Imipramine Is an Orally Active Drug against Both Antimony Sensitive and Resistant Leishmania donovani Clinical Isolates in Experimental Infection

Background: In an endeavor to find an orally active and affordable antileishmanial drug, we tested the efficacy of a cationic amphiphilic drug, imipramine, commonly used for the treatment of depression in humans. The only available orally active antileishmanial drug is miltefosine with long half life and teratogenic potential limits patient compliance. Thus there is a genuine need for an orally active antileishmanial drug. Previously it was shown that imipramine, a tricyclic antidepressant alters the protonmotive force in promastigotes, but its in vivo efficacy was not reported. Methodology/Principal Findings: Here we show that the drug is highly active against antimony sensitive and resistant Leishmania donovani in both promastigotes and intracellular amastigotes and in LD infected hamster model. The drug wasfound to decrease the mitochondrial transmembrane potential of Leishmania donovani (LD) promastigotes and purified amastigotes after 8 h of treatment, whereas miltefosine effected only a marginal change even after 24 h. The drug restores defective antigen presenting ability of the parasitized macrophages. The status of the host protective factors TNF a, IFN c and iNOS activity increased with the concomitant decrease in IL 10 and TGF b level in imipramine treated infected hamsters and evolution of matured sterile hepatic granuloma. The 10-day therapeutic window as a monotherapy, showing about 90% clearance of organ parasites in infected hamsters regardless of their SSG sensitivity. Conclusions: This study showed that imipramine possibly qualifies for a new use of an old drug and can be used as an effective orally active drug for the treatment of Kala-azar

KMP-11 DNA Immunization Significantly Protects against L. Donovani Infection but Requires Exogenous IL-12 as an Adjuvant for Comparable Protection against L. major

As vaccine potential of cross-species protection by a candidate antigen is less explored, in this study we compared cross-specific protective efficacy of KinetoplastidMembrane Protein-11 (KMP-11) as aDNAvaccine alone and in conjunction with exogenous IL-12 administration in experimental BALB/c model against two most widely prevalent forms of clinical diseases caused by Leishmania major (LM) and Leishmania donovani (LD). Whereas, KMP-11 DNA vaccination alone showed significant potential in terms of resolution of splenic and hepatic parasite burden against virulent LD challenge, it showed considerably less efficacy (<70% reduction) against virulent LM challenge in terms of presence of parasite in lymph node. Remarkably exogenous IL-12 administration in the form of IL-12 p35/p40 expression vectors or recombinant protein along with KMP-11 DNA had exactly opposing effect on protection against LM and LD. Exogenous IL-12 administration significantly increased residual LD-burden but enhanced the protective efficacy ofKMP-11DNAvaccine against LMcompared toKMP-11 immunization alone. Elucidation of effector mechanism showed KMP-11 DNA induced protection against LDwas associated with the generation of mixed Th1/Th2 response, while KMP-11/IL-12-induced comparable protection against LMwas associated with high IgG2a titre indicative of a polarized Th1 response. Exogenous IL-12 administration resulted in robust gamma interferon (IFN-�) production and suppression of IL-4 from CD4+ T cell against both LM and LD. Nevertheless protective immune responsewas only compromised against LD infection where frequency of anti-KMP-11 CTL responsewas significantly reduced after exogenous IL-12 administration. Our study provides a comparative evaluation of effector mechanisms in the assessment of cross-specific protection by KMP-11 and KMP-11/IL-12 immunization against these two prevalent forms of leishmaniasis

TLR4-mediated activation of MyD88 signaling induces protective immune response and IL-10 down-regulation in <i style="mso-bidi-font-style:normal">Leishmania donovani</i> infection

531-541In visceral leishmaniasis, a fragmentary IL-12 driven type 1 immune response along with the expansion of IL-10 producing T-cells correlates with parasite burden and pathogenesis. Successful immunotherapy involves both suppression of IL-10 production and enhancement of IL-12 and nitric oxide (NO) production. As custodians of the innate immunity, the toll-like receptors (TLRs) constitute the first line of defense against invading pathogens. The TLR-signaling cascade initiated following innate recognition of microbes shapes the adaptive immune response. Whereas numerous studies have correlated parasite control to the adaptive response in Leishmania infection, growing body of evidence suggests that the activation of the innate immune response also plays a pivotal role in disease pathogenicity. In this study, using a TLR4 agonist, a Leishmania donovani (LD) derived 29 kDa β 1,4 galactose terminal glycoprotein (GP29), we demonstrated that the TLR adaptor myeloid differentiation primary response protein-88 (MyD88) was essential for optimal immunity following LD infection. Treatment of LD-infected cells with GP29 stimulated the production of IL-12 and NO while suppressing IL-10 production. Treatment of <i style="mso-bidi-font-style: normal">LD-infected cells with GP29 also induced the degradation of IKB and the nuclear translocation of NF-<span style="font-family:Symbol; mso-ascii-font-family:" times="" new="" roman";mso-hansi-font-family:"times="" roman";="" mso-ansi-language:en-in;mso-char-type:symbol;mso-symbol-font-family:symbol"="">kB, as well as rapid phosphorylation of p38 MAPK and p54/56 JNK. <span style="mso-ansi-language: EN-IN">Knockdown of TLR4 or MYD88 using siRNA showed reduced inflammatory response to GP29 in LD-infected cells. Biochemical inhibition of p38 MAPK, JNK or NF-<span style="font-family:Symbol; mso-ascii-font-family:" times="" new="" roman";mso-hansi-font-family:"times="" roman";="" mso-ansi-language:en-in;mso-char-type:symbol;mso-symbol-font-family:symbol"="">kB, but not p42/44 ERK, reduced GP29-induced IL-12 and NO production in LD-infected cells. These results suggested a potential role for the TLR4-MyD88–IL-12 pathway to induce adaptive immune responses to LD infection that culminated in an effective control of intracellular parasite replication. </span

Kinetoplastid Membrane Protein-11 DNA Vaccination Induces Complete Protection against Both Pentavalent Antimonial-Sensitive and -Resistant Strains of Leishmania donovani That Correlates with Inducible Nitric Oxide Synthase Activity and IL-4 Generation: Evidence for Mixed Th1- and Th2-Like Responses in Visceral Leishmaniasis1

The emergence of an increasing number of Leishmania donovani strains resistant to pentavalent antimonials (SbV), the first line of treatment for visceral leishmaniasis worldwide, accounts for decreasing efficacy of chemotherapeutic interventions. A kinetoplastid membrane protein-11 (KMP-11)-encoding construct protected extremely susceptible golden hamsters from both pentavalent antimony responsive (AG83) and antimony resistant (GE1F8R) virulent L. donovani challenge. All the KMP-11 DNA vaccinated hamsters continued to survive beyond 8 mo postinfection, with the majority showing sterile protection. Vaccinated hamsters showed reversal of T cell anergy with functional IL-2 generation along with vigorous specific anti-KMP-11 CTL-like response. Cytokines known to influence Th1- and Th2-like immune responses hinted toward a complex immune modulation in the presence of a mixed Th1/Th2 response in conferring protection against visceral leishmaniasis. KMP-11 DNA vaccinated hamsters were protected by a surge in IFN-�, TNF-�, and IL-12 levels along with extreme down-regulation of IL-10. Surprisingly the prototype candidature of IL-4, known as a disease exacerbating cytokine, was found to have a positive correlation to protection. Contrary to some previous reports, inducible NO synthase was actively synthesized by macrophages of the protected hamsters with concomitant high levels of NO production. This is the first report of a vaccine conferring protection to both antimony responsive and resistant Leishmania strains reflecting several aspects of clinical visceral leishmaniasis

Sub-Optimal dose of Sodium Antimony Gluconate (SAG)-Diperoxovanadate Combination Clears Organ Parasites from BALB/c Mice Infected with Antimony Resistant Leishmania Donovani by Expanding Antileishmanial T-Cell Repertoire and Increasing IFN-c to IL-10 Ratio

We demonstrate that the combination of sub-optimal doses of Sodium Antimony Gluconate (SAG) and the diperoxovanadate compound K[VO(O2)2(H2O)], also designated as PV6, is highly effective in combating experimental infection of BALB/c mice with antimony resistant (SbR) Leishmania donovani (LD) as evident from the significant reduction in organ parasite burden where SAG is essentially ineffective. Interestingly, such treatment also allowed clonal expansion of antileishmanial T-cells coupled with robust surge of IFN-c and concomitant decrease in IL-10 production. The splenocytes from the treated animals generated significantly higher amounts of IFN-c inducible parasiticidal effector molecules like superoxide and nitric oxide as compared to the infected group. Our study indicates that the combination of sub-optimal doses of SAG and PV6 may be beneficial for the treatment of SAG resistant visceral leishmaniasis patients

Class II MHC/Peptide Interaction in Leishmania donovani Infection: Implications in Vaccine Design

We show that Leishmania donovani–infectedmacrophages (MFs) are capable of stimulatingMHC class II (MHC-II)–restricted T cells at 6 h of infection. At 48 h, infectedMFs (I-MFs) failed to stimulateMHC-II–restricted T cells but notMHC class I–restricted ones, in contrast to normalMFs. Such I-MFs could stimulate T cells at a higher Ag concentration, indicating that general Ag processing and trafficking of peptide–MHC-II complexes are not defective. Analysis of the kinetic parameters, like “kon” and “koff,” showed that peptide–MHC-II complex formation is compromised in I-MFs comparedwith normal MFs. This indicates interference in loading of the cognate peptide to MHC-II, which may be due to the presence of a noncognate molecule. This notion received support from the finding that exposure of I-MFs to low pH or treatment with 2-(1-adamantyl)-ethanol, a molecule that favors peptide exchange, led to T cell activation. When treated with 2-(1-adamantyl)-ethanol, splenocytes from 8 wk–infected BALB/c mice showed significantly higher antileishmanial T cell expansion in vitro compared with untreated controls. Hence, it is tempting to speculate that high, but not low, concentrations of cognate peptide may favor peptide exchange in I-MFs, leading to expansion of the antileishmanial T cell repertoire. The results suggest that a high Ag dose may overcome compromised T cell responses in visceral leishmaniasis, and this has an important implication in therapeutic vaccine desig

Hybrid Cell Vaccination Resolves Leishmania donovani Infection by Eliciting a Strong CD8+ Cytotoxic T-Lymphocyte Response with Concomitant Suppression of Interleukin-10 (IL-10) but Not IL-4 or IL-13▿ ‡

There is an acute dearth of therapeutic interventions against visceral leishmaniasis that is required to restore an established defective cell-mediated immune response. Hence, formulation of effective immunotherapy requires the use of dominant antigen(s) targeted to elicit a specific antiparasitic cellular immune response. We implemented hybrid cell vaccination therapy in Leishmania donovani-infected BALB/c mice by electrofusing dominant Leishmania antigen kinetoplastid membrane protein 11 (KMP-11)-transfected bone marrow-derived macrophages from BALB/c mice with allogeneic bone marrow-derived dendritic cells from C57BL/6 mice. Hybrid cell vaccine (HCV) cleared the splenic and hepatic parasite burden, eliciting KMP-11-specific major histocompatibility complex class I-restricted CD8+ cytotoxic T-lymphocyte (CTL) responses. Moreover, splenic lymphocytes of HCV-treated mice not only showed the enhancement of gamma interferon but also marked an elevated expression of the Th2 cytokines interleukin-4 (IL-4) and IL-13 at both transcriptional and translational levels. On the other hand, IL-10 production from splenic T cells was markedly suppressed as a result of HCV therapy. CD8+ T-cell depletion completely abrogated HCV-mediated immunity and the anti-KMP-11 CTL response. Interestingly, CD8+ T-cell depletion completely abrogated HCV-induced immunity, resulting in a marked increase of IL-10 but not of IL-4 and IL-13. The present study reports the first implementation of HCV immunotherapy in an infectious disease model, establishing strong antigen-specific CTL generation as a correlate of HCV-mediated antileishmanial immunity that is reversed by in vivo CD8+ T-cell depletion of HCV-treated mice. Our findings might be extended to drug-nonresponsive visceral leishmaniasis patients, as well as against multiple infectious diseases with pathogen-specific immunodominant antigens

Antimony-Resistant Leishmania donovani Exploits miR-466i to Deactivate Host MyD88 for Regulating IL-10/IL-12 Levels during Early Hours of Infection

Infection with antimony-resistant Leishmania donovani (SbRLD) induces aggressive pathology in the mammalian hosts as compared with ones with antimony-sensitive L. donovani (SbSLD) infection. SbRLD, but not SbSLD, interacts with TLR2/TLR6 to induce IL-10 by exploiting p50/c-Rel subunits of NF-kB in infected macrophages (Mfs). Most of the TLRs exploit the universal adaptor protein MyD88 to activate NF-kB. We now show that infection of Mfs from MyD882/2 mice with SbRLD gave rise to significantly higher intracellular parasite number coupled with elevated IL-10/IL-12 ratio in the culture supernatant as compared with infection in wild type (WT) Mfs. Τhese attributes were not seen with SbSLD in similar experiments. Further, SbRLD infection upregulated miR-466i, which binds with 39-untranslated region, leading to the downregulation of MyD88. Infection of MyD882/2 Mf or IL-122/2 Mf with SbRLD induced IL-10 surge at 4 h, whereas the same in WT Mf started from 12 h. Thus, absence of IL-12 in MyD882/2 mice favored early binding of NF-kB subunits to the IL-10 promoter, resulting in IL-10 surge. Infection of MyD882/2 mice with SbRLD showed significantly higher organ parasites coupled with ill-defined and immature hepatic granulomas, whereas in WT mice there were less organ parasites and the granulomas were well defined. From the survival kinetics it was observed that SbRLD-infected MyD882/2 mice died by 60 d postinfection, whereas the WT mice continued to survive. Our results demonstrate that SbRLD has evolved a unique strategy to evade host antileishmanial immune repertoire by manipulating host MyD88 to its advantage