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Nutr Hosp. 2013;28(6):2157-2164ISSN 0212-1611 CODEN NUHOEQ

S.V.R. 318

Original / Investigacin animalEffects of human milk on blood and bone marrow cells in a malnourishedmice model; comparative study with cow milk Isabel Garca1, Susana Salva2, Hortensia Zelaya1, Julio Villena2 and Graciela Agero1

1Instituto de Bioqumica Aplicada. Facultad de Bioqumica, Qumica y Farmacia. Universidad Nacional de Tucumn. Argentina.2Centro de Referencia para Lactobacilos (CERELA-CONICET). Argentina.

EFECTO DE LA LECHE HUMANA SOBRECLULAS DE SANGRE Y DE MDULA SEAEN UN MODELO DE RATOS DESNUTRIDOS;

ESTUDIO COMPARATIVO CON LECHE DE VACA

Resumen

Introduccin: Las alteraciones causadas por la defi-ciencia de nutrientes pueden ser revertidas por un aportenutricional adecuado.

Objetivos: Realizar estudios comparativos entre lechehumana y leche de vaca para evaluar su impacto en larecuperacin de las clulas de sangre y de mdula seaafectadas en ratones desnutridos.

Mtodos: Los ratones fueron desnutridos al recibir unadieta libre de protenas durante 21 das a partir del des-tete. Posteriormente, estos ratones desnutridos recibieronleche de vaca (LV) o leche humana (LH) durante 7 o 14das consecutivos, mientras continuaban consumiendo ladieta libre de protenas ad libitum. El grupo control dedesnutricin (CD) slo recibi la dieta libre de protenasmientras que los ratones controles bien nutridos (CBN)consumieron la dieta balanceada convencional.

Resultados y Discusin: Ambas leches normalizaron losniveles de albumina srica e incrementaron el peso deltimo. La leche humana fue menos efectiva que la leche devaca para incrementar el peso corporal y los niveles detransferrina en suero. Sin embargo, la leche humana fuems efectiva para incrementar el nmero de leucocitos(CBN: 6,90 1,60a; CD: 2,80 0,90b; LV 7d: 3,74 1,10b;LH 7d: 7,16 1,90a; LV 14d: 4,35 1,20b; LH 14d: 6,75 1,20a (109/L); p < 0,05) y linfocitos (CBN: 5,80 0,36a; CD:1,80 0,40b; LV 7d: 2,50 0,30b; LH 7d: 4,20 0,50c; LV14d: 3,30 0,31d; LH 14d: 4,70 0,28c (109/L); p < 0,05) ensangre perifrica. Ambas leches indujeron un incrementode las clulas del compartimiento mittico de mdula seay de las clulas -naftil butirato esterasa positivas en san-gre perifrica. Adems, normalizaron la funcin fagoc-tica en neutrfilos de sangre perifrica y el estallido oxi-dativo en las clulas peritoneales.

Conclusiones: Ambas leches fueron igualmente efecti-vas para ejercer efectos favorables en el nmero de lasclulas de la mdula sea y en las funciones de las clulasperitoneales y de la sangre involucradas en la respuestainmune. Sin embargo, slo la leche humana normaliz el

Abstract

Introduction: It has been demonstrated that the altera-tions caused by nutrient deficiency can be reverted byadequate nutritional repletion.

Objective: To perform comparative studies betweenhuman and cow milks in order to evaluate the impact ofboth milks on the recovery of blood and bone marrowcells affected in malnourished mice.

Method: Weaned mice were malnourished afterconsuming a protein free diet for 21 days. Malnourishedmice received cow or human milk (CM or HM) for 7 or14 consecutive days. During the period of administra-tion of milk, the mice consumed the protein free diet adlibitum. The malnourished control (MNC) groupreceived only protein free diet whereas the well-nourished control (WNC) mice consumed the balancedconventional diet.

Results and Discussion: Both milks normalized serumalbumin levels and improved thymus weight. Humanmilk was less effective than cow milk to increase bodyweight and serum transferrin levels. In contrast, humanmilk was more effective than cow milk to increase thenumber of leukocytes (WNC: 6.90 1.60a; MNC: 2.80 0.90b; CM 7d: 3.74 1.10b; HM 7d: 7.16 1.90a; CM 14d:4.35 1.20b; HM 14d: 6.75 1.20a (109/L); p < 0.05) andlymphocytes (WNC: 5.80 0.36a; MNC: 1.80 0.40b; CM7d: 2.50 0.30b; HM 7d: 4.20 0.50c; CM 14d: 3.30 0.31d; HM 14d: 4.70 0.28c (109/L); p < 0.05) in peripheralblood. Both milks induced an increment in mitotic poolcells in bone marrow and -naphthyl butyrate esterasepositive cells in peripheral blood. They also normalizedphagocytic function in blood neutrophils and oxidativeburst in peritoneal cells.

Conclusion: Both milks were equally effective to exertfavorable effects on the number of the bone marrow cellsand the functions of the blood and peritoneal cellsinvolved in immune response. However, only human milk

Correspondencia: Graciela Agero.Instituto de Bioqumica Aplicada.Facultad de Bioqumica, Qumica y Farmacia.Universidad Nacional de Tucumn.Balcarce, 747.4000 San Miguel de Tucumn. Argentina.E-mail: gaguero@fbqf.unt.edu.arRecibido: 17-IV-2013.1. Revisin: 24-VI-2013.Aceptado: 18-IX-2013.

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Abbreviations

NBE+: -naphthyl butyrate esterase positive cells.G+: -glucuronidase positive cells. BCD: balanced conventional diet.CM: cow milk.CM 7d: malnourished mice replete with CM for 7

consecutive days. CM 14d: malnourished mice replete with CM for 14

consecutive days.Hb: haemoglobin concentration. HCT: haematocrit. HM: human milk.HM 7d: malnourished mice replete with HM for 7

consecutive days. HM 14d: malnourished mice replete with HM for 14

consecutive days.MNC: malnourished control mice.MPO: myeloperoxidase. NBT: nitro blue tetrazolium. NBT+: NBT positive cells. PBS: phosphate buffer saline. PFD: protein free diet.WNC: well-nourished control mice.

Introduction

Protein energy deprivation alters cellular immunity,phagocyte function, the complement system, secretoryimmunoglobulin A concentrations, and cytokine pro-duction1. As a consequence, malnutrition increases thesusceptibility to infections. It was reported that proteinenergy deprivation also causes severe lesions in organswith high cellular proliferation such as the intestinalepithelium2 and the hematopoietic tissue3.

It has been demonstrated that the alterations caused bynutrient deficiency can be reverted by adequate nutritio-nal repletion. Numerous experiences suggest that milk issuperior to other repletion diets used in the treatment ofmalnutrition in terms of mortality, sepsis, improvementof intestinal permeability and weight gain4.

Human milk is a bodily fluid which, apart frombeing an excellent nutritional source for the growinginfant, also contains a variety of immune componentssuch as antibodies, growth factors, cytokines, antimi-crobial compounds, and specific immune cells5-7. Thesefactors help to support the immature immune system ofnewborns and to protect them against infectious risksduring the postnatal period while its own immune sys-

tem matures8. More recent clinical and experimentalobservations also suggest that human milk not onlyprovides passive protection, but also can directlymodulate the immunological development of the reci-pient infant9. In addition, the presence of growth factorsand hormones, both beneficial for the host, has beenobserved in milk from different species, includinghuman and bovine10-12. Proteins in human milk not onlyprovide amino acids but also bind to and facilitate theabsorption of nutrients, stimulate the growth and deve-lopment of the intestinal epithelium, and aid in thedigestion of other nutrients13.

On the other hand, cow milk is one of the mostimportant sources of dietary protein in humans. It isconstituted by high protein quality arises both from itsnutritional value and its physiological properties. Phy-siological properties of cow milk protein include acuteregulatory effects on nutrient bioavailability or onimmune mechanisms and longer term potential bene-fits for cardiovascular-system or tissue development14.

Considering these relevant antecedents, in the pre-sent work it was performed comparative studies bet-ween human and cow milks in order to evaluate theimpact of both milks on the recovery of blood and bonemarrow cells affected in malnourished mice.

Materials and methods

Animals

Balb/c mice were obtained from the closed colonykept at the bioterium of CERELA. They were housed inplastic cages in a controlled atmosphere (temperature 22 2 C; humidity 55 2%) with a 12 h light/dark cycle.

Feeding procedures

Weaned mice (3-week-old) were malnourished afterthey consumed a protein-free diet (PFD) for 21 days.

Milk from creole cows (CM) was provided by farmersfrom Tucumn, Argentina. Human milk (HM) wasobtained from healthy women with normal pregnancyand delivery. Informed consent was obtained from everymother. Exclusion criteria included gestation < 37weeks, birth weight < 2.5 kg, multiple pregnancy, majorillness requiring intensive care admission, and majorcongenital anomalies. The samples of mature milkswere expressed manually from each breast, collected in

normalized the number of leukocytes and increased thenumber of neutrophils in peripheral blood.

(Nutr Hosp. 2013;28:2157-2164)DOI:10.3305/nh.2013.28.6.6790

Key words: Malnourished mice. Human milk. Cow milk.Blood cells. Bone marrow cells.

nmero de leucocitos e increment el nmero de neutrfi-los en sangre perifrica.

(Nutr Hosp. 2013;28:2157-2164)DOI:10.3305/nh.2013.28.6.6790

Palabras clave: Ratones desnutridos. Leche humana. Lechede vaca. Clulas sanguneas. Clula de la mdula sea.

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sterile containers between 9.00-11.30 a.m. and then ali-quoted. Samples were stored at -70 C until use.

Malnourished mice received CM or HM for 7 (CM7d, HM 7d) or 14 (CM 14d, HM 14d) consecutive days.During the period of administration of milk, the miceconsumed the PFD ad libitum. The malnourished con-trol (MNC) group received only PFD whereas the well-nourished control (WNC) mice consumed the balancedconventional diet (BCD).

Samples from control (WNC and MNC), CM 7d,HM 7d, CM 14d, and HM 14d groups were obtained atthe end of each feeding period.

Animals were cared for in accordance with standardguidelines (Canadian Council on Animal Care, 1993).The experimental protocol was approved by the EthicalCommittee for Animal Care of CERELA and of theUniversidad Nacional de Tucumn, Argentina.

Study of nutritional state

Body weight and thymus weight were determined atthe beginning and end of each feeding period with anelectronic balance with a sensitivity of 0.01 g and 0.001g respectively. Body and thymus weight resulted fromthe mean of the values obtained in three different weig-hings performed alternately. Body weight was expres-sed as percentage of body weight increase with respectto the initial value.

Thymus weight was expressed in mg and could onlybe determined in the animals that received the milksupplementation since malnutrition caused extremeinvolution of the organ.

For the determination of serum parameters, bloodsamples were obtained by cardiac puncture fromsodium pentobarbital-anesthetized animals. The sam-ples were collected in glass tubes and the serum wasobtained.

Albumin concentration was determined using acolorimetric assay based on albumin binding to bromo-cresol green (Wiener Lab, Rosario, Argentina).

Serum transferrin was determined by radial immu-nodiffusion (Diffu-Plate; Biocientfica, S.A., BuenosAires, Argentina).

Study of blood cells

Basic haematological parameters: blood sampleswere collected in tubes containing EDTA as an anticoa-gulant. Haemoglobin concentration (Hb) was carried outusing the haemiglobincyanide method. Haematocrit(HCT) was determined manually by micro-haematocrittechniques. Total number of blood leukocytes was deter-mined with a haemocytometer. The results were expres-sed as 109/L. Differential blood leukocyte count wasdetermined by counting 200 cells in blood smears stai-ned with May Grnwald-Giemsa using a light micros-cope (100x) and absolute numbers were calculated.

Study of bone marrow cells

Anesthetized mice were killed by cervical disloca-tion and bone marrow samples were obtained by flus-hing the femoral cavity with phosphate buffer saline(PBS).

Differential cell counts were carried out by counting500 cells in bone marrow smears stained with MayGrnwald-Giemsa. Myeloid cells were grouped intothe mitotic pool, which includes cells capable of repli-cation (myeloblasts, promyelocytes and myelocytes),and the post-mitotic pool, whose cells usually do noreplicate but are able to evolve towards more matureand differentiated cells (metamyelocytes, band cellsand neutrophils). Lymphoid cells and erythroblastswere also counted. The results of myeloid cells, lymp-hoid cells, and erythroblast counts were expressed aspercentages of total white bone marrow cells.

To study T cells maturation, cytochemical assayswere performed according the maturation scheme pro-posed by Basso et al.15 It was determined the percen-tage of -glucuronidase positive (G+) cells and -naphthyl butyrate esterase positive (NBE+) cells inbone marrow and blood samples using commercialcytochemical assay kits (Sigma-Aldrich, St Louis,MO, USA). The cells were counted under a lightmicroscope (100x) and were regarded as positive cellsthat showed granular localized coarse positivity.

Study of myeloperoxidase (MPO) activity in bloodneutrophils and bone marrow myeloid cells

Myeloperoxidase (MPO) activity was determinedusing a cytochemical method (Washburn test) withbenzidine as a myeloperoxidase chromogen. Cellswere graded as negative or weakly, moderately orstrongly positive and were used to calculate the score.

Study of nitro blue tetrazolium (NBT) reduction testin peritoneal macrophages

The oxidative burst was studied in peritoneal lavage-derived macrophages using the nitro blue tetrazolium(NBT) reduction test (Sigma-Aldrich, St Louis, MO,USA). Samples were examined under a light micros-cope for blue precipitate. A hundred cells were countedand the percentage of NBT positive cells (NBT+) wasdetermined.

Statistical analysis

Experiments were performed in triplicate. For eachexperiment, 30-36 mice were used (5-6 animals eachgroup). Results were expressed as means SD. Afterverification of the normal distribution of data, ANOVAwas used. Tukeys test (for pairwise comparisons of the

Effect of human milk onmalnourished mice

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means) was used to test for differences between thegroups. Differences were considered significant at p