Suprabhat Mukherjee

TLR2 and TLR4 mediated host immune responses in major infectious diseases: a review

During the course of evolution, multicellular organisms have been orchestrated with an efficient and versatile immune system to counteract diverse group of pathogenic organisms. Pathogen recognition is considered as the most critical step behind eliciting adequate immune response during an infection. Hitherto Toll-like receptors (TLRs), especially the surface ones viz. TLR2 and TLR4 have gained immense importance due to their extreme ability of identifying distinct molecular patterns from invading pathogens. These pattern recognition receptors (PRRs) not only act as innate sensor but also shape and bridge innate and adaptive immune responses. In addition, they also play a pivotal role in regulating the balance between Th1 and Th2 type of response essential for the survivability of the host. In this work, major achievements rather findings made on the typical signalling and immunopathological attributes of TLR2 and TLR4 mediated host response against the major infectious diseases have been reviewed. Infectious diseases like tuberculosis, trypanosomiasis, malaria, and filariasis are still posing myriad threat to mankind. Furthermore, increasing resistance of the causative organisms against available therapeutics is also an emerging problem. Thus, stimulation of host immune response with TLR2 and TLR4 agonist can be the option of choice to treat such diseases in future.

Antifilarial effect of ursolic acid from Nyctanthes arbortristis: molecular and biochemical evidences.

A bio-assay guided fractionation and purification approach was used to examine in vitro antifilarial activities of the crude methanolic extract of Nyctanthes arbortristis as well as fractions and isolated compound. From ethyl-acetate fraction we isolated and identified a triterpenoid compound which has been characterized as ursolic acid (UA) by HPLC and NMR data. We are reporting for the first time isolation and identification of UA from the leaves of N. arbortristis. The crude extract and UA showed significant micro- as well as macrofilaricidal activities against the oocyte, microfilaria and adult of Setaria cervi (S. cervi) by dye exclusion test and MTT reduction assay. Significant microfilaricidal activity of UA was further proved against mf of W. bancrofti by viability assay. The findings thus provide a new lead for development of a suitable filaricide from natural products. The molecular mechanism of UA was investigated by performing TUNEL, Hoechst staining, Annexin V-Cy3, flow cytometric analysis and DNA fragmentation assay. Differential expressions of pro- and anti-apoptotic genes were observed at the transcription and translational levels in a dose-dependent manner. Depletion in the worm GSH level and elevation in the parasite GST, SOD and super oxide anion indicated the generation of ROS. In this investigation we are reporting for the first time that UA acts its antifilarial effect through induction of apoptosis and by downregulating and altering the level of some key antioxidants like GSH, GST and SOD of S. cervi.

Design and green synthesis of polymer inspired nanoparticles for the evaluation of their antimicrobial and antifilarial efficiency

Polymer inspired silver nano particles (AgNPs), endowed with high biochemical potency and very low cytotoxicity, were designed and synthesized through a tyrosine controlled green process. In contrary to other conventional techniques, the process was so optimized so that the least amount of reducing and stabilizing agents could generate the maximum possible amount of stable nanoparticles. The role of tyrosine as reducing as well as stabilizing agent was found to be dependent on the level of its concentration. Biocompatible polymers inspired the stability and biological activity of the synthesized nanoparticles to a great extent. The formation of AgNPs was monitored spectroscopically (UV-Vis) both in the presence and absence of biocompatible polymers. Biochemical effects of the polymer stabilized metal nano particles were studied in terms of antiparasitic (antifilarial), antibacterial and antifungal activities. The particles were found to be strongly interactive with the principle cellular bio-molecule (viz. DNA and protein) which might be the reason behind their bioactivity. Particularly against filarial parasites, the synthesized AgNPs were able to induce apoptosis through increasing the level of key regulator of cell death and fragmentation of genomic DNA. Among the biocompatible polymers used, chitosan improved the stability and bio-activity of the composites significantly as evident from the bioactivity and molecular studies like Circular Dichorism (CD) spectroscopic analysis as well.

Molecular evidence on the occurrence of co-infection with Pichia guilliermondii and Wuchereria bancrofti in two filarial endemic districts of India

BackgroundLymphatic filariasis (LF), a vector-borne parasitic disease, is endemic in several parts of India and mostly affects the poor or those with a low-income. The disease results in huge numbers of morbidities, disabilities, and deaths every year. Association of co-infection with other pathogens makes the condition more severe. Although co-infection is becoming a growing area of research, it is yet to emerge as a frontier research topic in filarial research specifically. This study reports the occurrence of a fungal infection in a large number of patients suffering from bancroftian filariasis in two districts of West Bengal, India.MethodsNocturnal blood samples from filarial patients containing parasites and fungus were initially co-cultured, and further the fungus was isolated and characterized. Molecular identification of the isolate was carried out by PCR-based selective amplification and sequencing of highly-conserved D1/D2 region of 26S rDNA, whereas pathogenicity was determined by amplification of the RPS0 gene. A phylogenetic tree was constructed to study the relationship between the isolate and common pathogenic yeasts. The isolate was studied for antibiotic sensitivity, whereas morphological characterization was performed by microscopic techniques.ResultsThe isolate was identified as Pichia guilliermondii and this fungus was found to exist in co-infection with Wuchereria bancrofti in filarial patients. The fungus showed resistance to azole antifungals, griseofulvin, and, amphotericin B, whereas significant susceptibility was evident in cases of nystatin and cycloheximide. A total of 197 out of 222 patients showed this co-infection.ConclusionThis study revealed, for the first time, that P. guilliermondii exists as a co-infection in microfilaraemic individuals living in a filarial endemic zone. The findings are important and have relevance to human health, especially for filarial patients.

Isolation and Characterization of Arsenic-Resistant Bacteria from Contaminated Water-Bodies in West Bengal, India

Bengal Basin is known for severe arsenic contamination. In the present study, we have isolated six bacteria from the arsenic contaminated surface water of Bengal Basin. 16S rDNA sequence analysis identified them as Microbacterium oleivorans, Acinetobacter soli, Acinetobacter venetianus, Acinetobacter junii, Acinetobacter baumannii, Acinetobacter calcoaceticus. All the isolates possess arsenic accumulation potential and high molecular weight plasmid (>10 kb). PCR amplification indicated the presence of arsenic-resistance genes (arsB and aoxB) either in the genome or plasmid or in both in the isolated bacteria (except in Acinetobacter venetianus). Exposure to arsenic affected bacterial growth and induced alteration in cytoplasmic membrane integrity.

In vitro antifilarial activity of Azadirachta indica aqueous extract through reactive oxygen species enhancement.

OBJECTIVE To evaluate an aqueous preparation from the Azadirachta indica leaves (AEA) against Setaria cervi (S. cervi), a model filarial parasite. METHOD In vitro efficacy of AEA was evaluated against S. cervi through estimation of relative motility value, dye exclusion test and MTT assay. Visible morphological alterations were monitored using conventional microscopic techniques in microfilariae and haematoxylin-eosin stained sections of AEA-treated adults. RESULTS Enhancement of reactive oxygen species in S. cervi treated with AEA was established through alteration in the activity of glutathione S-transferase, superoxide dismutase, catalase, peroxidase and level of superoxide anion and reduced glutathione. CONCLUSION In vitro filaricidal activity of AEA is possibly through disturbing redox homeostasis by down-regulating and altering the level of some key antioxidants and regulatory enzymes like reduced glutathione, glutathione S-transferase, superoxide dismutase, catalase and glutathione peroxidase of S. cervi.

A supramolecular hydrogel for generation of a benign DNA-hydrogel

A novel hydrogel has been synthesized using 2′,4′,6′-tri(4-pyridyl)pyridine in an acidic water medium. TEM analyses along with rheological experiments explore the supramolecular features of the hydrogel. In vitro and in vivo toxicity studies depict the non-toxic and bio-relevant nature of the pure supramolecular hydrogel and its aqueous solution. This bio-compatible supramolecular hydrogel is used for the synthesis of a DNA-hydrogel with calf thymus DNA. The DNA-hydrogel is intriguing for the effective stabilization of photochemically synthesized silver nanoparticles from Ag(I) under the direct exposure of sunrays. DNA-hydrogel capped Ag-NPs are also bio-compatible. The luminous features of the 2′,4′,6′-tri(4-pyridyl)pyridine based supramolecular hydrogel, DNA-hydrogel and DNA-hydrogel capped Ag-NPs and their aqueous solutions are explored through fluorescence microscopy and spectral analyses, respectively.

Green silver nanoparticles for drug transport, bioactivities and a bacterium (Bacillus subtilis)-mediated comparative nano-patterning feature

The ‘green’ synthetic aspects of functionally potent and biologically relevant nanomaterials are a crucial research objective. Pursuing this concept, we have investigated a green synthetic scheme for the sunlight-mediated generation of luminescent silver nanoparticles, which become stabilized via a supramolecular hydrogel (SHGel) network, as already reported by our group. In vitro and in vivo toxicity studies confirm the biologically relevant nature of SHGel-capped Ag NPs. Nontoxic SHGel-capped Ag NPs were intelligently used for the transport of drugs, including antifilarial and antibiotic agents, into cells. Apart from this activity, SHGel-capped Ag NPs and our previously reported nontoxic DNA hydrogel-capped Ag NPs are potent against pathogens and parasites. Most interestingly, the nanostructural patterns of SHGel- and DNA hydrogel-capped Ag NPs have been transformed into cotyledon- and flower bud-shaped forms of nanosilver, respectively, during their chemotherapeutic action against a particular bacterium, Bacillus subtilis. Transmission electron microscopy was used for the visualization of several patterns of nanosilver and the incorporation of Ag NPs into macrophages.

Phenolics and Terpenoids; the Promising New Search for Anthelmintics: A Critical Review.

Ailments caused by helminth parasites are global causing different types of clinical complications with permanent and long term morbidity in humans. Although huge advances have been made in medical sciences the effectiveness of available anthelmintics are still quite limited. Starting from the 50s, most importance was given to synthetic compounds for developing remedies from them, however, the traditional knowledge of medicine of different countries continued to provide us clues against this widespread health problem. Natural products or structural analogs with diverse structures are always been the major sources for discovering new therapeutics and in recent past different active compounds have also been identified form these plant sources having anthelmintic properties. Although compounds of diverse chemical nature and classes were identified most active ones belong to either phenol or terpene in broad chemical nature. The mechanism of action of these phytotherapeutics is usually multi-targeted and can act against the helminth parasites through diverse spectrum of activities. In this reviewwe summarized the effective anthelmintics belong to either phenolics or terpenoids and highlighted the major way of their effectiveness. This also highlights the recent development of new therapeutic strategies against helminth parasites in the light of recent advances of knowledge. In addition, developing efficient strategies to promote apoptosis and disturbing redox status in them by natural products can provide us a clue in antifilarial drug developmental research and crucial unmet medical need.

Surface proteins of Setaria cervi induces inflammation in macrophage through Toll- like Receptor 4 (TLR4) mediated signaling pathway.

Lymphatic filariasis is a vectorborne parasitic disease that results in morbidities, disabilities and socio‐economic loss each year globally. Inflammatory consequences associated with any form of filariasis have drawn special attention. However, the molecular insight behind the inflammation of host macrophage (MФ) is considered as one of the shaded areas in filarial research. Herein, major emphasis was given to study the signalling pathway of MФ inflammation induced by surface proteins (SPs) of filarial parasite through in vitro and in vivo approaches. Twenty‐four hours of in vitro stimulation of Raw MФs with endotoxin‐free SPs of Setaria cervi resulted in the secretion of pro‐inflammatory cytokines (TNF‐α and IL‐1β) that revealed induction of inflammation, which was found to be elicited from classical NF‐кB activation. Moreover, this NF‐кB activation was found to be signalled from TLR4 and mediated by the downstream signalling intermediates, viz. MyD88, pTAK1 and NEMO. In vivo studies in adult Wistar rats, experimentally injected with SPs, clearly supported the outcomes of in vitro experiments by showing higher degree of inflammation rather classical activation of the peritoneal MФs. Therefore, SPs from S. cervi cuticle could be responsible for the induction of pro‐inflammatory response in MФ, which appears to be propagated through TLR4‐NF‐кB route.