Impact of the hypoxic microenvironment on spermatogonial stem cells in culture.

The stem cell niche plays a crucial role in the decision to either self-renew or differentiate. Recent observations lead to the hypothesis that O2 supply by blood and local O2 tension could be key components of the testicular niche of spermatogonial stem cells (SSCs). In this study, we investigated the impact of different hypoxic conditions (3.5%, 1%, and 0.1% O2 tension) on murine and human SSCs in culture. We observed a deleterious effect of severe hypoxia (1% O2 and 0.1% O2) on the capacity of murine SSCs to form germ cell clusters when plated at low density. Severe effects on SSCs proliferation occur at an O2 tension ≤1% and hypoxia was shown to induce a slight differentiation bias under 1% and 0.1% O2 conditions. Exposure to hypoxia did not appear to change the mitochondrial mass and the potential of membrane of mitochondria in SSCs, but induced the generation of mitochondrial ROS at 3.5% and 1% O2. In 3.5% O2 conditions, the capacity of SSCs to form colonies was maintained at the level of 21% O2 at low cell density, but it was impossible to amplify and maintain stem cell number in high cell density culture. In addition, we observed that 3.5% hypoxia did not improve the maintenance and propagation of human SSCs. Finally, our data tend to show that the transcription factors HIF-1α and HIF-2α are not involved in the SSCs cell autonomous response to hypoxia.

Type II collagen peptide Coll2-1 is an actor of synovitis.

OBJECTIVE: We evaluated the ability of Coll2-1, a type II collagen peptide, to activate pro-inflammatory pathways in synovial cells and to induce arthritis in Lewis rats. METHOD: Human synoviocytes and chondrocytes from knee OA patients were cultured for 24 h with/without Coll2-1 and/or purified immunoglobulin G (AS0619) binding specifically this peptide, and/or CLI-095, a TLR-4 signaling inhibitor and/or apocynin and diphenyleneiodonium, Reactive oxygen species (ROS) production inhibitors. The Interleukin (IL)-8 and Vascular Endothelium Growth Factor (VEGF) expression, the IL-8 production, the IκB-α and p65 phosphorylation and ROS were evaluated. Coll2-1 peptide, bovine type II collagen (CIA), streptococcal cell wall (SCW) or saline solution were injected into Lewis rats. The Coll2-1 peptide was injected subcutaneously (SC; 20-200µg/100µl/animal) or intra-articularly (IA; 0.5-5µg/50µl/animal) and compared to CIA injected in SC (200µg/100µl/animal) and SCW in IA (5µg/50µl/animal). The animals were injected on day 0 and monitored for 28 days. Histological lesions assessment was performed using an arthritis score. RESULTS: Coll2-1 peptide significantly increased IL-8 gene expression and production by synoviocytes. AS0619 and CLI-095 significantly decreased IL-8 expression. Coll2-1 induced p65 and IκBα phosphorylation and oxidative stress inhibitors decreased it. In human chondrocytes culture, Coll2-1 significantly increased MMP-3 and VEGF gene expression. In Lewis rats, CIA, SCW or Coll2-1 injection triggered arthritis. Like CIA or SCW, Coll2-1 induced synovitis, loss of cartilage proteoglycans, cartilage structure lesion and subchondral bone remodeling. CONCLUSIONS: Coll2-1 activates synoviocytes to produce IL-8 and induces arthritis in rat. These findings suggest that neutralizing Coll2-1 could be a therapeutic approach of arthritis.

Absence of recognition of low alkaline phosphatase level in a tertiary care hospital.

SUMMARY: Low serum total alkaline phosphatase level (ALP), the hallmark for hypophosphatasia (HPP), must be recognized to provide appropriate care of the patients and to avoid antiresorptive treatment. The prevalence of persistent low ALP in a clinical setting is 0.13% and the recognition is very low (3%). INTRODUCTION: A low serum total alkaline phosphatase level is the hallmark for the diagnosis of hypophosphatasia. Although very rare, HPP must be recognized to provide appropriate treatment of non-union fractures and to avoid potentially harmful drugs, such as antiresorptive treatments. The aim of this study was to assess the recognition of persistent low ALP in a tertiary care hospital. METHODS: Between the 1st of January and the 31st of December 2013, 48,755 patients had ALP assessment in the Biochemistry Department of our hospital. Sixty-eight patients had all serum ALP values persistently below 40 IU/l. Among them, six had potential causes of secondary hypophosphatasia. We consulted the summary discharges of the 62 patients in order to check for the notation of low ALP. Patients from the departments of rheumatology and internal medicine were contacted to fulfill a questionnaire about clinical manifestations potentially related to HPP. RESULTS: 0.13% of hospitalized patients had persistently low value. They were 46.5 ± 17.7 years old, and 73% were females. The low ALP value was notified in the discharge summary for two patients (3%), without any comment. Twenty-four patients (46 + /-16 years old) were contacted. Eight patients had fractures; two had a diagnosis of rickets in the childhood; two had symptomatic chondrocalcinosis. Nine had dental abnormalities. Three were receiving a bisphosphonate; two of them had a fracture while being treated with bisphosphonate. CONCLUSION: Our study shows that low ALP is not recognized in a clinical setting in adults hospitalized in a tertiary care hospital.

Inter-relations between osteoarthritis and metabolic syndrome: A common link?

Osteoarthritis (OA) is a degenerative disorder of the joint, principally occurring during aging, and characterized by a focal degradation of cartilage. It is the most prevalent rheumatic disease in industrialized countries and represents the second cause of disability in France. However, the etiology of OA remains unclear. There is only one cell type found in cartilage, chondrocyte, which is responsible for its repair and the synthesis of the elements of the extra-cellular matrix. A dysfunction of these cells results in an imbalance between repair and degradation in cartilage, leading to its destruction. Recently, a link between OA and metabolic syndrome (MetS) has been suggested, introducing a notion of metabolic OA, and a new vision of the disease. MetS is characterized by a cluster of factors (insulin resistance, hypertension, dyslipidemia, visceral obesity), although there is still no clear definition of it. During the 20th century, MetS dramatically increased with changes in population lifestyle, becoming a major health issue in industrialized countries. MetS concerns 10-30% of the worldwide population, but is prevalent in 59% of OA patients. Patients with both OA and MetS have more severe symptoms, occurring sooner than in the general population. Indeed, OA is generally a disease concerning the population over 65 years old, but with an associated MetS the target population is around 50 years old. In this review, we will focus on common factors in OA and MetS, such as hypertension, obesity, dyslipidemia, mitochondrial dysfunction and hyperglycemia, linking one disease to the other.

Individual site-specific bone mineral density gain in normocalcemic primary hyperparathyroidism.

UNLABELLED: In this study, we show that successful parathyroidectomy is followed at 1 year by a significant individual bone mineral density (BMD) gain in nearly half of normocalcemic PHPT patients with reduced bone mass. Alkaline phosphatase levels above median were identified as an independent predictor of individual BMD gain in normocalcemic PHPT patients. INTRODUCTION: The aims of this study were to assess bone mineral density (BMD) gains after parathyroidectomy (PTX) in normocalcemic primary hyperparathyroidism (PHPT) at the individual level and to identify predictors of BMD gain after PTX in this context. METHODS: Longitudinal cohort study of 55 PHPT patients referred for low bone mass and mild abnormalities of calcium/phosphorus metabolism, and successfully treated by PTX. BMD gain at 1 year was considered significant if ≥0.030 g/cm(2) at one site or more, without any equivalent BMD loss at another site. A logistic regression analysis was performed to identify predictive factors of individual BMD gain. RESULTS: Among the 55 PHPT patients included, 29 patients with hypercalcemia, 36 patients with normocalcemic PHPT, defined by normal pre-PTX serum total (albumin-corrected) calcium (tCa), including 15 patients with normal ionized calcium (iCa), were identified. At 1 year of PTX, an individual BMD gain was observed in 73.7 % of hypercalcemic, 44.4 % of normocalcemic, and 46 % of PHPT patients with both normal tCa and iCa. Site-specific BMD gains were most important at the spine and hip in all subgroups including patients with normal iCa. Alkaline phosphatase activity above median, which reflects high bone turnover, was predictive of individual BMD gain, both in the overall cohort (OR = 4.9, 95 % CI 1.3-18.9), and in the normocalcemic group: OR = 8.4, 95 % CI 1.4-56.6. CONCLUSIONS: Successful PTX is followed at 1 year by a significant individual BMD gain in nearly half of normocalcemic PHPT patients with osteoporosis. ALP levels above median could contribute to the therapeutic decision in this context.

Effect of hypoxia/reoxygenation on the cytokine-induced production of nitric oxide and superoxide anion in cultured osteoarthritic synoviocytes.

OBJECTIVE: Hypoxia/reoxygenation (H/R) is an important feature in the osteoarthritis (OA) physiopathology. Nitric oxide (NO) is a significant proinflammatory mediator in the inflamed synovium. The purpose of this study was to investigate the effects of H/R on inducible NO synthase (iNOS) activity and expression in OA synoviocytes. In addition we studied the relationship between nitrosative stress and NADPH oxidase (NOX) in such conditions. METHODS: Human cultured synoviocytes from OA patients were treated for 24 h with interleukin 1-ß (IL-1ß), tumour necrosis factor α (TNF-α) or neither; for the last 6 h, they were submitted to either normoxia or three periods of 1-h of hypoxia followed by 1-h of reoxygenation. ·NO metabolism (iNOS expression, nitrite and peroxynitrite measurements) was investigated. Furthermore, superoxide anion O2(·-) production, NOX subunit expression and nitrosylation were also assessed. RESULTS: iNOS expression and nitrite (but not peroxynitrite) production were ~0.20 to ~0.12 nmol min(-1) mg proteins(-1) (P < 0.05), while NOXs' subunit expression and p47-phox phosphorylation were increased. NOXs and p47-phox were dramatically nitrosylated under H/R conditions (P < 0.05 vs normoxia). Using NOS inhibitors under H/R conditions, p47-phox nitrosylation was prevented and O2(·-) production was restored at normoxic levels (0.21 nmol min(-1) mg of proteins(-1)). CONCLUSIONS: Our results provide evidence for an up-regulation of iNOS activity in OA synoviocytes under H/R conditions, associated to a down-regulation of NOX activity through nitrosylation. These findings highlight the importance of radical production to OA pathogenesis, and appraise the metabolic modifications of synovial cells under hypoxia.

APRI and FIB-4 Scores Are Useful After Liver Transplantation Independently of Etiology.

Noninvasive liver fibrosis scores are evaluated in hepatitis C virus-infected patients but are less known in liver transplant recipients. Fibrosis is a frequent, multifactorial event in these patients. This preliminary retrospective study reviewed the diagnostic performance of 3 simple scores for liver fibrosis in transplant patients: namely, APRI (aspartate aminotransferase to platelet ratio index), FORNS (platelets, gamma-glutamyltransferase, patient age, and cholesterol), and FIB-4 (patient age, aspartate aminotransferase, alanine aminotransferase, and platelets). Ninety-four biopsies were collected from 50 liver transplant recipients at a mean period after orthotopic liver transplantation (OLT) of 30.7 months (range, 12-108 months). The indications for OLT were hepatitis C in 23% of cases, hepatitis B in 14%, alcoholic disease in 33%, cholestatic disease in 19%, and others in 11%. According to the Metavir classification, 72% of biopsies revealed no significant histological fibrosis (F0-1 = group 1) and 28% showed significant fibrosis (F2-4 = group 2). A correlation was observed between the histological stage of fibrosis and albumin, gamma-glutamyltransferase, aspartate aminotransferase, alanine aminotransferase, and hyaluronic acid levels. APRI and FIB-4 correlated significantly with the histological stage of fibrosis both globally and in the subgroup of nonhepatitis C liver recipients. When APRI and FIB-4 tests were applied to predict fibrosis (area under the receiver operating characteristic curve), the results were 0.87 and 0.78, respectively. Values were not significant with the FORNS test. In conclusion, APRI and FIB-4 enabled accurate prediction of significant fibrosis after OLT. In the nonhepatitis C subgroup, we found similar predictive performances. These simple scores may be applied in clinical practice in the context of follow-up after OLT independent of hepatitis C status.

N-terminal pro-brain natriuretic peptide in systemic sclerosis: a new cornerstone of cardiovascular assessment?

BACKGROUND: Cardiac involvement, a common and often fatal complication of systemic sclerosis (SSc), is currently detected by standard echocardiography enhanced by tissue Doppler echocardiography (TDE). OBJECTIVE: The performance of the biomarker of cardiovascular disease, N-terminal pro-brain natriuretic peptide (NT-proBNP), in the detection of cardiac involvement by SSc was examined. METHODS: A total of 69 consecutive patients with SSc (mean (SD) age 56 (13) years, 56 women) were prospectively studied with standard echocardiography and TDE measurements of longitudinal mitral and tricuspid annular velocities. Plasma NT-proBNP was measured in all patients. RESULTS: Overall, 18 patients had manifestations of cardiac involvement, of whom 7 had depressed left ventricular and 8 depressed right ventricular myocardial contractility, and 8 had elevated systolic pulmonary arterial pressure. Patients with reduced contractility had increased mean (SD) NT-proBNP (704 (878) pg/ml versus 118 (112) pg/ml in patients with normal myocardial contractility, p<0.001). Similarly, NT-proBNP was higher in patients with (607 (758) pg/ml) than in patients without (96 (78) pg/ml) manifestations of overall cardiac involvement (p<0.001). Receiver operating characteristic analysis showed NT-proBNP reliably detected depressed myocardial contractility and overall cardiac involvement (area under the curve 0.905 (95% CI 0.814 to 0.996) and 0.935 (95% CI 0.871 to 0.996), respectively). Considering patients with SSc with normal echocardiography and TDE as controls, and using a 125 pg/ml cut-off concentration, sensitivity and specificity were 92% and 71% in the detection of depressed myocardial contractility, and 94% and 78% for overall cardiac involvement. CONCLUSIONS: NT-proBNP reliably detected the presence of cardiac involvement and appears to be a very useful marker to risk stratify patients presenting with SSc.

Disturbed angiogenesis in systemic sclerosis: high levels of soluble endoglin.

OBJECTIVE: SSc is a CTD characterized by early generalized microangiopathy with disturbed angiogenesis. Soluble endoglin (sENG), a serum anti-angiogenic protein, has recently been described as a major actor in pre-eclampsia, another severe vascular disease with abnormal angiogenesis. The aim of this study was to investigate, in a cross-sectional study, sENG levels together with other serum vascular markers. METHODS: Serum levels of sENG were assessed by ELISA in consecutive SSc patients and controls matched for age and sex. We also measured by ELISA serum levels of VEGF and asymmetric dimethylarginine (ADMA), as respective markers of angiogenesis and endothelial dysfunction. RESULTS: We included 235 unrelated subjects: 187 SSc patients and 48 controls. Higher concentrations of sENG (P = 0.002) and sVEGF (P < 0.0001) were found in SSc patients compared with controls whereas there was no difference for ADMA. In multivariate analysis, sENG levels were significantly increased in SSc patients with cutaneous ulcerations (P = 0.0003), positive for ACAs (P = 0.009) and with abnormal diffusing capacity for carbon monoxide divided by alveolar volume (P = 0.03). Soluble ENG levels negatively correlated with ADMA, but no relationship was found between sENG and sVEGF. CONCLUSION: This study shows increased values of sENG in a large SSc cohort and a relevant association with a vascular phenotype. The predictive value of the biomarker sENG and its potential role on cellular endothelial disturbances remain to be determined.

High N-terminal pro-brain natriuretic peptide levels and low diffusing capacity for carbon monoxide as independent predictors of the occurrence of precapillary pulmonary arterial hypertension in patients with systemic sclerosis.

OBJECTIVE: To evaluate predictors of pulmonary arterial hypertension (PAH) in a prospective cohort of patients with systemic sclerosis (SSc). METHODS: Routine clinical assessments as well as measurements of the diffusing capacity for carbon monoxide/alveolar volume (DLCO/VA) ratio and N-terminal pro-brain natriuretic peptide (NT-proBNP) level were performed in a prospective cohort of 101 SSc patients who did not have PAH or severe comorbidities. After a planned 36-month followup, we evaluated the predictive value of these parameters for the development of precapillary PAH, as demonstrated by cardiac catheterization, disease progression, and death. Criteria for cardiac catheterization were a systolic pulmonary artery pressure (PAP) of >40 mm Hg on echocardiography, a DLCO value of <50% without pulmonary fibrosis, and unexplained dyspnea. RESULTS: Eight patients developed PAH, 29 had disease progression, and 10 died during a median followup of 29 months. Kaplan-Meier analysis identified the following baseline parameters as being predictors of PAH: DLCO/VA ratio <70% or <60% (P<0.01 for each comparison), elevated plasma NT-proBNP level (>97th percentile of normal; P = 0.005), echocardiographically estimated systolic PAP >40 mm Hg (P=0.08), and erythrocyte sedimentation rate >28 mm/hour (P=0.015). In multivariate analyses, an elevated baseline NT-proBNP level (hazard ratio [HR] 9.97 [95% confidence interval (95% CI) 1.69-62.42]) and a DLCO/VA ratio <60% (HR 36.66 [95% CI 3.45-387.6]) were predictors of the occurrence of PAH during followup. An increased NT-proBNP level together with a decreased DLCO/VA ratio of <70% was highly predictive of the occurrence of PAH during followup (HR 47.20 [95% CI 4.90-450.33]). CONCLUSION: This prospective study identified a decreased DLCO/VA ratio and an increased NT-proBNP as predictors of PAH in SSc. Use of these markers should result in improved PAH risk stratification and allow earlier initiation of therapy.

Lack of association between three vascular endothelial growth factor gene polymorphisms and systemic sclerosis: results from a multicenter EUSTAR study of European Caucasian patients.

INTRODUCTION: Systemic sclerosis (SSc) is characterised by disturbed vessel morphology and an overproduction of vascular endothelial growth factor (VEGF). The VEGF gene located on chromosome 6p21.3 has several polymorphisms. OBJECTIVE: To test the hypothesis that disturbed angiogenesis may be related to the genetic background of the VEGF gene. MATERIALS AND METHODS: EUSTAR centres included European Caucasian patients with SSc and matched controls with osteoarthritis. The VEGF gene was genotyped by polymerase chain reaction, followed by restriction enzyme analysis. The 634 C/T and 936 C/G mutations and an 18-base pair insertion/deletion at -2549 of the VEGF promoter region were tested. RESULTS: 416 patients with SSc and 249 controls were included in the study population. Of the patients with SSc, 42% had a diffuse cutaneous subtype, 16% had increased pulmonary arterial pressure and 61% had decreased carbon monoxide diffusion capacity. The genotype frequencies in the patients with SSc and in controls were in Hardy-Weinberg equilibrium. The allele and genotype frequencies of the polymorphisms did not differ between patients with SSc and controls. No association was found between these polymorphisms and disease phenotypes. CONCLUSION: This study shows that there is no association between the three selected functional VEGF polymorphisms and SSc.

Superoxide production and NADPH oxidase expression in human rheumatoid synovial cells: regulation by interleukin-1beta and tumour necrosis factor-alpha.

OBJECTIVES: to evaluate the rheumatoid synovial cell capacity to produce superoxide anion in response to interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha), and to study the NADPH oxidase involvement in this production. MATERIAL AND METHODS: Synovial cells obtained from 7 rheumatoid arthritis (RA), 5 osteoarthritic (OA) patients, and dermal fibroblasts, were stimulated (i) with IL-1beta and TNF-alpha, or (ii) with specific oxidase activators and inhibitors, before studying superoxide production; we also studied NADPH oxidase mRNAs and protein expression, and p47-phox phosphorylation. RESULTS: Constitutive superoxide production by RA cells was increased in comparison to OA cells and dermal fibroblasts, and was stimulated by PMA and ionomycin. This production was increased after cytokine treatment of RA synovial cells. Cytokine-induced superoxide production by RA cells was inhibited by iodonium diphenyl or apocynin, suggesting the involvement of NADPH oxidase. RT-PCR and western blot analysis revealed the presence of p47-phox, gp91-phox and Nox4 in RA and OA cells, and in dermal fibroblasts. P47-phox phosphorylation was enhanced after cytokine-treatment in RA and OA cells, suggesting a PKC-mediated up-regulation of NADPH oxidase. CONCLUSIONS: NADPH oxidase is involved in the superoxide release by RA synovial cells, constitutively and after cytokine up-regulation. These cells express two different homologues (gp91-phox and Nox4).

Serum protein oxidation in patients with rheumatoid arthritis and effects of infliximab therapy.

OBJECTIVE: To examine protein oxidation in rheumatoid arthritis (RA) and evaluate its evolution after infliximab therapy in a subgroup of patients. METHODS: Seventy-one consecutive patients with RA were included. Among them, 30 patients refractory to conventional therapy were treated with infliximab. Serum markers of oxidative stress were determined at baseline and before the infusions of infliximab at weeks 6 and 30. Baseline values were compared with those in 30 healthy volunteers. RESULTS: Mean levels of serum carbonyl groups were significantly higher in RA patients than in controls (1.29+/-0.76 versus 0.58+/-0.39 nmol/mg of protein, p<0.0001), whereas thiol levels were found to be lower (238.3+/-61.6 versus 316.5+/-54.8 micromol/L, p<0.0001). Thiol levels inversely correlated with the disease activity score (r=-0.42, p=0.004), and with CRP values (r=-0.45, p=0.001). Immunoblots showed that albumin and heavy chain immunoglobulin were oxidized more markedly than in healthy volunteers. Significantly lower levels of thiol groups were detected in patients with refractory RA disease (208.9+/-66.8 versus 264.2+/-43.0 micromol/L, p<0.0004) but concentrations of carbonyl groups were similar. Short-term treatment with infliximab significantly decreased carbonyl groups (0.97+/-0.47 nmol/mg protein, p=0.02) and increased thiol (231.2+/-48.7 micromol/L, p=0.02) levels. CONCLUSION: Our results highlight free radical protein damage in RA and a link with inflammation, as underlined by the beneficial effects of infliximab.

Increased plasma soluble CD40 ligand concentrations in systemic sclerosis and association with pulmonary arterial hypertension and digital ulcers.

BACKGROUND: Increased expression of CD40L has been reported on activated CD4+ T lymphocytes in systemic sclerosis. CD40L can be expressed in soluble form (sCD40L). OBJECTIVE: To compare sCD40L concentrations in patients with systemic sclerosis and healthy controls. METHODS: Quantitative sandwich ELISA was used to measure plasma sCD40L in systemic sclerosis (n = 50) and matched healthy controls (n = 20). Patients with systemic sclerosis had limited cutaneous disease (29), digital ulcers (14), pulmonary arterial hypertension (PAH) (10), pulmonary fibrosis on CT (23), positive anti-Scl70 (14), and anti-centromere antibodies (10). Calcium channel blockers were discontinued 72 hours before measurements. RESULTS: Median (range) sCD40L concentration (pg/ml) was higher in systemic sclerosis than in controls (495 (10 to 7720) v 79 (50 to 118); p = 0.003), in limited cutaneous disease v diffuse disease (620 (20 to 7720) v 250 (10 to 2690); p = 0.005), in patients with digital ulcers v those without (1430 (36 to 7720) v 370 (10 to 2320); p = 0.002), and in those with PAH v those without (995 (15 to 3850) v 400 (10 to 7720); p = 0.048). sCD40L correlated with pulmonary arterial pressure estimated by Doppler echocardiography (r = 0.41; p = 0.005). CONCLUSIONS: The soluble form of CD40L is increased in plasma in systemic sclerosis and may be associated with vascular complications of the disease.

N-terminal pro-brain natriuretic peptide as a diagnostic marker of early pulmonary artery hypertension in patients with systemic sclerosis and effects of calcium-channel blockers.

OBJECTIVE: To evaluate N-terminal pro-brain natriuretic peptide (NT-proBNP) as a marker of early pulmonary artery hypertension (PAH) and to study changes in the levels of this marker following treatment with dihydropyridine-type calcium-channel blocker (DTCCB) in patients with systemic sclerosis (SSc). METHODS: We evaluated 40 consecutive SSc patients who had been hospitalized for followup care (mean +/- SD age 56 +/- 11 years and mean +/- SD duration of cutaneous disease 9 +/- 9 years; 27 with limited cutaneous and 13 with diffuse cutaneous disease) but who had no clinical symptoms of heart failure and had a normal left ventricular ejection fraction. At baseline, 10 patients had PAH, defined as a systolic pulmonary artery pressure (sPAP) >40 mm Hg, as measured by echocardiography. Levels of NT-proBNP were determined at baseline (after discontinuation of DTCCB treatment for 72 hours), after taking 3 doses of DTCCB following treatment reinitiation (assessment 1), and after 6-9 months of continuous DTCCB treatment (assessment 2) in the 20 patients who attended regular appointments (including the 10 patients with PAH at baseline). RESULTS: At baseline, 13 patients had high NT-proBNP values for their ages. High NT-proBNP levels identified patients with PAH with a sensitivity of 90%, a specificity of 90.3%, a positive predictive value of 69.2%, and a negative predictive value of 96%. The NT-proBNP level correlated with the sPAP (r = 0.44; P = 0.006). By assessment 1, the number of patients with PAH and high levels of NT-proBNP had decreased from 9 of 10 to 2 of 10 (P = 0.02). This decrease was partially sustained at assessment 2 (4 of 10 patients; P = 0.06). CONCLUSION: NT-proBNP is a useful biologic marker that can be used to diagnose early PAH in SSc patients without clinical heart failure. Measurement of NT-proBNP may be valuable for the evaluation of treatment with DTCCB and vasodilators in patients with PAH.

Variability in urinary excretion of bone resorption markers: limitations of a single determination in clinical practice.

In this study we assessed the within and between-subject variability of the concentrations of two urinary markers, free deoxypyridinoline (DPD) and C telopeptide (CTX-I), in healthy patients with the aim of setting reliable thresholds to enable physicians to take decisions about individual patients with confidence.Between-subject variability for the women was 25.4% for DPD and 38.2% for CTX-I, and for the men was 12.9% for DPD and 23.8% for CTX-I. The coefficients of variation were similar for daily, weekly and monthly determinations, giving means of 13.8 and 28.1% for DPD and CTX-I respectively. Critical difference (CD) was lower for DPD than for CTX-I (about 44 and 80% respectively). The number of samples required to determine the true mean with a CD at the 5% level was 29 for DPD and more than 113 for CTX-I.DPD was the least biologically variable. One determination was not sufficient to determine bone resorption status and a 44% decrease in DPD levels and an 80% decrease in CTX-I levels were required to demonstrate the efficacy of antiresorptive therapy in individual patients.

Effects of bile acids on the humoral immune response: a mechanistic approach.

Whereas bile acids in excess depress the cell-mediated immune response, their effects on the humoral response have been little investigated. The aim of this study was to investigate the effects of bile acids on immunoglobulin production. Human peripheral blood mononuclear cells were stimulated for 5 days by Staphylococcus aureus Cowan I (SAC-I). Immunoglobulins were measured in the supernatants and cell lysates using ELISA. We found that bile acids inhibited IgM production in a dose-dependent manner. The inhibitory effects of 50 microM chenodeoxycholic acid (CDCA) and its glyco- and tauro-conjugates (62, 53 and 51%, respectively) were stronger than those of ursodeoxycholic acid (UDCA) and its conjugates (45, 40 and 34%, respectively). The inhibition of IgG production by CDCA and UDCA was weak (23 and 12%, respectively, at 50 microM). IgA production was not modified. The inhibition of intracellular IgM concentration paralleled that observed in the secreted compartment. By contrast, CDCA enhanced intracellular concentration of IgG. In the absence of significant necrosis or apoptosis, CDCA-mediated inhibition of SAC-I-induced IgM production was significantly correlated to the ability of the bile acid to inhibit cell proliferation (r=0.98; p<0.05). In conclusion, we showed that hydrophobic bile acids strongly depress the primary humoral response. This effect resulted from both an inhibition of cell proliferation, and to a lesser extent from a deficient exocytosis of immunoglobulins.

Low levels of nitric oxide (NO) in systemic sclerosis: inducible NO synthase production is decreased in cultured peripheral blood monocyte/macrophage cells.

OBJECTIVE: To investigate nitric oxide (NO) production and inducible NO synthase expression by cultured peripheral blood mononuclear cells (PBMC) in patients with systemic sclerosis (SSc). METHODS: Eighteen patients with SSc were compared with two control groups: 16 patients with rheumatoid arthritis (RA) and 23 patients with mechanical sciatica. Nitrate was determined by fluorimetry in plasma and by spectrophotometry in supernatants. Inducible NO synthase (iNOS) was detected in cultured PBMC by immunofluorescence, immunoblotting and flow cytometry with or without treatment of the cells with interleukin (IL) 1beta+ tumour necrosis factor alpha (TNF-alpha), IL-4 or interferon gamma (IFN-gamma) from day 1 to day 5. RESULTS: NO metabolite concentrations were lower in SSc patients (mean+/-s.e.m. 34.3+/-2.63 micromol/l) than in RA (48.3+/-2.82 micromol/l; P<0.02) and sciatica (43.3+/-5.24 micromol/l; P<0.03) patients. iNOS was detected in cultured monocytes in all three groups but induction occurred on day 1 in RA, day 2 in sciatica and only on day 3 in SSc, whatever the stimulus. CONCLUSIONS: The concentrations of NO metabolites are decreased in SSc patients and the metabolism of these compounds in PBMC is altered. Low levels of NO, a vasodilator, may be involved in vasospasm, which is critical in SSc. This may have therapeutic implications.

Tetracyclines inhibit nitrosothiol production by cytokine-stimulated osteoarthritic synovial cells.

OBJECTIVE AND DESIGN: To evaluate the capacity of doxycycline and minocycline to inhibit NO production and N-nitrosation reactions in vitro. METHODS: Synovial cells obtained from 6 patients with osteoarthritic joint disease were incubated for 24 hours with (i) or without (ii) IL-1beta (1 ng/ml), TNF-alpha (500 pg/ml), IFN-gamma (10(4) U/ml) plus minocycline or doxycycline (10(-4) to 10(-6) M), diclofenac (10(-5) M), or cortisol (10(-5) M). Nitrosothiols were determined by fluorimetry, nitrite by the Griess reaction, nitrate by a spectrophotometric assay using oxidation by nitrate reductase and iNOS by immunoblotting. RESULTS: After 24 hours of stimulation, the level of NO production was much higher than that in untreated cells: about 5.5 times higher for nitrosothiols, 5.2 times higher for nitrate and about 3.5 times higher for nitrite. Doxycycline and minocycline induced a dose-dependent decrease in the production of nitrosothiols, nitrate and nitrite, and inhibited the synthesis of the iNOS protein. Doxycycline and minocycline inhibited the N-nitrosation reaction of DAN effectively, with IC50 values close to 100 microM. Diclofenac and cortisol had no effect. CONCLUSION: This study provides new information on the mechanism by which tetracyclines exert anti-inflammatory effects, via inhibiting nitrosothiols.