Peripheral Lymphatic Organs Research Articles

Dysregulation and chronicity of pathogenic T cell responses in the pre-diseased stage of lupus.

In the normal immune system, T cell activation is tightly regulated and controlled at several levels to ensure that activation occurs in the right context to prevent the development of pathologic conditions such as autoimmunity or other harmful immune responses. CD4+FoxP3+ regulatory T cells (Treg) are crucial for the regulation of T cell responses in the peripheral lymphatic organs and thus for the prevention and control of autoimmunity. In systemic lupus erythematosus (SLE), a prototypic systemic autoimmune disease with complex etiology, a disbalance between Treg and pathogenic effector/memory CD4+ T cells develops during disease progression indicating that gradual loss of control over T cell activation is an important event in the immune pathogenesis. This progressive failure to adequately regulate the activation of autoreactive T cells facilitates chronic activation and effector/memory differentiation of pathogenic T cells, which are considered to contribute significantly to the induction and perpetuation of autoimmune processes and tissue inflammation in SLE. However, in particular in humans, little is known about the factors which drive the escape from immune regulation and the chronicity of pathogenic T cell responses in an early stage of autoimmune disease when clinical symptoms are still unapparent. Here we briefly summarize important findings and discuss current views and models on the mechanisms related to the dysregulation of T cell responses which promotes chronicity and pathogenic memory differentiation with a focus on the early stage of disease in lupus-prone individuals.

An In Vivo Mouse Model for Chronic Inflammation-Induced Immune Suppression: A "Factory" for Myeloid-Derived Suppressor Cells (MDSCs).

Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immature myeloid cells known to play a role in perpetuating a wide range of pathologies, such as chronic infections, autoimmune diseases, and cancer. MDSCs were first identified in mice by the markers CD11b+ Gr1+ , and later, based on their morphology, they were classified into two subsets: polymorphonuclear MDSCs, identified by the markers CD11b+ Ly6G+ Ly6CLow , and monocytic MDSCs, detected as being CD11b+ Ly6G- Ly6CHi . MDSCs are studied as immunosuppressive cells in various diseases characterized by chronic inflammation and are associated with disease causes/triggers such as pathogens, autoantigens, and cancer. Therefore, different diseases may diversely affect MDSC metabolism, migration, and differentiation, thus influencing the generated MDSC functional features and ensuing suppressive environment. In order to study MDSCs in a pathology-free environment, we established and calibrated a highly reproducible mouse model that results in the development of chronic inflammation, which is the major cause of MDSC accumulation and immune suppression. The model presented can be used to study MDSC phenotypes, functional diversity, and plasticity. It also permits study of MDSC migration from the bone marrow to peripheral lymphatic and non-lymphatic organs and MDSC crosstalk with extrinsic factors, both in vivo and ex vivo. Furthermore, this model can serve as a platform to assess the effects of anti-MDSC modalities. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Repetitive M.tb immunizations for the induction of chronic inflammation Alternate Protocol 1: Creating a lower grade of inflammation by changing the site of immunization Alternate Protocol 2: In vivo evaluation of immune status Support Protocol 1: Preparation of reconstituted M.tb aliquots and M.tb-IFA emulsions for each of the three injections Support Protocol 2: Preparation of an ovalbumin lentiviral expression vector Support Protocol 3: Fluorescence titering assay for the lentiviral expression vector Support Protocol 4: Spleen excision, tissue dissociation, and preparation of a single-cell suspension Support Protocol 5: Labeling of splenocytes with CFSE proliferation dye.

In vivo induction of regulatory T cells by anti-CD45RB antibody causes transferable tolerance to discordant human xenogenic islets.

The treatment of diabetes by islet cell transplantation has become an accepted therapy, with transplantation of xenogeneic islet cells an attractive alternative to the problem. Previous studies in mice have demonstrated that anti-CD45RB induce immune tolerance in human pancreatic islet cells. The current study was to define the mechanism of action of anti-CD45RB induced nonspecific immune tolerance to heteroantigens. A total of 1500 IEQ human islets were transplanted to diabetic B6μMT-/- mice, B6 mice, and μMT-/- diabetic mice undergoing thymectomy. These mice were treated short-term with doses of anti-CD45RB. CD4+Foxp3+Tregs were detected in the blood, peripheral lymphatic organs by flow cytometry, and immunohistochemistry. In addition, anti-CD25 mAb was administered to tolerant human islet cells B6μMT-/-mice. Mice then were transplanted with other human islet cells and received CD4+CD25+Tregs isolated from tolerant human islets mice to observe islet destruction. Anti-CD45RB treatment-induced tolerance to islets in both immunocompetent and B-cell-deficient mice (μMT-/- mice) by processes that were dependent on CD25+ Tregs, but not B cells. Anti-CD45RB treatment increased the number of CD4+Foxp3+Tregs cells. Anti-CD45RB treatment-induced immune tolerance that was antigen nonspecific, with Tregs playing an important role. Anti-CD45RB treatment-induced tolerance generated Tregs that could be transferred to another individual to manifest nonspecific immune tolerance. The results of the experiment suggest that anti-CD45RB induced tolerance to human islet xenografts is mediated by the proliferation of Tregs. These tolerogenic Tregs can be transferred to other mice and induce nonspecific immune tolerance.

Porcine reproductive and respiratory syndrome virus reinfection causes the distribution of porcine interleukin-4 in close proximity to B lymphocytes within lymphoid follicles and a reduction in B and T lymphocytes

Interleukin 4 (IL-4) plays a major role in T-lymphocyte development and is thought to be a central regulator as a cofactor in resting B-lymphocyte proliferation. Primary infection with porcine reproductive and respiratory syndrome virus (PRRSV) induces minimal IL-4 production, whereas an IL-4 response occurs in the peripheral blood of piglets reinfected by PRRSV. The locations and interaction partners for the massive volume of IL-4 triggered by PRRSV reinfection remain unclear. This study aimed to investigate the characteristics of IL-4 secretion and location changes in peripheral immune organs induced by PRRSV infection and reinfection. Our results show that PRRSV reinfection induced higher levels of IL-4 mRNA and protein expression in the peripheral immune organs (e.g., lymph node and spleen) and peripheral blood compared with PRRSV primary infection. Importantly, we found that, following PRRSV reinfection, an obvious large-scale migration of IL-4 occurred in the lymph nodes. During PRRSV primary infection, IL-4 was mainly concentrated around the lymphoid follicles and paracortical regions of the lymph node and also located in the marginal area and periarterial lymphatic sheath region of the spleen. During PRRSV reinfection, the now abundant IL-4 gathered into the lymphoid follicles of the lymph node and spleen. Notably, IL-4 changed its location state from scattered and sparse during primary infection to clinging to B lymphocytes in the lymphoid follicles during reinfection. During reinfection, IL-4 was often co-localized with T and B lymphocytes; furthermore, the percentages of several T lymphocyte subsets, N protein-specific antibody levels, and viral load in the peripheral blood or lymph tissues underwent remarkable variation. Another important finding of this study was that the numbers of B lymphocytes and T lymphocytes in the lymphoid nodes were significantly reduced after PRRSV infection or reinfection, presumably due to PRRSV-induced acute bone marrow failure and autophagy in thymic epithelial cells. This study revealed the characteristics of IL-4 migration and distribution in the peripheral lymph organs induced by PRRSV reinfection and provides valuable clues for further exploration of the interactions between IL-4, B lymphocytes, and T lymphocytes during PRRSV infection and reinfection.

DNA Engineered Lymphocyte-Based Homologous Targeting Artificial Antigen-Presenting Cells for Personalized Cancer Immunotherapy.

Artificial antigen-presenting cells (aAPCs) constructed by integrating T cell activation ligands on biocompatible materials hold great potential in tumor immunotherapy. However, it remains challenging to develop aAPCs, which could mimic the characteristics of natural APCs, thereby realizing antigen-specific T cells activation in vivo. Here, we report the first effort to construct natural lymphocyte-based homologous targeting aAPCs (LC-aAPCs) with lipid-DNA-mediated noninvasive live cell surface engineering. Through a predesigned bottom-up self-assembly path, we achieved natural-APC-mimicking distribution of T cell activation ligands on LC-aAPCs, which would enable the optimized T cell activation. Moreover, the lipid-DNA-mediated self-assembly occurring on lipid bilayers would not affect the functions of homing receptors expressed on lymphocyte. Therefore, such LC-aAPCs could actively migrate to peripheral lymphatic organs and then effectively activate antigen-specific T cells. Combined with an immune checkpoint inhibitor, such LC-aAPCs could effectively inhibit the growth of different tumor models. Thus, our work provides a new design of aAPCs for in vivo applications in tumor immunotherapy, and the lipid-DNA-mediated noninvasive live cell surface engineering would be a powerful tool for designing cell-based therapeutics.

IMMU-32. IDENTIFICATION OF TUMOR-ANTIGEN SPECIFIC T CELLS AND THE EFFICACY OF IMMUNOTHERAPY VACCINES FOR GLIOBLASTOMA ANTIGENS DETERMINED USING CANCER IMMUNOGENOMICS APPROACH

Abstract BACKGROUND Glioblastoma multiforme (GBM) remains a disease with debilitating survival outcomes. Owing to the heterogeneous nature and low mutation burden, identifying multiple antigens inherent to GBM that may serve as targets for immune-based therapies is attractive. Our aim is to develop a personalized immunotherapy approach using cancer immunogenomics for prospectively identifying neoantigens and uniquely expressed tumor proteins and then selectively expanding T cells against these truly tumor-specific antigens and dendritic cell vaccines to boost the T cell responses. METHODS RNAseq and WES was performed for murine KR158-luc GBM tumor. Using a cancer immunogenomics approach that we developed, called the O pen R eading Frame A ntigen N etwork (O.R.A.N.), we identified the immunogenic neoantigens and tumor-associated antigens (TAAs) including cancer testis and developmental antigens, that are aberrantly over-expressed in KR158-luc tumor. All predicted genes were subjected to a gene enrichment strategy and an mRNA library was generated containing predominantly only the target genes but had some background non-specific genes (validated by RNAseq). KR158-luc tumor bearing animals were then treated with dendritic cells loaded with the tumor antigen specific mRNA library. Tumor volume and thus progress was determined using in vivo luciferase imaging technique. Additionally, tetramers specific to several of the predicted antigens were manufactured and the frequency of antigen specific T cells was determined using flow cytometry. RESULTS The dendritic cell vaccines were effective in delaying the progression of KR158-luc tumors and we identified T cells targeting several of our predicted antigens in the tumor bearing animals. The antigen specific T cells were detected in the tumor infiltrating lymphocytes as well as in the peripheral lymph organs. CONCLUSION We developed a dendritic cell-based vaccination approach targeting all neoantigens and TAAs identified as being tumor-specific and validated our developed immunogenomics pipeline by identifying antigen-specific T cells in the tumor bearing animals against novel GBM antigens.

Constipation induced gut microbiota dysbiosis exacerbates experimental autoimmune encephalomyelitis in C57BL/6 mice

BackgroundConstipation is a common gastrointestinal dysfunction which has a potential impact on people's immune state and their quality of life. Here we investigated the effects of constipation on experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS).MethodsConstipation was induced by loperamide in female C57BL/6 mice. The alternations of gut microbiota, permeability of intestinal barrier and blood–brain barrier, and histopathology of colon were assessed after constipation induction. EAE was induced in the constipation mice. Fecal microbiota transplantation (FMT) was performed from constipation mice into microbiota-depleted mice. Clinical scores, histopathology of inflammation and demyelination, Treg/Th17 and Treg17/Teff17 imbalance both in the peripheral lymphatic organs and central nervous system, cytokines include TGF-β, GM-CSF, IL-10, IL-17A, IL-17F, IL-21, IL-22, and IL-23 in serum were assessed in different groups.ResultsCompared with the vehicle group, the constipation mice showed gut microbiota dysbiosis, colon inflammation and injury, and increased permeability of intestinal barrier and blood–brain barrier. We found that the clinical and pathological scores of the constipation EAE mice were severer than that of the EAE mice. Compared with the EAE mice, the constipation EAE mice showed reduced percentage of Treg and Treg17 cells, increased percentage of Th17 and Teff17 cells, and decreased ratio of Treg/Th17 and Treg17/Teff17 in the spleen, inguinal lymph nodes, brain, and spinal cord. Moreover, the serum levels of TGF-β, IL-10, and IL-21 were decreased while the GM-CSF, IL-17A, IL-17F, IL-22, and IL-23 were increased in the constipation EAE mice. In addition, these pathological processes could be transferred via their gut microbiota.ConclusionsOur results verified that constipation induced gut microbiota dysbiosis exacerbated EAE via aggravating Treg/Th17 and Treg17/Teff17 imbalance and cytokines disturbance in C57BL/6 mice.

Testing Cancer Immunotherapy in a Human Immune System Mouse Model: Correlating Treatment Responses to Human Chimerism, Therapeutic Variables and Immune Cell Phenotypes.

Over the past decade, immunotherapies have revolutionized the treatment of cancer. Although the success of immunotherapy is remarkable, it is still limited to a subset of patients. More than 1500 clinical trials are currently ongoing with a goal of improving the efficacy of immunotherapy through co-administration of other agents. Preclinical, small-animal models are strongly desired to increase the pace of scientific discovery, while reducing the cost of combination drug testing in humans. Human immune system (HIS) mice are highly immune-deficient mouse recipients rtpeconstituted with human hematopoietic stem cells. These HIS-mice are capable of growing human tumor cell lines and patient-derived tumor xenografts. This model allows rapid testing of multiple, immune-related therapeutics for tumors originating from unique clinical samples. Using a cord blood-derived HIS-BALB/c-Rag2nullIl2rγnullSIRPαNOD (BRGS) mouse model, we summarize our experiments testing immune checkpoint blockade combinations in these mice bearing a variety of human tumors, including breast, colorectal, pancreatic, lung, adrenocortical, melanoma and hematological malignancies. We present in-depth characterization of the kinetics and subsets of the HIS in lymph and non-lymph organs and relate these to protocol development and immune-related treatment responses. Furthermore, we compare the phenotype of the HIS in lymph tissues and tumors. We show that the immunotype and amount of tumor infiltrating leukocytes are widely-variable and that this phenotype is tumor-dependent in the HIS-BRGS model. We further present flow cytometric analyses of immune cell subsets, activation state, cytokine production and inhibitory receptor expression in peripheral lymph organs and tumors. We show that responding tumors bear human infiltrating T cells with a more inflammatory signature compared to non-responding tumors, similar to reports of “responding” patients in human immunotherapy clinical trials. Collectively these data support the use of HIS mice as a preclinical model to test combination immunotherapies for human cancers, if careful attention is taken to both protocol details and data analysis.

Morphogenesis of limph nodes in Muscovy ducks during early posnatal onthogenesis

The dynamics of mass and linear parameters of lymph nodes of ducks is a direct reflection of structural and functional transformations of their lymphatic parenchyma. The peculiarities of quantitative dynamics of tissue components of peripheral lymphatic organs at early stages of postnatal ontogenesis are to a great extent determined by advanced growth rates of their absolute mass against the background of sharp increase of their antigenic stimulation intensity. Parenchyma of lymph nodes of newborn ducklings is characterized by relatively low degree of differentiation and is represented by diffuse lymphatic tissue with no pronounced signs of its division into separate functional zones. Internally, the lymphatic channel is represented by only two lymphatic sinuses - the central one, which is located in the central part of the organ and occupies a large relative area and a discrete edge sinus, which borders on the node capsule and has a much smaller relative area and, accordingly, is located at its periphery. The organ parenchyma is a diffuse cluster of stromal and lymphatic cells in the enlarged lymphatic vessel between the central (inner) and edge (outer) lymphatic sinuses, without signs of its division into cortical and brain matter. Among the lymphatic tissue of the lymph node, reticular stroma cells and the population of small lymphocytes have the largest relative amount.

Chemotherapy but Not the Tumor Draining Lymph Nodes Determine the Immunotherapy Response in Secondary Tumors.

SummaryImmunotherapies are used as adjuvant therapies for cancers. However, knowledge of how traditional cancer treatments affect immunotherapies is limited. Using mouse models, we demonstrate that tumor-draining lymph nodes (TdLNs) are critical for tumor antigen-specific T cell response. However, removing TdLNs concurrently with established primary tumors did not affect the immune checkpoint blockade (ICB) response on localized secondary tumor due to immunotolerance in TdLNs and distribution of antigen-specific T cells in peripheral lymphatic organs. Notably, treatment response improved with sequential administration of 5-fluorouracil (5-FU) and ICB compared with concurrent administration of ICB with 5-FU. Immune profiling revealed that using 5-FU as induction treatment increased tumor visibility to immune cells, decreased immunosuppressive cells in the tumor microenvironment, and limited chemotherapy-induced T cell depletion. We show that the effect of traditional cytotoxic treatment, not TdLNs, influences immunotherapy response in localized secondary tumors. We postulate essential considerations for successful immunotherapy strategies in clinical conditions.

Blockade of PD-1 effectively inhibits in vivo malignant transformation of oral mucosa

ABSTRACTCurbing PD-1 immunosuppressive signaling represents an effective immune awakening or immune-reactivation approach for tumor eradication for many cancers. Yet, the potential involvement of this critical PD-1 immunosuppressive signaling in de novo malignant transformation of epithelial cells to pre-cancerous or cancerous lesions is largely unknown. In this study, we demonstrate that PD-1 signaling is critically involved in de novo malignant transformation of oral mucosa upon carcinogen exposure in vivo. Our findings revealed that 4NQO-treated mice had almost double the numbers of PD-1-positive CD4+ cells and PD-1-positive CD8+ cells in peripheral blood lymphocytes as well as elevated PD-1 expression in tumor infiltrating lymphocytes (when compared to that of control-treated mice), strongly supportive of a general immune-suppression induced by carcinogen challenges in vivo. Importantly, inhibition of PD-1 signaling during the carcinogenesis process (immediately after 4NQO challenge) significantly reduced and delayed de novo formation of both pre-cancerous and cancerous lesions in vivo, in conjunction with effective PD-1 down-modulation in the tumor infiltrating leukocyte and peripheral lymph organs. Lastly, reduction of carcinogen-induced lesions upon PD-1 mAb treatment in vivo was accompanied by reduction of potent immunosuppressive myeloid-derived suppressor cells (MDSCs), and increase in “activated” T cell accumulations in the lesion-microenvironment (127% increase) and peripheral lymph nodes (25% increase). These data support PD-1 blockade as a new approach to enhance the efficacy of T-cell immunotherapy and reduce canceration rate in premalignant lesions.

Bone marrow T cells populations are differently affected by sepsis than their splenic counterparts in the murine sepsis cecal ligation and puncture (CLP) model

Abstract Sepsis is known to induce lymphopenia which correlates with secondary infections and mortality. In sepsis survivors the lymphocyte count restores with time, although the T cell responses are impaired. So far, the mechanisms of T cells recovery are not fully understood and the murine studies were focused on the peripheral lymphatic organs in which the spleen was most common. Our study was designed to elucidate the recovery of bone marrow (BM) T cells which contain population of memory cells, after the CLP murine sepsis. In our model, mild CLP produced peripheral lymphopenia which was lowest at 48h after CLP but after 7 days circulating T cells count was rebounding. 7 days after CLP the BM frequencies of naïve and central memory (CD44+CD62L+) CD4 cells were unchanged while they were reduced in the spleen. Percentage of effector memory (CD44+CD62L−) CD4 and CD8 cells increased in the BM and at the same time they dramatically decreased in the spleen. On the contrary, the effector (CD44−CD62L+) CD4 and CD8 cells expanded at that time point in the spleen while they did not change in the BM. At D7 after CLP, the rates of BrdU+ proliferating CD4+ and CD8+ cells were markedly higher in the BM than spleen. Among the BM CD4+ cells most of the proliferating cells were the effector memory cells, while the effector CD4+ had highest proliferation rate in the spleen. Similarly, CD8 effector memory cells in the BM and CD8 effector cells were most extensively proliferating in the BM and the spleen, respectively. Taken together, our data point to the BM as the place of effector memory T cell proliferation during post-sepsis recovery.

Murine Cytomegalovirus Infection Induces Susceptibility to EAE in Resistant BALB/c Mice.

In contrast to C57BL/6 mice, BALB/c mice are relatively resistant to the induction of experimental autoimmune encephalomyelitis (EAE) after challenge with MOG35–55 peptide. Here, we provide the first evidence that infection with murine cytomegalovirus (MCMV) in adulthood abrogates this resistance. Infected BALB/c mice developed clinical and histological signs similar to those seen in susceptible C57BL/6 mice. In addition to CD4+ cells, large proportion of cells in the infiltrate of diseased BALB/c mice was CD8+, similar with findings in multiple sclerosis. CD8+ cells that responded to ex vivo restimulation with MOG35–55 were not specific for viral epitopes pp89 and m164. MCMV infection favors proinflammatory type of dendritic cells (CD86+CD40+CD11c+) in the peripheral lymph organs, M1 type of microglia in central nervous system, and increases development of Th1/Th17 encephalitogenic cells. This study indicates that MCMV may enhance autoimmune neuropathology and abrogate inherent resistance to EAE in mouse strain by enhancing proinflammatory phenotype of antigen-presenting cells, Th1/Th17, and CD8 response to MOG35–55.

Research Progress of Novel Small Molecule Drugs in the Treatment of Chronic Lymphocytic Leukemia -Review

Chronic lymphocytic leukemia (CLL), the most frequent adult leukemia in Western population, is characterized by accumulation of mature-looking CD5+/19+/23+ B cells in peripheral blood, bone marrow, and lymphatic organs. Over the last 20 years, there has been a dramatic change in therapy for CLL, the complete response rate increased from the initial <5% to the current 40%-50%, this remarkable improvement has been attributable to combination of chemoimmunotherapy agents that have contributed to the backbone of therapy for patients with CLL. Especially over the past 5 years, there has been an explosion of new active agents that provide a very effective solution for patients with recurrent/refractory disease as well as those who harbor poor cytogenetic abnormalities. This review focuses on some of the novel small molecule drugs that have either been approved or are at the forefront of clinical development in the treatment of patients with CLL, including tyrosine kinase inhibitior ibrutinib, PI3K inhibitor idelalisib, Syk inhibitor, BCL-2 inhibitor and so on.

Sleep influences the immune response and the rejection process alters sleep pattern: Evidence from a skin allograft model in mice

IntroductionSleep generally regulates immune functions in a supportive manner and can affect parameters that are directly involved in the rejection process. Study objectivesThe first objective was to assess whether sleep deprivation (SD) or sleep restriction (SR) affects the allograft rejection process in mice. The second objective was to investigate whether the rejection process itself modulates the sleep pattern of allografted mice. DesignAdult BALB/c and C57BL/6J male mice were used as the donors and recipients, respectively, except for the syngeneic group (ISOTX), which received skin from mice of the same strain (C57BL/6J). The recipients were randomly assigned to either one of two control groups – TX (allogenic) or ISOTX (syngeneic) – which underwent stereotaxic surgery to enable sleep recording prior to the allograft but were not sleep deprived; one of two paradoxical sleep deprived groups – SDTX and TXSD – which underwent 72h of continuous SD either before or after the allograft respectively, and one of two sleep restricted groups – SRTX and TXSR – which underwent 21h of SD and 3h of sleep for 15days either before or after the allograft respectively. InterventionsThe skin allograft was inspected daily to determine the survival time, expected as 8.0±0.4days in this transplant model under no treatment. The sleep pattern was controlled throughout the rejection process in the SD and SR groups. Draining lymph nodes, spleen, blood and skin grafts were harvested on the 5th day after transplantation for evaluation of the immune parameters related to allograft rejection. Measurements and resultsIn the control groups, we observed a reduction in paradoxical sleep throughout the entire allograft rejection process. Acute and chronic experimental sleep loss in the SD and SR groups produced marked alterations in the immune response. Both SD and SR prolonged allograft survival compared to the non-sleep-deprived group. There were reductions in the following parameters involved in the allograft rejection under sleep loss: CD4+ and CD8+ T cell subpopulations in the peripheral lymph organs and spleen, circulating sIL-2R levels, graft-infiltrating CD4+ T cells and skin allograft global gene expression. ConclusionsWe provide, as far as we are aware, the first evidence in vivo that the immune response can alter the normal sleep pattern, and that sleep loss can conversely affect the immune response related to graft rejection.

Attenuated flow-induced dilatation of middle cerebral arteries is related to increased vascular oxidative stress in rats on a short-term high salt diet.

Recent studies have shown that high salt (HS) intake leads to endothelial dysfunction and impaired vascular reactivity in different vascular beds in both animal and human models, due to increased oxidative stress. The objective of this study was to assess vascular response to flow-induced dilatation (FID) and to elucidate the role of vascular oxidative stress/antioxidative capacity in middle cerebral arteries (MCAs) of HS-fed rats in vitro. The novelty of this study is in demonstrating impaired flow-induced dilatation of MCAs and down-regulation of vascular antioxidant genes with HS intake, leading to increased levels of oxidative stress in blood vessels and peripheral lymph organs, which together contribute to impaired FID. In addition, results show increased oxidative stress in leukocytes of peripheral lymph organs, suggesting the occurrence of inflammatory processes due to HS intake. Recirculation of leukocytes might additionally increase vascular oxidative stress in vivo. The aim of this study was to determine flow-induced dilatation (FID) and the role of oxidative stress/antioxidative capacity in isolated, pressurized middle cerebral arteries (MCAs) of high salt (HS)-fed rats. Healthy male Sprague-Dawley rats (11weeks old) were fed low salt (0.4% NaCl; LS group) or high salt (4% NaCl; HS group) diets for 1week. Reactivity of MCAs in response to stepwise increases in pressure gradient (Δ10-Δ100mmHg) was determined in the absence or presence of the superoxide dismutase (SOD) mimetic TEMPOL and/or the nitric oxide synthases (NOS) inhibitor N(ω) -nitro-l-arginine methyl ester (l-NAME). mRNA levels of antioxidative enzymes, NAPDH-oxidase components, inducible (iNOS) and endothelial nitric oxide synthases (eNOS) were determined by quantitative real-time PCR. Blood pressure (BP), antioxidant enzymes activity, oxidative stress in peripheral leukocytes, lipid peroxidation products and the antioxidant capacity of plasma were measured for both groups. FID was reduced in the HS group compared to the LS group. The presence of TEMPOL restored dilatation in the HS group, with no effect in the LS group. Expression of glutathione peroxidase 4 (GPx4) and iNOS in the HS group was significantly decreased; oxidative stress was significantly higher in the HS group compared to the LS group. HS intake significantly induced basal reactive oxygen species production in the leukocytes of mesenteric lymph nodes and splenocytes, and intracellular production after stimulation in peripheral lymph nodes. Antioxidant enzyme activity and BP were not affected by HS diet. Low GPx4 expression, increased superoxide production in leukocytes, and decreased iNOS expression are likely to underlie increased oxidative stress and reduced nitric oxide bioavailability, leading to impairment of FID in the HS group without changes in BP values.

Antibodies from donor B cells perpetuate cutaneous chronic graft-versus-host disease in mice

AbstractCutaneous sclerosis is one of the most common clinical manifestations of chronic graft-versus-host disease (cGVHD). Donor CD4+ T and B cells play important roles in cGVHD pathogenesis, but the role of antibodies from donor B cells remains unclear. In the current studies, we generated immunoglobulin (Ig)Hµγ1 DBA/2 mice whose B cells have normal antigen-presentation and regulatory functions but cannot secrete antibodies. With a murine cGVHD model using DBA/2 donors and BALB/c recipients, we have shown that wild-type (WT) grafts induce persistent cGVHD with damage in the thymus, peripheral lymphoid organs, and skin, as well as cutaneous T helper 17 cell (Th17) infiltration. In contrast, IgHµγ1 grafts induced only transient cGVHD with little damage in the thymus or peripheral lymph organs or with little cutaneous Th17 infiltration. Injections of IgG-containing sera from cGVHD recipients given WT grafts but not IgG-deficient sera from recipients given IgHµγ1 grafts led to deposition of IgG in the thymus and skin, with resulting damage in the thymus and peripheral lymph organs, cutaneous Th17 infiltration, and perpetuation of cGVHD in recipients given IgHµγ1 grafts. These results indicate that donor B-cell antibodies augment cutaneous cGVHD in part by damaging the thymus and increasing tissue infiltration of pathogenic Th17 cells.

Effect of in ovo-delivered prebiotics and synbiotics on lymphoid-organs’ morphology in chickens

Prebiotics and probiotics, either alone or together (synbiotics), can influence the intestinal microbiota and modulate the immune response. We aimed to investigate the effects of prebiotic and synbiotic administration during the early stage of development on the histological structures of central (bursa of Fabricius and thymus) and peripheral (spleen) lymphatic organs in broilers. We used 800 hatching eggs from meat-type hens (Ross 308). Prebiotics and synbiotics were administered in ovo into the air chamber of chicken eggs at d 12 incubation, as follows: prebiotic inulin (Pre1), Bi2 tos (Pre2), a synbiotic composed of inulin and Lactococcus lactis subsp. lactis IBB SL1 (Syn1), a synbiotic composed of Bi2 tos and L. lactis subsp. cremoris IBB SC1 (Syn2), or physiological saline (control group, C). In ovo delivery of prebiotics and synbiotics had no adverse effect on the development of the immune system in exposed chickens. Administration of Bi2 tos with L. lactis subsp. cremoris (Syn2) decreased the cortex/medulla ratio in the thymus and slowed the development of the cortex in bursal follicles on d 21 posthatching, with consequent impacts on the primary lymphatic organs. The above treatment also stimulated germinal centers’ formation in the spleens of 21- and 35-day-old chickens, indicating enhanced B-cell proliferation in secondary lymphatic organs. Syn2 also caused an age-dependent increase in the spleen/bursa of Fabricius ratio. In conclusion, the in ovo administration of pre- and synbiotics at d 12 incubation can modulate the central and peripheral lymphatic organ development in broilers. This effect is more pronounced after synbiotic treatment than in prebiotic-treated groups.

The effect of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on lymphocyte subsets and humoral immune response in SRBC-immunized mice

5-Amino-3-methyl-4-isoxazolecarboxylic acid hydrazide is a non-cytotoxic synthetic isoxazole derivative with considerable immunomodulatory properties demonstrated in in vitro experiments. The aim of this study was to investigate the influence of this compound, depending on the dosage and schedule of treatment, on lymphocyte subsets in non-immunized mice and humoral immune response in SRBC (sheep red blood cells)-immunized mice. An analysis of lymphocyte subsets was carried out by flow cytometry, using specific monoclonal antibodies stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). In the SRBC-immunized mice, the influence of the compound on the humoral response was determined, depending on the time of administration relative to the antigen. The number of plaque forming cells (PFC) was determined by a local hemolysis technique in an agar gel. Total and 2-mercaptoethanol resistant serum agglutination titers were defined by active hemagglutination test carried out on microplates. The investigated hydrazide was able to modulate the percentage and absolute number of T lymphocyte subsets in the thymus, and T and B lymphocytes in the peripheral lymphatic organs. It also enhanced humoral immune response in SRBC-immunized mice by increasing the number of cells producing hemolytic anti-SRBC antibodies (PFC) and by augmenting the level of total and 2-mercaptoethanol resistant hemagglutinin. The present study showed modulatory effects of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on lymphocyte subsets and humoral immune response in mice. This compound could be potentially useful for the treatment of autoimmune diseases, infections or as an adjuvant for boosting the efficacy of vaccines.

Chemotactic signaling and beyond: link between interleukin-16 and axonal degeneration in multiple sclerosis.

Multiple sclerosis (MS) is a progressive inflammatory, and chronic demyelinating, neurodegenerative disease of central nervous system (CNS). Autoimmune responses to myelin and other CNS antigens mediated by cluster of differentiation 4 (CD4+) T cells are critical for initiation and progression of disease. Migration of autoimmune T cells from the peripheral lymph organs into CNS parenchyma leads to inflammation, demyelination and damage of axonal cytoskeleton, which manifest in decreased impulse conduction velocity of motor and sensory nerves. Myelin and axonal pathology causes motor, sensory and autonomic nerve dysfunction, including optic nerve damage leading to double or distorted vision; paresis and paralysis of extremities, painful sensations, and bladder sphincter dysfunction, manifested as bladder incontinence. Gray matter pathology in cortical and subcortical regions, including cerebellum and hippocampus underlies cognitive and behavioral dysfunction consisting of memory deficits, depression, and ataxic gait and tremor.