A modified flow cytometry method for objective estimation of human CD4+ regulatory T cells (CD4+ Tregs) in peripheral blood, via CD4/CD25/CD45RO/FoxP3 labeling.

BACKGROUND: Several methods exist for flow-cytometric estimation of human peripheral blood CD4+ T regulatory cells (CD4+ Tregs). METHODS: We report our experience with the estimation of human CD4+ Tregs via three different characterizations using flow cytometry (CD25high FoxP3+ , CD25high CD127low/- FoxP3+ , and CD4+ CD25high/int CD45ROFoxP3+ ) in normal subjects. We have used these methods on the control populations from two studies (32 and 36 subjects, respectively), the latter two methods retrospectively on the subjects of the first study. The six CD4+ T cell fractions obtained by the third method were differentially colored to ascertain the distribution of these cell fractions in the CD25/FoxP3, CD45RO/FoxP3, and CD25/CD127 dot plots from CD4/CD25/CD45RO/FoxP3 and CD4/CD25/CD45RO/CD127 panels. RESULTS: Each approach gives significantly different estimates of Tregs (expressed as percentage of CD4+ T cells), with the second almost invariably yielding higher percentages than the other two. Only the third approach can distinguish among effector and naïve Tregs and FoxP3+ non-Tregs. Analysis of CD25/CD127 dot plots reveals that Treg delineation via the widely used definition of CD4+ CD25high CD127low/- cells unavoidably yields a mixture of nearly all effector and most of naïve Tregs, as well as FoxP3+ non-Tregs plus other cells. Delineation of effector/naïve Tregs and FoxP3+ non-Tregs is possible via CD45RO/CD25 dot plots but not by CD45RO/FoxP3 counterparts (as done previously) because of overlapping FoxP3 intensities among Tregs and non-Tregs. CONCLUSION: Our comparison shows that CD4/CD25/CD45RO/FoxP3 panels are an objective means of estimating effector and naïve Tregs via colored dot plots, aiding thus in Treg delineation in health and detecting aberrations in disease.

Emerging Role of l-Dopa Decarboxylase in Flaviviridae Virus Infections.

l-dopa decarboxylase (DDC) that catalyzes the biosynthesis of bioactive amines, such as dopamine and serotonin, is expressed in the nervous system and peripheral tissues, including the liver, where its physiological role remains unknown. Recently, we reported a physical and functional interaction of DDC with the major signaling regulator phosphoinosite-3-kinase (PI3K). Here, we provide compelling evidence for the involvement of DDC in viral infections. Studying dengue (DENV) and hepatitis C (HCV) virus infection in hepatocytes and HCV replication in liver samples of infected patients, we observed a negative association between DDC and viral replication. Specifically, replication of both viruses reduced the levels of DDC mRNA and the ~120 kDa SDS-resistant DDC immunoreactive functional complex, concomitant with a PI3K-dependent accumulation of the ~50 kDa DDC monomer. Moreover, viral infection inhibited PI3K-DDC association, while DDC did not colocalize with viral replication sites. DDC overexpression suppressed DENV and HCV RNA replication, while DDC enzymatic inhibition enhanced viral replication and infectivity and affected DENV-induced cell death. Consistently, we observed an inverse correlation between DDC mRNA and HCV RNA levels in liver biopsies from chronically infected patients. These data reveal a novel relationship between DDC and Flaviviridae replication cycle and the role of PI3K in this process.

von Willebrand factor and procoagulant imbalance predict outcome in patients with cirrhosis and thrombocytopenia.

BACKGROUND & AIMS: Several lines of evidence suggest that the hemostatic disorders of cirrhosis may have a significant clinical impact. We investigated the independent predictive value of components of the hemostatic system on the occurrence of ascites, variceal bleeding (VB), and survival. METHODS: One hundred and two patients with thrombocytopenia (Child-Pugh class A/B/C: 34/34/34) were enrolled. Platelet counts, factors (F) II, V, VII, and VIII, antithrombin, protein C (PC), FVIII-to-PC ratio as an index of procoagulant imbalance, von Willebrand factor antigen (vWF-Ag), and model for end-stage liver disease (MELD) were evaluated. Two multivariate analyses were performed: one excluding (model 1) and one including MELD (model 2). RESULTS: Higher vWF-Ag levels and FVIII-to-PC ratios were the most prominent hemostatic disorders in patients with cirrhosis. Increased levels of vWF-Ag and FVIII, and higher FVIII-to-PC ratios independently predicted the presence of ascites and varices at baseline. Independent predictors of ascites and VB during follow-up were vWF-Ag (model 1/2: p=0.001/p=0.009 and p=0.008/p=0.01, respectively) and FVIII-to-PC ratio (model 1/2: p=0.003/p=0.02 and p=0.01/p=0.03, respectively). vWF-Ag (model 1/2: p=0.007/p=0.002), FVIII-to-PC ratio (model 1/2: p=0.001/p=0.01), and MELD (p=0.02) independently predicted mortality. Patient groups with significantly higher probability of new-onset ascites, VB, and mortality were identified by certain cut-offs of vWF-Ag (213%, 466%, and 321%, respectively) and FVIII-to-PC ratio (1.99, 3.29, and 2.36, respectively). vWF-Ag and FVIII-to-PC ratio equaled MELD in mortality prediction. CONCLUSIONS: Advanced cirrhosis is characterized by increased thrombotic potential. vWF-Ag and FVIII-to-PC ratio independently predict new-onset ascites, VB, and mortality. Targeting hypercoagulability could improve the outcome of patients with cirrhosis. LAY SUMMARY: Higher von Willebrand factor antigen (vWF-Ag) levels and factor VIII-to-protein C (FVIII-to-PC) ratio are the prominent hemostatic disorders in patients with cirrhosis. vWF-Ag and FVIII-to-PC ratio independently predict new-onset ascites, variceal bleeding, and mortality in these patients.

Thrombin generation measured as thrombin-antithrombin complexes predicts clinical outcomes in patients with cirrhosis.

AIM: Hypercoagulability has been detected in patients with cirrhosis yet its clinical significance remains unclear. We investigated the association of hypercoagulability with clinical outcomes in patients with cirrhosis. METHODS: Thrombin-antithrombin (TAT) complexes as thrombin generation (TG) marker, D-dimer, antithrombin (AT), protein C, protein S, international normalized ratio (INR), activated partial thromboplastin time, fibrinogen, Child-Pugh class and Model for End-Stage Liver Disease (MELD) were evaluated. Two different multivariate analyses were performed: one not including MELD (model 1) and one including MELD and excluding INR (model 2). RESULTS: Eighty-one patients (Child-Pugh class A/B/C: 27/27/27) and 40 healthy subjects were enrolled. Only ΤΑΤ and AT were independently associated with increasing liver disease severity. Increased TAT levels and MELD score were significantly associated with ascites and varices at baseline. Independent predictors of follow-up events were: TAT and MELD score for new-onset ascites; TAT and AT for variceal bleeding (VB); TAT and AT for portal vein thrombosis (PVT); and TAT and MELD for mortality. TAT equaled MELD in mortality prediction at 12 and 18 months. TAT cut-offs at 5.35, 14.6, 13.5 and 9.25 ng/mL identified patient groups with significantly higher probability of new-onset ascites, VB, PVT and mortality, respectively. CONCLUSION: Increased TG is strongly correlated with portal hypertension-related complications, PVT and mortality in patients with cirrhosis. Measuring TG by TAT could enable risk stratification and institution of preventive strategies to improve clinical outcomes.

Changes of serum adhesion molecules and cytokines in post-ERCP pancreatitis: adhesion molecules and cytokines in acute pancreatitis.

OBJECTIVES: To assess the early changes of soluble IFN-γ, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, TNF-α, TNF-ß, IL-17A, IL-22, soluble (s) P-Selectin, sE-Selectin and sICAM-1 in post-ERCP pancreatitis (PEP). METHODS: Single center, prospective study of 318 ERCP procedures. Serum samples were acquired from all patients prior to ERCP, 6 hours and 24 hours after the procedure. For every PEP case, another patient was chosen as a control, matched for gender, age and time period in which ERCP took place. RESULTS: Totally, 28 cases and 28 controls were studied. Except for significantly higher IL-1b levels in cases at baseline, no significant differences were observed between cases and controls after Bonferroni corrections. An increase in IL-6 was noted between baseline and 6 h in cases alone (p=0.016). There was a significant fall in sP-selectin levels at 6 and 24 hours compared to baseline in all patients (corrected p=0.008 and 0.016 for cases and 0.016 and 0.048 for controls respectively). An increase of sE-selectin in cases was observed between 6 and 24 hours post-ERCP (corrected p=0.03). CONCLUSIONS: Soluble forms of cytokines and adhesion molecules studied seem not to play a major role in PEP.

Genetic polymorphisms associated with retinal vein occlusion: a Greek case-control study and meta-analysis.

BACKGROUND: The genetic background of retinal vein occlusion (RVO) remains unclear. In the current study, we aimed to replicate polymorphisms related to thrombophilia/hypofibrinolysis in a Greek population and also systematically summarize current evidence available on the topic. MATERIALS AND METHODS: A total of 48 RVO patients and 53 controls were genotyped for factor V H1299R and V Leiden, ß-fibrinogen G455A, PAI-1 4G/5G, ACE I/D, HPA1, prothrombin G20210A, factor XIII Val34Leu, MTHFR A1298C and C677T polymorphisms. We examined the association between RVO and the above polymorphisms under a per-allele genetic model in a Greek unrelated case/control population. Additionally, searching PubMed up to January 2012, we identified existing evidence on these polymorphisms and performed meta-analyses. RESULTS: A total of three polymorphisms had nominally significant associations with RVO. These associations pertained to ACE D allele (odds ratio, OR, 2.08 [95% CI, 1.12-3.85], p = 0.02); factor XIII 34Leu allele (OR = 0.41 [95% CI, 0.18-0.95], p = 0.037] and MTHFR 677T variant (OR = 2.20 [95% CI 1.10-4.40], p = 0.026). We performed a meta-analysis on the associations between RVO and PAI-1 (n = 5), factor V Leiden (n = 21), MTHFR C677T (n = 19) and prothrombin G20210A (n = 21). We observed nominally significant associations only for PAI-1 (OR = 1.27 [95% CI, 1.02-1.60, p = 0.036]) (I(2) = 44.7%), and factor V Leiden (OR = 1.40 [95% CI, 1.07-1.84, p = 0.015]) (I(2) = 3.6%) using random effects model. CONCLUSIONS: Our results suggest that there may be an association between increased risk for RVO and ACE I/D, MTHFR C677T, PAI-1 4G/5G and factor V Leiden polymorphisms, whereas the Val34Leu variant may exert a protective effect.

Asymptomatic T-cell large granular lymphocyte leukemia with an unusual immunophenotype.

T-cell large granular lymphocyte (T-LGL) leukemia represents a clonal proliferation of cytotoxic T-cells which etiology has not been entirely elucidated. However, CD4(+), CD4(-), CD8(-), CD4(+), CD8(+) cases have been described. The disease is usually characterized by cytopenias and a modest lymphocytosis. The majority of patients with T-LGL leukemia remains asymptomatic for a long period and will require treatment later during the course of their disease. Hereby we describe a case of T-LGL leukemia diagnosed by flow cytometry, which presented indolent course and required no treatment so far.

Post-implantation syndrome following endovascular abdominal aortic aneurysm repair: preliminary data.

OBJECTIVES: Endovascular aneurysm repair (EVAR), may elicit an unexpected systemic inflammatory response, which has been named post-implantation syndrome (PIS). The aim of this study was to prospectively evaluate the association of PIS with clinical and laboratory parameters in patients who underwent EVAR for abdominal aortic aneurysms (AAA). METHODS: Forty consecutive patients who underwent EVAR for AAA were studied. Complete blood count, fibrinogen, high sensitivity C-reactive protein (hs-CRP), interleukin (IL)-6, IL-1, tumor necrosis factor-alpha were determined before and after surgery. Several parameters regarding the operation, as well as the hospitalization days were recorded. RESULTS: PIS was diagnosed in 35% of the patients. Patients with PIS showed significant greater changes of inflammation marker levels, including hs-CRP and IL-6, as compared with the non-PIS group. PIS was associated with longer hospitalization. CONCLUSION: PIS is a relatively common complication of EVAR used to treat AAAs and it is associated with features of a systemic inflammatory response and prolongation of hospitalization. Further studies are necessary towards understanding the underlying pathophysiology and evaluating effective preventive strategies.

Prothrombotic state, cardiovascular, and metabolic syndrome risk factors in prepubertal children born large for gestational age.

OBJECTIVE: To evaluate metabolic syndrome and cardiovascular disease risk factors in prepubertal children born large for gestational age (LGA) to nondiabetic, nonobese mothers. RESEARCH DESIGN AND METHODS: At 6-7 years of age, the comparison of various factors was made between 31 LGA and 34 appropriate-for-gestational-age (AGA) children: fibrinogen, antithrombin III, protein C and S, fasting insulin, glucose, homeostasis assessment model of insulin resistance (HOMA-IR) index, adiponectin, leptin, visfatin, IGF-1, IGF-binding protein (IGFBP)-1, IGFBP-3, lipids, and the genetic factors V Leiden G1691A mutation, prothrombin 20210A/G polymorphism, and mutation in the enzyme 5,10-methylenetetrahydrofolate-reductase gene (MTHFR-C677T). RESULTS: LGA children had higher levels of leptin (P<0.01), fasting insulin (P<0.01), and HOMA-IR (P<0.01), but lower IGFBP-3 (P=0.0001), fibrinogen (P=0.0001), and lipoprotein(a) (P<0.001) than AGA children. Significantly more LGA children were homozygous for the MTHFR-C677T mutation (P=0.0016). CONCLUSIONS: Being born LGA to nondiabetic, nonobese mothers is associated with diverse effects on cardiometabolic risk factors at prepuberty.

Thrombophilic risk factors in the pathogenesis of non-arteritic anterior ischemic optic neuropathy patients.

BACKGROUND: Non-arteritic anterior ischemic optic neuropathy (N-AION) is caused by acute ischemic infarction of the optic nerve head, supplied by the posterior ciliary arteries. Thrombophilia is the tendency/predisposition to vascular thromboses of arteries and veins, and the existence of thrombophilic risk factors leads to blood hypercoagulability and potentially increased risk for thromboses. OBJECTIVES: To investigate whether there is an association between N-AION and a wide spectrum of thrombophilic risk factors. PATIENTS AND METHODS: Seventy-seven consecutive cases of confirmed N-AION and 60 age- and sex-matched consecutive controls constituted the study group. Fibrinogen levels, deficiency of proteins C, S, ATIII, lupus anticoagulant, activated protein C resistance, factor V Leiden, factor V H1299R, factor II G20210A, MTHFR C677T, MTHFR A1298C, GPIIIa A1/A2, and ACE I/D polymorphisms were analysed. RESULTS: Statistical analysis of the plasma proteins in our study demonstrated that the only significant difference was the one concerning protein S levels. In particular, the mean value for N-AION patients was 78.8% +/- 21.2, and for the control group the mean value was 88% +/- 21.2 (p = 0.013). Despite the above-mentioned result, there was not any statistical difference between the two subgroups regarding actual protein S deficiency, as 9/77 (11.7%) patients and 4/60 (6.7%) controls had protein S levels below 60% (p = 0.32). In our study sample, homozygosity for MTHFR C677T polymorphism in the study group as a whole, and the presence of at least one A2 allele of GPIIIa in the subgroup of male patients as compared to healthy male controls, proved to be the most significant thrombophilic risk factors, with odds ratios of 16.78 (95% C.I 0.96-294.42, p = 0.054) and 4.6 (95% C.I 1.52-13.88, p = 0.007) respectively. CONCLUSION: Screening for these polymorphisms would probably constitute a valuable procedure in N-AION patients, as they may have an important contribution to the pathogenesis of the disease.

Serum levels of soluble fas in patients with multinodular goiter.

OBJECTIVE: Fas is a cell-surface receptor responsible for induction of apoptosis in human thyrocytes upon interaction with Fas Ligand. Fas protein expression on thyroid cells and Fas-mediated apoptosis is decreased in multinodular goiter (MNG) resulting in thyroid cell proliferation. The soluble form of Fas (sFas) produced by alternative mRNA splicing may inhibit Fas-Fas Ligand binding and apoptosis. The aim of this study was to examine whether sFas is differentially expressed in multinodular goiter (MNG), which is associated with decreased Fas-mediated apoptosis. METHOD: We determined serum sFas levels using enzyme-linked immunosorbent assay (ELISA) in 42 patients with MNG and 23 normal controls. RESULTS: Serum sFas levels were increased in patients with MNG (7.47 +/- 2.55 ng/ml) compared to normal controls (2.26 +/- 0.9 ng/ml). Levels of sFas were not significantly correlated with age, sex or clinical parameters, such as serum levels of FT4 or TSH. DISCUSSION: Increased sFas in MNG may indicate increased expression of alternatively spliced Fas mRNA variant and decreased expression of cell-surface Fas protein, and may enhance thyroid cell proliferation by protecting thyroid cells from Fas-mediated apoptosis.

Protein Z Plasma Levels are Not Elevated in Patients with Non-Arteritic Anterior Ischemic Optic Neuropathy.

BACKGROUND: Protein Z is a glycoprotein that acts as a co-factor for the inhibition of activated coagulation factor X. Protein Z circulating in abnormal levels has been associated with increased risk for acute ischemic events. Non-arteritic Anterior Ischemic Optic Neuropathy (N-AION) is caused by acute ischemic infarction of the optic nerve head, supplied by the posterior ciliary arteries. OBJECTIVES: The aim was to investigate whether there is an association between N-AION and plasma protein Z levels. PATIENTS AND METHODS: Twenty-six cases of confirmed N-AION and fifty-two controls were included in the study group. Protein Z was estimated in thawed citrate plasma on both N-AION cases and controls by an enzyme immunoassay. The imprecision of the estimation was satisfactory (CV = 4, 6%). RESULTS: The controls' protein Z values distributed within a range 340 to 4200 ng/ml (median = 1420, mean = 1673, SD = 1040 ng/ml). Patients' protein Z values distributed within a range 420 to 3600 ng/ml (median = 1030, mean = 1520, SD = 939 ng/ml). There was no statistical difference between the two distributions (Independent t-test, p=0.529). CONCLUSION: In our study, protein Z levels are not implicated in the pathogenesis of non-arteritic anterior ischemic optic neuropathy (N-AION).

Peripheral blood flow cytometry based diagnosis in primary cutaneous aggressive epidermotropic CD8+ T-cell lymphoma: implications for more extensive use.

Primary cutaneous epidermotropic CD8-positive T-cell lymphoma represents an aggressive form of T-cell cutaneous lymphomas. Diagnosis is based on biopsies obtained from skin lesions. Here, we would like to report a case diagnosed by using flow cytometry performed on peripheral blood mononuclear cells. Moreover, an important finding was the difference in the results on targeting the CD8 antigen by using two different commercially available monoclonal antibodies. Perhaps, more than one antibody should be used in primary cutaneous aggressive epidermotropic CD8-positive T-cell lymphomas, in order to be more accurate in the diagnosis and so in the classification of such diseases.

HLA-DR expressing peripheral T regulatory cells in newly diagnosed patients with different forms of autoimmune thyroid disease.

BACKGROUND: Several reports have claimed a role for T regulatory cells (Tregs) in the pathogenesis of various autoimmune diseases, including autoimmune thyroid disease (AITD). The aim of the present study was to examine whether changes in the number of peripheral CD4 + CD25highHLA-DR + lymphocytes, a subpopulation of Tregs, occur in patients with AITD. METHODS: Three-color flow cytometry was used to detect the proportion of CD4 cells expressing CD25, CD25high, and HLA-DR in 70 newly diagnosed and untreated AITD patients and 20 controls. The intensity of CD25 expression on these cells was also examined. RESULTS: The proportion of CD4 + CD25 + cells as well as the proportion of CD4 + CD25high cells among the population of CD4 lymphocytes was not different in AITD patients relative to controls. However, a significant increase in the proportion of CD4 + CD25highHLA-DR + cells among the population of CD4 lymphocytes was found in patients with Hashimoto's thyroiditis (HT) compared to controls. CONCLUSIONS: In HT patients there is a quantitative increase of CD4 + CD25highHLA-DR + cells that may indicate a compensatory expansion of this subpopulation of Tregs in an attempt to suppress the immune response.

Differential expression of Fas system apoptotic molecules in peripheral lymphocytes from patients with Graves' disease and Hashimoto's thyroiditis.

OBJECTIVE: To examine whether the Fas system apoptotic molecules are differentially expressed in Graves' disease (GD) and Hashimoto's thyroiditis (HT), the two opposite phenotypes of autoimmune thyroid disease (AITD). DESIGN: The expression of Fas and Fas ligand (FasL) on peripheral CD4 and CD8 lymphocytes, and non-lymphoid immune cells as well as their soluble forms in serum from untreated patients with GD and HT were evaluated. METHODS: Flow cytometry was performed for the study of peripheral immune cells from 70 newly diagnosed patients with AITD (55 with HT and 15 with GD) and 20 controls. ELISA was used for the measurement of soluble Fas (sFas) in serum samples from a subgroup of 35 AITD patients. RESULTS: An increase in the proportion of CD4 and CD8 cells expressing Fas was found in both GD and HT, albeit with some differences, when compared with controls. Importantly, in GD patients, the intensity of Fas expression on CD4 and CD8 lymphocytes was reduced and sFas levels in serum were simultaneously increased when compared with HT patients and controls. CONCLUSIONS: The Fas system apoptotic molecules appear to be differentially expressed on peripheral lymphocytes in the two opposite phenotypes of AITD.

Global profiling of EGFR gene mutation, amplification, regulation and tissue protein expression in unknown primary carcinomas: to target or not to target?

INTRODUCTION: Epidermal growth factor receptor (EGFR) signalling contributes to malignant transformation and survival. We studied molecular predictors of benefit from EGFR-modulating therapies in patients with cancer of unknown primary (CUP). MATERIALS AND METHODS: Tumours from paraffin-embedded biopsies of 50 patients with CUP were stained for EGFR protein by immunohistochemistry. Polymerase chain reaction amplification, single-strand conformational polymorphism and direct sequencing were used to study EGFR intron 1 cytosine-adenosine (CA) repeat length as well as exon 18, 19, 21 activating mutations and amplification. RESULTS: Thirty-seven tumours (74%) expressed EGFR protein but only six (12%) strongly. Regarding intron 1 CA repeat length, we detected five alleles with CA repeat numbers 16-20, allele 16 being the most common (39%). All samples were heterozygous, the commonest genotype consisting of 16/18 dinucleotides (78%). Five samples had three intron 1 alleles and were associated with EGFR overexpression in 40% of cases. There was no evidence of EGFR exon 18, 19, 21 amplification. Two mutations were detected: Exon 21 2508 C > T, a silent nucleotide polymorphism (R836R) and a G > A substitution in sequences flanking exon 19 (IVS19 + 24G > A) resulting in aberrant mRNA splicing. Neither EGFR protein expression nor CA repeat length were prognostic factors for survival. CONCLUSIONS: Our data depict absence of molecular predictors of benefit from EGFR modulation in patients with CUP. Study of its molecular pathophysiology and targeting other molecular pathways may be warranted instead.

Free functional muscle transfer failure and thrombophilic gene mutations as a potential risk factor: a case report.

The evolution of microsurgery popularized the free functioning muscle transfers as secondary procedures to reanimate paralyzed extremities after severance of the brachial plexus, especially when the surgeon deals with late cases. Studies considering transplantation, describe thrombophilic factors as a cause of severe complications after transplantation, such as acute or early rejection episodes, delayed graft function, or chronic graft dysfunction. It is the first time that thrombophilia associated with free muscle-graft rejection is reported. A young man who had two free functional muscle transfers for brachial plexus reconstruction in the same forearm within an interval of 6 months. The free functional muscle transfer was failed in both cases because of vein thrombosis and subsequent arterial clot. The possibility of thrombophilia was investigated and during the genetic investigation it was discovered that he was heterozygous for the mutations of factor V, G1691A-Leiden, A4070G and homozygous for the MTHFR C677T mutation.

Effect of barnidipine on blood pressure and serum metabolic parameters in patients with essential hypertension: a pilot study.

The effect of barnidipine, a calcium channel blocker, on metabolic parameters is not well known. The authors conducted the present pilot study to evaluate the possible effects of barnidipine on parameters involved in atherogenesis, oxidative stress, and clotting activity. This open-label intervention study included 40 adult patients with essential hypertension who received barnidipine 10 mg once daily. Barnidipine significantly reduced systolic and diastolic blood pressure as well as isoprostane levels, which represent a reliable marker of oxidative stress. In contrast, barnidipine had a neutral effect on lipid profile and apolipoprotein levels, did not influence glucose homeostasis, had no effect on renal function, and did not cause any changes in electrolyte levels. Moreover, barnidipine did not affect either the clotting/fibrinolytic status (evaluated by measurement of fibrinogen, total plasminogen activator inhibitor, tissue plasminogen activator, and a2 antiplasmin) or the enzymatic activity of the inflammatory/anti-inflammatory mediators lipoprotein-associated phospholipase A2 and paraoxonase 1, respectively. Barnidipine should be mainly considered as an antihypertensive agent with neutral effects on most of the studied metabolic parameters in hypertensive patients. Any antioxidant effect of barnidipine needs further investigation.

Genetic risk factors associated with thrombosis in children with congenital neurologic disorders.

Thromboembolic events during the perinatal period are responsible for irreversible brain damage owing to cerebral hypoxia and neuronal necrosis. We investigated the presence of thrombophilia risk factors in children with congenital neurologic disorders. Nineteen children (9 males and 10 females), aged 1 to 14 years (median 4.5 years), who had presented with symptoms and signs of congenital neurologic disorders were studied. Thirty-five age-matched healthy children recruited from the same geographic area served as controls. Three patients of 19 (15.8%) were carrying the factor V Leiden mutation compared with 2 children among the controls (5.7%). One patient was heterozygous for the prothrombin G20210A variant (5.2%) compared with one child who was heterozygous among the controls. Three patients were homozygous (15.8%) and 11 were heterozygous (57.9%) for the C677T 5,10-methylenetetrahydrofolate reductase gene mutation compared with 4 (11.5%) and 18 (51.4%), respectively, among the controls. Three patients of 19 (15.8%) were carrying more than one mutation. We found 18 mutations in 79% (15/19) of the patients and 25 mutations in 69% (24/35) of the healthy children. Among the individuals carrying the homozygous 677TT 5,10-methylenetetrahydrofolate reductase genotype, we found 7 mutations in 32% (6/19) of the patients and 7 mutations in 20% (7/35) of the healthy children (P > .05). In one patient, lupus anticoagulant and antiphospholipid antibodies of IgG isotype were detected. Reduced activities of protein C, protein S, or antithrombin III were not observed in either the patient or the control group. Although, among our cases, we found some well-known risk factors associated with thrombosis in adults, the pathogenesis of these clinical entities remains obscure.