Biology subjects

Prakash, H.

Publications and source records attributed to Prakash, H..

8 recordsLinked to original sources

bioRxiv2025

Dual Preconditioned Mesenchymal Stem Cells-Derived Culture Conditioned Media Augment Immunomodulation and Drive Metabolic Reprogramming in Acute Graft-versus-Host Disease

BackgroundThe toxicity associated with conventional conditioning regimens limits treatment outcomes in autoimmune disorders such as acute graft-versus-host disease (aGVHD). Developing effective adjunctive interventions to restore immune balance is therefore essential. This study investigated the immunomodulatory potential of mesenchymal stem cells (MSCs)-derived culture-conditioned media (CCM) from naive and preconditioned MSCs-hypoxic (MSCsHYP), apoptotic (MSCsAPO), and dual preconditioned (MSCsHYP+APO) in aGVHD. MethodsHuman MSCs isolated from bone marrow and Whartons Jelly were preconditioned under hypoxia (1% O2), apoptosis (1 {micro}M staurosporine, 24 h), or both. The immunoregulatory and antioxidant properties of their CCM were evaluated through T-cell proliferation assays, Treg induction, macrophage polarization, mitochondrial function, and T-cell bioenergetics. Comparative proteomic profiling of CCM from WJ-MSCs and WJ-MSCsHYP+APO co-cultured with aGVHD patient-derived activated PBMNCs was performed using LC-MS/MS, alongside in vivo validation in a chemotherapy-induced aGVHD murine model. ResultsWJ-MSCsHYP+APO-CCM exhibited superior immunomodulatory efficacy, suppressing T-cell proliferation, promoting Treg and Th2/Th9 differentiation, and driving M2 macrophage polarization. It reduced mitochondrial ROS, enhanced mitochondrial polarization, and shifted T-cell metabolism from glycolysis toward oxidative phosphorylation. Proteomic analysis revealed modulation of IL-12, IL-17, and JAK-STAT pathways, along with regulation of complement, coagulation, and metabolic cascades. Interaction with immune cells further enhanced its antioxidant and tissue-reparative properties via extracellular matrix remodeling. ConclusionDual preconditioning under hypoxia and apoptosis amplifies the immunomodulatory, antioxidant, and reparative efficacy of WJ-MSC-CCM, offering a potent non-cellular therapeutic strategy for aGVHD management. GRAPHICAL ABSTRACTImmunomodulatory and immune metabolic reprogramming potential of naive and preconditioned MSCs (MSCs, MSCsHYP, MSCsAPO, MSCsHYP+APO) derived CCM in Acute GVHD (created using Biorender.com) O_FIG O_LINKSMALLFIG WIDTH=95 HEIGHT=200 SRC="FIGDIR/small/686182v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@f7ed73org.highwire.dtl.DTLVardef@1063015org.highwire.dtl.DTLVardef@13ed827org.highwire.dtl.DTLVardef@e103c0_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Modulating p38MAPK recalibrates sensitivity of drug-resistant pancreatic adenoductal carcinoma cells toward chemotherapy and correlate with improved outcome in PDAC patients: Experimental and metadata evidence

Pancreatic cancer is one of the deadliest cancers and has very limited therapeutic options and a dismal prognosis. Among various signaling pathways which are activated during tumor development, hyperactivation of Mitogen-Activated Protein Kinase (MAPK) is responsible for high grade angiogenesis, polarization of Tumor Associated Macrophages, unfolded protein responses and exhaustion of T cells, which together contributes towards this therapeutic resistance. We therefore believe that MAPK targeting is expected to enhance sensitivity of highly resistant PDAC cells toward various cancer directed interventions. In this context, we investigated the impact of modulating p38MAPK on the sensitivity of pancreatic cancer cells towards gemcitabine. Supporting our hypothesis, our results convincely, demonstrated that indeed, p38 inhibition sensitizes both KRAS positive Panc-1 and MiaPaCa2 pancreatic carcinoma cells towards gemcitabine induced death. Interestingly p38MAPK targeting significantly reduced the cell viability, clonogenic potential of these cells and enhanced the early apoptosis. Our in-silico studies, supporting our in vitro data, potentially correlated that that high expression of p38 MAPK14 in PDAC patients is associated with poor prognosis and disease free survival. Deep miming of in silico data further demonstrated that MAPK14, in association with, hypoxia inducible factor-1 alpha and vascular endothelial growth factor signaling pathways promote angiogenic programming of PDAC which render these tumors refractory for cancer directed interventions. Based on our preliminary data, we believe that p38 MAPK based approach is potential approach for changing the faith of PDAC patients toward chemo and immunotherapy and believed to improve PDAC burden effectively in the host. HighlightsO_LIp38 MAPK inhibition enhances the sensitivity of KRAS+ pancreatic cancer cells for Gemcitabine C_LIO_LIP38MAPK knockdown cells are sensitive for Gemcitabine induced death C_LIO_LIMAPK14 (p38) is associated with angiogenesis and poor prognosis in pancreatic cancer C_LIO_LIMAPK14 Regulates Pro-Tumorigenic Pathways and immune infiltration in Pancreatic Cancer C_LI

cancer biology↗

bioRxiv2025

ATLS2021-PA (A Sphingolipid mimetic): Upcoming Host Directed Therapy for Acute and MDR Tuberculosis

Several reports have shown that reduced Sphingolipids content in the lungs largely contributes to the outcome of Tuberculosis diseases. In this context, our pioneer study has advocated that Sphingosine -1 phosphate (a central Sphingolipid metabolite) can help host in limiting Mycobacterium tuberculosis burden in lungs by their virtue of tweaking M1 retuning of infected macrophages. This indicated that S1P may serve as anti-tubercular regimen, however allergic and autoimmune manifestation of S-1P refrain its further use as anti-tubercular Drugs. In view of this limitation, and high demand of developing newer host directed anti tubercular regimen, we explored whether enhancing / boosting Sphingolipids levels via de novo pathways by our patented drugs ATLS2021 and PA would also help host in controlling mycobacterial infection. Our results prudently demonstrated that ATLS2021 was effective in controlling survival of both wild type and MDR strain of TB in various models we tested. Our results further demonstrated the influence of ATLS2021 on NO mediated killing of mycobacteria which indicated one of most possible anti-mycobacterial mechanism of ATLS2021. Additionally, ATLS2021 lowered the IC50 value of Rifampicin for both wild-type and MDR TB by sensitizing the MDR clinical isolate of Mycobacterium tuberculosis to Rifampicin-mediated killing. ATLS2021 induced immunogenic responses in blood derived CD14+ macrophages from both healthy donors as well as MDR-TB patient demonstrating immune adjuvant potential of ATLS2021. Real time PCR data demonstrated that ATLS2021 enhanced the expression of almost all key enzymes involved in the Sphingolipid biosynthesis pathways in the CD14 positive monocytes from healthy donors and MDR TB patients. Taken together these results potentially advocated that ATLS2021 based approach is potentially immunogenic interventions against TB.

pharmacology and toxicology↗

bioRxiv2025

Hypoxia-Mediated Molecular Interactions of Tissue-Specific Mesenchymal Stem Cells Drive Metabolic Reprogramming and Immunomodulation in Acute Graft-versus-Host Disease

BackgroundMesenchymal stem cells (MSCs) mediate immunomodulation through various mechanisms, including apoptosis, efferocytosis, and mitochondrial transfer. Our study investigates the impact of hypoxia preconditioning on the immune metabolic reprogramming and immunomodulatory potential of MSCs in acute graft-versus-host disease (aGVHD). Additionally, we explored the differential immunomodulatory effects of tissue-specific MSCs, specifically bone marrow (BM) and Whartons Jelly (WJ), and elucidated the mechanisms underlying variability in their therapeutic efficacy. MethodsMSCs were isolated from BM and WJ and subjected to hypoxia preconditioning. Their immunometabolic programming potential was assessed by evaluating T-cell proliferation, regulatory T-cell (Treg) induction, effector T-cell differentiation toward Th2, Th9 phenotypes, and macrophage polarization, T-cell bioenergetics in the direct co-culture systems. ResultsWJ-MSCsHYP exhibited superior immunomodulatory properties compared to BM-MSCsHYP, by inhibiting T-cell proliferation, enhancing Treg induction, and promoting anti-inflammatory macrophage polarization. WJ-MSCsHYP demonstrated enhanced mitochondrial transfer to T-cell, improving mitochondrial health, reducing ROS, and promoting oxidative phosphorylation, leading to immune homeostasis. Unlike BM-MSCs, WJ-MSCs exhibited higher rates of apoptosis, which facilitated immune modulation through mechanisms independent of efferocytosis. ConclusionOur findings highlight that WJ-MSCsHYP is a superior candidate for aGVHD by utilizing apoptosis, mitochondrial transfer, and metabolic reprogramming to achieve immune regulation.

immunology↗

bioRxiv2024

Caloric restriction: A potential approach for mitigating neuronal damage: Lesson from cellular model of Alzheimer Disease

Alzheimers disease is a neurodegenerative disorder and characterized by amyloid beta accumulation, synaptic dysfunction, and oxidative stress, lacks effective therapies. Caloric restriction mimetics such as fisetin and chlorogenic acid, natural polyphenols with antioxidant and autophagy-inducing properties, show promise in mitigating age-related diseases. This study investigates their neuroprotective effects against amyloid beta induced toxicity in differentiated human neuroblastoma SHSY5Y cells. Amyloid beta exposure disrupted redox homeostasis, impaired autophagy, induced mitochondrial dysfunction, and exacerbating neuronal degeneration. Fisetin and chlorogenic acid treatments reversed these deleterious effects by restoring redox balance, suppressing reactive oxygen species and upregulating critical antioxidant enzymes like SOD1, GSR, and catalase. These compounds also attenuated amyloid beta induced mitophagy via reduced PINK1 expression and restored mitochondrial fusion by upregulating Mfn2. Autophagy-related pathways were significantly modulated, evidenced by increased AMPK and decreased mTOR mRNA levels, alongside elevated expression of ATG101, ATG13, ULK1, P62 and reduced ATG5 levels. Docking studies also revealed binding of fisetin and CGA within the binding pockets of AMPK and FKBP12 supporting their interaction. Furthermore, fisetin and CGA improved synaptic integrity by upregulating PSD95 and synaptophysin and reducing acetylcholinesterase expression. These findings highlight their potential in ameliorating amyloid beta induced neuronal toxicity through autophagy activation, synaptic preservation, and mitochondrial function enhancement. While this study demonstrates the transcriptional impact and binding affinities of these caloric restriction mimetics further translational and biophysical analyses are required to elucidate their mechanisms and confirm their therapeutic viability. This research underscores the potential of fisetin and CGA as neuroprotective agents, offering promising therapeutic avenues for combating related Alzheimers disease neuropathies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/628076v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1253dd0org.highwire.dtl.DTLVardef@1fd3ffforg.highwire.dtl.DTLVardef@1749d1dorg.highwire.dtl.DTLVardef@f71415_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

MHC class I / II restricted T cell epitopes from clinical isolate of Mycobacterium tuberculosis: A potential candidate for vaccine development for Tuberculosis

Tuberculosis is major challenge to the health care system with TB associated death rates increasing annually. Optimum management of TB (particularly latent or MDR cases) warrants use of immunological approaches like subunit or peptide-based vaccination for tailoring effector immunity in patients. Since MHC class I is a potent enhancer element of host immunity and effective in clearing large variety of intracellular pathogens or tumors. In this context, we explore whether MHC-I restricted peptides from clinical isolates of M. tuberculosis can be used as an adjuvant for augmenting host immune responses. In the present study, we have synthesized various peptides from clinical isolates of M. tuberculosis which were having high affinity for Class I MHC molecules as potential immune enhancer for T cell or iNKT cell populations. We have evaluated the immunogenic potential of various MHC class I restricted epitopes (Rv2588c, Rv1357, Rv0148, Rv2973, Rv2557 and Rv2445) which were derived from clinical isolates of M. tuberculosis on increased proliferation of T or iNKT cells, release of IFN gamma secreted by T cells as well as NO as indicative parameters of immuno-stimulation. As expected, FACS and ELISA data clearly revealed that these peptides were potentially immunogenic for PBMCs from both healthy as well as 10 HC PTB patients. Our data clearly demonstrated a significant immune response in the PBMC from w PTB patients over healthy individuals which mimicked booster response. Our cytokine and nitric oxide data further revealed the influence of these peptides on sensitizing innate immune response as well. SignificanceOur study demonstrates the significance of MHC class I restricted peptides from M. tuberculosis for inducing potential immunogenic responses in host that may qualify them as potent vaccine candidate. To the best of our knowledge this is the first immune monitoring protocol describing the impact of synthetic novel MHC class I restricted T-cell epitope (Rv2588c, Rv0148) on cell mediated and innate immune response in PBMC populations and suggests their potential as vaccine candidate

immunology↗

bioRxiv2024

A Comparative assessment of T-Cell response of Healthy donors and Acute Graft-versus-Host-Disease Patients: Customizing immune monitoring platform

BackgroundT-cell activation and proliferation are critical for understanding immune responses in both healthy and pathological conditions such as acute graft-versus-host disease (aGVHD). Phytohemagglutinin (PHA) and interleukin-2 (IL-2) are commonly used in in vitro assays to study T-cell responses. In view of the discrete response of T cells from aGvHD patients cohorts, our study optimized PHA / IL-2 based T-cell response among healthy individuals versus aGVHD patients. MethodsPeripheral blood was collected from age- and sex-matched healthy individuals (n=10) and aGVHD patients (n=10). CD3+ T-cell were isolated and stimulated with varying concentrations of PHA (1-10g/ml) and IL-2 (50-500 IU/ml). Cell proliferation was assessed using MTS and CFSE assays, while their apoptosis was evaluated with Annexin V/7-AAD staining. ResultsWe observed enhanced proliferation of healthy individuals at higher PHA concentrations (5-10g/ml), whereas aGVHD patients exhibited heightened proliferation even at lower PHA concentrations (1-2.5g/ml) at 48 hours. Prolonged exposure of T cells from GvHD patients to PHA led to decreased proliferation while it increased in the T cells from healthy donors.IL-2 supplementation (50 IU/ml) of T-cells from healthy donors significantly enhanced their proliferation and survival, with the optimal concentration supporting robust proliferation over extended culture periods. ConclusionOur study optimized PHA and IL-2 concentrations required for T-cell proliferation studies among healthy individuals and aGVHD patients. and underscored experimental conditions required for studying T-cell behavior/dysregulation in aGVHD condition. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/611044v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@ab3c47org.highwire.dtl.DTLVardef@2786forg.highwire.dtl.DTLVardef@30c698org.highwire.dtl.DTLVardef@3e4821_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights of the studyO_LILower doses of PHA (1.0g/ml) and IL-2 (50IU/ml) are optimum conditions for aGVHD patients derived CD3+ T-cell proliferation under in vitro conditions. C_LIO_LIThe maximum T-cell proliferation in healthy individuals occurs with 7.5g/ml PHA and 50IU/ml IL-2. C_LIO_LIHigher doses of PHA induce cytotoxicity in both cohorts. C_LIO_LIIL-2 significantly enhances T-cell survival, with 50IU/ml maintaining robust proliferation over extended periods. C_LI

immunology↗

bioRxiv2024

Synergistic Hypoxia and Apoptosis Conditioning Unleashes Superior Mesenchymal Stem Cells Efficacy in Acute Graft-versus-Host-Disease

Mesenchymal stem cells (MSCs) have emerged as promising candidates for immune modulation in various diseases that are associated with dysregulated immune responses like Graft-versus-Host-Disease (GVHD). MSCs are pleiotropic and the fate of MSCs following administration is a major determinant of their therapeutic efficacy. In this context, we here demonstrate that hypoxia preconditioned apoptotic MSCs [bone marrow (BM), Whartons Jelly (WJ)] bear more immune programming ability in a cellular model of acute Graft-versus-Host-Disease (aGVHD). To this purpose, we programmed MSCs by exposing them to hypoxia and inducing apoptosis both sequentially as well as simultaneously. Our findings demonstrated that WJ MSCs that were conditioned with indicated approaches simultaneously induced the differentiation of CD4+T-cell towards Tregs, enhanced Th2 effector, and concomitantly mitigated Th1 and Th17, with polarization of M1 effector macrophages towards their M2 phenotype, and more interestingly enhanced efferocytosis by macrophages indicated Th2 programming ability of MSCs programmed by conjunctional approaches Overall, our study highlights the potential of WJ-MSCs conditioned with hypoxia and apoptosis concurrently, as a promising therapeutic strategy for aGVHD and underscores the importance of considering MSC apoptosis in optimizing MSCs-based cellular therapy protocols for enhanced therapeutic efficacy in aGvHD. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=123 HEIGHT=200 SRC="FIGDIR/small/588248v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@92c102org.highwire.dtl.DTLVardef@bd895forg.highwire.dtl.DTLVardef@185e041org.highwire.dtl.DTLVardef@45ef0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗