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Background: A major goal of the Target Enablement to Accelerate Therapy Development for Alzheimer's disease (TREAT-AD) program is to develop and identify high-quality tools to test target or mechanistic hypotheses. As part of this initiative, it is important that commercial reagents including research antibodies being used to interrogate drug targets have confirmed validation data in knock-out cell lines. Ideally, these antibodies should also have utility for both in vitro and in vivo studies such that the levels of target proteins in target tissues can be quantified., Methods: We evaluated commercial antibodies against TREAT-AD protein targets Moesin (Uniprot ID: P26038), CD44 (Uniprot ID: P16070), Midkine (Uniprot ID: P21741) and Secreted frizzled-related protein 1, referred to as "sFRP-1" (sFRP-1; Uniprot ID: Q8N474). Moesin, Midkine and sFRP-1, that were confirmed as selective based on data in knock-out cell lines. Western blot analysis was used to compare protein levels in brain homogenates from a mouse model with AD-relevant pathology (5XFAD) versus age-matched C57BL/6J control mice., Results: Anti-Moesin ab52490 reacted in mouse brain homogenate with a predicted molecular weight of 68 kDa. Moesin protein expression was 2.8 times higher in 5xFAD compared to WT. Anti-CD44 ab189524 reacted with a band at the predicted size of 82 kDa. CD44 protein expression was 1.9 times higher in 5xFAD compared to WT. Anti-Midkine AF7769 reacted with a band ~16 kDa and a 17.8 times greater expression in 5xFAD compared to WT. Anti-sFRP-1 ab267466 reacted with a band at 35 kDa as predicted. sFRP-1 protein expression was 11.9 times greater in 5xFAD compared to WT., Conclusions: These data confirm the utility of these selective commercially available antibodies against Moesin, CD44, Midkine, and sFRP-1 for in vivo studies in mice and provide insight into the use of 5XFAD mice for in vivo target engagement studies for these target proteins., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Doolen S et al.)

Naïve T cell activation in secondary lymphoid organs such as lymph nodes (LNs) occurs upon recognition of cognate antigen presented by antigen presenting cells (APCs). T cell activation requires cytoskeleton rearrangement and sustained interactions with APCs. Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are a family of cytoskeletal effector proteins responsible for actin polymerization and are frequently found at the leading edge of motile cells. Ena/VASP proteins have been implicated in motility and adhesion in various cell types, but their role in primary T cell interstitial motility and activation has not been explored. Our goal was to determine the contribution of Ena/VASP proteins to T cell-APC interactions, T cell activation, and T cell expansion in vivo . Our results showed that naïve T cells from Ena/VASP-deficient mice have a significant reduction in antigen-specific T cell accumulation following Listeria monocytogenes infection. The kinetics of T cell expansion impairment were further confirmed in Ena/VASP-deficient T cells stimulated via dendritic cell immunization. To investigate the cause of this T cell expansion defect, we analyzed T cell-APC interactions in vivo by two-photon microscopy and observed fewer Ena/VASP-deficient naïve T cells interacting with APCs in LNs during priming. We also determined that Ena/VASP-deficient T cells formed conjugates with significantly less actin polymerization at the T cell-APC synapse, and that these conjugates were less stable than their WT counterparts. Finally, we found that Ena/VASP-deficient T cells have less LFA-1 polarized to the T cell-APC synapse. Thus, we conclude that Ena/VASP proteins contribute to T cell actin remodeling during T cell-APC interactions, which promotes the initiation of stable T cell conjugates during APC scanning. Therefore, Ena/VASP proteins are required for efficient activation and expansion of T cells in vivo ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Waldman, Rahkola, Sigler, Chung, Willett, Kedl, Friedman and Jacobelli.)

Aging and reduced exposure to environmental microbes can both potentiate neuroinflammatory responses. Prior studies indicate that immunization with the immunoregulatory and anti-inflammatory bacterium, Mycobacterium vaccae (M. vaccae), in aged rats limits neuroimmune activation and cognitive impairments. However, the mechanisms by which M. vaccae immunization ameliorates age-associated neuroinflammatory "priming" and whether microglia are a primary target remain unclear. Here, we investigated whether M. vaccae immunization protects against microglia morphological changes in response to aging. Adult (3 mos) and aged (24 mos) Fisher 344 × Brown Norway rats were immunized with either M. vaccae or vehicle once every week for 3 weeks. Aging led to elevated Iba1 immunoreactivity, microglial density, and deramification of microglia processes in the hippocampus and amygdala but not other brain regions. Additionally, aged rats exhibited larger microglial somas in the dorsal hippocampus, suggestive of a more activated phenotype. Notably, M. vaccae treatment ameliorated indicators of microglia activation in both the amygdala and hippocampus. While changes in morphology appeared to be region-specific, gene markers indicative of microglia activation were upregulated by age and lowered in response to M. vaccae in all brain regions evaluated. Taken together, these data suggest that peripheral immunization with M. vaccae quells markers of age-associated microglia activation., (© 2022. The Author(s).)

Supervillin (SVIL) is an actin-binding and membrane-associated protein, which belongs to villin/gelsolin family. It has been reported that SVIL was involved in the regulation of macrophages' movement and lipopolysaccharide (LPS) increased the SVIL mRNA expression in neutrophils, but the underlying mechanisms remain unknown. This work investigated the underlying molecular mechanisms of LPS regulating SVIL expression in macrophages and hence the possible role of SVIL in LPS-induced inflammation. We found that in THP-1-derived macrophages, LPS obviously increased SVIL mRNA and protein expression. Inhibition of TLR4 by Resatorvid (Res) remarkably reversed the LPS-induced SVIL expression. Additionally, inhibition of ERK1/2 signaling pathway (by U0126 or GDC-0994) and NF-κB (by BAY) significantly reduced the LPS-induced SVIL expression. Interestingly, down-regulation of SVIL by SVIL-specific shRNAs significantly attenuated the expression of IL-6, IL-1β & TNF-α induced by LPS at both mRNA and protein levels. Furthermore, we also observed that SVIL knockdown decreased the proportion of cells in G 2 /M phase and increased the proportion of cells in S & G 0-1 phase of THP-1 derived macrophages, but did not influence the cell viability. Taken together, we demonstrated that LPS induced the expression of SVIL via activating TLR4/NF-κB and ERK1/2 MAPK pathways, and SVIL participated in the inflammatory response of LPS-induced IL-6, IL-1β and TNF-α upregulation in macrophages., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

X-linked moesin associated immunodeficiency (X-MAID) is a primary immunodeficiency disease in which patients suffer from profound lymphopenia leading to recurrent infections. The disease is caused by a single point mutation leading to a R171W amino acid change in the protein moesin (moesin R171W ). Moesin is a member of the ERM family of proteins, which reversibly link the cortical actin cytoskeleton to the plasma membrane. Here, we describe a novel mouse model with global expression of moesin R171W that recapitulates multiple facets of patient disease, including severe lymphopenia. Further analysis reveals that these mice have diminished numbers of thymocytes and bone marrow precursors. X-MAID mice also exhibit systemic inflammation that is ameliorated by elimination of mature lymphocytes through breeding to a Rag1-deficient background. The few T cells in the periphery of X-MAID mice are highly activated and have mostly lost moesin R171W expression. In contrast, single-positive (SP) thymocytes do not appear activated and retain high expression levels of moesin R171W . Analysis of ex vivo CD4 SP thymocytes reveals defects in chemotactic responses and reduced migration on integrin ligands. While chemokine signaling appears intact, CD4 SP thymocytes from X-MAID mice are unable to polarize and rearrange cytoskeletal elements. This mouse model will be a valuable tool for teasing apart the complexity of the immunodeficiency caused by moesin R171W , and will provide new insights into how the actin cortex regulates lymphocyte function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Avery, Robertson, Wu, Roy, Chauvin, Perkey, Vanderbeck, Maillard and Burkhardt.)

Moesin is a member of the ezrin-radixin-moesin (ERM) family of proteins that link plasma membrane proteins with actin filaments in the cell cortex. Hemizygous mutations in the X-linked moesin gene are associated with primary immunodeficiency with T and B cell lymphopenia, which also affects natural killer (NK) cells in most cases. We previously showed that moesin deficiency in mice substantially affects lymphocyte homeostasis, but its impact on NK cells remains unexplored. Here, we found that in moesin-deficient mice, NK cells were decreased in the peripheral blood and bone marrow but increased in the spleen. Analysis of female heterozygous mice showed a selective advantage for moesin-expressing NK cells in the blood. Moesin-deficient NK cells exhibited increased cell death and impaired signaling in response to IL-15, suggesting that moesin regulates NK cell survival through IL-15-mediated signaling. Our findings thus identify moesin as an NK cell homeostasis regulator in vivo., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: Small fiber neuropathy (SFN) is clinically and etiologically heterogeneous. Although autoimmunity has been postulated to be pathophysiologically important in SFN, few autoantibodies have been described. We aimed to identify autoantibodies associated with idiopathic SFN (iSFN) by a novel high-throughput protein microarray platform that captures autoantibodies expressed in the native conformational state., Methods: Sera from 58 SFN patients and 20 age- and gender-matched healthy controls (HCs) were screened against >1,600 immune-related antigens. Fluorescent unit readout and postassay imaging were performed, followed by composite data normalization and protein fold change (pFC) analysis. Analysis of an independent validation cohort of 33 SFN patients against the same 20 HCs was conducted to identify reproducible proteins in both cohorts., Results: Nine autoantibodies were screened with statistical significance and pFC criteria in both cohorts, with at least 50% change in serum levels. Three proteins showed consistently high fold changes in main and validation cohorts: MX1 (FC = 2.99 and 3.07, respectively, p = 0.003, q = 0.076), DBNL (FC = 2.11 and 2.16, respectively, p = 0.009, q < 0.003), and KRT8 (FC = 1.65 and 1.70, respectively, p = 0.043, q < 0.003). Further subgroup analysis into iSFN and SFN by secondary causes (secondary SFN) in the main cohort showed that MX1 is higher in iSFN compared to secondary SFN (FC = 1.61 vs 0.106, p = 0.009)., Interpretation: Novel autoantibodies MX1, DBNL, and KRT8 are found in iSFN. MX1 may allow diagnostic subtyping of iSFN patients. ANN NEUROL 2022;91:66-77., (© 2021 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Microbial penetration of the blood-brain barrier, a prerequisite for the development of central nervous system (CNS) infection, involves microbial invasion, intracellular traversal, and exocytosis. Microbial invasion of the blood-brain barrier has been investigated, but the molecular basis for microbial traversal and exit from the blood-brain barrier remains unknown. We performed transcriptome analysis of human brain microvascular endothelial cells (HBMEC) infected with Escherichia coli and Cryptococcus neoformans, representative bacterial and fungal pathogens common in CNS infections. Among the targets upregulated in response to E. coli and C. neoformans infection, PDLIM2 was knocked down by small hairpin RNA (shRNA) in HBMEC for further investigation. We demonstrated that Pdlim2 specifically regulated microbial traversal and exit from HBMEC by assessing microbial invasion, transcytosis, intracellular multiplication, and egression. Additionally, the defective exocytosis of internalized E. coli cells from the PDLIM2 shRNA knockdown cells was restored by treatment with a calcium ionophore (ionomycin). Moreover, we performed proximity-dependent biotin labeling with the biotin ligase BioID2 and identified 210 potential Pdlim2 interactors. Among the nine Pdlim2 interactors enriched in response to both E. coli and C. neoformans infection, we selected MPRIP and showed that HBMEC with knockdown of MPRIP mimicked the phenotype of PDLIM2 knockdown cells. These results suggest that the CNS-infecting microbes hijack Pdlim2 and Mprip for intracellular traversal and exocytosis in the blood-brain barrier.

Pancreatic cancer (PC) is a malignant tumor with poor prognosis. The poor effect of surgery and chemotherapy makes the research of immunotherapy target molecules significant. Therefore, identifying the new molecular targets of PC is important for patients. In our study, we systematically analyzed molecular correlates of pancreatic cancer by bioinformatic analysis. We characterized differentially expressed analysis based on the TCGA pancreatic cancer dataset. Then, univariate Cox regression was employed to screen out overall survival- (OS-) related DEGs. Based on these genes, we established a risk signature by the multivariate Cox regression model. The ICGC cohort and GSE62452 cohort were used to validate the reliability of the risk signature. The impact of T lymphocyte-related genes from risk signature was confirmed in PC. Here, we observed the correlation between the T lymphocyte-related genes and the expression level of targeted therapy. We established a five-mRNA (LY6D, ANLN, ZNF488, MYEOV, and SCN11A) prognostic risk signature. Next, we identified ANLN and MYEOV that were associated with T lymphocyte infiltrations ( P < 0.05). High ANLN and MYEOV expression levels had a poorer prognosis in decreased T lymphocyte subgroup in PC. Correlation analysis between ANLN and MYEOV and immunomodulators showed that ANLN and MYEOV may have potential value in pancreatic cancer immunotherapy., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Ziang Li et al.)

Aims: Because serous cystadenoma (SCA) does not usually require excision, it is critical to distinguish it from differential diagnoses which do, especially neuroendocrine tumour (NET). The gold standard for diagnosing SCA is assessment of endoscopic ultrasound-guided fine needle aspiration/biopsy (EUS-FNAB) material. Inhibin immunohistochemistry aids this assessment, but such positivity is not absolutely sensitive or specific to SCA. The following is the largest known study of SCA EUS-FNAB specimens and the first to compare four potential SCA immunomarkers between themselves and inhibin, compared against NET., Methods and Results: Immunohistochemistry for calponin, mucin 6 (MUC6), glucose transporter 1 (GLUT1) and vascular endothelial growth factor A (VEGFA) was performed on 30 EUS-FNAB and three resection specimens of SCA and 32 EUS-FNAB specimens of NET. GLUT1 and VEGFA were suboptimal as diagnostic immunomarkers of SCA, being expressed by 10 and 44% of NETs, respectively. Further, their expression by cellular constituents of blood which often contaminate EUS-FNAB specimens hampered identification of neoplastic cells, especially in hypocellular samples. While 19% of NETs showed nuclear MUC6 positivity, cytoplasmic expression of the protein showed 100% specificity and sensitivity as an SCA marker. However, assessing MUC6 in EUS-FNAB specimens must also consider the protein's focal expression in physiological pancreatic, gastric or duodenal tissues, which can contaminate these specimens. Calponin was less sensitive (71% versus 100%) but more specific (100% versus 91%) than inhibin, although easier to assess in EUS-FNAB specimens than MUC6., Conclusions: Of the four potential immunomarkers of SCA suggested by the existing literature, calponin and MUC6 are useful complementary studies to inhibin for application to EUS-FNAB specimens., (© 2021 John Wiley & Sons Ltd.)

Colorectal cancer (CRC) is one of the main causes of cancer death in the world. Post-translational modifications (PTMs) have been extensively studied in malignancies due to its relevance in tumor pathogenesis and therapy. This review is focused on the dysregulation of glycosyltransferase expression in CRC and its impact in cell function and in several biological pathways associated with CRC pathogenesis, prognosis and therapeutic approaches. Glycan structures act as interface molecules between cells and their environment and in several cases facilitate molecule function. CRC tissue shows alterations in glycan structures decorating molecules, such as annexin-1, mucins, heat shock protein 90 (Hsp90), β1 integrin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), insulin-like growth factor-binding protein 3 (IGFBP3), transforming growth factor beta (TGF-β) receptors, Fas (CD95), PD-L1, decorin, sorbin and SH3 domain-containing protein 1 (SORBS1), CD147 and glycosphingolipids. All of these are described as key molecules in oncogenesis and metastasis. Therefore, glycosylation in CRC can affect cell migration, cell-cell adhesion, actin polymerization, mitosis, cell membrane repair, apoptosis, cell differentiation, stemness regulation, intestinal mucosal barrier integrity, immune system regulation, T cell polarization and gut microbiota composition; all such functions are associated with the prognosis and evolution of the disease. According to these findings, multiple strategies have been evaluated to alter oligosaccharide processing and to modify glycoconjugate structures in order to control CRC progression and prevent metastasis. Additionally, immunotherapy approaches have contemplated the use of neo-antigens, generated by altered glycosylation, as targets for tumor-specific T cells or engineered CAR (Chimeric antigen receptors) T cells.

Asplenia imparts susceptibility to life-threatening sepsis with encapsulated bacteria, such as the pneumococcus. However, the cellular components within the splenic environment that guard against pneumococcal bacteremia have not been defined. The actin-bundling protein L-plastin (LPL) is essential for the generation of marginal zone B cells and for anti-pneumococcal host defense, as revealed by a mouse model of genetic LPL deficiency. In independent studies, serine phosphorylation of LPL at residue 5 (S5) has been described as a key "switch" in regulating LPL actin binding and subsequent cell motility, although much of the data are correlative. To test the importance of S5 phosphorylation in LPL function, and to specifically assess the requirement of LPL S5 phosphorylation in anti-pneumococcal host defense, we generated the "S5A" mouse, expressing endogenous LPL bearing a serine-to-alanine mutation at this position. S5A mice were bred to homozygosity, and LPL was expressed at levels equivalent to wild-type, but S5 phosphorylation was absent. S5A mice exhibited specific impairment in clearance of pneumococci following i.v. challenge, with 10-fold-higher bacterial bloodstream burden 24 h after challenge compared with wild-type or fully LPL-deficient animals. Defective bloodstream clearance correlated with diminished population of marginal zone macrophages and with reduced phagocytic capacity of multiple innate immune cells. Development and function of other tested leukocyte lineages, such as T and B cell motility and activation, were normal in S5A mice. The S5A mouse thus provides a novel system in which to elucidate the precise molecular control of critical immune cell functions in specific host-pathogen defense interactions., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Caspase-11 sensing of intracellular lipopolysaccharide (LPS) plays critical roles during infections and sepsis. However, the key cell types that sense intracellular LPS and their contributions to the host responses at the organismal level are not completely clear. Here, we show that macrophage/monocyte-specific caspase-11 plays a dominant role in mediating the pathological manifestations of endotoxemia, including gasdermin D (GSDMD) activation, interleukin (IL)-1β, IL-18, and damage-associated molecular pattern (DAMP) release, tissue damage, and death. Surprisingly, caspase-11 expression in CD11c + cells and intestinal epithelial cells (IECs) plays minor detrimental roles in LPS shock. In contrast, caspase-11 expression in neutrophils is dispensable for LPS-induced lethality. Importantly, caspase-11 sensing of intracellular LPS in LyzM + myeloid cells and MRP8 + neutrophils, but not CD11c + cells and IECs, is necessary for bacterial clearance and host survival during intracellular bacterial infection. Thus, we reveal hierarchical cell-type-specific roles of caspase-11 that govern the host-protective and host-detrimental functions of the cytosolic LPS surveillance., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Transgelin-2 is a 22 kDa actin-binding protein that has been proposed to act as an oncogenic factor, capable of contributing to tumorigenesis in a wide range of human malignancies. However, little is known whether this tiny protein also plays an important role in immunity, thereby keeping body from the cancer development and metastasis. Here, we investigated the functions of transgelin-2 in dendritic cell (DC) immunity. Further, we investigated whether the non-viral transduction of cell-permeable transgelin-2 peptide potentially enhance DC-based cancer immunotherapy., Methods: To understand the functions of transgelin-2 in DCs, we utilized bone marrow-derived DCs (BMDCs) purified from transgelin-2 knockout (Tagln2 -/- ) mice. To observe the dynamic cellular mechanism of transgelin-2, we utilized confocal microscopy and flow cytometry. To monitor DC migration and cognate T-DC interaction in vivo, we used intravital two-photon microscopy. For the solid and metastasis tumor models, OVA + B16F10 melanoma were inoculated into the C57BL/6 mice via intravenously (i.v.) and subcutaneously (s.c.), respectively. OTI TCR T cells were used for the adoptive transfer experiments. Cell-permeable, de-ubiquitinated recombinant transgelin-2 was purified from Escherichia coli and applied for DC-based adoptive immunotherapy., Results: We found that transgelin-2 is remarkably expressed in BMDCs during maturation and lipopolysaccharide activation, suggesting that this protein plays a role in DC-based immunity. Although Tagln2 -/- BMDCs exhibited no changes in maturation, they showed significant defects in their abilities to home to draining lymph nodes (LNs) and prime T cells to produce antigen-specific T cell clones, and these changes were associated with a failure to suppress tumor growth and metastasis of OVA + B16F10 melanoma cells in mice. Tagln2 -/- BMDCs had defects in filopodia-like membrane protrusion and podosome formation due to the attenuation of the signals that modulate actin remodeling in vitro and formed short, unstable contacts with cognate CD4 + T cells in vivo. Strikingly, non-viral transduction of cell-permeable, de-ubiquitinated recombinant transgelin-2 potentiated DC functions to suppress tumor growth and metastasis., Conclusion: This work demonstrates that transgelin-2 is an essential protein for both cancer and immunity. Therefore, transgelin-2 can act as a double-edged sword depending on how we apply this protein to cancer therapy. Engineering and clinical application of this protein may unveil a new era in DC-based cancer immunotherapy. Our findings indicate that cell-permeable transgelin-2 have a potential clinical value as a cancer immunotherapy based on DCs.

Primary membranous nephropathy (pMN) is a leading cause of nephrotic syndrome in adults. In most cases, this autoimmune kidney disease is associated with autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) expressed on kidney podocytes, but the mechanisms leading to glomerular damage remain elusive. Here, we developed a cell culture model using human podocytes and found that anti-PLA2R1-positive pMN patient sera or isolated IgG4, but not IgG4-depleted sera, induced proteolysis of the 2 essential podocyte proteins synaptopodin and NEPH1 in the presence of complement, resulting in perturbations of the podocyte cytoskeleton. Specific blockade of the lectin pathway prevented degradation of synaptopodin and NEPH1. Anti-PLA2R1 IgG4 directly bound mannose-binding lectin in a glycosylation-dependent manner. In a cohort of pMN patients, we identified increased levels of galactose-deficient IgG4, which correlated with anti-PLA2R1 titers and podocyte damage induced by patient sera. Assembly of the terminal C5b-9 complement complex and activation of the complement receptors C3aR1 or C5aR1 were required to induce proteolysis of synaptopodin and NEPH1 by 2 distinct proteolytic pathways mediated by cysteine and aspartic proteinases, respectively. Together, these results demonstrated a mechanism by which aberrantly glycosylated IgG4 activated the lectin pathway and induced podocyte injury in primary membranous nephropathy.

RNA helicases play roles in various essential biological processes such as RNA splicing and editing. Recent in vitro studies show that RNA helicases are involved in immune responses toward viruses, serving as viral RNA sensors or immune signaling adaptors. However, there is still a lack of in vivo data to support the tissue- or cell-specific function of RNA helicases owing to the lethality of mice with complete knockout of RNA helicases; further, there is a lack of evidence about the antibacterial role of helicases. Here, we investigated the in vivo role of Dhx15 in intestinal antibacterial responses by generating mice that were intestinal epithelial cell (IEC)-specific deficient for Dhx15 (Dhx15 f/f Villin1-cre, Dhx15 ΔIEC ). These mice are susceptible to infection with enteric bacteria Citrobacter rodentium ( C. rod ), owing to impaired α-defensin production by Paneth cells. Moreover, mice with Paneth cell-specific depletion of Dhx15 (Dhx15 f/f Defensinα6-cre, Dhx15 ΔPaneth ) are more susceptible to DSS (dextran sodium sulfate)-induced colitis, which phenocopy Dhx15 ΔIEC mice, due to the dysbiosis of the intestinal microbiota. In humans, reduced protein levels of Dhx15 are found in ulcerative colitis (UC) patients. Taken together, our findings identify a key regulator of Wnt-induced α-defensins in Paneth cells and offer insights into its role in the antimicrobial response as well as intestinal inflammation., Competing Interests: Competing interest statement: R.F. is a cofounder of Ventus, which studies inflammatory pathways.

Background: Previous studies have identified LCP1 as a diagnostic and prognostic marker in several cancers. However, the role of LCP1 in gastric cancer (GC) and its effect on tumor immune infiltration remain unclear., Objective: The aim was to explore the role of LCP1 in GC and its effect on tumor immune infiltration., Methods: We explored the expression of LCP1 relative to clinicopathology in GC patients by bioinformatics analysis and immunohistochemistry. Using cBioportal database, we analyzed the characteristic genetic variations of LCP1 in GC. In addition, we evaluated the correlation between LCP1 expression and tumor-infiltrating lymphocytes (TILs) using R software, TIMER and TISIDB databases. Finally, we analyzed the biological functions in which LCP1 may participate and the signaling pathways it may regulate., Results: Here, we showed that LCP1 expression is significantly correlated with tumor aggressiveness and poor prognosis in GC patients. Additionally, the results indicated that LCP1 was associated with TILs, including both immunosuppressive and immunosupportive cells, and was strongly correlated with various immune marker sets in GC. GSEA analysis demonstrated that LCP1 expression played an important role in lymphocyte formation and immune reaction., Conclusions: LCP1 may be a potential prognostic biomarker for GC patients and a marker for tumor immunotherapy.

Moesin is a member of the ezrin, radixin and moesin (ERM) proteins that are involved in the formation and/or maintenance of cortical actin organization through their cross-linking activity between actin filaments and proteins located on the plasma membranes as well as through regulation of small GTPase activities. Microglia, immune cells in the central nervous system, show dynamic reorganization of the actin cytoskeleton in their process elongation and retraction as well as phagocytosis and migration. In microglia, moesin is the predominant ERM protein. Here, we show that microglial activation after systemic lipopolysaccharide application is partly inhibited in moesin knockout (Msn-KO) mice. We prepared primary microglia from wild-type and Msn-KO mice, and studied them to compare their phenotypes accompanying morphological changes and reorganization of the actin cytoskeleton induced by UDP-stimulated phagocytosis and ADP-stimulated migration. The Msn-KO microglia showed higher phagocytotic activity in the absence of UDP, which was not further increased by the treatment with UDP. They also exhibited decreased ADP-stimulated migration activities compared with the wild-type microglia. However, the Msn-KO microglia retained their ability to secrete tumor necrosis factor α and nitric oxide in response to lipopolysaccharide.

There is increasing evidence from genetic, biochemical, pharmacological, neuroimaging and post-mortem studies that immunological dysregulation plays a crucial role in the pathogenesis of psychoses. The involvement of microglia in schizophrenia and bipolar disorder (BD) has remained controversial, however, since results from various post-mortem studies are still inconclusive. Here, we analyzed the estimated density of microglia of age-matched individuals with schizophrenia (n = 17), BD (n = 13), and non-psychiatric control subjects (n = 17) in the anterior midcingulate cortex (aMCC), a brain area putatively involved in the pathogenesis of psychoses, using ionized calcium binding adaptor molecule 1 (Iba1)-immunohistochemistry. The microglial cells displayed a homogenously distributed Iba1-staining pattern in the aMCC with slightly varying activation states in all three groups. The estimated microglial densities did not differ significantly between individuals with schizophrenia, BD and control subjects. Remarkably, when both hemispheres were investigated separately within the three groups, the density was significantly lateralized towards the right aMCC in schizophrenia (p = 0.01) and-even more evident-in BD subjects (p = 0.008). This left-right lateralization was not observed in the control group (p = 0.52). Of note, microglial density was significantly lower in BD individuals who did not commit suicide compared with BD individuals who died from suicide (p = 0.002). This difference was not observed between individuals with BD who committed suicide and controls. The results, tentatively interpreted, suggest a hitherto unknown increased lateralization of microglial density to the right hemisphere in both psychiatric groups. If confirmed in independent samples, lateralization should be considered in all post-mortem studies on microglia. Density differences between suicide and non-suicide individuals needs further elucidation.

Multiple myeloma is a hematologic cancer that disrupts normal bone marrow function and has multiple lines of therapeutic options, but is incurable as patients ultimately relapse. We developed a novel antibody-drug conjugate (ADC) targeting CS-1, a protein that is highly expressed on multiple myeloma tumor cells. The anti-CS-1 mAb specifically bound to cells expressing CS-1 and, when conjugated to a cytotoxic pyrrolobenzodiazepine payload, reduced the viability of multiple myeloma cell lines in vitro In mouse models of multiple myeloma, a single administration of the CS-1 ADC caused durable regressions in disseminated models and complete regression in a subcutaneous model. In an exploratory study in cynomolgus monkeys, the CS-1 ADC demonstrated a half-life of 3 to 6 days; however, no highest nonseverely toxic dose was achieved, as bone marrow toxicity was dose limiting. Bone marrow from dosed monkeys showed reductions in progenitor cells as compared with normal marrow. In vitro cell killing assays demonstrated that the CS-1 ADC substantially reduced the number of progenitor cells in healthy bone marrow, leading us to identify previously unreported CS-1 expression on a small population of progenitor cells in the myeloid-erythroid lineage. This finding suggests that bone marrow toxicity is the result of both on-target and off-target killing by the ADC., (©2020 American Association for Cancer Research.)

Selective suppression of graft rejection while maintaining anti-pathogen responses has been elusive. Thus far, the most successful strategies to induce suppression of graft rejection relies on inhibition of T-cell activation. However, the very same mechanisms that induce allograft-specific T-cell suppression are also important for immunity against microbial pathogens as well as oncogenically transformed cells, resulting in significant immunosuppression-associated comorbidities. Therefore, defining the pathways that differentially regulate anti-graft versus antimicrobial T-cell responses may allow the development of regimen to induce allograft-specific tolerance. Recent work has defined a molecular pathway driven by the immunoregulatory protein coronin 1 that regulates the phosphodiesterase/cyclic adenosine monophosphate pathway and modulates T cell responses. Interestingly, disruption of coronin 1 promotes allograft tolerance while immunity towards a range of pathogenic microbes is maintained. Here, we briefly review the work leading up to these findings as well as their possible implications for transplantation medicine.

Allograft inflammatory factor-1 (AIF1) is a calcium-responsive cytoplasmic scaffold protein that directs hematopoiesis and immune responses within dendritic cells (DC) and macrophages. Although the role of AIF1 in transplant rejection and rheumatoid arthritis has been explored, little is known about its role in type 1 diabetes. Here, we show that in vivo silencing of AIF1 in NOD mice restrained infiltration of immune cells into the pancreas and inhibited diabetes incidence. Analyses of FACS-sorted CD45neg nonleukocyte populations from resected pancreatic islets showed markedly higher expression of insulin in the AIF1-silenced groups. Evaluation of CD45+ leukocytes revealed diminished infiltration of effector T cells and DC in the absence of AIF1. Transcriptional profiling further revealed a marked decrease in cDC1 DC-associated genes CD103, BATF3, and IRF8, which are required for orchestrating polarized type 1 immunity. Reduced T cell numbers within the islets were observed, with concomitant lower levels of IFN-γ and T-bet in AIF1-silenced cohorts. In turn, there was a reciprocal increase in functionally suppressive pancreas-resident CD25+Foxp3+CD4+ Tregs. Taken together, results show that AIF1 expression in myeloid cells plays a pivotal role in promoting type 1 diabetes and that its suppression restrains insulitis by shifting the immune microenvironment toward tolerance.

Feline mammary tumors are usually malignant and aggressive carcinomas. Most cases are simple monophasic carcinomas (1 epithelial population), and additional phenotyping is usually not needed. In this study, we describe 10 malignant mammary tumors from 9 female cats that had unusual histomorphology: they appeared biphasic, with 2 distinct cell populations. Initially, they were morphologically diagnosed as either carcinosarcoma (1/10) or malignant pleomorphic tumor (9/10) of the mammary gland, as the latter did not match any previously described histological subtype. Immunohistochemistry (IHC) was performed for pancytokeratin, cytokeratins 8 and 18, cytokeratin 14, cytokeratins 5 and 6, vimentin, p63, calponin, alpha-smooth muscle actin, Ki-67, ERBB2, estrogen receptor alpha, and progesterone receptor. In 7 of 10 cases, the biphasic nature was confirmed and, on the basis of the IHC results, they were classified as carcinoma and malignant myoepithelioma (4/10), ductal carcinoma (1/10), and carcinosarcoma (2/10). The other 3 of 10 cases were monophasic based on IHC. In the cases of carcinoma and malignant myoepithelioma, the malignant myoepithelial cells were 100% positive for vimentin (4/4) and variably positive for p63, calponin, and cytokeratins (4/4). These findings show that, although rare, biphasic mammary carcinomas do occur in cats. In dogs and humans, tumors composed of malignant epithelial and myoepithelial cells have a less aggressive behavior than certain simple carcinomas, and therefore, their identification might also be clinically significant in the cat.

Why ocular mucosa is paucibacterial is unknown. Many different mechanisms have been suggested but the comprehensive experimental studies are sparse. We found that a deficiency in L-plastin (LCP1), an actin bundling protein, resulted in an ocular commensal overgrowth, characterized with increased presence of conjunctival Streptococcal spp. The commensal overgrowth correlated with susceptibility to P. aeruginosa -induced keratitis. L-plastin knock-out (KO) mice displayed elevated bacterial burden in the P. aeruginosa -infected corneas, altered inflammatory responses, and compromised bactericidal activity. Mice with ablation of LPL under the LysM Cre ( LysM. Cre pos LPL fl / fl ) and S100A8 Cre ( S100A8.Cre pos LPL fl / fl ) promoters had a similar phenotype to the LPL KOs mice. In contrast, infected CD11c.Cre pos LPL fl / fl mice did not display elevated susceptibility to infection, implicating the myeloid L-plastin-sufficient cells (e.g., macrophages and neutrophils) in maintaining ocular homeostasis. Mechanistically, the elevated commensal burden and the susceptibility to infection were linked to defects in neutrophil frequencies at steady state and during infection and compromised bactericidal activities upon priming. Macrophage exposure to commensal organisms primed neutrophil responses to P. aeruginosa , augmenting PMN bactericidal capacity in an L-plastin dependent manner. Cumulatively, our data highlight the importance of neutrophils in controlling ocular paucibacteriality, reveal molecular and cellular events involved in the process, and suggest a link between commensal exposure and resistance to infection., (Copyright © 2020 Lu, Kugadas, Smith-Page, Lamb, Lin, Ru, Morley, Fichorova, Mittal, Chauhan, Littleton, Saban and Gadjeva.)

Objective: To explore the underlying mechanism of action of Tongxieyaofang decoction in rats with visceral hypersensitivity using proteomics technology., Methods: Twenty-four female Sprague-Dawley rats were randomly divided into three groups: control group, irritable bowel syndrome (IBS) group and Tongxieyaofang treatment group. An IBS model, characterized as visceral hypersensitivity, was established using the odour of mothballs as conditional stimulation and colorectal distension combined with classic physical restraint as non-conditional stimulation. Rats were intragastrically treated with Tongxieyaofang (2 or 4 mL·kg－1·d－1) for 4 weeks. On the 45th day, the rats were dissected and the colonic mucosal proteins were extracted. Differential protein spots were screened by fluorescent two-dimensional differential gel electrophoresis (2D-DIGE), and identified by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS). Western blotting experiments were performed to verify the changes observed in 2D-DIGE and MALDI-TOF-MS., Results: It was found that the visceral sensitivity of rats in the Tongxieyaofang treatment group (4 mL/kg) was lower than that in the IBS group (P < 0.01). Sixty-one protein spots were differentially expressed between the IBS group and the Tongxieyaofang treatment group. Of these, 23 spots were upregulated in the Tongxieyaofang treatment group, while 38 spots were downregulated. Three specific proteins were successfully identified from the five protein spots with the most obvious changes. The two upregulated proteins were transgelin (TAGLN) and acetaldehyde dehydrogenase 2 (Aldh2) and the downregulated protein was cytokeratin 8 (CK8)., Conclusion: Tongxieyaofang can dose-dependently ameliorate visceral hypersensitivity in rats and the mechanism of action may involve the upregulation of TAGLN and Aldh2 and the downregulation of CK8.

A growing body of evidence has indicated that the release of nociceptive factors, such as interleukins and chemokines, by activated immune and glial cells has crucial significance for neuropathic pain generation and maintenance. Moreover, changes in the production of nociceptive immune factors are associated with low opioid efficacy in the treatment of neuropathy. Recently, it has been suggested that CC chemokine receptor type 1 (CCR1) signaling is important for nociception. Our study provides evidence that the development of hypersensitivity in rats following chronic constriction injury (CCI) of the sciatic nerve is associated with significant up-regulation of endogenous CCR1 ligands, namely, CCL2, CCL3, CCL4, CCL6, CCL7 and CCL9 in the spinal cord and CCL2, CCL6, CCL7 and CCL9 in dorsal root ganglia (DRG). We showed that single and repeated intrathecal administration of J113863 (an antagonist of CCR1) attenuated mechanical and thermal hypersensitivity. Moreover, repeated administration of a CCR1 antagonist enhanced the analgesic properties of morphine and buprenorphine after CCI. Simultaneously, repeated administration of J113863 reduced the protein levels of IBA-1 in the spinal cord and MPO and CD4 in the DRG and, as a consequence, the level of pronociceptive factors, such as interleukin-1β (IL-1β), IL-6 and IL-18. The data obtained provide evidence that CCR1 blockade reduces hypersensitivity and increases opioid-induced analgesia through the modulation of neuroimmune interactions., (© 2020 John Wiley & Sons Ltd.)

We identified the presence of AIF-1 (allograft inflammatory factor-1) in human peripheral blood mononuclear cells (PBMCs) from normal subjects by immunocytological methods. After isolation of different types of mononuclear cells by FACS (Fluorescence-activated cell sorting) with >95% purity, we studied the transcript levels of AIF-1 using qPCR. We observed the following order of AIF-1 mRNA expression in mononuclear cells: T-lymphocytes ˃ Monocytes ˃ B-lymphocytes ˃ NK. After T cell expansion of isolated PBMCs using anti-CD3-CD28 magnetic beads (Dynabeads®), AIF-1 increased intracellularly in the presence of brefeldin A; this finding, along with an increase in the medium in the absence of the drug, suggests that AIF-1 is processed in the Golgi apparatus and may be secreted extracellularly. In another set of experiments, interleukin-12 and anti-interleukin-4 were added to PBMCs during T cell expansion to promote Th1 polarization and to inhibit Th2 differentiation. In this case, the presence of 6 nM of rhAIF-1 (recombinant human AIF-1) increased the mRNA expression of interferon-ϒ and interleukin-2. In the same set of experiments, the incubation of PBMCs with rhAIF-1 (6 nM) promoted the decrease of mRNA expression of IL-10 and TGF-β, along with the decrease of CD25 and Foxp3 proteins. Furthermore, extracellular rhAIF-1 (6 nM) increased the survival of naive and effector T cells during Th1 polarization by inhibition of apoptosis, without causing changes in cell cycle rate and in retinoblastoma-cyclin-dependent kinase (Rb-CDK) activation. Taken together, rhAIF-1 treatment of PBMCs potentiates Th1 response and inhibits functionally suppressive CD25 + Foxp3 + Treg, which suggests an important immunomodulatory role in governing T cell response., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Protein 4.1R, a member of the 4.1 family, functions as a bridge between cytoskeletal and plasma membrane proteins. It is expressed in T cells, where it binds to a linker for activation of T cell (LAT) family member 1 and inhibits its phosphorylation and downstream signaling events after T cell receptor triggering. The role of the 4.1R protein in cell activation through other immunoreceptors is not known. In this study, we used 4.1R-deficient (4.1R-KO) and 4.1R wild-type (WT) mice and explored the role of the 4.1R protein in the high-affinity IgE receptor (FcεRI) signaling in mast cells. We found that bone marrow mast cells (BMMCs) derived from 4.1R-KO mice showed normal growth in vitro and expressed FcεRI and c-KIT at levels comparable to WT cells. However, 4.1R-KO cells exhibited reduced antigen-induced degranulation, calcium response, and secretion of tumor necrosis factor-α. Chemotaxis toward antigen and stem cell factor (SCF) and spreading on fibronectin were also reduced in 4.1R-KO BMMCs, whereas prostaglandin E 2 -mediated chemotaxis was not affected. Antibody-induced aggregation of tetraspanin CD9 inhibited chemotaxis toward antigen in WT but not 4.1R-KO BMMCs, implying a CD9-4.1R protein cross-talk. Further studies documented that in the absence of 4.1R, antigen-mediated phosphorylation of FcεRI β and γ subunits was not affected, but phosphorylation of SYK and subsequent signaling events such as phosphorylation of LAT1, phospholipase Cγ1, phosphatases (SHP1 and SHIP), MAP family kinases (p38, ERK, JNK), STAT5, CBL, and mTOR were reduced. Immunoprecipitation studies showed the presence of both LAT1 and LAT2 (LAT, family member 2) in 4.1R immunocomplexes. The positive regulatory role of 4.1R protein in FcεRI-triggered activation was supported by in vivo experiments in which 4.1R-KO mice showed the normal presence of mast cells in the ears and peritoneum, but exhibited impaired passive cutaneous anaphylaxis. The combined data indicate that the 4.1R protein functions as a positive regulator in the early activation events after FcεRI triggering in mast cells., (Copyright © 2020 Draberova, Draberova, Potuckova, Halova, Bambouskova, Mohandas and Draber.)

The dynamic organization of the actin cytoskeleton into bundles and networks is orchestrated by a large variety of actin-binding proteins. Among them, the actin-bundling protein L-plastin is normally expressed in hematopoietic cells, where it is involved in the immune response. However, L-plastin is also often ectopically expressed in malignant cancer cells of non-hematopoietic origin and is even considered as a marker for cancer progression. Post-translational modification modulates L-plastin activity. In particular, L-plastin Ser5 phosphorylation has been shown to be important for the immune response in leukocytes as well as for invasion and metastasis formation of carcinoma cells. This chapter discusses the physiological and pathological role of L-plastin with a special focus on the importance of L-plastin Ser5 phosphorylation for the protein functions. The potential use of Ser5 phosphorylated L-plastin as a biomarker and/or therapeutic target will be evoked., (© 2020 Elsevier Inc. All rights reserved.)

Understanding tissue structure and function requires tools that quantify the expression of multiple proteins while preserving spatial information. Here, we describe MIBI-TOF (multiplexed ion beam imaging by time of flight), an instrument that uses bright ion sources and orthogonal time-of-flight mass spectrometry to image metal-tagged antibodies at subcellular resolution in clinical tissue sections. We demonstrate quantitative, full periodic table coverage across a five-log dynamic range, imaging 36 labeled antibodies simultaneously with histochemical stains and endogenous elements. We image fields of view up to 800 μm × 800 μm at resolutions down to 260 nm with sensitivities approaching single-molecule detection. We leverage these properties to interrogate intrapatient heterogeneity in tumor organization in triple-negative breast cancer, revealing regional variability in tumor cell phenotypes in contrast to a structured immune response. Given its versatility and sample back-compatibility, MIBI-TOF is positioned to leverage existing annotated, archival tissue cohorts to explore emerging questions in cancer, immunology, and neurobiology., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Previous research has shown that CXCR5 -/- mice develop retinal degeneration (RD) with age, a characteristic related to age macular degeneration (AMD). RD in these mice is not well-understood, and in this study, we sought to characterize further the RD phenotype and to gain mechanistic insights into the function of CXCR5 in the retina. CXCR5 -/- and WT control mice were used. Fundus images demonstrated a significant ( p < 0.001) increase of hypo-pigmented spots in the retina of aged CXCR5 -/- mice compared with WT control mice. PAS staining indicated localization of deposits in the sub-retinal pigment epithelia (RPE) layer. AMD-associated proteins Cryab, amyloid beta, and C3d were detected within the RPE/sub-RPE tissues by immunofluorescence (IF). In addition, western blot analysis of COX-2, Arg1, and VEGF-a revealed an increase in the signaling of these molecules within the RPE/choroid complex. Transmission electron microscopy (TEM) indicated a drusen-like structure of sub-RPE deposits with an accumulation of vacuolated cellular debris. Loss of photoreceptors was detected by peanut lectin staining and was corroborated by a reduction in MAP2 signaling. Loss of blood-retinal barrier integrity was demonstrated by a reduction of ZO-1 expression. Inflammatory cells were detected in the sub-RPE space, with an increase in IBA-1 positive microglia cells on the surface of the RPE. Mass spectrometry analysis of CXCR5 -/- mouse RPE/choroid proteins extracts, separated by SDS-page and incubated with autologous serum, identified autoantibodies against AMD-associated proteins: Cryaa, Cryab, and Anxa2. In vitro evaluations in BV-2 cell culture indicated a significant increase in production of Arg-1 ( p < 0.001) and COX-2 ( p < 0.01) in the presence of anti-CXCR5 antibody when compared with Igg-treated control BV-2 cells stimulated with IL-4 and TNFα/IFNγ, respectively. Anti-CXCR5 antibody treatment without stimulating agents did not affect Arg-1 and COX-2 expression; this suggests that CXCR5 may have a regulatory role in microglia cells activation. These results indicate that with age, CXCR5 -/- mice develop RD characterized by microglia dysfunction, increased production of CXCL13 in the RPE progressive photoreceptor, neuronal loss, and sub-RPE deposition of cellular debris, resulting in the production of immunogenic proteins and autoimmune-mediated RD.

Protein 4.1R, an 80 000 MW membrane skeleton protein, is a vital component of the red blood cell membrane cytoskeleton that stabilizes the spectrin-actin network and regulates membrane properties of deformability and mechanical stability. It has been shown that 4.1R is expressed in T cells, including CD8 + T cells, but its role in CD8 + T cells remains unclear. Here, we have explored the role of 4.1R in CD8 + T cells using 4.1R -/- mice. Our results showed that cell activation, proliferation and secretion levels of interleukin-2 and interferon-γ were significantly increased in 4.1R -/- CD8 + T cells. Furthermore, the phosphorylation levels of linker for activation of T cells (LAT) and its downstream signaling molecule extracellular signal-regulated kinase were enhanced in the absence of 4.1R. In vitro co-immunoprecipitation experiments showed a direct interaction between 4.1R and LAT. Moreover, 4.1R -/- CD8 + T cells and mice exhibited an enhanced T-cell-dependent immune response. These data enabled the identification of a negative regulation function for 4.1R in CD8 + T cells by a direct association between 4.1R and LAT, possibly through inhibiting phosphorylation of LAT and then modulating intracellular signal transduction., (© 2019 John Wiley & Sons Ltd.)

Despite extensive investigation focused on both the molecular characteristics and the expression level of Toll-like receptors (TLRs) during the inflammatory response in vertebrates, few data are available in the literature on the role of these proteins in invertebrate's immune response. Here, we propose the medicinal leech as a valuable model to better elucidate the role of TLR4 and its related products, such as tumor necrosis factor (TNF-α), after activation of the leech peripheral immune system with the endogenous medicinal leech recombinant allograft inflammatory factor-1 (rHmAIF-1) or with an exogenous stimulus, such as lipopolysaccharide (LPS). Our results indicate that activated macrophages (HmAIF-1 + ) and granulocytes (CD11b + ) express both TLR4 and its coreceptor CD14. Moreover, functional studies performed by injecting a cyanobacterium selective TLR4 antagonist CyP demonstrated that only the TLR4 pathway was blocked, while the immune response caused by lipoteichoic acid (LTA) treatment is not affected. These results are consistent with literature on vertebrates, indicating that TLR4 functions as a LPS receptor while the recognition of LTA may involve other pathways.

Neutrophils are a major component of immune defense and are recruited through neutrophil chemotaxis in response to invading pathogens. However, the molecular mechanism that controls neutrophil chemotaxis remains unclear. Here, we report that PTENα, the first isoform identified in the PTEN family, regulates neutrophil deformability and promotes chemotaxis of neutrophils. A high level of PTENα is detected in neutrophils and lymphoreticular tissues. Homozygous deletion of PTENα impairs chemoattractant-induced migration of neutrophils. We show that PTENα physically interacts with cell membrane cross-linker moesin through its FERM domain and dephosphorylates moesin at Thr558, which disrupts the association of filamentous actin with the plasma membrane and subsequently induces morphologic changes in neutrophil pseudopodia. These results demonstrate that PTENα acts as a phosphatase of moesin and modulates neutrophil-mediated host immune defense. We propose that PTENα signaling is a potential target for the treatment of infections and immune diseases., (© 2019 by The American Society of Hematology.)

Gastric adenocarcinoma is usually diagnosed in late stages, necessitating the use of different therapeutic modalities. Currently, antibody-based therapies have also been approved through with limited clinical efficacy. Reinforcing antibody-based immunotherapy by using chimeric antigen receptor (CAR) T cells may enhance the approach. However, the cells can cause severe on-target and off-tumor toxicities owing to their higher sensitivity to low-level antigen expressions. To address the need for safe and reliable targets, we made a bioinformatics pipeline by which we screened overexpressed genes in the disease for off-tumor sites in many normal tissues. Our inspection showed that MSLN (Mesothelin), ANTXR1 (TEM8), and MUC3A are the probable targets of CAR T cell therapy in gastric adenocarcinoma. The proposed antigenic targets might respond to the need to simultaneously target multiple antigens in a tumor matrix to prevent resistance., (© 2018 Wiley Periodicals, Inc.)

Inflammation is a critical feature of aging and its related diseases, including cardiovascular diseases. Recent epidemiological studies demonstrated that abdominal aortic aneurysm (AAA), an aging-related vascular pathological condition, is associated with cognitive decline. However, the underlying mechanism, especially the role of vascular inflammation, is largely unknown because of lack of an available animal model. In this study, we examined whether vascular inflammation affects synaptic and cognitive dysfunction, using an AAA mouse model. In young (3 months) and middle-aged (12 months) C57BL/6J mice, AAA was induced by angiotensin II infusion with calcium chloride application. After 4 weeks of induction, aortic diameter was significantly increased and excessive Mac3-positive inflammatory cells infiltrated the destroyed aorta in middle-aged mice. AAA-induced middle-aged mice further exhibited cognitive impairment. Neuronal loss was observed in the CA3 region of the hippocampus. IBA1/MHCII-double-positive microglia activation was also seen in the hippocampus, suggesting that vascular inflammation drives neuroinflammation and subsequent cognitive dysfunction. Furthermore, we found that senescence-accelerated mice prone 8 exhibited robust AAA formation and a marked decrease of cognitive and synaptic function in the hippocampus mediated by inflammation. In conclusion, this novel murine model convincingly suggested the occurrence of vascular inflammation-derived cognitive dysfunction.

Gelsolin (GSN), one of the most abundant actin-binding proteins, is involved in cell motility, shape and metabolism. As a member of the GSN superfamily, GSN is a highly structured protein in eukaryotic cells that can be regulated by calcium concentration, intracellular pH, temperature and phosphatidylinositol-4,5-bisphosphate. GSN plays an important role in cellular mechanisms as well as in different cellular interactions. Because of its participation in immunologic processes and its interaction with different cells of the immune system, GSN is a potential candidate for various therapeutic applications. In this review, we summarise the structure of GSN as well as its regulating and functional roles, focusing on distinct diseases such as Alzheimer's disease, rheumatoid arthritis and cancer. A short overview of GSN as a therapeutic target in today's medicine is also provided.

Defining pathways that differentially regulate graft-reactive versus anti-microbial T cell responses could facilitate more precise manipulation of immune responses in the context of organ and tissue transplantation. Here, Jayachandran et al. (2019) identify a coronin-1-PDE4-cAMP axis that, when perturbed, results in the induction of transplantation tolerance while maintaining anti-microbial immunity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

To address the role of chronic antigenic stimulation in primary central nervous system lymphoma (PCNSL), we searched for autoantigens and identified sterile α-motif domain containing protein 14 (SAMD14) and neural tissue-specific F-actin binding protein I (neurabin-I) as autoantigenic targets of the B-cell receptors (BCRs) from 8/12 PCNSLs. In the respective cases, SAMD14 and neurabin-I were atypically hyper- N -glycosylated (SAMD14 at ASN339 and neurabin-I at ASN1277), explaining their autoimmunogenicity. SAMD14 and neurabin-I induced BCR pathway activation and proliferation of aggressive lymphoma cell lines transfected with SAMD14- and neurabin-I-reactive BCRs. Moreover, the BCR binding epitope of neurabin-I conjugated to truncated Pseudomonas exotoxin-killed lymphoma cells expressing the respective BCRs. These results support the role of chronic antigenic stimulation by posttranslationally modified central nervous system (CNS) driver autoantigens in the pathogenesis of PCNSL, serve as an explanation for their CNS tropism, and provide the basis for a novel specific treatment approach., (© 2018 by The American Society of Hematology.)

NK cells eliminate viral-infected and malignant cells through a highly orchestrated series of cytoskeletal rearrangements, resulting in the release of cytolytic granule contents toward the target cell. Central to this process is the convergence of cytolytic granules to a common point, the microtubule-organizing center (MTOC), before delivery to the synapse. In this study, we show that vasodialator-stimulated phosphoprotein (VASP), an actin regulatory protein, localizes to the cytolytic synapse, but surprisingly, shows no impact on conjugate formation or synaptic actin accumulation despite being required for human NK cell-mediated killing. Interestingly, we also find that a pool of VASP copurifies with lytic granules and localizes with lytic granules at the MTOC. Significantly, depletion of VASP decreased lytic granule convergence without impacting MTOC polarization. Using the KHYG-1 cell line in which lytic granules are in a constitutively converged state, we find that either VASP depletion or F-actin destabilization promoted spreading of formerly converged granules. Our results demonstrate a novel requirement for VASP and actin polymerization in maintaining lytic granule convergence during NK cell-mediated killing., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Transgelin-2 is a small 22-kDa actin-binding protein implicated in actin dynamics, which stabilizes actin structures and participates in actin-associated signaling pathways. Much curiosity regarding transgelin-2 has centered around its dysregulation in tumor development and associated diseases. However, recent studies have shed new light on the functions of transgelin-2, the only transgelin family member present in leukocytes, in the context of various immune responses. In this review, we outlined the biochemical properties of transgelin-2 and its physiological functions in T cells, B cells, and macrophages. Transgelin-2 regulates T cell activation by stabilizing the actin cytoskeleton at the immunological synapse. Transgelin-2 in B cells also participates in the stabilization of T cell-B cell conjugates. While transgelin-2 is expressed at trace levels in macrophages, its expression is highly upregulated upon lipopolysaccharide stimulation and plays an essential role in macrophage phagocytosis. Since transgelin-2 increases T cell adhesion to target cells via boosting the "inside-out" costimulatory activation of leukocyte function-associated antigen 1, transgelin-2 could be a suitable candidate to potentiate the antitumor response of cytotoxic T cells by compensating for the lack of costimulation in tumor microenvironment. We discussed the feasibility of using native or engineered transgelin-2 as a synergistic molecule in cell-based immunotherapies, without inducing off-target disturbance in actin dynamics in other cells., (©2018 The Authors. Society for Leukocyte Biology Published by Wiley Periodicals, Inc.)

Background: The actin-interacting protein WD repeat-containing protein 1 (WDR1) promotes cofilin-dependent actin filament turnover. Biallelic WDR1 mutations have been identified recently in an immunodeficiency/autoinflammatory syndrome with aberrant morphology and function of myeloid cells., Objective: Given the pleiotropic expression of WDR1, here we investigated to what extent it might control the lymphoid arm of the immune system in human subjects., Methods: Histologic and detailed immunologic analyses were performed to elucidate the role of WDR1 in the development and function of B and T lymphocytes., Results: Here we identified novel homozygous and compound heterozygous WDR1 missense mutations in 6 patients belonging to 3 kindreds who presented with respiratory tract infections, skin ulceration, and stomatitis. In addition to defective adhesion and motility of neutrophils and monocytes, WDR1 deficiency was associated with aberrant T-cell activation and B-cell development. T lymphocytes appeared to develop normally in the patients, except for the follicular helper T-cell subset. However, peripheral T cells from the patients accumulated atypical actin structures at the immunologic synapse and displayed reduced calcium flux and mildly impaired proliferation on T-cell receptor stimulation. WDR1 deficiency was associated with even more severe abnormalities of the B-cell compartment, including peripheral B-cell lymphopenia, paucity of B-cell progenitors in the bone marrow, lack of switched memory B cells, reduced clonal diversity, abnormal B-cell spreading, and increased apoptosis on B-cell receptor/Toll-like receptor stimulation., Conclusion: Our study identifies a novel role for WDR1 in adaptive immunity, highlighting WDR1 as a central regulator of actin turnover during formation of the B-cell and T-cell immunologic synapses., (Copyright © 2018 Ludwig Boltzmann Society - Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases. Published by Elsevier Inc. All rights reserved.)

Supervillin isoforms have been implicated in cell proliferation, actin filament-based motile processes, vesicle trafficking, and signal transduction. However, an understanding of the roles of these proteins in cancer metastasis and physiological processes has been limited by the difficulty of obtaining specific antibodies against these highly conserved membrane-associated proteins. To facilitate research into the biological functions of supervillin, monoclonal antibodies were generated against the bacterially expressed human supervillin N-terminus. Two chimeric monoclonal antibodies with rabbit Fc domains (clones 1E2/CPTC-SVIL-1; 4A8/CPTC-SVIL-2) and two mouse monoclonal antibodies (clones 5A8/CPTC-SVIL-3; 5G3/CPTC-SVIL-4) were characterized with respect to their binding sites, affinities, and for efficacy in immunoblotting, immunoprecipitation, immunofluorescence microscopy and immunohistochemical staining. Two antibodies (1E2, 5G3) recognize a sequence found only in primate supervillins, whereas the other two antibodies (4A8, 5A8) are specific for a more broadly conserved conformational epitope(s). All antibodies function in immunoblotting, immunoprecipitation and in immunofluorescence microscopy under the fixation conditions identified here. We also show that the 5A8 antibody works on immunohistological sections. These antibodies should provide useful tools for the study of mammalian supervillins., Competing Interests: We have the following interests: Research in the Antibody Characterization Program was overseen by Leidos Biomedical Research, Inc. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Objective: Autoantibodies are clinically useful for phenotyping patients across the spectrum of autoimmune rheumatic diseases. Using serum from a patient with Sjögren's syndrome (SS), we detected a new specificity by immunoblotting. This study was undertaken to identify this autoantibody and to evaluate its disease specificity., Methods: A prominent 40-kd band was detected when immunoblotting was performed using SS patient serum and lysate from rat dorsal root ganglia (DRGs). Using 2-dimensional gel electrophoresis and liquid chromatography tandem mass spectrometry peptide sequencing, the autoantigen was identified as calponin 3. Anti-calponin 3 antibodies were evaluated in sera from patients with primary SS (n = 209), patients with systemic lupus erythematosus (SLE; n = 138), patients with myositis (n = 138), patients with multiple sclerosis (MS; n = 44), and healthy controls (n = 46) by enzyme-linked immunosorbent assay. Expression of calponin 3 was assessed by immunohistochemistry., Results: Calponin 3 was identified as a new autoantigen. Anti-calponin 3 antibodies were detected in 23 (11.0%) of the 209 SS patients, 12 (8.7%) of the 138 SLE patients, 7 (5.1%) of the 138 myositis patients, 3 (6.8%) of the 44 MS patients, and 1 (2.2%) of the 46 healthy controls. Among SS patients, the frequency of anti-calponin 3 antibodies was highest in those with neuropathies (7 [17.9%] of 39). In this subset, the frequency of anti-calponin 3 antibodies differed significantly from that in the control group (P = 0.02). Calponin 3 was expressed primarily in rat DRG perineuronal satellite cells but not neurons., Conclusion: Calponin 3 is a novel autoantigen. Antibodies against this protein are found in SS and associate with the subset of patients experiencing neuropathies. Intriguingly, we found that calponin 3 is expressed in DRG perineuronal satellite cells, suggesting that these may be a target in SS., (© 2018, American College of Rheumatology.)

Multiple sclerosis (MS) is a chronic inflammatory disorder of the CNS characterized by demyelination and axonal injury. Current therapies that mainly target lymphocytes do not fully meet clinical need due to the risk of severe side effects and lack of efficacy against progressive MS. Evidence suggests that MS is associated with CNS inflammation, although the underlying molecular mechanism is poorly understood. Transient receptor potential melastatin 2 (TRPM2), a Ca 2+ -permeable nonselective cation channel, is expressed at high levels in the brain and by immune cells, including monocyte lineage cells. Here, we show that TRPM2 plays a pathological role in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Knockout (KO) or pharmacological inhibition of TRPM2 inhibited progression of EAE and TRPM2-KO mice showed lower activation of Iba1-immunopositive monocyte lineage cells and neutrophil infiltration of the CNS than WT mice. Moreover, CXCL2 production in TRPM2-KO mice was significantly reduced at day 14, although the severity of EAE was the same as that in WT mice at that time point. In addition, we used BM chimeric mice to show that TRPM2 expressed by CNS-infiltrating macrophages contributes to progression of EAE. Because CXCL2 induces migration of neutrophils, these results indicate that reduced expression of CXCL2 in the CNS suppresses neutrophil infiltration and slows progression of EAE in TRPM2-KO mice. Together, the results suggest that TRPM2 plays an important role in progression of EAE pathology and shed light on its putative role as a therapeutic target for MS. SIGNIFICANCE STATEMENT Current therapies for multiple sclerosis (MS), which mainly target lymphocytes, carry the risk of severe side effects and lack efficacy against the progressive form of the disease. Here, we found that the transient receptor potential melastatin 2 (TRPM2) channel, which is abundantly expressed in CNS-infiltrating macrophages, plays a crucial role in development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE progression was suppressed by Knockout (KO) or pharmacological inhibition of TRPM2; this was attributed to a reduction in CXCL2 chemokine production by CNS-infiltrating macrophages in TRPM2-KO mice, resulting in suppression of neutrophil infiltration into the CNS. These results reveal an important role of TRPM2 in the pathogenesis of EAE and shed light on its potential as a therapeutic target., (Copyright © 2018 the authors 0270-6474/18/388484-12$15.00/0.)

Animal models of maternal immune activation study the effects of infection, an environmental risk factor for schizophrenia, on brain development. Microglia activation and cytokine upregulation may have key roles in schizophrenia neuropathology. We hypothesised that maternal immune activation induces changes in microglia and cytokines in the brains of the adult offspring. Maternal immune activation was induced by injecting polyriboinosinic:polyribocytidylic acid into pregnant rats on gestational day (GD) 10 or GD19, with brain tissue collected from the offspring at adulthood. We observed no change in Iba1, Gfap, IL1-β and TNF-α mRNA levels in the cingulate cortex (CC) in adult offspring exposed to maternal immune activation. Prenatal exposure to immune activation had a significant main effect on microglial IBA1-positive immunoreactive material (IBA1+IRM) in the corpus callosum; post-hoc analyses identified a significant increase in GD19 offspring, but not GD10. No change in was observed in the CC. In contrast, maternal immune activation had a significant main effect on GFAP+IRM in the CC at GD19 (not GD10); post-hoc analyses only identified a strong trend towards increased GFAP+IRM in the GD19 offspring, with no white matter changes. This suggests late gestation maternal immune activation causes subtle alterations to microglia and astrocytes in the adult offspring., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

A 28-year-old man noticed sensory disturbance in the distal parts of his four extremities and muscle weakness of his hands two weeks after cytomegalovirus (CMV) infection. He had splenomegaly, impairment of hepatic function and peripheral neuropathy with decreased tendon reflexes. Protein-cell dissociation was observed in the cerebrospinal fluid, and the nerve conduction study (NCS) showed the changes due to demyelination. Intravenous immunoglobulin therapy was performed for 5 days after the diagnosis of Guillain-Barré syndrome. He did not show any severe symptoms such as bulbar palsy and was discharged on day 16. Anti-GM2 and anti-GalNAc-GD1a IgM antibodies were detected and acute inflammatory demyelinating polyneuropathy following the CMV infection was confirmed. NCS showed the abnormal changes were normalized after 4 months. The levels of antibodies against moesin, which is a protein existing in trace amounts in node of Ranvier, were increased. However, the antibodies were not detected 4 months after therapy. These changes were well correlated to his clinical course.

Fascin1 is an actin-bundling protein involved in cancer cell migration and has recently been shown also to have roles in virus-mediated immune cell responses. Because viral infection has been shown to activate immune cells and to induce interferon-β expression in human cancer cells, we evaluated the effects of fascin1 on virus-dependent signaling via the membrane- and actin-associated protein RIG-I (retinoic acid-inducible gene I) in colon cancer cells. We knocked down fascin1 expression with shRNA retrovirally transduced into a DLD-1 colon cancer and L929 fibroblast-like cell lines and used luciferase reporter assays and co-immunoprecipitation to identify fascin1 targets. We found that intracellular poly(I·C) transfection to mimic viral infection enhances the RIG-I/MDA5 (melanoma differentiation-associated gene 5)-mediated dimerization of interferon regulatory factor 3 (IRF-3). The transfection also significantly increased the expression levels of IRF-7, interferon-β, and interferon-inducible cytokine IP-10 in fascin1-deleted cells compared with controls while significantly suppressing cell growth, migration, and invasion. We also found that fascin1 constitutively interacts with IκB kinase ϵ (IKKϵ) in the RIG-I signaling pathway. In summary, we have identified fascin1 as a suppressor of the RIG-I signaling pathway associating with IκB kinase ϵ in DLD-1 colon cancer cells to suppress immune responses to viral infection., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Microbes have interacted with eukaryotic cells for as long as they have been co-existing. While many of these interactions are beneficial for both the microbe as well as the eukaryotic cell, several microbes have evolved into pathogenic species. For some of these pathogens, host cell invasion results in irreparable damage and thus host cell destruction, whereas others use the host to avoid immune detection and elimination. One of the latter pathogens is Mycobacterium tuberculosis , arguably one of the most notorious pathogens on earth. In mammalian macrophages, M. tuberculosis manages to survive within infected macrophages by avoiding intracellular degradation in lysosomes using a number of different strategies. One of these is based on the recruitment and phagosomal retention of the host protein coronin 1, that is a member of the coronin protein family and a mammalian homolog of coronin A, a protein identified in Dictyostelium . Besides mediating mycobacterial survival in macrophages, coronin 1 is also an important regulator of naïve T cell homeostasis. How, exactly, coronin 1 mediates its activity in immune cells remains unclear. While in lower eukaryotes coronins are involved in cytoskeletal regulation, the functions of the seven coronin members in mammals are less clear. Dictyostelium coronins may have maintained multiple functions, whereas the mammalian coronins may have evolved from regulators of the cytoskeleton to modulators of signal transduction. In this minireview, we will discuss the different studies that have contributed to understand the molecular and cellular functions of coronin proteins in mammals and Dictyostelium .