Chimeric Antigen Receptor-T Cell and Oncolytic Viral Therapies for Gastric Cancer and Peritoneal Carcinomatosis of Gastric Origin: Path to Improving Combination Strategies.

Precision immune oncology capitalizes on identifying and targeting tumor-specific antigens to enhance anti-tumor immunity and improve the treatment outcomes of solid tumors. Gastric cancer (GC) is a molecularly heterogeneous disease where monoclonal antibodies against human epidermal growth factor receptor 2 (HER2), vascular endothelial growth factor (VEGF), and programmed cell death 1 (PD-1) combined with systemic chemotherapy have improved survival in patients with unresectable or metastatic GC. However, intratumoral molecular heterogeneity, variable molecular target expression, and loss of target expression have limited antibody use and the durability of response. Often immunogenically "cold" and diffusely spread throughout the peritoneum, GC peritoneal carcinomatosis (PC) is a particularly challenging, treatment-refractory entity for current systemic strategies. More adaptable immunotherapeutic approaches, such as oncolytic viruses (OVs) and chimeric antigen receptor (CAR) T cells, have emerged as promising GC and GCPC treatments that circumvent these challenges. In this study, we provide an up-to-date review of the pre-clinical and clinical efficacy of CAR T cell therapy for key primary antigen targets and provide a translational overview of the types, modifications, and mechanisms for OVs used against GC and GCPC. Finally, we present a novel, summary-based discussion on the potential synergistic interplay between OVs and CAR T cells to treat GCPC.

Higher proinflammatory responses possibly contributing to suppressed cytotoxicity in patients with COVID-19 associated mucormycosis.

INTRODUCTION: COVID-19 Associated Mucormycosis (CAM), an opportunistic fungal infection, surged during the second wave of SARS Cov-2 pandemic. Since immune responses play an important role in controlling this infection in immunocompetent hosts, it is required to understand immune perturbations associated with this condition for devising immunotherapeutic strategies for its control. We conducted a study to determine different immune parameters altered in CAM cases as compared to COVID-19 patients without CAM. METHODOLOGY: Cytokine levels in serum samples of CAM cases (n = 29) and COVID-19 patients without CAM (n = 20) were determined using luminex assay. Flow cytometric assays were carried out in 20 CAM cases and 10 controls for determination of frequency of NK cells, DCs, phagocytes, T cells and their functionalities. The cytokine levels were analyzed for their association with each other as well as with T cell functionality. The immune parameters were also analyzed with respect to the known risk factors such as diabetes mellitus and steroid treatment. RESULTS: Significant reduction in frequencies of total and CD56 + CD16 + NK cells (cytotoxic subset) was noted in CAM cases. Degranulation responses indicative of cytotoxicity of T cell were significantly hampered in CAM cases as compared to the controls. Conversely, phagocytic functions showed no difference in CAM cases versus their controls except for migratory potential which was found to be enhanced in CAM cases. Levels of proinflammatory cytokines such as IFN-γ, IL-2, TNF-α, IL-17, IL-1ß, IL-18 and MCP-1 were significantly elevated in cases as compared to the control with IFN-γ and IL-18 levels correlating negatively with CD4 T cell cytotoxicity. Steroid administration was associated with higher frequency of CD56 + CD16- NK cells (cytokine producing subset) and higher MCP-1 levels. Whereas diabetic participants had higher phagocytic and chemotactic potential and had higher levels of IL-6, IL-17 and MCP-1. CONCLUSION: CAM cases differed from the controls in terms of higher titers of proinflammatory cytokines, reduced frequency of total and cytotoxic CD56 + CD16 + NK cell. They also had reduced T cell cytotoxicity correlating inversely with IFN-γ and IL-18 levels, possibly indicating induction of negative feedback mechanisms while diabetes mellitus or steroid administration did not affect the responses negatively.

Both N- and C-terminal domains of galectin-9 are capable of inducing HIV reactivation despite mediating differential immunomodulatory functionalities.

Background: The shock-and-kill strategy for HIV cure requires the reactivation of latent HIV followed by the killing of the reactivated cellular reservoir. Galectin-9, an immunomodulatory protein, is shown to induce HIV reactivation as well as contribute to non-AIDS- and AIDS-defining events. The protein is prone to cleavage by inflammatory proteases at its linker region separating the N- and C-terminal carbohydrate-binding domains (N- and C-CRDs) which differ in their binding specificities. It is important to study the activity of its cleaved as well as uncleaved forms in mediating HIV reactivation and immunomodulation in order to understand their role in HIV pathogenesis and their further utilization for the shock-and-kill strategy. Methodology: The PBMCs of HIV patients on virally suppressive ART (n = 11) were stimulated using 350 nM of the full-length protein and N- and C-CRDs of Gal-9. HIV reactivation was determined by analyzing gag RNA copies using qPCR using isolated CD4 cells and intracellular P24 staining of PBMCs by flow cytometry. Cytokine responses induced by the full-length protein and N- and C-CRDs of Gal-9 were also assessed by flow cytometry, Luminex, and gene expression assays. Changes in T helper cell gene expression pattern after the stimulation were also determined by real-time PCR array. Results: Both N- and C-CRDs of galectin-9 induced HIV reactivation in addition to the full-length galectin-9 protein. The two domains elicited higher cytokine responses than the full-length protein, possibly capable of mediating higher perturbations in the immune system if used for HIV reactivation. N-CRD was found to induce the development of Treg cells, whereas C-CRD inhibited the induction of Treg cells. Despite this, both domains elicited IL-10 secretory response although targeting different CD4 cell phenotypes. Conclusion: N- and C-CRDs were found to induce HIV reactivation similar to that of the full-length protein, indicating their possible usefulness in the shock-and-kill strategy. The study indicated an anti-inflammatory role of N-CRD versus the proinflammatory properties of C-CRD of galectin-9 in HIV infection.

Reduced Autophagy by a microRNA-mediated Signaling Cascade in Diabetes-induced Renal Glomerular Hypertrophy.

Autophagy plays a key role in the pathogenesis of kidney diseases, however its role in diabetic nephropathy (DN), and particularly in kidney glomerular mesangial cells (MCs) is not very clear. Transforming Growth Factor- ß1 (TGF-ß), a key player in the pathogenesis of DN, regulates expression of various microRNAs (miRNAs), some of which are known to regulate the expression of autophagy genes. Here we demonstrate that miR-192, induced by TGF-ß signaling, plays an important role in regulating autophagy in DN. The expression of key autophagy genes was decreased in kidneys of streptozotocin-injected type-1 and type-2 (db/db) diabetic mice and this was reversed by treatment with Locked Nucleic Acid (LNA) modified miR-192 inhibitors. Changes in autophagy gene expression were also attenuated in kidneys of diabetic miR-192-KO mice. In vitro studies using mouse glomerular mesangial cells (MMCs) also showed a decrease in autophagy gene expression with TGF-ß treatment. miR-192 mimic oligonucleotides also decreased the expression of certain autophagy genes. These results demonstrate that TGF-ß and miR-192 decrease autophagy in MMCs under diabetic conditions and this can be reversed by inhibition or deletion of miR-192, further supporting miR-192 as a useful therapeutic target for DN.

Inhibition of the processing of miR-25 by HIPK2-Phosphorylated-MeCP2 induces NOX4 in early diabetic nephropathy.

Phosphorylated methyl-CpG binding protein2 (p-MeCP2) suppresses the processing of several microRNAs (miRNAs). Homeo-domain interacting protein kinase2 (HIPK2) phosphorylates MeCP2, a known transcriptional repressor. However, it is not known if MeCP2 and HIPK2 are involved in processing of miRNAs implicated in diabetic nephropathy. p-MeCP2 and HIPK2 levels were significantly increased, but Seven in Absentia Homolog1 (SIAH1), which mediates proteasomal degradation of HIPK2, was decreased in the glomeruli of streptozotocin injected diabetic mice. Among several miRNAs, miR-25 and its precursor were significantly decreased in diabetic mice, whereas primary miR-25 levels were significantly increased. NADPH oxidase4 (NOX4), a target of miR-25, was significantly increased in diabetic mice. Protein levels of p-MeCP2, HIPK2, and NOX4 were increased in high glucose (HG)- or TGF-ß-treated mouse glomerular mesangial cells (MMCs). miR-25 (primary, precursor, and mature) and mRNA levels of genes indicated in the in vivo study showed similar trends of regulation in MMCs treated with HG or TGF-ß. The HG- or TGF-ß-induced upregulation of p-MeCP2, NOX4 and primary miR-25, but downregulation of precursor and mature miR-25, were attenuated by Hipk2 siRNA. These results demonstrate a novel role for the SIAH1/HIPK2/MeCP2 axis in suppressing miR-25 processing and thereby upregulating NOX4 in early diabetic nephropathy.

Epigenetic Histone Modifications Involved in Profibrotic Gene Regulation by 12/15-Lipoxygenase and Its Oxidized Lipid Products in Diabetic Nephropathy.

AIMS: Epigenetic mechanisms, including histone post-translational modifications and DNA methylation, are implicated in the pathogenesis of diabetic nephropathy (DN), but the mediators are not well known. Moreover, although dyslipidemia contributes to DN, epigenetic changes triggered by lipids are unclear. In diabetes, increased expression of 12/15-lipoxygenase (12/15-LO) enhances oxidized lipids such as 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], which promote oxidant stress, glomerular and mesangial cell (MC) dysfunction, and fibrosis, and mediate the actions of profibrotic growth factors. We hypothesized that 12/15-LO and its oxidized lipid products can regulate epigenetic mechanisms mediating profibrotic gene expression related to DN. RESULTS: 12(S)-HETE increased profibrotic gene expression and enrichment of permissive histone lysine modifications at their promoters in MCs. 12(S)-HETE also increased protein levels of SET7, a histone H3 lysine 4 methyltransferase, and promoted its nuclear translocation and enrichment at profibrotic gene promoters. Furthermore, SET7 (Setd7) gene silencing inhibited 12(S)-HETE-induced profibrotic gene expression. 12/15-LO (Alox15) gene silencing or genetic knockout inhibited transforming growth factor-ß1 (TGF-ß1)-induced expression of Setd7 and profibrotic genes and histone modifications in MCs. Furthermore, 12/15-LO knockout in mice ameliorated key features of DN and abrogated increases in renal SET7 and profibrotic genes. Additionally, 12/15-LO siRNAs in vivo blocked increases in renal SET7 and profibrotic genes in diabetic mice. INNOVATION AND CONCLUSION: These novel results demonstrate for the first time that 12/15-LO-derived oxidized lipids regulate histone modifications associated with profibrotic gene expression in MCs, and 12/15-LO can mediate similar actions of TGF-ß1 and diabetes. Targeting 12/15-LO might be a useful strategy to inhibit key epigenetic mechanisms involved in DN.

An endoplasmic reticulum stress-regulated lncRNA hosting a microRNA megacluster induces early features of diabetic nephropathy.

It is important to find better treatments for diabetic nephropathy (DN), a debilitating renal complication. Targeting early features of DN, including renal extracellular matrix accumulation (ECM) and glomerular hypertrophy, can prevent disease progression. Here we show that a megacluster of nearly 40 microRNAs and their host long non-coding RNA transcript (lnc-MGC) are coordinately increased in the glomeruli of mouse models of DN, and mesangial cells treated with transforming growth factor-ß1 (TGF- ß1) or high glucose. Lnc-MGC is regulated by an endoplasmic reticulum (ER) stress-related transcription factor, CHOP. Cluster microRNAs and lnc-MGC are decreased in diabetic Chop-/- mice that showed protection from DN. Target genes of megacluster microRNAs have functions related to protein synthesis and ER stress. A chemically modified oligonucleotide targeting lnc-MGC inhibits cluster microRNAs, glomerular ECM and hypertrophy in diabetic mice. Relevance to human DN is also demonstrated. These results demonstrate the translational implications of targeting lnc-MGC for controlling DN progression.

Anticlastogenic activity of flavonoid rich extract of Cassia auriculata Linn. on experimental animal [corrected].

OBJECTIVE: To determine antimutagenic activity of Cassia auriculata Linn. on chromosomal damage induced by cyclophosphamide (CP). MATERIAL AND METHODS: In the present investigation, four groups of six Swiss albino mice in each group were used. Excepting for the first group all the remaining groups were treated with CP (50 mg/kg). Mice of third and fourth group were treated with ethyl acetate extract of C. auriculata Linn. at 100 mg/kg and 200 mg/kg with CP. Metaphase of bone marrow cells of all animals were analyzed for qualitative and quantitative chromosomal aberrations. Break, fragment, deletion, Polyploidy, pulverized, ring and total aberration were observed. RESULTS: Flavonoids rich extracts of root of C. auriculata Linn. provided significant protection (P < 0.05) against CP induced chromosomal aberration. Total chromosomal aberration was found to be 12.16 and 7.33% in 100 and 200 mg/kg of extract treated animals respectively. CONCLUSION: From the present study it can was observed that ethyl acetate extract of C. auriculata Linn possess significant anti-mutagenic potential against CP induced chromosomal aberration.

Transforming growth factor-ß-induced cross talk between p53 and a microRNA in the pathogenesis of diabetic nephropathy.

Elevated p53 expression is associated with several kidney diseases including diabetic nephropathy (DN). However, the mechanisms are unclear. We report that expression levels of transforming growth factor-ß1 (TGF-ß), p53, and microRNA-192 (miR-192) are increased in the renal cortex of diabetic mice, and this is associated with enhanced glomerular expansion and fibrosis relative to nondiabetic mice. Targeting miR-192 with locked nucleic acid-modified inhibitors in vivo decreases expression of p53 in the renal cortex of control and streptozotocin-injected diabetic mice. Furthermore, mice with genetic deletion of miR-192 in vivo display attenuated renal cortical TGF-ß and p53 expression when made diabetic, and have reduced renal fibrosis, hypertrophy, proteinuria, and albuminuria relative to diabetic wild-type mice. In vitro promoter regulation studies show that TGF-ß induces reciprocal activation of miR-192 and p53, via the miR-192 target Zeb2, leading to augmentation of downstream events related to DN. Inverse correlation between miR-192 and Zeb2 was observed in glomeruli of human subjects with early DN, consistent with the mechanism seen in mice. Our results demonstrate for the first time a TGF-ß-induced feedback amplification circuit between p53 and miR-192 related to the pathogenesis of DN, and that miR-192-knockout mice are protected from key features of DN.

TGF-ß induces acetylation of chromatin and of Ets-1 to alleviate repression of miR-192 in diabetic nephropathy.

MicroRNAs (miRNAs), such as miR-192, mediate the actions of transforming growth factor-ß1 (TGF-ß) related to the pathogenesis of diabetic kidney diseases. We found that the biphasic induction of miR-192 expression by TGF-ß in mouse renal glomerular mesangial cells initially involved the Smad transcription factors, followed by sustained expression that was promoted by acetylation of the transcription factor Ets-1 and of histone H3 by the acetyltransferase p300, which was activated by the serine and threonine kinase Akt. In mesangial cells from Ets-1-deficient mice or in cells in which Ets-1 was knocked down, basal amounts of miR-192 were higher than those in control cells, but sustained induction of miR-192 by TGF-ß was attenuated. Furthermore, inhibition of Akt or ectopic expression of dominant-negative histone acetyltransferases decreased p300-mediated acetylation and Ets-1 dissociation from the miR-192 promoter and prevented miR-192 expression in response to TGF-ß. Activation of Akt and p300 and acetylation of Ets-1 and histone H3 were increased in glomeruli from diabetic db/db mice compared to nondiabetic db/+ mice, suggesting that this pathway may contribute to diabetic nephropathy. These findings provide insight into the regulation of miRNAs through signaling-mediated changes in transcription factor activity and in epigenetic histone acetylation under normal and disease states.

Involvement of a bacterial microcompartment in the metabolism of fucose and rhamnose by Clostridium phytofermentans.

BACKGROUND: Clostridium phytofermentans, an anaerobic soil bacterium, can directly convert plant biomass into biofuels. The genome of C. phytofermentans contains three loci with genes encoding shell proteins of bacterial microcompartments (BMC), organelles composed entirely of proteins. METHODOLOGY AND PRINCIPAL FINDINGS: One of the BMC loci has homology to a BMC-encoding locus implicated in the conversion of fucose to propanol and propionate in a human gut commensal, Roseburia inulinivorans. We hypothesized that it had a similar role in C. phytofermentans. When C. phytofermentans was grown on fucose, the major products identified were ethanol, propanol and propionate. Transmission electron microscopy of fucose- and rhamnose-grown cultures revealed polyhedral structures, presumably BMCs. Microarray analysis indicated that during growth on fucose, operons coding for the BMC locus, fucose dissimilatory enzymes, and an ATP-binding cassette transporter became the dominant transcripts. These data are consistent with fucose fermentation producing a 1,2-propanediol intermediate that is further metabolized in the microcompartment encoded in the BMC locus. Growth on another deoxyhexose sugar, rhamnose, resulted in the expression of the same BMC locus and similar fermentation products. However, a different set of dissimilatory enzymes and transport system genes were induced. Quite surprisingly, growth on fucose or rhamnose also led to the expression of a diverse array of complex plant polysaccharide-degrading enzymes. CONCLUSIONS/SIGNIFICANCE: Based on physiological, genomic, and microarray analyses, we propose a model for the fermentation of fucose and rhamnose in C. phytofermentans that includes enzymes encoded in the same BMC locus. Comparative genomic analysis suggests that this BMC may be present in other clostridial species.