Studies using a viral challenge and CD8 T cell depletions on the roles of cellular and humoral immunity in the control of an SHIV-89.6P challenge in DNA/MVA-vaccinated macaques

Here, we study immune responses in four DNA/MVA-vaccinated macaques following an SHIV-89.6P challenge and a subsequent CD8 cell depletion. Both post-challenge and post-depletion peaks of viremia contracted with the expansion, or re-emergence, of CD8 T cells. Post-depletion, CD8 cells expanded in the presence of higher levels of neutralizing Ab and CD4 help than post-challenge and had superior maturational characteristics as measured by expression of the anti-apoptotic protein Bcl-2, the IL-7 receptor CD127 and co-production of IFN-gamma and IL-2. Pre-challenge and pre-depletion titers of neutralizing Ab correlated inversely with peaks of viremia and directly with peaks for anti-viral CD4 cells. Thus, our results reveal CD8 cells playing a central role, and neutralizing Ab, a supporting role in SHIV-89.6P control. They also suggest a dynamic relationship between neutralizing Ab, antigen load and anti-viral CD4 cells in the maturation of high-quality anti-viral CD8 T cells.     

Retrospective-prospective study of safety and efficacy of sofosbuvir-based direct-acting antivirals in HIV/HCV-coinfected participants with decompensated liver disease pre– or post–liver transplant

Direct-acting antiviral (DAA) therapy has transformed the management of human immunodeficiency virus (HIV) and hepatitis C (HCV) coinfected patients with advanced liver disease. STOP-Coinfection was a multicenter prospective and retrospective, open-label study using sofosbuvir-based DAA therapy to treat HIV/HCV-coinfected participants pre– or post–liver transplant (LT). Sixty-eight participants with end-stage liver disease (Child-Turcotte-Pugh score ≥7 and Model for End-Stage Liver Disease score 6–29) were enrolled, 26 had hepatocellular carcinoma. Forty-two participants were treated pre–LT and 26 post–LT. All participants completed therapy without need for dose reduction or transfusion; eight required two or more courses of therapy. Ninety-three percent achieved a sustained virologic response and DAA therapy was well tolerated. Despite HCV cure, 12 end-stage liver disease participants required subsequent LT, 7 for decompensated liver disease. Thirteen participants died, 10 with decompensated liver disease pre–LT and three post–LT. Overall, transplant free survival was 42.8% at 4 years and post–LT survival was 87.9% at 5 years. We conclude that sofosbuvir-based DAA therapy is safe and highly effective in HCV-HIV patients with decompensated liver disease and post–LT, with post–LT survival rates comparable to other indications. This removes one of the last barriers to liver transplantation in this challenging cohort of recipients.     

Multicentricity in Renal Cell Carcinoma

Objective: To find the incidence of multicentric renal cell carcinoma and its possible relationship to the other clinical and pathologic findings. Methods: A total of 40 patients with renal cell carcinoma underwent radical nephrectomy between March 1994 and January 1996 at Hacettepe University, School of Medicine, Department of Urology. All of the materials were examined grossly and histologically by the same pathologist. Results: Among 40 kidneys 4 had satellite carcinoma (10%), 3 of them had been shown by preoperative imaging techniques, 1 was found histopathologically. Conclusion: If preoperative imaging techniques do not show additional lesion in the kidney besides the small early stage primary in incidentally discovered patients, the incidence of satellite renal cell carcinoma is low enough to justify nephron sparing surgery.     

Diffuse bipolar cells provide input to OFF parasol ganglion cells in the macaque retina

Parasol retinal ganglion cells are more sensitive to luminance contrast and respond more transiently at all levels of adaptation than midget ganglion cells. This may be due, in part, to differences between bipolar cells that provide their input, and the goal of these experiments was to study these differences. Midget bipolar cells are known to be presynaptic to midget ganglion cells. To identify the bipolar cells presynaptic to parasol cells, these ganglion cells were intracellularly injected with Neurobiotin, cone bipolar cells were immunolabeled, and the double-labeled material was analyzed. In the electron microscope, we found that DB3 diffuse bipolar cells labeled by using antiserum to calbindin D-28k were presynaptic to OFF parasol cells. In the confocal microscope, DB3 bipolars costratified with OFF parasol cell dendrites and made significantly more appositions with them than expected due to chance. Flat midget bipolar cells were labeled with antiserum to recoverin. Although they made a few appositions with parasol cells, the number was no greater than would be expected when two sets of processes have overlapping distributions in the inner plexiform layer. DB2 diffuse bipolar cells were labeled with antibodies to excitatory amino acid transporter 2, and they also made appositions with OFF parasol cells. These results suggest that DB2 bipolar cells are also presynaptic to OFF parasol ganglion cells, but midget bipolar cells are not. We estimate that midperipheral OFF parasol cells receive approximately 500 synapses from 50 DB3 bipolar cells that, in turn, receive input from 250 cones.     

Plackett–Burman randomization method for Bacterial Ghosts preparation form E. coli JM109

Plackett–Burman randomization method is a conventional tool for variables randomization aiming at optimization. Bacterial Ghosts (BGs) preparation has been recently established using methods other than the E lysis gene. The protocol has been based mainly on using critical concentrations from chemical compounds able to convert viable cells to BGs. The Minimum Inhibition Concentration (MIC) and the Minimum Growth Concentration (MGC) were the main guide for the BGs preparation. In this study, Escherichia coli JM109 DEC has been used to produce the BGs following the original protocol. The study contained a detail protocol for BGs preparation that could be used as a guide.     

Guidelines and considerations for conducting experiments using tissue microarrays

Tissue microarrays (TMAs) represent a powerful method for undertaking large‐scale tissue‐based biomarker studies. While TMAs offer several advantages, there are a number of issues specific to their use which need to be considered when employing this method. Given the investment in TMA‐based research, guidance on design and execution of experiments will be of benefit and should help researchers new to TMA‐based studies to avoid known pitfalls. Furthermore, a consensus on quality standards for TMA‐based experiments should improve the robustness and reproducibility of studies, thereby increasing the likelihood of identifying clinically useful biomarkers. In order to address these issues, the National Cancer Research Institute Biomarker and Imaging Clinical Studies Group organized a 1‐day TMA workshop held in Nottingham in May 2012. The document herein summarizes the conclusions from the workshop. It includes guidance and considerations on all aspects of TMA‐based research, including the pre‐analytical stages of experimental design, the analytical stages of data acquisition, and the postanalytical stages of data analysis. A checklist is presented which can be used both for planning a TMA experiment and interpreting the results of such an experiment. For studies of cancer biomarkers, this checklist could be used as a supplement to the REMARK guidelines.