Biology subjects

Khare, P.

Publications and source records attributed to Khare, P..

13 recordsLinked to original sources

bioRxiv2026

The CD19-4-1BBL antibody fusion protein unleashes the immune system against high-risk chronic lymphocytic leukemia

CD19-4-1BBL is a bispecific antibody fusion protein that targets CD19 and costimulates 4-1BB on T cells and other immune cells. Its antitumor activity has been reported in B-cell non-Hodgkin lymphoma with emphasis on its T-cell mediated cytotoxic activity. Its effect on other 4-1BB expressing immune cells is unexplored. Here, we investigated the molecular mechanisms and the antileukemic effect of CD19-4-1BBL in chronic lymphocytic leukemia (CLL), a B-cell malignancy profoundly marked by the immunosuppressive activity of myeloid-derived suppressor cells, tumor-associated macrophages and CD4+ regulatory T cells. We demonstrated that CD19-4-1BBL simultaneously mitigates the immunosuppressive phenotype and transcriptome machinery of these cells and promotes antitumor CD8+ T-cell immunity. Finally, in a preclinical, patient-derived xenograft model of CLL, we observed a favourable survival impact, especially in mice transplanted with immune cells from patients with high-risk/progressive leukemia. Our findings provide evidence that the CD19-4-1BBL treatment is a multifaceted, immune-based strategy that should be clinically explored in patients with chronic lymphocytic leukemia. KEY POINTSO_LICD19-4-1BBL sharpens the myeloid cell transcriptome and stimulates diverse memory CD8+ T cell clonotypic responses. C_LIO_LICD19-4-1BBL costimulation can be therapeutically exploited in high-risk chronic lymphocytic leukemia. C_LI

immunology↗

bioRxiv2026

Metabolic reprogramming by caloric restriction enhances acute phase virological control and reduces chronic inflammation in SIV-infected rhesus macaques

Nutrient metabolism influences HIV-1 replication, antiviral immunity, and chronic inflammation, yet is difficult to leverage for therapeutic gain. We sought to modulate metabolism in the non-human primate model of HIV-1 by caloric restriction (CR), a modality canonically known for its antiaging benefits. Four months of 30% CR was safe and resulted in broad and systemic metabolic reprogramming in healthy adult male and female rhesus macaques. Relative to that of ad libitum-fed animals, CR lowered the frequencies of target CCR5+ CD4 T cells in the gut mucosa. Upon infection with SIV, CR reduced acute phase viremia, dampened type I interferon signaling, and overall permitted a more vigorous cycling of CD8+ T cells in lymphoid tissues. CR-induced protection from SIV was associated with a robust up-regulation of glycolysis, which supported an early reduction in viremia that ultimately waned over time. During virologic suppression with antiretroviral therapy (ART), CR significantly limited gastrointestinal (GI) immune activation, improved tricarboxylic acid cycle flux, and lowered concentrations of soluble CD14 and several TNF-related molecules in plasma. Blood SIV DNA levels however were unchanged by CR, suggesting that residual GI dysfunction and inflammation can be decoupled from viral persistence. Our findings highlight that a dietary modality can limit pathology in a primate lentiviral infection. They also reveal the robust but temporally constrained nature of glycolysis in supporting an acute antiviral response. SIGNIFICANCECaloric restriction (CR) is a safe dietary intervention known to confer anti-aging and health benefits across diverse animal models. However, its application in the context of infectious diseases has yielded mixed outcomes and has largely been limited to murine systems. In this study, we therefore employed CR to examine the impact of dietary modulation on SIV infection outcomes. Our findings demonstrate that CR reduced acute-phase viremia and attenuated markers of chronic inflammation following ART, effects that were associated with distinct metabolic signatures. Collectively, these findings underscore the importance of diet and nutrition in shaping chronic viral infection outcomes, such as SIV, within a clinically relevant non-human primate model.

immunology↗

bioRxiv2026

Role of brown adipose tissue-specific sympathetic and sensory innervations in menthol-induced energy-expending phenotype

Menthol, a TRPM8 agonist and pharmacological cold mimic, activates brown adipose tissue (BAT) to increase adaptive thermogenesis and whole-body energy expenditure. BAT is highly innervated with sensory and sympathetic nerve fibers. There is existing literature that sympathetic nerves have a critical role in regulating menthols (or cold) effect on energy homeostasis, but there is minimal literature about the involvement of somatosensory innervations. Here, we ought to investigate whether short-term topical application (3 days) of menthol elicits sympathetic and sensory nerves. We used chemical denervation models of complete (100 mg/kg; 6-OHDA and 125 mg/kg; capsaicin, s.c.) and localized (in BAT, 20 {micro}L of 6-OHDA (20 mg/ml) and 20{micro}L of capsaicin (20{micro}g/{micro}L) in each BAT lobe) ablation of sympathetic (6-OHDA) and sensory (capsaicin) innervations to explore their roles in menthol induced BAT activation and energy expanding phenotype. In the present study we have shown that (i) short-term topical application of menthol (10 % menthol for 3 consecutive days) elicits sympathetic and sensory innervations, and induces thermogenesis, lipolysis and mitochondrial biogenesis in mice BAT; (ii) localized ablation of sympathetic innervation prevented menthol induced BAT activation and lipolysis (iii) localized ablation of sensory neurons augments sympathetic innervations induced BAT activation and modulated thermoregulation. Collectively, our results suggest that BAT activation following short-term topical application of menthol is dependent primarily but not exclusively on centrally mediated SNS activation. TRPV1-positive sensory neurons are partially necessary for thermoregulation and efficient BAT activation.

pharmacology and toxicology↗

bioRxiv2025

Mitochondria-containing large extracellular vesicles target mouse motor neurons upon intramuscular injection

Amyotrophic Lateral Sclerosis (ALS) is a neurological disorder that causes progressive degeneration of motor neurons. Mitochondrial dysfunction accelerates neurodegeneration aggravating the severity of ALS. We hypothesized that increasing the mitochondrial function of motor neurons may promote neuronal survival. Therefore, we investigated the potential of neuron-derived mitochondria containing extracellular vehicles (EVs) as a novel therapeutic approach for ALS using differentiated NSC-34 cells as a surrogate for neurons. Neuron derived-large EVs (lEVs) but not small EVs (sEVs) contained mitochondria. However, we observed increased cell viability and oxygen consumption rates in heat-stressed neurons treated with both sEVs and lEVs suggesting improved mitochondrial function in recipient neurons. The increased oxygen consumption rates in sEV-treated heat-stressed neurons was accompanied by a greater proton leak compared to lEV treatment. The greater proton leak observed with sEVs likely suggests a lower efficiency of oxidative phosphorylation compared to that achieved by cells treated with mitochondria-containing lEVs. These findings suggest that mitochondrial components present in sEVs, such as proteins and mitochondrial DNA, may too contribute to improving cellular respiration. Furthermore, we have demonstrated that lEV mitochondria are transported into the lumbar spinal cord motor neurons following intramuscular injection in C57BL/6 mice in a EV dose-dependent manner. Collectively, for the first time, we have demonstrated the therapeutic effects of neuronal EVs in recipient heat-stressed neurons and the delivery of lEV mitochondria to spinal cord motor neurons in vivo without any EV surface modifications for neuronal targeting. Further studies will determine the therapeutic efficacy of mitochondria-containing EVs in the SOD1G93A transgenic mouse model of ALS. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=166 HEIGHT=200 SRC="FIGDIR/small/675842v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@af590aorg.highwire.dtl.DTLVardef@1fed11dorg.highwire.dtl.DTLVardef@52d339org.highwire.dtl.DTLVardef@1a97182_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Purinergic receptor activation rectifies autism-associated endothelial dysfunction

Early cerebrovascular alterations affect brain maturation by impacting trophic support and energy supply. Recent evidence in a 16p11.2 deletion mouse model of autism spectrum disorder (ASD) revealed brain endothelial abnormalities postnatally. Yet, the endothelial alterations eliciting these changes remain unknown. Isolation of brain endothelial cells (ECs) from 14-day old male 16p11.2-deficient and wild-type mice revealed that 16p11.2 deletion-induced endothelial dysfunction is linked to a bioenergetic failure, with reduced intracellular ATP. Intra- or extra-cellular ATP supplementation rescued the function of 16p11.2-deficient ECs in vitro via P2 purinergic receptor activation, specifically P2Y2 receptors. Activating P2Y2 receptors restored cerebrovascular reactivity in 16p11.2-deficient parenchymal arterioles ex vivo and rescued 16p11.2 deletion-associated mouse behaviors. Taken together, this study demonstrates that metabolic reprogramming of brain ECs via purinergic receptor engagement represents a possible therapeutic avenue for ASD.

cell biology↗

bioRxiv2025

Ionic liquid-coated lipid nanoparticles promote enhanced red blood cell hitchhiking

Lipid nanoparticles (LNPs) have transformed the delivery of nucleic acid therapeutics; however, their natural tropism favors the liver resulting in clearance by the reticuloendothelial system, with less than 1% of the injected dose reaching challenging targets such as brain. Biocompatible ionic liquids (ILs) are tunable materials that can modulate nanoparticle interactions with blood components. Choline trans-2-hexenoate (C2HA) is an IL known to facilitate red blood cell (RBC) hitchhiking of PLGA polymeric nanoparticles and reduces hepatic uptake and therefore enabling transport to distant non-hepatic organs. We wanted to determine if C2HA coatings can show similar RBC hitchhiking effects with LNPs. We previously demonstrated that IL-coated LNPs reduce serum protein binding to LNPs--a key contributor to rapid clearance via the liver. While LNPs coated with choline trans-2-hexenoate at 1:1 and 1:2 cation: anion ratios decreased mouse serum protein binding and improved cellular uptake into brain endothelial cells (BECs) and motor neurons, they did not show hitchhiking behavior. To identify IL formulations capable of this behavior, we screened higher IL cation: anion ratios (1:3 and 1:5) for LNP coating and optimized IL volumes that allowed stable particle diameters. The resulting IL-coated LNPs successfully hitchhiked on both mouse and human RBCs and significantly enhanced uptake in b.End3 mouse BECs, and NSC-34 neuroblastoma cells compared to uncoated LNPs. 1:3 IL-coated LNPs demonstrated the most pronounced improvement in RBC binding. Whole blood pharmacokinetic and biodistribution analyses demonstrated that IL-coating significantly extends the circulation time of LNPs and results in reduced hepatic uptake alongside increased routing to the brain, compared to standard LNPs. These findings reveal that ILs can be leveraged to re-engineer clinically approved LNP platforms to promote RBC hitchhiking behavior and further be developed for drug delivery to challenging extra-hepatic targets such as the brain. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/649225v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@3fadceorg.highwire.dtl.DTLVardef@abc821org.highwire.dtl.DTLVardef@1e00156org.highwire.dtl.DTLVardef@177ba2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

A foundational atlas of autism protein interactions reveals molecular convergence

Translating high-confidence (hc) autism spectrum disorder (ASD) genes into viable treatment targets remains elusive. We constructed a foundational protein-protein interaction (PPI) network in HEK293T cells involving 100 hcASD risk genes, revealing over 1,800 PPIs (87% novel). Interactors, expressed in the human brain and enriched for ASD but not schizophrenia genetic risk, converged on protein complexes involved in neurogenesis, tubulin biology, transcriptional regulation, and chromatin modification. A PPI map of 54 patient-derived missense variants identified differential physical interactions, and we leveraged AlphaFold-Multimer predictions to prioritize direct PPIs and specific variants for interrogation in Xenopus tropicalis and human forebrain organoids. A mutation in the transcription factor FOXP1 led to reconfiguration of DNA binding sites and altered development of deep cortical layer neurons in forebrain organoids. This work offers new insights into molecular mechanisms underlying ASD and describes a powerful platform to develop and test therapeutic strategies for many genetically-defined conditions.

systems biology↗

bioRxiv2023

Effect of homotypic vs. heterotypic interactions on the cellular uptake of extracellular vesicles

Extracellular vehicles (EVs) are an emerging class of drug carriers and are primarily reported to be internalized into recipient cells via a combination of endocytic routes such as clathrin-mediated, caveolae-mediated and macropinocytosis pathways. In this work, (1) we investigated potential effects of homotypic vs. heterotypic interactions by studying the cellular uptake of homologous EVs (EV donor cells and recipient cells of the same type) vs. heterologous EVs (EV donor cells and recipient cells of different types) and (2) determined the route of EV internalization into low pinocytic/hard-to-deliver cell models such as brain endothelial cells (BECs). We used BECs and macrophages as low-pinocytic and phagocytic cell models, respectively, to study the effect of homotypic vs. heterotypic interactions on EV uptake in the recipient cells. Homotypic interactions led to a greater extent of uptake into the recipient BECs compared to heterotypic interactions. However, we did not see a complete reduction in EV uptake into recipient BECs when endocytic pathways were blocked using pharmacological inhibitors. Our results suggest that EVs primarily use membrane fusion to enter low-pinocytic recipient BECs instead of relying on endocytosis. Lipophilic PKH67 dye-labeled EVs but not intravesicular esterase-activated calcein ester-labeled EVs severely reduced particle uptake into BECs while phagocytic macrophages internalized both types of EV-labeled particles to comparable extents. Our results also highlight the importance of carefully choosing labeling dye chemistry to study EV uptake, especially in the case of low pinocytic cells such as BECs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=171 HEIGHT=200 SRC="FIGDIR/small/563628v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@d5cb50org.highwire.dtl.DTLVardef@172d1dorg.highwire.dtl.DTLVardef@192caf6org.highwire.dtl.DTLVardef@4b16ba_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

A novel multi-epitope peptide vaccine candidate targeting Hepatitis E virus: an in-silico approach

HEV is a foodborne virus transmitted through the fecal-oral route that causes viral hepatitis in humans worldwide. Ever since its discovery as a zoonotic agent, HEV was isolated from several species with an expanding range of hosts. HEV possesses several features of other RNA viruses but also has certain HEV-specific traits that make its viral-host interactions inimitable. HEV leads to severe morbidity and mortality in immunocompromised people and pregnant women across the world. The situation in underdeveloped countries is even more alarming. Even after creating a menace across the world, we still lack an effective vaccine against HEV. Till date, there is only one licensed vaccine for HEV available only in China. The development of an anti-HEV vaccine that can reduce HEV-induced morbidity and mortality is required. Live attenuated and killed vaccines against HEV are not accessible due to the lack of a tolerant cell culture system, slow viral replication kinetics and varying growth conditions. Thus, the main focus for anti-HEV vaccine development is now on the molecular approaches. In the current study, we have designed a multi-epitope vaccine against HEV through a reverse vaccinology approach (Figure 1). For the first time, we have used viral ORF3, capsid protein and polyprotein altogether for epitope prediction. These are crucial for viral replication and persistence and are major vaccine targets against HEV. The proposed in-silico vaccine construct comprises of highly immunogenic and antigenic T-cell and B-cell epitopes of HEV proteins and ORF3. The construct is capable of inducing an effective and long-lasting host immune response as evident from the simulations results. In addition, the construct is stable, non-allergic and antigenic for the host. Altogether, our findings suggest that the in-silico vaccine construct may be useful as a vaccine candidate for preventing HEV infections. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/520355v1_fig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@354f48org.highwire.dtl.DTLVardef@1e80119org.highwire.dtl.DTLVardef@a04ed5org.highwire.dtl.DTLVardef@97bab0_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO An overview of multi-epitope vaccine construct designing using different in-silico tools. C_FIG

immunology↗

bioRxiv2022

Lipidoid nanoparticles increase ATP uptake into hypoxic brain endothelial cells

Lipidoid nanoparticles (LNPs) are clinically successful carriers for nucleic acid delivery to liver and muscle targets. Their ability to load and deliver small molecule drugs has not been reported yet. We propose that the delivery of adenosine triphosphate (ATP) to brain endothelial cells (BECs) lining the blood-brain barrier may increase cellular energetics of the injured BECs. We formulated and studied the physicochemical characteristics of ATP-loaded LNPs using the C12-200 ionizable cationic lipid and other helper lipids. Polyethylene glycol-dimyristoyl glycerol (PEG-DMG), one of the helper lipids, played a crucial role in maintaining colloidal stability of LNPs over time whereas the inclusion of both ATP and PEG-DMG maintained the colloidal stability of LNPs in the presence of serum proteins. ATP-LNPs formulated with PEG-DMG resulted in a 7.7- and 6.6-fold increased uptake of ATP into normoxic and hypoxic BECs, respectively. Altogether, our results demonstrate the potential of LNPs as a novel carrier for the delivery of small molecular mass actives to BECs--a CNS target. HighlightsO_LILNPs were formulated with ATP, a small molecule drug C_LIO_LIPEG-DMG plays a critical role in maintaining particle stability over tim C_LIO_LIATP and PEG-DMG play a critical role in maintaining particle stability in 10% serum C_LIO_LIATP-LNPs were internalized by normoxic and hypoxic brain endothelial cells (BECs) C_LIO_LILNP delivery to BECs broadens its applicability to CNS targets C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/487513v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1a315eaorg.highwire.dtl.DTLVardef@1f3d649org.highwire.dtl.DTLVardef@17989fdorg.highwire.dtl.DTLVardef@526ce2_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Development of lipidoid nanoparticles for siRNA delivery to neural cells

Lipidoid nanoparticles (LNPs) are the delivery platform in Onpattro, the first FDA-approved siRNA drug. LNPs are also the carriers in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While these applications have demonstrated that LNPs effectively deliver nucleic acids to hepatic and muscle cells, it is unclear if LNPs could be used for delivery of siRNA to neural cells, which are notoriously challenging delivery targets. Therefore, the purpose of this study was to determine if LNPs could efficiently deliver siRNA to neurons. Because of their potential utility in either applications in the central nervous system and the peripheral nervous system, we used both cortical neurons and sensory neurons. We prepared siRNA-LNPs using C12-200, a benchmark ionizable cationic lipidoid along with helper lipids. We demonstrated using dynamic light scattering that the inclusion of both siRNA and PEG-lipid provided a stabilizing effect to the LNP particle diameters and polydispersity indices by minimizing aggregation. We found that siRNA-LNPs were safely tolerated by primary dorsal root ganglion neurons. Flow cytometry analysis revealed that Cy5 siRNA delivered via LNPs into rat primary cortical neurons showed uptake levels similar to Lipofectamine RNAiMAX--the gold standard commercial transfection agent. However, LNPs demonstrated a superior safety profile whereas the Lipofectamine-mediated uptake was concomitant with significant toxicity. Fluorescence microscopy demonstrated a time-dependent increase in the uptake of LNP-delivered Cy5 siRNA in a human cortical neuron cell line. Overall, our results suggest that LNPs are a viable platform that can be optimized for delivery of therapeutic siRNAs to neural cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/454207v2_ufig1.gif" ALT="Figure 1"> View larger version (72K): org.highwire.dtl.DTLVardef@1ac52acorg.highwire.dtl.DTLVardef@67dbb0org.highwire.dtl.DTLVardef@880526org.highwire.dtl.DTLVardef@a1693_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

bioengineering↗

bioRxiv2020

Immunofocusing humoral immunity potentiates the functional efficacy of the AnAPN1 malaria transmission-blocking vaccine antigen

Malaria transmission-blocking vaccines (TBVs) are a critical tool for disease elimination. TBVs prevent completion of the developmental lifecycle of malarial parasites within the mosquito vector, effectively blocking subsequent infections. The mosquito midgut protein Anopheline alanyl aminopeptidase N (AnAPN1) is the leading, mosquito-based TBV antigen and structure-function studies have identified two Class II epitopes that induce potent transmission-blocking (T-B) antibodies. Here, we functionally screened new immunogens and down-selected to the UF6b construct that has two glycine-linked copies of the T-B epitopes. We established a process for manufacturing UF6b and evaluated in outbred female CD1 mice the immunogenicity of the preclinical product with the human-safe adjuvant Glucopyranosyl Lipid Adjuvant in a liposomal formulation with saponin QS21 (GLA-LSQ). UF6b:GLA-LSQ was immunogenic and immunofocused the humoral response to one of the key T-B epitopes resulting in potent T-B activity and establishing UF6b as a prime TBV candidate to aid in malaria elimination and eradication efforts.

immunology↗

bioRxiv2020

Role of TRPV1 in colonic mucin production and gut microbiota profile

PURPOSEThis study focuses on exploring the role of sensory cation channel Transient Receptor Potential channel subfamily Vanilloid 1 (TRPV1) in gut health, specifically mucus secretion and microflora profile in gut. METHODS AND RESULTSWe employed resiniferatoxin (ultrapotent TRPV1 agonist) induced chemo-denervation model in rats and studied the effects of TRPV1 ablation on gut mucus secretion patterns. Histological and transcriptional analysis showed substantial decrease in mucus production as well as in expression of genes involved in goblet cells differentiation, mucin production and glycosylation. 16S metagenome analysis revealed changes in abundance of various gut bacteria, including decrease in beneficial bacteria like Lactobacillus spp and Clostridia spp. Also, TRPV1 ablation significantly decreased the levels of short chain fatty acids, i.e. acetate and butyrate. CONCLUSIONThe present study provides first evidence that systemic TRPV1 ablation leads to impairment in mucus secretion and causes dysbiosis in gut. Further, it suggests to address mucin production and gut microbiota related adverse effects during the development of TRPV1 antagonism/ablation-based therapeutic and preventive strategies.

physiology↗