bluuebunny/crossref_metadata_2025_split huggingface dataset:
Altered cellular organization of the splenic marginal zone in aged mice (132.24)
Authors: Birjandi Shirin , Ippolito Jill , Ramadorai Anand , Witte Pamela 
Published: January 2023

Abstract
Splenic marginal zones (MZ) are architecturally organized to generate a rapid response against blood borne antigens entering the spleen. Proper alignment includes MZ B cells, MARCO+ MZ macrophages (MZM), and MOMA1+ metallophilic macrophages positioned around a MAdCAM-1+ cell lined sinus. Our objective was to assess cell specific morphological changes of the MZ in young vs aged mice. We observed gross changes in the spleens of aged mice that reflect the improper alignment of cells in the MZ. Immunocytochemistry and immunofluorescence were used to reveal the positioning of MAdCAM-1+ sinus lining cells and MZM or MOMA1+ macrophages. As reported, in young mice, a continuous single cell layer of MAdCAM-1+ cells enveloped the white pulp, in addition to a cohesive line of MZM and MOMA1+ macrophages closely aligned with MAdCAM-1+ cells. In contrast, the MAdCAM-1+ cells of aged mice were more diffuse and less organized. MOMA1+ macrophages were also discontinuous and highly diffused into the white pulp and B-cell follicles. MAdCAM-1+ cells were surrounded by discontinuous, patchy groups of MZM. The reduction of MZM in aged spleens was confirmed by evaluating the ability of MZM to bind the FITC-labeled Dextran in vivo. By flow cytometry, the reduction in MZM in individual aged mice statistically correlated with reduced frequency of MZB cells. These changes may affect the ability of the MZ to properly respond to and clear pathogens in aged mice. NIH RO1AG013874 and T32AG031780
Internalization of BCR in EU12 µHC+ cells by BCR-Crosslinking cells by BCR-Crosslinking (132.27)
Authors: Marran-Nichol Kathleen , Lui Jing , Zhang Zhixin 
Published: January 2023

Abstract
Engagement of the B cell antigen receptor (BCR) on mature B cells activates signal transduction pathways leading to B cell activation. It has also been shown that crosslinking BCR on murine mature B cells results in partial BCR internalization and limited receptor recycling. The EU12 µHC+ cells are phenotypically and functionally similar to human bone marrow immature B cells. Stimulation of these cells with low concentrations of goat F(ab’)2 anti-IgM antibodies results in complete internalization of the BCR. The internalized BCRs travel to the early and late endosomes, presumably for degradation. Re-expression of the BCR on the cell surface can be detected as early as 2 hours and reach almost normal levels within 6 to 12 hours. However, the reappearance of the BCR, which is completely blocked by Cycloheximide and Brefeldin A treatment respectively, requires de novo protein synthesis and secretion. These results show that the EU12 µHC+ immature B cells do not recycle surface BCR after complete internalization of the BCR.
CD8 T cell response for H60 requires CD4 T cell help when the antigen is expressed as a viral antigen (132.36)
Authors: Kang Bora , Ryu Su Jeong , Choi Eun Young 
Published: January 2023

Abstract
CD4 helper T cell is critical for the generation of primary and memory CD8 T cells specific for non-inflammatory antigens. Minor histocompatibility antigens are representative non-inflammatory cellular antigens that require CD4 help for the response induction, and we have shown that CD8 T cell response for minor histocompatibility antigen H60 is dependent on CD4 help not only for the primary response but also for the secondary response inductions. In this study, we addressed a question whether H60 epitope as a viral antigen would require CD4 help for specific response induction. A recombinant vaccinia virus expressing H60-CD8 epitope and HY-CD4 epitope (rVV-H60HY) was used for immunization of B6 female mice. Immunization with the recombinant virus induced a significant amount of H60-specific CD8 T cell proliferation with a peak frequency around 10~20% of total blood CD8 T cells. The response was CD4 help dependent both after the primary and secondary viral challenges. The CD4 help could be substituted by combined treatment of TLR agonists and anti-CD40 antibody. Our results demonstrate that CD4 help is required for appropriate CD8 T cell responses specific for H60 not only as cellular antigen but also as viral antigen, and the signaling through TLR and CD40 would license antigen presenting cells to appropriately activate antigen-specific CD8 T cells.
Recombinant Rabbit Monoclonal Antibodies to Study Apoptosis and Apoptotic Pathways (132.4)
Authors: Lubkin Margaret , Shallice Matthew , Nyhus Julie , Leong Louis , Aggeler Birte 
Published: January 2023

Abstract
Flow cytometry a tool for studying apoptosis and apoptotic pathways. Using monoclonal antibodies for flow cytometry leads to high specificity for the detection of the target epitope of interest, limiting the use of flow cytometry to available mouse monoclonal antibodies. Here we present high quality recombinant rabbit monoclonal antibodies that do not rely on hybridoma cell lines, but are made with a proprietary recombinant technology to obtain cloned antibodies. These rabbit monoclonals were compared to other available antibodies to demonstrate high specificity and affinity to their targets. We examined lot-to-lot consistency using the same antibody for flow cytometry, western blot and immunocytochemistry. In this study two rabbit monoclonal antibodies involved in apoptotic pathways, Cleaved Caspase-3[Asp175] and p53[pS15] were examined. The p53 antibody is the phosphorylated form of p53 [pS15], which in turn induces p53 Upregulated Modulator of Apoptosis (PUMA), the result being apoptosis through mitochondrial degradation. We performed western blot, and immunocytochemistry studies to show high specificity for both antibodies as well as obtain spatial resolution within the cell.
Lymphopenia precedes the onset of intestinal inflammation in Gimap5 deficient mice (132.6)
Authors: Aksoylar Halil , Barnes Michael , Lampe Kristin , Hoebe Kasper 
Published: January 2023

Abstract
Using N-ethyl-N-nitrosourea (ENU) germline mutagenesis technique, our lab identified a recessive point mutation in a conserved coding region of Gimap5 gene which resulted in severe lymphopenia. The mutation, designated as sphinx, caused total absence of CD8+ T and natural killer cells in the secondary lymphoid organs and caused a progressive loss of peripheral CD4+ T and B cells of homozygous mutant mice with age. Gimap5sph/sph CD4+ T cells adopted a CD44hi CD62Llow effector/memory-like phenotype that is a characteristic of lymphopenia induced proliferation. Additionally, Gimap5sph/sph mice developed intestinal inflammation and wasting disease starting around 8-10 weeks of age which could be reversed by antibiotics treatment and adoptive transfer of wild type splenocytes. Prior to the initiation of colitis, Gimap5sph/sph mice exhibited a reduced total number of regulatory T cells but also a reduced frequency of Foxp3+ cells within the CD4+ T cell population in the spleen and mesenteric lymph nodes. Together, these results establish Gimap5 as a key regulator of lymphocyte development and homeostasis.
Bcl-2 maintains KLRG1low CD127high effector CD8+ and central memory CD8+ T cells by distinct mechanisms (132.9)
Authors: Kurtulus Sema , Tripathi Pulak , Sholl Allyson , Hildeman David 
Published: January 2023

Abstract
After acute infection, most of the effector CD8+ T cells die while some of them survive to become memory cells. Recent work has shown that effector CD8+ T cell subpopulations can be distinguished based on the reciprocal expression of KLRG1 and CD127. During viral infection, KLRG1lo CD127hi effector CD8+ T cells have increased ability to generate memory T cells compared to KLRG1hi CD127lo CD8+ T cells. Here, we assessed the role of Bim and Bcl-2 in controlling the survival of these effector CD8+ T cell subsets. We found that KLRG1lo CD127hi cells had higher levels of Bim and Bcl-2 than KLRG1hi CD127lo effector CD8+ T cells. Using both genetic and pharmacological approaches, we found that survival of most KLRG1lo CD127hi and some KLRG1hi CD127lo effector cells required Bcl-2 to combat the pro-apoptotic activity of Bim. Further, when Bcl-2 was blocked in effector T cells, their development into long-term memory cells was significantly decreased. Similar to subpopulations of effector T cells, we found differential expression of Bcl-2 and Bim in memory T cell subsets. Further, survival of central memory (TCM), but not effector memory (TEM) CD8+ T cells was significantly impaired when Bcl-2 was absent or inhibited. Interestingly, loss of TCM CD8+ T cells could not be rescued by loss of Bim suggesting that Bcl-2 combats molecules other than Bim in CD8+ TCM cells. Combined, these data show the critical role of Bcl-2 and Bim in the control of effector and memory T cell homeostasis.
Native low-density lipoprotein uptake by Macrophage Colony-Stimulating Factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis (134.12)
Authors: Anzinger Joshua , Chang Janet , Xu Qing , Buono Chiara , Li Yifu , Leyva Francisco , Park Bum-Chan , Greene Lois , Kruth Howard 
Published: January 2023

Abstract
Human macrophage colony-stimulating factor (M-CSF)-differentiated macrophages—the predominant macrophage phenotype in human atherosclerotic plaques—constitutively take up native low-density lipoprotein (LDL) by fluid-phase pinocytosis, but the pinocytotic pathways mediating LDL uptake by these macrophages have not been thoroughly examined. We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages was only partially inhibited (~40%) by these agents. We therefore investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (~40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. Our findings show that human M-CSF-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.
Identification and characterization of guinea pig interleukin-6 (IL-6) (134.15)
Authors: Brodey Mary , Chen Linda , Cheng Andy , Stollar Neil , Huuskonen Jarkko , Beernink Hans 
Published: January 2023

Abstract
The guinea pig (Cavia porcellus) is an important research model for a number of diseases of the lung, including chronic obstructive pulmonary disease (COPD), tuberculosis, and asthma. Inflammation appears to play an important role in the progression of these various diseases. An important focus in understanding the etiology of these various diseases is the detection and quantification of inflammatory mediators such as cytokines and prostaglandins. Measurements of these inflammatory mediators can also provide a method by which efficacy of potential therapeutics is assessed. A key mediator of inflammation in a number of experimental systems is interleukin-6 (IL-6). The sequence of guinea pig IL-6 has not been published. To enable the development of reagents for the detection and quantification of IL-6 in guinea pig samples, recombinant guinea pig IL-6 was developed through the isolation of mRNA from LPS-stimulated guinea pig splenocytes, amplification of the cDNA, and cloning and expression using appropriate vectors in E. coli. Following refolding and purification, the protein was found to be highly reactive in a B9 mouse hybridoma proliferation assay. The observed ED50 of guinea pig IL-6 was comparable to the observed ED50 of mouse IL-6 (4-8 pg/mL) and human IL-6 (8-20 pg/mL). Sequence analysis revealed that guinea pig IL-6 was approximately 55% homologous with mouse IL-6, and approximately 57% homologous with human IL-6.
DACS-DB: A curation and dissemination model for disease-associated cytokine SNPs (134.19)
Authors: Perumal Narayanan , Bhushan Sushant 
Published: January 2023

Abstract
Cytokines play valuable roles in immune cell growth and lineage specification, and in various disease pathologies. Both low and high throughput studies have implicated the association of cytokine single nucleotide polymorphisms (SNPs) with diseases. A preponderance of such experiments has not shown any causality of an identified SNP to the associated disease. Instead, they have identified statistically significant SNP-disease associations; it is likely that some of these cytokine gene variants may directly or indirectly cause the disease phenotype(s). To fill this knowledge gap and derive study parameters for SNP-disease causality relationships, we have designed and developed the Disease Associated Cytokine SNP Database (DACS-DB). DACS-DB includes data on 456 cytokine genes (derived from the ImmPort database), 10,000 SNPs (from dbSNP), and 330 SNP-associated diseases (from PubMed). We have manually curated functional annotation, heterozygosity and population frequency for the SNPs, and statistical significance of SNP-disease association for diseases. We are currently developing a front end to publish this data on the WWW for dissemination to immunologists and biomedical researchers. The curated data in the DB is to be employed to build a computational tool to predict the disease association potential of an uncharacterized cytokine SNP. This DB can be vital to understand the role of cytokine SNPs as markers in disease and may help identify drug targets for common diseases.
TNF secretion from human mast cells is regulated by mitochondrial dynamics and mitochondrial uncoupling protein 2 (UCP2) (135.11)
Authors: Zhang Bodi , Alysandratos Kostas , Angelidou Asimenia , Kempuraj Duraisamy , Tagen Michael , Vasiadi Magdalini , Asadi Shahrzad , Theoharides Theoharis 
Published: January 2023

Abstract
Tumor necrosis factor (TNF) is involved in T cell-dependent diseases such as psoriasis, and in T cell activation by mast cells, which can both store and de novo synthesize TNF. Yet, regulation of TNF secretion is unknown hampering our understanding of how mast cells regulate allergic, immune and inflammatory processes. Here we show that stimulated human LAD2 leukemic and umbilical cord-derived cultured mast cell (hCBMCs) by SP or IgE/anti-IgE induces degranulation and prestored TNF release, which is accompanied by mitochondrial fission. Images of Confocal microscopy and electronic transmitted microscopy show fragmented mitochondria and relocation from a perinuclear distribution to the cell surface. Moreover, treatment with a mitochondrial fission blocker inhibits degranulation, indicating the necessarily of mitochondria fission in mast cell secretion. Besides mitochondria morphological changes, SP stimulation of LAD2 and hCBMCs increased expression (8 hr) of the mitochondrial uncoupling protein 2 (UCP2), which regulates production of ROS and intracellular calcium, leading to inhibition of subsequent re-stimulation by SP, when is enough for mast cells to recover. Moreover, UCP2 gene expression was reduced, as compared to control normal skin, in affected psoriatic skin, an inflammatory diseases in which mast cells play important role. These findings indicate that mitochondria play a key regulatory role in mast cell TNF secretion and in psoriasis.
Inflammation fails to induce the vitamin D3 1α-hydroxylase. (135.13)
Authors: Ooi Jot Hui , Cantorna Margherita 
Published: January 2023

Abstract
The active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH) 2D3], is largely produced by a renally expressed enzyme 1α-hydroxylase, which is encoded by the Cyp27B1 gene. Using transgenic mice that have replaced the Cyp27B1 gene with the bacterial lacZ reporter gene (β-galactosidase), we wanted to investigate the conditions that induce extrarenal 1α-hydroxylase expression. Expression of the 1α-hydroxylase in extrarenal tissues including the immune system has been reported. We did not detect β-galactosidase expression in LPS-stimulated BMDM, splenocytes, and peritoneal cells. Moreover, in vivo challenges of LPS, α-galactosylceramide, and Listeria monocytogenes showed no detectable β-galactosidase expression in lung, liver, spleen, and blood. Cyp27B1 expression has been reported in the colon of both DSS-treated and control mice. Cyp27B1 KO mice were more susceptible to DSS colitis than WT mice and showed severe weight loss, inflammation, bleeding, and shrinkage of the colon. DSS treatment of Cyp27B1 KO mice resulted in spotty induction of β-galactosidase in the distal small intestine with a frequency of 3 positives for 22 tested and in the colon with a frequency of 6 positives for 22 tested. No β-galactosidase activity was detected in the intestines of untreated controls. The induction of Cyp27B1 expression was sporadic and not associated with the severity of disease. Our data does not support a role for the induction of Cyp27B1 following inflammation.
Ubiquitination system defects and abnormal tolerance in CD4+ T cells from systemic lupus erythematosus patients. (135.16)
Authors: Gomez-Martin Diana , Galindo-Campos Miguel , Díaz-Zamudio Mariana , Crispín José , Alcocer-Varela Jorge 
Published: January 2023

Abstract
Ubiquitin ligases, such as Cbl-b, GRAIL and Itch are included in the anergy genetic program, and their deficiency in murine models has been related to a resistant to anergy phenotype in T cells and lupus like disease. The aim was to analyze Cbl-b, GRAIL and Itch expression in T cells from SLE patients upon anergy induction. We included 14 SLE patients in clinical remission and 5 active untreated SLE patients. Cbl-b RNA expression was measured by RT-PCR in peripheral CD4+ T cells from SLE patients and controls upon anergy induction (ionomycin). Cell proliferation was measured by CFSE. Cytokine production was analyzed by luminometry and activation markers surface expression by flow cytometry We show that CD4+ T cells from SLE patients display decreased expression of Cbl-b RNA upon anergy induction vs resting (p=0.020) and ex vivo (p=0.030) conditions. Also, a resistant to anergy phenotype (increased proliferation and IL-2 production upon ionomycin) was evidenced. Decreased IL-17 and IFN-γ production as well as increased CD69 expression were found in CD4+ T cells from SLE patients after anergy induction. The expression of GRAIL and Itch was not significantly altered. Our data suggest that CD4+ T cells from SLE patients in clinical remission display abnormalities in the ubiquitination system, specifically, deficient Cbl-b expression which is related to the resistant to anergy phenotype, and should be considered as part of the intrinsic T cell defects in SLE.
Naïve T cells found in the islets of NOD mice do not cause Type I diabetes (135.17)
Authors: Young Ellen , Talmage Garrick , Larry Fatima , Buntzman Adam , Hess Paul , Tisch Roland , Frelinger Jeffrey 
Published: January 2023

Abstract
The islets of non obese diabetes-prone (NOD) mice become chronically inflamed during progression of autoimmune Type I diabetes. In a previous study we found that there are substantial numbers of naïve T cells in the islets of NOD mice. Up to 30% of CD4+ T cells and 40% of CD8+ T cells have a naïve phenotype of CD62Lhigh and CD44low in the islets of 12 to 14 week old female NOD mice. Since mice with higher levels of inflammation had higher levels of naïve T cells we wondered if the inflammation levels of individual islets would also affect the accumulation of naïve T cells. We examined the phenotype of T cells emerging from individual islets. We found an inflammation threshold of 100 to 200 CD4+ and 20 to 70 CD8+ T cells in individual islets. Above this threshold we found significant numbers of naïve T cells. To determine if these naïve T cells were contributing to diabetogenesis, we separated naïve and effector T cells from the islets of NOD mice and transferred either naïve or effector T cells to NOD scid mice. Islet effector T cells could transfer diabetes to NOD scid mice while islet naïve T cells could not. We conclude that individual islets progress as unique sites of inflammation that evolve individually to form environments of tertiary lymphoid structure that support the migration of naïve T cells into the islets. Given the large number of B cells also in the islet, the presence of naïve T cells may allow for epitope spreading to also occur in the islets.
A hematopoetic factor contributes to brain microhemorrhage in CD8 T cell induced blood brain barrier disruption (135.19)
Authors: Johnson Aaron , Chen Yi , Suidan Georgette , McDole Jeremiah , Lohrey Anne , Pirko Istvan 
Published: January 2023

Abstract
Blood brain barrier (BBB) disruption is a common feature of neurological disease. We developed a novel model of CD8 T cell mediated BBB disruption using the Theilers murine encephalomyelitis virus (TMEV) model of multiple sclerosis. At 7 days post TMEV infection, 50-70% of CNS infiltrating CD8 T cells are specific for an immunodominant virus peptide VP2 121-130 presented in the context of the Db class I molecule. Intravenous injection of VP2 121-130 peptide at 7 days post TMEV infection results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. Despite having an identical CD8 T cell response, 129 SvIm mice survive administration of the VP2 121-130 peptide. We performed bone marrow transplants to determine if a hematopoetic factor was responsible for this fatal syndrome. 129 SvIm mice were reconstituted with C57BL/6 or autologous 129 SvIm bone marrow. Similar brain infiltration and purity of transplanted CD8 T cells was observed during acute TMEV infection in reconstituted mice. However, 129 SvIm mice reconstituted with C57BL/6 bone marrow had increased microhemorrhage formation as measured by T2* weighted MRI and became moribund. Meanwhile, 129 SvIm mice receiving an autologous bone marrow transplant had significantly less microhemorrhage formation and remained resistant to becoming moribund. These studies demonstrate that a hematopoetic factor is responsible for the onset of brain microhemorrhage in immune mediated fatal BBB disruption.
Liver Th1 cells drive the accumulation of Myeloid Derived Suppressor Cells during acute hepatitis in an IFN-γ-dependent manner. (135.2)
Authors: Cripps James , Wang Jing , Maria Ann , Blumenthal Ian , Gorham James 
Published: January 2023

Abstract
Th1 responses in the liver are the basis for parenchymal damage in autoimmune hepatitis (AIH). The cellular pathways that inhibit liver Th1 responses are not well understood. BALB/c mice lacking TGF-β1 spontaneously and rapidly develop acute Th1 cell-mediated necroinflammatory hepatitis. Liver damage requires interferon gamma (IFN-γ) production by CD4+ T cells. We hypothesized that the Th1 response would also activate pathways capable of down-regulating T cell responses, specifically, a myeloid derived suppressor cell (MDSC) population. Myeloid cells were indeed abundant in liver parenchyma in histological sections of Tgfb1KO livers. Characterization of this cell type showed that they were CD11b+Gr-1+ cells that strongly suppressed T cell proliferation in vitro, thus MDSCs. By contrast, Tgfb1WT liver CD11b+Gr-1+ cells were much less abundant and exhibited no T cell suppression capabilities. Suppression required nitric oxide (NO) and cell-cell contact, but not arginase, PDL1, or H2O2. Tgfb1KO MDSCs suppressed T cell proliferation by inducing apoptosis. Further studies using Tgfb1KO/IfngKO mice showed that IFN-γ was required both for MDSC accumulation in liver as well as for MDSC suppressor activity. These results demonstrate that, during hepatitis, Th1 cells activate through IFN-γ production an MDSC negative feedback pathway that can inhibit T cell responses through NO. Why this negative feedback pathway ultimately fails in AIH remains critical to elucidate.