Nucleoside-Derived Metallohydrogel Induces Cell Death in Leishmania Parasites.

Self-assembled hydrogels by virtue of their unique 3D network and tunability have extensively been explored for bio-medical applications like tissue engineering, delivery and release of therapeutic agents, etc. Herein, we demonstrate for the first-time nucleoside-based biocompatible hydrogels with a remarkable leishmanicidal effect against both Leishmania major promastigotes and amastigotes and no cytotoxic effect on the macrophage cell line. In this work, a series of biocompatible hydrogels have been synthesized by silver ion-driven self-assembly of natural nucleoside and nucleotide-like cytidine and 5'-GMP. The supramolecular metallogel obtained from the assembly of cytidine and boronic acid is capable of inducing apoptotic-like cell death of protozoan parasite by causing damage to the membrane as well as DNA. These hydrogels could find promising applications in combating cutaneous leishmaniasis by topical treatment.

Antimony resistance mechanism in genetically different clinical isolates of Indian Kala-azar patients.

The central theme of this enterprise is to find common features, if any, displayed by genetically different antimony (Sb)-resistant viscerotropic Leishmania parasites to impart Sb resistance. In a limited number of clinical isolates (n = 3), we studied the breadth of variation in the following dimensions: (a) intracellular thiol content, (b) cell surface expression of glycan having N-acetyl-D-galactosaminyl residue as the terminal sugar, and (c) gene expression of thiol-synthesizing enzymes (CBS, MST, gamma-GCS, ODC, and TR), antimony-reducing enzymes (TDR and ACR2), and antimonial transporter genes (AQP1, MRPA, and PRP1). One of the isolates, T5, that was genotypically characterized as Leishmania tropica, caused Indian Kala-azar and was phenotypically Sb resistant (T5-LT-SSG-R), while the other two were Leishmania donovani, out of which one isolate, AG83, is antimony sensitive (AG83-LD-SSG-S) and the other isolate, T8, is Sb resistant (T8-LD-SSG-R). Our study showed that the Sb-resistant parasites, regardless of their genotype, showed significantly higher intracellular thiol compared with Sb-sensitive AG83-LD-SSG-S. Seemingly, T5-LT-SSG-R showed about 1.9-fold higher thiol content compared with T8-LD-SSG-R which essentially mirrored cell surface N-acetyl-D-galactosaminyl expression. Except TR, the expression of the remaining thiol-synthesizing genes was significantly higher in T8-LD-SSG-R and T5-LT-SSG-R than the sensitive one, and between the Sb-resistant parasites, the latter showed a significantly higher expression. Furthermore, the genes for Sb-reducing enzymes increased significantly in resistant parasites regardless of genotype compared with the sensitive one, and between two resistant parasites, there was hardly any difference in expression. Out of three antimony transporters, AQP1 was decreased with the concurrent increase in MRPA and PRP1 in resistant isolates when compared with the sensitive counterpart. Interestingly, no difference in expression of the above-mentioned transporters was noted between two Sb-resistant isolates. The enduring image that resonated from our study is that the genetically diverse Sb-resistant parasites showed enhanced thiol-synthesizing and antimony transporter gene expression than the sensitive counterpart to confer a resistant phenotype.

Role of Structural and Non-Structural Proteins and Therapeutic Targets of SARS-CoV-2 for COVID-19.

Coronavirus belongs to the family of Coronaviridae, comprising single-stranded, positive-sense RNA genome (+ ssRNA) of around 26 to 32 kilobases, and has been known to cause infection to a myriad of mammalian hosts, such as humans, cats, bats, civets, dogs, and camels with varied consequences in terms of death and debilitation. Strikingly, novel coronavirus (2019-nCoV), later renamed as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and found to be the causative agent of coronavirus disease-19 (COVID-19), shows 88% of sequence identity with bat-SL-CoVZC45 and bat-SL-CoVZXC21, 79% with SARS-CoV and 50% with MERS-CoV, respectively. Despite key amino acid residual variability, there is an incredible structural similarity between the receptor binding domain (RBD) of spike protein (S) of SARS-CoV-2 and SARS-CoV. During infection, spike protein of SARS-CoV-2 compared to SARS-CoV displays 10-20 times greater affinity for its cognate host cell receptor, angiotensin-converting enzyme 2 (ACE2), leading proteolytic cleavage of S protein by transmembrane protease serine 2 (TMPRSS2). Following cellular entry, the ORF-1a and ORF-1ab, located downstream to 5' end of + ssRNA genome, undergo translation, thereby forming two large polyproteins, pp1a and pp1ab. These polyproteins, following protease-induced cleavage and molecular assembly, form functional viral RNA polymerase, also referred to as replicase. Thereafter, uninterrupted orchestrated replication-transcription molecular events lead to the synthesis of multiple nested sets of subgenomic mRNAs (sgRNAs), which are finally translated to several structural and accessory proteins participating in structure formation and various molecular functions of virus, respectively. These multiple structural proteins assemble and encapsulate genomic RNA (gRNA), resulting in numerous viral progenies, which eventually exit the host cell, and spread infection to rest of the body. In this review, we primarily focus on genomic organization, structural and non-structural protein components, and potential prospective molecular targets for development of therapeutic drugs, convalescent plasm therapy, and a myriad of potential vaccines to tackle SARS-CoV-2 infection.

In vivo experiments demonstrate the potent antileishmanial efficacy of repurposed suramin in visceral leishmaniasis.

BACKGROUND: Treatment failure and resistance to the commonly used drugs remains a major obstacle for successful chemotherapy against visceral leishmaniasis (VL). Since the development of novel therapeutics involves exorbitant costs, the effectiveness of the currently available antitrypanosomatid drug suramin has been investigated as an antileishmanial, specifically for VL,in vitro and in animal model experiments. METHODOLOGY/PRINCIPAL: Leishmania donovani promastigotes were treated with suramin and studies were performed to determine the extent and mode of cell mortality, cell cycle arrest and other in vitro parameters. In addition, L. donovani infected BALB/c mice were administered suramin and a host of immunological parameters determined to estimate the antileishmanial potency of the drug. Finally, isothermal titration calorimetry (ITC) and enzymatic assays were used to probe the interaction of the drug with one of its putative targets namely parasitic phosphoglycerate kinase (LmPGK). FINDINGS: The in vitro studies revealed the potential efficacy of suramin against the Leishmania parasite. This observation was further substantiated in the in vivo murine model, which demonstrated that upon suramin administration, the Leishmania infected BALB/c mice were able to reduce the parasitic burden and also generate the host protective immunological responses. ITC and enzyme assays confirmed the binding and consequent inhibition of LmPGK due to the drug. CONCLUSIONS/SIGNIFICANCE: All experiments affirmed the efficacy of suramin against L. donovani infection, which could possibly lead to its inclusion in the repertoire of drugs against VL.

In vitro screening of known drugs identified by scaffold hopping techniques shows promising leishmanicidal activity for suramin and netilmicin.

OBJECTIVE: The rapid emergence of drug resistant Leishmanial strains makes it imperative to continue the development of cheap and effective drugs against the parasite. Due to the absence of effective vaccines against leishmaniasis, current therapeutic measures exclusively rely on chemotherapy. Here we attempt, to identify novel antileishmanial from a list of known drugs determined from a previous bioinformatics study. Synergism between various drug combinations (involving netilmicin, suramin, paromomycin and curcumin) have been estimated to identify potent multidrug therapies to combat the disease. RESULTS: The drugs were screened against Leishmania promastigotes by utilizing the MTT assay and against intracellular amastigotes using murine Macrophage like tumor cell, RAW 264.7 as a host. In vitro drug interactions were tested for several drug combinations with a modified fixed ratio isobologram method against both Leishmania major and Leishmania donovani. This work reports the in vitro antileishmanial activity for the aminoglycoside netilmicin (for some Leishmania parasites) and the anti-trypanosomatid suramin. Synergism was also observed between paromomycin-suramin and netilmicin-curcumin.

Miltefosine Resistant Field Isolate From Indian Kala-Azar Patient Shows Similar Phenotype in Experimental Infection.

Emergence of resistance to drugs used to treat the Indian Kala-azar patients makes control strategy shattered. In this bleak situation, Miltefosine (MIL) was introduced to treat mainly antimonial unresponsive cases. Within years, resistance to MIL has been reported. While checking the MIL sensitivity of the recent KA clinical isolates (n = 26), we came across one isolate which showed four times more EC50 for MIL than that of MIL-Sensitive (MIL-S) isolates and considered as putative MIL-Resistant (MIL-R). The expressions of LdMT and LdRos3 genes of this isolate were found down regulated. Th1/Th2 cytokines, ROS and NO, FACS dot plots and mitochondrial trans membrane potential measurement were performed. In vivo hamster model with this MIL-R isolate showed much lesser reduction in liver weight (17.5%) compared to average reduction in liver weight (40.2%) of the animals infected with MIL-S isolates. The splenic and hepatic stamps smears of MIL-R infected hamsters revealed the retention of parasite load of about 51.45%. The splenocytes of these animals failed to proliferate anti leishmanial T-cells and lack of cell mediated immunity hampered recovery. Thus, these phenotypic expressions of experimental model may be considered similar to that of the MIL unresponsive patients. This is first such kind of report.

Molecular identification of an old clinical isolate of Indian Kala-azar.

Molecular characterization is an important task for species identification of the isolates belonging to different Leishmania species. Clinical symptoms, tissue tropism, vector preference, reservoir and geographical distribution may act as distinguishing parameters but not always decisive. On the other hand, modern taxonomic tools employed to divulge characteristics of the genome or protein molecules of the parasite would be convincing and for Leishmania sp., they include nuclear and kDNA buoyant density, multi locus enzyme electrophoresis (MLEE), RAPD, RFLP or use of monoclonal antibodies etc. In the present study, we intend to establish the identification of an old clinical isolate of Indian Kala-azar, familiarly known as 'UR6' by MLEE, RAPD, RFLP and species specific monoclonal antibodies. UR6 has been isolated from a confirmed Kala-azar patient admitted in Calcutta School of Tropical Medicine, Kolkata in 1978. From then it is being regularly used for various scientific studies by the Leishmania Research Group of India and abroad. The isozyme profile of UR6 showed similar electrophoretic mobility that of WHO reference strain for Leishmania tropica, K27. Similar findings were obtained in the RAPD and RFLP assays. Screening with species specific monoclonal antibodies showed its strong reactivity towards L. tropica. The Jaccard's Similarity Indices were calculated.

Particle induced X-ray emission study of blood samples of Indian Kala-azar patients.

Visceral leishmaniasis (VL) or Kala-azar (KA) is a neglected tropical disease caused by protozoan parasite, Leishmania sp. and is fatal, if left untreated. In this study, we measured trace elements (K, Fe, Cu, Zn, Br, Cl, S, Ca, Mn, Cr, Ni, As, Se, Rb and Sr) in the blood of Indian VL patients (32) by particle-induced X-ray emission (PIXE) study. Blood was collected from 36 subjects including healthy controls from Rambagh Kala-azar Hospital, Muzaffarpur, Bihar, India. PIXE experiment was carried out at the Institute of Physics, Bhubaneswar, India and data were analyzed by GUPIXWIN software. We observed first time the association of bromine with the disease. The results showed 48.47 % decrease in Br, 35.16 % decrease in Zn and 29.05 % decrease in Fe in untreated state of the KA patients. In the same group, Cu has been increased by 16.73 %. Cu/Zn ratio has been altered in diseased state. The association of bromine with the disease is reported for the first time and altered levels of trace elements (Br, Cu, Fe and Zn) may come back to normal after completion of the treatment regimen with Amphotericin B.

Genetic markers for antimony resistant clinical isolates differentiation from Indian Kala-azar.

Visceral Leishmaniasis or Kala-azar is caused by the protozoan parasites belonging to the Genus Leishmania. Once thought eradicated from the Indian subcontinent, the disease came back with drug resistance to almost all prevalent drugs. Molecular epidemiological studies revealed the polymorphic nature of the population of the main player of the disease, Leishmania donovani and involvement of other species (L. tropica) and other genus (Leptomonas) with the disease. This makes control measures almost futile. It also strongly demands the characterization of each and every isolate mandatory which is not done. In this background, the present study has been carried out to assess the genetic attributes of each clinical isolates (n=26) of KA and PKDL patients from India and Bangladesh. All the isolates were characterized through Restriction Fragment Length Polymorphism (RFLP) analysis to ascertain their species identity. 46.2% of the isolates were found to be Sodium Stibogluconate (SSG) resistant by amastigote-macrophage model. When the clinical isolates were subjected to Single Stranded Conformation Polymorphism (SSCP) of Internal Transcribed Spacer 1 (ITS1), Internal Transcribed Spacer 2 (ITS2) and some anonymous markers, the drug resistant Leishmania isolates of SSG can be distinguished from the sensitive isolates distinctly. This study showed for the first time, the genetic markers for SSG drug resistance of Indian Kala-azar clinical isolates.

Evaluation of s.c. route of immunization by homologous radio attenuated live vaccine in experimental murine model of visceral leishmaniasis.

Our previous studies in BALB/c mice showed substantial protection against the experimental murine visceral leishmaniasis (MVL) when the animals were immunized with γ-irradiated live Leishmania donovani parasites through intra peritoneal (i.p.) and intra muscular (i.m.) routes respectively. The observations encouraged us to check the prophylactic efficacy of subcutaneous (s.c.) route as it is better alternative for human trial. The mice immunized with two subsequent doses of the radio attenuated homologous vaccine were challenged with virulent L. donovani parasites. Seventy-five days post infection, the animals were sacrificed. The extent of protection against the disease was evaluated by assessing the reduction of parasite burden in spleen and liver, the generation of free radicals (NO & ROS) and release of the cytokines from T-lymphocyte helper 1 (Th 1) and T-lymphocyte helper 2 (Th 2) along with the measurement of the serum immunoglobulins. The reductions in parasitic burden were observed up to 21 and 24 % in spleen and liver of the immunized groups with NO and ROS productions 27 and 34 % respectively. Whereas the increase in IFN gamma releases was between 19 and 34 %, the decrease in IL-10 release was not more than 22 %. This indicates the failure of the establishment of pronounced Th1 ambience which was further corroborated by the observed IgG2a and IgG1 ratio. The present study when compared with our previous observations with i.m. and i.p. routes revealed that s.c. route may not be a good choice for the use of radio attenuated vaccine.

RFLPs of ITS, ITS1 and hsp70 amplicons and sequencing of ITS1 of recent clinical isolates of Kala-azar from India and Bangladesh confirms the association of L. tropica with the disease.

Visceral Leishmaniasis or Kala-azar (KA) is a serious health concern in India. In the present study, Restriction Fragment Length Polymorphism (RFLP) of three genetic markers viz., Internal Transcribed Spacer (ITS), ITS1 and heat shock protein 70 (hsp70) have been employed for typing the clinical isolates [n=15] of KA and post Kala-azar Dermal Leishmaniosis (PKDL) collected from India and Bangladesh in the period of 2006-2010. Experimentally, ITS, ITS1 and hsp70 regions of genomes of all the clinical isolates were separately amplified by PCR and then digested with restriction enzymes: ITS with Alu1, EcoR1 and Msp1, ITS1 with Hae III and Rsa1 and hsp70 with Hae III. The resultant fragments were analyzed by agarose gel electrophoresis and the RFLP profiles of the clinical isolates were compared with that of the WHO reference strains for Leishmania donovani (DD8) and Leishmania tropica (K27), respectively. Also, the ITS1 regions of all the clinical isolates along with the two WHO reference strains were sequenced and a phylogram was constructed to ascertain the extent of similarity or dissimilarity. Interestingly, the RFLP profiles of one of the isolates showed a significant homology with K27 and the phylogram revealed its closeness with the same putting credence to our earlier typing of isolates by RAPD method. This observation also supported an earlier report claiming that both the species are responsible for KA in India and thus, emphasizes urgent need for thorough systematic characterization of the clinical isolates of Indian KA as appropriate treatment regime relies primarily on proper diagnosis.

Therapeutic immunization with radio-attenuated Leishmania parasites through i.m. route revealed protection against the experimental murine visceral leishmaniasis.

After our promising results from prophylactic and therapeutic study (i.p. route) with the radio-attenuated Leishmania donovani parasites against experimental murine visceral leishmaniasis, we prompted to check their therapeutic efficacy through i.m route. BALB/c mice were infected with highly virulent L. donovani parasites. After 75 days, mice were treated with gamma (γ)-irradiated parasites. A second therapeutic immunization was given after 15 days of first immunization. The protection against kala-azar was estimated with the reduction of Leishman-Donovan unit from spleen and liver that scored up to 80% and 93%, respectively, while a twofold increase in nitric oxide (NO) and reactive oxygen species (ROS) productions has been observed in the immunized groups of animals. These groups of mice also showed disease regression by skewing Th2 cytokines (IL-10) towards Th1 cytokine (IFN-γ) bias along with the increased generation of NO and ROS, while the infected control group of mice without such treatment surrendered to the disease. Establishment of Th1 ambience in the treated groups has also been supported from the measured antileishmanial antibody IgG subsets (IgG2a and IgG1) with higher anti-soluble Leishmania antigen-specific IgG2a titer. As seen in our previous studies, doses of attenuation by γ-radiation should be taken into serious consideration. Attenuation of parasites at 50 Gy of absorbed dose of gamma rays has not worked well. Thus, therapeutic use of L. donovani parasites radio-attenuated at particular doses can be exploited as a promising vaccine agent. Absence of any adjuvant may increase its acceptability as vaccine candidate further.

Characterization of the recent clinical isolates of Indian Kala-azar patients by RAPD-PCR method.

Leishmaniasis is one of the most important vector borne diseases caused by kinetoplastid protozoa Leishmania sp. Among all forms of Leishmaniasis, Visceral leishmaniasis (VL) or Kala-azar is the severest form of the illness. VL is characterized by fever, hepatosplenomegaly, anaemia, edema, weight loss and invariably fatal if left untreated. Characterization of Leishmania sp. is extremely necessary to understand the epidemiology, taxonomy and population genetics of the parasites which ultimately helps in designing appropriate drug regimen to combat the disease. In this study, we aimed to type the clinical isolates of Leishmania species collected in the period 2006-2010 from patients (n = 9) diagnosed with Kala-azar and post Kala-azar dermal leishmaniasis (PKDL) by RAPD-PCR method using eight selected primers. Genome of the clinical isolates were amplified and electrophoresed in agarose gel. These were compared with the RAPD PCR profiles of WHO reference strains for L. donovani (DD8) and L. tropica (K27) respectively. We calculated the Jaccard's Similarity Coefficient and found one (study code T5) out of nine isolates as L. tropica while the rest were L. donovani. This pilot study supports the earlier single report claiming that both the species are responsible for Kala-azar in India and it also emphasizes the need for more systematic typing of clinical isolates of Indian Kala-azar.