Linking membrane fluidity with defective antigen presentation in leishmaniasis.

Hampering-surface presentation of immunogenic peptides by class I/II MHCs is a key strategy opted by several intracellular protozoan pathogens including Leishmania to escape CD8/CD4 mediated host-protective T-cell response. Although Leishmania parasites (LP) primarily hijack/inhibit host lysosomal/proteasomal pathways to hamper antigen-processing/presentation machinery, recent pieces of evidence have linked host-membrane fluidity as a major cause of defective antigen presentation in leishmaniasis. Increased membrane fluidity severely compromised peptide-MHC stability in the lipid raft regions, thereby abrogating T-cell mediated-signalling in the infected host. LP primarily achieves this by quenching host cholesterol, which acts as cementing material in maintaining the membrane fluidity. In this review, we have particularly focused on several strategies opted by LP to hijack-host cholesterol resulting in lipid droplets accumulation around leishmania-containing parasitophorous vacuole favouring intracellular survival of LP. In fact, LP infection can result in altered cholesterol and lipid metabolism in the infected host, thereby favouring the establishment and progression of the infection. From our analysis of two genome-wide transcriptomics data sets of LP infected host, we propose a possible molecular network that connects these interrelated events of altered lipid metabolism with eventual compromised antigen presentation, still existing as a gap in our current understanding of Leishmania infection.

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 (Sb(R)LD) induces aggressive pathology in the mammalian hosts as compared with ones with antimony-sensitive L. donovani (Sb(S)LD) infection. Sb(R)LD, but not Sb(S)LD, interacts with TLR2/TLR6 to induce IL-10 by exploiting p50/c-Rel subunits of NF-κB in infected macrophages (Mϕs). Most of the TLRs exploit the universal adaptor protein MyD88 to activate NF-κB. We now show that infection of Mϕs from MyD88(-/-) mice with Sb(R)LD 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) Mϕs. Τhese attributes were not seen with Sb(S)LD in similar experiments. Further, Sb(R)LD infection upregulated miR-466i, which binds with 3'-untranslated region, leading to the downregulation of MyD88. Infection of MyD88(-/-) Mϕ or IL-12(-/-) Mϕ with Sb(R)LD induced IL-10 surge at 4 h, whereas the same in WT Mϕ started from 12 h. Thus, absence of IL-12 in MyD88(-/-) mice favored early binding of NF-κB subunits to the IL-10 promoter, resulting in IL-10 surge. Infection of MyD88(-/-) mice with Sb(R)LD 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 Sb(R)LD-infected MyD88(-/-) mice died by 60 d postinfection, whereas the WT mice continued to survive. Our results demonstrate that Sb(R)LD has evolved a unique strategy to evade host antileishmanial immune repertoire by manipulating host MyD88 to its advantage.

Copper salisylaldoxime (CuSAL) imparts protective efficacy against visceral leishmaniasis by targeting Leishmania donovani topoisomerase IB.

In vitro and in vivo anti-leishmanial efficacy of copper salisylaldoxime (CuSAL), a transition metal complex, was evaluated and the underlying mechanism was studied. In vitro studies revealed that 30 µM of CuSAL causes 96% reduction in parasite burden in infected macrophages. CuSAL is least toxic in host cells. A dose of 5 mg/kg bodyweight per mice on alternate days (5 doses) gives ∼97% protection in both liver and spleen. Moreover, CuSAL potentially inhibits the catalytic activity of LdTOPILS and causes apoptosis of Leishmania parasites through induction of intracellular ROS generation and activation of caspase-like proteases. Interestingly, CuSAL does not inhibit the catalytic activity of human topoisomerase I. The present study illuminated that CuSAL, has potent anti-leishmanial activity, which selectively targets LdTOPILS; and is a safe for human. Therefore, this compound might be highly promising candidate to develop the rational approaches for chemotherapy of human leishmaniasis.

Bioactivity guided fractionation of Moringa oleifera Lam. flower targeting Leishmania donovani.

Leishmaniases is a group of diseases caused by the protozoan parasite belonging to the genus Leishmania. At least 20 species of Leishmania are known to infect humans transmitted by female sandflies, Phlebotomus spp. Leishmania donovani causes visceral leishmaniasis, considered most lethal among the common three forms of leishmaniasis. Lack of appropriate vaccines, emergence of drug resistance and side effects of currently used drugs stress the need for better alternative drugs, particularly from natural sources. Here, we conducted in vitro and in vivo experiments to study the efficacy of different parts of Moringa oleifera Lam. against Leishmania donovani promastigotes. The flower extract of M. oliefera (MoF) was found to be the most potent antileishmanial agent when compared to other parts of the plant like leaf, root, bark and stem. It imparted significant reduction in parasite number in infected macrophages. The bioactivity guided fractionation of MoF showed ethyl acetate fraction (MoE) as the most active and gave significant parasite reduction in the infected macrophages. Further, growth kinetics studies revealed loss of L. donovani promastigotes viability in the presence of MoE in both time and dose dependent manner. In vivo experiment in Balb/c mouse model of leishmaniasis supported the in vitro findings with a remarkable reduction of the parasite burden in both liver and spleen.

TLR4 and NKT cell synergy in immunotherapy against visceral leishmaniasis.

NKT cells play an important role in autoimmune diseases, tumor surveillance, and infectious diseases, providing in most cases protection against infection. NKT cells are reactive to CD1d presented glycolipid antigens. They can modulate immune responses by promoting the secretion of type 1, type 2, or immune regulatory cytokines. Pathogen-derived signals to dendritic cells mediated via Toll like Receptors (TLR) can be modulated by activated invariant Natural Killer T (iNKT) cells. The terminal ß-(1-4)-galactose residues of glycans can modulate host responsiveness in a T helper type-1 direction via IFN-γ and TLRs. We have attempted to develop a defined immunotherapeutic, based on the cooperative action of a TLR ligand and iNKT cell using a mouse model of visceral leishmaniasis. We evaluated the anti-Leishmania immune responses and the protective efficacy of the ß-(1-4)-galactose terminal NKT cell ligand glycosphingophospholipid (GSPL) antigen of L. donovani parasites. Our results suggest that TLR4 can function as an upstream sensor for GSPL and provoke intracellular inflammatory signaling necessary for parasite killing. Treatment with GSPL was able to induce a strong effective T cell response that contributed to effective control of acute parasite burden and led to undetectable parasite persistence in the infected animals. These studies for the first time demonstrate the interactions between a TLR ligand and iNKT cell activation in visceral leishmaniasis immunotherapeutic.

TLR4-mediated activation of MyD88 signaling induces protective immune response and IL-10 down-regulation in Leishmania donovani infection.

In 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 ß 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 LD-infected cells with GP29 also induced the degradation of IKB and the nuclear translocation of NF-κB, as well as rapid phosphorylation of p38 MAPK and p54/56 JNK. 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-κB, 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.

Zoonotic human liver flukes, a type 1 biocarcinogen, in freshwater fishes: genetic analysis and confirmation of molluscan vectors and reservoir hosts in Bangladesh.

BACKGROUND: Opisthorchiid flukes, particularly Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, and Metorchis spp. are the most common fish-borne zoonotic human liver flukes (hLFs). Liver fluke infections are more prevalent in resource-deprived and underprivileged areas. We herein estimated the prevalence of the metacercariae (MC) of major hLFs in common large freshwater fishes (lFWF) marketed for human consumption from some selected areas of Bangladesh along with detection of their molluscan vectors and reservoirs. METHODS: The current status of fish-borne zoonotic hLF infections in lFWF was investigated along with their molluscan vectors and mammalian reservoir hosts in Mymensingh and Kishoreganj in Bangladesh from July 2018-June 2022 using conventional and multiple molecular techniques, such as PCR, PCR-restriction fragment length polymorphism (RFLP), sequencing, and bioinformatic analyses. The infection rate of fishes was analyzed using the Z-test and the loads of MC were compared using the chi-squared (χ2) test. RESULTS: The MC of C. sinensis, Opisthorchis spp., and Metorchis spp. were detected in 11 species of common and popular lFWF. In lFWF, the estimated prevalence was 18.7% and the mean load was 137.4 ± 149.8 MC per 100 g of fish. The prevalence was the highest (P < 0.05) in spotted snakehead fishes (Channa punctata, 63.6%). The highest rate of infection (P < 0.05) was observed with the MC of C. sinensis (11.8%). Metacercariae were almost equally (P > 0.05) distributed between the head and body of fishes. The infection rate was slightly higher in cultured (19.6%) fishes. The MC of C. sinensis, O. felineus, O. viverrini, and Metorchis orientalis in fishes were confirmed using PCR, PCR-RFLP and bioinformatics. The cercariae of opisthorchiid (Pleurolophocercus cercariae) flukes were only recovered from Bithynia spp. (3.9%, 42 out of 1089). The ova of hLFs from dogs (4.3%, 5 out of 116) and cats (6.0%, 6 out of 100), and adult flukes (M. orientalis) from ducks (41.1% 113 out of 275) were detected. CONCLUSIONS: The MC of hLFs are highly prevalent in fresh water fishes in Bangladesh. Reservoir hosts, such as street dogs, cats, and ducks carried the patent infection, and residents of Bangladesh are at risk.

TLR-mediated distinct IFN-γ/IL-10 pattern induces protective immunity against murine visceral leishmaniasis.

Resistance to murine visceral leishmaniasis (VL) correlates with the development of an IFN-γ predominant immune response. Beta1,4-galactose terminal glycans are potent inducers of IFN-γ. Here, we demonstrate the efficacy of a 29 kDa ß1,4-galactose terminal glycoprotein (GP29) of Leishmania donovani (LD) in an in vitro macrophage model and an in vivo mouse model of VL. GP29 induced splenic macrophages to release NO and ROS in appreciable amounts that resulted in effective parasite clearance from macrophages. This was associated with the toll-like receptor (TLR)-4 mediated IL-12 induction and inhibition of TLR2-mediated IL-10 production. Two subcutaneous injections of GP29 at fortnightly intervals resulted in dominant IL-12-mediated IFN-γ production and 100% animals were protected against a subsequent challenge with virulent LD parasites. Vaccinated mice showed a reversal of T-cell anergy, significantly elevated expression of iNOS and a type-1 IgG subclass response. Moreover, vaccinated mice downregulated arginase1 and IL-10 expression but did not alter IL-4 expression. The IFN-γ/IL-10 ratio regulated the intensity of the protective immune response. Experiments with IFN-γ and IL-10 knockout mice reiterated the role IL-10 and IFN-γ play in disease progression or resolution in the murine model of VL.

Present status with impacts and roles of miRNA on Soil Transmitted Helminthiosis control: A review.

Soil-Transmitted Helminthiasis (STH) is one of the most widespread Neglected Tropical Diseases (NTDs), and almost 1.5 billion of the global population is affected, mostly in the indigent, countryside sectors of tropics/subtropics. STH, commonly caused by various nematodes, adversely affects the hosts' growth, cognatic development, and immunity. Albendazole is most commonly used against STH (Soil-Transmitted Helminths) but resistance has already been reported in different countries. To date, no effective vaccine is present against STH. miRNAs are a unique class of small non-coding RNA, regulating various biological activities indulging host immune responses in host-pathogen interaction of STH. Dysregulation of miRNAs are being considered as one of the most important aspect of host-parasite interactions. Thus, it is the prime importance to identify and characterize parasite-specific as well as host-derived miRNAs to understand the STH infection at the molecular level. Systematic bibliometric analysis reveals a huge knowledge gap in understanding the disease by using both host and parasitic miRNAs as a potential biomarker. In this study, we addressed the present status of the STH prevalence, and therapy under the light of miRNAs. This would further help in designing new inhibitors and therapeutic strategies to control STH.

Clinical Profile and Outcomes of Epidemic Dropsy Patients Attending a Tertiary Care Centre in Assam, India.

Background The clinical condition of epidemic dropsy is caused by the consumption of edible oils contaminated with Argemone mexicana oil. Two of the most toxic alkaloids found in argemone oil are sanguinarine and dehydrosanguinarine, which cause capillary dilation, proliferation, and increased permeability. Extreme cardiac decompensation leading to congestive heart failure and glaucoma resulting in blindness are the most serious consequences of epidemic dropsy.  Materials and methods All patients attending the medicine department of Tezpur Medical College and Hospital with clinical features of epidemic dropsy were included in the study after obtaining informed consent. All patients, after a complete history, underwent a thorough clinical examination, and findings were recorded using a pre-formed proforma. Along with routine blood examination, patients were also evaluated with echocardiography, ECG, and chest X-ray. Cooking oil samples obtained from patients were investigated for the presence of sanguinarine in a standardized laboratory with the help of the district authority. The statistical analysis was done using MS Excel 2017. Results Out of 38 patients, 36 were male (94.7%), and only two were female (5.2%). Male to female ratio was 18:1. This difference in sex ratio may be due to the fact that only severely ill patients attended our tertiary care hospital. In contrast, moderate and mildly ill patients were treated in local hospitals. The mean age of patients was 28.1 years, and the mean length of hospital stay was eight days. Bilateral pitting type of ankle edema was the most common clinical manifestation, and all 38 patients (100%) exhibited edema. A total of 76% of patients had dermatological manifestations. Sixty-two percent of patients had gastrointestinal manifestations. In cardiovascular manifestation, persistent tachycardia was seen in 52% of patients, pansystolic murmur was best heard in the apical area in 42% of patients, and 21 percent had evidence of a raised jugular venous pressure (JVP). Five percent of patients had pleural effusion. Sixteen percent of patients had ophthalmological manifestations. Eight patients (21%) required ICU care. The in-hospital fatality rate was 10.53% (n=4). Of the expired patients, 100% were male. The most common cause of death was cardiogenic shock (75%), followed by septic shock (25%). Conclusion From our study, it was found that most of the patients were male, with an age group of 25-45 years. The most common clinical manifestation was dependent edema, along with signs of heart failure. Other common manifestations were dermatological and gastrointestinal. The severity and outcome were directly related to the delay in seeking medical consultation and diagnosis.

Leishmania donovani glycosphingolipid facilitates antigen presentation by inducing relocation of CD1d into lipid rafts in infected macrophages.

NKT cells respond to presentation of specific glycolipids with release of both Th1- and Th2-type cytokines. Leishmania donovani (LD)-infected splenic macrophages (sMϕ(I)) and bone marrow-derived dendritic cells (BMDC(I)) failed to activate NKT cells in response to α-galactosyl ceramide (α-GalCer). The defective antigen presentation could be corrected by treating the cells with the immunostimulating glycosphingophospholipid (GSPL) of LD parasites. In vitro pulsing of BMDC(I) or sMϕ(I) with GSPL, caused the activation of the Vα14(+) CD1d1-specific NKT cell hybridoma DN32.D3. Localization of MHC II and CD1d molecules to membrane lipid rafts has been suggested to play an important role in antigen presentation. Confocal analysis clearly demonstrated that LD infection changed the pattern of CD1d distribution to the non-lipid raft regions and this change could be reversed by GSPL treatment. Isoelectric focusing gel shift assay indicated that GSPL binds to CD1d. GSPL-treated but not untreated BMDC(I) formed immune synapses with NKT cells and this was associated with calcium mobilization. In conclusion, GSPL treatment was associated with modification of BMDC(I)/sMϕ(I) lipid raft structure, which is a site for immune regulation.

Asiaticoside induces tumour-necrosis-factor-α-mediated nitric oxide production to cure experimental visceral leishmaniasis caused by antimony-susceptible and -resistant Leishmania donovani strains.

OBJECTIVES: The aim of this study was to investigate and characterize the efficacy of asiaticoside in an experimental model of visceral leishmaniasis caused by antimony-susceptible (AG83) and -resistant (GE1F8R and K39) Leishmania donovani. METHODS: The effect of asiaticoside was evaluated by microscopic counting of intracellular amastigotes in cultured macrophages stained with Giemsa. The antileishmanial effect of the compounds was assessed in infected BALB/c mice by estimation of splenic and liver parasite burdens in Leishman Donovan units. Cytokines were measured by real-time PCR and ELISA. Intracellular tumour necrosis factor-α (TNF-α) was measured by fluorescence-activated cell sorting. Nitric oxide was measured by the Griess reaction. RESULTS: Besides effectively inhibiting in vitro replication of the parasite within macrophages, asiaticoside treatment resulted in almost complete clearance of the liver and splenic parasite burden when administered at a dose of 5 mg/kg × 10 starting on day +30 of challenge with antimony-susceptible (AG83) and -resistant (GE1F8R and K39) L. donovani. Asiaticoside treatment was associated with a switch in the host from a Th2- to a Th1-type immune response accompanied by the induction of TNF-α-mediated nitric oxide production, all of which are important elements for macrophage function in antileishmanial defence mechanisms. CONCLUSIONS: These results suggest that oral therapy with asiaticoside shows promising antileishmanial efficacy in animals infected by antimony-susceptible (AG83) and -resistant (GE1F8R and K39) L. donovani.

Activation of TLR-pathway to induce host Th1 immune response against visceral leishmaniasis: Involvement of galactosylated-flavonoids.

Immunotherapeutic strategies against visceral leishmaniasis (VL) are pertinent because of the emergence of resistance against existing chemotherapy, coupled with their toxicity and high costs. Various bioactive components with potential immunomodulatory activity, such as alkaloids, terpenes, saponins, flavonoids obtained primarily from medicinal plants, have been screened against different disease models. Reports suggested that glycans containing terminal ß-galactose can skew host immune response towards Th1 by engaging TLRs. In this study, two synthesized terminal galactose-containing flavones, Quercetin 3-d-galactoside (Q-gal) and Kaempferol 3-O-d-galactoside (K-gal), are profiled in terms of inducing host protective Th1 response in both in vitro & in vivo animal models of experimental VL individually against antimony-resistant & antimony-susceptible Leishmania donovani. Further, we explored that both Q-gal and K-gal induce TLR4 mediated Th1 response to encounter VL. Molecular docking analysis also suggested strong interaction with TLR4 for both the galactosides, with a slightly better binding potential towards Q-gal. Treatment with both Q-gal and K-gal showed significant antileishmanial efficacy. Each considerably diminished the liver and splenic parasite burden 60 days after post-infection (>90% in AG83 infected mice and >87% in GE1F8R infected mice) when administered at a 5 mg/kg/day body-weight dose for ten consecutive days. However, the treatments failed to clear the parasites in the TLR4 deficient C3H/HeJ mice. Treatment with these compounds favors the elevation of TLR4 dependent host protective Th1 cytokines and suppression of disease-promoting IL-10. Q-gal and K-gal also triggered sufficient ROS generation in macrophages to kill intracellular parasites directly.

TLR mediated GSK3ß activation suppresses CREB mediated IL-10 production to induce a protective immune response against murine visceral leishmaniasis.

During Leishmania donovani (LD) infection Interleukin (IL)-10 favors parasite replication and plays a central role as a target for immune-based therapy. Glycogen synthase kinase 3 (GSK3)ß differentially regulates TLR-mediated cytokine production. CREB, an important transcription factor that induces IL-10 production is negatively regulated by GSK3ß. However, down regulation of IL-10 via CREB suppression has not been well explored in controlling LD infection. Here we demonstrate that, the TLR4 agonist 29 KDa ß 1,4-galactose terminal glycoprotein (GP29) of LD activated GSK3ß through TLR4 to induce IL-12-mediated Nitric oxide (NO) production that resulted in effective parasite clearance from macrophages. GSK3ß activation abrogated both CREB phosphorylation and IL-10 production. Two subcutaneous injections of GP29 at fortnightly intervals in a 4-week infected mouse model of LD resulted in a dominant IL-12-mediated NO production and 100% animals were protected against a subsequent challenge with virulent LD parasites. Complete absence of GP29 mediated protection with down regulated NO and IL-12 production and dominant IL-10 production in presence of the GSK3ß inhibitor, Lithium chloride reiterated the role of GSK3ß in disease resolution in the murine model of visceral leishmaniasis.