Effect of sodium stibogluconate in recruiting and awakening immune cells in the pleural fluid of pancreatic cancer: preparation for immunotherapy.

Ascites and pleural effusion are recognized complications of pancreatic cancer. These diseases are accompanied by ascites and pleural effusion, and drug treatment is limited by high costs, long hospital stays, and failure rates. Immunotherapy may offer new option, but in most patients with late stages of cancer, immune cells may lose the ability to recognize tumor cells, how to activate their immune cells is a major problem, sodium glucosidate (SSG) is injected into ascites as a protein tyrosine phosphatase inhibitor to wake up immune cells and prepare for immunotherapy. We used single-cell RNA sequencing (scRNA-seq) to investigate whether and how SSG injected into ascites of pancreatic cancer elicits an immune response. Our study showed that the process of SSG fusion treatment of ascites and pleural effusion, the interaction between TandNK cells, MPs cells, monocytes and neutrophils was induced, and large numbers of genes were expressed, resulting in upregulation of immune response, which also approved that SSG is not only used as a protein tyrosine phosphatase inhibitor, but also it works as a protein tyrosine phosphatase inhibitor. It can also be used to regulate immune cell function, recruiting immune cells to the right place with the help of PD-1 or PD-L1 to fight cancer cells in ascites and pleural effusions in cancer patients.

Antileishmanial efficacy and tolerability of combined treatment with non-ionic surfactant vesicle formulations of sodium stibogluconate and paromomycin in dogs.

Infection with Leishmania infantum causes the disease visceral leishmaniasis (VL), which is a serious clinical and veterinary problem. The drugs used to treat canine leishmaniasis (CanL) do not cause complete parasite clearance; they can be toxic, and emerging drug resistance in parasite populations limits their clinical utility. Therefore, in this study we have evaluated the toxicity and efficacy of joint treatment with a 1:1 mixture of sodium stibogluconate-NIV (SSG-NIV, 10 mg Sbv/day) and paromomycin-NIV (PMM-NIV, 10 mg PMM/kg/day), given intravenously daily for seven days from day 270 post-infection, to nine-month-old female beagle dogs (n = 6) experimentally infected with Leishmania infantum. Treatment significantly improved the clinical symptoms of VL infection in all the treated dogs, reduced parasite burdens in lymph nodes and bone marrow, and all symptomatic treated dogs, were asymptomatic at 90 days post-treatment. Treatment was associated with a progressive and significant decrease in specific IgG anti-Leishmania antibodies using parasite soluble antigen (p < 0.01) or rK39 (p < 0.01) as the target antigen. In addition, all dogs were classified as parasite negative based on Leishmania nested PCR and quantitative real time PCR tests and as well as an inability to culture of promastigote parasites from lymph nodes and bone marrow tissue samples taken at day 90 post-treatment. However, treatment did not cure the dogs as parasites were detected at 10 months post-treatment, indicating that a different dosing regimen is required to cause long term cure or prevent relapse.

Immunomodulator mediated changes in plasma membrane calcium ATPase in controlling visceral leishmaniasis.

Immunomodulation is an emerging concept to combat infection in recent years. Immunomodulators like arabinosylated-lipoarabinomannan (Ara-LAM) and glycyrrhizic-acid (GA) possess anti-leishmanial property, whereas sodium-antimony-gluconate (SAG) is still considered as the first choice for chemotherapy against leishmaniasis. During infection, invasion of Leishmania donovani needs the potential requirement of Ca2+, which is further responsible for apoptosis in intracellular amastigotes. However, suppression of elevated intracellular calcium by the activation of plasma-membrane-calcium-ATPase (PMCA4) facilitates survival of L. donovani in the host. In the present study, SAG, Ara-LAM, and GA were found to evoke significant increase in intracellular Ca2+ in L. donovani infected macrophages by inhibiting PMCA4. Moreover, PMCA4 inhibition by TFP or PMCA4 siRNA elevated the level of PKCß, whereas calcium-independent upregulation of PKCζ remained unchanged in infected macrophages. Furthermore, application of immunomodulators in infected macrophages resulted in down-regulation of PKCζ, conversion of anti-inflammatory to pro-inflammatory cytokines and inhibition of PMCA4. Plasma membrane-associated ceramide which is known to be elevated during leishmaniasis, triggered upregulation of PMCA4 via PKCζ activation. Interestingly, immunomodulators attenuated ceramide generation, which resulted into reduced PKCζ activation leading to the decreased PMCA expression in infected macrophages. Therefore, our study elucidated the efficacy of SAG, Ara-LAM, and GA in the reduction of parasite burden in macrophages by suppressing PMCA activation through inhibition of ceramide mediated upregulation of PKCζ.

Chemogenomic Profiling of Antileishmanial Efficacy and Resistance in the Related Kinetoplastid Parasite Trypanosoma brucei.

The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited and beset by toxicity and emergent resistance. Furthermore, our understanding of drug mode of action and potential routes to resistance is limited. Forward genetic approaches have revolutionized our understanding of drug mode of action in the related kinetoplastid parasite Trypanosoma brucei Therefore, we screened our genome-scale T. brucei RNA interference (RNAi) library against the current antileishmanial drugs sodium stibogluconate (antimonial), paromomycin, miltefosine, and amphotericin B. Identification of T. brucei orthologues of the known Leishmania antimonial and miltefosine plasma membrane transporters effectively validated our approach, while a cohort of 42 novel drug efficacy determinants provides new insights and serves as a resource. Follow-up analyses revealed the antimonial selectivity of the aquaglyceroporin TbAQP3. A lysosomal major facilitator superfamily transporter contributes to paromomycin-aminoglycoside efficacy. The vesicle-associated membrane protein TbVAMP7B and a flippase contribute to amphotericin B and miltefosine action and are potential cross-resistance determinants. Finally, multiple phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania miltefosine transporter, a putative ß-subunit/CDC50 cofactor, and additional membrane-associated hits, affect amphotericin B efficacy, providing new insights into mechanisms of drug uptake and action. The findings from this orthology-based chemogenomic profiling approach substantially advance our understanding of antileishmanial drug action and potential resistance mechanisms and should facilitate the development of improved therapies as well as surveillance for drug-resistant parasites.

Decanethiol functionalized silver nanoparticles are new powerful leishmanicidals in vitro.

We evaluated, for the first time, the leishmanicidal potential of decanethiol functionalized silver nanoparticles (AgNps-SCH) on promastigotes and amastigotes of different strains and species of Leishmania: L. mexicana and L. major isolated from different patients suffering from localized cutaneous leishmaniasis (CL) and L. mexicana isolated from a patient suffering from diffuse cutaneous leishmaniasis (DCL). We recorded the kinetics of promastigote growth by daily parasite counting for 5 days, promastigote mobility, parasite reproduction by CFSE staining's protocol and promastigote killing using the propidium iodide assay. We also recorded IC50's of promastigotes and amastigotes, therapeutic index, and cytotoxicity by co-culturing macrophages with AgNps-SCH or sodium stibogluconate (Sb) used as reference drug. We used Sb as a reference drug since it is used as the first line treatment for all different types of leishmaniasis. At concentrations 10,000 times lower than those used with Sb, AgNps-SCH had a remarkable leishmanicidal effect in all tested strains of parasites and there was no toxicity to J774A.1 macrophages since > 85% were viable at the concentrations used. Therapeutic index was about 20,000 fold greater than the corresponding one for Sb treated cells. AgNps-SCH inhibited > 80% promastigote proliferation in all tested parasites. These results demonstrate there is a high leishmanicidal potential of AgNps-SCH at concentrations of 0.04 µM. Although more studies are needed, including in vivo testing of AgNps-SCH against different types of leishmaniasis, they can be considered a potential new treatment alternative.

Integrating genomics and proteomics permits identification of immunodominant antigens associated with drug resistance in human visceral leishmaniasis in India.

Resistance of human pathogens like Leishmania to drugs is a growing concern where the multidrug-resistant phenotype renders chemotherapy ineffective. The acquired resistance of Leishmania to antimony has promoted intense research on the mechanisms involved but the question has not been resolved yet. In this study we have explored host-pathogen- drug interactions leading to identification of pharmacological determinants of host macrophages that resist the sodium antimony gluconate (SAG) mediated intracellular parasite killing. mRNA profiling of mammalian host stage amastigotes of sodium antimony gluconate (SAG) 'sensitive' and 'resistant' parasite lines was carried out using Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array. Patient sera was used to identify immunogenic proteins by two-dimensional gel analysis (2DE) and mass spectrometric analysis (LC-MS/MS). Immunofluorescence microscopy confirmed the identities on 'sensitive' and 'resistant' parasite lines. A total of nine immunogenic proteins whose intensities changed significantly and consistently in multiple experiments were detected, suggesting that a cohort of proteins are altered in expression levels in the 'resistant' parasites. Global expression profiling using microarrays revealed this regulation was not reflected by changes in the levels of the cognate mRNAs. Following identification of proteins by mass spectrometry, one such regulated protein, enolase, was chosen for more detailed analysis. Immunofluorescence microscopy employing antisera against this enzyme confirmed that its level was differentially regulated in the 'resistant' isolate. We show that high serum level of immunoreactive protein is associated with 'resistant' phenotype. Differentially expressed proteins with immunomodulatory activities were found to be associated with the 'resistant phenotype'.

Antimonial drugs entrapped into phosphatidylserine liposomes: physicochemical evaluation and antileishmanial activity

Abstract: INTRODUCTION: Leishmaniasis is a disease caused by the protozoan Leishmania that resides mainly in mononuclear phagocytic system tissues. Pentavalent antimonials are the main treatment option, although these drugs have toxic side effects and high resistance rates. A potentially alternative and more effective therapeutic strategy is to use liposomes as carriers of the antileishmanial agents. The aims of this study were to develop antimonial drugs entrapped into phosphatidylserine liposomes and to analyze their biological and physicochemical characteristics. METHODS: Liposomes containing meglumine antimoniate (MA) or pentavalent antimony salt (Sb) were obtained through filter extrusion (FEL) and characterized by transmission electron microscopy. Promastigotes of Leishmania infantum were incubated with the drugs and the viability was determined with a tetrazolium dye (MTT assay). The effects of these drugs against intracellular amastigotes were also evaluated by optical microscopy, and mammalian cytotoxicity was determined by an MTT assay. RESULTS: Liposomes had an average diameter of 162nm. MA-FEL showed inhibitory activity against intracellular L. infantum amastigotes, with a 50% inhibitory concentration (IC50) of 0.9μg/mL, whereas that of MA was 60μg/mL. Sb-FEL showed an IC50 value of 0.2μg/mL, whereas that of free Sb was 9μg/mL. MA-FEL and Sb-FEL had strong in vitro activity that was 63-fold and 39-fold more effective than their respective free drugs. MA-FEL tested at a ten-times higher concentration than Sb-FEL did not show cytotoxicity to mammalian cells, resulting in a higher selectivity index. CONCLUSIONS: Antimonial drug-containing liposomes are more effective against Leishmania-infected macrophages than the non-liposomal drugs.

In-vitro sensitivity of Pakistani Leishmania tropica field isolate against buparvaquone in comparison to standard anti-leishmanial drugs.

In this study, in vitro anti-leishmanial activity of buparvaquone was evaluated against promastigotes and intracellular amastigotes of Pakistani Leishmania tropica isolate KWH23 in relation to the current standard chemotherapy for leishmaniasis (sodium stibogluconate, sodium stibogluconate, amphotericin B and miltefosine). For buparvaquone, mean % inhibition in intracellular amastigotes at four different concentrations (1.35 µM, 0.51 µM, 0.17 µM and 0.057 µM) was 78%, 44%, 20% and 14% respectively, whereas, against promastigotes it was 89%, 77%, 45% and 35% respectively. IC50 values calculated to estimate the anti-leishmanial activity of buparvaquone against intra-cellular amastigotes and promastigotes was 0.53 µM (95% C.I. = 0.32-0.89) and 0.15 µM (95% C.I. = 0.01-1.84) respectively. Amphotericin B was the most potent in-vitro drug tested, with an IC50 of 0.075 µM (95% C.I. = 0.006-0.907) against promastigotes, and 0.065 µM (95% C.I. = 0.048-0.089) against intra-cellular amastigotes. Amphotericin B was more cytotoxic against THP1 cells, with an IC50 of 0.15 µM (95% C.I. = 0.01-0.95) and an apparent in-vitro therapeutic index of 2.0, than was buparvaquone, with an IC50 of 12.03 µM (95% C.I. = 5.36-26.96) against THP1 cells and a therapeutic index of 80.2. The study proposes that buparvaquone may be further investigated as a candidate drug for treatment of cutaneous leishmaniasis.

Cationic liposomal sodium stibogluconate (SSG), a potent therapeutic tool for treatment of infection by SSG-sensitive and -resistant Leishmania donovani.

Pentavalent antimonials have been the first-line treatment for leishmaniasis for decades. However, the development of resistance to sodium stibogluconate (SSG) has limited its use, especially for treating visceral leishmaniasis (VL). The present work aims to optimize a cationic liposomal formulation of SSG for the treatment of both SSG-sensitive (AG83) and SSG-resistant (GE1F8R and CK1R) Leishmania donovani infections. Parasite killing was determined by the 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) assay and microscopic counting of Giemsa-stained macrophages. Macrophage uptake studies were carried out by confocal microscopic imaging. Parasite-liposome interactions were visualized through transmission electron microscopy. Toxicity tests were performed using assay kits. Organ parasite burdens were determined by microscopic counting and limiting dilution assays. Cytokines were measured by enzyme-linked immunosorbent assays (ELISAs) and flow cytometry. Although all cationic liposomes studied demonstrated leishmanicidal activity, phosphatidylcholine (PC)-dimethyldioctadecylammonium bromide (DDAB) vesicles were most effective, followed by PC-stearylamine (SA) liposomes. Since entrapment of SSG in PC-DDAB liposomes demonstrated enhanced ultrastructural alterations in promastigotes, PC-DDAB-SSG vesicles were further investigated in vitro and in vivo. PC-DDAB-SSG could effectively alleviate SSG-sensitive and SSG-resistant L. donovani infections in the liver, spleen, and bone marrow of BALB/c mice at a dose of SSG (3 mg/kg body weight) not reported previously. The parasiticidal activity of these vesicles was attributed to better interactions with the parasite membranes, resulting in direct killing, and generation of a strong host-protective environment, necessitating a very low dose of SSG for effective cures.

Chemotherapy of leishmaniasis part XIII: design and synthesis of novel heteroretinoid-bisbenzylidine ketone hybrids as antileishmanial agents.

Some novel heteroretinoid-bisbenzylidine ketone hybrids have been synthesized and evaluated for their in vitro antileishmanial activity against intramacrophagic amastigotes of Leishmania donovani. Among all the nine synthetic compounds, five compounds (7c, 7d and 7f-h) have shown significant (less than 7µM) activity against intramacrophagic amastigotes. The IC50 values of these compounds were found better than the reference drugs sodium stibogluconate (SSG) and miltefosine. This study helped us in identifying the new class of compounds that could be exploited as potent antileishmanial agents.

Imipramine exploits histone deacetylase 11 to increase the IL-12/IL-10 ratio in macrophages infected with antimony-resistant Leishmania donovani and clears organ parasites in experimental infection.

The efflux of antimony through multidrug resistance protein (MDR)-1 is the key factor in the failure of metalloid treatment in kala-azar patients infected with antimony-resistant Leishmania donovani (Sb(R)LD). Previously we showed that MDR-1 upregulation in Sb(R)LD infection is IL-10-dependent. Imipramine, a drug in use for the treatment of depression and nocturnal enuresis in children, inhibits IL-10 production from Sb(R)LD-infected macrophages (Sb(R)LD-Mϕs) and favors accumulation of surrogates of antimonials. It inhibits IL-10-driven nuclear translocation of c-Fos/c-Jun, critical for enhanced MDR-1 expression. The drug upregulates histone deacetylase 11, which inhibits acetylation of IL-10 promoter, leading to a decrease in IL-10 production from Sb(R)LD-Mϕs. It abrogates Sb(R)LD-mediated p50/c-Rel binding to IL-10 promoter and preferentially recruits p65/RelB to IL-12 p35 and p40 promoters, causing a decrease in IL-10 and overproduction of IL-12 in Sb(R)LD-Mϕs. Histone deacetylase 11 per se does not influence IL-12 promoter activity. Instead, a imipramine-mediated decreased IL-10 level allows optimal IL-12 production in Sb(R)LD-Mϕs. Furthermore, exogenous rIL-12 inhibits intracellular Sb(R)LD replication, which can be mimicked by the presence of Ab to IL-10. This observation indicated that reciprocity exists between IL-10 and IL-12 and that imipramine tips the balance toward an increased IL-12/IL-10 ratio in Sb(R)LD-Mϕs. Oral treatment of infected BALB/c mice with imipramine in combination with sodium stibogluconate cleared organ Sb(R)LD parasites and caused an expansion of the antileishmanial T cell repertoire where sodium stibogluconate alone had no effect. Our study deciphers a detailed molecular mechanism of imipramine-mediated regulation of IL-10/IL-12 reciprocity and its impact on Sb(R)LD clearance from infected hosts.

Studies on the protective efficacy of second-generation vaccine along with standard antileishmanial drug in Leishmania donovani infected BALB/c mice.

It is well established that visceral leishmaniasis (VL; also known as Kala azar) causes immunosuppression, and a successful drug treatment is associated with the development of cell-mediated immunity. Therefore combining a drug with an immune enhancer can provide a better approach for the treatment of the disease. Keeping this in mind, the in vivo antileishmanial efficacy of immunochemotherapy was evaluated with the use of a 78 kDa antigen with or without monophosphoryl lipid A (MPL-A) along with a traditional drug sodium stibogluconate (SSG) in Leishmania donovani infected BALB/c mice. Mice were infected intracardially with promastigotes of L. donovani, and 30 days after infection, these animals were given specific immunotherapy (78 kDa/78 kDa+MPL-A) or chemotherapy (SSG) or immunochemotherapy (SSG+78 kDa/SSG+78 kDa+MPL-A). Animals were euthanased on 1, 15 and 30 post-treatment days. The antileishmanial potential of the immunochemotherapy was revealed by significant reduction in the parasite burden (P<0·001). These animals were also found to exhibit increased delayed type hypersensitivity (DTH) responses, higher IgG2a levels, lower IgG1 levels and greater cytokine (IFN-γ and IL-2) concentrations compared with chemotherapy or immunotherapy alone, pointing towards the generation of a strong protective (Th1) type of immune response. Immunochemotherapy with SSG+78 kDa+MPL-A was found to be most effective in protecting mice against VL and therefore can be an alternative option for treatment of VL.

Characterization of the proliferating cell nuclear antigen of Leishmania donovani clinical isolates and its association with antimony resistance.

Previously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate of Leishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance in L. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by ≥ 3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ~5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate. L. donovani PCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate of L. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (Sb(V)) and trivalent antimonial (Sb(III)) drugs was assessed in vitro against promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of Sb(V) (from 41.2 ± 0.6 µg/ml to 66.5 ± 3.9 µg/ml) and Sb(III) (from 24.0 ± 0.3 µg/ml to 43.4 ± 1.8 µg/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon Sb(III) treatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance in L. donovani clinical isolates, which warrants detailed investigations regarding its mechanism.

Increased parasite surface antigen-2 expression in clinical isolates of Leishmania donovani augments antimony resistance.

Resistance to sodium antimony gluconate (SAG) is a major cause of therapeutic failure in a large proportion of visceral leishmaniasis (VL) cases. Determinants of SAG resistance have been widely studied; however, the mechanism operating in clinical isolates is poorly understood. In the present study, expression of parasite surface antigen-2 (PSA-2) gene was studied in clinical isolates of Leishmania donovani comprising of antimony resistant (n=10) and sensitive (n=4) parasites. The expression of PSA-2 gene was found to be consistently high in SAG resistant clinical isolates (≥1.5-fold) at both transcript and protein level. Further, over-expression of PSA-2 in L. donovani isolates (LdPSA-2(++)) resulted in conversion of SAG sensitive phenotype to resistant. The LdPSA-2(++) parasites showed significantly decreased susceptibility towards SAG (>12-fold), amphotericin B (>4-fold) and miltefosine (>2.5-fold). Marked decrease in antimony accumulation and enhanced tolerance towards complement mediated lysis was evident in LdPSA-2(++) parasites. The study established the role of PSA-2 gene in SAG resistance and its potential as a biomarker to distinguish resistant and sensitive clinical isolates of L. donovani.

In vivo and in vitro antileishmanial activity of Bungarus caeruleus snake venom through alteration of immunomodulatory activity.

Leishmaniasis threatens more than 350 million people worldwide specially in tropical and subtropical region. Antileishmanial drugs that are currently available have various limitations. The search of new drugs from natural products (plants, animals) possessing antileishmanial activity is ventured throughout the world. The present study deals with the antileishmanial activity of Bungarus caeruleus snake venom (BCV) on in vitro promastigotes and amastigotes of Leishmania donovani parasite and leishmania infected BALB/c mice. The effect of BCV on peritoneal macrophage, release of cytokines from the activated macrophages, production of nitric oxide, reactive oxygen species and cytokines were studied in vivo and in vitro. IC50 value of BCV on L. donovani promastigote was 14.5 µg/ml and intracellular amastigote was 11.2 µg/ml. It activated peritoneal macrophages, significantly increased cytokines and interleukin production. BCV (20 µg/kg and 40 µg/kg body weight of mice) decreased parasite count by 54.9% and 74.2% in spleen and 41.4% and 60.4% in liver of infected BALB/c mice. BCV treatment significantly increased production of TNF-α, IFN-γ, ROS, NO in infected mice. Histological studies showed decreased granuloma formation in treated liver as compared with control. Liver and spleen structure was partially restored due to BCV treatment in infected mice. The present study revealed that BCV possessed antileishmanial activity against L. donovani parasite in vivo and in vitro and this activity was partly mediated through immunomodulatory activity involving macrophages.

The endothelial tyrosine phosphatase SHP-1 plays an important role for vascular haemostasis in TNFα -induced inflammation in vivo.

INTRODUCTION: Inflammation and endothelium-derived superoxides are important pathomechanisms in atherothrombotic diseases. We could previously show that the tyrosine phosphatase SHP-1 acts as a negative regulator in endothelial superoxide production. In this study we investigated the influence of SHP-1 on platelet-endothelium interaction and arterial thrombosis in TNFα -induced endothelial inflammation in vivo. METHODS: Arteriolar thrombosis and platelet rolling in vivo were investigated in C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. RESULTS: Inhibition of SHP-1 by the specific pharmacological inhibitor sodium stibogluconate did not significantly enhance platelet-endothelium interaction in vivo under physiological conditions but led to an augmented fraction of rolling platelets in TNFα -induced systemic inflammation. Accordingly, ferric-chloride-induced arteriolar thrombus formation, which was already increased by SHP-1 inhibition, was further enhanced in the setting of TNFα -induced inflammation. Platelet aggregation in vitro as well as ex vivo was not influenced by SHP-1-inhibition. In cultured endothelial cells, sodium stibogluconate increased TNFα -induced surface expression of p-selectin and von Willebrand factor. Additionally, TNFα increased SHP-1 activity and protein expression. CONCLUSIONS: The endothelial tyrosine phosphatase SHP-1 plays an important role for vascular hemostasis in vivo, which is crucial in TNF α -induced endothelial inflammation where it may serve as an autoinhibitory molecule to prevent excess inflammatory response and thrombus formation.

A new ABC half-transporter in Leishmania major is involved in resistance to antimony.

The characterization of ABCI4, a new intracellular ATP-binding cassette (ABC) half-transporter in Leishmania major, is described. We show that ABCI4 is involved in heavy metal export, thereby conferring resistance to Pentostam, to Sb(III), and to As(III) and Cd(II). Parasites overexpressing ABCI4 showed a lower mitochondrial toxic effect of antimony by decreasing reactive oxygen species production and maintained higher values of both the mitochondrial electrochemical potential and total ATP levels with respect to controls. The ABCI4 half-transporter forms homodimers as determined by a coimmunoprecipitation assay. A combination of subcellular localization studies under a confocal microscope and a surface biotinylation assay using parasites expressing green fluorescent protein- and FLAG-tagged ABCI4 suggests that the transporter presents a dual localization in both mitochondria and the plasma membrane. Parasites overexpressing ABCI4 present an increased replication in mouse peritoneal macrophages. We have determined that porphyrins are substrates for ABCI4. Consequently, the overexpression of ABCI4 confers resistance to some toxic porphyrins, such as zinc-protoporphyrin, due to the lower accumulation resulting from a significant efflux, as determined using the fluorescent zinc-mesoporphyrin, a validated heme analog. In addition, ABCI4 has a significant ability to efflux thiol after Sb(III) incubation, thus meaning that ABCI4 could be considered to be a potential thiol-X-pump that is able to recognize metal-conjugated thiols. In summary, we have shown that this new ABC transporter is involved in drug sensitivity to antimony and other compounds by efflux as conjugated thiol complexes.

SHP-1 phosphatase activity counteracts increased T cell receptor affinity.

Anti-self/tumor T cell function can be improved by increasing TCR-peptide MHC (pMHC) affinity within physiological limits, but paradoxically further increases (K(d) < 1 µM) lead to drastic functional declines. Using human CD8(+) T cells engineered with TCRs of incremental affinity for the tumor antigen HLA-A2/NY-ESO-1, we investigated the molecular mechanisms underlying this high-affinity-associated loss of function. As compared with cells expressing TCR affinities generating optimal function (K(d) = 5 to 1 µM), those with supraphysiological affinity (K(d) = 1 µM to 15 nM) showed impaired gene expression, signaling, and surface expression of activatory/costimulatory receptors. Preferential expression of the inhibitory receptor programmed cell death-1 (PD-1) was limited to T cells with the highest TCR affinity, correlating with full functional recovery upon PD-1 ligand 1 (PD-L1) blockade. In contrast, upregulation of the Src homology 2 domain-containing phosphatase 1 (SHP-1/PTPN6) was broad, with gradually enhanced expression in CD8(+) T cells with increasing TCR affinities. Consequently, pharmacological inhibition of SHP-1 with sodium stibogluconate augmented the function of all engineered T cells, and this correlated with the TCR affinity-dependent levels of SHP-1. These data highlight an unexpected and global role of SHP-1 in regulating CD8(+) T cell activation and responsiveness and support the development of therapies inhibiting protein tyrosine phosphatases to enhance T cell-mediated immunity.

TTAS a new stilbene derivative that induces apoptosis in Leishmania infantum.

Leishmania parasites are able to undergo apoptosis (programmed cell death), similarly to mammalian cells. Recently it was demonstrated in vitro the anti-leishmanial effect of some natural and synthetic stilbenoids including resveratrol and piceatannol. In this study we evaluated the Leishmanicidal activity of a pool of stilbene derivatives which had previously shown high apoptotic efficacy against neoplastic cells. All the compounds tested were capable to decrease the parasite viability in a dose-dependent manner. Trans-stilbenes proved to be markedly more effective than cis-isomers. This was different from that observed in tumor cells in which cis-stilbenes were more potent cytotoxic agents. Trans-3,4',5-trimethoxy-3'-amino-stilbene (TTAS) was the most active stilbene showing in Leishmania infantum a LD(50) value of 2.6 µg/mL. In contrast TTAS showed a low toxicity when tested on normal hemopoietic cells. This compound induced apoptosis in parasites by disrupting the mitochondrial membrane potential. Moreover it shows the ability to block Leishmania parasites in G(2)-M phase of cell cycle in agreement with the data obtained by affinity chromatography that identify tubulin as the putative target of TTAS. In conclusion, our results indicate that some stilbene derivatives are highly effective as anti-leishmanial agents and TTAS represents a pro-apoptotic agent in Leishmania parasites that merit further in vivo investigation.

Increased metacyclogenesis of antimony-resistant Leishmania donovani clinical lines.

Mathematical models predict that the future of epidemics of drug-resistant pathogens depends in part on the competitive fitness of drug-resistant strains. Considering metacyclogenesis (differentiation process essential for infectivity) as a major contributor to the fitness of Leishmania donovani, we tested its relationship with pentavalent antimony (SbV) resistance in clinical lines. Different methods for the assessment of metacyclogenesis were cross-validated: gene expression profiling (META1 and SHERP), morphometry (microscopy and FACS), in vitro infectivity to macrophages and resistance to complement lysis. This was done on a model constituted by 2 pairs of reference strains cloned from a SbV-resistant and -sensitive isolate. We selected the most adequate parameter and extended the analysis of metacyclogenesis diversity to a sample of 20 clinical lines with different in vitro susceptibility to the drug. The capacity of metacyclogenesis, as measured by the complement lysis test, was shown to be significantly higher in SbV-resistant clinical lines of L. donovani than in SbV-sensitive lines. Together with other lines of evidence, it is concluded that L. donovani constitutes a unique example and model of drug-resistant pathogens with traits of increased fitness. These findings raise a fundamental question about the potential risks of selecting more virulent pathogens through massive chemotherapeutic interventions.