Molecular analysis of CYP21A2 can optimize the follow-up of positive results in newborn screening for congenital adrenal hyperplasia

Neonatal screening for congenital adrenal hyperplasia (CAH) is useful in diagnosing salt wasting form (SW). However, there are difficulties in interpreting positive results in asymptomatic newborns. The main objective is to analyze genotyping as a confirmatory test in children with neonatal positive results. Patients comprised 23 CAH children and 19 asymptomatic infants with persistently elevated 17-hydroxyprogesterone (17OHP) levels. CYP21A2 gene was sequenced and genotypes were grouped according to the enzymatic activity of the less severe allele: A1 null, A2 < 2%, B 3-7%, C > 20%. Twenty-one children with neonatal symptoms and/or 17OHP levels > 80 ng/ml carried A genotypes, except two virilized girls (17OHP < 50 ng/ml) without CAH genotypes. Patients carrying SW genotypes (A1, A2) and low serum sodium levels presented with neonatal 17OHP > 200 ng/ml. Three asymptomatic boys carried simple virilizing genotypes (A2 and B): in two, the symptoms began at 18 months; another two asymptomatic boys had nonclassical genotypes (C). The remaining 14 patients did not present CAH genotypes, and their 17OHP levels were normalized by 14 months of age. Molecular analysis is useful as a confirmatory test of CAH, mainly in boys. It can predict clinical course, identify false-positives and help distinguish between clinical forms of CAH.

Discontinuation of continuous renal replacement therapy: A post hoc analysis of a prospective multicenter observational study

Objectives: To describe current practice for the discontinuation of continuous renal replacement therapy in a multinational setting and to identify variables associated with successful discontinuation. The approach to discontinue continuous renal replacement therapy may affect patient outcomes. However, there is lack of information on how and under what conditions continuous renal replacement therapy is discontinued. Design: Post hoc analysis of a prospective observational study. Setting. Fifty-four intensive care units in 23 countries. Patients: Five hundred twenty-nine patients (52.6%) who survived initial therapy among 1006 patients treated with continuous renal replacement therapy. Interventions: None. Measurements and Main Results., Three hundred thirteen patients were removed successfully from continuous renal replacement therapy and did not require any renal replacement therapy for at least 7 days and were classified as the ""success"" group and the rest (216 patients) were classified as the ""repeat-RRT"" (renal replacement therapy) group. Patients in the ""success"" group had lower hospital mortality (28.5% vs. 42.7%, p < .0001) compared with patients in the ""repeat-RRT"" group. They also had lower creatinine and urea concentrations and a higher urine output at the time of stopping continuous renal replacement therapy. Multivariate logistic regression analysis for successful discontinuation of continuous renal replacement therapy identified urine output (during the 24 hrs before stopping continuous renal replacement therapy: odds ratio, 1.078 per 100 mL/day increase) and creatinine (odds ratio, 0.996 per mu mol/L increase) as significant predictors of successful cessation. The area under the receiver operating characteristic curve to predict successful discontinuation of continuous renal replacement therapy was 0.808 for urine output and 0.635 for creatinine. The predictive ability of urine output was negatively affected by the use of diuretics (area under the receiver operating characteristic curve, 0.671 with diuretics and 0.845 without diuretics). Conclusions. We report on the current practice of discontinuing continuous renal replacement therapy in a multinational setting. Urine output at the time of initial cessation (if continuous renal replacement therapy was the most important predictor of successful discontinuation, especially if occurring without the administration of diuretics. (Crit Care Med 2009; 37:2576-2582)

Analysis of the PTPN11 gene in idiopathic short stature children and Noonan syndrome patients

Background Mutations in the PTPN11 gene are the main cause of Noonan syndrome (NS). The presence of some NS features is a frequent finding in children with idiopathic short stature (ISS). These children can represent the milder end of the NS clinical spectrum and PTPN11 is a good candidate for involvement in the pathogenesis of ISS. Objective To evaluate the presence of mutations in PTPN11 in ISS children who presented NS-related signs and in well-characterized NS patients. Patients and methods We studied 50 ISS children who presented at least two NS-associated signs but did not fulfil the criteria for NS diagnosis. Forty-nine NS patients diagnosed by the criteria of van der Burgt et al. were used to assess the adequacy of these criteria to select patients for PTPN11 mutation screening. The coding region of PTPN11 was amplified by polymerase chain reaction (PCR), followed by direct sequencing. Results No mutations or polymorphisms were found in the coding region of the PTPN11 gene in ISS children. Nineteen of the 49 NS patients (39%) presented mutations in PTPN11. No single characteristic enabled us to distinguish between NS patients with or without PTPN11 mutations. Conclusion Considering that no mutations were found in the present cohort with NS-related signs, it is unlikely that mutations would be found in unselected ISS children. The van der Burgt et al. criteria are adequate in attaining NS diagnosis and selecting patients for molecular studies. Mutations in the PTPN11 gene are commonly involved in the pathogenesis of NS but are not a common cause of ISS.

Immunohistochemical analysis of p53, APE1, hMSH2 and ERCC1 proteins in actinic cheilitis and lip squamous cell carcinoma

Aims: This study has compared the tissue expression of the p53 tumour suppressor protein and DNA repair proteins APE1, hMSH2 and ERCC1 in normal, dysplastic and malignant lip epithelium. Methods and results: Morphological analysis and immunohistochemistry were performed on archived specimens of normal lip mucosa (n = 15), actinic cheilitis (AC) (n = 30), and lip squamous cell carcinoma (LSCC) (n = 27). AC samples were classified morphologically according to the severity of epithelial dysplasia and risk of malignant transformation. LSCC samples were morphologically staged according to WHO and invasive front grading (IFG) criteria. Differences between groups and morphological stages were determined by bivariate statistical analysis. Progressive increases in the percentage of epithelial cells expressing p53 and APE1 were associated with increases in morphological malignancy from normal lip mucosa to LSCC. There was also a significant reduction in epithelial cells expressing hMSH2 and ERCC1 proteins in the AC and LSCC groups. A higher percentage of malignant cells expressing APE1 was found in samples with an aggressive morphological IFG grade. Conclusions: Our data showed that epithelial cells from premalignant to malignant lip disease exhibited changes in the expression of p53, APE1, hMSH2 and ERCC1 proteins; these molecular change might contribute to lip carcinogenesis.

Thirty-Day Morbidity and Mortality of the Laparoscopic Ileal Interposition Associated with Sleeve Gastrectomy for the Treatment of Type 2 Diabetic Patients with BMI < 35: An Analysis of 454 Consecutive Patients

Background The objective of this study was to evaluate the early results of the laparoscopic interposition of a segment of ileum associated with a sleeve gastrectomy (LII-SG) in order to treat patients with type 2 diabetes mellitus (T2DM) and BMI <35. Data regarding morbidly obese diabetic patients subjected to surgery has consistently been validated. To date, there is scarce information about morbidity and mortality related to the surgical treatment of a ""true"" typical diabetic population with BMI <35. Methods The procedures were performed in 454 patients (322 male, 132 female). Mean age was 53.6 +/- 8 years (range = 27-75). Mean BMI was 29.7 +/- 3.6 kg/m(2) (range = 19-34.8). All patients had the diagnosis of T2DM for at least 3 years. Insulin therapy was used by 45.6% of patients. Mean duration of T2DM was 10.8 +/- 5.9 years (range = 3-35). Mean hemoglobin A(1c) was 8.8 +/- 1.9%. Dyslipidemia was observed in 78.4%, hypertension in 64.8%, nephropathy in 28.6%, retinopathy in 32.6%, neuropathy in 34.6%, and coronary heart disease in 13%. Results There was no conversion to open surgery. All patients were evaluated postoperatively. Mortality was 0.4%. There were 29 major complications (6.4%) in 22 patients (4.8%) and 51 minor complications (11.2%). Reoperations were performed on 8 patients (1.7%). Twenty patients (4.4%) were readmitted to the hospital. Mean postoperative BMI was 25.8 +/- 3.5 kg/m(2). Mean fasting plasma glucose decreased from 198 +/- 69 to 128 +/- 67 mg/dl and mean postprandial plasma glucose decreased from 262 +/- 101 to 136 +/- 43 mg/dl. Conclusions The laparoscopic ileal interposition associated with a sleeve gastrectomy was considered a safe operation with low rates of morbidity and mortality in a diabetic population with BMI < 35. An early control of postprandial glycemia was observed.

Meta-analysis of infrapopliteal angioplasty for chronic critical limb ischemia

Background: Percutaneous transluminal angioplasty has been used with increasing frequency in the treatment of infrainguinal arterial occlusive disease. This meta-analysis aimed to assess the middle-term outcomes after crural angioplasty in patients with chronic critical limb ischemia and compare results with a meta-analysis of popliteal-to-distal vein bypass graft. Methods: Data were retrieved from 30 articles published from 1990 through 2006 (63% of articles published between 2000 and 2006). All studies used survival analysis, reported a 12-month cumulative rate of patency or limb salvage, and included at least 15 infrapopliteal angioplasties. The outcome measures were immediate technical success, primary and secondary patency, limb salvage, and patient survival. Data from life-tables, survival curves, and texts were used. Results. The pooled estimate of success was 89.0% +/- 2.2% for immediate technical result. Results at 1 and 36 months were 77.4% +/- 4.1% and 48.6% +/- 8.0% for primary patency, 83.3% +/- 1.4% and 62.9% +/- 11.0% for secondary patency, 93.4% +/- 2.3% and 82.4% +/- 3.4% for limb salvage, and 98.3% +/- 0.7% and 68.4% +/- 5.5% for patient survival, respectively. Studies with >75% of the limbs with tissue loss fared worse than their respective comparative subgroup for technical success and patency but not for limb salvage or survival. No publication bias was detected. Conclusion: The technical success and subsequent durability of crural angioplasty are limited compared with bypass surgery, but the clinical benefit is acceptable because limb salvage rates are equivalent to bypass surgery. Further studies are necessary to determine the proper role of infrapopliteal angioplasty.

cDNA microarray analysis of differentially expressed genes in penile tissue after treatment with tadalafil

To evaluate differential gene expression in penile tissue after treatment with the phosphodiesterase 5 (PDE5) inhibitor tadalafil, as of the three clinically available PDE5 inhibitors (sildenafil, tadalafil, and vardenafil) used for the treatment of erectile dysfunction (ED), tadalafil has a long half-life and low incidence of side-effects. In all, 32 adult rats were divided into two groups. The control group received 0.5 mL of drinking water alone, while the tadalafil group was treated with tadalafil at a dose of 0.27 mg/kg. At 4 h after treatment with water or tadalafil the rats were killed and the penile tissue was removed. The total RNA was isolated from the penile tissue from both groups and differentially expressed genes were identified by cDNA microarray analysis. To validate the expression data from the microarray analysis, quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry were used. In all, 153 genes were differentially expressed between the control group and the tadalafil group. We validated the microarray results by quantitative PCR for the insulin-like growth factor binding protein 6 (IGFBP-6) gene and the neuronal calcium sensor 1 (NCS-1) gene, both of which were up-regulated in the tadalafil group, and for the natriuretic peptide receptor 1 (NPR-1) gene that was down-regulated in this group. Immunohistochemistry showed localization of the NCS-1 protein in sinusoid trabeculae of the corpus cavernosum in control and tadalafil-treated rats. There was differential expression in 153 genes after tadalafil treatment. Some of these genes such as IGFBP-6, NPR-1 and NCS-1, might result in new targets in the treatment of ED.

Biomechanical analysis of sutures used for mesh fixation in the donor area after removal of the rectus abdominis muscle

Background: The use of synthetic mesh for abdominal wall closure after removal of the rectus abdominis is established but not standardised. This study compares two forms of mesh fixation: a simple suture, which fixes the mesh to the edges of the defect on the anterior rectus abdominis fascia; and total fixation, which incorporates the fasciae of the internal oblique, external oblique and transverse muscles in the suture, anchoring the mesh in the position of the removed muscle. Method: A total of 16 fresh cadavers were dissected. Two sutures were compared: simple and total. Three different sites were analysed: 5 cm above, 5 cm below and at the level of the umbilicus. The two sutures compared were tested in each region using a standardised technique. All sutures were performed with nylon 0, perpendicular to the linea alba. Each suture was secured to a dynamometer, which was pulled perpendicularly towards the midline until the rupture of the aponeurosis. `Rupture resistance` was measured in kilogram force. The mean among the groups was compared using the paired Student`s t-test to a significance level of 1% (p < 0.01). Results: The mean rupture resistance of the total suture was 160% higher than that of the simple suture. Conclusion: The total suture includes the external oblique, internal oblique and transverse fasciae, which are multi-directional, and creates a much higher resistance when compared with the simple suture. Total suture may reduce the incidence of bulging and hernias of the abdominal wall after harvesting the rectus abdominis muscle, but comparative clinical studies are necessary. (C) 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

Analysis of the Strength of the Abdominal Fascia in Different Sutures Used in Abdominoplasties

Protrusion of the abdominal wall secondary to abdominoplasty may occur in patients with weakness of the aponeurotic structures. The anterior layer of the rectus abdominis muscle consists of fibers that are transverse rather than vertical. Based on this anatomical feature, vertical sutures are suggested for the correction of diastasis recti, since they include a greater amount of fascial fibers and thus would be more resistant to tensile strength than horizontal ones. The anterior layers of the rectus abdominis muscles of 15 fresh cadavers were dissected. Two vertical lines were marked on each side of the linea alba, corresponding to the site where plication is usually performed in abdominoplasties. Three abdominal levels were evaluated: the supraumbilical, umbilical, and infraumbilical levels. A simple suture was placed in the vertical direction in one group and in the horizontal direction in the other group, at each of the three levels previously described. These sutures were connected to a dynamometer, which was pulled medially toward the linea alba until rupture of the aponeurosis occurred. The mean strength required to rupture the aponeurotic structures in which the vertical sutures had been placed was greater than for the horizontal ones (p < 0.0001). The vertical suture of the rectus abdominis sheaths was stronger than the horizontal suture because of the more transversal arrangement of its aponeurotic fibers. Thus, routine use of the vertical suture in plications of the aponeurosis of the rectus abdominis muscles is suggested.

Comparative Analysis of the Influence of Perichondrium on Conjunctival Epithelialization on Conchal Cartilage Grafts in Eyelid Reconstruction: Experimental Study in Rabbits

Background: Although the role of cartilage grafts in reconstruction of the posterior eyelid lamella is well established, spontaneous conjunctival epithelialization on such grafts has yet to be fully proven. The aim of this study was to perform a comparative analysis of the influence of perichondrium on conjunctival epithelialization over conchal cartilage grafts used in eyelid reconstruction in rabbits. Methods: The posterior lamellae of 100 lower eyelids from 50 rabbits were reconstructed with autogenous grafts of conchal ear cartilage. In the right eyelids, cartilage was grafted with the perichondrium in direct contact with the eyeball, and the left eyelids were reconstructed in a similar manner but using cartilage grafts without perichondrium. The animals were killed after 1, 2, 3, 4, and 5 weeks, and their lower eyelids were analyzed macroscopically and histologically. Results: The percentage difference in conjunctival epithelialization on the cartilage with perichondrium and that without perichondrium was 11.41 percent in the first week of the experiment, 13.64 percent in the second week, 18.69 percent in the third week, 10.38 percent in the fourth week, and 6.17 percent in the fifth week. The average percentage conjunctival epithelialization in the eyelids reconstructed with a cartilage graft with perichondrium was significantly higher throughout the 5 weeks of the experiment than in the eyelids reconstructed with cartilage without perichondrium (p < 0.0002). Conclusion: It was found that the perichondrium had an important role in conjunctival epithelialization in eyelids reconstructed with a cartilage graft in the present study. (Plast. Reconstr. Surg. 123: 55, 2009.)

Mutational analysis of the necdin gene in patients with congenital isolated hypogonadotropic hypogonadism

Context: Necdin activates GNRH gene expression and is fundamental for the development, migration, and axonal extension of murine GNRH neurons. In humans, necdin plays a potential role in the hypogonadotropic hypogonadism phenotype in patients with Prader-Willi syndrome. Aim: To investigate necdin gene (NDN) variants in patients with isolated hypogonadotropic hypogonadism (IHH). Patients and methods: We studied 160 Brazilian patients with IHH, which includes 92 with Kallmann syndrome and 68 with normosmic IHH. Genomic DNA was extracted and the single NDN exon was amplified and sequenced. To measure GNRH transcriptional activity, luciferase reporter plasmids containing GNRH regulatory regions were transiently transfected into GT1-7 cells in the presence and absence of overexpressed wild-type or mutant necdin. Results: A heterozygous variant of necdin, p.V318A, was identified in a 23-year-old male with Kallmann syndrome. The p.V318A was also present in affected aunt and his father and was absent in 100 Brazilian control subjects. Previous FGFR1 gene analysis revealed a missense mutation (p.P366L) in this family. Functional studies revealed a minor difference in the activation of GNRH transcription by mutant protein compared with wild type in that a significant impairment of the necdin protein activity threshold was observed. Conclusion: A rare variant of necdin (p.V318A) was described in a family with Kallmann syndrome associated with a FGFR1 mutation. Familial segregation and in vitro analysis suggested that this non-synonymous variant did not have a direct causative role in the hypogonadism phenotype. NDN mutations are not a frequent cause of congenital IHH.

Analysis of Different Staining Techniques for C4d Detection in Renal Allograft Biopsies

Capillary C4d deposition has been recognized as a marker of antibody-mediated rejection (AMR). Although the detection of capillary C4d by means of immunofluorescence (IF) in cryostat sections is well established, frozen tissue is not always available, thus limiting the diagnosis of AMR. The aim of the present study was to analyze different techniques for C4d staining and the prevalence of C4d in renal allograft biopsies. Detection of C4d was carried out using IF or immunohistochemistry (IHC) on frozen and paraffin sections of renal allograft biopsies available from the same patients. Biopsies obtained from 20 patients were classified into 3 groups: no rejection, acute rejection, and chronic allograft nephropathy (CAN). The capillary C4d deposition prevalence in frozen-IF, considered the gold standard technique for C4d detection, was 45% (9/20 cases). Compared with frozen-IF, the frozen-IHC technique presented an 85% concordance rate (17/20 cases; r =.70; P <.001; sensitivity = 77.8%; specificity = 90.9%). The paraffin-IF technique showed similar results, with an 80% concordance rate (16/20 cases; r =.64; P <.005; sensitivity = 55.6%; specificity = 100%), whereas C4d detection occurred in only 65% of paraffin-IHC cases (13/20; r =.30; not significant; sensitivity = 66.7%; specificity = 63.6%). No capillary C4d deposition was detected in cases without evidence of rejection. However, 4/7 cases (57%) of acute rejection were C4d positive. In the CAN group, 5111 cases (45%) were C4d positive. In conclusion, these results demonstrated that frozen-IHC and paraffin-IF can be considered alternative techniques to frozen-IF for C4d detection. The paraffin-IHC technique displayed the lowest concordance rate for C4d detection.

Factors affecting hematopoietic progenitor cell mobilization: An analysis of 307 patients

We reviewed the data of 307 patients treated with autologous bone marrow transplantation with the aim to identify factors associated with poor hematopoietic stern cell (HSC) mobilization after administration of cyclophosphamide and granulocyte-colony stimulating factor. Success in mobilization was defined when >= 2.0 x 10(6) CD34+ cells/kg weight could be collected with <= 3 leukapheresis procedures. Success was observed in 260 patients (84.7%) and nonsuccess in 47 patients (15.3%). According to the stepwise regression model: diagnosis, chemotherapy load, treatment with mitoxantrone and platelet count before mobilization were found to be independent predictive factors for HSC mobilization. These results could help in the previous recognition of patients at risk for non response to mobilization and allow to plan an alternative protocol for this group of patients. (C) 2008 Elsevier Ltd. All rights reserved.

Cost analysis of drug therapy in rheumatoid arthritis

With the aim to compare the cost of treatment for rheumatoid arthritis therapy with desease-modifying antirheumatic drugs (DMARDS) for a 48-month period, were studied five different treatment stage based on clinical protocols recommended by the Brazilian Society of Rheumatology, and then five therapy cycles. The analytical model based on the Markov Analysis, considered chaces for the patient continue in some stages or change between them according with a positive effect on outcomes. Only direct costs were comprised in the analyzed data, like drugs, materials and tests used for monitoring these patients. The results of the model show that the stage in with metotrexato drug is used like monotherapy was cost-effective (R$ 113,900,00 for patient during 48 months), followed by refractory patient (R$ 1,554,483,43), those that use therapy triplicate followed by infleximable drug (R$ 1, 701, 286.76), the metotrexato intolearant patient (R$ 2,629,919,14), and final the result from that use metotrexato and infliximable in the beginning (R$ 9,292,879,31). The sensitivity analysis confirm this results, when alternate the efficacy of metotrexato and infliximabe.

Meta-analysis of the association of 4 angiotensinogen polymorphisms with essential hypertension - A role beyond M235T?

Angiotensinogen (AGT) gene polymorphisms have been linked to increased risk of hypertension, but the data remain controversial. In this study we review the most commonly investigated polymorphisms at the AGT locus (other than M235T) and provide summary estimates regarding their association with essential hypertension, while addressing heterogeneity, as well as publication biases. Data on 26 818 subjects from 46 studies for the 4 most-studied AGT variants (T174M in exon 2 and 3 promoter variants: A-6G, A-20C, and G-217A) were meta-analyzed. Statistically significant associations with hypertension were identified for the T174M ( odds ratio [OR]: 1.19; 95% CI: 1.07 to 1.33; P = 0.002) and G-217A (OR: 1.37; 95% CI: 1.17 to 1.59; P = 0.00006) polymorphisms. A dual but consistent effect was observed for the -20C allele, which was associated with a decreased risk of hypertension in populations of mixed and European ancestries (OR: 0.64; 95% CI: 0.44 to 0.92; P = 0.02 and OR: 0.77; 95% CI: 0.65 to 0.91; P = 0.003, respectively), but with a 24% increase in the odds of hypertension in Asian subjects (OR: 1.24; 95% CI: 1.04 to 1.48; P = 0.02). No association of the A-6G variant with hypertension was detected. Current studies support the notion that single variants at the AGT might modulate the risk of hypertension but indicate caution in interpreting these results because of the putative presence of publication bias and gene-environment interactions.