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.

Comparative Analysis of the Performance of Various Crystalloid Cardioplegic Solutions on Myocardial Protection After Prolonged Cold Ischemia

Introduction. The quality and effectiveness of myocardial protection are fundamental problems to expand the use of and consequently good outcomes of donated hearts for transplantation. Objective. The purpose of this investigation was to compare the cardioprotective effects of Krebs-Henseleit, Bretschneider-HTK, St Thomas, and Celsior solutions using a modified nonrecirculating Langendorff column model of isolated perfused rat heart during prolonged cold storage. Materials and Methods. After removal 36 rat hearts underwent isolated perfusion into a Langendorff apparatus using Krebs-Henseleit solution for a 15-minute period of recovery; we excluded organs that did not maintain an aortic pressure above 100 m Hg. Subsequently, we equally distributed the hearts into four groups according to the cardioprotection solution; group 1, Krebs-Henseleit (control); group II, Bretschneider-HTK; group III, St Thomas; and group IV, Celsior. Each heart received the specific cardioplegic solution at 10 C for 2-hour storage at 20 C, before a 15 minutes perfusion with Krebs-Henseleit solution for recovery and stabilization. After 60 additional minutes of perfusion, every 5 minutes we determined heart rate (HR), coronary flow (CF), left ventricular systolic pressure (LVSP), and positive and negative peak of the first derivative of left ventricular pressure (+dP/dt and dP/dt, respectively). Results. Comparative analysis by Turkey`s test showed the following performances among the groups at 60 minutes of reperfusion: HR: II = IV > III > I; CF: II = IV > I = III; LVSP: IV > I = II = III; +dP/dt: IV > I = II = III; and dP/dt: IV = II > I = II. Conclusion. Cardioprotective solutions generally used in clinical practice are not able to avoid hemodynamic alterations in hearts exposed to prolonged ischemia. Celsior solution showed better performance than Bretschneider-HTK, St Thomas, and Krebs-Henseleit.

Reference Values of Tissue Doppler Imaging and Pulsed Doppler Echocardiography for Analysis of Left Ventricular Diastolic Function in Healthy Adults

To determine reference values for tissue Doppler imaging (TDI) and pulsed Doppler echocardiography for left ventricular diastolic function analysis in a healthy Brazilian adult population. Observations were based on a randomly selected healthy population from the city of Vitoria, Espirito Santo, Brazil. Healthy volunteers (n = 275, 61.7% women) without prior histories of cardiovascular disease underwent transthoracic echocardiography. We analyzed 175 individuals by TDI and evaluated mitral annulus E`- and A`-waves from the septum (S) and lateral wall (L) to calculate E`/A` ratios. Using pulsed Doppler echocardiography, we further analyzed the mitral E- and A-waves, E/A ratios, isovolumetric relaxation times (IRTs), and deceleration times (DTs) of 275 individuals. Pulsed Doppler mitral inflow mean values for men were as follows: E-wave: 71 +/- 16 cm/sec, A-wave: 68 +/- 15 cm/sec, IRT: 74.8 +/- 9.2 ms, DT: 206 +/- 32.3 ms, E/A ratio: 1.1 +/- 0.3. Pulsed Doppler mitral inflow mean values for women were as follows: E-wave: 76 +/- 17, A-wave: 69 +/- 14 cm/sec, IRT: 71.2 +/- 10.5 ms, DT: 197 +/- 33.3 ms, E/A ratio: 1.1 +/- 0.3. IRT and DT values were higher in men than in women (P = 0.04 and P = 0.007, respectively). TDI values in men were as follows: E`S: 11 +/- 3 cm/sec, A`S: 13 +/- 2 cm/sec, E`S/A`S: 0.89 +/- 0.2, E`L: 14 +/- 3 cm/sec, A`L: 14 +/- 2 cm/sec, E`L/A`L: 1.1 +/- 0.4. E-wave/ E`S ratio: 6.9 +/- 2.2; E-wave / E`L ratio: 4.9 +/- 1.7. In this study, we determined pulsed Doppler and TDI derived parameters for left ventricular diastolic function in a large sample of healthy Brazilian adults. (Echocardiography 2010;27:777-782).

Observer agreement of spine stenosis on magnetic resonance imaging analysis of patients with cervical spine myelopathy

Objectives: The purpose of this study was to measure the intraobserver and interobserver reliability of magnetic resonance detection of cervical spondylotic myelopathy with and without operational guidelines. Methods: Seven radiologists examined images from 10 patients with cord signal abnormalities and clinical signs of myelopathy. Radiologist examined films twice, with and without operational guidelines designed to define stenotic changes, while blinded to the clinical findings of the patients. Analyses included a Fleiss kappa assessment of intraobserver and interobserver reliability. Results: Results demonstrated high percentage of agreement and strong intraobserver reliability and variable Fleiss kappa, values for interobserver assessment. Operational guidelines did not improve the intraobserver or interobserver agreement. Conclusion: Although the percentage of agreement was high in some cases, the kappa agreement was low-most likely a result of the base rate problem of a kappa analysis. Sample bias toward severe degenerative changes resulted in highly prevalent selections and kappa adjusted values. Nonetheless, the results do suggest that substantial intraobserver kappa agreement and a wide range of interobserver kappa agreement exists among trained radiologists during detection of stenotic changes associated with cervical spondylotic myelopathy.

Arg72Pro TP53 polymorphism and cancer susceptibility: a comprehensive meta-analysis of 302 case-control studies

Arg72Pro is a common polymorphism in TP53, showing differences in its biological functions. Case-control studies have been performed to elucidate the role of Arg72Pro in cancer, although the results are conflicting and heterogeneous. Here, we analyzed pooled data from case-control studies to determine the role of Arg72Pro in different cancer sites. We performed a systematic review and meta-analysis of 302 case-control studies that analyzed Arg72Pro in cancer susceptibility. Odds ratios were estimated for different tumor sites using distinct genetic models, and the heterogeneity between studies was explored using I(2) values and meta-regression. We adopted quality criteria to classify the studies. Subgroup analyses were done for tumor sites according to ethnicity, histological, and anatomical sites. Results indicated that Arg72Pro is associated with higher susceptibility to cancer in some tumor sites, mainly hepatocarcinoma. For some tumor sites, quality of studies was associated with the size of genetic association, mainly in cervical, head and neck, gastric, and lung cancer. However, study quality did not explain the observed heterogeneity substantially. Meta-regression showed that ethnicity, allelic frequency and genotyping method were responsible for a substantial part of the heterogeneity observed. Our results suggest ethnicity and histological and anatomical sites may modulate the penetrance of Arg72Pro in cancer susceptibility. This meta-analysis denotes the importance for more studies with good quality and that the covariates responsible for heterogeneity should be controlled to obtain a more conclusive response about the function of Arg72Pro in cancer.

COST-EFFECTIVENESS ANALYSIS OF CISPLATIN-BASED CHEMORADIATION TO TREAT PATIENTS WITH UNRESECTABLE, NONMETASTATIC HEAD AND NECK CANCER IN BRAZIL

Background. The purpose of this study was to analyze the cost-effectiveness of cisplatin-based chemoradiation compared to radiation therapy (RT) alone to treat patients with advanced head and neck cancer in Brazil. Methods. Data were collected retrospectively from the medical records of 33 patients treated with RT alone (strategy 1) and from 29 patients treated with cisplatin-based chemoradiation (strategy 2). The Brazilian National Health System (Sistema Unico de Saude [SUS]) reimbursement parameters perspective was considered, and the effectiveness was measured in years of disease-free life gained. One-way sensitivity analysis was performed to determine robustness of this study. Results. In strategy 1, there were 31% of the patients who lived without disease progression for more than 13 months after treatment, compared to 58% of patients in strategy 2. According to SUS parameters, the total cost per patient in strategy 1 was $1167.00 U.S. dollars and in strategy 2, it was $2058.00 U.S. dollars. Incremental cost-effectiveness ratio (ICER) was $3303.00 U.S. dollars per life-year gained. Conclusion. Cisplatin-based chemoradiation proved to be more cost-effective than RT alone. (C) 2010 Wiley Periodicals, Inc. Head Neck 33: 1199-1205, 2011

Quantitative ferromagnetic resonance analysis of CD133 stem cells labeled with iron oxide nanoparticles

The aim of this work is to provide a quantitative method for analysis of the concentration of superparamagnetic iron oxide nanoparticles (SPION), determined by means of ferromagnetic resonance (FMR), with the nanoparticles coupled to a specific antibody (AC133), and thus to express the antigenic labeling evidence for the stem cells C D133(+). The FMR efficiency and sensitivity were proven adequate for detecting and quantifying the low amounts of iron content in the C D133(+) cells (similar to 6.16 x 10(5) pg in the volume of 2 mu l containing 4.5 x 1011 SPION). The quantitative method led to the result of 1.70 x 10(-13) mol of Fe (9.5 pg), or 7.0 x 10(6) nanoparticles per cell. For the quantification analysis via the FMR technique it was necessary to carry out a preliminary quantitative visualization of iron oxide-labeled cells in order to ensure that the nanoparticles coupled to the antibodies are indeed tied to the antigen at the stem cell surface and that the cellular morphology was conserved, as proof of the validity of this method. The quantitative analysis by means of FMR is necessary for determining the signal intensity for the study of molecular imaging by means of magnetic resonance imaging (MRI).

Atopic dermatitis in adults: evaluation of peripheral blood mononuclear cells proliferation response to Staphylococcus aureus enterotoxins A and B and analysis of interleukin-18 secretion

Atopic dermatitis (AD) is a chronic, inflammatory skin disease with a high prevalence and complex pathogenesis. The skin of AD patients is usually colonized by Staphylococcus aureus (S. aureus); its exotoxins may trigger or enhance the cutaneous inflammation. Several mediators are related to the AD immune imbalance and interleukin-18 (IL-18), an inflammatory cytokine, may play a role in the atopic skin inflammation. To evaluate peripheral blood mononuclear cells (PBMC) proliferation response to staphylococcal enterotoxins A (SEA) and B (SEB) and the levels of IL-18 in adults with AD. Thirty-eight adult patients with AD and 33 healthy controls were analysed. PBMC were stimulated with SEA and SEB, phytohemaglutinin (PHA), pokeweed (PWM), tetanus toxoid (TT) and Candida albicans (CMA). IL-18 secretion from PBMC culture supernatants and sera were measured by ELISA. A significant inhibition of the PBMC proliferation response to SEA, PHA, TT and CMA of AD patients was detected (P <= 0.05). Furthermore, increased levels of IL-18 were detected both in sera and non-stimulated PBMC culture supernatants from AD patients (P <= 0.05). A decreased PBMC proliferation response to distinct antigens and mitogens (TT, CMA, SEA and PHA) in adults with AD suggest a compromised immune profile. IL-18 secretion from AD upon stimulation was similar from controls, which may indicate a diverse mechanism of skin inflammation maintained by Staphylococcus aureus. On the other hand, augmented IL-18 secretion from AD sera and non-stimulated cell culture may enhance the immune dysfunction observed in AD, leading to constant skin inflammation.

Comparative analysis of the expression of cytokeratins (1,10,14,16,4), involucrin, filaggrin and e-cadherin in plane warts and epidermodysplasia verruciformis plane wart-type lesions

Background: Epidermodysplasia verruciformis (EV) is a rare genodermatosis with susceptibility to human papillomavirus (HPV) infection, and high risk of skin cancer considered a model of viral oncogenesis. Methods: Fifteen cases of EV plane wart (PW)-type lesions (EV) and 14 cases of PW in healthy individuals were subjected to immunohistochemical technique for cytokeratins (K) 1, 10, 14, 16, 4, involucrin, filaggrin and e-cadherin. Results: K1/10 showed retarded or negative expression in EV, being substituted by K14. Expression of K14 occurred in the basal and suprabasal layers in both groups, but in EV, its expression was observed up to the more superficial layers. Both groups showed positivity for K16 and K4, involucrin expression in lower levels of the spinous layer and unaltered filaggrin expression. E-cadherin expression was diminished at the koilocytotic foci of both lesions, more superficially in EV. Conclusion: Infection by HPV may alter the differentiation status of the epidermis, leading to a major expression of K14, delayed or absent expression of K1/10 and earlier involucrin expression, especially in EV. It also stimulates the expression of K16 and K4. Filaggrin expression is not altered, and e-cadherin is diminished in superficial koilocytotic cells` foci in EV.

Phylogenetic Analysis of Hepatitis B Virus Genotype F Complete Genome Sequences From Chilean Patients With Chronic Infection

Molecular epidemiological data concerning the hepatitis B virus (HBV) in Chile are not known completely. Since the HBV genotype F is the most prevalent in the country, the goal of this study was to obtain full HBV genome sequences from patients infected chronically in order to determine their subgenotypes and the occurrence of resistance-associated mutations. Twenty-one serum samples from antiviral drug-naive patients with chronic hepatitis B were subjected to full-length PCR amplification, and both strands of the whole genomes were fully sequenced. Phylogenetic analyses were performed along with reference sequences available from GenBank (n = 290). The sequences were aligned using Clustal X and edited in the SE-AL software. Bayesian phylogenetic analyses were conducted by Markov Chain Monte Carlo simulations (MCMC) for 10 million generations in order to obtain the substitution tree using BEAST. The sequences were also analyzed for the presence of primary drug resistance mutations using CodonCode Aligner Software. The phylogenetic analyses indicated that all sequences were found to be the HBV subgenotype F1b, clustered into four different groups, suggesting that diverse lineages of this subgenotype may be circulating within this population of Chilean patients. J. Med. Virol. 83: 1530-1536, 2011. (C) 2011 Wiley-Liss, Inc.

Seven-year trend analysis of nosocomial candidemia and antifungal (fluconazole and caspofungin) use in Intensive Care Units at a Brazilian University Hospital

Candidemia is associated with high morbidity and mortality resulting in significant increases in the length of patients` hospitalization and in healthcare costs. Critically ill patients are at particular risk for candidemia because of their debilitated condition and frequent need for invasive procedures. The aim of this study was to characterize the incidence and epidemiology of candidemia over a seven-year period in intensive care units (ICUs) and the use of fluconazole and caspofungin in a large university-affiliated hospital. All cases of candidemia were identified by surveillance, using the Centers for Diseases Control and Prevention criteria. Demographic variables, use of antifungal (fluconazole and caspofungin) and patient outcomes were evaluated. The 2 test for linear trend was employed to evaluate the distribution of Candida spp. and the use of fluconazole and caspofungin by defined daily dose (DDD) per 1,000 patients-days during the study period. One hundred and eight episodes of candidemia were identified. The overall incidence of candidemia (P=0.20) and incidence of non-Candida albicans Candida infections (P=0.32) remained stable over the study period and ranged from 0.3-0.9 episodes per 1,000 catheter-days and 0.39-0.83 episodes per 1,000 patients-days. However, the use of fluconazole and caspofungin increased significantly (P0.001). While there were no reports of the use of fluconazole for prophylaxis in 1999, its use for this purpose increased from 3% in 2000 to 7.0% (P=0.07) in 2006. C. albicans was the most frequent specie isolated and burns and cancer were the most frequent underlying conditions. The overall mortality was 76%. There was no difference between C. albicans and non-C. albicans Candida infections when the crude and 14-day mortality rates were compared. Our data demonstrated that C. albicans is still the most frequent species causing candidemia in our intensive care units. Our rates of candidemia are lower than those reported from the region and similar to American and European hospitals. Although the incidence of blood stream infections (BSI) and candidemia remained stable, the use of fluconazole and caspofungin increased significantly over the years included in this study but had no impact on the incidence of infections caused by non-C. albicans Candida species.

An integrated genomic analysis of human glioblastoma Multiforme

Glioblastoma multiforme ( GBM) is the most common and lethal type of brain cancer. To identify the genetic alterations in GBMs, we sequenced 20,661 protein coding genes, determined the presence of amplifications and deletions using high- density oligonucleotide arrays, and performed gene expression analyses using next- generation sequencing technologies in 22 human tumor samples. This comprehensive analysis led to the discovery of a variety of genes that were not known to be altered in GBMs. Most notably, we found recurrent mutations in the active site of isocitrate dehydrogenase 1 ( IDH1) in 12% of GBM patients. Mutations in IDH1 occurred in a large fraction of young patients and in most patients with secondary GBMs and were associated with an increase in overall survival. These studies demonstrate the value of unbiased genomic analyses in the characterization of human brain cancer and identify a potentially useful genetic alteration for the classification and targeted therapy of GBMs.