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Many processing problems still occur, causing the low quality of raw rubber materials and farmers low-income. This can be seen from the use of coagulants, storage methods and the level of cleanliness. This study was aimed at analyzing the quality and added value of raw rubber materials based on the use of coagulant types. This study was conducted in October - November 2021. Fresh latex was obtained from the rubber plantation in Mulyaguna Village, OKI Regency. Completely randomized design was used with 5 treatments and 4 replications. The coagulants used were the recommended ones (liquid smoke, formic acid), and the commonly used by the farmers (sulfuric acid, aluminum sulfate, and TSP fertilizer). The results of the study showed that liquid smoke and formic acid were better than the coagulants commonly used by farmers, which could be seen from the ash content, initial plasticity, and plasticity retention index. The dry rubber contents (DRCs) using liquid smoke, formic acid, sulfuric acid, aluminum sulfate, and TSP fertilizer were 66.081; 62.978; 59.067; 56.202 and 60.796 respectively. Liquid smoke and formic acid provided the added value for raw rubber materials of 1,203/kg IDR and 792/kg IDR compared to sulfuric acid commonly used by farmers.

The information on rice genetic material is needed to create new high-yielding varieties adaptive to dryland. Local rice genetic resources have been tested to be tolerant under conditions affected by global climate change (increased temperature, pest/disease attacks, droughts). This study aims to identify and characterize the agronomic character of local rice in Musi Rawas (MURA) and North MURA Regencies, South Sumatra. The results of identification and characterization, as well as correlation and regression analysis, revealed that three local varieties have agronomic characteristics that determine high-yield local rice productivity. This information can be utilized by breeders to create new superior varieties of rice in dryland. The agronomic characteristics of three local rice varieties that determine productivity are the area of the flag leaf and the number of filled grain/panicles. Local varieties with superior agronomic characteristics are Siam, Bolouh, and Gel. These three varieties have the potential to be used as parents in the creation of new high-yielding varieties that are specific, namely having broad flag leaves and a high number of filled grains/panicles. The acquisition of new high-yielding varieties specific to dryland through a breeding program will increase the choice of varieties for stakeholders and support increased rice production in dryland.

Shallow swamps can be used for rice production during the dry season. The problem that occurs is lack of water. The technology needed to overcome these problems is drought tolerant superior varieties. The purpose of this study is to obtain superior varieties tolerant to drought on shallow lebak swampy. The study was conducted from April 2019 to August 2019. The varieties used were Situ bagendit, Rindang 1, Rindang 2, and the Siam (local) as comparison. Data collected included: plant height, number of productive tillers, number of grains per panicle, number of filled grains per panicle, number of empty grains per panicle and production. The method used is direct observation in the field. The data obtained were compiled by tabulation and analyzed by statistical tests namely the similarity test of the middle value (t-test). The results showed that the varieties Situ Bagendit, Rindang 1, and Rindang 2 had better numbers of productive tillers, number of grains per panicle, and number of filled grains per panicle than Siam varieties. The highest production was achieved by the Situ Bagendit variety, namely 4.6 tonnes/ha. Meanwhile, the production of Rindang 2, Rindang 1, and Siam varieties were 4.4; 4.0; and 2.0 tonnes/ha.

Objective: Janus kinase family members are essential for signaling by multiple cytokines, including many implicated in systemic lupus erythematosus (SLE) pathogenesis. To test whether inhibition of JAK1 signaling can be efficacious in SLE, we used a JAK1-selective inhibitor (ABT-317) and evaluated its ability to ameliorate disease in murine SLE., Methods: Efficacy of ABT-317 was evaluated using NZB/W-F 1 mice treated prophylactically and therapeutically. Primary endpoints were proteinuria, survival, and saliva production. Other endpoints included histological analysis of kidneys and salivary glands, flow cytometric analysis of splenic cell populations, and gene expression analysis by RNA sequencing in the kidneys, salivary glands, and blood. Publicly available human kidney gene transcription data were used to assess the translatability of the mouse findings., Results: ABT-317 was efficacious when dosed prophylactically and prevented disease for up to two months after treatment cessation. When dosed therapeutically, ABT-317 quickly reversed severe proteinuria and restored saliva production, as well as diminished kidney and salivary gland inflammation. ABT-317-induced changes in glomerular morphology coincided with normalization of a human nephrotic gene signature, suggesting translatability to human lupus nephritis (LN)., Conclusion: JAK1 inhibition prevented and reversed kidney and salivary gland manifestations of murine lupus with long-lasting effects after treatment cessation. These data, along with the presence of JAK1 and nephrotic gene signatures in human LN glomeruli, suggest that a JAK1-selective inhibitor may be an effective therapeutic in the treatment of human SLE and LN., (© 2024 AbbVie Inc. ACR Open Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Idiopathic pulmonary fibrosis (IPF) is an age-related disease with poor prognosis and limited therapeutic options. Activation of lung fibroblasts and differentiation to myofibroblasts are the principal effectors of disease pathology, but damage and senescence of alveolar epithelial cells, specifically type II (ATII) cells, has recently been identified as a potential trigger event for the progressive disease cycle. Targeting ATII senescence and the senescence-associated secretory phenotype (SASP) is an attractive therapeutic strategy; however, translatable primary human cell models that enable mechanistic studies and drug development are lacking. Here, we describe a novel system of conditioned medium (CM) transfer from bleomycin-induced senescent primary alveolar epithelial cells (AEC) onto normal human lung fibroblasts (NHLF) that demonstrates an enhanced fibrotic transcriptional and secretory phenotype compared to non-senescent AEC CM treatment or direct bleomycin damage of the NHLFs. In this system, the bleomycin-treated AECs exhibit classical hallmarks of cellular senescence, including SASP and a gene expression profile that resembles aberrant epithelial cells of the IPF lung. Fibroblast activation by CM transfer is attenuated by pre-treatment of senescent AECs with the senolytic Navitoclax and AD80, but not with the standard of care agent Nintedanib or senomorphic JAK-targeting drugs (e.g., ABT-317, ruxolitinib). This model provides a relevant human system for profiling novel senescence-targeting therapeutics for IPF drug development.

Fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic scleroderma (SSc), are commonly associated with high morbidity and mortality, thereby representing a significant unmet medical need. Interleukin 11 (IL11)-mediated cell activation has been identified as a central mechanism for promoting fibrosis downstream of TGFβ. IL11 signaling has recently been reported to promote fibroblast-to-myofibroblast transition, thus leading to various pro-fibrotic phenotypic changes. We confirmed increased mRNA expression of IL11 and IL11Rα in fibrotic diseases by OMICs approaches and in situ hybridization. However, the vital role of IL11 as a driver for fibrosis was not recapitulated. While induction of IL11 secretion was observed downstream of TGFβ signaling in human lung fibroblasts and epithelial cells, the cellular responses induced by IL11 was quantitatively and qualitatively inferior to that of TGFβ at the transcriptional and translational levels. IL11 blocking antibodies inhibited IL11Rα-proximal STAT3 activation but failed to block TGFβ-induced profibrotic signals. In summary, our results challenge the concept of IL11 blockade as a strategy for providing transformative treatment for fibrosis., Competing Interests: Authors YT, KM, QZ, SO’B, MC, SP, SWi, SC, JW, MA, HA-S, DB, AC, KO, BD, BH, JM, CN, DW, CT, AB, FD, VM-T, XL, GS, MW, SA, HK, SWe, LP, TC, LR-B, RSi, KW, LR, BS, YH, LH, AM, WS, IC, SG, GV, CH, TR, RSa, and EF are employees of the company AbbVie. JX, JK, and ML were employees of AbbVie at the time of the study. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Tan, Mosallanejad, Zhang, O’Brien, Clements, Perper, Wilson, Chaulagain, Wang, Abdalla, Al-Saidi, Butt, Clabbers, Ofori, Dillon, Harvey, Memmott, Negron, Winarta, Tan, Biswas, Dong, Morales-Tirado, Lu, Singh, White, Ashley, Knight, Westmoreland, Phillips, Carr, Reinke-Breen, Singh, Xu, Wu, Rinaldi, Stoll, He, Hazelwood, Karman, McCluskey, Stine, Correia, Gauld, Levesque, Veldman, Hubeau, Radstake, Sadhukhan and Fiebiger.)

Background: Treatment for the debilitating disease hidradenitis suppurativa (HS) is inadequate in many patients. Despite an incidence of approximately 1%, HS is often under-recognized and underdiagnosed, and is associated with a high morbidity and poor quality of life., Objectives: To gain a better understanding of the pathogenesis of HS, in order to design new therapeutic strategies., Methods: We employed single-cell RNA sequencing to analyse gene expression in immune cells isolated from involved HS skin vs. healthy skin. Flow cytometry was used to quantify the absolute numbers of the main immune populations. The secretion of inflammatory mediators from skin explant cultures was measured using multiplex and enzyme-linked immunosorbent assays., Results: Single-cell RNA sequencing analysis identified a significant enrichment in the frequency of plasma cells, T helper (Th) 17 cells and dendritic cell subsets in HS skin, and the immune transcriptome was distinct and more heterogeneous than healthy skin. Flow cytometry revealed significantly increased numbers of T cells, B cells, neutrophils, dermal macrophages and dendritic cells in HS skin. Genes and pathways associated with Th17 cells, interleukin (IL)-17, IL-1β and the NLRP3 inflammasome were enhanced in HS skin, particularly in samples with a high inflammatory load. Inflammasome constituent genes principally mapped to Langerhans cells and a subpopulation of dendritic cells. The secretome of HS skin explants contained significantly increased concentrations of inflammatory mediators, including IL-1β and IL-17A, and culture with an NLRP3 inflammasome inhibitor significantly reduced the secretion of these, as well as other, key mediators of inflammation., Conclusions: These data provide a rationale for targeting the NLRP3 inflammasome in HS using small-molecule inhibitors that are currently being tested for other indications., Competing Interests: Conflicts of interest: R.H. has received honoraria from AbbVie, Janssen and UCB, and has acted as a subinvestigator in clinical trials for AbbVie and UCB. K.H.G.M. is the co-founder of and a shareholder in a startup biotechnology company involved in the development of anti-inflammatory therapeutics. B.K. has received grants and/or honoraria from AbbVie, Almirall, AstraZeneca, Biogen, Bristol Myers Squibb, Celgene, Janssen, Lilly, LEO Pharma, Merck, Moonlake, Novartis, Pfizer and UCB Pharma.​ J.M.F. has received honoraria from Novartis and Moonlake. M.R. is a former employee of AbbVie. B.M., C.M.S., A.Z., J.K., R.W.D., K.M.S., J.M., A.P., A.F., M.O’D., K.H., W.J.H. and D.C.W. declare no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Association of Dermatologists.)

Immune cells are fundamental regulators of extracellular matrix (ECM) production by fibroblasts and have important roles in determining extent of fibrosis in response to inflammation. Although much is known about fibroblast signaling in fibrosis, the molecular signals between immune cells and fibroblasts that drive its persistence are poorly understood. We therefore analyzed skin and lung samples of patients with diffuse cutaneous systemic sclerosis, an autoimmune disease that causes debilitating fibrosis of the skin and internal organs. Here, we define a critical role of epiregulin-EGFR signaling between dendritic cells and fibroblasts to maintain elevated ECM production and accumulation in fibrotic tissue. We found that epiregulin expression marks an inducible state of DC3 dendritic cells triggered by type I interferon and that DC3-derived epiregulin activates EGFR on fibroblasts, driving a positive feedback loop through NOTCH signaling. In mouse models of skin and lung fibrosis, epiregulin was essential for persistence of fibrosis in both tissues, which could be abrogated by epiregulin genetic deficiency or a neutralizing antibody. Therapeutic administration of epiregulin antibody reversed fibrosis in patient skin and lung explants, identifying it as a previously unexplored biologic drug target. Our findings reveal epiregulin as a crucial immune signal that maintains skin and lung fibrosis in multiple diseases and represents a promising antifibrotic target.

Background: Neutrophils are present in the early phases of spondyloarthritis-related uveitis, skin and intestinal disease, but their role in enthesitis, a cardinal musculoskeletal lesion in spondyloarthritis, remains unknown. We considered the role of neutrophils in the experimental SKG mouse model of SpA and in human axial entheses., Methods: Early inflammatory infiltrates in the axial and peripheral entheseal sites in SKG mice were evaluated using immunohistochemistry and laser capture microdissection of entheseal tissue. Whole transcriptome analysis was carried out using Affymetrix gene array MTA 1.0, and data was analyzed via IPA. We further isolated neutrophils from human peri-entheseal bone and fibroblasts from entheseal soft tissue obtained from the axial skeleton of healthy patients and determined the response of these cells to fungal adjuvant., Results: Following fungal adjuvant administration, early axial and peripheral inflammation in SKG mice was characterized by prominent neutrophilic entheseal inflammation. Expression of transcripts arising from neutrophils include abundant mRNA for the alarmins S100A8 and S100A9. In normal human axial entheses, neutrophils were present in the peri-entheseal bone. Upon fungal stimulation in vitro, human neutrophils produced IL-23 protein, while isolated human entheseal fibroblasts produced chemokines, including IL-8, important in the recruitment of neutrophils., Conclusion: Neutrophils with inducible IL-23 production are present in uninflamed human entheseal sites, and neutrophils are prominent in early murine spondyloarthritis-related enthesitis. We propose a role for neutrophils in the early development of enthesitis., (© 2022. The Author(s).)

Infections with pathogenic mycobacteria are controlled by the formation of a unique structure known as a granuloma. The granuloma represents a host-pathogen interface where bacteria are killed and confined by the host response, but also where bacteria persist. Previous work has demonstrated that the T cell repertoire is heterogenous even at the single granuloma level. However, further work using pigeon cytochrome C (PCC) epitope-tagged BCG (PCC-BCG) and PCC-specific 5CC7 RAG -/- TCR transgenic (Tg) mice has demonstrated that a monoclonal T cell population is able to control infection. At the chronic stage of infection, granuloma-infiltrating T cells remain highly activated in wild-type mice, while T cells in the monoclonal T cell mice are anergic. We hypothesized that addition of an acutely activated non-specific T cell to the monoclonal T cell system could recapitulate the wild-type phenotype. Here we report that activated non-specific T cells have access to the granuloma and deliver a set of cytokines and chemokines to the lesions. Strikingly, non-specific T cells rescue BCG-specific T cells from anergy and enhance the function of BCG-specific T cells in the granuloma in the chronic phase of infection when bacterial antigen load is low. In addition, we find that these same non-specific T cells have an inhibitory effect on systemic BCG-specific T cells. Taken together, these data suggest that T cells non-specific for granuloma-inducing agents can alter the function of granuloma-specific T cells and have important roles in mycobacterial immunity and other granulomatous disorders.

Aims: Ulcerative colitis and Crohn's disease, collectively known as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the intestine for which key elements in disease initiation and perpetuation are defects in epithelial barrier integrity. Achieving mucosal healing is essential to ameliorate disease outcome and so new therapies leading to epithelial homeostasis and repair are under investigation. This study was designed to determine the mechanisms by which IL-22 regulates intestinal epithelial cell function., Main Methods: Human intestinal organoids and resections, as well as mice were used to evaluate the effect of IL-22 on stem cell expansion, proliferation and expression of mucus components. IL-22 effect on barrier function was assessed in polarized T-84 cell monolayers. Butyrate co-treatments and organoid co-cultures with immune cells were performed to monitor the impact of microbial-derived metabolites and inflammatory environments on IL-22 responses., Key Findings: IL-22 led to epithelial stem cell expansion, proliferation, barrier dysfunction and anti-microbial peptide production in human and mouse models evaluated. IL-22 also altered the mucus layer by inducing an increase in membrane mucus but a decrease in secreted mucus and goblet cell content. IL-22 had the same effect on anti-microbial peptides and membrane mucus in both healthy and IBD human samples. In contrast, this IL-22-associated epithelial phenotype was different when treatments were performed in presence of butyrate and organoids co-cultured with immune cells., Significance: Our data indicate that IL-22 promotes epithelial regeneration, innate defense and membrane mucus production, strongly supporting the potential clinical utility of IL-22 as a mucosal healing therapy in IBD., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Idiopathic pulmonary fibrosis is a progressive and debilitating lung disease with large unmet medical need and few treatment options. We describe an analysis connecting single cell gene expression with bulk gene expression-based subsetting of patient cohorts to identify IPF patient subsets with different underlying pathogenesis and cellular changes. We reproduced earlier findings indicating the existence of two major subsets in IPF and showed that these subsets display different alterations in cellular composition of the lung. We developed classifiers based on the cellular changes in disease to distinguish subsets. Specifically, we showed that one subset of IPF patients had significant increases in gene signature scores for myeloid cells versus a second subset that had significantly increased gene signature scores for ciliated epithelial cells, suggesting a differential pathogenesis among IPF subsets. Ligand-receptor analyses suggested there was a monocyte-macrophage chemoattractant axis (including potentially CCL2-CCR2 and CCL17-CCR4) among the myeloid-enriched IPF subset and a ciliated epithelium-derived chemokine axis (e.g. CCL15) among the ciliated epithelium-enriched IPF subset. We also found that these IPF subsets had differential expression of pirfenidone-responsive genes suggesting that our findings may provide an approach to identify patients with differential responses to pirfenidone and other drugs. We believe this work is an important step towards targeted therapies and biomarkers of response., Competing Interests: All authors are employees of AbbVie, Inc. This does not alter our adherence to PLoS ONE policies on sharing data and materials.

CD40 is a costimulatory receptor on APCs that is critical for the induction and maintenance of humoral and cell-mediated immunity. Accordingly, CD40 and its ligand, CD40L, have long been considered targets for the treatment of autoimmune diseases. We developed a rat/mouse chimeric anti-mouse CD40 antagonist mAb, 201A3, and evaluated its ability to alleviate murine lupus. Treatment of NZB/W-F 1 mice with 201A3 after the onset of severe proteinuria rapidly reversed established severe proteinuria and nephritis and largely restored normal glomerular and tubular morphology. This coincided with a normalization of the expression of genes associated with proteinuria and injury by kidney parenchymal cells. Anti-CD40 treatment also prevented and reversed loss of saliva production and sialadenitis. These effects on kidney and salivary gland function were confirmed using mice of a second strain, MRL/Mp- lpr / lpr , and extended to alleviating joint inflammation. Immunologically, anti-CD40 treatment disrupted multiple processes that contribute to the pathogenesis of systemic lupus erythematosus (SLE), including autoreactive B cell activation, T effector cell function in target tissues, and type I IFN production. This ability to disrupt disease-critical immunological mechanisms, to reverse glomerular and tubular injury at the cellular and gene expression levels, and to confer exceptional therapeutic efficacy suggests that CD40 is a central disease pathway in murine SLE. Thus, a CD40 antagonist Ab could be an effective therapeutic in the treatment of SLE., (Copyright © 2019 by The American Association of Immunologists, Inc.)

T cells continuously sample CNS-derived antigens in the periphery, yet it is unknown how they sample and respond to CNS antigens derived from distinct brain areas. We expressed ovalbumin (OVA) neoepitopes in regionally distinct CNS areas (Cnp-OVA and Nes-OVA mice) to test peripheral antigen sampling by OVA-specific T cells under homeostatic and neuroinflammatory conditions. We show that antigen sampling in the periphery is independent of regional origin of CNS antigens in both male and female mice. However, experimental autoimmune encephalomyelitis (EAE) is differentially influenced in Cnp-OVA and Nes-OVA female mice. Although there is the same frequency of CD45 high CD11b+ CD11c+ CX3CL1+ myeloid cell-T-cell clusters in neoepitope-expressing areas, EAE is inhibited in Nes-OVA female mice and accelerated in CNP-OVA female mice. Accumulation of OVA-specific T cells and their immunomodulatory effects on EAE are CX3C chemokine receptor 1 (CX3CR1) dependent. These data show that despite similar levels of peripheral antigen sampling, CNS antigen-specific T cells differentially influence neuroinflammatory disease depending on the location of cognate antigens and the presence of CX3CL1/CX3CR1 signaling. SIGNIFICANCE STATEMENT Our data show that peripheral T cells similarly recognize neoepitopes independent of their origin within the CNS under homeostatic conditions. Contrastingly, during ongoing autoimmune neuroinflammation, neoepitope-specific T cells differentially influence clinical score and pathology based on the CNS regional location of the neoepitopes in a CX3CR1-dependent manner. Altogether, we propose a novel mechanism for how T cells respond to regionally distinct CNS derived antigens and contribute to CNS autoimmune pathology., (Copyright © 2018 the authors 0270-6474/18/387058-14$15.00/0.)

Central nervous system (CNS) immune privilege is complex, and it is still not understood how CNS antigens are sampled by the peripheral immune system under steady state conditions. To compare antigen sampling from immune-privileged or nonprivileged tissues, we created transgenic mice with oligodendrocyte or gut epithelial cell expression of an EGFP-tagged fusion protein containing ovalbumin (OVA) antigenic peptides and tested peripheral anti-OVA peptide-specific sentinel OT-I and OT-II T cell activation. We report that oligodendrocyte or gut antigens are sampled similarly, as determined by comparable levels of OT-I T cell activation. However, activated T cells do not access the CNS under steady state conditions. These data show that afferent immunity is normally intact as there is no barrier at the antigen sampling level, but that efferent immunity is restricted. To understand how this one-sided surveillance contributes to CNS immune privilege will help us define mechanisms of CNS autoimmune disease initiation.

Crosslinking ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) with a bispecific fusion protein (BsB) comprised of a mutant mouse CD80 and lymphocyte activation antigen-3 (LAG-3) has been shown to attenuate TCR signaling and to direct T-cell differentiation toward Foxp3(+) regulatory T cells (Tregs) in an allogenic mixed lymphocyte reaction (MLR). Here, we show that antigen-specific Tregs can also be induced in an antigen-specific setting in vitro. Treatment of non-obese diabetic (NOD) female mice between 9-12 weeks of age with a short course of BsB elicited a transient increase of Tregs in the blood and moderately delayed the onset of autoimmune type 1 diabetes (T1D). However, a longer course of treatment (10 weeks) of 4-13 weeks-old female NOD animals with BsB significantly delayed the onset of disease or protected animals from developing diabetes, with only 13% of treated animals developing diabetes by 35 weeks of age compared to 80% of the animals in the control group. Histopathological analysis of the pancreata of the BsB-treated mice that remained non-diabetic revealed the preservation of insulin-producing β-cells despite the presence of different degrees of insulitis. Thus, a bifunctional protein capable of engaging CTLA-4 and MHCII and indirectly co-ligating CTLA-4 to the TCR protected NOD mice from developing T1D.

T cell immunoglobulin and mucin domain–containing 3 (Tim-3) is an inhibitory receptor that is expressed on exhausted T cells during infection with HIV-1 and hepatitis C virus. By contrast, Tim-3 expression and function are defective in multiple human autoimmune diseases. However, the molecular mechanisms modulating Tim-3 function are not well understood. Here we show that human leukocyte antigen B (HLA-B)-associated transcript 3 (Bat3) binds to, and represses the function of, Tim-3. Bat3 protects T helper type 1 (TH1) cells from galectin-9–mediated cell death and promotes both proliferation and proinflammatory cytokine production. Bat3-deficient T cells have elevated expression of exhaustion-associated molecules such as Tim-3, Lag3, Prdm1 and Pbx3, and Bat3 knockdown in myelin-antigen–specific CD4+ T cells markedly inhibits the development of experimental autoimmune encephalomyelitis while promoting the expansion of a dysfunctional Tim-3hi, interferon-γ (IFN-γ)loCD4+ cell population. Furthermore, expression of Bat3 is reduced in exhausted Tim-3+ T cells from mouse tumors and HIV-1–infected individuals. These data indicate that Bat3 acts as an inhibitor of Tim-3–dependent exhaustion and cell death. Bat3 may thus represent a viable therapeutic target in autoimmune disorders, chronic infections and cancers.

Cross-linking of ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) during the early phase of T cell activation attenuates TCR signaling, leading to T cell inhibition. To promote this event, a bispecific fusion protein comprising a mutant mouse CD80 (CD80w88a) and lymphocyte activation antigen-3 was engineered to concurrently engage CTLA-4 and cross-link it to the TCR. Cross-linking is expected to be attained via ligation of CTLA-4 first to MHCII and then indirectly to the TCR, generating a CTLA-4-MHCII-TCR trimolecular complex that forms between T cells and antigen-presenting cells during T cell activation. Treating T cells with this bispecific fusion protein inhibited T cell activation. In addition, it induced the production of IL-10 and TGF-β and attenuated AKT and mTOR signaling. Intriguingly, treatment with the bispecific fusion protein also directed early T cell differentiation into Foxp3-positive regulatory T cells (Tregs). This process was dependent on the endogenous production of TGF-β. Thus, bispecific fusion proteins that engage CTLA-4 and co-ligate it to the TCR during the early phase of T cell activation can negatively regulate the T cell response. Bispecific biologics with such dual functions may therefore represent a novel class of therapeutics for immune modulation. These findings presented here also reveal a potential new role for CTLA-4 in Treg differentiation.

Pre-existing immunity against adeno-associated virus (AAV) remains a major challenge facing the clinical use of systemic administration of recombinant AAV vectors for the treatment of genetic and acquired diseases using gene therapy. In this study, we evaluated the potential of bortezomib (marketed under trade name Velcade) to abrogate a pre-existing immunity to AAV in mice, thereby allowing subsequent transduction by a recombinant AAV vector of the same serotype. We demonstrate that bortezomib efficiently reduces AAV-specific IgG titres and moderates the cytotoxic T cell response in mice that have a pre-existing immunity to AAV2/8. Significant depletion of AAV2/8-specific IgG-producing plasma cells in secondary lymphoid organs and bone marrow was observed. However, this inhibition of the immune response by bortezomib was insufficient to allow subsequent re-infection with a recombinant AAV vector of a similar serotype. We show that this shortcoming is probably due to the combination of residual antibody levels and the inability of bortezomib to completely deplete the memory B cells that are re-activated in response to a repeated infection with a recombinant AAV vector. Taken together, the results of this study argue for the use of immunosuppressive therapies that target both plasma and memory B cells for the efficient elimination of pre-existing immunity against AAV2/8 vectors.

Lipid rafts reportedly have a role in coalescing key signaling molecules into the immunological synapse during T cell activation, thereby modulating T cell receptor (TCR) signaling activity. Recent findings suggest that a correlation may exist between increased levels of glycosphingolipids (GSLs) in the lipid rafts of T cells and a heightened response of those T cells toward activation. Here, we show that lowering the levels of GSLs in CD4(+) T cells using a potent inhibitor of glucosylceramide synthase (Genz-122346) led to a moderation of the T cell response toward activation. TCR proximal signaling events, such as phosphorylation of Lck, Zap70 and LAT, as well as early Ca(2+) mobilization, were attenuated by treatment with Genz-122346. Concomitant with these events were significant reductions in IL-2 production and T cell proliferation. Similar findings were obtained with CD4(+) T cells isolated from transgenic mice genetically deficient in GM3 synthase activity. Interestingly, lowering the GSL levels in CD4(+) T cells by either pharmacological inhibition or disruption of the gene for GM3 synthase also specifically inhibited the differentiation of T cells to the Th(17) lineage but not to other Th subsets in vitro. Taken together with the recently reported effects of Raftlin deficiency on Th(17) differentiation, these results strongly suggest that altering the GSL composition of lipid rafts modulates TCR signaling activity and affects Th(17) differentiation.

IFN-gamma plays a central role in antitumor immunity. T cell Ig and mucin domain (Tim-3) is expressed on IFN-gamma-producing Th1 cells; on interaction with its ligand, galectin-9, Th1 immunity is terminated. In this study, we show that transgenic overexpression of Tim-3 on T cells results in an increase in CD11b(+)Ly-6G(+) cells and inhibition of immune responses. Molecular characterization of CD11b(+)Ly-6G(+) cells reveals a phenotype consistent with granulocytic myeloid-derived suppressor cells. Accordingly, we find that modulation of the Tim-3/galectin-9 (Gal-9) pathway impacts on tumor growth. Similarly, overexpression of Tim-3 ligand, Gal-9, results in an increase in CD11b(+)Ly-6G(+) cells and inhibition of immune responses. Loss of Tim-3 restores normal levels of CD11b(+)Ly-6G(+) cells and normal immune responses in Gal-9 transgenic mice. Our data uncover a novel mechanism by which the Tim-3/Gal-9 pathway regulates immune responses and identifies this pathway as a therapeutic target in diseases where myeloid-derived suppressor cells are disadvantageous.

Background: Memantine, an NMDA antagonist, is effective for moderate to severe Alzheimer's disease., Objective: Determine whether memantine improves cognitive performance (CP) among subjects with multiple sclerosis (MS) and cognitive impairment (CI)., Methods: This double-blind, randomized, placebo-controlled trial (Clinicaltrials.gov NCT00300716) compared memantine 10 mg twice a day (4 week titration followed by 12 weeks on the highest tolerated dose) with placebo. The primary outcome was the change from baseline to exit on the Paced Auditory Serial Addition Test (PASAT) and the California Verbal Learning Test-II (CVLT-II) Long Delay Free Recall (LDFR). Secondary outcomes included additional neuropsychological tests; self-report measures of quality of life, fatigue, and depression; and family/caregiver reports of subjects' CI and neuropsychiatric symptoms., Results: The differences between the groups on the change on the PASAT (placebo-memantine = 0.0 correct responses, 95% CI 3.4, 3.4; p = 0.9) and on CVLT-II LDFR (placebo-memantine =-0.6 words, 95% CI -2.1, 0.8; p = 0.4) as well as on the other cognitive tests were not significant. Subjects on memantine had no serious adverse events (AEs) but had more fatigue and neurological AEs as well as, per family members' reports, less cognitive improvement and greater neuropsychiatric symptoms than subjects on placebo., Conclusion: Memantine 10 mg twice a day does not improve CP in subjects with MS, ages 18-65, without major depression, who have subjective cognitive complaints and perform worse than one SD below the mean on the PASAT or on the California Verbal Learning Test-II (total recall or delayed free recall).

CD4+ T helper 1 (TH1) cells are important mediators of inflammation and are regulated by numerous pathways, including the negative immune receptor Tim-3. We found that Tim-3 is constitutively expressed on cells of the innate immune system in both mice and humans, and that it can synergize with Toll-like receptors. Moreover, an antibody agonist of Tim-3 acted as an adjuvant during induced immune responses, and Tim-3 ligation induced distinct signaling events in T cells and dendritic cells; the latter finding could explain the apparent divergent functions of Tim-3 in these cell types. Thus, by virtue of differential expression on innate versus adaptive immune cells, Tim-3 can either promote or terminate TH1 immunity and may be able to influence a range of inflammatory conditions.

The homophilic cell adhesion molecule PECAM-1 is a major participant in the migration of leukocytes across endothelium. We examined the ability of a chimeric soluble PECAM-1 fused to human IgG-Fc to impair leukocyte entry through the blood-brain barrier and reduce CNS autoimmunity. sPECAM-Fc impaired migration of lymphocytes across brain endothelial monolayers and diminished the severity of EAE, an experimental model of MS, when administered at the onset of symptoms. However, in mice transgenic for sPECAM-Fc, the chronically elevated levels of sPECAM-Fc hastened onset of EAE disease without significantly changing clinical score severity. Our data suggest that short-term treatment of diseases like MS with sPECAM-Fc has therapeutic potential.

The effect of secondary infections on CD4 T-cell-regulated chronic granulomatous inflammation is not well understood. Here, we have investigated the effect of an acute viral infection on the cellular composition and bacterial protection in Mycobacterium bovis strain bacille Calmette-Guérin (BCG)-induced granulomas using an immunocompetent and a partially immunodeficient murine model. Acute lymphocytic choriomeningitis virus (LCMV) coinfection of C57BL/6 mice led to substantial accumulation of gamma interferon (IFN-gamma)-producing LCMV-specific T cells in liver granulomas and increased local IFN-gamma. Despite traffic of activated T cells that resulted in a CD8 T-cell-dominated granuloma, the BCG liver organ load was unaltered from control levels. In OT-1 T-cell-receptor (TCR) transgenic mice, ovalbumin (OVA) immunization or LCMV coinfection of BCG-infected mice induced CD8 T-cell-dominated granulomas containing large numbers of non-BCG-specific activated T cells. The higher baseline BCG organ load in this CD8 TCR transgenic animal allowed us to demonstrate that OVA immunization and LCMV coinfection increased anti-BCG protection. The bacterial load remained substantially higher than in mice with a more complete TCR repertoire. Overall, the present study suggests that peripherally activated CD8 T cells can be recruited to chronic inflammatory sites, but their contribution to protective immunity is limited to conditions of underlying immunodeficiency.

Dendritic cells (DCs) accumulate in the CNS during inflammatory diseases, but the exact mechanism regulating their traffic into the CNS remains to be defined. We now report that MIP-1alpha increases the transmigration of bone marrow-derived, GFP-labeled DCs across brain microvessel endothelial cell monolayers. Furthermore, occludin, an important element of endothelial tight junctions, is reorganized when DCs migrate across brain capillary endothelial cell monolayers without causing significant changes in the barrier integrity as measured by transendothelial electrical resistance. We show that DCs produce matrix metalloproteinases (MMP) -2 and -9 and GM6001, an MMP inhibitor, decreases both baseline and MIP-1alpha-induced DC transmigration. These observations suggest that DC transmigration across brain endothelial cell monolayers is partly MMP dependent. The migrated DCs express higher levels of CD40, CD80, and CD86 costimulatory molecules and induce T cell proliferation, indicating that the transmigration of DCs across brain endothelial cell monolayers contributes to the maintenance of DC Ag-presenting function. The MMP dependence of DC migration across brain endothelial cell monolayers raises the possibility that MMP blockers may decrease the initiation of T cell recruitment and neuroinflammation in the CNS.

CD4(+) T cells are central in mediating granuloma formation and limiting growth and dissemination of mycobacterial infections. To determine whether T cells responding to influenza infection can interact with T cells responding to Mycobacterium bovis bacille Calmette-Guérin (BCG) infection and disrupt granuloma formation, we infected mice containing two monoclonal T cell populations specific for the model Ags pigeon cytochrome c (PCC) and hen egg lysozyme (HEL). These mice were chronically infected with PCC epitope-tagged BCG (PCC-BCG) and acutely infected with HEL epitope-tagged influenza virus (HEL-flu). In these mice, PCC-BCG infection is much more abundant in the liver than the lung, whereas HEL-flu infection is localized to the lung. We observe that both T cells have access to both inflammatory sites, but that PCC-specific T cells dominate the PCC-BCG inflammatory site in the liver, whereas HEL-specific T cells dominate the HEL-flu inflammatory site in the lung. Influenza infection, in the absence of an influenza-specific T cell response, is able to increase the activation state and IFN-gamma secretion of PCC-BCG-specific T cells in the granuloma. Activation of HEL-specific T cells allows them to secrete IFN-gamma and contribute to protection in the granuloma. Ultimately, infection with influenza has little effect on bacterial load, and bacteria do not disseminate. In summary, these data illustrate complex interactions between T cell responses to infectious agents that can affect effector responses to pathogens.

Neuroinflammation often starts with the invasion of T lymphocytes into the CNS leading to recruitment of macrophages and amplification of inflammation. In this study, we show that dendritic cells (DCs) facilitate T-T cell help in the CNS and contribute to the amplification of local neuroinflammation. We adoptively transferred defined amounts of naive TCR-transgenic (TCR) recombination-activating gene-1-deficient T cells into another TCR-transgenic mouse strain expressing different Ag specificity. Following adoptive transfers, we coinjected DCs that presented one or multiple Ags into the brain and followed the activation of T cells with defined specificities simultaneously. Injection of DCs presenting both Ags simultaneously led to significantly higher infiltration of T cells into the brain compared with injection of a mixture of DCs pulsed with two Ags separately. DCs mediated either cooperative or competitive interactions between T cell populations with different specificities depending upon their MHC-restricting element usage. These results suggest that DC-mediated cooperation between brain-infiltrating T cells of different Ag specificities in the CNS plays an important role in regulation of neuroinflammation. This work also implies that blocking Ag-specific responses may block not only the targeted specificities, but may also effectively block their cooperative assistance to other T cells. Therefore, these data justify more attention to Ag-specific therapeutic approaches for neuroinflammation.

Substance P (SP) is a modulatory, pro-inflammatory neuropeptide. We investigated the role of the SP receptor, neurokinin-1 (NK-1), in EAE. Our data show that in the chronic phase, mice lacking NK-1 have improved mobility and decreased numbers of LFA-1 high CD4+ T cells and MOG-specific, IFN-gamma producing CD4+ T cells. SR140333, an NK-1 antagonist, administered alone during the chronic phase of EAE was not sufficient to ameliorate symptoms. These results indicate that SP, through NK-1, contributes to maintenance of CNS inflammation, and combining NK-1 antagonists with conventional anti-inflammatory treatments may enhance the success of treatments for diseases like multiple sclerosis.

Rising rates of Histoplasma capsulatum infection are an emerging problem among the rapidly growing population of immune-compromised individuals. Although there is a growing understanding of systemic immunity against Histoplasma, little is known about the local granulomatous response, which is an important component in the control of infection. The focus of this article is the characterization of Histoplasma-induced granulomas. Five days after i.p. infection, infected macrophage appear in the liver and lung; however, no granulomas are apparent. Two days later, well-formed sarcoid granulomas are abundant in the lung and liver of infected mice, which contain all visible Histoplasma. Granulomas are dominated by macrophage and lymphocytes. Most of the Histoplasma and most of the apoptotic cells are found in the center of the lesions. We isolated liver granulomas at multiple time points after infection and analyzed the cellular composition, TCR gene usage, and cytokine production of granuloma-infiltrating cells. The lesions contain both CD4+ and CD8+ T cell subsets, and T cells are the primary source of IFN-gamma and IL-17. The main source of local TNF-alpha is macrophage. Chemokines are produced by both infiltrating macrophage and lymphocytes. Dendritic cells are present in granulomas; however, T cell expansion seems to occur systemically because TCR usage is very heterogeneous even at the level of individual lesions. This study is the first direct examination of host cellular responses in the Histoplasma-induced granuloma representing the specific interface between host and pathogen. Our studies will allow further analysis of key elements of host Histoplasma interactions at the site of chronic infection.

The contribution of dendritic cells (DCs) to initiating T cell-mediated immune response in and T cell homing into the CNS has not yet been clarified. In this study we show by confocal microscopy and flow cytometry that cells expressing CD11c, CD205, and MHC class II molecules and containing fluorescently labeled, processed Ag accumulate at the site of intracerebral Ag injection. These cells follow a specific pattern upon migrating out of the brain. To track their pathway out of the CNS, we differentiated DCs from bone marrow of GFP-transgenic mice and injected them directly into brains of naive C57BL/6 mice. We demonstrate that DCs migrate from brain to cervical lymph nodes, a process that can be blocked by fixation or pertussis toxin treatment of the DCs. Injection of OVA-loaded DCs into brain initiates a SIINFEKL (a dominant OVA epitope)-specific T cell response in lymph nodes and spleen, as measured by specific tetramer and LFA-1 activation marker staining. Additionally, a fraction of activated SIINFEKL-specific T cells home to the CNS. Specific T cell homing to the CNS, however, cannot be induced by i.v. injection of OVA-loaded DCs alone. These data suggest that brain-emigrant DCs are sufficient to support activated T cells to home to the tissue of DC origination. Thus, initiation of immune reactivity against CNS Ags involves the migration of APCs from nervous tissue to peripheral lymphoid tissues, similarly to that in other organs.

Background: Quantitative DNA analysis of fresh biopsy material can contribute to a more accurate diagnosis and prognosis. The authors aimed to develop and test a mechanical, nuclear preparation protocol for quantitative DNA analysis., Patients and Methods: Altogether 32 gastric (10 healthy, 17 gastritis, 7 adenocarcinoma) and 48 colon (21 healthy, 20 colitis ulcerosa, 7 adenocarcinoma) biopsy specimens were evaluated. The mechanical disruption was performed by Medimachine (DAKO, Denmark). The flow cytometry analysis was performed on a BD FACSStar flow cytometer., Results: DNA Aneuploidy was found in gastric samples only in tumours. The S-phase fraction of the normal cases was 5.9 +/- 2.1%, 5.1% +/- 1.2% in gastritis and 10.7 +/- 1.6% in carcinomas. Seven out of 20 colitis ulcerosa and 4 out of 7 colon cancer samples were aneuploid. The S-phase fraction of normal colon cases was 5.7 +/- 3.4%, in colitis 8.1 +/- 4.2% and 15.1 +/- 5.7% in carcinomas, respectively., Conclusion: Mechanical nuclear isolation is a useful method for flow cytometric DNA ploidy analysis of fresh biopsy samples.

Background: The majority of liver transplant centers require a 6-month abstinence period before listing candidates for liver transplantation with alcoholic cirrhosis and a persistent sobriety thereafter. We attempted to identify risk factors for failure to comply with these requirements., Methods: Ninety-nine consecutive patients with alcoholic cirrhosis were referred for liver transplant evaluation between September 1996 and May 1998. The mean age was 49 years, 74% were male, and 54% were hepatitis C virus positive. To be listed, patients had to meet the following requirements. All patients received extensive psychosocial evaluations and were frequently monitored with random urine and blood alcohol tests; patients found positive were excluded or removed from the liver transplant waiting list. Detailed patient information was entered into a computerized database, and 36 discreet variables were analyzed in relation to success (patient listed and remained on the list) or failure (not listed or removed from the list based on noncompliance)., Results: Forty-nine patients were successfully listed. Nineteen received a transplant, with a 95% 1-year patient and graft survival rate and 21% alcohol relapse rate after transplantation. Twenty-two patients had either medical contraindication and/or died before transplant listing. Twenty-four patients were never listed and four were removed from the list due to recurrent alcoholism, for a total of 28 failures. Our statistical analysis identified five significant risk factors for failure: (I) living arrangement (alone/family versus community/friend), P=0.006; (II) history of suicide ideation, P=0.03; (III) history of previous alcohol-related hospitalization, P=0.01; (IV) lack of previous alcoholic rehabilitation before transplant evaluation, P=0.001; and (V) failure to accept further alcoholic rehabilitation before orthotopic liver transplantation, P=0.01., Conclusions: Our experience confirms that transplantation can be extremely successful in properly selected patients with alcoholic cirrhosis. We identified several predictive psychosocial factors of early alcoholic recidivism in transplant candidates.