Preserved Antigen-Specific Immune Response in Patients with Multiple Sclerosis Responding to IFNβ-Therapy

Background

Interferon-beta (IFNβ) regulates the expression of a complex set of pro- as well as anti-inflammatory genes. In cohorts of MS patients unstratified for therapeutic response to IFNβ, normal vaccine-specific immune responses have been observed. Data capturing antigen-specific immune responses in cohorts of subjects defined by response to IFNβ-therapy are not available.

Objective

To assess antigen-specific immune responses in a cohort of MS patients responding clinically and radiologically to IFNβ.

Methods

In 26 MS patients, clinical and MRI disease activity were assessed before and under treatment with IFNβ. Humoral and cellular immune response to influenza vaccine was prospectively characterized in these individuals, and 33 healthy controls by influenza-specific Enzyme-Linked Immunosorbent Assay (ELISA) and Enzyme Linked Immuno Spot Technique (ELISPOT).

Results

Related to pre-treatment disease activity, IFNβ reduced clinical and radiological MS disease-activity. Following influenza vaccination, frequencies of influenza-specific T cells and concentrations of anti-influenza A and B IgM and IgG increased comparably in MS-patients and in healthy controls.

Conclusions

By showing in a cohort of MS-patients responding to IFNβ vaccine-specific immune responses comparable to controls, this study indicates that antigen-specific immune responses can be preserved under successful IFNβ-therapy.     

Extracellular HSP70/HSP70-PCs Promote Epithelial-Mesenchymal Transition of Hepatocarcinoma Cells

Background

Extracellular heat shock protein 70 and peptide complexes (eHSP70/HSP70-PCs) regulate a variety of biological behaviors in tumor cells. Whether eHSP70/HSP70-PCs are involved in the epithelial-mesenchymal transition (EMT) of tumor cells remains unclear.

Aims

To determine the effects of eHSP70/HSP70-PCs on EMT of hepatocarcinoma cells.

Methods

The expressions of E-cadherin, HSP70, α-smooth muscle actin protein (α-SMA) and p-p38 were detected immunohistochemically in liver cancer samples. Immunofluorescence, western blotting and real-time RT-PCR methods were used to analyze the effects of eHSP70/HSP70-PCs on the expressions of E-cadherin, α-SMA and p38/MAPK in vivo.

Results

HSP70, E-cadherin, α-SMA and p-p38 were elevated in hepatocellular carcinoma tissues. The expression of HSP70 was positively correlated with malignant differentiated liver carcinoma. The expressions of HSP70, α-SMA and p-p38 correlated with recurrence-free survival after resection. eHSP70/HSP70-PCs significantly promoted the expressions of α-SMA and p-p38 and reduced the expressions of E-cadherin in vivo. The effect was inhibited by SB203580.

Conclusion

The expressions of HSP70, E-cadherin, α-SMA and p-p38 may represent indicators of malignant potential and could discriminate the malignant degree of liver cancer. eHSP70/HSP70-PCs play an important role in the EMT of hepatocellular carcinoma via the p38/MAPK pathway.     

HELP LDL Apheresis Reduces Plasma Pentraxin 3 in Familial Hypercholesterolemia

Background

Pentraxin 3 (PTX3), a key component of the humoral arm of innate immunity, is secreted by vascular cells in response to injury, possibly aiming at tuning arterial activation associated with vascular damage. Severe hypercholesterolemia as in familial hypercholesterolemia (FH) promotes vascular inflammation and atherosclerosis; low-density lipoprotein (LDL) apheresis is currently the treatment of choice to reduce plasma lipids in FH. HELP LDL apheresis affects pro- and antiinflammatory biomarkers, however its effects on PTX3 levels are unknown. We assessed the impact of FH and of LDL removal by HELP apheresis on PTX3.

Methods

Plasma lipids, PTX3, and CRP were measured in 19 patients with FH undergoing chronic HELP LDL apheresis before and after treatment and in 20 control subjects. In the patients assessment of inflammation and oxidative stress markers included also plasma TNFα, fibrinogen and TBARS.

Results

At baseline, FH patients had higher (p = 0.0002) plasma PTX3 than matched control subjects. In FH PTX3 correlated positively (p≤0.05) with age, gender and CRP and negatively (p = 0.01) with HELP LDL apheresis vintage. The latter association was confirmed after correction for age, gender and CRP. HELP LDL apheresis acutely reduced (p≤0.04) plasma PTX3, CRP, fibrinogen, TBARS and lipids, but not TNFα. No association was observed between mean decrease in PTX3 and in LDL cholesterol. PTX3 paralleled lipids, oxidative stress and inflammation markers in time-course study.

Conclusion

FH is associated with increased plasma PTX3, which is acutely reduced by HELP LDL apheresis independently of LDL cholesterol, as reflected by the lack of association between change in PTX3 and in LDL levels. These results, together with the finding of a negative relationship between PTX3 and duration of treatment suggest that HELP LDL apheresis may influence both acutely and chronically cardiovascular outcomes in FH by modulating PTX3.     

Adipocytokine Profile,Cytokine Levels and Foxp3 Expression in Multiple Sclerosis: a Possible Link to Susceptibility and Clinical Course of Disease

Background

Adipocytokines may be involved in multiple sclerosis (MS) as well as other autoimmune and inflammatory-related diseases. This study aims to compare levels of resistin, visfatin and leptin in three subgroups of MS patients with healthy subjects and also to study their relationship with Foxp3 expression and levels of several pro-inflammatory mediators such as interleukine-1 β(IL-1 β),tumor necrosis factor-α (TNF-α) and human sensitive C-reactive protein (hs-CRP).

Methods

A total of 391 subjects including 200 healthy controls and 191 MS patients were recruited for this case-control study. Circulating adipocytokines and inflammatory mediators were measured using immunoassay methods. Foxp3 gene expression in peripheral blood mononuclear cells (PBMC) was determined by quantitative real-time PCR. Fat tissue mass was evaluated by using dual energy X-ray absorptiometery (DEXA).

Results

A significant difference was observed in levels of inflammatory mediators, adipocytokines, Foxp3 gene expression and adipose tissue mass between MS patients and healthy controls. All adipocytokines were positively correlated with levels of inflammatory mediators and negatively correlated with Foxp3 expression in MS patients. In controls, there were positive correlations between circulating leptin and resistin with TNF-α and IL-1β in subgroup analysis, the highest levels of TNF-α, IL-1β, hs-CRP, resistin and leptin were observed in primary progressive-MS (PP-MS) patients. Also, expression of Foxp3 and levels of visfatin in relapsing remitting-MS(RR-MS) patients were higher compared with the other subgroups.

Conclusions

Our findings suggest the potential role of adipocytokines in pathogenesis and severity of MS. Notably, the relationship of adipocytokines levels with inflammatory cytokines as well as clinical features of MS could be considerable in translational medicine and biomarker studies.     

The Quality of Life of Men Who Have Sex with Men in China: Reliability and Validity Testing of the SF-36 Questionnaire

Objective

The aim of the study was to assess the psychometric properties of the 36-Item Short Form Health Survey (SF-36) in the men who have sex with men (MSM) population in China.

Methods

A cross-sectional survey was conducted among 373 MSM from September to December, 2012, in Zhengzhou and Huludao City, China. Internal reliability of the questionnaire was calculated by Cronbach’s α coefficient. Validity was analyzed through construct validity, divisional validity, and collective validity testing.

Results

The overall Cronbach’s α coefficient of the SF-36 questionnaire was 0.943, while the Cronbach’s α coefficients for each of the dimensions were all > 0.70. Results showed that the SF-36 questionnaire was reliable and valid.

Conclusions

This study provided evidence that the SF-36 is an acceptable, valid and reliable instrument in evaluating the quality of life of MSM in Mainland China.     

Linezolid Has Unique Immunomodulatory Effects in Post-Influenza Community Acquired MRSA Pneumonia

Introduction

Post influenza pneumonia is a leading cause of mortality and morbidity, with mortality rates approaching 60% when bacterial infections are secondary to multi-drug resistant (MDR) pathogens. Staphylococcus aureus, in particular community acquired MRSA (cMRSA), has emerged as a leading cause of post influenza pneumonia.

Hypothesis

Linezolid (LZD) prevents acute lung injury in murine model of post influenza bacterial pneumonia

Methods

Mice were infected with HINI strain of influenza and then challenged with cMRSA at day 7, treated with antibiotics (LZD or Vanco) or vehicle 6 hours post bacterial challenge and lungs and bronchoalveolar lavage fluid (BAL) harvested at 24 hours for bacterial clearance, inflammatory cell influx, cytokine/chemokine analysis and assessment of lung injury.

Results

Mice treated with LZD or Vanco had lower bacterial burden in the lung and no systemic dissemination, as compared to the control (no antibiotic) group at 24 hours post bacterial challenge. As compared to animals receiving Vanco, LZD group had significantly lower numbers of neutrophils in the BAL (9×10³ vs. 2.3×10⁴, p < 0.01), which was associated with reduced levels of chemotactic chemokines and inflammatory cytokines KC, MIP-2, IFN-γ, TNF-α and IL-1β in the BAL. Interestingly, LZD treatment also protected mice from lung injury, as assessed by albumin concentration in the BAL post treatment with H1N1 and cMRSA when compared to vanco treatment. Moreover, treatment with LZD was associated with significantly lower levels of PVL toxin in lungs.

Conclusion

Linezolid has unique immunomodulatory effects on host inflammatory response and lung injury in a murine model of post-viral cMRSA pneumonia.     

Effects of Syzygium aromaticum-Derived Triterpenes on Postprandial Blood Glucose in Streptozotocin-Induced Diabetic Rats Following Carbohydrate Challenge

Purpose

Recent reports suggest that the hypoglycaemic effects of the triterpenes involve inhibition of glucose transport in the small intestine. Therefore, the effects of Syzygium spp-derived triterpenes oleanolic acid (OA) and maslinic acid (MA) were evaluated on carbohydrate hydrolyzing enzymes in STZ-induced diabetic rats and consequences on postprandial hyperglycaemia after carbohydrate loading.

Methods

We determined using Western blot analysis the expressions of α-amylase and α-glucosidase and glucose transporters SGLT1 and GLUT2 in the small intestine intestines isolated from diabetic rats treated with OA/MA for 5 weeks. In vitro assays were used to assess the inhibitory activities of OA and MA against α-amylase, α-glucosidase and sucrase.

Results

OA and MA ameliorated postprandial hyperglycemia in carbohydrate loaded diabetic rats as indicated by the significantly small glucose area under the curve (AUC) in treated diabetic animals compared with that in untreated diabetic rats. Western blotting showed that OA and MA treatment not only down-regulated the increase of SGLT1 and GLUT2 expressions in the small intestine of STZ-induced diabetic rats, but also inhibited small intestine α-amylase, sucrase and α-glucosidase activity. IC₅₀ values of OA against α-amylase (3.60 ± 0.18 mmol/L), α-glucosidase (12.40 ± 0.11 mmol/L) and sucrase (11.50 ± 0.13 mmol/L) did not significantly differ from those of OA and acarbose.

Conclusions

The results of suggest that OA and MA may be used as potential supplements for treating postprandial hyperglycemia.

Novelty of the Work

The present observations indicate that besides improving glucose homeostasis in diabetes, OA and MA suppress postprandial hyperglycaemia mediated in part via inhibition of carbohydrate hydrolysis and reduction of glucose transporters in the gastrointestinal tract. Inhibition of α-glucosidase and α-amylase can significantly decrease the postprandial hyperglycaemia after a mixed carbohydrate diet and therefore can be an important strategy in the management of postprandial blood glucose levels in NIDDM patients.     

Hepatitis C Virus NS3/4A Protease Inhibits Complement Activation by Cleaving Complement Component 4

Background

It has been hypothesized that persistent hepatitis C virus (HCV) infection is mediated in part by viral proteins that abrogate the host immune response, including the complement system, but the precise mechanisms are not well understood. We investigated whether HCV proteins are involved in the fragmentation of complement component 4 (C4), composed of subunits C4α, C4β, and C4γ, and the role of HCV proteins in complement activation.

Methods

Human C4 was incubated with HCV nonstructural (NS) 3/4A protease, core, or NS5. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then subjected to peptide sequencing. The activity of the classical complement pathway was examined using an erythrocyte hemolysis assay. The cleavage pattern of C4 in NS3/4A-expressing and HCV-infected cells, respectively, was also examined.

Results

HCV NS3/4A protease cleaved C4γ in a concentration-dependent manner, but viral core and NS5 did not. A specific inhibitor of NS3/4A protease reduced C4γ cleavage. NS3/4A protease–mediated cleavage of C4 inhibited classical pathway activation, which was abrogated by a NS3/4A protease inhibitor. In addition, co-transfection of cells with C4 and wild-type NS3/4A, but not a catalytic-site mutant of NS3/4A, produced cleaved C4γ fragments. Such C4 processing, with a concomitant reduction in levels of full-length C4γ, was also observed in HCV-infected cells expressing C4.

Conclusions

C4 is a novel cellular substrate of the HCV NS3/4A protease. Understanding disturbances in the complement system mediated by NS3/4A protease may provide new insights into the mechanisms underlying persistent HCV infection.     

Decrease in Urinary Creatinine Excretion in Early Stage Chronic Kidney Disease

Background

Little is known about muscle mass loss in early stage chronic kidney disease (CKD). We used 24-hour urinary creatinine excretion rate to assess determinants of muscle mass and its evolution with kidney function decline. We also described the range of urinary creatinine concentration in this population.

Methods

We included 1072 men and 537 women with non-dialysis CKD stages 1 to 5, all of them with repeated measurements of glomerular filtration rate (mGFR) by ⁵¹Cr-EDTA renal clearance and several nutritional markers. In those with stage 1 to 4 at baseline, we used a mixed model to study factors associated with urinary creatinine excretion rate and its change over time.

Results

Baseline mean urinary creatinine excretion decreased from 15.3±3.1 to 12.1±3.3 mmol/24 h (0.20±0.03 to 0.15±0.04 mmol/kg/24 h) in men, with mGFR falling from ≥60 to <15 mL/min/1.73 m², and from 9.6±1.9 to 7.6±2.5 (0.16±0.03 to 0.12±0.03) in women. In addition to mGFR, an older age, diabetes, and lower levels of body mass index, proteinuria, and protein intake assessed by urinary urea were associated with lower mean urinary creatinine excretion at baseline. Mean annual decline in mGFR was 1.53±0.12 mL/min/1.73 m² per year and that of urinary creatinine excretion rate, 0.28±0.02 mmol/24 h per year. Patients with fast annual decline in mGFR of 5 mL/min/1.73 m² had a decrease in urinary creatinine excretion more than twice as big as in those with stable mGFR, independent of changes in urinary urea as well as of other determinants of low muscle mass.

Conclusions

Decrease in 24-hour urinary creatinine excretion rate may appear early in CKD patients, and is greater the more mGFR declines independent of lowering protein intake assessed by 24-hour urinary urea. Normalizing urine analytes for creatininuria may overestimate their concentration in patients with reduced kidney function and low muscle mass.     

PKCδ Localization at the Membrane Increases Matrix Traction Force Dependent on PLCγ1/EGFR Signaling

Introduction

During wound healing, fibroblasts initially migrate into the wound bed and later contract the matrix. Relevant mediators of transcellular contractility revealed by systems analyses are protein kinase c delta/myosin light chain-2 (PKCδ/MLC-2). PKCδ is activated by growth factor-driven PLCγ1 hydrolysis of phosphoinositide bisphosphate (PIP₂) hydrolysis when it becomes tranlocated to the membrane. This leads to MLC-2 phosphorylation that regulates myosin for contractility. Furthermore, PKCδ n-terminus mediates PKCδ localization to the membrane in relative proximity to PLCγ1 activity. However, the role this localization and the relationship to its activation and signaling of force is not well understood. Therefore, we investigated whether the membrane localization of PKCδ mediates the transcellular contractility of fibroblasts.

Methods

To determine PKCδ activation in targeted membrane locations in mouse fibroblast cells (NR6-WT), two PKCδ constructs were generated; PKCδ-CaaX with farnesylation moiety targeting PKCδ to the membrane and PKCδ-SaaX a non-targeting control.

Results

Increased mean cell force was observed before and during EGF stimulation in fibroblasts expressing membrane-targeted PKCδ (PKCδ-CaaX) when analyzed with 2D cell traction force and 3D compaction of collagen matrix. This effect was reduced in cells deficient in EGFR/PLCy1 signaling. In cells expressing non-membrane targeted PKCδ (PKCδ-SaaX), the cell force exerted outside the ECM (extracellular matrix) was less, but cell motility/speed/persistence was increased after EGF stimulation. Change in cell motility and increased force exertion was also preceded by change in cell morphology. Organization of actin stress fibers was also decreased as a result of increasing membrane targeting of PKCδ.

Conclusion

From these results membrane tethering of PKCδ leads to increased force exertion on ECM. Furthermore, our data show PLCγ1 regulation of PKCδ, at least in part, drives transcellular contractility in fibroblasts.     

Development of a One-Step Probe Based Molecular Assay for Rapid Immunodiagnosis of Infection with M. tuberculosis Using Dried Blood Spots

Background

Antigen specific release of IP-10 is the most promising alternative marker to IFN-γ for infection with M. tuberculosis. Compared to Interferon-γ release assays (IGRA), IP-10 is released in high levels enabling novel approaches such as field friendly dried blood spots (DBS) and molecular detection.

Aim

To develop a robust IP-10 based molecular assay for the diagnosis of infection with M. tubercuolsis from whole blood and DBS.

Method

We developed a one-step probe based multiplex RT-qPCR assay for detecting IP-10 and IFN-γ mRNA expression from whole blood and DBS samples. The assay was validated and applied for the diagnosis of M. tuberculosis infection in DBS samples from 43 patients with confirmed TB, 13 patients with latent TB and 96 presumed uninfected controls. In parallel, IP-10 and INF-γ levels were measured in Quantiferon (QFT-TB) plasma supernatants.

Results

IP-10 mRNA upregulation was detectable at 4 hours after stimulation (6 fold upregulation) peaking at 8 hours (108 fold upregulation). IFN-γ expression occurred in concert but levels were lower (peak 6.7 fold upregulation). IP-10 gene expression level was significantly higher in patients with tuberculosis (median 31.2, IQR 10.7–67.0) and persons with latent tuberculosis infection (LTBI) (41.2, IQR 9.8–64.9) compared to healthy controls (1.6, IQR 1.1–2.4; p<0.0001). The IP-10 mRNA and protein based tests had comparable diagnostic accuracy to QFT-TB, sensitivity (85% and 88% vs 85%) and specificity (96% and 96% vs 97%, p = ns.).

Conclusion

We developed a rapid, robust and accurate molecular immunodiagnostic test for M. tuberculosis infection. By combining DBS based sample acquisition, mail or currier based sample transport with centralized molecular detection, this immunodiagnostic test concept can reduce the local technological requirements everywhere and make it possible to offer highly accurate immunodiagnostic tests in low resource settings.     

Pegylated Interferon-α2a Inhibits Proliferation of Human Liver Cancer Cells In Vitro and In Vivo

Purpose

We investigated the effects of pegylated interferon-α2a (PEG-IFN-α2a) on the growth of human liver cancer cells.

Methods

The effect of PEG-IFN-α2a on the proliferation of 13 liver cancer cell lines was investigated in vitro. Cells were cultured with medium containing 0–4,194 ng/mL of PEG-IFN-α2a, and after 1, 2, 3, or 4 days of culture, morphologic observation and growth assay were performed. After hepatocellular carcinoma (HCC) cells (HAK-1B and KIM-1) were transplanted into nude mice, various doses of PEG-IFN-α2a were subcutaneously administered to the mice once a week for 2 weeks, and tumor volume, weight, and histology were examined.

Results

PEG-IFN-α2a inhibited the growth of 8 and 11 cell lines in a time- and dose-dependent manner, respectively, although the 50% growth inhibitory concentrations of 7 measurable cell lines on Day 4 were relatively high and ranged from 253 ng/mL to 4,431 ng/mL. Various levels of apoptosis induction were confirmed in 8 cell lines. PEG-IFN-α2a induced a dose-dependent decrease in tumor volume and weight, and a significant increase of apoptotic cells in the tumor. Subcutaneous administration of clinical dose for chronic hepatitis C (3 μg/kg, 0.06 μg/mouse) was effective and induced about 30-50% reduction in the tumor volume and weight as compared with the control.

Conclusions

Although in vitro anti-proliferative effects of PEG-IFN-α2a were relatively weak, PEG-IFN-α2a induced strong anti-tumor effects on HCC cells in vivo. The data suggest potential clinical application of PEG-IFN-α2a for the prevention and treatment of HCC.     

Cytokine Profiles at Birth Predict Malaria Severity during Infancy

Background

Severe malaria risk varies between individuals, and most of this variation remains unexplained. Here, we examined the hypothesis that cytokine profiles at birth reflect inter-individual differences that persist and influence malaria parasite density and disease severity throughout early childhood.

Methods and Findings

Cytokine levels (TNF-α, IFN-γ, IL-1β, IL-4, IL-5, IL-6 and IL-10) were measured at birth (cord blood; N=783) and during subsequent routine follow-up visits (peripheral blood) for children enrolled between 2002 and 2006 into a birth cohort in Muheza, Tanzania. Children underwent blood smear and clinical assessments every 2-4 weeks, and at the time of any illness. Cord blood levels of all cytokines were positively correlated with each other (Spearman’s rank correlation). Cord levels of IL-1β and TNF-α (but not other cytokines) correlated with levels of the same cytokine measured at routine visits during early life (P < 0.05). Higher cord levels of IL-1β but not TNF-α were associated with lower parasite densities during infancy (P=0.003; Generalized Estimating Equation (GEE) method), with an average ~40% reduction versus children with low cord IL-1β levels, and with decreased risk of severe malaria during follow-up (Cox regression): adjusted hazard ratio (95% CI) 0.60 (0.39-0.92), P = 0.02.

Conclusion

IL-1β levels at birth are related to future IL-1β levels as well as the risk of severe malaria in early life. The effect on severe malaria risk may be due in part to the effect of inflammatory cytokines to control parasite density.     

Aromatized to Find Mates: α-Pinene Aroma Boosts the Mating Success of Adult Olive Fruit Flies

Background

Contrary to other Tephritidae, female but also male olive flies, Bactrocera oleae release pheromones during their sexual communication. Alpha-pinene, a common plant volatile found in high amounts in unripe olive fruit and leaves has been detected as one of the major components of the female pheromone. However, possible effects of α-pinene and that of other host volatiles on the mating behavior of the olive fly have not been investigated.

Methodology

Using wild olive flies, reared on olive fruit for 3 generations in the laboratory, we explored whether exposure of male and female olive flies to α-pinene affects their sexual performance.

Results

Exposure of sexually mature adult olive flies to the aroma of α-pinene significantly increases the mating performance over non-exposed individuals. Interestingly, exposure to α-pinene boosts the mating success of both males and female olive flies.

Conclusions

This is the first report of such an effect on the olive fly, and the first time that a single plant volatile has been reported to induce such a phenomenon on both sexes of a single species. We discuss the possible associated mechanism and provide some practical implications.     

The Contribution of Non-Conventional T Cells and NK Cells in the Mycobacterial-Specific IFNγ Response in Bacille Calmette-Guérin (BCG)-Immunized Infants

Background

The Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccine is given to >120 million infants each year worldwide. Most studies investigating the immune response to BCG have focused on adaptive immunity. However the importance of TCR-gamma/delta (γδ) T cells and NK cells in the mycobacterial-specific immune response is of increasing interest.

Methods

Participants in four age-groups were BCG-immunized. Ten weeks later, in vitro BCG-stimulated blood was analyzed for NK and T cell markers, and intracellular IFNgamma (IFNγ) by flow cytometry. Total functional IFNγ response was calculated using integrated median fluorescence intensity (iMFI).

Results

In infants and children, CD4 and CD4-CD8- (double-negative (DN)) T cells were the main IFNγ-expressing cells representing 43-56% and 27-37% of total CD3+ IFNγ+ T cells respectively. The iMFI was higher in DN T cells compared to CD4 T cells in all age groups, with the greatest differences seen in infants immunized at birth (p=0.002) or 2 months of age (p<0.0001). When NK cells were included in the analysis, they accounted for the majority of total IFNγ-expressing cells and, together with DN Vδ2 γδ T cells, had the highest iMFI in infants immunized at birth or 2 months of age.

Conclusion

In addition to CD4 T cells, NK cells and DN T cells, including Vδ2 γδ T cells, are the key populations producing IFNγ in response to BCG immunization in infants and children. This suggests that innate immunity and unconventional T cells play a greater role in the mycobacterial immune response than previously recognized and should be considered in the design and assessment of novel tuberculosis vaccines.     

Syndecan-3 and TFPI Colocalize on the Surface of Endothelial-, Smooth Muscle-, and Cancer Cells

Background

Tissue factor (TF) pathway inhibitor (TFPI) exists in two isoforms; TFPIα and TFPIβ. Both isoforms are cell surface attached mainly through glycosylphosphatidylinositol (GPI) anchors. TFPIα has also been proposed to bind other surface molecules, like glycosaminoglycans (GAGs). Cell surface TFPIβ has been shown to exert higher anticoagulant activity than TFPIα, suggesting alternative functions for TFPIα. Further characterization and search for novel TFPI binding partners is crucial to completely understand the biological functions of cell associated TFPI.

Methods and Results

Potential association of TFPI to heparan sulphate (HS) proteoglycans in the syndecan family were evaluated by knock down studies and flow cytometry analysis. Cell surface colocalization was assessed by confocal microscopy, and native PAGE or immunoprecipitation followed by Western blotting was used to test for protein interaction. Heparanase was used to enzymatically degrade cell surface HS GAGs. Anticoagulant potential was evaluated using a factor Xa (FXa) activity assay. Knock down of syndecan-3 in endothelial,- smooth muscle- and breast cancer cells reduced the TFPI surface levels by 20-50%, and an association of TFPIα to syndecan-3 on the cell surface was demonstrated. Western blotting indicated that TFPIα was found in complex with syndecan-3. The TFPI bound to syndecan-3 did not inhibit the FXa generation. Removal of HS GAGs did not release TFPI antigen from the cells.

Conclusions

We demonstrated an association between TFPIα and syndecan-3 in vascular cells and in cancer cells, which did not appear to depend on HS GAGs. No anticoagulant activity was detected for the TFPI associated with syndecan-3, which may indicate coagulation independent functions for this cell associated TFPI pool. This will, however, require further investigation.     

Valproic Acid Treatment Inhibits Hypoxia-Inducible Factor 1α Accumulation and Protects against Burn-Induced Gut Barrier Dysfunction in a Rodent Model

Objective

Burn-induced gut dysfunction plays an important role in the development of sepsis and multiple organ dysfunction. Emerging evidence suggests that hypoxia-inducible factor-1α (HIF-1α) is critical in paracelluar barrier functions via regulating vascular endothelial growth factor (VEGF) and myosin light chain kinase (MLCK) expression. Previous studies have also demonstrated that histone deacetylase inhibitors (HDACIs) can repress HIF-1α. This study aims to examine whether valproic acid (VPA), a HDACI, protects against burn-induced gut barrier dysfunction via repressing HIF-1α-dependent upregulation of VEGF and MLCK expression.

Methods

Rats were subjected to third degree 55% TBSA burns and treated with/ without VPA (300mg/kg). Intestinal barrier dysfunction was evaluated by permeability of intestinal mucosa to fluorescein isothiocyanate (FITC)-dextran and histologic evaluation. Histone acetylation, tight junction protein zonula occludens 1 (ZO-1), VEGF, MLCK and HIF-1α were measured. In addition, CaCO₂ cells were transfected with siRNA directed against HIF-1α and were stimulated with CoCl2 (1mM) for 24 hours with/without VPA (2mM) followed by analysis of HIF-1α, MLCK, VEGF and ZO-1.

Results

Burn insults resulted in a significant increase in intestinal permeability and mucosal damage, accompanied by a significant reduction in histone acetylation, ZO-1, upregulation of VEGF, MLCK expression, and an increase in HIF-1α accumulation. VPA significantly attenuated the increase in intestinal permeability, mucosa damage, histone deacetylation and changes in ZO-1 expression. VPA also attenuated the increased VEGF, MLCK and HIF-1α protein levels. VPA reduced HIF-1α, MLCK and VEGF production and prevented ZO-1 loss in CoCl2-stimulated Caco-2 cells. Moreover, transfection of siRNA directed against HIF-1α led to inhibition of MLCK and VEGF production, accompanied by upregulation of ZO-1.

Conclusions

These results indicate that VPA can protect against burn-induced gut barrier dysfunction. These protective effects may be due to its inhibitory action on HIF-1α, leading to a reduction in intestinal VEGF and MLCK expression and minimizing ZO-1 degradation.