Characterization of the thrombin generation profile in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multisystemic inflammatory autoimmune disorder. Thrombotic events occur at a higher incidence among SLE patients. The investigation of thrombin generation (TG) with calibrated automated thrombogram (CAT) test as a global hemostasis assay is applicable for the overall functional assessment of the hemostasis. The aim of this study was to characterize the hemostatic alterations observed in SLE by CAT assay. In this study, CAT parameters and basic coagulation parameters of SLE patients (n = 22) and healthy control subjects (n = 34) were compared. CAT area under the curve (i.e., endogenous thrombin potential) was lower than normal in SLE (807 vs. 1,159 nM*min, respectively), whereas other CAT parameters (peak, lag time, time to peak, and velocity index) and the basic coagulation tests were within the normal range. The presence of anti-phospholipid antibodies and the applied therapy was not associated with hemostasis parameters in SLE. We concluded that the reported high risk of thrombosis is not related to TG potential.

Relation of circulating T cell profiles to airway inflammation and asthma control in asthmatic pregnancy.

Asthmatic inflammation during pregnancy poses a risk for maternal and fetal morbidities. Circulating T cell immune phenotype is known to correlate with airway inflammation (detectable by fractional concentration of nitric oxide present in exhaled breath (FENO)) in non-pregnant allergic asthmatics. The aim of this study was to assess the relationship of peripheral T cell phenotype to FENO and clinical variables of asthma during pregnancy.We examined 22 pregnant women with allergic asthma in the 2nd/3rd trimester. The prevalence of Th1, Th2, regulatory T (Treg) and natural killer (NK) cell subsets was identified with flow cytometry using cell-specific markers. FENO, Asthma Control Test (ACT) total score and lung function were evaluated.Peripheral blood Th1, Th2, Treg, and NK cell prevalence were not significantly correlated to airway inflammation assessed by FENO in asthmatic pregnant women (all cells p > 0.05; study power > 75%). However, an inverse correlation was detected between Th2 cell prevalence and ACT total scores (p = 0.03) in asthmatic pregnancy.Blunted relationship between T cell profile and airway inflammation may be the result of pregnancy induced immune tolerance in asthmatic pregnancy. On the other hand, increased Th2 response impairs disease control that supports direct relationship between symptoms and cellular mechanisms of asthma during pregnancy.

Factors influencing skin autofluorescence of patients with peritoneal dialysis.

Skin autofluorescence (SAF) measurement is a simple, noninvasive method to assess tissue advanced glycation end products (AGE). In patients with end-stage renal disease and in those on hemodialysis AGE production is increased. Less is known about those treated with peritoneal dialysis (PD). In this study we tested if SAF is influenced by clinical and treatment characteristics in PD patients.This cross-sectional study included 198 PD patients (of those, 128 were on traditional glucose-based solutions and 70 patients were partially switched to icodextrin-based PD). SAF measurements were done with a specific AGE Reader device. The impact of patients' age, gender, current diabetes, duration of PD, cumulative glucose exposure, body mass index, smoking habits and use of icodextrin on SAF values were tested with multiple regression analysis.Our analysis revealed that patients' age, current diabetes and icodextrin use significantly increase patients' SAF values (p = 0.015, 0.012, 0.005, respectively). AGE exposure of PD patients with diabetes and on icodextrin solution is increased. Further investigation is required whether this finding is due to the icodextrin itself or for a still unspecified clinical characteristic of PD population treated with icodextrin.

Resistance of human regulatory Foxp3+ T cells to normobaric hyperoxia exposure under resting and stimulating conditions.

Oxygen tension levels may modulate immune responses. Evidence shows that hyperoxia influences the risk of infection, autoimmunity and alloreactivity and hence is a possible therapeutic option in a number of disorders. Regulatory T cells (Tregs) play a central role in tolerance maintenance, but their behaviour under hyperoxia is largely unknown. We investigated in vitro the impact of normobaric hyperoxia on human Tregs and their cellular network. Peripheral blood mononuclear cells isolated from six healthy men were cultured under normoxia and escalating duration of normobaric hyperoxia (10 min, 1, 16, 88 h) under resting conditions and at the presence of anti-CD3/CD28 beads. Foxp3+ Tregs' and other T cell subsets' survival, proliferation, activation, maturation and Th1/Th2 markers were assessed by flow cytometry. We observed decreasing CD4+ cell survival with increasing duration of hyperoxia irrespectively of the presence of stimulators. The prevalence of CD4+ CD45RA+ cells increased under stimulation (P=0.001). In stimulated samples, the proliferation and induced Foxp3 expression decreased after 88 h of hyperoxia (both P=0.001). In conclusion, normobaric hyperoxia up to 16 h does not induce significant changes in basic human T cell subsets, including the prevalence naturally occurring Tregs. Prolonged exposure to hyperoxia likely affects all unstimulated T cell subsets in a similar way. In stimulated T lymphocytes, the proliferation is hampered and cell death increases more evidently after prolonged hyperoxia (several days). Inducible Foxp3 expression is likely closely related to these processes. Naive CD4+ T cells are maintained by stimulation during exposure to hyperoxia.

Maternal cytokine balance on the third postpartum day is not affected by the mode of delivery after healthy pregnancies.

Previous reports have suggested that delivery is associated with the induction of inflammatory cytokines. The present study was designed to investigate whether increased cytokine production was present on postpartum day 3 after a normal pregnancy and whether any changes were associated with the mode of delivery. In total, 33 pregnant women were enrolled; 18 delivered vaginally and 15 underwent an elective caesarean section (C-section). The levels of 17 cytokines and growth hormones were measured at the beginning of delivery or before anaesthesia and on postpartum day 3. While interleukin (IL)-6 and IL-8 levels decreased significantly postpartum, other cytokine concentrations were comparable before and after delivery. Only IL-7 levels were significantly increased in the C-section patients compared with the vaginal birth patients postpartum. In conclusion, there was no risk of a prolonged maternal inflammatory reaction after an uncomplicated vaginal birth or elective C-section, so it is probably not necessary to consider this as an issue when making a decision on the mode of delivery following uncomplicated pregnancy.

Effect of 3 months of doxazosin therapy on T-cell subsets in type 2 diabetic patients.

Doxazosin, an alpha(1)-adrenergic receptor inhibitor, is commonly administered to patients with type 2 diabetes, hypertension and nephropathy. The impact of 3 months' doxazosin therapy on the prevalence of activated and regulatory T lymphocytes was analysed in this pilot study of men with type 2 diabetes (n = 10) who received doxazosin 4 mg/day in addition to their ongoing therapy. The prevalence of CD4(+), CD8(+), CD25(+) and CD69(+) cells at baseline and after 3 months of add-on therapy was determined. The prevalence of regulatory T-cells was detected by two different approaches: forkhead box P3 (FoxP3) positivity; and the number of CD4(+)CD25(+high) cells. During 3 months of doxazosin therapy, patients' blood pressure, blood glucose control and lipid profiles all significantly improved. Simultaneously, the prevalence of activated T-cells (CD4(+)CD69(+) and CD8(+)CD69(+) cells) decreased, whereas that of regulatory T-cells increased. These results indicate an immunomodulatory action of doxazosin in type 2 diabetic patients.

Cytometry-acquired calcium-flux data analysis in activated lymphocytes.

Flow cytometry enables the sequential determination of calcium levels in millions of stimulated lymphocytes over a short period of time. Current algorithms available are not suitable for the statistical analysis of this large amount of data. The authors aimed to develop a robust algorithm that fits a function to median values of measured data and provides an opportunity for statistical comparison between different calcium-flux measurements. The alteration of calcium signal was monitored in CD4+ cells loaded with calcium binding fluorescent dyes and stimulated with phytohemagglutinin; the alteration of calcium signal was monitored for 10 minutes. The authors also reanalyzed published calcium-flux data of CD3+ cells and Jurkat cells stimulated with different concentrations of anti-CD3 and thapsigargin. The authors fitted different functions to the medians of data per time unit and identified hormesis function as the best fitting one. On the basis of the optimally fitting function, the authors calculated the most relevant biological descriptors such as starting value, peak, time to reach the maximum, and time to reach 50% of maximum before and after the peak. Statistically significant differences in cell activation kinetics at different stimulatory concentrations were also demonstrated. This approach enables us to characterize the kinetics and distribution of calcium-flux data derived by flow cytometry and may be a reliable tool for the characterization of lymphocyte activation (for details see: http://calciumflux.intralab.eu).

Do regulatory T cells contribute to Th1 skewness in obesity?

BACKGROUND: Recent data suggest that an increased prevalence of interferon-gamma (IFN-gamma) producing CD4 (+) cells is present in obesity. Regulatory T cells (Tregs) have a strong impact on activation and proliferation of CD4 (+) lymphocytes. Data are not available about Tregs and their possible contribution to chronic mild inflammation in obesity. DESIGN: We investigated the prevalence of Tregs in obese children. We also collected data about dendritic cells and monocytes (so-called antigen presenting cells, APCs), important modulators of Tregs and we determined the cytokine production of CD4 (+) lymphocytes, the main target cells of Tregs. METHODS: Twelve obese children and 10 healthy age-matched controls have been enrolled. For flow cytometric analyses, peripheral blood mononuclear cells were used. We determined the prevalence of Tregs by Foxp3 expression of CD4 (+) cells; prevalence of myeloid and plasmacytoid dendritic cells (DCs); prevalence of tumor necrosis factor (TNF)-alpha and interleukin(IL)-12 producing monocytes; and prevalence of IL-2, IL-4 and IFN-gamma producing CD4 (+) cells. RESULTS: The prevalence of Tregs, DCs, TNF-alpha and IL-12 producing macrophages, IL-2 and IFN-gamma producing CD4 (+) cells was similar in both groups. The prevalence of IL-4 producing CD4 (+) cells was lower in obese children than in healthy controls (p=0.028). The ratio of IFN-gamma (+)/ IL-4 (+) CD4 (+) cells was higher in obese children than in those with normal weight (p=0.046). CONCLUSIONS: CD4 (+) reactions are polarized toward Th1 direction in obesity. The unaltered number of Treg and APCs suggests that these immune regulator cells do not contribute to altered immune status in obese children.

Prohepcidin levels during human perinatal adaptation.

The aim of this study was to test for the presence of prohepcidin in cord blood, to gauge its alteration during the early postnatal period, and to look for a possible association with neonatal iron homeostasis. Cord blood and postnatal venous blood samples were taken from 20 healthy neonates. In both kinds of samples the presence prohepcidin could be detected. No association was found between cord blood and postnatal samples prohepcidin and iron homeostasis. However, an association is demonstrated between cord blood prohepcidin values and mean cell hemoglobin concentration (MCHC). Prohepcidin increased postnatally in half of the neonates, indicating the active synthesis of the molecule. Interestingly, neonates with detectable non-protein-bound iron levels in cord blood were presented with lower prohepcidin concentrations. Association between cord blood prohepcidin and MCHC may suggest a possible link between hepcidin and fetal iron homeostasis.

Investigation of regulatory T cells in anorexia nervosa.

OBJECTIVE: The aim of our study was to determine, how severe calorie restriction in anorexia nervosa (AN) may influence regulatory T (Treg) cells and their cellular networks, that is, their main inducers (dendritic cells (DC) and monocytes) and their target cells, CD4+ lymphocytes. DESIGN: We measured the prevalence of Tregs, myeloid and plasmocytoid DC. The prevalence of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-12-positive monocytes, IL-2, IL-4 and interferon (IFN)-gamma positive CD4+ cells was determined by intracellular staining after activation. SETTING AND SUBJECTS: In total, 21 AN patients and 19 healthy age-matched controls (body mass index values, median (range): 14.9 (11.1-17.4) vs 23.2 (19.5-27.4) kg/m(2)) have been recruited. RESULTS: Prevalence of Tregs, DCs, TNF-alpha and IL-12-positive monocytes, IL-4 and IFN-gamma-producing CD4+ cells were similar in AN and controls. The prevalence of IL-2-positive CD4+ cells was somewhat lower in AN (% value, median (range): 12.05 (7.50-16.70) vs 14.40 (12.00-22.00), P<0.05). None of these parameters correlated with the patients' clinical characteristics. CONCLUSIONS: Our results suggest that the antigen presenting cell - regulatory T cell - CD4+ lymphocyte axis is not affected by calorie and nutritional deficiency.

Association of interferon gamma T+874A and interleukin 12 p40 promoter CTCTAA/GC polymorphism with the need for respiratory support and perinatal complications in low birthweight neonates.

BACKGROUND: Data support the role of interferon (IFN)gamma and interleukin (IL)12 in perinatal complications. IFNgamma T(+874)A and IL12 p40 promoter CTCTAA/GC polymorphisms may have an effect on cytokine production. METHODS: DNA was extracted from dried blood samples of 153 low birthweight (LBW) infants and 172 healthy term infants. IFNgamma and IL12 genetic polymorphisms were determined to investigate the association between polymorphisms and ventilation characteristics, bronchopulmonary dysplasia (BPD) and other perinatal disorders. RESULTS: The IFNgamma(+874)A allele was over-represented in LBW infants. Carriers of the IFNgamma(+874)T allele required mechanical ventilation and oxygen supplementation for time periods 41% and 35%, respectively, shorter than those required by those not carrying the IFNgamma(+874)T allele. Stepwise logistic regression analysis showed that carriers of the IFNgamma(+874)T allele were protected against BPD (odds ratio (OR) 0.35 (95% confidence interval (CI) (0.12 to 0.99))) and patent ductus arteriosus (OR 0.43 (95% CI 0.19 to 0.97)), whereas carriers of the IFNgamma(+874)A allele were at higher risk of severe hypotension (OR 3.40 (95% CI 1.01 to 11.52)) and respiratory distress syndrome (OR 4.03 (95% CI 1.30 to 12.50)). Carriers of the IL12 GC allele were protected against pneumonia (OR 0.32 (95% CI 0.14 to 0.75)). Carriers of the IL12 CTCTAA allele were at higher risk of developing necrotising enterocolitis (NEC; OR 2.37 (95% CI 1.01 to 5.53)). CONCLUSIONS: Carrier state of the IFNgamma(+874)A allele presents an increased risk for premature birth and lung damage, as well as other perinatal complications. The risks of pneumonia and NEC are higher in heterozygotic carriers of the IL12 CTCTAA/GC polymorphism. Further studies are needed to determine whether these associations are the result of altered cytokine-producing capacity in infants carrying the tested alleles.

Three mechanisms in the pathogenesis of pre-eclampsia suggested by over-represented transcription factor-binding sites detected with comparative promoter analysis.

Microarray studies generating lists of genes with altered expression in placentas from pregnancies complicated with pre-eclampsia (PE) have so far been published in several different studies. Working under the assumption that altered gene expression in PE may be the result of altered expression of regulatory transcription factors (TFs), we looked for over-represented TF-binding sites (TFBSs)-which indicate the involvement of TFs in gene regulatory networks-in lists of genes (n = 143) compiled in these studies. We compared the prevalence of TFBSs in the promoter regions of 68 genes with the background prevalence of TFBSs in promoters of the human genome. The prevalence of the E47, sterol regulatory element binding protein (SREBP) and NFKB-p50 TFBSs was higher (P < 0.005) in the promoter sequences of the PE gene lists than in the background model. Each of these TFBSs could be implicated in the development of PE. The E47 protein is an E-protein or basic helix-loop-helix (bHLH) TF. Data support the role of bHLHs in the differentiation of placental tissue. SREBP-1, a lipid-sensing sterol regulatory element-binding protein, is a critical regulator of fatty acid homeostasis in the placenta. The target genes of NFKB-p50 determine inflammatory response, and aberrant cytokine homeostasis is a further sign of PE. These TFs may provide an insight into the pathogenesis of the disease.

Bilateral striatal lesion associated with varicella.

Bilateral striatal lesion is characterised by a specific clinical syndrome (encephalopathy with rigidity, irritability, variable pyramidal, and extrapyramidal symptoms, speech abnormalities) and symmetrical lesion of the basal ganglia including the caudate nucleus, the putamen, and occasionally other nuclei. We report three cases in whom bilateral striatal lesion developed in association with varicella. Each patient recovered completely and showed no signs of cognitive deficiency, chorea or hyperkinetic syndrome, all of which have been reported as sequelae of BSL associated with other conditions. These cases suggest that bilateral striatal lesion may be an immune-mediated complication of varicella.

Prevalence of alpha1-antitrypsin phenotypes in patients with IgA nephropathy.

BACKGROUND: alpha1-antitrypsin (AAT) is the main protease inhibitor in the blood. Several different AAT phenotypes exist. The most common variant is the MM phenotype, which is also associated with normal AAT levels. The less common phenotypes with Z and S variants are associated with low AAT levels. AAT deficiency is a risk factor for pulmonary emphysema, liver impairment and some immune-mediated diseases, some of which are also associated with IgA nephropathy (IgAN). In fact, liver impairment resulting from AAT deficiency may directly contribute to renal abnormalities resembling IgAN. PATIENTS AND METHODS: We investigated AAT phenotype and AAT levels in 100 IgAN patients who did not have end-stage liver disease. Fifteen patients in our sample had secondary IgAN. We also tested for the presence of renal deposition of AAT in patients heterozygous for AAT variants as well as in a randomly chosen group of patients with MM phenotype. We checked for any association between AAT phenotype and the progression of IgAN as well as the prevalence of diseases associated with IgAN (i.e. secondary IgAN). RESULTS: Twelve patients in our sample were heterozygous for AAT variants. Phenotypes were MZ in 5 patients, MS in 3, MF in 1, ML in 2 and ME in 1 patient. AAT levels were lower in these 12 patients than in those homozygous for the M variant (1.17+/-0.46 vs. 1.44+/-0.34 g/l, p < 0.05). We found renal deposition of AAT in 2 heterozygous patients and in 1 of the 12 patients which were randomly chosen. End-stage renal (ESRF) failure developed in 3 of the 12 heterozygous patients and in 6 of the 88 homozygous patients (p = 0.07) during the follow-up. The prevalence of heterozygosity was significantly higher in patients with secondary IgAN than in those with primary IgAN ((5/15 vs. 7/85; p < 0.02). CONCLUSIONS: AAT phenotype is not associated with the risk of primary IgA nephropathy, but might have an impact on disease outcome as well as on the risk of secondary IgAN.

HbA1c levels and erythrocyte transport functions in complication-free type 1 diabetic children and adolescents.

Higher erythrocyte sodium-lithium countertransport activity (SLC) is implicated in the development of diabetic nephropathy. Altered glucose homeostasis and genetic susceptibility are claimed to play a role in the elevation of SLC. We aimed to test whether metabolic control or the genetic variants of G protein beta 3 (Gb3) subunits determine SLC and other erythrocyte transport activities in complication-free stage of type 1 diabetes. A total of 96 complication-free type 1 diabetic children and adolescents were enrolled. SLC, Na(+)/K(+)-ATPase (NAK) and Ca(2+)-ATPase (CA) were measured by functional assays in erythrocytes. Gb3-C825T polymorphism was determined by PCR-RFLP. Results were related to HbA(1c) and were compared to those of 97 healthy controls. SLC activity was higher in diabetics (387+/-146 vs. 280+/-65 mmol/RBC. hour) and correlated with HbA(1c) levels (y=0.004x+6.42, r=0.33, n=96, p<0.01). NAK and CA activities were unaltered. The prevalence of (825)T allele was similar in the patient and control groups (0.34 vs 0.37) and no differences in enzyme activities were observed between the (825)T allele-positive and negative subjects. Although metabolic control correlated with SLC, other membrane functions were not affected. Therefore we hypothesize that the relationship between advanced glycation and SLC elevation is not causative. Rather, a genetic susceptibility for the coexistence of poor metabolic control and higher SLC is more likely. However, the presence of Gb3-C825T variant is not likely to be a risk factor for SLC-elevation and altered metabolic control diabetes.