Further characterization of HDAC and SIRT gene expression patterns in pancreatic cancer and their relation to disease outcome.

Ductal adenocarcinoma of the pancreas is ranking 4 for patient' death from malignant disease in Western countries, with no satisfactory treatment. We re-examined more precisely the histone deacetylases (HDAC) and Sirtuin (SIRT) gene expression patterns in pancreatic cancer with more pancreatic tumors and normal tissues. We also examined the possible relationship between HDAC gene expression levels and long term disease outcome. Moreover, we have evaluated by using an in vitro model system of human pancreatic tumor cell line whether HDAC7 knockdown may affect the cell behavior. We analyzed 29 pancreatic adenocarcinoma (PA), 9 chronic pancreatitis (CP), 8 benign pancreatic (BP) and 11 normal pancreatic tissues. Concerning pancreatic adenocarcinoma, we were able to collect biopsies at the tumor periphery. To assess the possible involvement of HDAC7 in cell proliferation capacity, we have generated recombinant human Panc-1 tumor which underexpressed or overexpressed HDAC7. The expression of HDAC1,2,3,4,7 and Nur77 increased in PA samples at levels significantly higher than those observed in the CP group (p = 0.0160; 0.0114; 0.0227; 0.0440; 0.0136; 0.0004, respectively). The expression of HDAC7, was significantly greater in the PA compared with BP tissue samples (p = 0.05). Mean mRNA transcription levels of PA for HDAC7 and HDAC2 were higher when compared to their counterpart biopsies taken at the tumor periphery (p = 0.0346, 0.0053, respectively). Moreover, the data obtained using confocal microscopy and a quantitative method of immunofluorescence staining strongly support the HDAC7 overexpression in PA surgical specimens. The number of deaths and recurrences at the end of follow up were significantly greater in patients with overexpression of HDAC7. Interestingly, the rate of growth was significantly reduced in the case of cell carrying shRNA construct targeting HDAC7 encoding gene when compared to the parental Panc-1 tumor cells (p = 0.0015) at 48 h and 96 h (p = 0.0021). This study strongly support the notion that HDAC7play a role in pancreatic adenocarcinoma progression.

Abortive T follicular helper development is associated with a defective humoral response in Leishmania infantum-infected macaques.

Leishmania infantum causes a chronic infectious disease named visceral leishmaniasis (VL). We employed a non-human primate model to monitor immune parameters over time and gain new insights into the disease. Rhesus macaques were infected with L. infantum and the T helper and B cell immunological profiles characterized during acute and chronic phases of infection. Parasite detection in visceral compartments during the acute phase was associated with differentiation of effector memory CD4 T cells and increased levels of Th1 transcripts. At the chronic phase, parasites colonized novel lymphoid niches concomitant with increased expression of IL10. Despite the occurrence of hypergammaglobulinemia, the production of parasite-specific IgG was poor, being confined to the acute phase and positively correlated with the frequency of an activated memory splenic B cell population. We noticed the expansion of a splenic CD4 T cell population expressing CXCR5 and Bcl-6 during acute infection that was associated with the differentiation of the activated memory B cell population. Moreover, the number of splenic germinal centers peaked at one month after infection, hence paralleling the production of specific IgG. However, at chronic infection these populations contracted impacting the production of parasite-specific IgG. Our study provides new insights into the immune events taking place in a physiologically relevant host and a mechanistic basis for the inefficient humoral response during VL.

Impairment of T cell function in parasitic infections.

In mammals subverted as hosts by protozoan parasites, the latter and/or the agonists they release are detected and processed by sensors displayed by many distinct immune cell lineages, in a tissue(s)-dependent context. Focusing on the T lymphocyte lineage, we review our present understanding on its transient or durable functional impairment over the course of the developmental program of the intracellular parasites Leishmania spp., Plasmodium spp., Toxoplasma gondii, and Trypanosoma cruzi in their mammalian hosts. Strategies employed by protozoa to down-regulate T lymphocyte function may act at the initial moment of naïve T cell priming, rendering T cells anergic or unresponsive throughout infection, or later, exhausting T cells due to antigen persistence. Furthermore, by exploiting host feedback mechanisms aimed at maintaining immune homeostasis, parasites can enhance T cell apoptosis. We will discuss how infections with prominent intracellular protozoan parasites lead to a general down-regulation of T cell function through T cell anergy and exhaustion, accompanied by apoptosis, and ultimately allowing pathogen persistence.

Characterization and evaluation of BNIPDaoct-loaded PLGA nanoparticles for visceral leishmaniasis: in vitro and in vivo studies.

OBJECTIVE: To overcome the limitation of bisnaphthalimidopropyldiaaminooctane (BNIPDaoct) low physiological solubility and potentially increase its efficiency against visceral leishmaniasis (VL), a delivery system based on poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles was developed. MATERIALS & METHODS: BNIPDaoct-PLGA nanoparticles were prepared by nanoprecipitation and characterized. Anti-Leishmania activity was evaluated using in vitro and in vivo VL infection models. RESULTS: BNIPDaoct-PLGA nanoparticles were successfully produced and were sized at 156.0 ± 2.8 nm with an encapsulation efficiency of approximately 85%. The PLGA nanoparticles reduced BNIPDaoct cellular toxicity, retained its in vitro anti-leishmanial activity and led to a significant reduction (∼80%) in the parasite burden in the infected mice spleen when compared with the free drug or amphotericin B. In the liver the effect was less pronounced, with a 30-50% reduction observed between the nanoformulation and the BNIPDaoct per se or the amphotericin B, respectively. CONCLUSION: PLGA nanoparticles provide controlled and effective delivery of BNIPDaoct for treatment of VL.

Impact of continuous axenic cultivation in Leishmania infantum virulence.

Experimental infections with visceral Leishmania spp. are frequently performed referring to stationary parasite cultures that are comprised of a mixture of metacyclic and non-metacyclic parasites often with little regard to time of culture and metacyclic purification. This may lead to misleading or irreproducible experimental data. It is known that the maintenance of Leishmania spp. in vitro results in a progressive loss of virulence that can be reverted by passage in a mammalian host. In the present study, we aimed to characterize the loss of virulence in culture comparing the in vitro and in vivo infection and immunological profile of L. infantum stationary promastigotes submitted to successive periods of in vitro cultivation. To evaluate the effect of axenic in vitro culture in parasite virulence, we submitted L. infantum promastigotes to 4, 21 or 31 successive in vitro passages. Our results demonstrated a rapid and significant loss of parasite virulence when parasites are sustained in axenic culture. Strikingly, the parasite capacity to modulate macrophage activation decreased significantly with the augmentation of the number of in vitro passages. We validated these in vitro observations using an experimental murine model of infection. A significant correlation was found between higher parasite burdens and lower number of in vitro passages in infected Balb/c mice. Furthermore, we have demonstrated that the virulence deficit caused by successive in vitro passages results from an inadequate capacity to differentiate into amastigote forms. In conclusion, our data demonstrated that the use of parasites with distinct periods of axenic in vitro culture induce distinct infection rates and immunological responses and correlated this phenotype with a rapid loss of promastigote differentiation capacity. These results highlight the need for a standard operating protocol (SOP) when studying Leishmania species.

Anti-leishmanial activity of the bisnaphthalimidopropyl derivatives.

Bisnaphthalimidopropyl (BNIP) derivatives were recently identified as inhibitors of the Leishmania Silent Information Regulator 2 (SIR2) NAD(+)-dependent deacetylase. In this report we have for the first time, determined the potential of these compounds to treat visceral leishmaniasis using BALB/c mice chronically infected with Leishmania infantum as a model. These experiments led to the identification of BNIPdiaminooctane (BNIPDaoct) as an effective compound able to induce significant reduction of the parasite load in the spleen and in the liver. Indeed, at a dose of 1mg/kg, BNIPDaoct was more effective to treat leishmaniasis in a short course treatment (3 or 6 drug administrations) than the standard amphotericin B. Moreover, no indications of hematological toxicity were detected as evaluated by the hemoglobin, hematocrit, white and red blood cell counts, hence making BNIPDaoct a potential therapeutic agent against leishmaniasis.

In vitro activity of nicotinamide/antileishmanial drug combinations.

To improve the management of leishmaniasis, new drugs and/or alternative therapeutic strategies are required. Combination therapy of antileishmanial drugs is currently considered as one of the most rational approaches to lower treatment failure rate and limit drug resistance spreading. Nicotinamide (NAm), also known as vitamin B3 that is already is used in human therapy, exerts in vitro antileishmanial activity. Drug combination studies, performed on L. infantum axenic amastigotes, revealed that NAm significantly improves the antileishmanial activity of trivalent antimony in a synergistic manner while it shows additive activity with amphotericin B and slightly antagonizes pentamidine activity. NAm also significantly increases the toxicity of pentavalent antimony against the intracellular forms of L. infantum, L. amazonensis and L. braziliensis. The potential of NAm to be used as adjuvant during leishmaniasis chemotherapy is further discussed.

Rationale for possible targeting of histone deacetylase signaling in cancer diseases with a special reference to pancreatic cancer.

There is ongoing interest to identify signaling pathways and genes that play a key role in carcinogenesis and the development of resistance to antitumoral drugs. Given that histone deacetylases (HDACs) interact with various partners through complex molecular mechanims leading to the control of gene expression, they have captured the attention of a large number of researchers. As a family of transcriptional corepressors, they have emerged as important regulators of cell differentiation, cell cycle progression, and apoptosis. Several HDAC inhibitors (HDACis) have been shown to efficiently protect against the growth of tumor cells in vitro as well as in vivo. The pancreatic cancer which represents one of the most aggressive cancer still suffers from inefficient therapy. Recent data, although using in vitro tumor cell cultures and in vivo chimeric mouse model, have shown that some of the HDACi do express antipancreatic tumor activity. This provides hope that some of the HDACi could be potential efficient anti-pancreatic cancer drugs. The purpose of this review is to analyze some of the current data of HDACi as possible targets of drug development and to provide some insight into the current problems with pancreatic cancer and points of interest for further study of HDACi as potential molecules for pancreatic cancer adjuvant therapy.

Activation of phosphatidylinositol 3-kinase/Akt and impairment of nuclear factor-kappaB: molecular mechanisms behind the arrested maturation/activation state of Leishmania infantum-infected dendritic cells.

Understanding the complex interactions between Leishmania and dendritic cells (DCs) is central to the modulation of the outcome of this infection, given that an effective immune response against Leishmania is dependent on the successful activation and maturation of DCs. We report here that Leishmania infantum promastigotes successfully infect mouse bone marrow-derived DCs without triggering maturation, as shown by a failure in the up-regulation of CD40 and CD86 expression, and that parasites strongly counteract the lipopolysaccharide-triggered maturation of DCs. A small increase in interleukin (IL)-12 and IL-10 transcription and secretion and a decrease in IL-6 were observed in infected cells. This arrested DC maturation state is actively promoted by parasites because heat-killed or fixed parasites increased cytokine and costimulatory molecule expression. At a molecular level, L. infantum rapidly induced activation of phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2, whereas no effect was observed in the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase proinflammatory pathways. Moreover, parasites actively promoted cleavage of the nuclear factor-κB p65(RelA) subunit, causing its impairment. The blockade of phosphatidylinositol 3-kinase/Akt by either treatment of bone marrow-derived DCs with wortmannin or transfection with an Akt dominant-negative mutant resulted in a strong decrease in infection rates, revealing for the first time a crucial role of this pathway on Leishmania engulfment by DCs. Overall, our data indicate that activation of Akt and impairment of nuclear factor-κB are responsible for immunogenicity subversion of L. infantum-infected DCs.

In vitro and in vivo anticancer activity of a novel nano-sized formulation based on self-assembling polymers against pancreatic cancer.

PURPOSE: To evaluate the in vitro and in vivo pancreatic anticancer activity of a nano-sized formulation based on novel polyallylamine grafted with 5% mole cholesteryl pendant groups (CH(5)-PAA). METHODS: Insoluble novel anticancer drug, Bisnaphthalimidopropyldiaaminooctane (BNIPDaoct), was loaded into CH(5)-PAA polymeric self-assemblies by probe sonication. Hydrodynamic diameters and polydispersity index measurements were determined by photon correlation spectroscopy. The in vitro cytotoxicity evaluation of the formulation was carried out by the sulforhodamine B dye assay with human pancreatic adenocarcinoma BxPC-3 cells, while for the in vivo study, Xenograff mice were used. In vitro apoptotic cell death from the drug formulation was confirmed by flow cytometric analysis. RESULTS: The aqueous polymer-drug formulation had a mean hydrodynamic size of 183 nm. The drug aqueous solubility was increased from negligible concentration to 0.3 mg mL(-1). CH(5)-PAA polymer alone did not exhibit cytotoxicity, but the new polymer-drug formulation showed potent in vitro and in vivo anticancer activity. The mode of cell death in the in vitro study was confirmed to be apoptotic. The in vivo results revealed that the CH(5)-PAA alone did not have any anti-proliferative effect, but the CH(5)-PAA-drug formulation exhibited similar tumour reduction efficacy as the commercial drug, gemcitabine. CONCLUSIONS: The proposed formulation shows potential as pancreatic cancer therapeutics.

Bisnaphthalimidopropyl derivatives as inhibitors of Leishmania SIR2 related protein 1.

The NAD(+)-dependent deacetylases, namely sirtuins, are involved in the regulation of a variety of biological processes such as gene silencing, DNA repair, longevity, metabolism, apoptosis, and development. An enzyme from the parasite Leishmania infantum that belongs to this family, LiSIR2RP1, is a NAD(+)-dependent tubulin deacetylase and an ADP-ribosyltransferase. This enzyme's involvement in L. infantum virulence and survival underscores its potential as a drug target. Our search for selective inhibitors of LiSIR2RP1 has led, for the first time, to the identification of the antiparasitic and anticancer bisnaphthalimidopropyl (BNIP) alkyl di- and triamines (IC(50) values in the single-digit micromolar range for the most potent compounds). Structure-activity studies were conducted with 12 BNIP derivatives that differ in the length of the central alkyl chain, which links the two naphthalimidopropyl moieties. The most active and selective compound is the BNIP diaminononane (BNIPDanon), with IC(50) values of 5.7 and 97.4 microM against the parasite and human forms (SIRT1) of the enzyme, respectively. Furthermore, this compound is an NAD(+)-competitive inhibitor that interacts differently with the parasite and human enzymes, as determined by docking analysis, which might explain its selectivity toward the parasitic enzyme.

The contribution of Toll-like receptor 2 to the innate recognition of a Leishmania infantum silent information regulator 2 protein.

We have characterized a Leishmania protein belonging to the silent information regulator 2 (SIR2) family [SIR2 related protein 1 (SIR2RP1)] that might play an immunoregulatory role during infection through its capacity to trigger B-cell effector functions. We report here that SIR2RP1 leads to the proliferation of activated B cells, causing increased expression of major histocompatibility complex (MHC) II and the costimulatory molecules CD40 and CD86, which are critical ligands for T-cell cross-talk during the development of adaptive immune responses. In contrast, B cells isolated from Toll-like receptor 2 (TLR2) knockout mice were unable to respond to the SIR2RP1 stimulus. Similarly, SIR2RP1 induced the maturation of dendritic cells (DCs) in a TLR2-dependent manner with the secretion of pro-inflammatory cytokines [interleukin (IL)-12 and tumour necrosis factor (TNF)-alpha] and enhanced the costimulatory properties of DCs. Nevertheless, immunization assays demonstrated that TLR2-deficient mice were able to mount a specific humoral response to SIR2RP1. Interestingly, further investigations showed that macrophages were activated by SIR2RP1 even in the absence of TLR2. Therefore, a different type of interplay between SIR2RP1 and the major antigen-presenting cells in vivo could explain the immune response observed in TLR2-deficient mice. Together, these results demonstrate that TLR2 signalling contributes to SIR2RP1 recognition by innate immune host cells.

The Leishmania infantum cytosolic SIR2-related protein 1 (LiSIR2RP1) is an NAD+ -dependent deacetylase and ADP-ribosyltransferase.

Proteins of the SIR2 (Silent Information Regulator 2) family are characterized by a conserved catalytic domain that exerts unique NAD(+)-dependent deacetylase activity on histones and various other cellular substrates. Previous reports from us have identified a Leishmania infantum gene encoding a cytosolic protein termed LiSIR2RP1 (Leishmania infantum SIR2-related protein 1) that belongs to the SIR2 family. Targeted disruption of one LiSIR2RP1 gene allele led to decreased amastigote virulence, in vitro as well as in vivo. In the present study, attempts were made for the first time to explore and characterize the enzymatic functions of LiSIR2RP1. The LiSIR2RP1 exhibited robust NAD(+)-dependent deacetylase and ADP-ribosyltransferase activities. Moreover, LiSIR2RP1 is capable of deacetylating tubulin, either in dimers or, when present, in taxol-stabilized microtubules or in promastigote and amastigote extracts. Furthermore, the immunostaining of parasites revealed a partial co-localization of alpha-tubulin and LiSIR2RP1 with punctate labelling, seen on the periphery of both promastigote and amastigote stages. Isolated parasite cytoskeleton reacted with antibodies showed that part of LiSIR2RP1 is associated to the cytoskeleton network of both promastigote and amastigote forms. Moreover, the Western blot analysis of the soluble and insoluble fractions of the detergent of promastigote and amastigote forms revealed the presence of alpha-tubulin in the insoluble fraction, and the LiSIR2RP1 distributed in both soluble and insoluble fractions of promastigotes as well as amastigotes. Collectively, the results of the present study demonstrate that LiSIR2RP1 is an NAD(+)-dependent deacetylase that also exerts an ADP-ribosyltransferase activity. The fact that tubulin could be among the targets of LiSIR2RP1 may have significant implications during the remodelling of the morphology of the parasite and its interaction with the host cell.

High histone deacetylase 7 (HDAC7) expression is significantly associated with adenocarcinomas of the pancreas.

BACKGROUND: Alterations in HDACs gene expression have been reported in a number of human cancers. No information is available concerning the status of HDACs in pancreatic cancer tumors. The aim of the present study was to evaluate the expression levels of members of class I (HDAC1, 2,, 3), class II (HDAC4, 5, 6, and 7), and class III (SIRT1, 2, 3, 4, 5, and 6) in a set of surgically resected pancreatic tissues. METHODS: Total RNA was isolated from 11 pancreatic adenocarcinomas (PA): stage 0 (n = 1), IB (n = 1), IIB (n = 6), III (n = 1), IV (n = 2), one serous cystadenoma (SC), one intraductal papillary mucinous tumor of the pancreas (IMPN), one complicating chronic pancreatitis (CP), and normal pancreas (NP) obtained during donor liver transplantation. Moreover, six other control pancreatic were included. HDACs gene expression was conducted using quantitative real-time polymerase chain reaction (qPCR). Protein expression levels were analyzed by Western blot and their localization by immunohistochemistry analyses of cancer tissues sections. RESULTS: Remarkably, 9 of the 11 PA (approximately 81%) showed significant increase of HDAC7 mRNA levels. In contrast to PA samples, message for HDAC7 was reduced in CP, SC, and IMPN specimens. The Western blot analysis showed increased expression of HDAC7 protein in 9 out of 11 PA samples, in agreement with the qPCR data. Most of the PA tissue sections examined showed intense labeling in the cytoplasm when reacted against antibodies to HDAC7. CONCLUSION: The data showed alteration of HDACs gene expression in pancreatic cancer. Increased expression of HDAC7 discriminates PA from other pancreatic tumors.

Live attenuated Leishmania vaccines: a potential strategic alternative.

Leishmaniasis causes significant morbidity and mortality worldwide, constituting an important public health problem. Leishmania infections cause a wide spectrum of diseases, ranging in severity from spontaneously healing skin lesions to fatal visceral disease. Attempts to develop an effective vaccine to control leishmaniasis have been shown to be feasible, but no vaccine is in active clinical use. The ability to create genetically modified parasites by eliminating virulence or essential genes is considered a powerful alternative in the development of an effective protective vaccine. Here, recent findings related to genetically defined live attenuated Leishmania parasites as promising vaccine candidates are reviewed.

Structure function analysis of Leishmania sirtuin: an ensemble of in silico and biochemical studies.

Novel anti-leishmanial target LmSir2 has few subtle but prudent structural differences in ligand binding and catalytic domain as compared to its human counterpart. In silico screening and validation followed by in vitro deacetylation and cell killing assays described herein give a proof of concept for development of strategies exploiting such minor differences for screening libraries of small molecules to identify selective inhibitors.

Histone deacetylase (HDAC) encoding gene expression in pancreatic cancer cell lines and cell sensitivity to HDAC inhibitors.

PURPOSE: Multiple biochemical and molecular alterations occur in pancreatic cancer cells. In the present study, attempts were made for the first time, to explore the level of expression of members of histone deacetylase encoding genes (HDACs) in four pancreatic tumor cell lines: Panc-1, BxPC-3, SOJ-6 and MiaPaCa-2; and two non-related tumor cells: Jurkat and HeLa. Furthermore, we examined the possible relationship between the levels of HDACs expression and the sensitivity/resistance to HDAC inhibitors (TSA, Nicotinamide and Sirtinol). RESULTS: We have found that although a slight variation in the profiles of gene expression among cell lines could be evidenced, HDACs protein synthesis seem to be similar. Furthermore, the cells were equally sensitive to inhibition by Sirtinol whereas some variation in the IC(50) could be seen in the case of TSA. We also demonstrate that the drugs had the capacity to induce the death of cells by apoptosis. METHODS: We have used four human pancreatic tumor cell lines and two-non related tumor cells, to evaluate the expression of HDAC encoding genes by RT-PCR and Western blot analysis. We also measured the effect of certain HDAC inhibitors (HDIs) on cell growth, cell cycle alteration, membrane phosphatidyl-serine exposure, DNA fragmentation and mitochondrial membrane potential loss. CONCLUSIONS: Taken together, our data support the notion that the level of cell sensitivity to the HDIs might be related to the level of expression of genes such as those encoding proteins playing a role in cell cycle checkpoints control but not HDAC per se.

Exploring the FL-160-CRP gene family through sequence variability of the complement regulatory protein (CRP) expressed by the trypomastigote stage of Trypanosoma cruzi.

The complement regulatory protein (CRP) of Trypanosoma cruzi is a surface glycoprotein which confers to the infectious trypomastigote forms a protection against the lytic activity of the host complement. CRP belongs to the large family of the trans-sialidase-like proteins and its sequence is highly similar to those of the flagellar FL-160 and chronic exoantigen proteins, encoded by a multigene family. To further define the gene family encoding the CRP, we investigated the protein diversity among several strains of T. cruzi through the sequencing of trypomastigote transcripts, and used a phylogenetic analysis based on the multiple alignment of these proteins with the top scoring sequences detected by a database sequence homology search. Intrastrain variations in CRP sequences revealed the existence of several copies per strain. The interstrain variability of CRP was consistent with the genetic subdivisions of T. cruzi into lineages and discrete typing units. The phylogenetic analysis based on a 227 amino acid alignment of CRP sequences with the 200 putative proteins retrieved from the protein databases (including the sequences from the T. cruzi genome project) revealed that the CRP sequences clustered with the FL-160 proteins into a monophyletic group characterized by the presence of the 12 amino acid mimicry epitope that mimics nervous tissues. The phylogeny did not differentiate between the CRP and the FL-160 proteins. The identification of this group of CRP-like proteins and the high sequence similarity observed within it open up new prospects for the exploration of the localization, structure and function of these proteins and a better understanding of their involvement in key aspects of host-parasite interactions, such as the resistance to the complement. This work provides also information for the T. cruzi genome annotation of the trans-sialidase-like putative proteins.

SIR2-deficient Leishmania infantum induces a defined IFN-gamma/IL-10 pattern that correlates with protection.

The ability to manipulate the Leishmania genome to create genetically modified parasites by introducing or eliminating genes is considered a powerful alternative for developing a new generation vaccine against leishmaniasis. Previously, we showed that the deletion of one allele of the Leishmania infantum silent information regulatory 2 (LiSIR2) locus was sufficient to dramatically affect amastigote axenic proliferation. Furthermore, LiSIR2 single knockout (LiSIR2(+/-)) amastigotes were unable to replicate in vitro inside macrophages. Because this L. infantum mutant persisted in BALB/c mice for up to 6 wk but failed to establish an infection, we tested its ability to provide protection toward a virulent L. infantum challenge. Strikingly, vaccination with a single i.p. injection of LiSIR2(+/-) single knockout elicits complete protection. Thus, vaccinated BALB/c mice showed a reversal of T cell anergy with specific anti-Leishmania cytotoxic activity and high levels of NO production. Moreover, vaccinated mice simultaneously generated specific anti-Leishmania IgG Ab subclasses suggestive of both type 1 and type 2 responses. A strong correlation was found between the elimination of the parasites and an increased Leishmania-specific IFN-gamma/IL-10 ratio. Therefore, we propose that the polarization to a high IFN-gamma/low IL-10 ratio after challenge is a clear indicator of vaccine success. Furthermore these mutants, which presented attenuated virulence, represent a good model to understand the correlatives of protection in visceral leishmaniasis.