Single-cell analysis reveals isotype-specific autoreactive B cell repertoires in Sjögren's syndrome.

Microengraving is a novel technology that uses an array of microfabricated subnanoliter wells to isolate and characterize secreted proteins from larger number of single cells. This printing technique permits the capture and characterization of secreted antibodies on glass slides. Here, we profiled the antigenic repertoires of B cells reacting against salivary gland tissues in Sjögren's syndrome (SjS), an autoimmune disease targeting the exocrine glands. Single-cell suspensions of spleen and cervical lymph node cells prepared from normal C57BL/6 and SjS-susceptible (SjS(s)) C57BL/6.NOD-AecAec2 mice were dispersed into subnanoliter wells (nanowells). Capture slides preincubated with mouse immunoglobulins were used for printing. Detection antibodies included fluorescence conjugated anti-IgG1, salivary gland lysates of C57BL/6 and SjS(s) mice. Results indicate an increase in the frequency of IgG1-secreting cells in the spleen of SjS(s) mice compared to C57BL/6 mice. Cells from the lymph node of SjS(s) mice yield higher instances of IgG1 reactive against salivary gland antigens than cells from the lymph nodes of C57BL/6 mice. These data demonstrate the isotype-specific reactivity of antibodies during the autoimmune process, and further reveals significant differences in the non-autoimmune and autoimmune antibody repertoires. These results support the generation of self-reactive B cell repertoires during the autoimmune process, at the same time, verifying that microengraving of single cells might allow for identification of novel biomarkers in SjS.

Endothelial protein C receptor and pediatric arterial stroke.

OBJECTIVE: The aim of this study was to investigate the endothelial protein C receptor (EPCR) gene A3 haplotype and plasma soluble EPCR (sEPCR) levels in Turkish pediatric arterial stroke patients. MATERIALS AND METHODS: We analyzed 44 pediatric arterial stroke patients and 75 healthy controls. Following DNA isolation, genotyping of the A3 haplotype was determined via PCR and RFLP. Additionally, fasting sEPCR levels were determined via ELISA. RESULTS: There wasn't a significant difference in the sEPCR level between the control and patient groups, although the sEPCR level was higher in the patient group. We didn't observe a difference in the distribution of the CC and CG/GG genotypes between the control and patient groups. CONCLUSION: Further study on sEPCR levels at the onset of pediatric stroke is needed in order to reach a more definitive conclusion. CONFLICT OF INTEREST: None declared.

Zinc transporter ZIP14 functions in hepatic zinc, iron and glucose homeostasis during the innate immune response (endotoxemia).

ZIP14 (slc39A14) is a zinc transporter induced in response to pro-inflammatory stimuli. ZIP14 induction accompanies the reduction in serum zinc (hypozincemia) of acute inflammation. ZIP14 can transport Zn(2+) and non-transferrin-bound Fe(2+) in vitro. Using a Zip14(-/-) mouse model we demonstrated that ZIP14 was essential for control of phosphatase PTP1B activity and phosphorylation of c-Met during liver regeneration. In the current studies, a global screening of ZIP transporter gene expression in response to LPS-induced endotoxemia was conducted. Following LPS, Zip14 was the most highly up-regulated Zip transcript in liver, but also in white adipose tissue and muscle. Using ZIP14(-/-) mice we show that ZIP14 contributes to zinc absorption from the gastrointestinal tract directly or indirectly as zinc absorption was decreased in the KOs. In contrast, Zip14(-/-) mice absorbed more iron. The Zip14 KO mice did not exhibit hypozincemia following LPS, but do have hypoferremia. Livers of Zip14-/- mice had increased transcript abundance for hepcidin, divalent metal transporter-1, ferritin and transferrin receptor-1 and greater accumulation of iron. The Zip14(-/-) phenotype included greater body fat, hypoglycemia and higher insulin levels, as well as increased liver glucose and greater phosphorylation of the insulin receptor and increased GLUT2, SREBP-1c and FASN expression. The Zip14 KO mice exhibited decreased circulating IL-6 with increased hepatic SOCS-3 following LPS, suggesting SOCS-3 inhibited insulin signaling which produced the hypoglycemia in this genotype. The results are consistent with ZIP14 ablation yielding abnormal labile zinc pools which lead to increased SOCS-3 production through G-coupled receptor activation and increased cAMP production as well as signaled by increased pSTAT3 via the IL-6 receptor, which inhibits IRS 1/2 phosphorylation. Our data show the role of ZIP14 in the hepatocyte is multi-functional since zinc and iron trafficking are altered in the Zip14(-/-) mice and their phenotype shows defects in glucose homeostasis.

The Effects of Endothelial Protein C Receptor Gene Polymorphisms on the Plasma sEPCR Level in Venous Thrombosis Patients.

OBJECTIVE: The aim of this study was to investigate variations in the endothelial cell protein C receptor gene (EPCRgene) that may play a role in thrombosis and the effects of these variations on the plasma soluble endothelial cell proteinC receptor (sEPCR) level in Turkish patients with venous thrombosis. MATERIAL AND METHODS: This study included 111 thrombosis patients and 73 healthy controls. Following DNAextraction, PCR, SSCP, and DNA sequencing analysis of 4 exons of the EPCR gene was performed. Plasma sEPCR wasmeasured via enzyme-linked immunosorbent assay (ELISA). RESULTS: In all, 3 polymorphisms were detected in exons 1-4. C3998T (SNP no: rs2069952) polymorphism was detectedin intron 2 and C4678G (A1 haplotype) (SNP no: rs9574) in the 3' untranslated region (3'UTR). There weren't anysignificant differences in C3998T polymorphism between the control and patient groups. There wasn't a significantdifference in plasma sEPCR levels between both controls and patients that carried the A1 allele. A4600G substitution (A3haplotype) (SNP no: rs867186) was observed in exon 4 and was associated with a 2.04-fold higher risk of thrombosis.A3 allele carriers had higher sEPCR levels than those without the allele. Mean sEPCR level in the patients with thehomozygous A3 allele was 289 ng µL-1, versus 113.42 ng µL-1 in those with the homozygous A1 allele. CONCLUSION: The A1 haplotype might offer protection against thrombosis and the A3 haplotype might be associatedboth with elevated plasma sEPCR and elevated risk of venous thrombosis in the Turkish population. Plasma sEPCRlevels were significantly higher in those that carried the A3 allele (4600A>G) (both patients and controls). Among theparticipants that carried the A1 allele (4678C>G), plasma sEPCR did not differ significantly.

The Dual Diverse Dynamic Reversible Effects of Ankaferd Blood Stopper on EPCR and PAI-1 Inside Vascular Endothelial Cells With and Without LPS Challenge.

OBJECTIVE: Ankaferd blood stopper (ABS) is comprised of a mixture of the plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica. ABS is used as a topical hemostatic agent due to its antihemorrhagic effect, yet its hemostatic mechanism of action remains to be investigated. ABS does not affect the levels of coagulation factors II, V, VII, VIII, IX, X, XI and XII. The aim of this study was to investigate the effects of ABS on endothelium and immune response. As such, we evaluated changes in endothelial cell protein C receptor (EPCR) and plasminogen activator inhibitor type-1 (PAI-1) expression inside human umbilical vein endothelial cells (HUVECs) in the presence and absence of lipopolysaccharides (LPSs). MATERIAL AND METHODS: We exposed HUVECs to 10 µL and 100 µL of ABS for 5 min, 25 min, 50 min, 6 h, and 24 h. Additionally, 10 µg mL-1 of LPS was administered for 1 h to observe the effects of LPS challenge on HUVECs, and then the cells were treated with ABS for 5 min, 25 min, 50 min, and 6 h to observe the effects of ABS on HUVECs. Total RNA was isolated from HUVECs and then the level of expression of EPCR and PAI-1 mRNA was measured. RESULTS: Cells were microscopically observed to arise from the surface and adhere to each other following the administration of ABS to HUVECs. Additionally, after 24 h the cells had normal growth and physiology, which suggests that the adhesive cellular effects of ABS might be reversible. ABS had a negative effect on EPCR and PAI-1 expression; the effect in response to 100 µL was greater than that to 10 µL. EPCR and PAI-1 expression increased over time in response to LPS and 10 µL of ABS. EPCR and PAI-1 expression was very low during the first hour of exposure to LPS and 100 µL of ABS, but after 6 h increased to levels similar to those observed in response to LPS and 10 µL of ABS. CONCLUSION: It was observed that ABS had dual diverse dynamic reversible effects on EPCR and PAI-1 expression in HUVECs, which were dependent on dose and concentration. ABS might play a role in numerous cellular mechanisms, in addition to having hemostatic effects. CONFLICT OF INTEREST: None declared.

The association of protease activated receptor 1 gene -506 I/D polymorphism with disease-free survival in breast cancer patients.

BACKGROUND: Recent data have shown that tumor development and dissemination may be regulated by procoagulant/anticoagulant axis. The aim of the present study was to search for an association of the protease activated receptor (PAR)1 gene -506 insertion/deletion (I/D), factor V Leiden (FVL), prothrombin (PT) G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms with disease-free survival (DFS) in breast cancer. METHODS: Genotyping of -506 I/D in the promoter region of PAR1 gene was performed by single-strand conformation polymorphism analysis and sequencing. FVL, PT G20210A, and MTHFR C677T were also determined by the method of polymerase chain reaction-based DNA analysis. Data regarding patient's age, menopausal status, tumor size, lymph node status, disease stage, tumor grade, estrogen and progesterone receptor, c-erb B2 expression, PAR1 -506 I/D, MTHFR C677T, FVL, and PT G20210A polymorphisms were examined by the univariate and multivariate analyses. RESULTS: Recurrent disease occurred in 29 patients (19.6 %) within a median of 20 months. It was found that tumor size, lymph node status, tumor stage, tumor grade, c-erbB2 expression, and PAR1 -506 I/D polymorphism were associated with DFS when Kaplan-Meier method was applied (P<.05). By Cox proportional hazards model, the presence of allele D at -506 locus (P=.0249) and small tumor size (P=.0001) were significant favorable prognostic factor, but c-erbB2 expression was an adverse prognostic factor (P=.0049). CONCLUSION: Our study suggested the protective effect of the allele D at -506 locus of PAR1 gene on the recurrence of breast cancer.

Reversible protease-activated receptor 1 downregulation mediated by Ankaferd blood stopper inducible with lipopolysaccharides inside the human umbilical vein endothelial cells.

Ankaferd Blood Stopper (ABS) is a novel topical hemostatic agent with pleiotropic actions indicated in clinical hemorrhages. Protease-activated receptor 1 (PAR-1) is located in the crossroads of hemostasis, inflammation, infection, apoptosis and tumorigenesis. ABS-induced formation of the protein network with vital erythroid aggregation covers the entire physiological hemostatic process. The aim of this study is to assess the effects of ABS on PAR-1 in the Human Umbilical Vein Endothelial Cells (HUVEC) model, in relation to the "ipopolysaccharides (LPS)-challenge" to endothelium. For this purpose, ABS 10 µL and 100 µL, had been applied to HUVEC within the time periods of 5 minutes (min), 25 min, 50 min, 6 hours (h) and 24 h. The cells have lifted from the plastic surface and adhered to each other during theABSapplication to the HUVECs. After 24 hours the cells returned to normal baseline level. We observed dose-dependent reversible PAR-1 down-regulation mediated by ABS inside the human umbilical vein endothelial cells. ABS-induced sustained PAR-1 down-regulation in the presence of LPS. Those findings indicated that ABS hemostatic agent may act as a topical biological response modifier by acting on PAR-1 at the vascular endothelial and cellular level.