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A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8+ T Cells
Get PDFThe most effective strategy for protection against intracellular infections such as Leishmania is
vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live
vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8+cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4+ and CD8+ T cell responses for longterm protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very
efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to
efficient presentation of antigen to specific CD4+ and CD8+ T cells. Furthermore, lymphoid CD8+ T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8+ T cell responses when compared to free and liposomal ntigen. These liposomal formulations presented to CD8+ T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine
DNA damage-induced ephrin-B2 reverse signalling promotes chemoresistance and drives EMT in colorectal carcinoma harboring mutant p53
No full textMutation in the TP53 gene positively correlates with increased incidence of chemoresistance in different cancers. In this study, we investigated the mechanism of chemoresistance and epithelial-to-mesenchymal transition (EMT) in colorectal cancer involving the gain-of-function (GOF) mutant p53/ephrin-B2 signaling axis. Bioinformatic analysis of the NCI-60 data set and subsequent hub
prediction identified EFNB2 as a possible GOF mutant p53 target gene, responsible for chemoresistance. We show that the mutant p53-NF-Y complex transcriptionally upregulates EFNB2 expression in response to DNA damage. Moreover, the acetylated form of mutant p53 protein is recruited on the EFNB2 promoter and positively regulates its expression in conjunction with coactivator p300. In vitro cell line and in vivo nude mice data show that EFNB2 silencing restores chemosensitivity in mutant p53-harboring tumors. In addition, we observed high expression of EFNB2 in patients having neoadjuvant non-responder colorectal carcinoma
compared with those having responder version of the disease. In the course of deciphering the drug resistance mechanism, we also show that ephrin-B2 reverse signaling induces ABCG2 expression after drug treatment that involves JNK-c-Jun signaling in mutant p53 cells. Moreover, 5-fluorouracil-induced ephrin-B2 reverse signaling promotes tumorigenesis through the Src-ERK pathway, and drives EMT via the Src-FAK pathway. We thus conclude that targeting ephrin-B2 might enhance the therapeutic potential of DNA-damaging chemotherapeutic agents in mutant p53-bearing human tumor
Functional and Structural Analyses of CYP1B1 Variants Linked to Congenital and Adult-Onset Glaucoma to Investigate the Molecular Basis of These Diseases
Get PDFGlaucoma, the leading cause of irreversible blindness, appears in various forms. Mutations in CYP1B1 result in primary congenital glaucoma (PCG) by an autosomal recessive mode of inheritance while it acts as a modifier locus for primary open angle glaucoma (POAG). We investigated the molecular basis of the variable phenotypes resulting from the defects in CYP1B1 by using subclones of 23 CYP1B1 mutants reported in glaucoma patients, in a cell
based system by measuring the dual activity of the enzyme to metabolize both retinol and 17β-estradiol. Most variants linked to POAG showed low steroid metabolism while null or
very high retinol metabolism was observed in variants identified in PCG. We examined the translational turnover rates of mutant proteins after the addition of cycloheximide and observed that the levels of enzyme activity mostly corroborated the translational turnover
rate. We performed extensive normal mode analysis and molecular-dynamics-simulationsbased structural analyses and observed significant variation of fluctuation in certain segmental parts of the mutant proteins, especially at the B-C and F-G loops, which were previously shown to affect the dynamic behavior and ligand entry/exit properties of the cytochrome P450 family of proteins. Our molecular study corroborates the structural analysis,and suggests that the pathologic state of the carrier of CYP1B1 mutations is determined by the allelic state of the gene. To our knowledge, this is the first attempt to dissect biological
activities of CYP1B1 for correlation with congenital and adult onset glaucomas
Cholesterol Corrects Altered Conformation of MHC-II Protein in Leishmania donovani Infected Macrophages: Implication in Therapy
Get PDFPreviously we reported that Kala-azar patients show progressive decrease in serum cholesterol as a function of splenic parasite burden. Splenic macrophages (MΦ) of Leishmania donovani (LD) infected mice show decrease in membrane cholesterol, while LD infected macrophages (I-MΦ) show defective T cell stimulating ability that could be corrected by liposomal delivery of cholesterol. T helper cells recognize peptide antigen in the context of
class II MHC molecule. It is known that the conformation of a large number of membrane proteins is dependent on membrane cholesterol. In this investigation we tried to understand the influence of decreased membrane cholesterol in I-MΦ on the conformation of MHC-II protein and peptide-MHC-II stability, and its bearing on the antigen specific T-cell activatio
Importance of calcium in Parkinson’s disease pathophysiology
No full textParkinson Disease (PD) is the most common progressive neurodegenerative movement disorder
characterized by preferential loss of ventral midbrain dopaminergic neurons of substantia nigra
pars compacta. It has been recently suggested that high Ca2+ levels due to Cav1.3 channeldependent
pacemaking activity may contribute to SNpc susceptibility in PD. Based on this, in the
present study, we hypothesized that perturbed neuronal calcium homeostasis is one of the major
factors in the genesis of PD, and examined the mechanism of dopaminergic neuronal protection
provided by nimodipine, an L-type calcium channel blocker in cellular and animal models of the
disease. We used the pakinsonian neurotoxin, MPP+ in SH-SY5Y, MPTP in mice to induce PD
pathology and/or behavioral phenotypes and employed parkinsonian cybrids to study the PD
phenotype. Cell viability via MTT assay, calcium imaging employing Fura-2AM, mitochondrila
morphology by Mitotracker Green and mitochondrial membrane potential using TMRM were
investigated in SH-SY5Y cells. Animal behaviors were monitored, striatal dopamine levels were
measured employing HPLC-electrochemical detection procedure and nigral neuronal loss was
determined using tyrosine hydroxylase immunohistochemistry. Mitochondrial integrity and their
functions were examined using DCFDA fluorescence and oxygen consumption analysis. In SHSY5Y
cells, pretreatment with nimodipine attenuated MPP+-mediated increase in intracellular
calcium, reduction in cell viability, decrease mitochondrial membrane potential and corrected the
aberrations in mitochondrial morphology. In mice, MPTP-induced behavioral dysfunctions
(reduced swimming ability, akinesia and catalepsy), striatal dopamine depletion, increases in
mitochondrial reactive oxygen species generation and loss in mitochondrial respiration were
corrected by nimodipine administration. The level of intracellular calcium was elevated in PD
cybrids. Further, the expression of calcium binding protein calbindin as measured by real time
PCR and Immunoblot, was found to be decreased while that of calcium activated phosphatase
calcineurin B and protease calpain protein levels were found to be increased after the neurotoxin
treatment, while this trend was significantly attenuated following nimodipine treatment (both in
vitro and in vivo). These results suggest that the L-type calcium channel blocker, nimodipine
protects against the parkinsonian neurotoxin-induced cell death by correcting the defects in
mitochondrial integrity and functions, caused by increased intracellular calcium in dopaminergic
neurons
A Mechanistic Approach to Determine the Anticancer and Antimetastatic Potential of Attenuated Strain of L. Donovani and Its Membrane Lipoprotein Via Regulation of Different Immunological Factors
No full textEach year, millions of people are diagnosed worldwide with cancer, and more than half of these patients eventually die from this disease. In 2015, 27.9 million new cancer cases were diagnosed, and the number of deaths caused by this disease reached 16.7 million.Conventional cancer treatments, such as surgery, chemotherapy, and radiotherapy, often fail to achieve a complete cancer remission. Moreover, it has been widely recognized that radiotherapy and/or chemotherapy are likely to cause significant side effects. This has prompted the
development of many new approaches for the treatment of cancer. One such example involves the use of live or heat-killed attenuated microbes or their purified products. The use of microbial products for the treatment of cancer was pioneered by Dr. William B. Coley, who in 1891 treated patients with cancer with intra-tumoural injections of live Streptococcus pyogenes and,
subsequently, with mixtures of S. pyogenes and Serratia marcescens. The use of biotherapy reduced the cytotoxic side effects of the chemotherapeutic drugs and also was found to raise an immune response. For example, BCG which was earlier only used as a vaccine against
mycobacterium was successfully used in different cancers including acute lymphoblastic leukemia, melanoma, bladder cancer and etc. In the late 90s, biotherapy became very popular when different microbial cellular components mainly the membrane proteins and lipids were also
found to be highly potent against different cancers. Azurin and FTY720 are two membrane components that are already being patented and being approved by FDA as drugs against cancer and immune suppressive disease
A Mechanistic Study of the Estrogenic Regulation of Casein Kinase 2 alpha (CK2α) in Breast Cancer
No full textCasein kinase 2 alpha (CK2α) is a ubiquitously expressed and highly conserved
serine/threonine kinase. Protein kinase CK2α is frequently upregulated in different cancers.
Alteration of CK2α expression and its activity is sufficient to induce dramatic changes in cell
fate. Though the expression level of this vital kinase is strictly maintained under normal
cellular condition yet its deregulation has frequently been observed in colorectal, prostate and
lung cancer. It has been established that CK2α induces oncogenesis through modulation of
both AKT and PML. CK2α has been found to be overexpressed in breast cancer. In contrary,
statistical reports have shown low level of PML. However, the cause and consequences of its
overexpression are not fully understood. On the other hand, a majority of breast cancer cases
are estrogen receptor alpha (ERα) mediated where ER acting as a transcription factor plays a
key role. In recent years, it has been established that PML, a tumour suppressor is degraded
upon CK2 mediated phosphorylation. These prompted us to investigate the relationship that
the two important molecules (ERα and CK2α) share in breast cancer cells. In the current
study, we found CK2α and activated AKT positively correlate with ERα, whereas PML follows
an inverse correlation in human breast cancer tissues. Modulation of ERα signaling leads to
recruitment of activated ERα on the ERE sites of CK2α promoter, resulting in CK2α
transactivation. Furthermore, the DMBA induced tumours in rat showed elevated level of
active CK2α. Consequently it mediates enhancement of AKT activity and PML degradation,
resulting in increased cellular proliferation, migration and metastasis. Syngeneic ERα
overexpressing stable mouse 4T1 cells produce larger primary tumours and metastatic lung
nodules in mice, corroborating our in vitro findings. Hence, our study provides a novel route
of ERα dependent CK2α mediated oncogenesis that causes upregulation and consequent
AKT activation along with degradation of tumour suppressor PML
Leishmania donovani Exploits Myeloid Cell Leukemia 1(MCL-1) Protein to Prevent Mitochondria-dependent Host Cell Apoptosis
No full textApoptosis is one of the mechanisms used by host cells to
remove unwanted intracellular organisms, and often found to be
subverted by pathogens through use of host anti-apoptotic proteins.
In the present study, with the help of in vitro and in vivo
approaches, we documented that the macrophage anti-apoptotic
protein myeloid cell leukemia 1 (MCL-1) is exploited by the
intra-macrophage parasite Leishmania donovani to protect
their “home” from actinomycin D-induced mitochondria-dependent apoptosis. Among all the anti-apoptotic BCL-2 family members, infection preferentially up-regulated expression of MCL-1 at both themRNAand protein levels and compared with infected control, MCL-1-silenced infected macrophages documented enhanced caspase activity and increased apoptosis when subjected to actinomycin D treatment. Phosphorylation kinetics and ChIP assay demonstrated that infection-induced MCL-1 expression was regulated by transcription factor CREB(cAMP-response element-binding protein) and silencing of
CREB resulted in reduced expression of MCL-1 and increased
apoptosis. During infection, MCL-1 was found to be localized in mitochondria and this was significantly reduced in Tom70-silencedMCL-1 transport. In the mitochondria, MCL-1 interacts with the major pro-apoptotic protein BAK and prevents BAK-BAK
homo-oligomer formation thereby preventing cytochrome c
release-mediated mitochondrial dysfunction. Silencing of MCL-1 in the spleen of infected mice showed decreased parasite burden and increased induction of splenocyte apoptosis. Collectively our results showed that L. donovani exploited the macrophage anti-apoptotic protein MCL-1 to prevent BAK-mediated mitochondria-dependent apoptosis thereby protecting its niche, which is essential for disease progression.
macrophages, suggesting the active role of TOM70 i
Evolutionary genomics of epidemic visceral leishmaniasis in the Indian subcontinent
Get PDFLeishmania donovani causes visceral leishmaniasis (VL), the second most deadly vector-borne parasitic disease. A recent epidemic in the Indian subcontinent (ISC) caused up to 80% ofglobal VL and over 30,000 deaths per year. Resistance against antimonial drugs has probably been a contributing factor in the persistence of this epidemic. Here we use whole genome sequences from 204 clinical isolates to track the evolution and epidemiology of L. donovani from the ISC. We identify independent radiations that have emerged since a bottleneck coincident with 1960s DDT spraying campaigns. A genetically distinct population frequently resistant to antimonials has a two base-pair insertion in the aquaglyceroporin gene LdAQP1 that prevents the transport of trivalent antimonials. We find evidence of genetic exchange between ISC populations, and show that the mutation in LdAQP1 has spread by recombination. Our results reveal the complexity of L. donovani evolution in the ISC in response to drug treatment
Molecular mechanisms of virulence gene regulation in gastric cell adhered helicobacter pylori and development of therapeutic nano-particles
No full textHelicobacter pylori is a major human pathogen and is associated with chronic gastric inflammation, peptic ulcer disease and gastric cancer. In this study, it was observed
that adherence of H. pylori to the gastric epithelial cell line AGS strongly induces expression of the genes HP0102, fliK and cagA. HP0102 was identified to be involved
in the glycosylation of bacterial lipopolysachharides (LPS), whereas fliK is known to be required for flagellar biogenesis. One of the striking features of gastric epithelial cells (AGS) infected with ΔHP0102 and ΔfliK strains is their reduced “hummingbird” appearance and IL-8 secretion, a phenomenon attributed to the expression and translocation of the bacterial CagA protein, a major virulence factor. Interestingly, upregulation of cagA expression was not observed in AGS adhered H.pylori ΔHP0102 and ΔfliK mutants. Subsequently, it was identified that both HP0102 and fliK independently influence the expression of the alternative factor 28. Induction of fliA, encoding 28 was also impaired in the mutants following
adherence. Furthermore, it was identified that direct binding of σ28-RNAP initiated transcription of cagA from an alternative downstream promoter active specifically in
AGS-adhered wild type H. pylori but not in ΔHP0102 and ΔfliK strains. Thus, in this study we have identified that HP0102 and FliK are involved in the regulation of
cagA expression in host cell adhered H. pylori. Further investigation suggested that the chemotaxis and the acid escape response of the ΔHP0102 strain was affected, phenotypes that might account for its previously
reported deficiency in mice colonization. Finally, proteome analysis suggested that HP0102 is not only involved in the regulation of cagA but also in the fine tuning of
the expression of several other genes. A small non-coding RNA, HPnc2630 was also one of the genes strongly upregulated following adherence of H. pylori to AGS cells. Transcriptome analysis revealed that HPnc2630 regulates the expression of several genes, thereby influencing the
pathogenesis of the bacteria. Affinity capture studies suggested that this sRNA interacts with specific proteins CytC peroxidase & γ-glutamyl transpeptidase (GGT)
and probably sequesters them. GGT is a known positive modulator of VacA activity. Interestingly, a ΔsRNA mutant strain exhibited increased ability to adhere to AGS
cells and induced more vacuolation in these cells, thereby confirming the role of the sRNA in the regulation of VacA.
In the final part of the study, Polyethyleneinime (PEI) capped ZnO nano-particles (NPs) have been developed as a putative therapeutic. These have better dispersion,
stability under low pH conditions and are more potent when compared to the conventional ZnO NPs in their ability to inhibit the growth of antibiotic resistant H. pylori. Furthermore, they internalize into the bacterial cells, generate huge amounts of reactive oxygen species (ROS), leading to membrane damage and morphological
transition from the rod to coccoid form along with concomitant degradation of stable RNA molecules in these cells. Toxicity studies suggest that a dose < 25 μg/ml
is safe for use in human cells. At this concentration, ZnO-PEI NPs show notable synergy in combination with antibiotics to inhibit H. pylor