SEMA5A Research Articles

Genetic and Clinical Evaluation of Retinitis Pigmentosa

Background: The aim of this study was to evaluate the most common underlying genetic and clinical etiologies of retinitis pigmentosa (RP) disease in our geographical area.
 Material and Method: In our archive, there are about 3000 patients who applied to our clinic between the years 2015-2021. The files of approximately 700 patients with a definitive genetic diagnosis were retrospectively scanned. A definitive genetic diagnosis was made in 22 of these patients. During our research, we collected some clinical parameters including the prenatal, natal, and postnatal history of the patients, history of surgery and seizures, and family history. In family history, we did a detailed pedigree with at least 3 generational analyses, questioned parental kinship, looked for similar members in families, and identified inheritance patterns of their disorder. We draw 3 generations pedigree and we collected peripheral venous blood samples from patients and sent them to a commercial lab for gene panels or WES. After obtaining the definitive genetic diagnosis of all patients, we compiled a table with the other parameters we questioned.
 Results: As a result of our WES analysis in patients 1 and 2, homozygous c.1331_1332 dupAG/p. Thr445ArgfsTer10 Class 2 variant was detected in the POC1B gene of patient #2.In the RP panel 1 reports of patients 3 and 4, the genomic alteration of c.2254dupA (p.Ser752Lysfs*14) was detected in exon 15 of the ABCA4 (NM_000350) gene. Patient 5, EYS c.4964T>C heterozygous. Patient 6. SEMA4A C.1168A>G (heterozygous). Patient 7, SEMA4A C.1168A>G (heterozygous), RP1 c.5402C>T (heterozygous), CGNB1 c.1382C>T (heterozygous).Patient #8, . Heterozygous variation of p.Thr390Ala (c.1168A>G) in the SEMA4A gene is present.As a result of our WES analysis, a homozygous c.2021C>A/p.Pro674His Class 2 variant was detected in the RPGRIP1 gene of patient #9. Heterozygous c.119-2A>C Class 1 mutation was detected in the NR2E3 gene of patient 10. Homozygous c.271C>T/p.Gln91* Class 1 mutation was detected in the MFRP gene in patient 11. Patient #12 was diagnosed at the age of 7-8 years. When we look at the exome sequencing results, a homozygous mutation in the CNGB1 gene c.413-1G> of patient 13 was detected. Heterozygous p.Ser361Tyr (c.1082C>A) change detected in the ABCA4 gene of patient #14 was detected. The heterozygous p.Glu150Lys (c.448G>A) change detected in the RHO gene of patient #15 was pathogenic according to ClinVar database and in silico analysis. rated as. Prediagnosis was Bardet-Biedle Syndrome in patient 16. P.Gly244Asp change was detected in RPE65 gene of patients 17 and 18. Automated DNA sequencing of patient #19 and patient #20 results in a homozygous sequence variation in the coding sequence of the NR2E3 genes, a homozygous CGG>CAG nucleotide substitution, and an amino acid replacement of Arg311Gln. Heterozygous mutation was detected in the same gene region in patient 21 (fathers). Variation in NR2E3 is the most likely cause of these patients' eye condition, as it is a complete genotype and is strongly associated with RP in many published families. Genetic results on an allele of the BBS1 gene of patient 22 (chr11:66.278.121-66.291.364 (13.2kb)/ISCN: seq [GRCH37]11q13.2(66.278).121-66.291.364)x1). The other allele has a heterozygous point mutation (c.1424dupT p.Ser476fs-rs886039798).
 Conclusıons: As determined in our study, the disease can be encountered with many different genetic etiologies. In this regard, patients undergoing genetic testing should be carefully examined for both SNP (single nucleotide polymorphism) and CNV (copy number variation).In addition, before genetic tests are performed, it should be well determined whether there is an isolated RP or an accompanying RP. In this respect, patients should be evaluated by making a detailed anamnesis and physical examination and drawing a pedigree containing at least 3 generations. Therefore, it was concluded that accompanying abnormalities should also be examined in the evaluation of retinitis pigmentosa anomalies.

Relationship between disease and disease severity and semaphorin 5A and hemogram level in obsessive–compulsive disorder

Objective Semaphorin 5A (SEMA 5A) is a neuroprotein that regulates the formation of excitatory synapses between neurons, important in autoimmunity, inflammatory processes and behaviors. This study aimed to investigate the SEMA 5A levels in patients with obsessive–compulsive disorder (OCD) diagnosed for the first time and evaluate the relationship of disease and disease severity with the blood SEMA 5A level and hemogram. Methods More than 41,465 patients who applied to the psychiatry clinic from January 2018 to December 2020 were evaluated according to the DSM-5 criteria; 57 patients diagnosed with OCD for the first time, who met the inclusion criteria, were included in the study. Disease severity was investigated administering the Yale Brown Obsessive Compulsion Scale. The peripheral blood SEMA 5A level and hemogram were measured and evaluated in relation to platelet (PLT) activity, neutrophil–lymphocyte ratio (NLR), PLT–lymphocyte ratio (PLR), monocyte–lymphocyte ratio (MLR), C-reactive protein (CRP), and compared with control group of 26 people. Results The comparison of the groups revealed a significant difference in SEMA 5A and CRP level, neutrophil count and percentage, lymphocyte count, PLT activity. A significant correlation was found between disease and SEMA 5A level, NLR, PLR, and PLT parameters in diagnosis of OCD. As the severity of OCD increased, the SEMA 5A level and PLT count decreased, while the PDW and MLR values increased. Conclusion In patients with OCD, a relationship was found between plasma SEMA 5A, PLT activity, NLR, PLR, and MLR activity levels with disease and the disease severity.

Semaphorin 4A antibody alleviates arsenic-induced hepatotoxicity in mice via inhibition of AKT2/NF-κB inflammatory signaling

Semaphorin (Sema) 3A and Sema 4A are immunomodulatory molecules with a common receptor, neuropilin-1 (NRP-1), on the immune cells. Sema 3A binds to NRP-1 and inhibits T cell activation and inflammation, while Sema 4A binds to NRP-1 and promotes T cell activation and inflammation. These molecules are associated closely with the regulation of protein kinase B (AKT)/nuclear factor-kappaB (NF-κB) signaling, which are poorly understood in arsenic toxicity. The present study explored the role of Sema 3A or Sema 4A in arsenic-induced hepatotoxicity in mice. Arsenic exposure induced hepatic injury and resulted in the activations of p-AKT2, NF-κB p65, and NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, downregulation of Sema 3A, and upregulation of Sema 4A or NRP-1. Interestingly, intervention with anti-Sema 4A antibody showed the mitigation of arsenic-induced hepatotoxicity, accompanied by the downregulation of Sema 4A, rebound of Sema 3A, and upregulation of NRP-1. And, the inflammatory signaling p-AKT2 or NF-κB p65, and NLRP3 inflammasome showed a downregulation compared with arsenic treatment group. In contrast, anti-Sema 3A antibody intervention did not show the significant effect in the histopathological features compared with arsenic treatment group. In conclusion, the anti-Sema 4A antibody antagonizes arsenic-induced hepatotoxicity in mice and may be involved in the inhibitions of AKT2/NF-κB and NLRP3 inflammatory signaling mediated synergistically by Sema 4A or Sema 3A and their receptor NRP-1.

Semaphorin4A-Plexin D1 Axis Induces Th2 and Th17 While Represses Th1 Skewing in an Autocrine Manner

Semaphorin (Sema)4A is a transmembrane glycoprotein that is elevated in several autoimmune diseases such as systemic sclerosis, rheumatoid arthritis and multiple sclerosis. Sema4A has a key role in the regulation of Thelper Th1 and Th2 differentiation and we recently demonstrated that CD4+ T cell activation induces the expression of Sema4A. However, the autocrine role of Sema4A on Th cell differentiation remains unknown. Naïve Th cells from healthy controls were cell sorted and differentiated into Th1, Th2 and Th17 in the presence or absence of a neutralizing antibody against the Sema4A receptor PlexinD1. Gene expression was determined by quantitative PCR and protein expression by ELISA and flow cytometry. We found that the expression of Sema4A is induced during Th1, Th2 and Th17 differentiation. PlexinD1 neutralization induced the differentiation of Th1 cells, while reduced the Th2 and Th17 skewing. These effects were associated with an upregulation of the transcription factor T-bet by Th1 cells, and to downregulation of GATA3 and RORγt in Th2 cells and Th17 cells, respectively. Finally, PlexinD1 neutralization regulates the systemic sclerosis patients serum-induced cytokine production by CD4+ T cells. Therefore, the autocrine Sema4A-PlexinD1 signaling acts as a negative regulator of Th1 skewing but is a key mediator on Th2 and Th17 differentiation, suggesting that dysregulation of this axis might be implicated in the pathogenesis of CD4+ T cell-mediated diseases.

Reciprocal changes in CD11c+CD11b+ and CD11c+CD8α+ dendritic cell subsets determine protective or permissive immune response in murine experimental VL

CD11c+CD8α+ and CD11c+CD11b+ dendritic cells are two major subsets of murine splenic CD11c+ DCs which play a crucial role in T cell priming and shaping Th1/Th2 responses, but their role in the context of experimental visceral leishmaniasis (VL) is poorly understood. Herein, we showed that L. donovani infection in Balb/c mice preferentially decreased the population abundance of CD11c+CD11b+ DCs and increased relative abundance of splenic CD11c+CD8α +DCs. During infection, splenic CD11c+CD11b+ DCs induced Th1 differentiation whereas CD11c+CD8α+ DCs promoted Th2 differentiation. Additionally, treatment of infected mice with miltefosine as experimental control exhibited host defense allowing the restoration of CD11c+CD11b+ population and decrease in CD11c+CD8α+ subset. Furthermore, reciprocal regulation of immune accessory surface molecules, Sema4A and OX40L critically determined Th1/Th2 response induced by these DC subsets during VL. L. donovani infection significantly induced OX40L expression and slightly downregulated SEMA 4A expression in CD11c+CD8α+ DCs whereas miltefosine treatment significantly downregulated OX40L expression along with pronounced upregulation of SEMA 4A expression in CD11c+CD11b+ DCs. SiRNA mediated knockdown of SEMA 4A markedly reduced CD11c+CD11b+ driven IFN-γ, TNF-α and IL-12 synthesis in miltefosine treated mice whereas functional blocking of OX40L decreased CD11c+CD8α+ induced IL-10, IL-4 and TGF-β synthesis in L. donovani infected group. Vaccination of Balb/c mice with antigen-pulsed + CpG-ODN-activated DC subsets revealed that only antigen-pulsed CD11c+CD11b+ DCs eliminated parasite load in visceral organ and restored protective Th1 cytokine response. Collectively, our results suggest that differential regulation of splenic CD11c+ subsets by L. donovani is essential for disease progression and specific subtypes may be exploited as prophylactic measures against visceral leishmaniasis.

A Genome-Wide Association Study of Sprint Performance in Elite Youth Football Players

Pickering, C, Suraci, B, Semenova, EA, Boulygina, EA, Kostryukova, ES, Kulemin, NA, Borisov, OV, Khabibova, SA, Larin, AK, Pavlenko, AV, Lyubaeva, EV, Popov, DV, Lysenko, EA, Vepkhvadze, TF, Lednev, EM, Leońska-Duniec, A, Pająk, B, Chycki, J, Moska, W, Lulińska-Kuklik, E, Dornowski, M, Maszczyk, A, Bradley, B, Kana-ah, A, Cięszczyk, P, Generozov, EV, and Ahmetov, II. A genome-wide association study of sprint performance in elite youth football players. J Strength Cond Res 33(9): 2344-2351, 2019-Sprint speed is an important component of football performance, with teams often placing a high value on sprint and acceleration ability. The aim of this study was to undertake the first genome-wide association study to identify genetic variants associated with sprint test performance in elite youth football players and to further validate the obtained results in additional studies. Using micro-array data (600 K-1.14 M single nucleotide polymorphisms [SNPs]) of 1,206 subjects, we identified 12 SNPs with suggestive significance after passing replication criteria. The polymorphism rs55743914 located in the PTPRK gene was found as the most significant for 5-m sprint test (p = 7.7 × 10). Seven of the discovered SNPs were also associated with sprint test performance in a cohort of 126 Polish women, and 4 were associated with power athlete status in a cohort of 399 elite Russian athletes. Six SNPs were associated with muscle fiber type in a cohort of 96 Russian subjects. We also examined genotype distributions and possible associations for 16 SNPs previously linked with sprint performance. Four SNPs (AGT rs699, HSD17B14 rs7247312, IGF2 rs680, and IL6 rs1800795) were associated with sprint test performance in this cohort. In addition, the G alleles of 2 SNPs in ADRB2 (rs1042713 & rs1042714) were significantly over-represented in these players compared with British and European controls. These results suggest that there is a genetic influence on sprint test performance in footballers, and identifies some of the genetic variants that help explain this influence.

Plexin B1 expression on human Treg cells is required for regulation of their phenotypic stability by semaphorin 4A

Abstract We previously reported that neuroimmune semaphorin (Sema) 4A regulates the severity of experimental allergic asthma and increases regulatory T-cell (Treg) numbers in vivo, however the mechanisms of Sema4A action remain unknown. It was also reported that Sema4A controls murine Treg cell function and survival acting through Neuropilin 1 (NRP-1) receptor. To clarify Sema4A action on human T cells, we employed T-cell lines, HuT78 and HuT102, as well as human PBMCs and CD4+ T cells in phenotypic and functional assays. We found that HuT78 demonstrated a T-effector-like phenotype (CD4+CD25lowFoxp3-), while HuT102 expressed a Treg-like phenotype (CD4+CD25hi Foxp3+). Neither cell line expressed NRP-1. However, HuT102 cells expressed high levels of the Sema4A-counter receptor, Plexin B1 whereas HuT78 cells demonstrated high cell-surface expression of Sema4A. All human peripheral blood CD4+ T cells, including Treg cells, expressed PlexinB1 and lacked both, NRP-1 and −2. Culture of HuT cells with soluble Sema4A led to an upregulation of CD25 and Foxp3 markers on HuT102 cells. Addition of soluble Sema4A increased the relative numbers of CD4+CD25+Foxp3+ cells in PBMC and CD4+ T cells which did not express NRP-1 but were PlexinB1+, suggesting the role of this receptor in Treg cell stability. The inclusion of anti-PlexinB1 blocking Ab in cultures before rSema4A addition significantly decreased Treg cell numbers as compared to cultures with rSema4A alone. Sema4A was as effective as TGF-b in iTreg induction from CD4+CD25depleted cells but did not affect Treg cell suppressive activity in vitro. These results suggest strategies for the development of new Sema4A-based therapeutic measures to combat allergic inflammatory diseases.

Semaphorin 4A Stabilizes Human Regulatory T Cell Phenotype via Plexin B1.

We previously reported that neuroimmune semaphorin (Sema) 4A regulates the severity of experimental allergic asthma and increases regulatory T (Treg) cell numbers in vivo; however, the mechanisms of Sema4A action remain unknown. It was also reported that Sema4A controls murine Treg cell function and survival acting through neuropilin 1 (NRP-1) receptor. To clarify Sema4A action on human T cells, we employed T cell lines (HuT78 and HuT102), human PBMCs, and CD4+ T cells in phenotypic and functional assays. We found that HuT78 demonstrated a T effector-like phenotype (CD4+CD25lowFoxp3-), whereas HuT102 expressed a Treg-like phenotype (CD4+CD25hi Foxp3+). Neither cell line expressed NRP-1. HuT102 cells expressed Sema4A counter receptor Plexin B1, whereas HuT78 cells were Sema4A+. All human peripheral blood CD4+ T cells, including Treg cells, expressed PlexinB1 and lacked both NRP-1 and -2. However, NRP-1 and Sema4A were detected on CD3negativeCD4intermediate human monocytes. Culture of HuT cells with soluble Sema4A led to an upregulation of CD25 and Foxp3 markers on HuT102 cells. Addition of Sema4A increased the relative numbers of CD4+CD25+Foxp3+ cells in PBMCs and CD4+ T cells, which were NRP-1negative but PlexinB1+, suggesting the role of this receptor in Treg cell stability. The inclusion of anti-PlexinB1 blocking Ab in cultures before recombinant Sema4A addition significantly decreased Treg cell numbers as compared with cultures with recombinant Sema4A alone. Sema4A was as effective as TGF-β in inducible Treg cell induction from CD4+CD25depleted cells but did not enhance Treg cell suppressive activity in vitro. These results suggest strategies for the development of new Sema4A-based therapeutic measures to combat allergic inflammatory diseases. ImmunoHorizons, 2019, 3: 71-87.

Sema4A Responds to Hypoxia and Is Involved in Breast Cancer Progression.

Semaphorin4A (Sema4A) is a family member of semaphorins expressed in immune cells and is also related with disease progression of tumor disease. In this study, we investigate the expression and pathological role of Sema4A in breast cancer (BCa). Our data showed that the expression of Sema4A increased in the tissues and serum of BCa patients when compared with normal controls. The expression of Sema4A in BCa cells could be induced by hypoxic treatment, whereas silencing hypoxia-inducible factor (HIF)-1α could attenuate the above induced. Furthermore, chromatin immunoprecipitation (ChIP) analysis demonstrated that HIF-1α could regulate the expression of Sema4A through directly binding to the promoter of Sema4A gene, whose enrichment could be further enhanced by hypoxic stimulation. In addition, silencing Sema4A could inhibit the proliferation, vascular endothelial growth factor (VEGF) production and the phosphorylation of Akt, extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) and signal transduction and activator of transcription (STAT)3, but induce apoptosis of BCa cells in the presence of hypoxia. In contrast, recombinant human Sema4A treatment showed the opposite effects. Taken together, these results suggest that Sema4A could promote progression of BCa in the presence of hypoxia and it may hold potential for treatment target for BCa.

The expression of Semaphorin 5A in patients with rheumatoid arthritis and its effect on osteoclastogenesis

Objective: To investigate the clinical significance of soluble Semaphorin 5A(Sema 5A) in patients of rheumatoid arthritis(RA) and the effect of Sema 5A on osteoclastogenesis in RAW264.7 cells. Methods: (1)Soluble Sema 5A was detected in the serum of 62 RA patients, 30 osteoarthritis(OA) patients and 48 healthy controls(HC) by enzyme-linked immunosorbent assay(ELISA).The relationships between serum Sema 5A and disease activity, radiographic severity and laboratory parameters were investigated in RA patients.(2)RAW264.7 cells were treated with different concentrations of Sema 5A(0,0.5,1,2.5,5 μg/ml).After 7 days, tartrate-resistant acid phosphate(TRAP) staining was performed. The mRNA levels of TRAP, cathepsin-K and matrix metallopeptidase 9(MMP-9) were tested using real-time polymerase chain reaction (RT-PCR).(3)Bone resorption area of dentine slides cultured with RAW264.7 cells was calculated after Sema 5A(5μg/ml) treatment. Results: (1)The serum Sema 5A in RA patients[(5.24±0.59)μg/L] was significantly higher than those in healthy controls[(2.93±0.34)μg/L,P<0.01] and OA patients[(2.68±0.47)μg/L,P<0.05]. The Sema 5A level in RA patients was positively correlated with disease activity score with 28 joint using C-reactive protein(DAS28-CRP), clinical disease activity index (CDAI),C-reactive protein(CRP) and sharp scores(P<0.05 or P<0.01).In addition,the serum Sema 5A in RA patients with positive anti-cyclic citrullinated peptide(CCP) antibody was significantly greater than that of anti-CCP antibody-negative patients(P<0.05).(2)After RAW264.7 cells were treated with Sema 5A, the number of TRAP positive osteoclasts increased according to the increase of Sema 5A concentration with maximal effect at 5 μg/ml. Meanwhile, Sema 5A promoted mRNA expression of TRAP,Cathepsin-K and MMP-9.(3)Bone resorption area increased when RAW264.7 cells were treated with Sema 5A(5 μg/ml). Conclusions: Serum Sema 5A is elevated in RA patients and correlated with disease activity and radiographic severity. Sema 5A promotes osteoclastogenesis of RAW264.7 cells.

Elevated semaphorin 5A in patients with Hashimoto's thyroiditis: a case-control study.

ObjectiveHashimoto’s thyroiditis (HT) is characterized by elevated specific auto-antibodies, including TgAb and TPOAb. Increasing evidence has demonstrated the essential role of Th17 cells in HT. However, the underlying mechanism is still unclear. Semaphorin 5A (Sema 5A) is involved in several autoimmune diseases through the regulation of immune cells. The aim of the present study was to explore the role of Sema 5A in HT.MethodsWe measured serum Sema 5A levels in HT (n = 92) and healthy controls (n = 111) by enzyme-linked immunosorbent assay (ELISA). RNA levels of Sema 5A and their receptors (plexin-A1 and plexin-B3), as well as several cytokines (IFN-γ, IL-4 and IL-17), were detected by real-time polymerase chain reaction in peripheral blood mononuclear cells from 23 patients with HT and 31 controls. In addition, we investigated the relationship between serum Sema 5A and HT.ResultsSerum Sema 5A in HT increased significantly compared with healthy controls (P < 0.001). Moreover, serum Sema 5A levels were positively correlated with TgAb (r = 0.511, P < 0.001), TPOAb (r = 0.423, P < 0.001), TSH (r = 0.349, P < 0.001) and IL-17 mRNA expression (r = 0.442, P < 0.001). Increased Sema 5A RNA expression was observed (P = 0.041) in HT compared with controls. In receiver-operating characteristic (ROC) analysis, serum Sema 5A predicted HT with a sensitivity of 79.35% and specificity of 96.40%, and the area under the curve of the ROC curve was 0.836 (95% CI: 0.778–0.884, P < 0.001).ConclusionsThese data demonstrated elevated serum Sema 5A in HT patients for the first time. Serum Sema 5A levels were correlated with thyroid auto-antibodies and IL-17 mRNA expression. Sema 5A may be involved in immune response of HT patients.

Pathogenesis of immune thrombocytopenic purpura

Immune thrombocytopenic purpura(ITP) is a common autoimmune disorder characterized by isolated thrombocytopenia.Previous studies showed that autoantibodies bind to platelets and megakaryocytes playing a significant role in phagocytosis and depressing their maturation, which was the classic humoral immune mechanism. At present, the role of cellular immunity in the pathogenesis of ITP is more and more important. The abnormality of apoptosis of platelets and megakaryocytes also affects the incidence of ITP, abnormal protein of some kinds of cell subsets and abnormal levels of cytokines are confirmed in patients with ITP, and related researches on the gene of ITP patients has also made some progress. In addition, semaphorin (Sema) 5A and vitamin D3 abnormalities are also involved in pathogenesis of ITP. This article summarizes the research progress of ITP pathogenesis mainly from the humoral immunity, cellular immunity, apoptosis, gene expression, oxidative stress and other aspects. Key words: Purpura, thrombocytopenic, idiopathic; Immunity, humoral; Immunity, cellular; DNA methylation; Oxidative stress

Semaphorin 5A, Anti-Nucleosome Antibodies and Ferritin: Disease Activity Markers in Patients with Systemic Lupus Erythematosus

Background: Systemic Lupus Erythematosus (SLE) is a multi-factorial, chronic autoimmune disorder characterized by dysfunction of T and B lymphocytes. Although the prognosis of SLE has improved in the past few decades, the absence of biomarkers for residual activity in various organs and the early detection of disease flares hamper further management. Conventional serologic markers of SLE, such as anti-dsDNA and complement levels, are not ideal, as they are not sufficiently sensitive and specific for the diagnosis of the disease and monitoring of disease activity. Thus, novel biomarkers for SLE activity have to be developed. Semaphorin 5A (Sema 5A), antinuclosome antibody (Anu A) and ferritin may fall into this category of novel bio markers.Objective: To evaluate the role of serum Sema 5A, Anu A and ferritin in detection of SLE activity.Methods: The present study included 40 patients with SLE divided according to Systemic Lupus Erythematous Disease Activity Index (SLEDAI) into two groups: 20 active SLE and 20 inactive SLE patients. They were compared with 20, sex and age matched healthy individuals for control. Levels of Sema 5A, Anu A were measured by Enzyme- Linked Immunosorbent Assay (ELISA) and serum ferritin levels were measured by Electrochemiluminescence immunoassay (ECLIA).Results: There was highly significant increase in Sema 5A, Anu A and ferritin levels inactive when compared within active SLE patients and control subjects (P=0.000 and P=0.000 and P=0.000 respectively). There was a significant increase in ferritin level in inactive SLE patients when compared with control subjects (P=0.045) with no significant difference regarding Sema 5A, Anu A (P3=0.089 and 0.225 respectively). There were positive correlations between Sema 5A, Anu A, ferritin and each of SLEDAI, and CRP. Positive correlation also found between Sema 5A and each of Anu A and ferritin and between AnuA and ferritin. Negative correlation was found between Sema 5A and C3 and between Anu A, ferritin and C4. Significant relation was found between Sema 5A, Anu A, ferritin and each of ANA and anti-dsDNA in active SLE patients. Area under the curve (AUC) for ferritin, Sema 5A and Anu A were (0.861, 1.0 and 1.0 respectively).Conclusion: Our study showed that the serum proteins semaphorin 5A, antinuclosome antibodies and ferritin may be useful markers in monitoring disease activity in patients with SLE. Regarding area under the curve (AUC), these serum protein markers result in even better sensitivity and specificity profiles in picking up early relapse of SLE.

Doxorubicin-induced epithelial–mesenchymal transition through SEMA 4A in hepatocellular carcinoma

Semaphorins are essential for the functions in the regulation of cell migration. SEMA 4A has been proven to play a prominent role in immune function and angiogenesis. However, whether SEMA 4A is involved in HCC chemoresistance is unclear. We investigated the role of SEMA 4A in HCC chemoresistance and the underlying mechanisms. We tested the doxorubicin sensitivity of the Huh7, and Hep-G2 HCC cell lines. Immunofluorescence and Western blot were used to detect the location and expression of EMT-related protein, such as, E-cadherin, Vimentin, and SEMA4A expression. Microarray data showed that SEMA 4A and SEMA 3F increased most dramatically under DOX treatment. Kncokdown of SEMA 4A in hepatoma cells can reduce EMT process. Expectedly, depletion of SEMA 4A also reversed EMT and increased the DOX sensitivity. SEMA 4A confers doxorubicin resistance on HCC by inducing epithelial–mesenchymal transition (EMT).

Germline variants in the SEMA4A gene predispose to familial colorectal cancer type X.

Familial colorectal cancer type X (FCCTX) is characterized by clinical features of hereditary non-polyposis colorectal cancer with a yet undefined genetic background. Here we identify the SEMA4A p.Val78Met germline mutation in an Austrian kindred with FCCTX, using an integrative genomics strategy. Compared with wild-type protein, SEMA4AV78M demonstrates significantly increased MAPK/Erk and PI3K/Akt signalling as well as cell cycle progression of SEMA4A-deficient HCT-116 colorectal cancer cells. In a cohort of 53 patients with FCCTX, we depict two further SEMA4A mutations, p.Gly484Ala and p.Ser326Phe and the single-nucleotide polymorphism (SNP) p.Pro682Ser. This SNP is highly associated with the FCCTX phenotype exhibiting increased risk for colorectal cancer (OR 6.79, 95% CI 2.63 to 17.52). Our study shows previously unidentified germline variants in SEMA4A predisposing to FCCTX, which has implications for surveillance strategies of patients and their families.

A point mutation in Semaphorin 4A associates with defective endosomal sorting and causes retinal degeneration

Semaphorin 4A (Sema4A) has an essential role in photoreceptor survival. In humans, mutations in Sema4A are thought to contribute to retinal degenerative diseases. Here we generate a series of knock-in mouse lines with corresponding mutations (D345H, F350C or R713Q) in the Sema4A gene and find that Sema4AF350C causes retinal degeneration phenotypes. The F350C mutation results in abnormal localization of the Sema4A protein, leading to impaired endosomal sorting of molecules indispensable for photoreceptor survival. Additionally, protein structural modelling reveals that the side chain of the 350th amino acid is critical to retain the proper protein conformation. Furthermore, Sema4A gene transfer successfully prevents photoreceptor degeneration in Sema4AF350C/F350C and Sema4A−/− mice. Thus, our findings not only indicate the importance of the Sema4A protein conformation in human and mouse retina homeostasis but also identify a novel therapeutic target for retinal degenerative diseases.

Sema 4D/CD100-plexin B is a multifunctional counter-receptor

The discovery of Sema 4D can be dated to 1992, when Boumsell's team reported a novel 150-kDa glycoprotein dimer. It was expressed on T lymphocytes that had been activated with CD3 monoclonal antibody or phytohaemagglutinin.1 Hence, this molecule has been designated CD100 by the International Workshop on Human Leukocyte Differentiation Antigen. In 1996, 3 years after the discovery of the semaphorin family,2 the CD100 gene was cloned and identified as the first semaphorin with immune system functions.3 Unlike the prototypical semaphorins, which are neuronal chemorepellents, Sema 4D/CD100 together with Sema 3A, Sema 4A, Sema 6D and Sema 7A have been described as ‘immune semaphorins', because they are expressed on T cells, B cells, natural killer cells and dendritic cells, and exhibit a variety of immunological functions.4 The Sema 4D/CD100 molecule can bind to several receptors. CD72, a member of the C-type lectin family, is a low-affinity Sema 4D/CD100 receptor that is expressed on immune cells, such as B cells, dendritic cells, macrophages and mast cells (Figure 1a).5 At least three Sema 4D/CD100 receptors are plexin family members. The high-affinity Sema 4D/CD100 receptor plexin B1 is mainly expressed on endothelial cells and epithelial cells (Figure 1b),6 whereas plexin B2 and plexin C1 are low-affinity receptors for Sema 4D/CD100.7 Figure 1 Multiple functions mediated by Sema 4D/CD100-plexin B counter-receptors. (a) As a ligand, the ligation of Sema 4D/CD100 with the low-affinity receptor CD72 stimulates the activation of, proliferation of and antibody production by B cells, promotes the ... Recently, Witherden et al.8 demonstrated that the interaction of Sema 4D/CD100 with plexin B2 plays a key role in activating skin-resident γδ T cells (DETCs) in mice to respond to tissue damage (Figure 1c). The morphological changes in DETCs that precede cutaneous wound healing appear to be associated with the activation of key actin regulators in response to Sema 4D/CD100's binding with its ligand. This is the first example indicating that Sema 4D/CD100 is not only a ligand for plexin B1 or CD72 but also a direct signaling receptor in mediating Sema 4D/CD100-plexin B's biological functions. It is not surprising to identify Sema 4D/CD100-plexin B as a counter-receptor, because many counter-receptors have already been well documented in the B7-CD28 family and TNF–TNFR superfamilies. Another important point in Witherden's paper8 is that the Sema 4D/CD100 molecule on γδ T cells mediates a function involved in innate immunity. Bonneville9 pointed out that plexin B2 is broadly expressed on many epithelial tissues where resident CD100-expressing γδ T cells are located, suggesting that the Sema 4D/CD100–plexin B2 interaction may play a more general role in the immune control of the integrity of epithelial barriers. It is thought that, similar to αβ T cells, complete activation of DETCs requires costimulatory signals. DETCs do not express many of the usual coreceptors that are important for αβ T-cell activation, such as CD4 or CD8, or the costimulatory molecule CD28. In 2010, Witherden et al.10 identified a receptor–ligand pair, junctional adhesion molecule-like molecule and the cocksackie adenovirus receptor (CAR), which plays a crucial role in the DETC response. In addition, DETCs can recognize a stress- or damage-induced keratinocyte self-antigen through their γδ T-cell receptor (TCR), without the requirement for antigen presentation by MHC class I or class II molecules. More recently, Komori et al.11 demonstrated the presence of an as yet-uncharacterized antigen that is expressed on the surface of damaged keratinocytes and is recognized by the canonical TCR expressed by DETCs. Furthermore, Witherden et al.8 proposed that when keratinocytes are damaged, they express a self-antigen, plexin B2 and CAR, which can be recognized by the γδ TCR, Sema 4D/CD100 and junctional adhesion molecule-like molecule, respectively, on neighboring γδ T cells (Figure 1c). Therefore, these interactions may play a key role in regulating the ERK and cofilin signaling pathways of activated γδ T cells, leading to morphologic changes and the release of keratinocyte growth factor type 1 (KGF1) and KGF2. These growth factors can induce the proliferation and migration of keratinocytes and the restoration of epithelial integrity.

Abstract LB-101: A novel role of semaphorin4a (sema4a) in genotoxin-induced DNA damage response in human lung fibroblasts

Abstract Dysregulation of DNA repair signaling after genotoxin exposure contributes to genomic instability and carcinogenesis. Hexavalent chromium [Cr(VI)] is a genotoxin of occupational and environmental concern. Protein tyrosine phosphatase (PTP) inhibitors (i.e., sodium orthovanadate (SOV)) activate key survival pathways. We reported that SOV enhances clonogenic survival after Cr(VI) exposure by overriding Cr-induced growth arrest in human lung fibroblasts (HLFs). PTP inhibition with SOV increased expression of DNA damage/repair genes as determined by microarray analysis. Kinetic analysis of DNA double-strand breaks (DSBs) by comet assay showed equal DSB formation 15 min after Cr(VI) exposure in the absence and presence of SOV. However, PTP inhibition significantly decreased DSBs 0.5-24 hr after Cr(VI) treatment. Aim of the present study was to determine the molecular mechanism(s) by which PTP inhibition affects DNA damage response during genotoxic stress. Gene expression was examined by DNA Damage Signaling Pathway PCR Array after 1 μM Cr(VI) treatment for 24 hr in the absence or presence of SOV. The PTP inhibitor reversed Cr-mediated changes in expression of several unique genes. Interestingly, the sema4a gene, known to be involved in T cell activation and endothelial cell proliferation and migration, was strongly induced by co-treatment of SOV and Cr(VI). Gene expression correlated with increased sema4a protein expression after co-treatment of SOV and Cr(VI). A potential role of sema4a in Cr-induced DNA DSB formation/repair in the absence or presence of SOV was assessed after sema4a siRNA transfection. Silencing of sema4a did not alter the effect of SOV on Cr-induced DSBs. However, sema4a silencing completely blocked Cr-induced DSBs 4 hr after exposure. These results suggest a novel role of sema4a in DNA DSB formation/repair in normal HLFs. Taken together, these data shed light on the emerging role of semaphorins as mediators of cell survival as well as potential therapeutic targets in carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-101.

Severe retinal degeneration associated with disruption of semaphorin 4A.

Semaphorin 4A (Sema4A) is a member of the transmembrane class 4 family of semaphorins. It has recently been shown to participate in cell-cell communication in the immune system. High levels of sema4A are also present in brain and eye, but its function in the central nervous system has not been studied. To investigate the function of Sema4A, we generated mice deficient in this transmembrane signaling molecule. An embryonic stem (ES) cell clone with a retroviral gene-trap insertion in the sema4A gene was used to generate mice lacking this transmembrane semaphorin. Fundus photography, fluorescein angiography, and electroretinography were used to evaluate retinal anatomy and physiology in mice lacking Sema4A. Electron microscopy and immunohistochemistry with cell-type-specific markers were used to characterize retinal development. In situ hybridization with sema4A-specific riboprobes was used to localize expression of this gene in the developing and adult eye. Fundus photography performed at 14 weeks of age revealed severe retinal degeneration, attenuated retinal vessels, and depigmentation in mice lacking Sema4A. At this age, the outer nuclear layer was reduced to a single row of photoreceptor cells, and the outer plexiform layer was thin and disorganized. Disruption of Sema4A also compromised the physiological function of both rod and cone photoreceptors. Developmental studies in Sema4A-deficient mice revealed abnormal morphology of photoreceptor outer segments during the time at which they establish contacts with apical microvilli of the retinal pigment epithelium (RPE). Sema4A is expressed in the inner retina and RPE during the time at which photoreceptor outer segments elongate. These findings identify a previously unknown function of Sema4A in the developing visual system and provide a useful model for understanding cell-cell interactions that occur between photoreceptors and the RPE.

Semaphorin 4A Enhances T Cell Activation

Semaphorins, originally identified as axon guidance regulators in the nervous system, are also involved in various aspects of immune cell function (see Bismuth and Boumsell). Kumanagoh et al. report that Sema 4A enhanced T cell activation, measured as proliferation and stimulation of interleukin and interferon production, in vitro and in vivo. In a mouse model of autoimmune encephalomyelitis (EAE), EAE was suppressed if the mice were injected with an antibody against Sema 4A. The Tim-2 protein, a member of a family of proteins expressed on T cells that contain immunoglobulin and mucin domains, was identified by expression cloning as a receptor for Sema 4A. Unlike the CD100 (also known as Sema 4D), Sema 4A stimulated tyrosine phosphorylation of Tim-2 in transfected COS7 cells--CD100 promotes dephosphorylation of its receptor CD72. G. Bismuth, L. Boumsell, Controlling the immune system through semaphorins. Science's STKE (2002), http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2002/128/re4. [Abstract] [Full Text] A. Kumanogoh, S. Marukawa, K. Suzuki, N. Takegahara, C. Watanabe, E. Ch'ng, I. Ishida, H. Fujimura, S. Sakoda, K. Yoshida, H. Kikutani, Class IV semaphorin Sema4A enhances T-cell activation and interacts with Tim-2. Nature 419 , 629-633 (2002). [Online Journal]