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Introduction: Bluetongue (BT) poses a significant threat to the livestock industry, affecting various animal species and resulting in substantial economic losses. The existence of numerous BT virus (BTV) serotypes has hindered control efforts, highlighting the need for broad-spectrum vaccines., Methodology: In this study, we evaluated the conserved amino acid sequences within key non-structural (NS) proteins of BTV and identified numerous highly conserved murine- and bovine-specific MHC class I-restricted (MHC-I) CD8+ and MHC-II-restricted CD4+ epitopes. We then screened these conserved epitopes for antigenicity, allergenicity, toxicity, and solubility. Using these epitopes, we developed in silico-based broad-spectrum multiepitope vaccines with Toll-like receptor (TLR-4) agonists. The predicted proinflammatory cytokine response was assessed in silico using the C-IMMSIM server. Structural modeling and refinement were achieved using Robetta and GalaxyWEB servers. Finally, we assessed the stability of the docking complexes through extensive 100-nanosecond molecular dynamics simulations before considering the vaccines for codon optimization and in silico cloning., Results: We found many epitopes that meet these criteria within NS1 and NS2 proteins and developed in silico broad-spectrum vaccines. The immune simulation studies revealed that these vaccines induce high levels of IFN-γ and IL-2 in the vaccinated groups. Protein-protein docking analysis demonstrated promising epitopes with strong binding affinities to TLR-4. The docked complexes were stable, with minimal Root Mean Square Deviation and Root Mean Square Fluctuation values. Finally, the in silico-cloned plasmids have high % of GC content with > 0.8 codon adaptation index, suggesting they are suitable for expressing the protein vaccines in prokaryotic system., Discussion: These next-generation vaccine designs are promising and warrant further investigation in wet lab experiments to assess their immunogenicity, safety, and efficacy for practical application in livestock. Our findings offer a robust framework for developing a comprehensive, broad-spectrum vaccine, potentially revolutionizing BT control and prevention strategies in the livestock industry., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kolla, Dutt, Kumar, Hebbandi Nanjunadappa, Karakach, Singh, Kelvin, Clement Mertens and Umeshappa.)

Cardiac fibrosis can be mitigated by limiting fibroblast-to-myofibroblast differentiation and proliferation. Human antigen R (HuR) modulates messenger RNA stability and expression of multiple genes. However, the direct role of cardiac myofibroblast HuR is unknown. Myofibroblast-specific deletion of HuR limited cardiac fibrosis and preserved cardiac functions in pressure overload injury. Knockdown of HuR in transforming growth factor-β1-treated cardiac fibroblasts suppressed myofibroblast differentiation and proliferation. HuR deletion abrogated the expression and messenger RNA stability of cyclins D1 and A2, suggesting a potential mechanism by which HuR promotes myofibroblast proliferation. Overall, these data suggest that inhibition of HuR could be a potential therapeutic approach to limit cardiac fibrosis., Competing Interests: This work was supported by National Institutes of Health grants HL116729 (Dr Krishnamurthy) and HL138023 (Drs Krishnamurthy and Zhang), an American Heart Association Transformational Project Award (19TPA34850100, Dr Krishnamurthy), and an American Heart Association postdoctoral fellowship (916497, Dr Singh). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2024 The Authors.)

As emerging pollutants in the environment, nanoplastics (NPs) can cross biological barriers and be enriched in organisms, posing a greatest threat to the health of livestock and humans. However, the size-dependent toxic effects of NPs in higher mammals remain largely unknown. To determine the size-dependent potential toxicities of NPs, we exposed mouse (AML-12) and human (L02) liver cell lines in vitro, and 6-week-old C57BL/6 mice (well-known preclinical model) in vivo to five different sizes of polystyrene NPs (PS-NPs) (20, 50, 100, 200 and 500 nm). We found that ultra-small NPs (20 nm) induced the highest cytotoxicity in mouse and human liver cell lines, causing oxidative stress and mitochondrial membrane potential loss on AML-12 cells. Unexpectedly in vivo, after long-term oral exposure to PS-NPs (75 mg/kg), medium NPs (200 nm) and large NPs (500 nm) induced significant hepatotoxicity, evidenced by increased oxidative stress, liver dysfunction, and lipid metabolism disorders. Most importantly, medium or large NPs generated local immunotoxic effects via recruiting and activating more numbers of neutrophils and monocytes in the liver or intestine, which potentially resulted in increased proinflammatory cytokine secretion and the tissue damage. The discrepancy in in vitro-in vivo toxic results might be attributed to the different properties of biodistribution and tissue accumulation of different sized NPs in vivo. Our study provides new insights regarding the hepatotoxicity and immunotoxicity of NPs on human and livestock health, warranting us to take immense measures to prevent these NPs-associated health damage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are 2 major liver autoimmune diseases. PBC is common in women and primarily affects intrahepatic small bile duct epithelial cells, known as cholangiocytes. In contrast, PSC is dominant in men and primarily affects medium and big intrahepatic and extrahepatic bile duct epithelial cells. Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes, and its incidence is increasing worldwide in both men and women. Numerous retrospective and clinical studies have suggested that PBC patients rarely develop CCA compared to PSC patients. CCA is accountable for the higher deaths in PSC patients due to ineffective therapies and our inability to diagnose the disease at an early stage. Therefore, it is paramount to understand the differences in immune surveillance mechanisms that render PBC patients more resistant while PSC patients are susceptible to CCA development. Here, we review several potential mechanisms contributing to differences in the susceptibility to CCA in PBC versus PSC patients., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

Invariant NKT (iNKT) cells comprise a heterogeneous group of non-circulating, tissue-resident T lymphocytes that recognize glycolipids, including alpha-galactosylceramide (αGalCer), in the context of CD1d, but whether peripheral iNKT cell subsets are terminally differentiated remains unclear. Here we show that mouse and human liver-resident αGalCer/CD1d-binding iNKTs largely correspond to a novel Zbtb16 + Tbx21 + Gata3 + Maf low Rorc - subset that exhibits profound transcriptional, phenotypic and functional plasticity. Repetitive in vivo encounters of these liver iNKT (LiNKT) cells with intravenously delivered αGalCer/CD1d-coated nanoparticles (NP) trigger their differentiation into immunoregulatory, IL-10+IL-21-producing Zbtb16 high Maf high Tbx21 + Gata3 + Rorc - cells, termed LiNKTR1, expressing a T regulatory type 1 (TR1)-like transcriptional signature. This response is LiNKT-specific, since neither lung nor splenic tissue-resident iNKT cells from αGalCer/CD1d-NP-treated mice produce IL-10 or IL-21. Additionally, these LiNKTR1 cells suppress autoantigen presentation, and recognize CD1d expressed on conventional B cells to induce IL-10+IL-35-producing regulatory B (Breg) cells, leading to the suppression of liver and pancreas autoimmunity. Our results thus suggest that LiNKT cells are plastic for further functional diversification, with such plasticity potentially targetable for suppressing tissue-specific inflammatory phenomena., (© 2022. The Author(s).)

Pathological fibrosis contributes to progression of various diseases, for which the therapeutic options are limited. Idiopathic pulmonary fibrosis (IPF) is one such progressive and fatal interstitial fibrotic disease that is often characterized by excessive accumulation of extracellular matrix (ECM) proteins leading to stiff lung tissue and impaired gas exchange. However, the molecular mechanisms underlying IPF progression remain largely unknown. In this study, we determined the role of Runt-related transcription factor 1 (RUNX1), an evolutionarily conserved transcription factor, in the differentiation of human lung fibroblasts (HLFs) in vitro and in an animal model of bleomycin (BLM)-induced lung fibrosis. We observed that the expression of RUNX1 was significantly increased in the lungs of BLM-injected mice as compared to saline-treated mice. Furthermore, HLFs stimulated with transforming growth factor β (TGF-β) showed significantly higher RUNX1 expression at both mRNA and protein levels, and compartmentalization in the nucleus. Inhibition of RUNX1 in HLFs (using siRNA) showed a significant reduction in the differentiation of fibroblasts into myofibroblasts as evidenced by reduced expression of alpha-smooth muscle actin (α-SMA), TGF-β and ECM proteins such as fibronectin 1 (FN1), and collagen 1A1 (COL1A1). Mechanistic studies revealed that the increased expression of RUNX1 in TGF-β-stimulated lung fibroblasts is due to enhanced mRNA stability of RUNX1 through selective interaction with the RNA-binding profibrotic protein, human antigen R (HuR). Collectively, our data demonstrate that increased expression of RUNX1 augments processes involved in lung fibrosis including the differentiation of fibroblasts into collagen-synthesizing myofibroblasts. Our study suggests that targeting RUNX1 could limit the progression of organ fibrosis in diseases characterized by abnormal collagen deposition., (© 2022 Wiley Periodicals LLC.)

Nanovaccine-based immunotherapy (NBI) has received greater attention recently for its potential to prime tumor-specific immunity and establish a long-term immune memory that prevents tumor recurrence. Despite encouraging results in the recent studies, there are still numerous challenges to be tackled for eliciting potent antitumor immunity using NBI strategies. Based on the principles that govern immune response, here it is proposed that these challenges need to be addressed at the five critical cascading events: Loading tumor-specific antigens by nanoscale drug delivery systems (L); Draining tumor antigens to lymph nodes (D); Internalization by dendritic cells (DCs) (I); Maturation of DCs by costimulatory signaling (M); and Presenting tumor-peptide-major histocompatibility complexes to T cells (P) (LDIMP cascade in short). This review provides a detailed and objective overview of emerging NBI strategies to improve the efficacy of nanovaccines in each step of the LDIMP cascade. It is concluded that the balance between each step must be optimized by delicate designing and modification of nanovaccines and by combining with complementary approaches to provide a synergistic immunity in the fight against cancer., (© 2021 Wiley-VCH GmbH.)

Neutrophils with immunoregulatory properties, also referred to as type-2 neutrophils (N2), myeloid-derived suppressor cells (MDSCs), or tumor-associated neutrophils (TANs), comprise a heterogeneous subset of cells that arise from unknown precursors in response to poorly understood cues. Here, we find that, in several models of liver autoimmunity, pharmacologically induced, autoantigen-specific T regulatory type-1 (TR1) cells and TR1-cell-induced B regulatory (B reg ) cells use five immunoregulatory cytokines to coordinately recruit neutrophils into the liver and program their transcriptome to generate regulatory neutrophils. The liver-associated neutrophils from the treated mice, unlike their circulating counterparts or the liver neutrophils of sick mice lacking antigen-specific TR1 cells, are proliferative, can transfer disease protection to immunocompromised hosts engrafted with pathogenic effectors, and blunt antigen-presentation and local autoimmune responses via cathelin-related anti-microbial peptide (CRAMP), a cathelicidin, in a CRAMP-receptor-dependent manner. These results, thus, identify antigen-specific regulatory T cells as drivers of tissue-restricted regulatory neutrophil formation and CRAMP as an effector of regulatory neutrophil-mediated immunoregulation., Competing Interests: Declaration of interests P. Santamaria is founder, scientific officer, and stockholder of Parvus Therapeutics. He is an inventor on patents on pMHC-based nanomedicines and receives funding from the company. D.A.A.V. is cofounder and stockholder in Novasenta, Tizona, and Potenza; a stockholder in Oncorus and Werewolf; has patents licensed and royalties received from Astellas and BMS; is a scientific advisory board member at Tizona, Werewolf, F-Star, and Bicara; is a consultant at Astellas, BMS, Almirall, and Incyte; and receives research funding from BMS, Astellas, and Novasenta., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

To achieve safe and effective antitumor immunity, we constructed the M1-macrophage-membrane-coated nanoparticles [(C/I)BP@B-A(D)&M1m] having laser-responsive, size-changeable, on-demand drug release and prolonged circulation retention properties. (C/I)BP@B-A(D)&M1m delayed clearance by the phagocytic system and homed to tumor efficiently. Upon 650 nm laser irradiation, the hydrophobic core of the PEGylated bilirubin nanoparticles (BP) got disrupted, releasing small-sized deep-penetrating B-A(D) particles, photosensitive chlorin e6 (C), and tolerance-inducing indoleamine 2,3-dioxygenase inhibitor, indoximode (I). Treatment-induced immunogenic cell death and antitumor immunity, suppressing primary tumor growth in both 4T1 and B16F10 models without causing any adverse effects. Most importantly, it inhibited primary tumor recurrence as well as metastasis. Thus, this study provides a promising combinatorial strategy to trigger antitumor immunity in malignancies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Application of cancer vaccines is limited due to their systemic immunotoxicity and inability to satisfy all the steps, including loading of tumor antigens, draining of antigens to lymph nodes (LNs), internalization of antigens by dendritic cells (DCs), DC maturation, and cross-presentation of antigens for T cell activation. Here, we present a combinatorial therapy, based on a α-cyclodextrin (CD)-based gel system, DOX/ICG/CpG-P-ss-M/CD, fabricated by encapsulating doxorubicin (DOX) and the photothermal agent indocyanine green (ICG). Upon irradiation, the gel system exhibited heat-responsive release of DOX and vaccine-like nanoparticles, CpG-P-ss-M, along with chemotherapy- and phototherapy-generated abundant tumor-specific antigen storage in situ. The released CpG-P-ss-M acted as a carrier adsorbed and delivered antigens to LNs, promoting the uptake of antigens by DCs and DC maturation. Notably, combined with PD-L1 blocking, the therapy effectively inhibited primary tumor growth and induced tumor-specific immune response against tumor recurrence and metastasis., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Peptide MHC class II-based (pMHCII-based) nanomedicines trigger the formation of multicellular regulatory networks by reprogramming autoantigen-experienced CD4+ T cells into autoimmune disease-suppressing T regulatory type 1 (TR1) cells. We have shown that pMHCII-based nanomedicines displaying liver autoimmune disease-relevant yet ubiquitously expressed antigens can blunt various liver autoimmune disorders in a non-disease-specific manner without suppressing local or systemic immunity against infectious agents or cancer. Here, we show that such ubiquitous autoantigen-specific T cells are also awakened by extrahepatic tissue damage and that the corresponding TR1 progeny can suppress experimental autoimmune encephalomyelitis (EAE) and pancreatic β cell autoreactivity. In mice having EAE, nanomedicines displaying either ubiquitous or CNS-specific epitopes triggered the formation and expansion of cognate TR1 cells and their recruitment to the CNS-draining lymph nodes, sparing their liver-draining counterparts. Surprisingly, in mice having both liver autoimmunity and EAE, liver inflammation sequestered these ubiquitous or even CNS-specific TR1 cells away from the CNS, abrogating their antiencephalitogenic activity. In these mice, only the ubiquitous antigen-specific TR1 cells suppressed liver autoimmunity. Thus, the scope of antigen spreading in autoimmune disorders is larger than previously anticipated, involving specificities expected to be silenced by mechanisms of tolerance; the regulatory activity, but not the retention of autoreactive TR1 cells, requires local autoantigen expression.

Aging represents an independent risk factor for the development of cardiovascular disease, and is associated with complex structural and functional alterations in the vasculature, such as endothelial dysfunction. Small- and intermediate-conductance, Ca 2+ -activated K + channels (KCa2.3 and KCa3.1, respectively) are prominently expressed in the vascular endothelium, and pharmacological activators of these channels induce robust vasodilation upon acute exposure in isolated arteries and intact animals. However, the effects of prolonged in vivo administration of such compounds are unknown. In our study, we hypothesized that such treatment would ameliorate aging-related cardiovascular deficits. Aged (∼18 months) male Sprague Dawley rats were treated daily with either vehicle or the KCa channel activator SKA-31 (10 mg/kg, intraperitoneal injection; n = 6/group) for 8 weeks, followed by echocardiography, arterial pressure myography, immune cell and plasma cytokine characterization, and tissue histology. Our results show that SKA-31 administration improved endothelium-dependent vasodilation, reduced agonist-induced vascular contractility, and prevented the aging-associated declines in cardiac ejection fraction, stroke volume and fractional shortening, and further improved the expression of endothelial KCa channels and associated cell signalling components to levels similar to those observed in young male rats (∼5 months at end of study). SKA-31 administration did not promote pro-inflammatory changes in either T cell populations or plasma cytokines/chemokines, and we observed no overt tissue histopathology in heart, kidney, aorta, brain, liver and spleen. SKA-31 treatment in young rats had little to no effect on vascular reactivity, select protein expression, tissue histology, plasma cytokines/chemokines or immune cell properties. Collectively, these data demonstrate that administration of the KCa channel activator SKA-31 improved aging-related cardiovascular function, without adversely affecting the immune system or promoting tissue toxicity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Peptide-major histocompatibility complex class II (pMHCII)-based nanomedicines displaying tissue-specific autoantigenic epitopes can blunt specific autoimmune conditions by re-programming cognate antigen-experienced CD4+ T-cells into disease-suppressing T-regulatory type 1 (TR1) cells. Here, we show that single pMHCII-based nanomedicines displaying epitopes from mitochondrial, endoplasmic reticulum or cytoplasmic antigens associated with primary biliary cholangitis (PBC) or autoimmune hepatitis (AIH) can broadly blunt PBC, AIH and Primary Sclerosing Cholangitis in various murine models in an organ- rather than disease-specific manner, without suppressing general or local immunity against infection or metastatic tumors. Therapeutic activity is associated with cognate TR1 cell formation and expansion, TR1 cell recruitment to the liver and draining lymph nodes, local B-regulatory cell formation and profound suppression of the pro-inflammatory capacity of liver and liver-proximal myeloid dendritic cells and Kupffer cells. Thus, autoreactivity against liver-enriched autoantigens in liver autoimmunity is not disease-specific and can be harnessed to treat various liver autoimmune diseases broadly.

Nanomedicine is at the forefront of targeted drug delivery for cancer therapy. An improved combinatorial approach is highlighted for breast cancer treatment by Hu et al. in this issue of ACS Applied Materials and Interfaces. The authors demonstrated that, by combining multistage-responsive nanoparticles carrying a therapeutic drug, doxorubicin, a photothermal agent, indocyanine green, and a nitric oxide donor with photothermal therapy and intravenous injection of a tumor-homing iRGD peptide, one could achieve efficient therapeutics distribution deep inside the tumor and nearly eradicate primary tumor growth. An in-depth understanding of this approach in combination with other strategies such as the use of immunomodulators would facilitate treating metastasis  in distant organs, and clinical translation of this platform, benefiting cancer patients by providing long-lasting efficacy.

The extracellular signal-regulated kinase (ERK) pathway has been shown to regulate pathogenesis of many viral infections, but its role during rabies virus (RV) infection in vivo is not clear. In the present study, we investigated the potential role of MEK-ERK1/2 signalling pathway in the pathogenesis of rabies in mouse model and its regulatory effects on pro-inflammatory cytokines and other mediators of immunity, and kinetics of immune cells. Mice were infected with 25 LD 50 of challenge virus standard (CVS) strain of RV by intracerebral (i.c.) inoculation and were treated i.c. with U0126 (specific inhibitor of MEK1/2) at 10 μM/mouse at 0, 2, 4 and 6 days post-infection. Treatment with U0126 resulted in delayed disease development and clinical signs, increased survival time with lesser mortality than untreated mice. The better survival of inhibitor-treated and RV infected mice was positively correlated with reduced viral load and reduced viral spread in the brain as quantified by real-time PCR, direct fluorescent antibody test and immunohistochemistry. CVS-infected/mock-treated mice developed severe histopathological lesions with increased Fluoro-Jade B positive degenerating neurons in brain, which were associated with higher levels of serum nitric oxide, iNOS, TNF-α, and CXCL10 mRNA. Also CVS-infected/U0126-treated mice revealed significant decrease in caspase 3 but increase in Bcl-2 mRNA levels and less TUNEL positive apoptotic cells. CVS-infected/U0126-treated group also showed significant increase in CD4 + , CD8 + T lymphocytes and NK cells in blood and spleen possibly due to less apoptosis of these cells. In conclusion, these data suggest that MEK-ERK1/2 signalling pathway play critical role in the pathogenesis of RV infection in vivo and opens up new avenues of therapeutics., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We have shown that nanoparticles (NPs) can be used as ligand-multimerization platforms to activate specific cellular receptors in vivo. Nanoparticles coated with autoimmune disease-relevant peptide-major histocompatibility complexes (pMHC) blunted autoimmune responses by triggering the differentiation and expansion of antigen-specific regulatory T cells in vivo. Here, we define the engineering principles impacting biological activity, detail a synthesis process yielding safe and stable compounds, and visualize how these nanomedicines interact with cognate T cells. We find that the triggering properties of pMHC-NPs are a function of pMHC intermolecular distance and involve the sustained assembly of large antigen receptor microclusters on murine and human cognate T cells. These compounds show no off-target toxicity in zebrafish embryos, do not cause haematological, biochemical or histological abnormalities, and are rapidly captured by phagocytes or processed by the hepatobiliary system. This work lays the groundwork for the design of ligand-based NP formulations to re-program in vivo cellular responses using nanotechnology.

Microgravity has been known to induce cell death. However, its underlying mechanism is less studied. In this study, BL6-10 melanoma cells were cultured in flasks under simulated microgravity (SMG). We examined cell apoptosis, and assessed expression of genes associated with apoptosis and genes regulating apoptosis in cells under SMG. We demonstrate that SMG induces cell morphological changes and microtubule alterations by confocal microscopy, and enhances apoptosis by flow cytometry, which was associated with up- and down-regulation of pro-apoptotic and anti-apoptotic genes, respectively. Moreover, up- and down-regulation of pro-apoptotic (Caspases 3, 7, 8) and anti-apoptotic (Bcl2 and Bnip3) molecules was confirmed by Western blotting analysis. Western blot analysis also indicates that SMG causes inhibition of an apoptosis suppressor, pNF-κB-p65, which is complemented by the predominant localization of NF-κB-p65 in the cytoplasm. SMG also reduces expression of molecules regulating the NF-κB pathway including Uev1A, TICAM, TRAF2, and TRAF6. Interestingly, 10 DNA repair genes are down-regulated in cells exposed to SMG, among which down-regulation of Parp, Ercc8, Rad23, Rad51, and Ku70 was confirmed by Western blotting analysis. In addition, we demonstrate a significant inhibition of molecules involved in the DNA-damage response, such as p53, PCNA, ATM/ATR, and Chk1/2. Taken together, our work reveals that SMG promotes the apoptotic response through a combined modulation of the Uev1A/TICAM/TRAF/NF-κB-regulated apoptosis and the p53/PCNA- and ATM/ATR-Chk1/2-controlled DNA-damage response pathways. Thus, our investigation provides novel information, which may help us to determine the cause of negative alterations in human physiology occurring at spaceflight environment. J. Cell. Biochem. 117: 2138-2148, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The greatest hurdle in cancer treatment is the metastasis of primary tumors to distant organs. Our knowledge on how different immune cells, in the absence of exogenous stimulation, prevent tumor metastasis in distant organs is poorly understood. Using a highly metastatic murine lung B16 melanoma cell line BL6-10, we employed naive mice that genetically lack CD4(+) or CD8(+) T cells, or are depleted of dendritic cells (DCs) or natural killer (NK) cells to understand the relative importance of these cells in metastasis prevention. Irrespective of the presence of naïve CD4(+) T, CD8(+) T, DCs, or NK cells, lungs, which act as primary site of predilection for B16 melanoma, readily developed numerous lung BL6-10 melanoma colonies. However, their absence led to B16 melanoma metastasis in variable proportions to distant organs, particularly livers, kidneys, adrenals, ovaries, and hearts. NK cells mediate prevention of BL6-10 metastasis to various organs, especially to livers. Mechanistically, CD40L signaling, a critical factor required for DC licensing and CD8(+) cytotoxic T lymphocyte (CTL) responses, was required for CD4(+) T cell-mediated prevention of systemic BL6-10 metastasis. These results suggest that the composition and functions of different immune cells in distant tissue microenvironments (distant organs other than primary sites of predilection) robustly mediate natural resistance against melanoma metastasis. Thus, harnessing these immune cells' responses in immunotherapeutics would considerably limit organ metastasis.

Involvement of CD4(+) helper T (Th) cells is crucial for CD8(+) cytotoxic T lymphocyte (CTL)-mediated immunity. However, CD4(+) Th's signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4(+) Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4(+) T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA)-pulsed dendritic cell (DCova). CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2K(b)/OVA257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I) complex signaling, CD4(+) Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4(+) Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7) molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4(+) Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy.

Increased CD8(+) T-cell precursor frequency (PF) precludes the requirement of CD4(+) helper T (Th) cells for primary CD8(+) cytotoxic T-lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) -pulsed dendritic cells (DC(OVA)) derived from C57BL/6, CD40 knockout (CD40(-/-)) or CD40 ligand knockout (CD40L(-/-)) mice were used to immunize C57BL/6, Ia(b-/-), CD40(-/-) or CD40L(-/-) mice, whose PF was previously increased with transfer of 1 × 10(6) CD8(+) T cells derived from OVA-specific T-cell receptor (TCR) transgenic OTI, OTI(CD40(-/-)) or OTI(CD40L(-/-)) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4(+) T-cell help and Th-provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA-expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4(+) T-cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4(+) T-cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4(+) T-cell functions., (© 2012 Blackwell Publishing Ltd.)

Mononuclear leukocytes of peripheral blood mononuclear cells (PBMCs) and regional lymphoid organs (RLOs) play a critical role in primary BTV replication and subsequent viral dissemination to distant systemic organs. The lesions in animals develop primarily as a result of vascular insult, presumably induced by the activity of viral and/or proinflammatory vasoactive mediators. Hence, the current study was designed in sheep to investigate the responses of potent vasoactivators, inducible nitric oxide synthase (iNOS) and/or nitric oxide (NO) in mononuclear leukocytes of PBMCs and RLOs. The results show that BTV infection of sheep led to enhanced transcription of iNOS in PBMCs and in particular RLOs. The BTV RNAs and/or antigens were readily demonstrable in these mononuclear leukocytes, suggesting the possible role of BTV in iNOS induction. Moreover, upon in vitro infection of PBMCs with BTV-23, iNOS was up-regulated in time-dependent fashion and correlated with increased NO production. The results from these in vivo and in vitro studies thus suggest iNOS and NO produced by mononuclear leukocytes may potentially contribute to vascular-related pathology of BT.

The limitations of highly active anti-retroviral therapy have necessitated the development of alternative therapeutics for human immunodeficiency virus type-1 (HIV-1)-infected patients with dysfunctional dendritic cells (DCs) and CD4(+) T cell deficiency. We previously demonstrated that HIV-1 Gp120-specific T cell-based Gp120-Texo vaccine by using ConA-stimulated C57BL/6 (B6) mouse CD8(+) T (ConA-T) cells with uptake of pcDNA(Gp120)-transfected B6 mouse DC line DC2.4 (DC2.4(Gp120))-released exosomes (EXO(Gp120)) was capable of stimulating DC and CD4(+) T cell-independent CD8(+) cytotoxic T lymphocyte (CTL) responses detected in wild-type B6 mice using non-specific PE-anti-CD44 and anti-IFN-γ antibody staining by flow cytometry. To assess effectiveness of Gp120-Texo vaccine in transgenic (Tg) HLA-A2 mice mimicking the human situation, we constructed adenoviral vector AdV(Gp120) expressing HIV-1 GP120 by recombinant DNA technology, and generated Gp120-Texo vaccine by using Tg HLA-A2 mouse CD8(+) ConA-T cells with uptake of AdV(Gp120)-transfected HLA-A2 mouse bone marrow DC (DC(Gp120))-released EXO(Gp120). We then performed animal studies to assess Gp120-Texo-induced stimulation of Gp120-specific CTL responses and antitumor immunity in Tg HLA-A2 mice. We demonstrate that Gp120-Texo vaccine stimulates Gp120-specific CTL responses detected in Tg HLA-A2 mice using Gp120-specific PE-HLA-A2/Gp120 peptide (KLTPLCVTL) tetramer staining by flow cytometry. These Gp120-specific CTLs are capable of further differentiating into functional effectors with killing activity to Gp120 peptide-pulsed splenocytes in vivo. In addition, Gp120-Texo vaccine also induces Gp120-specific preventive, therapeutic (for 6 day tumor lung metastasis) and CD4(+) T cell-independent long-term immunity against B16 melanoma BL6-10(Gp120/A2Kb) expressing both Gp120 and A2Kb (α1 and α2 domains of HLA-A2 and α3 domain of H-2K(b)) in Tg HLA-A2 mice. Taken together, the novel CD8(+) Gp120-Texo vaccine capable of stimulating efficient CD4(+) T cell-independent Gp120-specific CD8(+) CTL responses leading to therapeutic and long-term immunity in Tg HLA-A2 mice may represent a new immunotherapeutic vaccine for treatment of HIV-1 patients with CD4(+) T cell deficiency., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Bluetongue disease has been causing variable morbidity and mortality in sheep in India and other countries of the world. The experimental infection with Indian BTV-1 strain induced mild to sub-acute infection in native sheep. There were low induction of IL-12, IFN-γ and TNF-α cytokine mRNA expressions determined by real time RT-PCR in draining lymph nodes (DLN), spleen, and PBMCs during the initial stages of infection (8 days post inoculation, DPI) and higher around 15 DPI. The reduced pro-inflammatory cytokine (IFN-γ and TNF-α) responses during the initial stage of infection (8 DPI) was also accompanied by similarly decreased T-cell populations and overt clinical symptoms. Later up regulation of these cytokines and substantial increase in the proportion of CD8(+) T-cells occurred with reduction of clinical signs and disappearance of BTV-1 antigen from tissues as determined by immunohistochemistry and RT-PCR. Thus there is definite involvement of pro-inflammatory cytokines and CD8(+) T cell activity in disease induced by BTV-1 strain., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The pathogenesis of bluetongue (BT) could vary with route of inoculation. Using laboratory-passaged moderately virulent bluetongue virus serotype 23 (BTV-23), one of the most prevalent Indian serotype, we investigated the pathogenesis of BT in intradermally (ID) and intravenously (IV) inoculated native sheep. The ID inoculation resulted in relatively increased clinical signs and lesions in many organs as compared to IV inoculation. BTV-23 detection by real-time RT-PCR and isolation studies revealed that ID inoculation can be more efficient than IV ones in disseminating and spreading virus to systemic organs, including pre-scapular draining lymph node, spleen, lungs and pulmonary artery. Furthermore, the ID inoculation resulted in early onset and increased humoral response with significant increase (P<0.01) in antibody titre at various intervals. Taken together, these data suggest that ID inoculation can be more potent in reproducing many aspects of natural infection, including clinical disease, viral and immune responses, and may be useful route in setting up experimental infections for challenge or pathogenesis studies using laboratory passaged BTVs., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutics. In this study, we generated ovalbumin (OVA)-pulsed and pcDNAgp120-transfected dendritic cell (DC)-released exosomes (EXOova and EXOgp120) and ConA-stimulated C57BL/6 CD8(+) T cells. OVA- and Gp120-Texo vaccines were generated from CD8(+) T cells with uptake of EXOova and EXOgp120, respectively. We demonstrate that OVA-Texo stimulates in vitro and in vivo OVA-specific CD4(+) and CD8(+) cytotoxic T lymphocyte (CTL) responses leading to long-term immunity against OVA-expressing BL6-10(OVA) melanoma. Interestingly, CD8(+) T cell responses are DC and CD4(+) T cell independent. Importantly, Gp120-Texo also stimulates Gp120-specific CTL responses and long-term immunity against Gp120-expressing B16 melanoma. Therefore, this novel HIV-1-specific EXO-targeted Gp120-Texo vaccine may be useful in induction of efficient CTL responses in AIDS patients with DC dysfunction and CD4(+) T cell deficiency., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Bluetongue (BT), a serious disease of sheep and some wild ruminants, is caused by bluetongue virus (BTV), a member of the family, Reoviridae. The current research thrust for controlling BT is on development of efficient vaccines, necessitating clear understanding of ovine immunology. At present, comparative studies on cytokine gene expression profiles of naïve and BTV-sensitized peripheral blood mononuclear cells (PBMC) in sheep have not been clearly understood. In the present study, PBMC from naïve and BEI-inactivated-saponin-adjuvanted BTV-1 vaccinated sheep were stimulated in vitro with heterologous BTV-23. At various intervals, RT-qPCR was carried out to estimate cytokine (interferon-gamma, interleukin-12 and interleukin-2) mRNA expressions that are linked to cell-mediated immunity. The results showed that PBMC cytokine profiles were relatively increased both temporally and quantitatively in immunized sheep PBMC compared to naïve ones, suggesting that BTV-1 vaccination may prime immune system that can cross-react with BTV-23 antigens., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The role of apoptosis in pathogenesis of bluetongue (BT) has been suggested from various in vitro studies. However, to date, no clear data are available regarding BTV-induced apoptosis and its consequences in natural host, sheep. In the present study, bluetongue virus (BTV)-induced apoptosis was studied in sheep blood and splenic mononuclear cells by analyzing annexin(+)-propidium iodide(-) early apoptotic cells, DNA ladder pattern, and caspase-3 gene expression. The onset of apoptosis and lymphocyte depletion in viraemic phase and IFN-alpha response indicated the involvement of BTV and IFN-alpha in the pathogenesis of BT. The development of Pasteurella pneumonia in 4 of 7 infected sheep during the experiment pointed to possible BTV-induced immuno-suppression and predisposition to secondary microbial infections. These results have significant implications not only in understanding immuno-pathological consequences but also in studying interactions of BTV with host cells., (Copyright 2010 Elsevier B.V. All rights reserved.)

Bluetongue is a serious hemorrhagic disease of sheep, cattle and other ruminants causing economic losses worldwide. Recent invasion of multiple bluetongue virus serotypes (BTV) in various countries warrants immediate development of efficacious vaccine that targets more than one serotype. In the present study, the cross-protective efficacy of binary ethylenimine (BEI)-inactivated BTV-1 vaccine was evaluated in Indian native sheep against virulent heterologous BTV-23 serotype challenge. BTV-1 vaccination induced significant cell-mediated immunity (CMI) as determined by lymphoproliferative responses, and increased CD8 T cell, IL-2 and IFN-gamma responses. Both naïve and immunized sheep also showed increased CD4 T cell, IL-12 and IFN-alpha responses. Collectively, these data suggested that inactivated BTV-1 vaccine induced appreciable CMI and greatly reduced the severity of heterologous BTV-23 infection., (Copyright 2010 Elsevier Ltd. All rights reserved.)

T cell-T cell Ag presentation is increasingly attracting attention. We previously showed that the in vitro OVA-pulsed dendritic cell (DC(OVA))-activated CD4(+) Th cells acquired OVA peptide/MHC (pMHC) class I and costimulatory molecules such as CD54 and CD80 from DC(OVA) and acted as CD4(+) Th-APC capable of stimulating OVA-specific CD8(+) CTL responses. In this study, we further applied the OVA-specific TCR-transgenic OT I and OT II mice with deficiency of various cytokines or costimulatory molecule genes useful for studying the molecular mechanisms underlying in Th-APC's stimulatory effect. We demonstrated that DC(OVA)-stimulated OT II CD4(+) Th-APC also acquired costimulatory molecules such as CD40, OX40L, and 4-1BBL and the functional pMHC II complexes by DC(OVA) activation. CD4(+) Th-APC with acquired pMHC II and I were capable of stimulating CD4(+) Th1 and central memory CD8(+)44(+)CD62L(high)IL-7R(+) T cell responses leading to antitumor immunity against OVA-expressing mouse B16 melanoma. Their stimulatory effect on CD8(+) CTL responses and antitumor immunity is mediated by IL-2 secretion, CD40L, and CD80 signaling and is specifically targeted to CD8(+) T cells in vivo via acquired pMHC I. In addition, CD4(+) Th-APC expressing OVA-specific TCR, FasL, and perforin were able to kill DC(OVA) and neighboring Th-APC expressing endogenous and acquired pMHC II. Taken together, we show that CD4(+) Th-APC can modulate immune responses by stimulating CD4(+) Th1 and central memory CD8(+) T cell responses and eliminating DC(OVA) and neighboring Th-APC. Therefore, our findings may have great impacts in not only the antitumor immunity, but also the regulatory T cell-dependent immune tolerance in vivo.