Membrane-bound Molecules Research Articles

A High Content Screen for Mucin-1-Reducing Compounds Identifies Fostamatinib as a Candidate for Rapid Repurposing for Acute Lung Injury During the COVID-19 Pandemic

Drug repurposing is the only method capable of delivering treatments on the shortened time-scale required for patients afflicted with lung disease arising from SARS-CoV-2 infection. Mucin-1 (MUC1), a membrane-bound molecule expressed on the apical surfaces of most mucosal epithelial cells, is a biochemical marker whose elevated levels predict the development of acute lung injury (ALI) and respiratory distress syndrome (ARDS), and correlate with poor clinical outcomes. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce MUC1 protein abundance. Our screen identified Fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo, Fostamatinib reduced MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro, SYK inhibition by Fostamatinib promoted MUC1 removal from the cell surface. Our work reveals Fostamatinib as a repurposing drug candidate for ALI and provides the rationale for rapidly standing up clinical trials to test Fostamatinib efficacy in patients with COVID-19 lung injury.Drug repurposing is the only method capable of delivering treatments on the shortened time-scale required for patients afflicted with lung disease arising from SARS-CoV-2 infection. Mucin-1 (MUC1), a membrane-bound molecule expressed on the apical surfaces of most mucosal epithelial cells, is a biochemical marker whose elevated levels predict the development of acute lung injury (ALI) and respiratory distress syndrome (ARDS), and correlate with poor clinical outcomes. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce MUC1 protein abundance. Our screen identified Fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo, Fostamatinib reduced MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro, SYK inhibition by Fostamatinib promoted MUC1 removal from the cell surface. Our work reveals Fostamatinib as a repurposing drug candidate for ALI and provides the rationale for rapidly standing up clinical trials to test Fostamatinib efficacy in patients with COVID-19 lung injury.Funding: FWKT has received research project grants from Rigel Pharmaceuticals, and has consultancy agreements with Rigel Pharmaceuticals, and is the Chief Investigator of an international clinical 290trial of a SYK inhibitor in IgA nephropathy (ClinicalTrials.gov NCT02112838), funded by Rigel Pharmaceuticals. Conflict of Interest: The authors declare no competing interests.

Implication of Soluble HLA-G and HLA-G +3142G/C Polymorphism in Breast Cancer Patients Receiving Adjuvant Therapy in Tanzania.

Background:During cancer growth, immunosuppressive microenvironment is created that enables tumour cells to evade an eliminative immune response and hence manage to grow into malignancy. HLA-G, existing as either membrane-bound (mHLA-G) or soluble (sHLA-G) molecule is thought to be immunosuppressive and produced more by tumor cells. The +3142G/C polymorphism in HLA-G gene affects its expression, and G allele is considered to be a protective mutant allele associated with less expression of HLA-G. The implication of HLA-G in cancer development has been reported in different cancers and populations. But, its implication in most African populations has not yet been investigated. The aim of this study was to determine the possible associations of soluble HLA-G and HLA-G +3142G/C SNP with breast cancer. Materials and Methods:75 breast cancer patients and 84 normal controls were recruited in this study. The genotyping of HLA-G +3142G/C polymorphism was determined by LightSNiP typing assay using quantitative Real-Time PCR and sHLA-G levels were determined by ELISA. Results:The sHLA-G levels were significantly lower in breast cancer patients than in controls (p<0.001). Also, they were significantly lower in mastectomized patients compared to non-mastectomized patients (p=0.018). The ROC analysis revealed a significant ability of sHLA-G to differentiate breast cancer patients versus normal controls (AUC=0.697, 95% CI= 0.619-0.767, p<0.001) and identify mastectomized patients (AUC=0.667, 95% CI= 0.549 to 0.772, p=0.041). The assessment of +3142G/C polymorphism revealed a relatively similar distribution of frequencies of genotypes and alleles between breast cancer patients and normal controls (p>0.05) and was neither associated with sHLA-G levels. Conclusion:While the +3142G/C SNP was found not to be relevant to breast cancer, the changes of sHLA-G levels in response to medical interventions such as mastectomy may be translated into its potential prognostic utility for breast cancer. More studies are needed to provide clear evidence of sHLA-G as a diagnostic and prognostic marker of breast cancer in Tanzania.

Soluble RANKL is physiologically dispensable but accelerates tumour metastasis to bone.

Receptor activator of NF-κB ligand (RANKL) is a multifunctional cytokine known to affect immune and skeletal systems, as well as oncogenesis and metastasis1-4. RANKL is synthesized as a membrane-bound molecule, and cleaved into its soluble form by proteases5-7. As the soluble form of RANKL does not contribute greatly to bone remodelling or ovariectomy-induced bone loss8, whether soluble RANKL has a role in pathological settings remains unclear. Here we show that soluble RANKL promotes the formation of tumour metastases in bone. Mice that selectively lack soluble RANKL (Tnfsf11ΔS/ΔS)5-7,9 have normal bone homoeostasis and develop a normal immune system but display markedly reduced numbers of bone metastases after intracardiac injection of RANK-expressing melanoma and breast cancer cells. Deletion of soluble RANKL does not affect osteoclast numbers in metastatic lesions or tumour metastasis to non-skeletal tissues. Therefore, soluble RANKL is dispensable for physiological regulation of bone and immune systems, but has a distinct and pivotal role in the promotion of bone metastases.

In vitro affinity maturation of antibody against membrane-bound GPCR molecules

G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors, are among the most important targets against which many small molecule drugs have been developed. However, only two antibody drugs targeting GPCRs have been approved for clinical use although many antibody drugs against non-GPCR protein targets have been successfully developed for various disease indications. One of the challenges for developing anti-GPCR drugs is the high difficulty to perform affinity maturation due to their insolubility in aqueous solutions. To address this issue, CHO cell display libraries of single-chain variable fragments (scFvs) and full-length antibodies were maturated directly against vesicle probes prepared from CHO cells displaying the endothelin A receptor (ETaR) GPCR. The probe in the vesicle form ensures the physiological conformation and functional activity of the protein and avoids issues with membrane protein insolubility. The size of the vesicle had a clear effect on protein-ligand interaction; we used small-sized vesicles with low expression levels of GPCRs for the affinity maturation. Four rounds of affinity maturation combining vesicles as probes with the CHO cell display platform improved affinity by 13.58-fold for scFvs and 5.05-fold for full-length antibodies. We expect that this method will not only be used for the affinity maturation of antibodies against GPCRs but will also be used to mature antibodies for other types of proteins where the conformation/activity of which depends on the proper membrane environment.

Platelet Serotonin Aggravates Myocardial Ischemia/Reperfusion Injury via Neutrophil Degranulation.

Platelets store large amounts of serotonin that they release during thrombus formation or acute inflammation. This facilitates hemostasis and modulates the inflammatory response. Infarct size, heart function, and inflammatory cell composition were analyzed in mouse models of myocardial reperfusion injury with genetic and pharmacological depletion of platelet serotonin. These studies were complemented by in vitro serotonin stimulation assays of platelets and leukocytes in mice and men, and by measuring plasma serotonin levels and leukocyte activation in patients with acute coronary syndrome. Platelet-derived serotonin induced neutrophil degranulation with release of myeloperoxidase and hydrogen peroxide (H2O2) and increased expression of membrane-bound leukocyte adhesion molecule CD11b, leading to enhanced inflammation in the infarct area and reduced myocardial salvage. In patients hospitalized with acute coronary syndrome, plasmatic serotonin levels correlated with CD11b expression on neutrophils and myeloperoxidase plasma levels. Long-term serotonin reuptake inhibition-reported to protect patients with depression from cardiovascular events-resulted in the depletion of platelet serotonin stores in mice. These mice displayed a reduction in neutrophil degranulation and preserved cardiac function. In line, patients with depression using serotonin reuptake inhibition, presented with suppressed levels of CD11b surface expression on neutrophils and lower myeloperoxidase levels in blood. Taken together, we identify serotonin as a potent therapeutic target in neutrophil-dependent thromboinflammation during myocardial reperfusion injury.

The Correlation between the Pool of Free Adhesion Molecule Receptors and the Activity of the Immune System in the Murmansk Oblast Residents

The molecules of intercellular adhesion are the surface cellular glycoproteins responsible for interaction between cells of different types with one another and with the components of intercellular matrix. They play the primary role in the formation of tissue structure and participate in a number of physiological processes, such as morphogenesis, embryogenesis, organogenesis, wound healing, and inflammation. Together with the other free receptors and their ligands, free intercellular adhesion molecules are formed mainly through shedding, resulting from the proteolytic cleavage of the transmembrane region under physiological conditions or cell destruction. There are almost no literature data on the influence of free intercellular adhesion molecules on the functional activity of T and B lymphocytes and the contents of immunoglobulins, cytokines, and circulating immune complexes. Therefore, the purpose of this study was to identify the interrelationship between the content of free and membrane-bound intercellular adhesion molecules and the level of immunocompetent cell activity in the residents of the Arctic settlement of Revda (Murmansk oblast), who form one of special populations of residents living in the Arctic region of the Russian Federation. It has been found that the contents of sCD54, sCD62L, and sCD324 free intercellular adhesion molecules vary widely, which results from an increase in the concentration of cells with the corresponding membrane forms. An increase in the levels of sCD54 and sCD62 is accompanied by the activation of migration processes with increasing concentrations of circulating neutrophils and total lymphocyte count. The formation of a pool of membrane-bound and free adhesion molecules occurs together with an increase in the content of activated T cells of the HLADRII and CD8 + phenotypes. An increase in the serum levels of sCD54 and L-selectin (sCD62L) adhesion molecules is accompanied by a decrease in the level of immunocompetent cell activity and a twofold increase in the serum level of a natural immunosuppressant IL-10. Low activity of the sCD54 and sCD62L is associated with an increase in the IgE concentration and in immune responses caused by the basophil and eosinophil activation.

The role of endothelial glycocalyx in sepsis

The surface of endothelial cells is filled with various membrane-bound molecules that form the glycocalyx. The endothelial glycocalyx is a surface layer mainly consisted of glycosaminoglycans that include heparan sulfate, chondroitin sulfate, and hyaluronic acid and its core proteins. Previous studies suggest that endothelial surface glycocalyx shedding could play a role in endothelial dysfunction and inflammation. This article will review the endothelial glycocalyx and its role in sepsis.

Compartments with predominant ephrin-B1 and EphB2/B4 expression are present alternately along the excurrent duct system in the adult mouse testis and epididymis.

Ephrin receptors (Eph) and ligands are membrane-bound cell-cell communication molecules that regulate the spatial organization of various tissues and organs by repulsive or adhesive signals arising from contact between Eph- and ephrin-bearing cells. However, the expression and functions of Eph receptors in the testis and epididymis are virtually unknown. We aimed to investigate the expression of several EphB receptors and ephrin-B ligands in the testis and epididymis of adult mice. mRNA and protein expression was detected via reverse transcription-polymerase chain reaction amplification and immunostaining, respectively. Complementary expression patterns were observed in the epithelia along the excurrent duct system in the testis and epididymis; ephrin-B1 was strongly expressed in the epithelia of the rete testis and segment I in the ductus epididymis, whereas EphB2 and/or EphB4 were strongly expressed in the epithelia of the straight tubules and efferent ductules. Moreover, ephrin-B1 was expressed in the spermatogonia, Leydig cells, and peritubular myoid cells in the testis, whereas EphB2 was expressed in elongated spermatids and EphB4 was expressed in the spermatogonia and Leydig cells. Furthermore, these receptors were found to be tyrosine-phosphorylated in the testis and/or epididymis. Receptor localization and phosphorylation patterns suggested that EphB/ephrin-B signaling might occur in the seminiferous tubules and epithelial junctions among the straight tubules, rete testis, efferent ductules, and ductus epididymis. Therefore, we propose that EphB/ephrin-B signaling may regulate epithelial boundary formation in the excurrent tubule/ductule/duct system as well as modulate spermatogenesis and spermiation. Overall, this study represents the first analysis of EphB receptor and ephrin-B ligand expression in the normal adult testis and epididymis.

Natural Killer Cells Exhibit a Peculiar Phenotypic Profile in Systemic Sclerosis and Are Potent Inducers of Endothelial Microparticles Release.

The pathophysiology of systemic sclerosis (SSc) involves early endothelial and immune activation, both preceding the onset of fibrosis. We previously identified soluble fractalkine and circulating endothelial microparticles (EMPs) as biomarkers of endothelial inflammatory activation in SSc. Fractalkine plays a dual role as a membrane-bound adhesion molecule expressed in inflamed endothelial cells (ECs) and as a chemokine involved in the recruitment, transmigration, and cytotoxic activation of immune cells that express CX3CR1, the receptor of fractalkine, namely CD8 and γδ T cells and natural killer (NK) cells. We aimed to quantify circulating cytotoxic immune cells and their expression of CX3CR1. We further investigated the expression profile of NK cells chemokine receptors and activation markers and the potential of NK cells to induce EC activation in SSc. We performed a monocentric study (NCT 02636127) enrolling 15 SSc patients [15 females, median age of 55 years (39–63), 11 limited cutaneous form and 4 diffuse] and 15 healthy controls. Serum fractalkine levels were significantly increased in SSc patients. Circulating CD8 T cells numbers were decreased in SSc patients with no difference in their CX3CR1 expression. Circulating γδ T cells and NK cells numbers were preserved. CX3CR1 expression in CD8 and γδ T cells did not differ between SSc patients and controls. The percentage and level of CX3CR1 expression in NK cells were significantly lowered in SSc patients. Percentages of CXCR4, NKG2D, CD69-expressing NK cells, and their expression levels were decreased in NK cells. Conversely, CD16 level expression and percentages of CD16+ NK cells were preserved. The exposure of human microvascular dermic EC line (HMVEC-d) to peripheral blood mononuclear cells resulted in similar NK cells degranulation activity in SSc patients and controls. We further showed that NK cells purified from the blood of SSc patients induced enhanced release of EMPs than NK cells from controls. This study evidenced a peculiar NK cells phenotype in SSc characterized by decreased chemokine and activation receptors expression, that might reflect NK cells involvement in the pathogenic process. It also highlighted the role of NK cells as a potent mechanism inducing endothelial activation through enhanced EMPs release.

Soluble CD83 Alleviates Experimental Autoimmune Uveitis by Inhibiting Filamentous Actin-Dependent Calcium Release in Dendritic Cells.

Soluble CD83 (sCD83) is the extracellular domain of the membrane-bound CD83 molecule, and known for its immunoregulatory functions. Whether and how sCD83 participates in the pathogenesis of uveitis, a serious inflammatory disease of the eye that can cause visual disability and blindness, is unknown. By flow cytometry and imaging studies, we show that sCD83 alleviates experimental autoimmune uveitis (EAU) through a novel mechanism. During onset and recovery of EAU, the level of sCD83 rises in the serum and aqueous humor, and CD83+ leukocytes infiltrate the inflamed eye. Systemic or topical application of sCD83 exerts a protective effect by decreasing inflammatory cytokine expression, reducing ocular and splenic leukocyte including CD4+ T cells and dendritic cells (DCs). Mechanistically, sCD83 induces tolerogenic DCs by decreasing the synaptic expression of co-stimulatory molecules and hampering the calcium response in DCs. These changes are caused by a disruption of the cytoskeletal rearrangements at the DC–T cell contact zone, leading to altered localization of calcium microdomains and suppressed T-cell activation. Thus, the ability of sCD83 to modulate DC-mediated inflammation in the eye could be harnessed to develop new immunosuppressive therapeutics for autoimmune uveitis.

Abstract 2638: Overexpressions of cortisol, annexin V and Lost in expression of PD-L1 and DARC proteins are associated with aggressive prostate cancer phenotypes in Black men: A CaPTC Cohort Study

Abstract Prostate cancer (CaP) is the most common male gender cancer and present with a 5-year prevalence, incidence and mortality rate in Nigerian Black men. There is disproportionate prevalence and poor understanding of CaP in Black men globally. The Prostate Cancer Transatlantic Consortium (CaPTC) has documented significant CaP burden among Nigerian black men in Nigeria and in the Diaspora. Notably, African ancestry is one of the most important risk factors of CaP globally. Increased levels of cortisol, a stress-related catabolic hormone is significantly associated to prostate specific antigen and poor prognosis in prostate cancer patients. Cortisol causes degradation of muscle proteins into amino-acids. Amino acids such as leucine, serves as a source of fuel for aggressive prostate cancer cells in a Warburg effect model through the L-amino acid transporters (LATs). The annexins are calcium and phospholipid binding proteins implicated in cancer development and progression. Overexpression of annexin 1 is one of the mechanisms by which cortisol inhibit inflammation. Annexin V has a high affinity to phosphatidylserine and play important role of inhibiting engulfment of apoptotic cells by macrophages to increase the immunogenicity of tumor cells undergoing apoptosis. PD-L1 is an important membrane-bound costimulatory molecule that inhibits immune responses through its receptor, PD-1. Overexpression of PD-L1 results to cancer cells aggressively evading the host immune system. The Duffy Antigen/Receptor for Chemokines (DARC) is a 7-transmembrane protein atypical chemokine receptor expressed on erythrocytes and vascular endothelial cells that binds to and clears angiogenic chemokines. Expression pattern of DARC is associated to cancer growth and metastasis. In this study, we report the expression pattern of tissue Cortisol, Annexin V, PD-L1 and DARC proteins in relationship to Gleason score in Nigerian Black men with Prostate cancer using immunohistochemistry and immunofluorescence. In addition, we assessed salivary cortisol levels by enzyme immunoassay and physical activity by a standardized CaPTC-AC3-MADCaP (C.A.M) CaP measures from 500 subjects recruited by via CaPTC cohort. Results showed significant expression of tissue and salivary cortisol in the CaP patients compared to the normal subjects (p &amp;gt; 0.05). The Tissue cortisol protein expression was higher in CaP cases with Gleason score 8. There was significant overexpression of Annexin protein in virtually the prostate cancer samples studied. We found a negative expression of PD-L1 and DARC proteins in all the CaP tissues studied. Cortisol and Annexin may serve as an important biomarker for prostate cancer diagnosis and prognosis in Nigerian Blacks. Citation Format: Faruk Mohammed, Folakemi T. Odedina, Sani Ibrahim, Abdulmumini Hassan Rafindadi, Ahmed Adamu, Abdullahi Adamu, Ahmad Bello, Surajo Mohammed Aminu, John Idoko, Aishatu Maude Suleiman, Solomon O. Rotimi, Ernie Kaninjing, Getachew A. Dagne, Clayton Yates, Yawale Iliyasu, Nissa Askins, Iya Eze Bassey, Renee Reams, Abdullahi Mohammad, Hussaini Yusuf Maitama, Dauda Maigatari, Mohammed Sani Shehu, Cheh Augustine Awasum, Abdulkadir Lawal Rafinadadi, Danladi Amodu Ameh, Serah Adewunmi, Ruth Agaba, Haruna Mohammad Muktar, Ahmad Mai, Saad Aliyu Ahmed, Ahmad Bello Kumo, Kasimu Umar Adoke, Ahmad Tijjani Lawal, Ahmad Muhammad, Omolora Fatiregun, Sunday Atawodi, Shehu Akuyam, Yusuf Abdulrashid, Mubarak Liman, Aliyu Muhammad, Abidemi Omonisi, Rebecca Gali, Hassan Dogo, Nkegoum Blaise, Anthonia Sowunmi, Titilola Akinremi, Emeka J. Iweala, Jigo Dangude Yaro, Badejo Adebukola Catherine, Akinwumi Oluwole Komolafe. Overexpressions of cortisol, annexin V and Lost in expression of PD-L1 and DARC proteins are associated with aggressive prostate cancer phenotypes in Black men: A CaPTC Cohort Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2638.

A disintegrin and metalloproteinase 15-mediated glycocalyx shedding contributes to vascular leakage during inflammation.

AimsEndothelial hyperpermeability exacerbates multiple organ damage during inflammation or infection. The endothelial glycocalyx, a protective matrix covering the luminal surface of endothelial cells (ECs), undergoes enzymatic shedding during inflammation, contributing to barrier hyperpermeability. A disintegrin and metalloproteinase 15 (ADAM15) is a sheddase capable of cleaving the ectodomains of membrane-bound molecules. Herein, we tested whether and how ADAM15 is involved in glycocalyx shedding and vascular leakage during sepsis.Methods and resultsDextran-150kD exclusion assay revealed lipopolysaccharide (LPS) significantly reduced glycocalyx thickness in mouse cremaster microvessels. Consistently, shedding products of glycocalyx constituents, including CD44 ectodomain, were detected with an increased plasma level after cecal ligation and puncture (CLP)-induced sepsis. The direct effects of CD44 ectodomain on endothelial barrier function were evaluated, which revealed CD44 ectodomain dose-dependently reduced transendothelial electrical resistance (TER) and caused cell–cell adherens junction disorganization. Furthermore, we examined the role of ADAM15 in CD44 cleavage and glycocalyx shedding. An in vitro cleavage assay coupled with liquid chromatography-tandem mass spectrometry confirmed ADAM15 cleaved CD44 at His235-Thr236 bond. In ECs with ADAM15 knockdown, LPS-induced CD44 cleavage and TER reduction were greatly attenuated, whereas, ADAM15 overexpression exacerbated CD44 cleavage and TER response to LPS. Consistently, ADAM15 knockout in mice attenuated CLP-induced increase in plasma CD44. Intravital and electron microscopic images revealed ADAM15 deficiency prevented LPS-induced glycocalyx injury in cremaster and pulmonary microvasculatures. Functionally, ADAM15−/− mice with better-preserved glycocalyx exhibited resistance to LPS-induced vascular leakage, as evidenced by reduced albumin extravasation in pulmonary and mesenteric vessels. Importantly, in intact, functionally vital human lungs, perfusion of LPS induced a significant up-regulation of ADAM15, accompanied by elevated CD44 in the effluent and increased vascular permeability to albumin.ConclusionTogether, our data support the critical role of ADAM15 in mediating vascular barrier dysfunction during inflammation. Its mechanisms of action involve CD44 shedding and endothelial glycocalyx injury.

Evaluation of membrane-bound and soluble forms of human leucocyte antigen-G in systemic sclerosis.

Systemic sclerosis (SSc) is a complex disease characterized by immune dysregulation, extensive vascular damage and widespread fibrosis. Human leucocyte antigen-G (HLA-G) is a non-classic class I major histocompatibility complex (MHC) molecule characterized by complex immunomodulating properties. HLA-G is expressed on the membrane of different cell lineages in both physiological and pathological conditions. HLA-G is also detectable in soluble form (sHLA-G) deriving from the shedding of surface isoforms (sHLA-G1) or the secretion of soluble isoforms (HLA-G5). Several immunosuppressive functions have been attributed to both membrane-bound and soluble HLA-G molecules. The plasma levels of sHLA-G were higher in SSc patients (444·27±304·84 U/ml) compared to controls (16·74±20·58U/ml) (P<0·0001). The plasma levels of transforming growth factor (TGF)-β were higher in SSc patients (18937±15217 pg/ml) compared to controls (11099±6081 pg/ml; P=0·003), and a significant correlation was found between TGF-β and the plasma levels of total sHLA-G (r=0·65; P<0·01), sHLA-G1 (r=0·60; P=0·003) and HLA-G5 (r=0·47; P=0·02). The percentage of HLA-G-positive monocytes (0·98±1·72), CD4+ (0·37±0·68), CD8+ (2·05±3·74) and CD4+ CD8+ double-positive cells (14·53±16·88) was higher in SSc patients than in controls (0·11±0·08, 0·01±0·01, 0·01±0·01 and 0·39±0·40, respectively) (P<0·0001). These data indicate that in SSc the secretion and/or shedding of soluble HLA-G molecules and the membrane expression of HLA-G by peripheral blood mononuclear cells (PBMC) is clearly elevated, suggesting an involvement of HLA-G molecules in the immune dysregulation of SSc.

Particle-based simulations of polarity establishment reveal stochastic promotion of Turing pattern formation.

Polarity establishment, the spontaneous generation of asymmetric molecular distributions, is a crucial component of many cellular functions. Saccharomyces cerevisiae (yeast) undergoes directed growth during budding and mating, and is an ideal model organism for studying polarization. In yeast and many other cell types, the Rho GTPase Cdc42 is the key molecular player in polarity establishment. During yeast polarization, multiple patches of Cdc42 initially form, then resolve into a single front. Because polarization relies on strong positive feedback, it is likely that the amplification of molecular-level fluctuations underlies the generation of multiple nascent patches. In the absence of spatial cues, these fluctuations may be key to driving polarization. Here we used particle-based simulations to investigate the role of stochastic effects in a Turing-type model of yeast polarity establishment. In the model, reactions take place either between two molecules on the membrane, or between a cytosolic and a membrane-bound molecule. Thus, we developed a computational platform that explicitly simulates molecules at and near the cell membrane, and implicitly handles molecules away from the membrane. To evaluate stochastic effects, we compared particle simulations to deterministic reaction-diffusion equation simulations. Defining macroscopic rate constants that are consistent with the microscopic parameters for this system is challenging, because diffusion occurs in two dimensions and particles exchange between the membrane and cytoplasm. We address this problem by empirically estimating macroscopic rate constants from appropriately designed particle-based simulations. Ultimately, we find that stochastic fluctuations speed polarity establishment and permit polarization in parameter regions predicted to be Turing stable. These effects can operate at Cdc42 abundances expected of yeast cells, and promote polarization on timescales consistent with experimental results. To our knowledge, our work represents the first particle-based simulations of a model for yeast polarization that is based on a Turing mechanism.

Could OX40 agonist antibody promote activation of the anti-tumor immune response in gastric cancer?

OX40, a membrane-bound molecule of the tumor-necrosis-factor-receptor superfamily, is a critical costimulatory receptor during the immune response, especially to T cells, but studies described their presence of OX-40 on neutrophils and monocytes, suggesting a potential role in the activation of immune response. Our aim was to characterize costimulatory receptors OX40 expression on circulating leukocytes in gastric cancer to identify novel targets for immunotherapy. Peripheral blood mononuclear cells were isolated from 24 gastric cancer patients and 34 healthy controls and the expression of costimulatory (OX40) receptors were analyzed on T cells, neutrophil and monocyte using monoclonal antibodies by flow cytometry. We found that the higher levels of OX40 + T cells, monocytes/OX40+ and neutrophils/OX40+ from gastric cancer patients when compared to controls (P < 0.0001), and also higher levels of OX40+ T cells when compared to stages III and IV (P = 0.02). Percentage levels of total T cells were similar between patients and controls. OX40 as a therapeutic agent has been investigated in many preclinical tumor models. Our findings suggest that of levels of costimulatory in T cells in GC will direct future studies on the role that costimulatory receptors play in the failure of T cell-mediated immunity.

Single-Molecule Study Reveals How Receptor and Ras Synergistically Activate PI3Kα and PIP3 Signaling

Cellular pathways controlling chemotaxis, growth, survival, and oncogenesis are activated by receptor tyrosine kinases and small G-proteins of the Ras superfamily that stimulate specific isoforms of phosphatidylinositol-3-kinase (PI3K). These PI3K lipid kinases phosphorylate the constitutive lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to produce the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Progress has been made in understanding direct, moderate PI3K activation by receptors. In contrast, the mechanism by which receptors and Ras synergistically activate PI3K to much higher levels remains unclear, and two competing models have been proposed: membrane recruitment versus activation of the membrane-bound enzyme. To resolve this central mechanistic question, this study employs single-molecule imaging to investigate PI3K activation in a six-component pathway reconstituted on a supported lipid bilayer. The findings reveal that simultaneous activation by a receptor activation loop (from platelet-derived growth factor receptor, a receptor tyrosine kinase) and H-Ras generates strong, synergistic activation of PI3Kα, yielding a large increase in net kinase activity via the membrane recruitment mechanism. Synergy requires receptor phospho-Tyr and two anionic lipids (phosphatidylserine and PIP2) to make PI3Kα competent for bilayer docking, as well as for subsequent binding and phosphorylation of substrate PIP2 to generate product PIP3. Synergy also requires recruitment to membrane-bound H-Ras, which greatly speeds the formation of a stable, membrane-bound PI3Kα complex, modestly slows its off rate, and dramatically increases its equilibrium surface density. Surprisingly, H-Ras binding significantly inhibits the specific kinase activity of the membrane-bound PI3Kα molecule, but this minor enzyme inhibition is overwhelmed by the marked enhancement of membrane recruitment. The findings have direct impacts for the fields of chemotaxis, innate immunity, inflammation, carcinogenesis, and drug design.

Semaphorin4D-PlexinB1 Signaling Attenuates Photoreceptor Outer Segment Phagocytosis by Reducing Rac1 Activity of RPE Cells.

Semaphorins form a family of secreted and membrane-bound molecules that were identified originally as axonal guidance factors during neuronal development. Given their wide distribution in many including mature tissues, semaphorin 4D (sema4D) and its main functional receptor plexin B1 (plxnB1) are expected to fulfill additional functions that remain to be uncovered. A main characteristic of the plexin receptor family is its ability to reorganize the cytoskeleton. PlxnB1 specifically may directly interact with Rho family GTPases to regulate F-actin driven pathways in cells in culture. Diurnal clearance phagocytosis by the retinal pigment epithelium (RPE) of photoreceptor outer segment fragments (POS) is critical for photoreceptor function and longevity. In this process, rearrangement of RPE cytoskeletal F-actin via activation of the Rho family GTPase Rac1 is essential for POS internalization. Here, we show a novel role in POS phagocytosis by RPE cells in culture and in vivo for plexin B1 and its ligand sema4D. Exogenous sema4D abolishes POS internalization (but not binding) by differentiated RPE cells in culture by decreasing the GTP load of Rac1. In the rat eye, sema4D localizes to retinal photoreceptors, while PlxnB1 is expressed by neighboring RPE cells. At the peak of diurnal retinal phagocytosis after light onset, plxnB1 phosphorylation and sema4D levels are reduced in wild-type rat retina in situ but not in mutant RCS rat retina in which the RPE lacks phagocytic activity. Finally, increased POS phagosome content after light onset is observed in the RPE in situ of mice with either plxnB1 or sema4D gene deletion. Altogether, our results demonstrate a novel physiological function for sema4D/plxnB1 signaling in RPE phagocytosis serving as attenuating brake prior to light onset whose release enables the diurnal phagocytic burst.

Soluble PD-1 and PD-L1: predictive and prognostic significance in cancer.

The membrane-bound molecules programmed death 1 (PD-1) and its ligand PD-L1 (PD-1/PD-L1) belong to the immune checkpoint pathway. PD-1 pathway downregulates effector T cells in immune response, thereby causing immune suppression. Recent studies have revealed that membrane-bound PD-1 and PD-L1 also have soluble forms. These soluble forms increase the complexity and diversity of the composition and function of the PD-1/PD-L1 signaling pathway. However, the exact roles of these molecules remain unknown. The objective of this systematic review was to elucidate the biological significance of soluble PD-1/PD-L1 in human cancers and evaluate whether they are potential diagnostic, therapeutic, or prognostic biomarkers. We expect to provide new clues for future research on soluble PD-1/PD-L1 pathway in human malignant tumors.

Oxidative Stress: Promoter of Allergic Sensitization to Protease Allergens?

Allergies arise from aberrant T helper type 2 responses to allergens. Several respiratory allergens possess proteolytic activity, which has been recognized to act as an adjuvant for the development of a Th2 response. Allergen source-derived proteases can activate the protease-activated receptor-2, have specific effects on immune cells by cleaving cell membrane-bound regulatory molecules, and can disrupt tight junctions. The protease activity can induce a non-allergen-specific inflammatory response in the airways, which will set the stage for an allergen-specific Th2 response. In this review, we will discuss the evidence for the induction of oxidative stress as an underlying mechanism in Th2 sensitization to proteolytic allergens. We will discuss recent data linking the proteolytic activity of an allergen to its potential to induce oxidative stress and how this can facilitate allergic sensitization. Based on experimental data, we propose that a less proficient anti-oxidant response to allergen-induced oxidative stress contributes to the susceptibility to allergic sensitization. Besides the effect of oxidative stress on the immune response, we will also discuss how oxidative stress can increase the immunogenicity of an allergen by chemical modification.

Production, characterization and application of monoclonal antibody against immunoglobulin D heavy chain of flounder (Paralichthys olivaceus)

Immunoglobulin D (IgD) is considered to be an enigmatic Ig molecule because of the lack understanding of its immunological functions. In the present study, a partial δ region of the flounder IgD was recombinantly expressed, purified and used as an immunogen to produce monoclonal antibodies (MAbs) against the H chain of flounder IgD. After fusion, a total of 97 hybridomas were generated and observed under an inverted microscope One of the hybridomas, designated 5G7, gave strong positive results in an indirect enzyme-linked immunosorbent assay (ELISA) and was cloned and subcloned by limiting dilution. Western blot analysis showed that MAb 5G7 could specifically recognize a 118 kDa protein from peripheral blood lymphocytes (PBLs), which was identified to be the H chain of flounder IgD by mass spectrometric analysis. Indirect immunofluorescence assay tests (IIFAT) showed that specific fluorescence signals were observed on the membranes of the PBLs, which suggests that MAb 5G7 could recognize the membrane-bound IgD molecule. Moreover, only the subset of IgD+/IgM + B cells were observed in the PBLs of healthy flounder when tested by flow cytometry analysis. Consistent with the results of flow cytometry, a double immunofluorescence assay test (DIFAT) showed that the positive lymphocytes were stained with both green and red fluorescence signals, which represent the IgM+/IgD + lymphocytes subset. These results demonstrate that the produced MAb 5G7 could specifically recognize the flounder IgD, which provides a useful tool to study the functions of flounder IgD.