Neem leaf glycoprotein binding to Dectin-1 receptors on dendritic cell induces type-1 immunity through CARD9 mediated intracellular signal to NFκB.

BACKGROUND: A water-soluble ingredient of mature leaves of the tropical mahogany 'Neem' (Azadirachta indica), was identified as glycoprotein, thus being named as 'Neem Leaf Glycoprotein' (NLGP). This non-toxic leaf-component regressed cancerous murine tumors (melanoma, carcinoma, sarcoma) recurrently in different experimental circumstances by boosting prime antitumor immune attributes. Such antitumor immunomodulation, aid cytotoxic T cell (Tc)-based annihilation of tumor cells. This study focused on identifying and characterizing the signaling gateway that initiate this systemic immunomodulation. In search of this gateway, antigen-presenting cells (APCs) were explored, which activate and induce the cytotoxic thrust in Tc cells. METHODS: Six glycoprotein-binding C-type lectins found on APCs, namely, MBR, Dectin-1, Dectin-2, DC-SIGN, DEC205 and DNGR-1 were screened on bone marrow-derived dendritic cells from C57BL/6 J mice. Fluorescence microscopy, RT-PCR, flow cytometry and ELISA revealed Dectin-1 as the NLGP-binding receptor, followed by verifications through RNAi. Following detection of ß-Glucans in NLGP, their interactions with Dectin-1 were explored in silico. Roles of second messengers and transcription factors in the downstream signal were studied by co-immunoprecipitation, western blotting, and chromatin-immunoprecipitation. Intracellularization of FITC-coupled NLGP was observed by processing confocal micrographs of DCs. RESULTS: Considering extents of hindrance in NLGP-driven transcription rates of the cytokines IL-10 and IL-12p35 by receptor-neutralization, Dectin-1 receptors on dendritic cells were found to bind NLGP through the ligand's peripheral ß-Glucan chains. The resulting signal phosphorylates PKCδ, forming a trimolecular complex of CARD9, Bcl10 and MALT1, which in turn activates the canonical NFκB-pathway of transcription-regulation. Consequently, the NFκB-heterodimer p65:p50 enhances Il12a transcription and the p50:p50 homodimer represses Il10 transcription, bringing about a cytokine-based systemic-bias towards type-1 immune environment. Further, NLGP gets engulfed within dendritic cells, possibly through endocytic activities of Dectin-1. CONCLUSION: NLGP's binding to Dectin-1 receptors on murine dendritic cells, followed by the intracellular signal, lead to NFκB-mediated contrasting regulation of cytokine-transcriptions, initiating a pro-inflammatory immunopolarization, which amplifies further by the responding immune cells including Tc cells, alongside their enhanced cytotoxicity. These insights into the initiation of mammalian systemic immunomodulation by NLGP at cellular and molecular levels, may help uncovering its mode of action as a novel immunomodulator against human cancers, following clinical trials.

NLGP regulates RGS5-TGFß axis to promote pericyte-dependent vascular normalization during restricted tumor growth.

Altered RGS5-associated intracellular pericyte signaling and its abnormal crosstalk with endothelial cells (ECs) result chaotic tumor-vasculature, prevent effective drug delivery, promote immune-evasion and many more to ensure ultimate tumor progression. Moreover, the frequency of lethal-RGS5high  pericytes within tumor was found to increase with disease progression, which signifies the presence of altered cell death pathway within tumor microenvironment (TME). In this study, we checked whether and how neem leaf glycoprotein (NLGP)-immunotherapy-mediated tumor growth restriction is associated with modification of pericytes' signaling, functions and its interaction with ECs. Analysis of pericytes isolated from tumors of NLGP treated mice suggested that NLGP treatment promotes apoptosis of NG2+ RGS5high -fuctionally altered pericytes by downregulating intra-tumoral TGFß, along with maintenance of more matured RGS5neg  pericytes. NLGP-mediated inhibition of TGFß within TME rescues binding of RGS5 with Gαi and thereby termination of PI3K-AKT mediated survival signaling by downregulating Bcl2 and initiating pJNK mediated apoptosis. Limited availability of TGFß also prevents complex-formation between RGS5 and Smad2 and rapid RGS5 nuclear translocation to mitigate alternate immunoregulatory functions of RGS5high  tumor-pericytes. We also observed binding of Ang1 from pericytes with Tie2 on ECs in NLGP-treated tumor, which support re-association of pericytes with endothelium and subsequent vessel stabilization. Furthermore, NLGP-therapy- associated RGS5 deficiency relieved CD4+  and CD8+ T cells from anergy by regulating 'alternate-APC-like' immunomodulatory characters of tumor-pericytes. Taken together, present study described the mechanisms of NLGP's effectiveness in normalizing tumor-vasculature by chiefly modulating pericyte-biology and EC-pericyte interactions in tumor-host to further strengthen its translational potential as single modality treatment.

Tumor activated platelets induce vascular mimicry in mesenchymal stem cells and aid metastasis.

Extent of metastasis influences activation of platelets in tumor-microenvironment. Activated platelets potentiate mesenchymal-stem-cells (MSCs) to migrate in secondary metastatic sites without participation in process of invasion. Presence of higher percentage of MSCs along with activated-platelets induces formation of vascular-mimicry (VM). The pathophysiology, VM, has already been reported in multiple types of cancer including lung, ovary, melanoma etc. and related to poor-prognosis. Interaction of MSCs with platelets in cell-to-cell contact dependent manner is essential for their migration, thereby, VM. Evidences are obtained suggesting that under influence of tumor-associated-activated-platelets, expressions of vimentin, ve-cadherin are increased, along with decrease in e-cadherin on CD105+ MSCs in both mRNA and protein levels that may help in formation of vessel like structure in VM. Adoptive transfer of MSCs along with tumor-activated-platelets causes greater B16 melanoma metastasis at lungs in comparison to MSCs with non-activated platelets. Presence of CD105+Vimentin+ MSCs in vessel like structure in the metastatic lung confirms the involvement of platelet-activated-MSCs in VM, thereby, in metastasis.

Replica symmetry breaking in coherent and incoherent random lasing modes.

We investigate intensity fluctuations of a weakly scattering optofluidic random laser having coherent and incoherent emission fractions. The coherent part comprises random spikes, whereas the incoherent part forms a broad pedestal in the emission spectra. Evaluating the fractional ratio of the coherent and incoherent parts of the emission, a replica symmetry breaking phase transition is observed independently in both coherent and incoherent parts of the intensity. Also, the incoherent component has higher non-zero correlation values compared to those of the coherent part, implying a larger contribution to mode coupling. Moreover, survival function analysis reveals a significant contribution of the incoherent part on determining the decay profile of lasing intensity.

Quantitative analysis of hidden particles diffusing behind a scattering layer using speckle correlation.

Speckle-correlation imaging is a family of methods that makes use of the "memory effect" to image objects hidden behind visually opaque layers. Here, we show that a correlation analysis can be applied to quantitative imaging of an ensemble of dynamic fluorescent beads diffusing on a 2D surface. We use an epi-fluorescence microscope where both the illumination and detection light patterns are speckled, due to light scattering by a thin disordered layer. The spatio-temporal cross-correlation of the detection speckle pattern is calculated as a function of lag time and spatial shift and is used to determine the diffusion constant and number of fluorescent particles in the sample without requiring any phase retrieval procedure. It is worth to note that the "memory effect" range is not required to extend beyond a distance of few speckle grains, thus making our method potentially useful for nearly arbitrary values of the thickness of the scattering layer.

Confocal fluorescence correlation spectroscopy through a sparse layer of scattering objects.

In the presence of strong light scattering, as often encountered in biological tissue, optical microscopy becomes challenging and technical demanding. Beside image quality, the quantitative determination of molecular properties is also strongly affected by scattering. We have carried out fluorescence correlation spectroscopy (FCS) experiments, in a solution of fluorophores, through a sparse scattering layer made of dielectric beads. We observe that the fluorescence signal steadily decreases as the focus is moved away from the scattering layer. By contrast, the estimated number of molecules recovers its normal value beyond a characteristic distance of about twice the bead diameters, below which it is strongly biased. Accompanying theoretical modeling demonstrates how diffraction and refraction by the scattering layer and their impact on FCS measurements depend on size and refractive index of the beads.

Carbapenem Resistance in Clonally Distinct Clinical Strains of Vibrio fluvialis Isolated from Diarrheal Samples.

Carbapenems have been used for many years to treat severe nosocomial Enterobacteriaceae infections. The spread of resistance to these drugs among other bacterial families is an emerging problem worldwide, mostly caused by New Delhi metallo-ß-lactamase (NDM-1). We screened for the prevalence of NDM-1-expressing enteric pathogens from hospitalized patients with acute diarrhea in Kolkata, India, and identified 27 Vibrio fluvialis-harboring blaNDM-1 (NDM-VF) strains. These isolates were also resistant to all the tested antimicrobial drugs except doxycycline. The large plasmid of V. fluvialis harboring blaNDM-1 could be easily transferred to other enteric pathogens. Genes flanking the blaNDM-1 were found to be identical to the reported sequence from an Escherichia coli isolate. Analyses showed that the V. fluvialis possessing the NDM-VF region belonged to different clones. The pathogenicity of V. fluvialis to humans and its ubiquitous presence in the environment call for constant monitoring of this species for emerging antimicrobial drug resistance.

Molecular evolution and functional divergence of Vibrio cholerae.

PURPOSE OF REVIEW: The purpose of this review is to synopsize and highlight the recent subtle genetic changes in cholera causing toxigenic Vibrio cholerae with special reference to their virulence, integrating and conjugative elements and toxin-antitoxin systems. It is not intended to cover issues on the whole genome sequence and epidemiology of cholera. RECENT FINDINGS: Analyses have been made using major published works on genetic changes associated with potential virulence, integrating and conjugative elements and toxin-antitoxin systems of toxigenic V. cholerae. During the course of evolution, V. cholerae strains show evidence of genetic selection for the expression of additional virulence, better survival in the environment, colonization ability and antimicrobial resistance. Some of the critical modifications that occurred at the molecular level include CTXϕ genome, cholera toxin B-subunit, integrating and conjugative elements and toxin-antitoxin systems. Frequent changes in the genome of V. cholerae appear to be an ongoing dynamic process that is assisting the pathogen to subtly change during or after epidemics of cholera. SUMMARY: Cholera is a reemerging public health problem. Continued basic research is important to understand the changing dynamics of bacterial virulence, survival strategies and disease pathogenesis for efficient therapeutic intervention and to abort transmission of the disease.

Optofluidic two-dimensional grating volume refractive index sensor.

We present an optofluidic reservoir with a two-dimensional grating for a lab-on-a-chip volume refractive index sensor. The observed diffraction pattern from the device resembles the analytically obtained fringe pattern. The change in the diffraction pattern has been monitored in the far-field for fluids with different refractive indices. Reliable measurements of refractive index variations, with an accuracy of 6×10-3 refractive index units, for different fluids establishes the optofluidic device as a potential on-chip tool for monitoring dynamic refractive index changes.

Signatures of periodicity and randomness in the angular emission profile of a 2-D on-average periodic optofluidic random laser.

Angle-dependent emission from a dye infiltrated 2-D on-average periodic structured optofluidic random laser is studied. Distinct signatures of periodicity and randomness are observed in the angle-resolved emission spectra of the device. Emission patterns composed of concentric ellipses are observed on transverse excitation of the device, attributed to the in-plane diffraction of light by a 2-D square lattice. The effect of randomness on the emission spectra is demonstrated by a highly resolved angle-dependent spectral scan of a single diffraction fringe. Finally, we conclude that the randomness in the size of the scatterers resolves the random lasing modes angularly.

Ultrathin MgO diffusion barriers for ferromagnetic electrodes on GaAs(001).

Ultrathin MgO(100) films serving as a diffusion barrier between ferromagnetic electrodes and GaAs(001) semiconductor templates have been investigated. Using Fe as an exemplary ferromagnetic material, heterostructures of Fe/MgO/GaAs(001) were prepared at 200 °C with the MgO thickness ranging from 1.5 to 3 nm. Structural characterization reveals very good crystalline ordering in all layers of the heterostructure. Auger electron spectroscopy depth-profiling and cross-sectional high-resolution transmission electron microscopy evidence diffusion of Fe into MgO and-for too thin MgO barriers-further into GaAs(001). Our results recommend a MgO barrier thickness larger than or equal to 2.6 nm for its application as a reliable diffusion barrier on GaAs(001) in spintronics devices.

Detection of integron-associated gene cassettes and other antimicrobial resistance genes in enterotoxigenic Bacteroides fragilis.

Twenty seven Enterotoxigenic Bacteroides fragilis (ETBF) strains isolated from children in Kolkata, India, were tested for their antimicrobial resistance, presence of integrons and resistance encoding genes. Almost all the strains (>90%) were resistant to two or more antimicrobials. About 59-92% of the strains were resistant to ampicillin, amoxicillin, streptomycin, tetracycline, ciprofloxacin and norfloxacin. Most of these antimicrobial agents have been used in the treatment of diarrhea and other infectious diseases. In addition, about half a number of strains (48-55%) were resistant to clindamycin, cefotaxime, ceftazidime, ampicillin/sulbactam and trimethoprim/sulfamethoxazole. Moxifloxacin and metronidazole resistance ranged from 30 to 40%. All strains however, were found to be susceptible to chloramphenicol and imipenem. Class 1 integrase (intI1) was detected in seven and class 2 integrase (intI2) in one of the twenty seven ETBF strains. Resistance gene cassettes carried by these integrons had different alleles of dfr or aad genes. Beside these integron-borne genes, other genes encoding different antimicrobial resistance were also detected. Resistance genes such as cep(A) and tet(Q) were detected in most of the ETBF strains. To the best of our knowledge, this work constituted the first extensive report from India on the detection of integrons and antimicrobial resistance genes in ETBF.

Neem Leaf Glycoprotein Disrupts Exhausted CD8+ T-Cell-Mediated Cancer Stem Cell Aggression.

Targeting exhausted CD8+ T-cell (TEX)-induced aggravated cancer stem cells (CSC) holds immense therapeutic potential. In this regard, immunomodulation via Neem Leaf Glycoprotein (NLGP), a plant-derived glycoprotein immunomodulator is explored. Since former reports have proven immune dependent-tumor restriction of NLGP across multiple tumor models, we hypothesized that NLGP might reprogram and rectify TEX to target CSCs successfully. In this study, we report that NLGP's therapeutic administration significantly reduced TEX-associated CSC virulence in in vivo B16-F10 melanoma tumor model. A similar trend was observed in in vitro generated TEX and B16-F10/MCF7 coculture setups. NLGP rewired CSCs by downregulating clonogenicity, multidrug resistance phenotypes and PDL1, OCT4, and SOX2 expression. Cell cycle analysis revealed that NLGP educated-TEX efficiently pushed CSCs out of quiescent phase (G0G1) into synthesis phase (S), supported by hyper-phosphorylation of G0G1-S transitory cyclins and Rb proteins. This rendered quiescent CSCs susceptible to S-phase-targeting chemotherapeutic drugs like 5-fluorouracil (5FU). Consequently, combinatorial treatment of NLGP and 5FU brought optimal CSC-targeting efficiency with an increase in apoptotic bodies and proapoptotic BID expression. Notably a strong nephron-protective effect of NLGP was also observed, which prevented 5FU-associated toxicity. Furthermore, Dectin-1-mediated NLGP uptake and subsequent alteration of Notch1 and mTOR axis were deciphered as the involved signaling network. This observation unveiled Dectin-1 as a potent immunotherapeutic drug target to counter T-cell exhaustion. Cumulatively, NLGP immunotherapy alleviated exhausted CD8+ T-cell-induced CSC aggravation. Implications: Our study recommends that NLGP immunotherapy can be utilized to counter ramifications of T-cell exhaustion and to target therapy elusive aggressive CSCs without evoking toxicity.

The immunomodulatory impact of naturally derived neem leaf glycoprotein on the initiation progression model of 4NQO induced murine oral carcinogenesis: a preclinical study.

Introduction: Murine tumor growth restriction by neem leaf glycoprotein (NLGP) was established in various transplanted models of murine sarcoma, melanoma and carcinoma. However, the role of NLGP in the sequential carcinogenic steps has not been explored. Thus, tongue carcinogenesis in Swiss mice was induced by 4-nitroquinoline-1-oxide (4NQO), which has close resemblance to human carcinogenesis process. Interventional role of NLGP in initiation-promotion protocol established during 4NQO mediated tongue carcinogenesis in relation to systemic immune alteration and epithelial-mesenchymal transition (EMT) is investigated. Methods: 4NQO was painted on tongue of Swiss mice every third day at a dose of 25µl of 5mg/ml stock solution. After five consecutive treatment with 4NQO (starting Day7), one group of mice was treated with NLGP (s.c., 25µg/mice/week), keeping a group as PBS control. Mice were sacrificed in different time-intervals to harvest tongues and studied using histology, immunohistochemistry, flow-cytometry and RT-PCR on different immune cells and EMT markers (e-cadherin, vimentin) to elucidate their phenotypic and secretory status. Results: Local administration of 4NQO for consecutive 300 days promotes significant alteration in tongue mucosa including erosion in papillae and migration of malignant epithelial cells to the underlying connective tissue stroma with the formation of cell nests (exophytic-hyperkeratosis with mild dysplasia). Therapeutic NLGP treatment delayed pre-neoplastic changes promoting normalization of mucosa by maintaining normal structure. Flow-cytometric evidences suggest that NLGP treatment upregulated CD8+, IFNγ+, granzyme B+, CD11c+ cells in comparison to 4NQO treated mice with a decrease in Ki67+ and CD4+FoxP3+ cells in NLGP treated cohort. RT-PCR demonstrated a marked reduction of MMP9, IL-6, IL-2, CD31 and an upregulation in CCR5 in tongues from 4NQO+NLGP treated mice in comparison to 4NQO treated group. Moreover, 4NQO mediated changes were associated with reduction of e-cadherin and simultaneous up-regulation of vimentin expression in epithelium that was partially reversed by NLGP. Discussion: Efficacy of NLGP was tested first time in sequential carcinogenesis model and proved effective in delaying the initial progression. NLGP normalizes type 1 immunity including activation of the CD8+T effector functions, reduction of regulatory T cell functions, along with changes in EMT to make the host systemically alert to combat the carcinogenic threat.

An outbreak of foodborne gastroenteritis caused by dual pathogens, Salmonella enterica serovar Weltevreden and Vibrio fluvialis in Kolkata, India.

Salmonella enterica serovar Weltevreden and Vibrio fluvialis were identified as etiological agents of a foodborne gastroenteritis outbreak after an Iftar feast in North Dumdum. Of the 278 cases admitted to the Infectious Diseases Hospital, Kolkata, 44 stool samples were tested for the enteric pathogens. Six were positive for Salmonella Weltevreden, 5 for Vibrio fluvialis, and 8 contained both of the pathogens. Consumption of mutton-ghogni might have been the likely vehicle of this outbreak. In the pulsed-field gel electrophoresis, Salmonella Weltevreden was identified as a single clone but the V. fluvialis strains were heterogeneous.

Terminally Exhausted CD8+ T Cells Resistant to PD-1 Blockade Promote Generation and Maintenance of Aggressive Cancer Stem Cells.

SIGNIFICANCE: Cross-talk with TTEX CD8+ T cells mediated by the VEGFR2 axis induces aggressive properties in cancer stem cells to promote tumor progression.

Cancer stem cell-immune cell crosstalk in breast tumor microenvironment: a determinant of therapeutic facet.

Breast cancer (BC) is globally one of the leading killers among women. Within a breast tumor, a minor population of transformed cells accountable for drug resistance, survival, and metastasis is known as breast cancer stem cells (BCSCs). Several experimental lines of evidence have indicated that BCSCs influence the functionality of immune cells. They evade immune surveillance by altering the characteristics of immune cells and modulate the tumor landscape to an immune-suppressive type. They are proficient in switching from a quiescent phase (slowly cycling) to an actively proliferating phenotype with a high degree of plasticity. This review confers the relevance and impact of crosstalk between immune cells and BCSCs as a fate determinant for BC prognosis. It also focuses on current strategies for targeting these aberrant BCSCs that could open avenues for the treatment of breast carcinoma.

High monocytic MDSC signature predicts multi-drug resistance and cancer relapse in non-Hodgkin lymphoma patients treated with R-CHOP.

Introduction: Non-Hodgkin Lymphoma (NHL) is a heterogeneous lymphoproliferative malignancy with B cell origin. Combinatorial treatment of rituximab, cyclophsphamide, hydroxydaunorubicin, oncovin, prednisone (R-CHOP) is the standard treatment regimen for NHL, yielding a complete remission (CR) rate of 40-50%. Unfortunately, considerable patients undergo relapse after CR or initial treatment, resulting in poor clinical implications. Patient's response to chemotherapy varies widely from static disease to cancer recurrence and later is primarily associated with the development of multi-drug resistance (MDR). The immunosuppressive cells within the tumor microenvironment (TME) have become a crucial target for improving the therapy efficacy. However, a better understanding of their involvement is needed for distinctive response of NHL patients after receiving chemotherapy to design more effective front-line treatment algorithms based on reliable predictive biomarkers. Methods: Peripheral blood from 61 CD20+ NHL patients before and after chemotherapy was utilized for immunophenotyping by flow-cytometry at different phases of treatment. In-vivo and in-vitro doxorubicin (Dox) resistance models were developed with murine Dalton's lymphoma and Jurkat/Raji cell-lines respectively and impact of responsible immune cells on generation of drug resistance was studied by RT-PCR, flow-cytometry and colorimetric assays. Gene silencing, ChIP and western blot were performed to explore the involved signaling pathways. Results: We observed a strong positive correlation between elevated level of CD33+CD11b+CD14+CD15- monocytic MDSCs (M-MDSC) and MDR in NHL relapse cohorts. We executed the role of M-MDSCs in fostering drug resistance phenomenon in doxorubicin-resistant cancer cells in both in-vitro, in-vivo models. Moreover, in-vitro supplementation of MDSCs in murine and human lymphoma culture augments early expression of MDR phenotypes than culture without MDSCs, correlated well with in-vitro drug efflux and tumor progression. We found that MDSC secreted cytokines IL-6, IL-10, IL-1ß are the dominant factors elevating MDR expression in cancer cells, neutralization of MDSC secreted IL-6, IL-10, IL-1ß reversed the MDR trait. Moreover, we identified MDSC secreted IL-6/IL-10/IL-1ß induced STAT1/STAT3/NF-κß signaling axis as a targeted cascade to promote early drug resistance in cancer cells. Conclusion: Our data suggests that screening patients for high titre of M-MDSCs might be considered as a new potential biomarker and treatment modality in overcoming chemo-resistance in NHL patients.

Lack of correlation between catalytic efficiency and basicity of amines during the reaction of aryl methyl ketones with DMF-DMA: an unprecedented supramolecular domino catalysis.

1-Methylimidazole exhibits an unusually high efficiency in promoting the reaction of aryl methyl ketones with DMF-DMA to form (2E)-1-aryl-3-dimethylamino-2-propenones which lacks correlation between the catalytic efficiency and the basicity of 1-methylimidazole in comparison to other amines. An unprecedented supramolecular domino catalysis rationalises the lack of correlation between the catalytic efficiency and basicity of the amines. The supramolecular assemblies have been characterized by mass-spectrometric ion fishing. The time-dependent increase/decrease in the concentration (ion current) of the supramolecular species consolidated the mechanism.