haug et al 1996Gazzinelli et al 1995 Medrano et al1998b) support the hypothesis that variousopportunistic infections may trigger the pro-FIG 5 Changes in the serum concentrations of

tumour necrosis factor-a (a) the numbers of CD4+ duction of pro-in ammatory cytokines duringcellsml blood (b) and the HIV-1 viraemia (c) in eight immunode ciency and in this way accelerateHIV-1-infected patients before and after they were the course of HIV-1 disease Again howeverfound to have visceral leishmaniasis (VL) For each

a more extensive knowledge of the immuno-experiment the results shown are mean values withlogical status induced by the co-infectionse shown as error barswill be necessary to determine the bestprocedures for treatment and control

et al 1996) HIV-negative patients with VLhave high serum concentrations of TNF-a

HUMORAL AND CELLULARfor example that normalize after successfulIMMUNE RESPONSEStreatment (Barral-Netto et al 1991 Cenini

et al 1993) and similar ndings exist forother pathogens such as mycobacteria (Haug In immunocompetent individuals VL is

associated with a clear humoral responseet al 1996) Consequently an enhancementin TNF-a production may be expected when the result of polyclonal B-cell activation and

the suppression of cellular responses to thethe immune system of HIV-1-infected patientsis activated by opportunistic pathogens parasite mdash although these are restored after

successful chemotherapy (Carvalho et alIn an in-vitro study Bernier et al (1995)demonstrated that Leishmania can trigger 1981) The T-cell-mediated immune response


is in fact critical to the recovery process pentavalent antimonial compounds Thiswould explain why in HIV-positives theand to protection against other infections

(Murray et al 1989) HIV-infected patients clinical and parasitological response to anti-leishmanial treatment is so poor (Medranowith VL have lower titres of antileishmanial

antibodies (Mary et al 1992) and much et al 1992) and the frequency of post-treatment relapse is so high (Alvar 1994)higher frequencies of clinical relapse follow-

ing antileishmanial treatment (Alvar 1994) Although the results of IFAT indicatedthat ve out of the 14 cases of co-infectionthan the immunocompetent cases Despite

the numerous cases of co-infection that have investigated recently had high titres of anti-Leishmania antibodies (unpubl obs seenow been reported there appear to have been

only two detailed investigations of the immune Table) these antibodies were not able tocontrol the spread of the parasite (Liew andresponses of such patients one on two AIDS

cases with American tegumentary leish- OrsquoDonnell 1993) The other nine casesinvestigated had titres of speci c antibodiesmaniasis caused by L braziliensis (Coutinho

et al 1996) and one on the changes in that were low or undetectable in IFATindicating why IFAT are of such limitedblood-cell subsets during L donovaniHIV

co-infection (Cenini et al 1994) value in the diagnosis of leishmaniasis inHIV-positive patients (unpubl obs) MoreSuccessful chemotherapy of VL in non-

immunocompromised patients reduces the (11 of 13) of the cases appeared seropositivewhen they were checked by immunoblottingantigenic load through parasite destruction

resulting in decreases in the serum titres of which is a more sensitive technique thanIFAT (Fig 6) and clearly preferable for thespeci c and non-speci c antibodies and the

restoration of speci c T-cell-mediated early diagnosis and therefore early treat-ment of VL in HIV-positive subjects (Kubarimmunity (Haldar et al 1983) There seems

to be no protective speci c immune response et al 1998) Immunoblotting also con rmedthat the co-infected cases had a partial orin most of those with L infantumHIV

co-infection even following treatment with weak humoral response in comparison to that

TABLE Details of the 17 cases of Leishmania infantumHIV co-infection investigated

Patient Risk group for HIV Anti-Leishmania antibody titre Immunoblot result Stimulation index

1 IVDU 1640 Polyclonal 0922 Homosexual 1640 Polyclonal 2033 Homosexual 1640 Oligoclonal 1114 IVDU 1640 Polyclonal 1025 Homosexual 1640 Oligoclonal 0896 IVDU 1320 Oligoclonal 0717 Homosexual 1160 Oligoclonal 0938 IVDU 1160 ND 3129 IVDU lt140 Oligoclonal 094

10 Haemophiliac lt140 Oligoclonal 22011 IVDU lt140 Oligoclonal 12312 IVDU lt140 Negative 09113 Blood transfusion lt140 Oligoclonal 15914 Blood transfusion lt140 Negative 28915 IVDU lt140 ND 10216 Unknown lt140 ND 14617 Unknown lt140 ND 129

The -fold increase in lymphocyte proliferation resulting from mitogen induction compared with that seen inunstimulated cellsIVDU Intravenous-drug user ND not determined

90 OLIVIER ET AL

however because of the preferential deathof memory cells upon activation in HIV-positive patients ( Janossy et al 1993) Theobservation that the four patients whoappeared responsive to SLA in the recentSpanish study all relapsed (unpubl obs)supports the loss of speci c cell-mediatedimmunity

Characterization of blast cells after stimu-lation showed that both CD4+ and CD8+cells are able to respond to SLA (Fig 7)CD4+ cells may have a protecting orexacerbating role in leishmaniasis (Liew andOrsquoDonnell 1993) and there is increasingevidence that CD8+ also have a speci crole in the disease (Da-Cruz et al 1994) Inexperimental VL it has been shown thatacquisition of resistance to infection with

FIG 6 Immunoblots showing the antibody reactivity L donovani requires both CD4+ and CD8+to soluble Leishmania infantum antigens (SLA) in sera cells but CD8+ cells alone contribute tofrom patients 1ndash7 ( lanes 3ndash9) and 9ndash14 (lanes 10ndash15) the hostrsquos defences against other infectionswith L infantumHIV co-infection (see Table) from a

(Murray et al 1992) Cure in patientshealthy donor (lane 1) or from an immunocompetentwith cutaneous leishmaniasis caused bypatient with visceral leishmaniasis caused by L infantum

( lane 2)

in immunocompetent cases of VL (Fig 6)as previously reported by Mary et al (1992)The low antibody titres generally observedare not the consequence of therapy as theymay have been in immunocompetent sub-jects but the result of the impaired immunityinduced by HIV infection Oligoclonal B-cellresponses may be consequent to the absenceof T cells that are able to recognize speci cLeishmania antigens or to stimulate B cellsThis loss of speci c antibodies has beendescribed for other infections in HIV-infectedpatients (Biggar et al 1987 Chamot et al1990)

Only one of the two AIDS caseswith American tegumentary leishmaniasisdescribed by Coutinho et al (1996) showeda lymphoproliferative response after anti-leishmanial treatment Clinical recovery of

FIG 7 The percentages of peripheral-blood mono-such patients and a positive response tonuclear cells (PBMC) that were CD4+ or CD8+soluble Leishmania antigen (SLA) in someas measured in patients co-infected with Leishmaniaof them would con rm that they are ableinfantum and HIV-1 (LeishHIV) or infected only with

to generate a speci c T-cell response This HIV-1 (HIV) The mean values are indicated byhorizontal linestype of cellular response is probably lost


L braziliensis is associated with a high fre- The immunosuppression induced byHIV infection may allow latent Leishmaniaquency of L braziliensis reactivity among

their CD8+ T cells in antigen-stimulated infections to re-activate and may facilitatethe development of VL Since both HIV andcultures (Da-Cruz et al 1994) and in

patients with the cutaneous leishmaniasis the Leishmania parasite can invade and repli-cate within macrophages it is possible that theand AIDS (Coutinho et al 1996) For the

four patients who were found to have lympho- interactions between these pathogens couldexacerbate both infections Wolday et alproliferative responses to SLA in the recent

study the ability of their CD4+ or CD8+ (1998) found for example that the additionof live or killed HIV-1 virions increased thecells to proliferate clearly did not induce

acquired resistance since all four suVered multiplication of L donovani in monocyte-derived cells It has also been proposed thatrelapses (unpubl obs) Although more

relevant data are needed it is probable that leishmanial infection can induce HIV repli-cation in macrophages (Tremblay et althe phenotype of the proliferating cells

depends on the CD4+ or CD8+ phenotype 1996) In vitro the infection with Leishmaniaof macrophage lines already infected withof the recall cells that have not been lost

( Janossy et al 1993) HIV produces an increase in viral replicationmediated by TNF-a (Bernier et al 1995)HIV and L infantum infections share

common features in terms of the changes Although the HIV-positive patients investi-gated in the recent study were found to havethey induce in PBMC subpopulations such

as increasing the numbers of activated T- similar serum concentrations of TNF-awhether they had VL or not (Fig 8 unpubland B-cells while decreasing the numbers of

CD4+ and natural-killer (NK) cells An obs) this does not preclude a speci c rolefor this cytokine in vivo given the autocrineincrease in the number of CD8+ cells only

appears to occur with HIV infection how- and paracrine eVects of TNF-a on Leish-mania-infected macrophages In the studyever and increased expression of human

leucocyte antigen-DR (HLA-DR) in lympho- by Medrano et al (1998b) TNF-a serumconcentrations were found to be higher incytes only happens with VL (De Martini

et al 1988 Cenini et al 1993) In those co-infected patients than in HIV-positiveLeishmania-negative patients or HIV-negativewith both VL and HIV infection the com-

mon eVects often show synergy but the HIV- cases of VL although the levels in thecases of co-infection declined after treat-speci c eVects tend to predominate over the

VL-speci c (Cenini et al 1994) For the ment while HIV expression was signi cantlyelevated These results do not seem to agreesubjects of the recent study the percentages

of PBMC that were CD4+ were similar for with a TNF-a-mediated induction of HIVexpression especially as Medrano et althose with L infantumHIV co-infection as

for the HIV-positive patients without leish- (1998b) found that 6 months before theirVL was diagnosed their co-infected casesmanial infection (although the counts were

made after antileishmanial treatment Fig 7 had levels of HIV RNA serum transcriptsthat were similar to those recorded at theunpubl obs) That the percentage of PBMC

that are CD8+ is relatively high in the cases time of VL diagnosisTumour growth factor-b (TGF-b) a potentof co-infection after antileishmanial treat-

ment might indicate that these cells have a inhibitor of the immune response is over-expressed in HIV infection and thus couldspeci c role in the post-treatment response

to the parasite or may simply re ect the contribute to the immune defects observed(Kekow et al 1990) This cytokine how-replacement of the decreased circulating

pool of CD4+ T-cells (Coutinho et al ever is a suppressor of HIV expression inchronically infected promonocytic cells and1996)

92 OLIVIER ET AL

a decrease in the serum concentrations ofTGF-b lead to increased viral replication inmacrophages (Medrano et al 1998a)

In both Leishmania and HIV infectionit has been proposed that susceptibility isassociated with a Th2-type response andresistance with a Th1-type response Theseassociations have been con rmed for leish-maniasis in murine models (Reiner andLocksley 1995) and in the cutaneous (Kempet al 1994) and visceral (Kemp et al 1993Miralles et al 1994) forms of the humandisease In HIV-positives progression toAIDS may be related to a Th1ndashTh2 switch(Clerici et al 1993) although this remainsa matter of controversy (Graziosi et al 1994Maggi et al 1994) In HIV-positive patientsa defect in the functioning of antigen-presenting cells would certainly inducethe secretion of Th2 cytokines (Borthwicket al 1994 Chehimi et al 1994) Since anindividualsrsquos protective response to infectionwith Leishmania usually depends on thepresence of cytokines during early T-cellactivation (Scott 1991) the immunological

FIG 8 The serum concentrations of tumour necrosisstatus of HIV-infected patients is particularlyfactor-a (TNF-a) and tumour growth factor-b (TGF-b)favourable for the multiplication and spreadobserved in patients co-infected with Leishmania infantum

and HIV-1 (e ) or infected only with HIV-1 (f ) The of leishmanial parasites (Cacopardo et alresults shown are the mean values with se shown as 1996)error bars

CONCLUSIONS

primary monocyte-derived macrophages (PoliHumans co-infected with HIV and L infantumet al 1991) TGF-b is also produced byare able to mount a T-cell response againstmurine macrophages exposed to L braziliensisthe parasite after antileishmanial treatmentand causes susceptibility to symptomaticbut this response is lost as the viral infectionleishmaniasis (Barral et al 1993) In theoryprogresses and almost always followed bytherefore individuals infected with HIV andrelapse of the leishmanial infection The co-

Leishmania who seem to have much lowerinfection may induce both an uncontrollableserum concentrations of TGF-b than thosespread of the parasite and an increase in viralinfected with HIV alone (Fig 8) shouldreplication Treatments against leishmaniasishave less TGF-b-related suppression of theirthat are eVective but do not promote HIVHIV but also less likelihood of developingreplication need to be developedsymptomatic leishmaniasis More research

is necessary to elucidate the interactionsbetween HIV and Leishmania parasites at acknowledgements The authors would

like to thank all the national and inter-least in monocytes and macrophages Thequestion that arises is can antileishmanial national granting agencies that have sup-

ported their investigations on LeishmaniaHIVtreatment of the co-infected cases by inducing


anism for Leishmania braziliensis Proceedings of theco-infection They would like to apologizeNational Academy of Sciences of the United States offor any relevant references that due to lackAmerica 90 3442ndash3446

of space have not been included in this Barral-Netto M Badaro R Pedral-Sampaio Dreview MO is a Burroughs Wellcome Fund Carvalho E M FalcoV E amp FalcoV R (1991)

Tumor necrosis factor (cachectin) in human visceralAwardee in Molecular Parasitology andleishmaniasis Journal of Infectious Diseases 163a Canadian Institute of Health Research853ndash857Investigator

Barral-Netto M Machado P amp Barral A (1995)Human cutaneous leishmaniasis recent advances inphysiopathology and treatment European Journal ofDermatology 5 104ndash113

REFERENCES Bernier R Turco S J Olivier M amp Tremblay M J(1995) Activation of human immunode ciency virustype 1 in monocytoid cells by the protozoan parasite

Abel P M McSharry C Galloway E Ross CLeishmania donovani Journal of Virology 69 7282ndash

Severn A Toner G Gruer L amp Wilkinson P C7285

(1992) Heterogeneity of peripheral blood monocyteBernier R Barbeau B Tremblay M J amp

populations in human immunode ciency virus-1 sero-Olivier M (1998) The lipophosphoglycan of Leish-

positive patients FEMS Microbiology and Immunologymania donovani up-regulates HIV-1 transcription in

5 317ndash323T cells through the nuclear factor-kappa B elements

Alvar J (1994) Leishmaniasis and AIDS co-infectionJournal of Immunology 160 2881ndash2888

the Spanish example Parasitology Today 10 160ndash163Biggar R J Goedert J J amp Hoofnagle J (1987)

Alvar J Verdejo J Osuna A amp Najera R (1987)Accelerated loss of antibody to hepatitis B surface

Visceral leishmaniasis in a patient seropositive forantigens among immunode cient homosexual men

HIV European Journal of Clinical Microbiology 6 infected with HIV New England Journal of Medicine604ndash606 316 630ndash631

Aukrust P Liabakk N B Muller F Lien E Borthwick N J Bo ll M Gombert W M AkbarEspevik T amp Froland S S (1994) Serum levels of A N Medina E Sagawa K Lipman M Ctumor necrosis factor-alpha (TNF-a) and soluble Johnson M A amp Janossy G (1994) LymphocyteTNF receptors in human immunode ciency virus activation in HIV-infection Functional defects oftype 1 infection mdash correlations to clinical immuno- CD28-T cells AIDS 8 431ndash441logic and virologic parameters Journal of Infectious Cacopardo B Nigro L Preiser W Fama ADiseases 169 420ndash424 Satariano M I Braner J Celesia B M

Badaro R (1997) When Leishmania and HIV interact Weber B Russo R amp Doerr H W (1996)a new broad spectrum of leishmaniasis occurs British Prolonged Th2 cell activation and increased viralJournal of Infectious Diseases 1 145ndash148 replication in HIVndashLeishmania co-infected patients

Badaro R Carvalho E M Rocha H Queiroz A C despite treatment Transactions of the Royal Societyamp Jones T C (1986a) Leishmania donovani an and Tropical Medicine and Hygiene 90 434ndash435opportunistic microbe associated with progressive Carvalho E M Teixeira R S amp Johnson Jr W Ddisease in three immunocompromised patients (1981) Cell mediated immunity in American visceralLancet i 647ndash649 leishmaniasis reversible immunosupression during

Badaro R Jones T C Carvalho E M acute infection Infection and Immunity 33 498ndash502Sampaio D Reed S G Barral A Teixeira R amp Cenini P Berhe N Hailu A McGinnes K ampJohnson Jr W D (1986b) New perspectives on a Frommel D (1993) Mononuclear cell subpopulationssubclinical form of visceral leishmaniasis Journal of and cytokine levels in human visceral leishmaniasisInfectious Diseases 154 1003ndash1011 before and after chemotherapy Journal of Infectious

Barbeau B Bernier R Dumais N Briand G Diseases 168 986ndash993Olivier M Faure R Posner B I amp Tremblay M Cenini P Berhe N Hailu A amp Frommel D(1997) Activation of HIV-1 LTR transcription (1994) Mononuclear cell subpopulations in humanand virus replication via NF-kB-dependent and immunode ciency virus (HIV)-positive versus HIV--independent pathways by potent phosphotyrosine negative patients with visceral leishmaniasis Journalphosphatase inhibitors the peroxovanadium com- of Infectious Diseases 169 707ndash708pounds Journal of Biological Chemistry 272 12968ndash Chamot E Hirschel B Wintsch J Robert C F12977 Gabriel V Deglon J J Yerly S amp Perrin L

Barral A Barral-Netto M Yong E C Brownell (1990) Loss of antibodies against hepatitis C virusC E Twardzik D R amp Reed S G (1993) in HIV-seropositive intravenous drug users AIDS 4

1275ndash1277Transforming growth factor b as a virulence mech-

94 OLIVIER ET AL

Chehimi J Starr S E Frank I drsquoAndrea A Dumais N Barbeau B Olivier M amp Tremblay MMa X MacGregor R R Sennelier J amp (1998) Prostaglandin E2 upregulates HIV-1 LTR-Trinchieri G (1994) Impaired interleukin 12 pro- driven gene activity in T cells via NF-k-dependentduction in human immunode ciency virus-infected and -independent signaling pathways Journal ofpatients Journal of Experimental Medicine 179 Biological Chemistry 273 27306ndash273141361ndash1366 Easterbrook M D Levy M H Gomez K M

Clerici M Stocks N I Zajac R A Boswell R D Turco S J Epand R M amp Rosenthal K L (1995)Lucey D R Via C S amp Shearer G M (1989) Inhibition of HIV-1-induced syncytia formation andDetection of three distinct patterns of T helper cell infectivity by lipophosphoglycan from Leishmaniadysfunction in asymptomatic HIV-seropositive patients Journal of AIDS and Human Retrovirology 10Journal of Clinical Investigation 84 1892ndash1899 496ndash505

Clerici M Hakim F T D Venzon D J Blatt S Fauci A S (1988a) The human immunode ciencyHendrix C W Wynn T A amp Shearer G M virus infectivity and mechanisms of pathogenesis(1993) Changes in interleukin 2 and interleukin 4 Science 239 617ndash622in asymptomatic human immunode ciency virus- Fauci A S (1988b) Immunopathogenic mechanismsseropositive individuals Journal of Clinical Investi- of human immunode ciency virus (HIV) infectiongation 91 759ndash765 In Human Retroviruses Cancer and AIDS Approaches

Coutinho S G Da-Cruz A M Pereira de to Prevention and Therapy ed Bolegnesi D p 187Oliveira M Mendonca S C F Bertho A L New York Alan R Lissamp de Luca P M (1996) CD4+ and CD8+ Fauci A S (1996) Host factors and the pathogenesisT cell immune response of immunocompetent and of HIV-induced disease Nature 384 529ndash534immunocompromised (AIDS) patients with American Fillola G Corberand J X Laharrague P Ftegumentary leishmaniasis Memorias do Institudo Levenes H Massip P amp Recco P (1992)Oswaldo Cruz 91 381ndash384 Peripheral intramonocytic leishmaniasis in an

Da-Cruz A M Conceicao-Silva F Bertho A L amp AIDS patient Journal of Clinical Microbiology 30Coutinho S G (1994) Leishmania-reactive CD4+ 3284ndash3285and CD8+ T cells associated with cure of human Foley P Kazazi F Biti R Sorrell T C ampcutaneous leishmaniasis Infection and Immunity 62

Cunningham A L (1992) HIV infection of mono-2614ndash2618

cytes inhibits the T-lymphocyte proliferative responseDalgleish A G Beverley P C L Clapham P R

to recall antigens via production of eicosanoidsCrawford D H Greaves M F amp Weiss R A

Immunology 75 391ndash397(1984) The CD4(T4) antigen is an essential com-

Folks T M Justement J Kinter A Dinarello C Aponent of the receptor for the AIDS retrovirus

amp Fauci A S (1987) Cytokine-induced expressionNature 312 763ndash767

of HIV-1 in a chronically infected promonocyte cellDe Martini R M Turner R R Formenti S C

line Science 238 800ndash802Boone D C Bishop P C Levine A MFrank Y Pavlakis S Black K amp Bakshi S (1998)amp Parker J W (1988) Peripheral blood mono-

Reversible occipitalndashparietal encephalopathy syndromenuclear cell abnormalities and their relationshipin an HIV-infected child Neurology 51 915ndash916to clinical course in homosexual men with HIV

Frantz B Nordby E Bren G SteVan Ninfection Clinical Immunology and ImmunopathologyPaya C Kincaid R Tocci M J OrsquoKeefe S J amp46 258ndash271OrsquoNeill E A (1994) Calcineurin acts in synergyDe Souza E Souza I NaiV R D Guimaraes T Cwith PMA to inactivate IkBMAD-3 an inhibitor ofNaiV M F Cupolillo E Rodrigues W A ampNF-kB EMBO Journal 13 861ndash870Schettini A P (1998) American cutaneous leish-

Gazzinelli R T Bala S Stevens R Baseler Mmaniasis due to Leishmania ( Viannia) guyanensis asWahl L Kovacs J amp Sher A (1995) HIV infectionan initial clinical presentation of human immuno-supresses type 1 lymphokine and IL-12 responses tode ciency virus infection Journal of the EuropeanToxoplasma gondii but fails to inhibit the synthesisAcademy of Dermatology and Venereology 10 214ndash217of other parasite-induced monokines Journal ofDesjeux P amp Alvar J (2003) LeishmaniaHIVImmunology 155 1565ndash1574co-infections epidemiology in Europe Annals of

Gradoni L amp Gramiccia M (1994) LeishmaniaTropical Medicine and Parasitology 97 (Suppl) 3ndash15infantum tropism strain genotype or host immuneDuh E J Maury W J Folks T M Fauci A S ampstatus Parasitology Today 10 264ndash267Rabson A B (1989) Tumor necrosis factor alpha

Graziosi C Pantaleo G Gantt K R Fortin J Pactivates human immunode ciency virus type 1Demarest J F Cohen O J Sekaly R Pampthrough induction of nuclear factor binding to theFauci A S (1994) Lack of evidence for the dicho-NF-kappa B sites in the long terminal repeattomy of Th1 and Th2 predominance in HIV-infectedProceeding of the National Academy of Sciences of the

United States of America 86 5974ndash5978 individuals Science 265 248ndash252


Haldar J P Ghose S Saha K C amp Ghose A C Kemp M Hey A S Kurtzhals J A Christensen(1983) Cell-mediated immune response in Indian C B Gaafar A Mustafa MD Kordona A Akala-azar and post kala-azar dermal leishmaniasis Ismail A Kharazmi A amp Theander T G (1994)Infection and Immunity 42 702ndash707 Dichotomy of the human T cell response to Leish-

Handman E Schnur L F Spithill T W amp mania antigens I Th1-like response to LeishmaniaMitchell G H (1986) Passive transfer of Leishmania major promastigotes antigens in individuals recoveredlipopolysaccharide confers parasite survival in macro- from cutaneous leishmaniasis Clinical and Experi-phages Journal of Immunology 137 3608ndash3613 mental Immunology 96 410ndash415

Haug C H J Aukrust P Lien E Miller F Klatzmann D Barre-Sinoussi F Nugeyre M TEspevik T amp Froland S S (1996) Disseminated Dauguet C Vilmer E Griscelli C Brun-Mycobacterium avium complex infection in AIDS Veziret F- Rouzioux C Gluckman J Cimmunopathogenic signi cance of an activated tumor Chermann J C amp Montagnier L (1984) Selectivenecrosis factor system and depressed serum levels tropism of lymphadenopathy associated virus (LAV)of 125 dihydroxyvitamin D Journal of Infectious for helper-inducer T lymphocytes Science 225Diseases 173 259ndash262 59ndash63

Ho D D Neumann A U Perelson A S Chen W Klein S A Dobmeyer J M Dobmeyer T SLeonard J M amp Markowitz M (1995) Rapid turn- Pape M Ottmann O G Helm E B Hoelzer Dover of plasma virions and CD4 lymphocytes in amp Rossol R (1997) Demonstration of the Th1HIV-1 infection Nature 373 123ndash126 to Th2 cytokine shift during the course of HIV-1

Janossy G Borthwick N Lomnitzer R Medina E infection using cytoplasmic cytokine detection on singleSquire S B Phillips A N Lipman M Johnson cell level by ow cytometry AIDS 11 1111ndash1118M A Lee C amp Bo ll M (1993) Lymphocyte Kubar J Marty P Lelievre A Quaranta J Factivation in HIV-1 infection I Predominant pro- Staccini P Caroli-Bosc C amp Le Fichoux Yliferative defects among CD45R0+ cells of the CD4 (1998) Visceral leishmaniosis in HIV-positive patientsand CD8 lineages AIDS 7 613ndash624 primary infection reactivation and latent infection

Jeannel D Tuppin P Bruckler G Danis M Impact of the CD4+ T-lymphocyte counts AIDSamp Gentilini M (1989) Leishmaniasis in France 12 2147ndash2153Lancet ii 804

Levy J A (1993) Pathogenesis of human immuno-Jeronimo S M Oliveira R M Mackay S

de ciency virus infection Microbiology Reviews 57Costa R M Sweet J Nacimiento E T

183ndash289Luz K G Fernandez M Z Jernigan J amp

Lewis R S amp Cahalan M D (1995) Potassium andPearson R D (1994) An urban outbreak of visceral

calcium channels in lymphocytes Annual Review ofleishmaniasis in Natal Brazil Transactions of the

Immunology 13 623ndash653Royal Society of Tropical Medicine and Hygiene 88

Liew F Y amp OrsquoDonnell C A (1993) Immunology386ndash388

of leishmaniasis Advances in Parasitology 32 161ndash259Jimenez M I Gutierrez-Solar B Benito AMaggi E Mazzetti M Ravina A Annunziato FAguiar A Garcotilde a E Cercenado E amp Alvar J

de Carli M Piccini M P Manetti R(1991) Cutaneous Leishmania infantum zymodemesCarbonari M Pesce A M del Prete G ampisolated from bone marrow in AIDS patientsRomagnani S (1994) Ability of HIV to promote aResearch and Reviews in Parasitology 51 95ndash99Th1 to Th0 shift and to replicate preferentially inJimenez M I Lopez-Velez R Molina RTh2 and Th0 cells Science 265 244ndash248Cavanate C amp Alvar J (1996) HIV coinfection

Mans eld J M amp Olivier M (2002) Immune evasionwith a currently non-pathogenic agellate Lancetby parasites In Immunology of Infectious Diseases edsi 264ndash265Kaufmann S H E Sher A amp Ahmed RKekow J Wachsman W McCutchan J App 379ndash392 Washington DC American Society forCronin M Carson D A amp Lotz M (1990)MicrobiologyTransforming growth factor b and noncytopathic

Martotilde nez P de la Vega E Laguna F Soriano Vmechanisms of immunode ciency in human immuno-Puente S Moreno V Sentchordi M J Garcia-de ciency virus infection Proceedings of the NationalAguado C amp Gonzalez-Lahoz J (1993) DiagnosisAcademy of Sciences of the United States of Americaof visceral leishmaniasis in HIV-infected individuals87 8321ndash8325using peripheral blood smears AIDS 7 227ndash230Kemp M Kurtzhals J A L Bendtzen K

Marty P Le Fichoux Y Pratlong F amp Gari-Poulsen L K Hansen M B Koech D KToussaint M (1994) Human visceral leishmaniasisKharazmi A amp Theander T G (1993) Leishmaniain Alpes-Maritimes France epidemiological charac-donovani-reactive Th1- and Th2-like T-cell clonesteristics for the period 1985ndash1992 Transactions of thefrom individuals who have recovered from visceralRoyal Society of Tropical Medicine and Hygiene 88leishmaniasis Infection and Immunity 61 1069ndash

1073 33ndash34

96 OLIVIER ET AL

Mary C Lamouroux D Dunan S amp Quilici M Meltzer M S Skillman D R Gomatos P JKalter D C amp Gendelman H E (1990) Role(1992) Western blot analysis of antibodies to

Leishmania infantum antigens potential of the 14-kD of mononuclear phagocytes in the pathogenesis ofhuman immunode ciency virus infection Annualand 16-kD antigens for diagnosis and epidemiologic

purposes American Journal of Tropical Medicine and Review of Immunology 8 169ndash194Miralles G D Stoeckle M Y McDermott D FHygiene 47 764ndash771

Matte C Maion G Mourad W amp Olivier M Finkelman F D amp Murray H W (1994) Th1 andTh2 cell-associated cytokines in experimental visceral(2001) Leishmania donovani-induced macrophages

cyclooxygenase-2 and prostaglandin E2 synthesis leishmaniasis Infection and Immunity 62 1058ndash1063Moreno-Camacho A Lopez-Velez R MunozParasite Immunology 23 177ndash184

McDougal J S Nicholson J K A Cross G D Sanz A amp Labarga-Echevarria P (1998) Intestinalparasitic infections and leishmaniasis in patients withCort S P Kennedy M S amp Mawle A C (1986)

Binding of the human retrovirus HTLV-IIILAV HIV infection Enfermedades Infecciosas y MicrobiologiaClinica 16 (Suppl 1) 52ndash60ARVHIV to the CD4 (T4) molecule conformation

dependence epitope mapping antibody inhibition Morrow R H Colebunders R L amp Chin J (1989)Interactions of HIV infection with endemic tropicaland potential for idiotypic mimicry Journal of

Immunology 137 2937ndash2944 diseases AIDS 3 (Suppl 1) S79ndashS87Munoz-Rodrotilde guez F J Padro S Pastor PMedrano F J Hernandez-Quero J Jimenez E

Pineda J A Rivero A Sanchez-Quijano A Rosa-Re D Valls M E Miro J M amp Gatell M(1997) Pleural and peritoneal leishmaniasis in anVelez I D Viciana P Castillo R Reyes M J

Carvajal F Leal M amp Lissen E (1992) Visceral AIDS patient European Journal of Clinical Micro-biology and Infectious Diseases 16 246ndash248leishmaniasis in HIV-1-infected individuals a com-

mon opportunistic infection in Spain AIDS 6 Murray H W Oca M J Granger A M ampSchreiber R D (1989) Requirement for T cells and1499ndash1503

Medrano F J Leal M Arienti D Rey C eVect of lymphokines in successful chemotherapyfor an intracellular infection Experimental visceralZagliani A Torres Y Sanchez-Quijano Lissen E

amp Clerici M (1998a) Tumor necrosis factor b leishmaniasis Journal of Clinical Investigation 831253ndash1257and soluble APO-1Fas independently predict pro-

gression to AIDS in HIV-seropositive patients AIDS Murray H W Squires K E Miralles C DStoeckle M Y Granger A M Granelli-Research and Human Retrovirology 14 833ndash844

Medrano F J Rey C Leal M Canavate C Piperno A Bogdan C (1992) Acquired resistanceand granuloma formation in experimental visceralRubio A Sanchez-Quijano A Alvar J amp

Lissen E (1998b) Dynamics of serum cytokines leishmaniasis DiVerential T cell and lymphokineroles in initial versus established immunity Journalin patients with visceral leishmaniasis and HIV-1

co-infection Clinical and Experimental Immunology of Immunology 148 1858ndash1863Nabors G S amp Farrell J P (1996) Successful chemo-114 733ndash737

Melbye M Biggar R J Ebbesen P Nealand C therapy in experimental leishmaniasis is in uencedby the polarity of the T cell response before treat-Goedert J J Faber V Lorenzen I Skinhoj P

Gallo R C amp Blattner W A (1986) Long- ment Journal of Infectious Diseases 173 979ndash986Nigro L Cacopardo B Preiser W Braner Jterm seropositivity for HTLV-III in homosexual

men without the acquired immunode ciency syn- Cinatl J Palermo F Russo R Doerr H W ampNunnari A (1999) In vitro production of type 1 anddrome development of immunological and clinical

abnormalities a longitudinal study Annals of Internal type 2 cytokines by peripheral blood mononuclearcells from subjects coinfected with human immuno-Medicine 104 496ndash500

Mellors J W GriYth B P Ortiz M A Landry de ciency virus and Leishmania infantum AmericanJournal of Tropical Medicine and Hygiene 60M L amp Ryan J L (1991) Tumor necrosis factor

alphacachectin enhances human immunode ciency 142ndash145Olivier M (1996) Modulation of host cell intracellularvirus type 1 replication in primary macrophages

Journal of Infectious Diseases 163 78ndash82 Ca2+ Parasitology Today 12 145ndash150Olivier M Baimbridge K G amp Reiner N EMellors J W Rinaldo C Gupta P White R M

Todd J A amp Kingsley L A (1996) Prognosis in (1992a) Inhibition of N-formyl-methionyl-leucyl-phenylalanine-induced stimulus-response coupling inHIV-1 infection predicted by the quantity of virus

in plasma Science 272 1167ndash1170 monocytes infected with Leishmania evidence for adefect in agonist-induced calcium release Journal ofMellors J W Munoz A Giorgi J V Margolick

J B Tassoni C J Gupta P Detels R Phair J P Immunology 148 1188ndash1196Olivier M Brownsey R W amp Reiner N E (1992b)amp Rinaldo Jr C R (1997) Plasma viral load and

CD4+ lymphocytes as prognostic morkers of HIV-1 Defective stimulusndashresponse coupling in humanmonocytes infected with Leishmania donovani isinfection Annals of Internal Medicine 126 946ndash954


associated with altered activation of protein kinase Reiner S L amp Locksley R M (1995) The regulationC Proceedings of the National Academy of Sciences of of immunity to Leishmania major Annual Review ofthe United States of America 89 7481ndash7485 Immunology 13 151ndash177

Olivier M Romero-Gallo B J Matte C Russo R Nigro L Minniti S Montinieri ABlanchette J Posner B I Tremblay M amp Gradoni L Caldeira L amp Davidson R N (1996)Faure R (1998) Modulation of interferon-c- Visceral leishmaniasis in HIV infected patients treat-induced macrophage activation by phosphotyrosine ment with high dose liposomal amphotericin Bphosphatases inhibition EVect on murine leish- (AmBisome) Journal of Infection 32 133ndash137maniasis progression Journal of Biological Chemistry Scott P (1991) IFN-c modulates the early develop-273 13944ndash13949 ment of Th1 and Th2 responses in a murine model

Pantaleo G Graziosi C Demarest J F Butini L of cutaneous leishmaniasis Journal of ImmunologyMontroni M Fox C H Orestein J M 147 3149ndash3155Kotler D P amp Fauci A S (1993) HIV infection Scott P (1993) IL-12 initiation cytokine for cellis active and progressive in lymphoid tissue during mediated immunity Science 260 496ndash497the clinically latent stage of disease Nature 362 Sebastian J J Garcia S Soria M T Pac J355ndash358 Vicente J amp Uribarrena R (1997) Visceral

Pantaleo G Graziosi C Demarest J F leishmaniasis diagnosed by colonoscopy Journal ofCohen O J Vaccarezza M Gantt K Muro Clinical Gastroenterology 25 691ndash692Cacho C amp Fauci A S (1994) Role of lymphoid Tremblay M Olivier M amp Bernier R (1996)organs in the pathogenesis of human immuno- Leishmania and the pathogenesis of HIV infectionde ciency virus (HIV) infection Immunology Reviews Parasitology Today 12 257ndash261140 105ndash130 Turco S J (1999) Adversial relationship between the

Perelson A S Neumann A U Markowitz M Leishmania lipophosphoglycan and protein kinase CLeonard J M amp Ho D D (1996) HIV-1 dynamics of host macrophages Parasite Immunology 21in vivo virion clearance rate infected cell life-span 597ndash600and viral generation time Science 271 1582ndash1586 Vazquez-Pineiro T Fernandez Alvarez J M

Pirmez C Yamamura M Uyemura K Paes- Gonzalo Lafuente J C Cano J Gimeno M ampOliveira M Conceicao-Silva F amp Modlin R L Berenguer J (1998) Visceral leishmaniasis a lingual(1993) Cytokine patterns in the pathogenesis of presentation in a patient with HIV infection Oralhuman leishmaniasis Journal of Clinical Investigation

Surgery Oral Medicine Oral Pathology and Oral91 1390ndash1395

Radiology 86 179ndash182Poli G Kinter A L Justement J S Bressler P

Weissman D amp Fauci A S (1997) Role of dendriticKehrl J H amp Fauci A S (1991) Transforming

cells in immunopathogenesis of human immuno-growth factor b suppresses human immunode ciency

de ciency virus infection Clinical Microbiology Reviewsvirus expression and replication in infected cells of

10 358ndash367the monocytemacrophage lineage Journal of Experi-

Wolday D AkuVo H Fessahaye G Valantine Amental Medicine 173 589ndash597

amp Britton S (1998) Live and killed human immuno-Pomerantz R J Feinberg M B Trono D ampde ciency virus type-1 increases the intracellularBaltimore D (1990) Lipopolysaccharide is a potentgrowth of Leishmania donovani in monocyte-derivedmonocytemacrophage-speci c stimulator of humancells Scandinavian Journal of Infectious Diseases 39immunode ciency virus type 1 expression Journal of29ndash34Experimental Medicine 172 253ndash261

World Health Organization (1997) LeishmaniaHIVPopovic M Sarngadharan M G Read E amp Galloco-infection Epidemiological analysis of 692 retro-R C (1984) Detection isolation and continuousspective cases Weekly Epidemiological Record 72production of cytopathic retroviruses (HTLV-III)49ndash54from patients with AIDS and pre-AIDS Science

Zaitseva M B Lee S Rabin R L TiVany H L224 497ndash500Farber J M Peden K W Murphy P M ampPreiser W Cacopardo B Nigro L Braner JGolding H (1998) CXCR4 and CCR5 on humanNunnariI A Doerr H W amp Weber B (1996)thymocytes biological function and role in HIV-1Immunological ndings in HIVLeishmania co-infectioninfection Journal of Immunology 161 3103ndash3113Intervirology 39 285ndash288

Zhang Y Nakata K Weiden M amp Rom W NReed S G amp Scott P (1993) T cells and cyto-(1995) Mycobacterium tuberculosis enhances humankine response in Leishmania Current Opinion inimmunode ciency virus type-1 replication by tran-Immunology 5 524ndash531scriptional activation at the long terminal repeatReiner N E amp Malemud C J (1984) ArachidonicJournal of Clinical Investigation 95 2324ndash2331acid metabolism in murine leishmaniasis (donovani )

Zijlstra E E El-Hassan A M Ismael A ampex-vivo evidence for increased cyclooxygenase andGhalib H W (1994) Endemic kala-azar in eastern5-lipoxygenase activity in spleen cells Cellular

Immunology 88 501ndash510 Sudan a longitudinal study on the incidence of

98 OLIVIER ET AL

clinical and subclinical infection and post-kala-azar Zumla A amp Croft S L (1992) Chemotherapy andimmunity in opportunistic parasitic infections indermal leishmaniasis American Journal of Tropical

Medicine and Hygiene 51 826ndash836 AIDS Parasitology 105 S93ndashS101

80 OLIVIER ET AL

between time of infection and onset of full- Bernier et al (1995) observed thatL donovani its surface lipophosphoglycanblown AIDS (Melbye et al 1986 Fauci

1988a) are indeed the principal clinical (LPG) and diverse structural componentsof the parasite could each induce viralcharacteristics of HIV-1 infection Co-

infection with certain opportunistic micro- replication in several monocytoid cell lineschronically infected with HIV-1 [Fig 1(a)]organisms including the protozoan parasites

of the genus Leishmania may lead not The LPG molecule a glycoconjugate isone of the major constituents expressed ononly to direct pathogenesis and morbidity

but may also play an important and active the surface of the Leishmania promastigoteWhen a promastigote is inoculated into arole in the progression of HIV-1 infection

toward AIDS Such opportunistic infections human host by an infected sand y it isengulfed into the phagolysosome of a macro-induce in ammatory responses and cellular-

signalling events in their host that could phage where it rapidly diVerentiates intoan amastigote During its engulfment thepromote viral replication (Tremblay et al

1996) Interestingly it is known that certain parasite loses most of its structural LPG atthe surface of the phagocyte and retainsstimuli can activate regulatory elements

located within the long-terminal-repeat (LTR) only the intramembrane component Thiscomponent the phosphatidyl inositol coresequences of HIV-1 and may induce HIV-1

gene expression from latent proviruses thus (core-PI) is solely encountered on the sur-face of the amastigote The LPG surfaceaccelerating viral replication and disease

progression (Fauci 1988a b) It seems likely molecule has been recognized as favouringthe intracellular survival and establishment ofthat cellular activation induced in the course

of infection by opportunistic pathogens the parasite (Turco 1999) The alteration ofsignalling events dependent on the secondcould lead to the activation of the regulatory

elements of HIV-1 messenger protein kinase C (PKC) has beenimplicated in the LPG-mediated protectionAlthough the role of protozoan parasites

as possible co-factors favouring the pro- of the parasite within the macrophage phago-lysosome and the LPG-induced inhibitiongression of HIV-1 infection towards AIDS

still needs much study Tremblay et al of several important macrophage functionssuch as the generation of oxygen radicals(1996) discussed the putative participation

of Leishmania in the pathogenesis of HIV-1 (Handman et al 1986) Among other host-cell dysfunctions induced by Leishmaniainfection Their ideas were based on the

observation that the geographical distributions are inhibiting mechanisms (possibly LPG-independent) that can lead to the alterationof HIV and Leishmania overlap in several

countries Many cases of L infantumHIV-1 of several signalling pathways in which pro-tein tyrosine kinase (PTK) PKC and calciumco-infection have already been recorded

around the Mediterranean basin particularly (Ca2+ ) participate (Olivier et al 1992a b1998)in France Spain and Italy ( Jeannel et al

1989 Fillola et al 1992) There are clini- In a recent Spanish study which is dis-cussed at length below L infantum LPG wascal and epidemiological reports implicating

Leishmania as an opportunistic pathogen found to be not only an excellent activatorof HIV-1 replication within latently infectedin immunosuppressed AIDS patients (Alvar

et al 1987) As leishmanial parasites con- monocytoid cells but also a potent inducerof HIV-1 LTR transcription and viral repli-stitute a major public-health risk through-

out subtropical and tropical areas of the cation in T-cells [Fig 1(b) unpubl obs]In vitro rapid upregulation of HIV-1 LTRworld ( Jeronimo et al 1994 Marty et al

1994 Zijlstra et al 1994) it should come activity was seen following LPG additionpossibly indicating a direct eVect on theas no surprise if LeishmaniaHIV co-infection

emerges in patients living in these regions LTR-dependent gene expression of HIV-1










82 OLIVIER ET AL



























FIG

2

Sch

emat

icre

pres

enta

tion

ofth

esi

gnal

ling

mec

han

ism

sin

volv

edin

the

Lei

shm

ania

-lip

opho

spho

glyc

an-m

edia

ted

HIV

-1ex

pres

sion

by

mon

ocyt

esm

acro

phag

esan

dT

lym

phoc

ytes

Ca2

+c

alci

um

EP

4pr

osta

glan

din

-E2

rece

ptor

ofty

pe4

IkB

nu

clea

r-fa

ctor

-kB

inhi

bit

ory

mol

ecu

leL

PG

lip

opho

spho

glyc

anL

TR

lon

gte

rmin

alre

pea

tP

GE

2p

rost

agla

ndin

E2

PK

A

prot

ein

kin

ase

A

PK

C

prot

ein

kin

ase

C

p65

p50

ac

tive

elem

ents

ofn

ucl

ear

fact

or-k

B

PT

K

prot

ein

tyro

sin

eki

nas

eR

re

cept

orfo

rL

PG

and

or

its

phos

pha

tid

ylin

osit

olco

re

TN

F-a

tu

mou

rn

ecro

sis

fact

or-a

T

NF

R

TN

F-a

rece

pto

r

84 OLIVIER ET AL



















































86 OLIVIER ET AL





































88 OLIVIER ET AL































90 OLIVIER ET AL





( lane 2)






























92 OLIVIER ET AL





CONCLUSIONS






























94 OLIVIER ET AL







































1073 33ndash34

96 OLIVIER ET AL


















































98 OLIVIER ET AL












82 OLIVIER ET AL



























FIG

2

Sch

emat

icre

pres

enta

tion

ofth

esi

gnal

ling

mec

han

ism

sin

volv

edin

the

Lei

shm

ania

-lip

opho

spho

glyc

an-m

edia

ted

HIV

-1ex

pres

sion

by

mon

ocyt

esm

acro

phag

esan

dT

lym

phoc

ytes

Ca2

+c

alci

um

EP

4pr

osta

glan

din

-E2

rece

ptor

ofty

pe4

IkB

nu

clea

r-fa

ctor

-kB

inhi

bit

ory

mol

ecu

leL

PG

lip

opho

spho

glyc

anL

TR

lon

gte

rmin

alre

pea

tP

GE

2p

rost

agla

ndin

E2

PK

A

prot

ein

kin

ase

A

PK

C

prot

ein

kin

ase

C

p65

p50

ac

tive

elem

ents

ofn

ucl

ear

fact

or-k

B

PT

K

prot

ein

tyro

sin

eki

nas

eR

re

cept

orfo

rL

PG

and

or

its

phos

pha

tid

ylin

osit

olco

re

TN

F-a

tu

mou

rn

ecro

sis

fact

or-a

T

NF

R

TN

F-a

rece

pto

r

84 OLIVIER ET AL



















































86 OLIVIER ET AL





































88 OLIVIER ET AL































90 OLIVIER ET AL





( lane 2)






























92 OLIVIER ET AL





CONCLUSIONS






























94 OLIVIER ET AL







































1073 33ndash34

96 OLIVIER ET AL


















































98 OLIVIER ET AL




FIG

2

Sch

emat

icre

pres

enta

tion

ofth

esi

gnal

ling

mec

han

ism

sin

volv

edin

the

Lei

shm

ania

-lip

opho

spho

glyc

an-m

edia

ted

HIV

-1ex

pres

sion

by

mon

ocyt

esm

acro

phag

esan

dT

lym

phoc

ytes

Ca2

+c

alci

um

EP

4pr

osta

glan

din

-E2

rece

ptor

ofty

pe4

IkB

nu

clea

r-fa

ctor

-kB

inhi

bit

ory

mol

ecu

leL

PG

lip

opho

spho

glyc

anL

TR

lon

gte

rmin

alre

pea

tP

GE

2p

rost

agla

ndin

E2

PK

A

prot

ein

kin

ase

A

PK

C

prot

ein

kin

ase

C

p65

p50

ac

tive

elem

ents

ofn

ucl

ear

fact

or-k

B

PT

K

prot

ein

tyro

sin

eki

nas

eR

re

cept

orfo

rL

PG

and

or

its

phos

pha

tid

ylin

osit

olco

re

TN

F-a

tu

mou

rn

ecro

sis

fact

or-a

T

NF

R

TN

F-a

rece

pto

r

84 OLIVIER ET AL



















































86 OLIVIER ET AL





































88 OLIVIER ET AL































90 OLIVIER ET AL





( lane 2)






























92 OLIVIER ET AL





CONCLUSIONS






























94 OLIVIER ET AL







































1073 33ndash34

96 OLIVIER ET AL


















































98 OLIVIER ET AL



84 OLIVIER ET AL



















































86 OLIVIER ET AL





































88 OLIVIER ET AL































90 OLIVIER ET AL





( lane 2)






























92 OLIVIER ET AL





CONCLUSIONS






























94 OLIVIER ET AL







































1073 33ndash34

96 OLIVIER ET AL


















































98 OLIVIER ET AL





























86 OLIVIER ET AL





































88 OLIVIER ET AL































90 OLIVIER ET AL





( lane 2)






























92 OLIVIER ET AL





CONCLUSIONS






























94 OLIVIER ET AL







































1073 33ndash34

96 OLIVIER ET AL


















































98 OLIVIER ET AL















88 OLIVIER ET AL































90 OLIVIER ET AL





( lane 2)






























92 OLIVIER ET AL





CONCLUSIONS






























94 OLIVIER ET AL







































1073 33ndash34

96 OLIVIER ET AL


















































98 OLIVIER ET AL























90 OLIVIER ET AL





( lane 2)






























92 OLIVIER ET AL





CONCLUSIONS






























94 OLIVIER ET AL







































1073 33ndash34

96 OLIVIER ET AL


















































98 OLIVIER ET AL




























92 OLIVIER ET AL





CONCLUSIONS






























94 OLIVIER ET AL







































1073 33ndash34

96 OLIVIER ET AL


















































98 OLIVIER ET AL



























94 OLIVIER ET AL







































1073 33ndash34

96 OLIVIER ET AL


















































98 OLIVIER ET AL






















1073 33ndash34

96 OLIVIER ET AL


















































98 OLIVIER ET AL



96 OLIVIER ET AL


















































98 OLIVIER ET AL























98 OLIVIER ET AL



The pathogenesis of Leishmania/HIV co-infection: cellular ... · PATHOGENESIS OF Leishmania/HIV...

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