Mesenchymal stem cells exert differential effects on alloantigen and virus-specific T-cell responses

Mesenchymal stem cells (MSCs) suppress alloantigen-induced T-cell functions in vitro and infusion of third-party MSCs seems to be a promising therapy for graft-versus-host disease (GVHD). Little is known about the specificity of immunosuppression by MSCs, in particular the effect on immunity to pathogens. We have studied how MSCs affect T-cell responses specific to Epstein-Barr virus (EBV) and cytomegalovirus (CMV). We found that EBV- and CMV-induced proliferation and interferon- (IFN-) production from peripheral blood mononuclear cells (PBMCs) was less affected by third-party MSCs than the response to alloantigen and that MSCs had no effect on expansion of EBV and CMV pentamer-specific T cells. Established EBV-specific cytotoxic T cells (CTL) or CMV-CTL cultured with MSCs retained the ability to proliferate and produce IFN- in response to their cognate antigen and to kill virally infected targets. Finally, PBMCs from 2 patients who received MSCs for acute GVHD showed persistence of CMV-specific T cells and retained IFN- response to CMV after MSC infusion. In summary, MSCs have little effect on T-cell responses to EBV and CMV, which contrasts to their strong immunosuppressive effects on alloreactive T cells. These data have major implications for immunotherapy of GVHD with MSCs and suggest that the effector functions of virus-specific T cells may be retained after MSC infusion.     

Cardiovascular risk factors and blood pressure in a primary care unit: Yugoslav Study of the Precursors of Atherosclerosis in School Children (YUSAD)

OBJECTIVES: The presence of cardiovascular risk factors in children may be important in the development of atherosclerosis in adulthood. Adequate control of blood pressure is a cornerstone in atherosclerosis prevention. The aim of the Yugoslav Study of the Precursors of Atherosclerosis in School Children (YUSAD) was to identify risk factors for elevated blood pressure in school children. METHODS: The YUSAD study is a multicentre follow-up study comprised of two cross-sectional surveys conducted five years apart. At baseline, 10-year-old children (3226 boys and 3074 girls [n=6300]) were randomly selected during periodical visits to primary health care centres. The risk factors measured were heart rate, weight, body mass index (BMI), waist-to-hip ratio, grade point average and current smoking status. RESULTS: Significant age and sex differences were identified in systolic blood pressure, diastolic blood pressure and all investigated independent variables. In a multivariate analysis, diastolic blood pressure in 10-year-old boys was directly and significantly related to total cholesterol and height, whereas it was inversely related to weight. At follow-up, in the multivariate model, only BMI was a significant predictor of diastolic blood pressure in boys. In girls at baseline in the multivariate regression analysis, the only significant predictor of diastolic blood pressure was total cholesterol. In 15-year-old girls, diastolic blood pressure was significantly and directly related to BMI and heart rate, whereas it was inversely related to weight. For both 10- and 15-year-old male and female participants, none of the variables by multivariate analysis were a significant predictor of systolic blood pressure. CONCLUSIONS: Age, sex, heart rate, cholesterol and weight are the most important predictors of blood pressure in school children.     

The Variants in the 3′ Untranslated Region of the Matrix Metalloproteinase 9 Gene as Modulators of Treatment Outcome in Children with Asthma

Purpose

The maintaining of asthma control is difficult due to high variability in response to therapy among patients. Since matrix metalloproteinase 9 (MMP9) is implicated in inflammation and remodeling of asthmatic airways, it could be associated with adequate response to asthma therapy. The aim of this study was to investigate whether variants in 3′ end of the MMP9 gene are associated with clinical phenotype and responsiveness to treatment in children with asthma.

Methods

The study included 127 asthmatic children from Slovenia. Variants in the 3′ end of the MMP9 gene were analyzed by direct DNA sequencing and the obtained results were correlated with clinical parameters.

Results

Two variants were detected, rs13925 and rs20544. For the variant rs20544, statistically significant difference in airway hyperresponsiveness (p?=?0.011) and asthma control (p?=?0.049) between genotypes was found. Patients with TT genotype had lower airway sensitivity, and after 12 months of treatment showed significant improvement in Asthma Control Test (ACT) scores compared to CC and CT genotype. For the variant rs13925, the association with lung function was observed. The carriers of A allele showed noticeable improvement of lung function after the first 6 months of treatment in comparison to the carriers of G allele (p?=?0.046).

Conclusion

The main finding of our study is the association of MMP9 genotypes rs20544 TT and rs13925 AA and AG with better asthma control, and indirectly better response to treatment. Based on these results, MMP9 deserves further research as a potential predictive biomarker for asthma.

     

Thioredoxin-related protein of 14 kDa is an efficient L-cystine reductase and S-denitrosylase

Thioredoxin-related protein of 14 kDa (TRP14, also called TXNDC17 for thioredoxin domain containing 17, or TXNL5 for thioredoxin-like 5) is an evolutionarily well-conserved member of the thioredoxin (Trx)-fold protein family that lacks activity with classical Trx1 substrates. However, we discovered here that human TRP14 has a high enzymatic activity in reduction of l-cystine, where the catalytic efficiency (2,217 min⁻¹⋅µM⁻¹) coupled to Trx reductase 1 (TrxR1) using NADPH was fivefold higher compared with Trx1 (418 min⁻¹⋅µM⁻¹). Moreover, the l-cystine reduction with TRP14 was in contrast to that of Trx1 fully maintained in the presence of a protein disulfide substrate of Trx1 such as insulin, suggesting that TRP14 is a more dedicated l-cystine reductase compared with Trx1. We also found that TRP14 is an efficient S-denitrosylase with similar efficiency as Trx1 in catalyzing TrxR1-dependent denitrosylation of S-nitrosylated glutathione or of HEK293 cell-derived S-nitrosoproteins. Consequently, nitrosylated and thereby inactivated caspase 3 or cathepsin B could be reactivated through either Trx1- or TRP14-catalyzed denitrosylation reactions. TRP14 was also, in contrast to Trx1, completely resistant to inactivation by high concentrations of hydrogen peroxide. The oxidoreductase activities of TRP14 thereby complement those of Trx1 and must therefore be considered for the full understanding of enzymatic control of cellular thiols and nitrosothiols.The redox or nitrosylation state of reactive cysteine (Cys) residues in proteins can affect a multitude of intracellular events, either beneficial or harmful, depending upon biological context (1, 2). Two major cellular systems that control the redox states of Cys residues are the thioredoxin (Trx) and the glutathione (GSH) systems. The Trx system includes isoforms of Trx, Trx reductase (TrxR), and NADPH together with several Trx-dependent enzymes and proteins (3). The GSH/GSH disulfide redox couple is kept reduced by the NADPH-dependent activity of GSH reductase (GR) and donates electrons to isoforms of glutaredoxin (Grx) and other GSH-dependent enzymes (4).In addition to Trx, many proteins have a Trx fold and a Trx-like active-site sequence. One such protein is thioredoxin-related protein of 14 kDa (TRP14, also known as TXNDC17 or TXNL5), which is an evolutionarily well-conserved cytosolic and widely expressed Trx-fold protein that can be reduced by TrxR1 (5). Its crystal structure, compared with Trx1, shows additional structural features in the active site, thereby explaining its lack of activity with most classical Trx1 protein disulfide substrates including ribonucleotide reductase, insulin, peroxiredoxins, or methionine sulfoxide reductase (5–7). TRP14 was suggested to have evolved to exert specific signaling roles in cells and was identified as a modulator of TNFα/NFκB signaling pathways through interactions with the dynein light chain LC8 protein (6, 8). We previously found that treatment of cells with cisplatin triggered the formation of covalent cross-links between TrxR1 and either Trx1 or TRP14, which suggests that TRP14 is tightly linked to TrxR1 within the cellular context (9). Recently, we also found that TRP14 is able to reactivate oxidized phosphotyrosine phosphatase 1B, thereby indeed implicating specific functions in modulation of cellular signaling pathways (10).In addition to having general protein disulfide reductase activities, Trx1 is also a denitrosylase for a broad spectrum of nitrosoproteins and nitrosothiols (11, 12). Substrates include S-nitrosocaspase 3 (13, 14), S-nitrosocaspase 8 (15), S-nitrosoglutathione (GSNO) (16, 17), and S-nitrosocysteine (l-CysSNO) (12). Nitrosylation and denitrosylation reactions provide a regulatory mechanism for protein function and are thereby also involved in a variety of cellular signal transduction pathways. For example, S-nitrosylation of caspases can inhibit their activity and thus regulate apoptosis in resting cells (18, 19). A full understanding of NO homeostasis and its pathways is of medical importance because an aberrant formation of nitrosylated proteins has been implicated in a variety of diseases. However, protein denitrosylation is a hitherto less studied part in NO-mediated signaling (20, 21). In addition to Trx1, another enzyme mediating cellular denitrosylation reactions is GSNO reductase (GSNOR). GSNOR is the same enzyme as class III alcohol dehydrogenase, mainly catalyzing denitrosylation of GSNO using NADH as an electron donor (22, 23). In addition, S-denitrosylation activities are supported by protein disulfide isomerase (PDI) (24), carbonyl reductase 1 (25), and lipoic acid (17).The high intracellular concentrations of GSH are also important in NO metabolism because of facilitated formation of GSNO by reaction of GSH with NO or by denitrosylation of cellular nitrosothiols (20, 26). Because the synthesis of GSH depends upon availability, cellular uptake and reduction of sulfur-containing precursors such as l-cystine, l-cystine homeostasis is also important for GSH functions (27). l-Cystine is taken up into cells using different transport systems, e.g., the oxidative stress-inducible cystine/glutamate antiporter (system ) (28). The mechanism behind the reduction of l-cystine still has not been fully elucidated, but has been implicated to include GSH itself or also TrxR1-dependent systems (29).In the present study, we wanted to further characterize the enzymatic properties of TRP14, which revealed that the protein is at least as efficient as Trx1 in supporting reduction of specific redox substrates, such as l-cystine. In that assay, TRP14 is a fivefold better substrate for TrxR1 than Trx1 itself and, furthermore, more dedicated as its activity is not diminished in the presence of other Trx1 substrates that are not reduced by TRP14. Furthermore, we discovered that TRP14 is yet another cytosolic oxidoreductase that can catalyze S-denitrosylation reactions.     

Selective apoptosis of multiple myeloma cells in primary samples induced by arsenic trioxide

Objectives

Currently, multiple myeloma (MM) is an incurable disease. Despite the fact that arsenic trioxide (ATO) shows promising results in vitro, data from treatment of patients with MM are disappointing. Due to these discrepancies, we compared the efficacy and selectivity of ATO at two different concentrations in samples from MM patients.

Methods

The extent of apoptosis induced by 2 and 5 µM ATO was evaluated by flow cytometry using annexin V. 34 diagnostic bone marrow samples obtained from MM patients were analysed.

Results

5 µM ATO efficiently induced apoptosis in primary samples. Besides efficacy, also selectivity of action on MM cells in comparison to remaining haematopoietic cells was demonstrated for 5 µM ATO but not for 2 µM ATO.

Discussion

Our study on primary samples confirmed that ATO has a potential role in therapeutic management of MM. Further controlled studies on MM patients are needed.     

Increased activation of blood coagulation in pregnant women with the Factor V Leiden mutation

Background

The risk of venous thromboembolism is enhanced in pregnant carriers of the Factor V Leiden mutation. The primary aim of the study was to compare prothrombin fragments 1 + 2, soluble fibrin and D-dimer levels in pregnant Factor V Leiden mutation carriers with those in non-carriers. Secondary aims were to evaluate whether these biomarkers could predict placenta-mediated complications or venous thromboembolism, and to study blood coagulation after caesarean section with thromboprophylaxis and after vaginal delivery without thromboprophylaxis.

Material/Methods

Prothrombin fragments 1 + 2, soluble fibrin and D-dimer levels were studied longitudinally in 476 carriers with singleton pregnancies from gestational weeks 23–25 until 8–10 weeks postpartum.

Results

Prothrombin fragments 1 + 2 and D-dimer levels gradually increased during pregnancy. D-dimer levels were higher in carriers, both during pregnancy and puerperium, compared to non-carriers. D-dimer levels above 0.5 mg/l were found in about 30% and 20% of the heterozygous carriers at 4–5 and 8–10 weeks postpartum, respectively. Soluble fibrin levels were mainly unchanged during pregnancy, with no difference between carriers and non-carriers. Biomarker levels were similar in carriers with uncomplicated and complicated pregnancies.

Conclusion

Higher D-dimer levels indicate increased blood coagulation and fibrinolysis activity in carriers. The high proportion of carriers with D-dimer levels exceeding 0.5 mg/l postpartum must be considered when assessing the probability of venous thromboembolism. Large overlaps in biomarker levels in normal and complicated pregnancies suggest that these biomarkers cannot be used as predictors. Thromboprophylaxis following caesarean section may prevent increased activation of blood coagulation.