Somenath Roy

Artesunate-sulfadoxine-pyrimethamine (ASSP) is the frontline artemisinin ombination therapy (ACT) in India. Random, irrational, sub-therapeutic artemisinin doses and self-medication with ACT along with predominance of sulfadoxine-pyrimethamine resistance parasite invoked a strong possibility of emerging artemisinin-resistant malaria parasites. This study involved 226 patients with uncomplicated P. falciparum infection who had successfully completed the 42 days follow up after artesunate-sulfadoxine-pyrimethamine combination therapy from April, 2014 to January, 2016. We assessed the ASSP treatment efficacy by evaluating parasite clearance half-life, pfkelch13 and other (pfdhfr, pfdhps, pfmdr1, pfcrt) gene mutations and survival of parasites as detected by an ex-vivo ring-stage survival assay (RSA). Slow-clearing infections with longer parasite clearance half-lives (>5hrs) were observed in 12% isolates. Cure rate after ASSP treatment was declining to about 84.1%. ASSP failure was recorded in 15.9% (ETF,7.9%; LTF,7.9%) of isolates. 24 patients (10.6%) had parasite clearance half-lives greater than 5hrs with pfkelch13 polymorphism after 441 codon; in 15 of those patients (6.6%), parasites had not cleared by 72 hrs after initiation of therapy. Median ex-vivo ring-stage survival rate of these isolates was very high (14.6%; 95%CI, 12.9 to 15.2) from that of cured patients (1.1%; 95%CI, 0.9 to 1.7). Of these 15 patients, 13 patients had pfkelch13 G625R polymorphism while 2 patients contained R539T polymorphism. As per the WHO guideline, these 15 isolates were true artemisinin-resistant isolates. Identification of artemisinin-resistant isolates in India together with new mutations and increasing combination therapy failures blow alarms for urgent malaria control.

Emergence of chloroquine resistant Plasmodium vivax is a serious obstacle towards malaria control in India. This study elucidates the temporal pattern of antifolate [sulfadoxine–pyrimethamine (SP)] resistance in P. vivax infection by means of genetic polymorphisms, especially analysing the single nucleotide polymorphisms of dihydrofolate reductase (pvdhfr) and dihydropteroate synthase (pvdhps) gene among the field isolates of urban Kolkata Municipal Corporation and rural Purulia region of West Bengal, India. Blood samples were collected from 99 microscopically diagnosed P. vivax patients (52 from Kolkata Municipal Corporation and 47 from Purulia). Parasitic DNA was extracted followed by polymerase chain reaction and sequencing of different codons of pvdhfr gene (15, 33, 50, 57, 58, 61, 64, 117, and 173 codons) and pvdhps gene (373, 380, 382, 383, 384, 512, 553, 585, and 601 codons) were performed to identify the mutations. Prevalence of double mutant dhfr A15P33N50F57 R 58 T61V64 N 117 I173 allele (53.85 %) was observed in Kolkata Municipal Corporation (KMC) whereas in Purulia, wild dhfr A15P33N50F57S58T61V64S117I173 allele was predominated (48.94 %). In pvdhps gene a significant number of isolates (17.31 %) in KMC contained the double mutant S373E380S382 G 383 P384K512 G 553 V585M601 allele. pvdhfr and pvdhps combination haplotype revealed the emergence of quadruple (13.46 %) and quintuple (3.84 %) mutant allele in KMC, which might result in poor clinical response against antifolate drugs. The study reveals that P. vivax parasites in rural Purulia may still be susceptible to SP but additional caution should be taken for treatment of vivax malaria in KMC to limit the blooming of quadruple and quintuple mutant allele in the remainder of the West Bengal, India.

The aim of this work is to understand the potential health effects of metal nanoparticles by exposing human leukemic cell lines (jurkat, K562 and KG1A cells) to nanosize phosphonomethyl iminodiacetic acid coated cobalt oxide (PMIDA-CoO) NPs. The synthesized PMIDA-CoO NPs were characterized by XRD, dynamic light scattering, transmission electron microscopy and scanning electron microscopy. Our results showed that exposure of leukemic cell lines to PMIDA-CoO NPs caused reactive oxygen species (ROS) generation by increasing the concentration of free Co⁺⁺ ions in cancer microenvironment. But at physiological pH, PMIDA-CoO liberates little amount of Co⁺⁺ ions into media and exerts lower toxicity to normal cells up to a certain dose. PMIDA-CoO NPs caused DNA damage in leukemic cell lines, which was reflected by an increase in apoptosis of jurkat, KG-1A and K562 cells. PMIDA-CoO NPs induced apoptosis by increasing pro-inflammatory cytokines, primarily TNF-α. The in vivo study shows that PMIDA-CoO NPs were efficiently killed DLA cells. These findings have important implications for understanding the potential anticancer property induced by surface-modified cobalt oxide nanoparticles.

Malaria is a major public health problem in tropical and subtropical countries, including India. This study elucidates the cause of chloroquine treatment failure (for Plasmodium falciparum infection) before the introduction of artemisinin combination therapy. One hundred twenty-six patients were randomized to chloroquine treatment, and the therapeutic efficacy was monitored from days 1 to 28. An in vitro susceptibility test was performed with all isolates. Parasitic DNA was isolated, followed by PCR and restriction digestion of different codons of the pfcrt gene (codons 72 to 76) and the pfmdr1 gene (N86Y, Y184F, S1034C, N1042D, and D1246Y). Finally, sequencing was done to confirm the mutations. Forty-three (34.13%) early treatment failure cases and 16 (12.69%) late treatment failure cases were observed after chloroquine treatment. In vitro chloroquine resistance was found in 103 isolates (81.75%). Twenty-six (60.47%) early treatment failure cases and 6 (37.5%) late treatment failure cases were associated with the CVMNK- Y YSN Y allele (the underlined amino acids are those that were mutated). Moreover, the CV IE K- Y YSN Y allele was found in 8 early treatment failure (18.60%) and 2 late treatment failure (12.5%) cases. The presence of the wild-type pfcrt (CVMNK) and pfmdr1 ( Y YSN Y ) double mutant allele in chloroquine-nonresponsive cases was quite uncommon. In vivo chloroquine treatment failure and in vitro chloroquine resistance were strongly correlated with the CVMNK- Y YSN Y and CV IE K- Y YSN Y haplotypes ( P < 0.01).

To evaluate the free radical generation and status of the antioxidant enzymes in murine peritoneal macrophage during in vitro vancomycin sensitive Staphylococcus aureus (VSSA) treatment with different time interval. Peritoneal macrophages were treated with 5×10(6) CFU/mL VSSA cell suspension in vitro for different time interval (1, 2, 3, 6, 12, and 24 h) and superoxide anion generation, NADPH oxidase activity, myeloperoxidase activity, nitric oxide generation, antioxidant enzyme status and components of glutathione cycle were analyzed. Superoxide anion generation, NADPH oxidase activity, myeloperoxidase activity and nitric oxide generation got peak at 3 h, indicating maximum free radical generation through activation of NADPH oxidase in murine peritoneal macrophages during VSSA infection. Reduced glutathione level, glutathione peroxidase, glutathione reductase, and glutathione-s-transferase activity were decreased significantly (P<0.05) with increasing time of VSSA infection. But the oxidized glutathione level was time dependently increased significantly (P<0.05) in murine peritoneal macrophages. All the changes in peritoneal macrophages after 3 h in vitro VSSA treatment had no significant difference. From this study, it may be summarized that in vitro VSSA infection not only generates excess free radical but also affects the antioxidant status and glutathione cycle in murine peritoneal macrophages.

The objective of this study was to develop chitosan-based delivery of cobalt oxide nanoparticles to human leukemic cells and investigate their specific induction of apoptosis. The physicochemical properties of the chitosan-coated cobalt oxide nanoparticles were characterized using transmission electron microscopy, dynamic light scattering, X-ray diffraction, and Fourier transform infrared spectroscopy. The solubility of chitosan-coated cobalt oxide nanoparticles was higher at acidic pH, which helps to release more cobalt ions into the medium. Chitosan-coated cobalt oxide nanoparticles showed good compatibility with normal cells. However, our results showed that exposure of leukemic cells (Jurkat cells) to chitosan-coated cobalt oxide nanoparticles caused an increase in reactive oxygen species generation that was abolished by pretreatment of cells with the reactive oxygen species scavenger N-acetyl-l-cysteine. The apoptosis of Jurkat cells was confirmed by flow-cytometric analysis. Induction of TNF-α secretion was observed from stimulation of Jurkat cells with chitosan-coated cobalt oxide nanoparticles. We also tested the role of TNF-α in the induction of Jurkat cell death in the presence of TNF-α and caspase inhibitors. Treatment of leukemic cells with a blocker had a greater effect on cancer cell viability. From our findings, oxidative stress and caspase activation are involved in cancer cell death induced by chitosan-coated cobalt oxide nanoparticles.

The purpose of this study is to evaluate the prospect of using surface modified cobalt oxide(CoO) nanoparticles as carriers of cancerantigens to human macrophages. N-Phosnomethyliminodiacetic acid (PMIDA) was used for surface modification to overcome the toxic effect of CoO nanoparticles. Here, the phosphonate group of the PMIDA acts as a surface-anchoring agent and the remaining –COOH groups bind nonspecifically with tumor associated antigens. This modification allows the conjugation of human oral carcinoma (KB) cell lysate (CL) as an antigen with PMIDA coated CoO nanoparticles (CL–PMIDA–CoO). Particle characterization was performed by dynamic light scattering, atomic force microscopy, and scanning electron microscopy studies. Fourier transform IR spectroscopy was used to investigate conjugation of the protein with nanoparticles. Protein encapsulation was confirmed by protein gel electrophoresis. Active uptake of antigen-conjugated nanoparticles by macrophages was confirmed by fluorescence microscopy. The antitumor activity of the nanocomplex pulsed macrophages was investigated on a human oral carcinoma cell line (KB) in vitro. The modified nanocomplexes upregulate IFN-γ and TNF-α and induce an anticancer immune response by activating macrophages. The use of TNF-α inhibitor confirmed the ability of the CL–PMIDA–CoO nanocomplex to stimulate TNF-α mediated immunostimulation. CL–PMIDA–CoO nanoparticles efficiently increased the CD4⁺ population. Thus, our findings provide insight into the use of PMIDA coated CoO nanoparticles as antigen delivery vehicles. Graphical abstract