Myostatin Expression,Lymphocyte Population,and Potential Cytokine Production Correlate with Predisposition to High-Fat Diet Induced Obesity in Mice

A strong relationship exists between increased inflammatory cytokines and muscle insulin resistance in obesity. This study focused on identifying a relationship between metabolic propensity and myostatin expression in muscle and spleen cells in response to high-fat diet intake. Using a comparative approach, we analyzed the effects of high-fat diet intake on myostatin and follistatin expression, spleen cell composition, and potential cytokine expression in high-fat diet induced obesity (HFDIO) resistant (SWR/J) and susceptible (C57BL/6) mice models. Results demonstrated overall increased myostatin expression in muscle following high-fat diet intake in HFDIO-susceptible mice, while myostatin expression levels decreased initially in muscle from high-fat diet fed resistant mice. In HFDIO-resistant mice, myostatin expression decreased in spleen, while myostatin increased in spleen tissue from HFDIO-susceptible mice. Proinflammatory cytokine (IL-17, IL-1β, and IFNγ) potential increased in splenocytes from HFDIO-susceptible mice. In comparison, C57BL/6 mice fed a high-fat diet exhibited higher frequencies of CD4⁺/CD44^hi and CD8⁺/CD44^hi cells in the spleen compared to control fed mice. Together, these results suggest that susceptibility to high-fat diet induced obesity could be influenced by local myostatin activity in a tissue-specific manner and that splenocytes exhibit differential cytokine production in a strain-dependent manner. This study sets the stage for future investigations into the interactions between growth, inflammation, and metabolism.     

Mimotope-Based Vaccines of Leishmania infantum Antigens and Their Protective Efficacy against Visceral Leishmaniasis

Background

The development of cost-effective prophylactic strategies to prevent leishmaniasis has become a high-priority. The present study has used the phage display technology to identify new immunogens, which were evaluated as vaccines in the murine model of visceral leishmaniasis (VL). Epitope-based immunogens, represented by phage-fused peptides that mimic Leishmania infantum antigens, were selected according to their affinity to antibodies from asymptomatic and symptomatic VL dogs'' sera.

Methodology/Main Findings

Twenty phage clones were selected after three selection cycles, and were evaluated by means of in vitro assays of the immune stimulation of spleen cells derived from naive and chronically infected with L. infantum BALB/c mice. Clones that were able to induce specific Th1 immune response, represented by high levels of IFN-γ and low levels of IL-4 were selected, and based on their selectivity and specificity, two clones, namely B10 and C01, were further employed in the vaccination protocols. BALB/c mice vaccinated with clones plus saponin showed both a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with individual clones or L. infantum extracts. Additionally, these animals, when compared to control groups (saline, saponin, wild-type phage plus saponin, or non-relevant phage clone plus saponin), showed significant reductions in the parasite burden in the liver, spleen, bone marrow, and paws'' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, mainly by CD8⁺ T cells, against parasite proteins. These animals also presented decreased parasite-mediated IL-4 and IL-10 responses, and increased levels of parasite-specific IgG2a antibodies.

Conclusions/Significance

This study describes two phage clones that mimic L. infantum antigens, which were directly used as immunogens in vaccines and presented Th1-type immune responses, and that significantly reduced the parasite burden. This is the first study that describes phage-displayed peptides as successful immunogens in vaccine formulations against VL.     

Responsiveness of Thymus Cells to Syngeneic and Allogeneic Lymphoid Cells

THE thymus is necessary for the normal development of cell-mediated immunity in mice as shown by the immunological defects after neonatal thymectomy¹. Thymus cells themselves can be stimulated by allogeneic lymphoid cells in mixed leucocyte reaction (MLR)² and become killer cells or cytotoxic lymphocytes after stimulation with allogeneic spleen cells in vitro (H. Wagner and M. Feldmann, unpublished work) and in vivo^3,4. This suggests that the thymus as well as peripheral lymphoid tissues contain T cells which can be stimulated by foreign histocompatibility antigen to divide and differentiate into the cytotoxic lymphocytes which mediate cellular immunity. There have been suggestions that thymus cells might be stimulated to divide by “self” antigen, as well as foreign cells: incorporation of ³H-thymidine above background levels has been found in cultures with syngeneic spleen and thymus cells of adult rats⁵, although the experiments do not determine whether thymus or spleen cells have been stimulated. In contrast to these experiments, Howe et al. reported that only thymus cells of neonatal CBA mice reacted to allogeneic and syngeneic spleen cells of adult animals in “one way” MLR cultures^6,7. Whether the reaction of neonatal thymus cells to syngeneic adult spleen cells is recognition of “self” antigens is uncertain, since spleens of adult mice could carry antigens which do not occur in neonatal animals and are therefore “unknown” for neonatal thymus cells. We demonstrate here that neonatal thymus cells do not react to 4-day-old CBA spleen cells, but adult thymus cells do react against both allogeneic and syngeneic adult spleen cells.     

Protein Malnutrition Induces Bone Marrow Mesenchymal Stem Cells Commitment to Adipogenic Differentiation Leading to Hematopoietic Failure

Protein malnutrition (PM) results in pathological changes that are associated with peripheral leukopenia, bone marrow (BM) hypoplasia and alterations in the BM microenvironment leading to hematopoietic failure; however, the mechanisms involved are poorly understood. In this context, the BM mesenchymal stem cells (MSCs) are cells intimately related to the formation of the BM microenvironment, and their differentiation into adipocytes is important because adipocytes are cells that have the capability to negatively modulate hematopoiesis. Two-month-old male Balb/c mice were subjected to protein-energy malnutrition with a low-protein diet containing 2% protein, whereas control animals were fed a diet containing 12% protein. The hematopoietic parameters and the expression of CD45 and CD117 positive cells in the BM were evaluated. MSCs were isolated from BM, and their capability to produce SCF, IL-3, G-CSF and GM-CSF were analyzed. The expression of PPAR-γ and C/EBP-α as well as the expression of PPAR-γ and SREBP mRNAs were evaluated in MSCs together with their capability to differentiate into adipocytes in vitro. The malnourished animals had anemia and leukopenia as well as spleen and bone marrow hypoplasia and a reduction in the expression of CD45 and CD117 positive cells from BM. The MSCs of the malnourished mice presented an increased capability to produce SCF and reduced production of G-CSF and GM-CSF. The MSCs from the malnourished animals showed increased expression of PPAR-γ protein and PPAR-γ mRNA associated with an increased capability to differentiate into adipocytes. The alterations found in the malnourished animals allowed us to conclude that malnutrition committed MSC differentiation leading to adipocyte decision and compromised their capacity for cytokine production, contributing to an impaired hematopoietic microenvironment and inducing the bone marrow failure commonly observed in protein malnutrition states.     

Effect of zinc deficiency on autologous rosette-forming cells

Ability to form autologous rosettes (A-rosettes) is characteristic of a certain population of immature T cells, present in the thymus of various species including man. In mice, few A-rosettes are normally found in peripheral blood and spleen but their number increases markedly in spleen following thymectomy. In our studies, A-rosette formation could be demonstrated to be significantly enhanced in the spleen of C57B1/Ks mice after the animals had been maintained 3, 6, and 8 weeks on a zinc-deficient diet (Zn^?) and to increase progressively with duration of feeding the Zn^? diet. These changes were quickly reversed by feeding a normal diet containing zinc but could not be eliminated by complete adrenalectomy, a finding that ruled out their dependence on pituitary-adrenal as is function attributable to stress. Pair-fed controls and mice fed a zinc-supplemented diet ad libitum showed few A-rosettes, as expected.     

Pyridoxine deficiency and cytotoxicity of T lymphocytes in vitro.

The effect of pyridoxine deficiency on the proliferation and cytotoxicity of BALB/c mouse lymphocytes stimulated in vitro with irradiated spleen cells from C3H mice was studied. Cytotoxicity was measured by Na⁵¹CrO₄ release from L cells which have the same histocompatibility loci as C3H mouse cells. Pyridoxal-5′-phosphate (PLP) content in the spleen and liver of pyridoxine-deficient animals was determined with Escherichia coli B/1 t7A apotryptophanase. Maintenance of animals on a pyridoxine-deficient diet for 1 to 3 weeks affected neither proliferation of lymphocytes in vitro nor their cytotoxicity. Lymphocytes from mice fed a pyridoxine-deficient diet for 5 to 6 weeks had a reduced capacity to respond to foreign lymphoid cells in vitro. The Cytotoxicity of these lymphocytes was also significantly decreased. PLP, but not pyridoxal, added directly to the medium in vitro partially restored the impaired functions of T lymphocytes.     

Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein

Background

Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria.

Methods

Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested.

Results

Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65.

Conclusion

20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs.

     

Antigenicity and Protective Efficacy of a Leishmania Amastigote-specific Protein,Member of the Super-oxygenase Family,against Visceral Leishmaniasis

Background

The present study aimed to evaluate a hypothetical Leishmania amastigote-specific protein (LiHyp1), previously identified by an immunoproteomic approach performed in Leishmania infantum, which showed homology to the super-oxygenase gene family, attempting to select a new candidate antigen for specific serodiagnosis, as well as to compose a vaccine against VL.

Methodology/Principal Findings

The LiHyp1 DNA sequence was cloned; the recombinant protein (rLiHyp1) was purified and evaluated for its antigenicity and immunogenicity. The rLiHyp1 protein was recognized by antibodies from sera of asymptomatic and symptomatic animals with canine visceral leishmaniasis (CVL), but presented no cross-reactivity with sera of dogs vaccinated with Leish-Tec, a Brazilian commercial vaccine; with Chagas'' disease or healthy animals. In addition, the immunogenicity and protective efficacy of rLiHyp1 plus saponin was evaluated in BALB/c mice challenged subcutaneously with virulent L. infantum promastigotes. rLiHyp1 plus saponin vaccinated mice showed a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with the recombinant protein. Immunized and infected mice, as compared to the control groups (saline and saponin), showed significant reductions in the number of parasites found in the liver, spleen, bone marrow, and in the paws'' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, produced mainly by CD4 T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 response could also be observed.

Conclusions/Significance

The present study showed that this Leishmania oxygenase amastigote-specific protein can be used for a more sensitive and specific serodiagnosis of asymptomatic and symptomatic CVL and, when combined with a Th1-type adjuvant, can also be employ as a candidate antigen to develop vaccines against VL.     

Dietary supplementation with probiotics improves hematopoiesis in malnourished mice

Background

Lactobacillus rhamnosus CRL1505 (Lr) administered during the repletion of immunocompromised-malnourished mice improves the resistance against intestinal and respiratory infections. This effect is associated with an increase in the number and functionality of immune cells, indicating that Lr could have some influence on myeloid and lymphoid cell production and maturation.

Objective

This study analyzed the extent of the damage caused by malnutrition on myeloid and lymphoid cell development in the spleen and bone marrow (BM). We also evaluated the impact of immunobiotics on the recovery of hematopoiesis affected in malnourished mice.

Methods

Protein malnourished mice were fed on a balanced conventional diet for 7 or 14 consecutive d with or without supplemental Lr or fermented goat''s milk (FGM). Malnourished mice and well-nourished mice were used as controls. Histological and flow cytometry studies were carried out in BM and spleen to study myeloid and lymphoid cells.

Results

Malnutrition induced quantitative alterations in spleen B and T cells; however, no alteration was observed in the ability of splenic B cells to produce immunoglobulins after challenge with LPS or CpG. The analysis of BM B cell subsets based on B220, CD24, IgM and IgD expression showed that malnutrition affected B cell development. In addition, BM myeloid cells decreased in malnourished mice. On the contrary, protein deprivation increased BM T cell number. These alterations were reverted with Lr or FGM repletion treatments since normal numbers of BM myeloid, T and B cells were observed in these groups.

Conclusions

Protein malnutrition significantly alters B cell development in BM. The treatment of malnourished mice with L. rhamnosus CRL1505 was able to induce a recovery of B cells that would explain its ability to increase immunity against infections. This work highlights the possibility of using immunobiotics to accelerate the recovery of lymphopoyesis in immunocompromised-malnourished hosts.     

Antigenicity,Immunogenicity and Protective Efficacy of Three Proteins Expressed in the Promastigote and Amastigote Stages of Leishmania infantum against Visceral Leishmaniasis

In the present study, two Leishmania infantum hypothetical proteins present in the amastigote stage, LiHyp1 and LiHyp6, were combined with a promastigote protein, IgE-dependent histamine-releasing factor (HRF); to compose a polyproteins vaccine to be evaluated against L. infantum infection. Also, the antigenicity of the three proteins was analyzed, and their use for the serodiagnosis of canine visceral leishmaniasis (CVL) was evaluated. The LiHyp1, LiHyp6, and HRF DNA coding sequences were cloned in prokaryotic expression vectors and the recombinant proteins were purified. When employed in ELISA assays, all proteins were recognized by sera from visceral leishmaniasis (VL) dogs, and presented no cross-reactivity with either sera from dogs vaccinated with a Brazilian commercial vaccine, or sera of Trypanosoma cruzi-infected or Ehrlichia canis-infected animals. In addition, the antigens were not recognized by antibodies from non-infected animals living in endemic or non-endemic areas for leishmaniasis. The immunogenicity and protective efficacy of the three proteins administered in the presence of saponin, individually or in combination (composing a polyproteins vaccine), were evaluated in a VL murine model: BALB/c mice infected with L. infantum. Spleen cells from mice inoculated with the individual proteins or with the polyproteins vaccine plus saponin showed a protein-specific production of IFN-γ, IL-12, and GM-CSF after an in vitro stimulation, which was maintained after infection. These animals presented significant reductions in the parasite burden in different evaluated organs, when compared to mice inoculated with saline or saponin. The decrease in parasite burden was associated with an IL-12-dependent production of IFN-γ against parasite total extracts (produced mainly by CD4⁺ T cells), correlated to the induction of parasite proteins-driven NO production. Mice inoculated with the recombinant protein-based vaccines showed also high levels of parasite-specific IgG2a antibodies. The polyproteins vaccine administration induced a more pronounced Th1 response before and after challenge infection than individual vaccines, which was correlated to a higher control of parasite dissemination to internal organs.     

Effects of a protein-free diet on the DNA-synthesizing and dividing cells in the lymphoid organs of rats. Changes induced by phytohemagglutinin and cortisone invivo

Levels of ³H-thymidine-labeled lymphocytes on autoradiographs of cell smears and mitotic indices (M.I.) were determined in the popliteal lymph nodes (PLN), spleen and thymus of rats fed with a balanced or a protein deprived (PD) diet for 7 weeks. The latter diet reduced the number of labeled cells per 10³ in the spleen but not in the PLN's and the thymus. The M.I.'s were reduced 50% in the spleen and dropped drastically in the thymus. Subplantar injection of phytohemagglutinin (PHA) provoked a sharp increase in the M.I.'s of the PLN's and the spleen, and also moderately increased the labeling index in the PLN's in normal rats. In PD rats PHA no longer influenced the mitoses in the PLN's and the spleen while it considerably increased the proportion of labeled cells in the latter. These discrepancies between DNA-synthesis and mitoses in the thymus and the spleen suggest a premitotic block of the cell cycle after protein deprivation. The effects of a 5 day-cortisone treatment resembled in a large measure the changes induced by a protein-free diet including the arrest of the cell cycle. The thymic lymphocytes were more sensitive to the hormone in PD than in normal rats. On the contrary, in the PLN's and the spleen mainly cortisone-resistant cells remained alive among the DNA-synthesizing lymphocytes after protein deprivation. All these data agree with the hypothesis of an intervention of endogenous glucocorticoids in the protein deficiency-induced lymphoid involution.     

Changes in lymphocyte populations in protein—Calorie-deficient mice

Changes in lymphocyte subpopulations induced by postweaning malnutrition were studied in C57BL/6 mice kept on a protein-restricted diet (D), by weekly assessment of the “homing” properties and the response to mitogens of thymus and spleen lymphocytes during the first 2 months of diet. Cell loss in the lymphoid organs during the early phase of protein restriction was mainly due to depletion of nonrecirculating cells. This resulted in relative enrichment of medullary cells in the thymus and T₂ cells in the periphery as shown by the rise in the percentage migration of D lymphocytes to the lymph nodes as well as in their response to optimal doses of PHA and Con A and PHA:Con A response ratio. Reversion of the distribution pattern of D lymphocytes, with depressed homing to the lymph nodes and decrease in the response to mitogens, was observed concomitantly with a second phase of partial recovery in the whole-body weight and cell content of the thymus and spleen. The [³H]thymidine uptake by D spleen cells stimulated with supraoptimal doses of mitogen was significantly increased during the whole length of the experiment. The suppression of DNA synthesis induced by high doses of mitogen reappeared after short-term nutritional rehabilitation.     

Dietary omega-3 fatty acids enhance the B1 but not the B2 cell immune response in mice with antigen-induced peritonitis

The effects of omega-3 fatty acids on the adaptive immune response have mainly been analysed in vitro with varying results. How omega-3 fatty acids affect the adaptive immune response in vivo is largely unknown. This study examined the effects of dietary fish oil on the adaptive immune response in antigen-induced inflammation in mice, focusing on its effects on B cells and B cell subsets. Mice were fed a control diet with or without 2.8% fish oil, immunized twice with methylated BSA (mBSA) and peritonitis induced by intraperitoneal injection of mBSA. Serum, spleen and peritoneal exudate were collected prior to and at different time points after induction of peritonitis. Serum levels of mBSA-specific antibodies were determined by ELISA and the number of peritoneal and splenic lymphocytes by flow cytometry. The levels of germinal center B cells and IgM⁺, IgG⁺ and CD138⁺ cells in spleen were evaluated by immunoenzyme staining. Mice fed the fish oil diet had more peritoneal B1 cells, more IgM⁺ cells in spleen and higher levels of serum mBSA-specific IgM antibodies compared with that in mice fed the control diet. However, dietary fish oil did not affect the number of peritoneal B2 cells, splenic IgG⁺ or CD138⁺ cells or serum levels of mBSA-specific IgG antibodies in mice with mBSA-induced peritonitis. These results indicate that dietary fish oil can enhance the adaptive immune response, specifically the B1 cell response, which may lead to better protection against secondary infection as well as improvement in reaching homeostasis following antigenic challenge.     

Low CXCL13 expression, splenic lymphoid tissue atrophy and germinal center disruption in severe canine visceral leishmaniasis

Visceral leishmaniasis is associated with atrophy and histological disorganization of splenic compartments. In this paper, we compared organized and disorganized splenic lymphoid tissue from dogs naturally infected with Leishmania infantum assessing the size of the white pulp compartments, the distribution of T, B and S100⁺ dendritic cells, using immunohistochemistry and morphometry and the expression of CCR7 and the cytokines, CXCL13, lymphotoxin (LT)-α, LT-β, CCL19, CCL21, TNF-α, IL-10, IFN-γ and TGF-β, using by real time RT-PCR. The lymphoid follicles and marginal zones were smaller (3.2 and 1.9 times, respectively; Mann-Whitney, P<0.02) in animals with disorganized splenic tissue in comparison to those with organized splenic lymphoid tissue. In spleens with disorganized lymphoid tissue, the numbers of T cells and S100⁺ dendritic cells were decreased in the follicles, and the numbers of B cells were reduced in both the follicles and marginal zones. CXCL13 mRNA expression was lower in animals with disorganized lymphoid tissue (0.5±0.4) compared to those with organized lymphoid tissue (2.7±2.9, both relative to 18S expression, P = 0.01). These changes in the spleen were associated with higher frequency of severe disease (7/12) in the animals with disorganized than in animals with organized (2/13, Chi-square, P = 0.01) splenic lymphoid tissue. The data presented herein suggest that natural infection with Leishmania infantum is associated with the impairment of follicular dendritic cells, CXCL13 expression, B cell migration and germinal center formation and associates these changes with severe clinical forms of visceral leishmaniasis. Furthermore the fact that this work uses dogs naturally infected with Leishmania infantum emphasizes the relevance of the data presented herein for the knowledge on the canine and human visceral leishmaniasis.     

Trafficking and Replication Patterns Reveal Splenic Macrophages as Major Targets of Dengue Virus in Mice

Human postmortem studies of natural dengue virus (DENV) infection have reported systemically distributed viral antigen. Although it is widely accepted that DENV infects mononuclear phagocytes, the sequence in which specific tissues and cell types are targeted remains uncharacterized. We previously reported that mice lacking alpha/beta and gamma interferon receptors permit high levels of DENV replication and show signs of systemic disease (T. R. Prestwood et al., J. Virol. 82:8411–8421, 2008). Here we demonstrate that within 6 h, DENV traffics to and replicates in both CD169⁺ and SIGN-R1⁺ macrophages of the splenic marginal zone or draining lymph node, respectively, following intravenous or intrafootpad inoculation. Subsequently, high levels of replication are detected in F4/80⁺ splenic red pulp macrophages and in the bone marrow, lymph nodes, and Peyer''s patches. Intravenously inoculated mice begin to succumb to dengue disease 72 h after infection, at which time viral replication occurs systemically, except in lymphoid tissues. In particular, high levels of replication occur in CD68⁺ macrophages of the kidneys, heart, thymus, and gastrointestinal tract. Over the course of infection, proportionately large quantities of DENV traffic to the liver and spleen. However, late during infection, viral trafficking to the spleen decreases, while trafficking to the liver, thymus, and kidneys increases. The present study demonstrates that macrophage populations, initially in the spleen and other lymphoid tissues and later in nonlymphoid tissues, are major targets of DENV infection in vivo.     

Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB/c mice against Leishmania chagasi and Leishmania amazonensis challenge

Leishmania chagasi and Leishmania amazonensis are the etiologic agents of different clinical forms of human leishmaniasis in South America. In an attempt to select candidate antigens for a vaccine protecting against different Leishmania species, the efficacy of vaccination using Leishmania ribosomal proteins and saponin as adjuvant was examined in BALB/c mice against challenge infection with both parasite species. Mice vaccinated with parasite ribosomal proteins purified from Leishmania infantum plus saponin showed a specific production of IFN-γ, IL-12 and GM-CSF after in vitro stimulation with L. infantum ribosomal proteins. Vaccinated mice showed a reduction in the liver and spleen parasite burdens after L. chagasi infection. After L. amazonensis challenge, vaccinated mice showed a decrease of the dermal pathology and a reduction in the parasite loads in the footpad and spleen. In both models, protection was correlated to an IL-12-dependent production of IFN-γ by CD4⁺ and CD8⁺ T cells that activate macrophages for the synthesis of NO. In the protected mice a decrease in the parasite-mediated IL-4 and IL-10 responses was also observed. In mice challenged with L. amazonensis, lower levels of anti-parasite-specific antibodies were detected. Thus, Leishmania ribosomal proteins plus saponin fits the requirements to compose a pan-Leishmania vaccine.     

Dual Immune Modulatory Effect of Vitamin A in Human Visceral Leishmaniasis

Vitamin A supplementation has shown to prevent mortality by diarrheal and respiratory diseases in several countries. Nevertheless, there are few studies investigating the effect of vitamin A in visceral leishmaniasis (VL), although there are reports of its deficiency in children with symptomatic VL in Brazil and Bangladesh. This study analyzed the effect of vitamin A on a subset of Treg cells and monocytes isolated from symptomatic VL and from healthy children residing in an endemic area for VL in Northeast Brazil. Serum retinol concentrations correlated inversely with IL-10 and TGF-β productions in CD4⁺CD25^highFoxp3⁺ T cells isolated from children with VL stimulated with leishmanial antigens. All-trans retinoic acid in vitro induced IL-10 in CD4⁺CD25^highFoxp3⁺ T cells; IL-10 and TGF-β production in CD4⁺CD25⁻Foxp3⁻ T cells, and IL-10 in monocytes isolated from healthy children. However, the use of all-trans retinoic acid together with leishmanial antigens in vitro prevented increases in IL-10 production in Treg cells and monocytes isolated from VL children. Strikingly, those results show a potential dual role of vitamin A in the immune system: improvement of a regulatory profile in cells from healthy children after leishmanial stimulation and down modulation of IL-10 in Treg cells and monocytes during symptomatic VL. Therefore, the use of vitamin A concomitant to VL therapy might be useful in improving recovery from disease status caused by Leishmania infantum infection and warrants additional study.     

Parasite Burden in Hamsters Infected with Two Different Strains of Leishmania (Leishmania) infantum: “Leishman Donovan Units” versus Real-Time PCR

To develop and test new therapeutics and immune prophylaxis strategies for visceral leishmaniasis (VL), understanding tissue parasitism evolution after experimental infection with Leishmania infantum is important. Experimental infection in a hamster model (Mesocricetus auratus) reproduces several typical aspects of canine and human VL that are closely related to the inoculum’s route. We quantified the parasitism in the liver and spleen of hamsters experimentally infected by various routes (intradermal, intraperitoneal, and intracardiac [IC]) and different strains of L. infantum (MHOM/BR/74/PP75 and Wild) and compared two different methodologies to evaluate tissue parasitism (Leishman Donovan units [LDU] and real-time qPCR). In addition, the quantification of specific total-IgG in the serum of uninfected and infected hamsters was determined by ELISA. The animals were followed for 1, 3, 6 and 9 months post-infection for survival analysis. We found that infection with the Wild strain by the IC route resulted in higher mortality. Positive antibody (IgG) responses were detected with higher peaks at 6 and 9 months in the IC group inoculated with PP75 strain. However, in animals infected with the Wild strain the IgG levels were elevated in all infected groups during all the time evaluated. We also observed by LDU analysis that the IC route lead to higher parasitism in the liver and spleen with both strains. Furthermore, qPCR showed higher sensitivity for identifying animals with low parasitic burden. In conclusion, qPCR can be useful for assessing parasitism in the spleen and liver of a hamster model infected with L. infantum independent of the route of infection, and this technique may become an essential tool for assessing parasite density in the hamster model after experimental treatment or immunization with potential vaccine candidates.