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.

Frequency of concurrent autoimmune disorders in patients with autoimmune hepatitis - Effect of age, gender, and genetic background

Background: Concurrent autoimmune disorders (CAIDs) have been shown to occur in 22% to 34% of the patients with autoimmune hepatitis (AIH). Their presence has been linked to female gender, older age, and to certain HLA antigens, namely HLA-A11. DRB1*04, and DRB4*01. Aims: To assess the frequency and nature of CAID in Brazilian patients with AIH types 1 (AIH-1) and 2 (AIH-2) and to investigate the influence of age, gender, and genetic background in their occurrence. Patients and Methods: The presence and nature of CAID was studied in 143 patients [117 females, median age 11 (1.3 to 69)] with AIH-1 (n = 125) and AIH-2 (n = 28). HLA typing and tumor necrosis factor a gene promoter and exon I cytotoxic T lymphocyte associated antigen 4 (CTLA-4) gene polymorphisms were determined by polymerase chain reaction-based techniques. Results: The frequency of CAID was similar in patients with AIH-1 (14%) and AIH-2 (18%), but their nature was shown to vary. Arthritis was seen in half of the patients (n = 8) with CAID and AIH-1 and in none of those with AIH-2. Subjects with AIH-1 and CAID were shown to be older [24 (1.3 to 6 1) vs. 11 (1.3 to 69) y P = 0.02] and to have more often circulating antinuclear antibody (76% vs. 40%, P = 0.008) and less frequently antiactin antibodies (33% vs. 75%, P = 0.008) when compared with their counterparts without CAID. No particular HLA-DR and DQ alleles, as well as tumor necrosis factor a and CTLA-4 genotypes, were associated with CAID. Conclusions: The nature, but not the frequency, of CAID was shown to vary in AIH-1 and AIH-2. In subjects with AIH-1, CAID was linked to older subjects and to the presence of antinuclear antibody. No predisposition to CAID was associated to HLA-DRB1*04 or DDB4*01 alleles. The observed lower frequency of CAID could be attributed to the lower age of disease onset in Brazilians and to differences in HLA-encoded susceptibility to AIH-1 observed in South America.

Intracardiac Cavopulmonary Connection in Patients with Univentricular Heart Using Intra-atrial Lateral Tunnel and Intra-atrial Conduit Techniques

Background: In this study, we analyzed the time course of hemodynamic efficiency and follow-up in Fontan candidates who underwent the bidirectional Glenn procedure for staged intracardiac cavopulmonary connection (ICPC). Methods: Between 1991 and 2008, 52 patients with univentricular heart (mean age, 3.3 years; range, 2-8 years; 27 female patients [51.9%]) underwent ICPC. The cardiac malformations were as follows: tricuspid atresia, 25 cases (48.0%); common ventricle, 16 cases (30.7%); and pulmonary atresia with intact ventricular septum, 11 cases (21.1%). The intracardiac cavopulmonary procedure was indicated for all 52 cases. In 42 patients (80.7%), an intra-atrial lateral tunnel was constructed with a bovine pericardium patch. In the last 10 consecutive cases (19.3%), we performed a modified surgical technique in which we implanted an intra-atrial corrugated bovine pericardium tube sutured around the superior and inferior vena cava ostium. In all cases, a 4-mm fenestration was made to reduce the intratunnel pressure. All 52 patients had previously undergone a Glenn operation. Results: There were 2 hospital deaths (3.8%) and no recorded late deaths. During the follow-up, all patients were medicated with antiplatelet drugs. To evaluate the hemodynamic performance, we used Doppler echocardiography, computed tomography, and magnetic nuclear resonance studies. There were no prosthesis thromboses during this follow-up period. To evaluate cardiac arrhythmias, we conducted a Holter study. The last 10 patients with an intra-atrial conduit (IAC) presented with sinus rhythm and no arrhythmias during the last 4 years. The 50 surviving patients (96.1%) have been followed up for 6 to 204 months; all these patients are free of reoperation. Conclusion: The Glenn operation, which is performed at an early age, prepares the pulmonary bed to receive the ICPC. The midterm results of the intracardiac Fontan procedure seem to be good. The modified surgical procedure (IAC) can be a good alternative technique to the Fontan procedure in suitable patients.

Effects of semen storage and separation techniques on sperm DNA fragmentation

Objective: To determine the effect of semen storage and separation techniques on sperm DNA fragmentation. Design: Controlled clinical study. Setting: An assisted reproductive technology laboratory. Patient(s): Thirty normoozospermic semen samples obtained from patients undergoing infertility evaluation. Intervention(s): One aliquot from each sample was immediately prepared (control) for the sperm chromatin dispersion assay (SCD). Aliquots used to assess storage techniques were treated in the following ways: snap frozen by liquid nitrogen immersion, slow frozen with Tris-yolk buffer and glycerol, kept on ice for 24 hours or maintained at room temperature for 4 and 24 hours. Aliquots used to assess separation techniques were processed by the following methods: washed and centrifuged in media, swim-up from washed sperm pellet, density gradient separation, density gradient followed by swim-up. DNA integrity was then measured by SCD. Main Outcome Measure(s): DNA fragmentation as measured by SCD. Result(s): There was no significant difference in fragmentation among the snap frozen, slow frozen, and wet-ice groups. Compared to other storage methods short-term storage at room temperature did not impact DNA fragmentation yet 24 hours storage significantly increased fragmentation. Swim-up, density gradient and density gradient/swim-up had significantly reduced DNA fragmentation levels compared with washed semen. Postincubation, density gradient/swim-up showed the lowest fragmentation levels. Conclusion(s): The effect of sperm processing methods on DNA fragmentation should be considered when selecting storage or separation techniques for clinical use. (Fertil Steril (R) 2010;94:2626-30. (C) 2010 by American Society for Reproductive Medicine.)

Frequency of human metapneumovirus infection in hematopoietic SCT recipients during 3 consecutive years

Respiratory viruses can cause significant morbidity in immunocompromised hosts. Human metapneumovirus (hMPV) has been increasingly associated with lower respiratory tract infection in hematopoietic SCT (HSCT) recipients, with mortality rates up to 50%. No data on the occurrence of hMPV infection in HSCT recipients have been reported in the southern hemisphere. We conducted a retrospective study including 228 nasal wash samples from 153 HSCT recipients with respiratory symptoms during 2001, 2002 and 2003. hMPV was detected by real-time PCR with primers complementary to the nucleocapsid region of hMPV genome. Eleven of the 153 patients (7.2%) acquired hMPV infection during the study period (6.4% in 2001, 4.7% in 2002 and 11.1% in 2003). Among the 11 HSCT recipients with hMPV infection, 1 died 8 days after the diagnosis, but the role of hMPV in the patient`s death could not be established. In 2001 and 2003, hMPV group A prevailed over group B. In 2002, both groups were detected equally. hMPV infections were diagnosed in late winter and spring. The frequency of hMPV infection in HSCT recipients living in Brazil was similar to those observed in the northern hemisphere. Sensitive techniques to detect hMPV should be included in the diagnostic assessment of HSCT recipients with respiratory symptoms.

Supporting content-based image retrieval and computer-aided diagnosis systems with association rule-based techniques

In this work, we take advantage of association rule mining to support two types of medical systems: the Content-based Image Retrieval (CBIR) systems and the Computer-Aided Diagnosis (CAD) systems. For content-based retrieval, association rules are employed to reduce the dimensionality of the feature vectors that represent the images and to improve the precision of the similarity queries. We refer to the association rule-based method to improve CBIR systems proposed here as Feature selection through Association Rules (FAR). To improve CAD systems, we propose the Image Diagnosis Enhancement through Association rules (IDEA) method. Association rules are employed to suggest a second opinion to the radiologist or a preliminary diagnosis of a new image. A second opinion automatically obtained can either accelerate the process of diagnosing or to strengthen a hypothesis, increasing the probability of a prescribed treatment be successful. Two new algorithms are proposed to support the IDEA method: to pre-process low-level features and to propose a preliminary diagnosis based on association rules. We performed several experiments to validate the proposed methods. The results indicate that association rules can be successfully applied to improve CBIR and CAD systems, empowering the arsenal of techniques to support medical image analysis in medical systems. (C) 2009 Elsevier B.V. All rights reserved.

Abnormal cell-cycle expression of the proteins p27, mdm2 and cathepsin B in oral squamous-cell carcinoma infected with human papillomavirus

Since the role of human papillomavirus (HPV) infection in oral carcinogenesis is still unclear, the purpose of this study was to verify the association between the expression of p27, mdm2 and cathepsin B and by HPV-related oral lesions. Fifty-five oral biopsies were studied and HPV detection and typing (6/11, 16, 18, 31 and 33) were performed using polymerase chain reaction techniques. The distribution p27, mdm2 and cathepsin B was determined by immunohistochemistry. Twenty-one (38%) out of the 55 oral lesions tested positive for HPV, of which 6(33%) were HPV 6/11, 1 (5%) was HPV 16,14 (72%) were HPV 18 and none was HPV 33/31. Among the 55 biopsies, immunopostivity for p27, mdm2 and cathepsin B was observed in 17 (30.9%), 37 (67.2%) and 37 (67.2%), respectively. Among 21 HPV-positive oral lesions, immunopostivity of mdm2, p27 and cathepsin B was found, respectively, in 6 (33%) out of 18 benign lesions (BL), 4(22%) out of 18 potential malignant epithelial lesions (PMEL) and 11(57.9%) out of 19 malignant lesions (ML). High-risk HPV types may be associated with oral carcinoma, by cell-cycle control dysregulation, contributing to oral carcinogenesis and the overexpression of mdm2, p27 and cathepsin B. (C) 2009 Elsevier GmbH. All rights reserved.