LmjF.36.3850, a novel hypothetical Leishmania major protein, contributes to the infection.

Leishmania is a protozoan parasite that resides in mammalian macrophages and inflicts the disease known as leishmaniasis. Although prevalent in 88 countries, an anti-leishmanial vaccine remains elusive. While comparing the virulent and avirulent L. major transcriptomes by microarray, PCR and functional analyses for identifying a novel virulence-associated gene, we identified LmjF.36.3850, a hypothetical protein significantly less expressed in the avirulent parasite and without any known function. Motif search revealed that LmjF.36.3850 protein shared phosphorylation sites and other structural features with sucrose non-fermenting protein (Snf7) that shuttles virulence factors. LmjF.36.3850 was predicted to bind diacylglycerol (DAG) with energy value similar to PKCα and PKCß, to which DAG is a cofactor. Indeed, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue, enhanced the phosphorylation of PKCα and PKCßI. We cloned LmjF.36.3850 gene in a mammalian expression vector and primed susceptible BALB/c mice followed by challenge infection. We observed a higher parasite load, comparable antibody response and higher anti-inflammatory cytokines such as IL-4 and IL-10, while expression of major anti-leishmanial cytokine, IFN-γ, remained unchanged in LmjF.36.3850-vaccinated mice. CSA restimulated LN cells from vaccinated mice after challenge infection secreted comparable IL-4 and IL-10 but reduced IFN-γ, as compared to controls. These observations suggest a skewed Th2 response, diminished IFN-γ secreting Th1-TEM cells and increased central and effector memory subtype of Th2, Th17 and Treg cells in the vaccinated mice. These data indicate that LmjF.36.3850 is a plausible virulence factor that enhances disease-promoting response, possibly by interfering with PKC activation and by eliciting disease-promoting T cells.

A novel nano-anti-malarial induces redox damage and elicits cytokine response to the parasite.

Emergence and spread of resistant parasites to the newest chemotherapeutic anti-malarial agents are the biggest challenges against malaria control programs. Therefore, developing a novel effective treatment to reduce the overgrowing burden of multidrug resistant malaria is a pressing need. Herein, we have developed a biocompatible and biodegradable, non-toxic chitosan-tripolyphosphate-chloroquine (CS-TPP CQ) nanoparticle. CS-TPP CQ nanoparticles effectively kill the parasite through redox generation and induction of the pro- and anti-inflammatory cytokines in both sensitive and resistant parasite in vitro. The in vitro observations showed a strong inhibitory effect (p < 0.01) on pro-inflammatory cytokines more specifically on TNF-α and IFN-γ whereas CS-TPP CQ nanoparticles significantly elevated the anti-inflammatory cytokines- IL-10 and TGF-ß. In addition, CS-TPP CQ nanoparticle significantly increased NO generation (p < 0.01) and altered the GSH/GSSG ratio 72 h after parasite co-culture with peripheral blood mononuclear cells culminating in the free radical induced parasite killing. CS-TPP CQ nanoparticle had an effective dose of 100 ng/ml against CQ-sensitive parasite lines (p < 0.001) whereas effective dose against CQ-resistant parasite line was 200 ng/ml CS-TPP CQ with an effective duration of 72 h (p < 0.001). Our studies suggest that CS-TPP CQ nanoparticle has a potential to modulate the pro- and anti-inflammatory responses, and to trigger the redox-mediated parasite killing. It can be a novel nano-based futuristic approach towards malaria control.

Immunostimulatory effect of chitosan conjugated green copper oxide nanoparticles in tumor immunotherapy.

Current study demonstrates the immunogenic role of biopolymer coated green synthesized copper oxide nanoparticles by the induction of cellular immunity through the activation immune cells. Alongside humoral immunity response was triggered by the surface coated NPs through IgG response which indicate the adjuvanic role of the nano conjugate. Th1 (Type 1 and Type 2 helper T cells) and Th2 cells were activated after the treatment with nano conjugate and act as an immunostimulant which would inhibit the proliferation of breast cancer (MCF-7) and cervical cancer (HeLa) cells in in vitro. Solid tumor induced by 4 T1 cells were also inhibited in in vivo Balb/C mice model. Secretion of pro-inflammatory cytokines and the increase in CD + 4 populations indicate the activation of immune cells in the current study. Immunotherapy by the help of metal nano conjugate can be an effective tool to eradicate the cancer cells from the system.

Leishmania major adenylate kinase immunization offers partial protection to a susceptible host.

Leishmania major causes mild-to-severe cutaneous lesions resulting in significant disfigurations, if untreated. The drugs are toxic, and drug-resistance parasites are emerging. Therefore, a prophylactic vaccination is an urgent need. As no vaccine is available, we compared the genes expressed by virulent and avirulent parasites. We identify L major adenylate kinase (AdeK) as a probable vaccine candidate after a series of experimentations. We cloned the gene in mammalian pcDNA6/HisA and pet28a+ vector for in vivo expression following immunization and in vitro protein expression for booster, respectively. We observed that immunization of susceptible BALB/c mice with AdeK resulted in significant protection against L major challenge infection. The protection was accompanied by increased IFN-γ producing lymphocytes and reduced IL-4, IL-17 and IL-10 secreting central and effector Th2, Th17 and Treg memory cells, respectively. These observations indicate L major AdeK as a potential vaccine candidate.

LmjMAPK10 offers protection against Leishmania donovani infection.

AIMS: This study aimed at evaluating the DNA vaccination efficacy of Leishmania major-derived MAPK10 against Leishmania donovani infection. METHODS AND RESULTS: MAPK10 is one of the 15 mitogen-activated protein kinases (MAPKs) of Leishmania major. Herein, we expressed the gene through a mammalian vector and tested whether priming with this gene would offer protection against L donovani infection. We report that LmjMAPK10 DNA vaccination using a mammalian expression vector significantly reduces the parasite burden. The protection is accompanied by host-protective T-cell functions, TH 1-type cytokines and elevated leishmanial antigen-specific IgG2a isotype response. T-cell response to the L donovani/challenge infection is associated with increase in IL-12 and IFN-γ, but reduced IL-10 and IL-4 production. CONCLUSIONS: LmjMAPK10 is cross-protective against L donovani infection.

Novel pfkelch13 Gene Polymorphism Associates With Artemisinin Resistance in Eastern India.

BACKGROUND: Artesunate-sulfadoxine-pyrimethamine (ASSP) is the frontline artemisinin combination therapy (ACT) in India. Random, irrational, subtherapeutic 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. METHODS: This study involved 226 patients with uncomplicated Plasmodium falciparum infection who had successfully completed the 42 days follow-up after ASSP 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). FINDINGS: Slow-clearing infections with longer parasite clearance half-lives (>5 hours) were observed in 12% isolates. Cure rate after ASSP treatment was declining to about 84.1%. ASSP failure was recorded in 15.9% (early treatment failure, 7.9%; late treatment failure, 7.9%) of isolates. In sum, 24 patients (10.6%) had parasite clearance half-lives greater than 5 hours with pfkelch13 polymorphism after 441 codon; in 15 of those patients (6.6%), parasites had not cleared by 72 hours after initiation of therapy. Median ex vivo ring-stage survival rate of these isolates was very high (12.2%; 95% confidence interval [CI], 10.9-13.8) from that of cured patients (0.9%; 95% CI, 0.09-1.07). Of these 15 patients, 13 patients had pfkelch13 G625R polymorphism, whereas 2 patients contained R539T polymorphism. As per the World Health Organization guideline, these 15 isolates were true artemisinin-resistant isolates. INTERPRETATION: Identification of artemisinin-resistant isolates in India together with new mutations and increasing combination therapy failures blow alarms for urgent malaria control.

Antigen conjugated nanoparticles reprogrammed the tumor-conditioned macrophages toward pro-immunogenic type through regulation of NADPH oxidase and p38MAPK.

Tumor associated macrophages (TAMs) are pertinent to cancer cell growth in the tumor microenvironment. Indeed, TAMs differentiate from monocytes (MΦ) due to specific growth factors present in the tumor microenvironment. TAMs show mostly an M2-like phenotype is due to the absence of pro-inflammatory signals and supply fuel to tumor growth. Several attempts have been taken to switch TAMs into a pro-immunogenic type. To address context, we used a tumor microenvironment by in vitro coculturing human blood MΦ with cancer cell conditioned media (TC-MΦ). We showed that the antigen cobalt oxide nanoparticles (Ag-NPs) can reprogram TC-MΦ to pro-immunogenic type to build up an antitumor immune response. Our results demonstrate that NPs-Ag induced a marked activation of NADPH oxidase in TC-MΦ, likely through stimulation of ROS linked to activation of p38 MAPK. These activated p38 MAPK up-regulated the IFN-γ, TNF-α and initial IL-12 production, in turn, the activation of IFN-γ prolonged IL-12 production.

Surface modification minimizes the toxicity of silver nanoparticles: an in vitro and in vivo study.

Currently toxicological research in Silver nanoparticle is a leading issue in medical science. The surface chemistry and physical dimensions of silver nanoparticles (Ag-NPs) play an important role in toxicity. The aim of this present study was to evaluate the in vitro and in vivo toxicity of Ag-NPs as well as the alteration of toxicity profile due to surface functionalization (PEG and BSA) and the intracellular signaling pathways involved in nanoparticles mediated oxidative stress and apoptosis in vitro and in vivo system. Ag-NPs released excess Ag+ ions leads to activation of NADPH oxidase and helps in generating the reactive oxygen species (ROS). Silver nanoparticles elicit the production of excess amount of ROS results activation of TNF-α. Ag-NPs activates caspase-3 and 9 which are the signature of mitochondrial pathway. Ag-NPs are responsible to decrease the antioxidant enzymes and imbalance the oxidative status into the cells but functionalization with BSA and PEG helps to protect the adverse effect of Ag-NPs on the cells. This study suggested that Ag-NPs are toxic to normal cells which directly lead with human health. Surface functionalization may open the gateway for further use of Ag-NPs in different area such as antimicrobial and anticancer therapy, industrial use or in biomedical sciences.

One-pot synthesis of multifunctional nanoscale metal-organic frameworks as an effective antibacterial agent against multidrug-resistant Staphylococcus aureus.

Drug-resistant bacteria are an increasingly serious threat to global public health. In particular, infections from multidrug-resistant (MDR) Gram-positive bacteria (i.e. Staphylococcus aureus) are growing global health concerns. In this work, we report the first use of nanoscale metal-organic frameworks (NMOFs) coencapsulating an antibiotic (vancomycin) and targeting ligand (folic acid) in one pot to enhance therapeutic efficacy against MDR S. aureus. Zeolitic imidazolate framework (ZIF-8) NMOFs, which have globular morphologies coencapsulating vancomycin and folic acid, are characterized by transmission electron microscopy, field-emission scanning electron microscopy, powder x-ray diffraction, ulltraviolet-visible spectroscopy, and dynamic light-scattering techniques. We determined that the presence of folic acid on the surface of the NMOFs is significant in the sense of effective uptake by MDR S. aureus through endocytosis. The functionalized NMOFs transport vancomycin across the cell wall of MDR S. aureus and enhance antibacterial activity, which has been confirmed from studies of the minimum inhibitory concentration, minimum bactericidal concentration, cytotoxicity of bacterial cells, and generation of reactive oxygen species. This work shows that functionalized NMOFs hold great promise for effective treatment of MDR S. aureus.

In vitro dose and duration dependent approaches for the assessment of ameliorative effects of nanoconjugated vancomycin against Staphylococcus aureus infection induced oxidative stress in murine peritoneal macrophages.

AIMS: The present study was aimed to evaluate the in vitro ameliorative effect of nanoconjugated vancomycin (NV) against vancomycin sensitive and resistant strains of Staphylococcus aureus infection-induced oxidative stress in murine peritoneal macrophage. METHODS: Peritoneal macrophages from mice were treated with VSSA and VRSA (5 × 10(6) CFU/mL), VSSA + NV (5-250 µg/ml) and VRSA + NV (5-250 µg/ml) for 18 h, having 3 h interval in culture media; and the superoxide anion generation, lipid peroxidation, protein oxidation, antioxidant enzymes status and glutathione enzymes activity were monitored. RESULTS: The significantly increased free radical generation, lipid peroxidation, protein carbonyls and oxidized glutathione levels were observed in VSSA and VRSA treated group as compared to control group; where as reduced glutathione level, antioxidant enzymes status and glutathione dependent enzymes were decreased significantly. All these changes come near to control in NV treated group in a dose and duration dependent fashion. Among the different doses and duration intervals of NV, maximum ameliorative effect was observed by 100 µg/ml for 12 h treatment which does not produce any damage to the cell. CONCLUSIONS: These findings suggest the potential use and beneficial role of nanoconjugated vancomycin as a modulator of S. aureus infection-induced cellular damage in murine peritoneal macrophage.

Low prevalence of dihydro folate reductase (dhfr) and dihydropteroate synthase (dhps) quadruple and quintuple mutant alleles associated with SP resistance in Plasmodium vivax isolates of West Bengal, India.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Correction: Pyrido[1,2-a]pyrimidinium ions - a novel bridgehead nitrogen heterocycles: synthesis, characterisation, and elucidation of DNA binding and cell imaging properties.

Correction for 'Pyrido[1,2-a]pyrimidinium ions - a novel bridgehead nitrogen heterocycles: synthesis, characterisation, and elucidation of DNA binding and cell imaging properties' by Susanta Kumar Manna et al., Org. Biomol. Chem., 2015, 13, 8037-8047.

SHP-1 plays a crucial role in CD40 signaling reciprocity.

CD40 plays dual immunoregulatory roles in Leishmania major infection and tumor regression. The functional duality emerges from CD40-induced reciprocal p38MAPK and ERK-1/2 phosphorylations. Because phosphotyrosine-based signaling in hematopoietic cells is regulated by the phosphotyrosine phosphatase SHP-1, which is not implied in CD40 signaling, we examined whether SHP-1 played any roles in CD40-induced reciprocal signaling and anti-leishmanial function. We observed that a weaker CD40 stimulation increased SHP-1 activation. ERK-1/2 inhibition or p38MAPK overexpression inhibited CD40-induced SHP-1 activation. An ultra-low-dose, CD40-induced p38MAPK phosphorylation was enhanced by SHP-1 inhibition but reduced by SHP-1 overexpression. A reverse profile was observed with ERK-1/2 phosphorylation. SHP-1 inhibition reduced syk phosphorylation but increased lyn phosphorylation; syk inhibition reduced but lyn inhibition enhanced CD40-induced SHP-1 phosphorylation. Corroborating these findings, in L. major-infected macrophages, CD40-induced SHP-1 phosphorylation increased and SHP-1 inhibition enhanced CD40-induced p38MAPK activation and inducible NO synthase expression. IL-10 enhanced SHP-1 phosphorylation and CD40-induced ERK-1/2 phosphorylation but reduced the CD40-induced p38MAPK phosphorylation, whereas anti-IL-10 Ab exhibited reverse effects on these CD40-induced functions, identifying IL-10 as a crucial element in the SHP-1-MAPK feedback system. Lentivirally overexpressed SHP-1 rendered resistant C57BL/6 mice susceptible to the infection. Lentivirally expressed SHP-1 short hairpin RNA enhanced the CD40-induced L. major parasite killing in susceptible BALB/c mice. Thus, we establish an SHP-1-centered feedback system wherein SHP-1 modulates CD40-induced p38MAPK activation threshold and reciprocal ERK-1/2 activation, establishing itself as a critical regulator of CD40 signaling reciprocity and mechanistically re-emphasizing its role as a potential target against the diseases where CD40 is involved.

Phosphonomethyl iminodiacetic acid-conjugated cobalt oxide nanoparticles liberate Co(++) ion-induced stress associated activation of TNF-α/p38 MAPK/caspase 8-caspase 3 signaling in human leukemia cells.

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.

Pyrido[1,2-a]pyrimidinium ions--a novel bridgehead nitrogen heterocycles: synthesis, characterisation, and elucidation of DNA binding and cell imaging properties.

A novel class of bridgehead nitrogen heterocycles, pyrido[1,2-a]pyrimidinium ions, has been readily synthesized by a two-step one-pot reaction in high yields (up to 93%). These ionic compounds are bench stable and moisture tolerant and have highly fluorescent properties (quantum yield up to 0.65). A characteristic bright bluish fluorescence was observed in polar solvents such as acetonitrile and fluorescent intensity gradually diminishes with decreasing the polarity of the medium, which becomes almost negligible in toluene. These compounds also show interesting bioactivity. DNA interaction, imaging, and viability experiments with human leukemic Jurkat and KG-1A cells revealed that they are potential candidates for cancer diagnosis.

Self assembled nano fibers of betulinic acid: A selective inducer for ROS/TNF-alpha pathway mediated leukemic cell death.

The main complication in betulinic acid (BA) based drug delivery has been observed due to its bulk structure. The present study demonstrates the potential effects of self assembled nano size betulinic acid (SA-BA) by treating human leukemic cell lines (KG-1A and K562) and its normal counterpart. Self assembly property of BA was investigated using SEM and DLS study which showed that the BA forms fibrous structure having few nanometers in diameter. Selective anti-leukemic efficacy study of SA-BA was investigated by cell viability assay. Mode of leukemic cell death and probable pathway of apoptosis was monitored by measuring ROS level, pro and anti-inflammatory cytokine status and expression of caspase-8 and caspage-3 by immunocytochemistry. Higher efficacy of SA-BA over non-assemble BA was monitored toward cancer cells, with no relevant toxicity to normal blood cells. SA-BA facilitated reactive oxygen species (ROS) mediated leukemic cell death, confirmed by pre-treatment of N-acetyl-L-cysteine. Induction of apoptosis by SA-BA treatment increased pro-inflammatory cytokines, specifically potentiated TNF-α mediated cell death, confirmed by expression of caspase-8 and caspage-3 by immunocytochemistry. This study explored the better anti-leukemic efficacy of SA-BA over BA and this modification will enrich the use of BA in cancer therapy.

Cobalt oxide nanoparticles induced oxidative stress linked to activation of TNF-α/caspase-8/p38-MAPK signaling in human leukemia cells.

The purpose of this study was to determine the intracellular signaling transduction pathways involved in oxidative stress induced by nanoparticles in cancer cells. Activation of reactive oxygen species (ROS) has some therapeutic benefits in arresting the growth of cancer cells. Cobalt oxide nanoparticles (CoO NPs) are an interesting compound for oxidative cancer therapy. Our results showed that CoO NPs elicited a significant (P <0.05) amount of ROS in cancer cells. Co-treatment with N-aceyltine cystine (an inhibitor of ROS) had a protective role in cancer cell death induced by CoO NPs. In cultured cells, the elevated level of tumor necrosis factor-alpha (TNF-α) was noted after CoO NPs treatment. This TNF-α persuaded activation of caspase-8 followed by phosphorylation of p38 mitogen-activated protein kinase and induced cell death. This study showed that CoO NPs induced oxidative stress and activated the signaling pathway of TNF-α-Caspase-8-p38-Caspase-3 to cancer cells.

Redox sensing and signaling by malaria parasite in vertebrate host.

Plasmodium parasites, which is responsible to cause malaria, are also exceedingly receptive to oxidative stress during their intraerythrocytic life stage as they devour haemoglobin inside their food vacuoles and engender toxic haem moieties and reactive oxygen species (ROS). Other than, several studies suggest that the generation of reactive oxygen and nitrogen species (ROS and RNS) associated with oxidative stress, plays a decisive role in the ripeness of systemic complications caused by malaria. Malaria infection provokes the generation of hydroxyl radicals (OH(•)), which most probably is the main reason for the induction of oxidative stress and apoptosis. In this study, it has been described to understand how redox molecules and NO carry out their diverse functions in both parasites and host. It is very important to understand the chemical reactions that produce those outcomes and how its regulation carried out by parasite during erythrocytic phase.