Large oncosomes contain distinct protein cargo and represent a separate functional class of tumor-derived extracellular vesicles

Large oncosomes (LO) are atypically large (1-10 mu m diameter) cancer-derived extracellular vesicles (EVs), originating from the shedding of membrane blebs and associated with advanced disease. We report that 25% of the proteins, identified by a quantitative proteomics analysis, are differentially represented in large and nano-sized EVs from prostate cancer cells. Proteins enriched in large EVs included enzymes involved in glucose, glutamine and amino acid metabolism, all metabolic processes relevant to cancer. Glutamine metabolism was altered in cancer cells exposed to large EVs, an effect that was not observed upon treatment with exosomes. Large EVs exhibited discrete buoyant densities in iodixanol (OptiPrep (TM)) gradients. Fluorescent microscopy of large EVs revealed an appearance consistent with LO morphology, indicating that these structures can be categorized as LO. Among the proteins enriched in LO, cytokeratin 18 (CK18) was one of the most abundant (within the top 5th percentile) and was used to develop an assay to detect LO in the circulation and tissues of mice and patients with prostate cancer. These observations indicate that LO represent a discrete EV type that may play a distinct role in tumor progression and that may be a source of cancer-specific markers.1182Ysciescopu

Rising burden of Hepatitis C Virus in hemodialysis patients

<p>Abstract</p> <p>Aim</p> <p>High prevalence of Hepatitis C virus (HCV) has been reported among the dialysis patients throughout the world. No serious efforts were taken to investigate HCV in patients undergoing hemodialysis (HD) treatment who are at great increased risk to HCV. HCV genotypes are important in the study of epidemiology, pathogenesis and reaction to antiviral therapy. This study was performed to investigate the prevalence of active HCV infection, HCV genotypes and to assess risk factors associated with HCV genotype infection in HD patients of Khyber Pakhtunkhwa as well as comparing this prevalence data with past studies in Pakistan.</p> <p>Methods</p> <p>Polymerase chain reaction was performed for HCV RNA detection and genotyping in 384 HD patients. The data obtained was compared with available past studies from Pakistan.</p> <p>Results</p> <p>Anti HCV antibodies were observed in 112 (29.2%), of whom 90 (80.4%) were HCV RNA positive. In rest of the anti HCV negative patients, HCV RNA was detected in 16 (5.9%) patients. The dominant HCV genotypes in HCV infected HD patients were found to be 3a (n = 36), 3b (n = 20), 1a (n = 16), 2a (n = 10), 2b (n = 2), 1b (n = 4), 4a (n = 2), untypeable (n = 10) and mixed (n = 12) genotype.</p> <p>Conclusion</p> <p>This study suggesting that i) the prevalence of HCV does not differentiate between past and present infection and continued to be elevated ii) HD patients may be a risk for HCV due to the involvement of multiple routes of infections especially poor blood screening of transfused blood and low standard of dialysis procedures in Pakistan and iii) need to apply infection control practice.</p

Clinicopathologic features of incidental prostatic adenocarcinoma in radical cystoprostatectomy specimens

<p>Abstract</p> <p>Background</p> <p>The aim of this study is to review all features of incidentally discovered prostate adenocarcinoma in patients undergoing radical cystoprostatectomy for bladder cancer.</p> <p>Methods</p> <p>The medical charts of 300 male patients who underwent radical cystoprostatectomy for bladder cancer between 1997 and 2005 were retrospectively reviewed. The mean age of the patients was 62 (range 51-75) years.</p> <p>Results</p> <p>Prostate adenocarcinoma was present in 60 (20%) of 300 specimens. All were acinar adenocarcinoma. Of these, 40 (66.7%) were located in peripheral zone, 20 (33.3%) had pT2a tumor, 12 (20%) had pT2b tumor, 22(36.7%) had pT2c and, 6 (10%) had pT3a tumor. Gleason score was 6 or less in 48 (80%) patients. Surgical margins were negative in 54 (90%) patients, and tumor volume was less than 0.5 cc in 23 (38.3%) patients. Of the 60 incidentally detected cases of prostate adenocarcinoma 40 (66.7%) were considered clinically significant.</p> <p>Conclusion</p> <p>Incidentally detected prostate adenocarcinoma is frequently observed in radical cystoprostatectomy specimens. The majority are clinically significant.</p

Mechanism Underlying Defective Interferon Gamma-Induced IDO Expression in Non-obese Diabetic Mouse Fibroblasts

Indoleamine 2,3-dioxygenase (IDO) can locally suppress T cell-mediated immune responses. It has been shown that defective self-tolerance in early prediabetic female non-obese diabetic (NOD) mice can be attributed to the impaired interferon-gamma (IFN-γ)- induced IDO expression in dendritic cells of these animals. As IFN-γ can induce IDO in both dendritic cells and fibroblasts, we asked the question of whether there exists a similar defect in IFN-γ-induced IDO expression in NOD mice dermal fibroblasts. To this end, we examined the effect of IFN-γ on expression of IDO and its enzymatic activity in NOD dermal fibroblasts. The results showed that fibroblasts from either prediabetic (8 wks of age) female or male, and diabetic female or male (12 and 24 wks of age respectively) NOD mice failed to express IDO in response to IFN-γ treatment. To find underlying mechanisms, we scrutinized the IFN- γ signaling pathway and investigated expression of other IFN-γ-modulated factors including major histocompatibility complex class I (MHC-I) and type I collagen (COL-I). The findings revealed a defect of signal transducer and activator of transcription 1 (STAT1) phosphorylation in NOD cells relative to that of controls. Furthermore, we found an increase in MHC-I and suppression of COL-I expression in fibroblasts from both NOD and control mice following IFN-γ treatment; indicating that the impaired response to IFN-γ in NOD fibroblasts is specific to IDO gene. Finally, we showed that an IFN-γ-independent IDO expression pathway i.e. lipopolysaccharide (LPS)-mediated-c-Jun kinase is operative in NOD mice fibroblast. In conclusion, the findings of this study for the first time indicate that IFN-γ fails to induce IDO expression in NOD dermal fibroblasts; this may partially be due to defective STAT1 phosphorylation in IFN-γ-induced-IDO signaling pathway

3D Bioprinted Human Skeletal Muscle Constructs for Muscle Function Restoration

A bioengineered skeletal muscle tissue as an alternative for autologous tissue flaps, which mimics the structural and functional characteristics of the native tissue, is needed for reconstructive surgery. Rapid progress in the cell-based tissue engineering principle has enabled in vitro creation of cellularized muscle-like constructs; however, the current fabrication methods are still limited to build a three-dimensional (3D) muscle construct with a highly viable, organized cellular structure with the potential for a future human trial. Here, we applied 3D bioprinting strategy to fabricate an implantable, bioengineered skeletal muscle tissue composed of human primary muscle progenitor cells (hMPCs). The bioprinted skeletal muscle tissue showed a highly organized multi-layered muscle bundle made by viable, densely packed, and aligned myofiber-like structures. Our in vivo study presented that the bioprinted muscle constructs reached 82% of functional recovery in a rodent model of tibialis anterior (TA) muscle defect at 8 weeks of post-implantation. In addition, histological and immunohistological examinations indicated that the bioprinted muscle constructs were well integrated with host vascular and neural networks. We demonstrated the potential of the use of the 3D bioprinted skeletal muscle with a spatially organized structure that can reconstruct the extensive muscle defects

Force Generation upon T Cell Receptor Engagement

T cells are major players of adaptive immune response in mammals. Recognition of an antigenic peptide in association with the major histocompatibility complex at the surface of an antigen presenting cell (APC) is a specific and sensitive process whose mechanism is not fully understood. The potential contribution of mechanical forces in the T cell activation process is increasingly debated, although these forces are scarcely defined and hold only limited experimental evidence. In this work, we have implemented a biomembrane force probe (BFP) setup and a model APC to explore the nature and the characteristics of the mechanical forces potentially generated upon engagement of the T cell receptor (TCR) and/or lymphocyte function-associated antigen-1 (LFA-1). We show that upon contact with a model APC coated with antibodies towards TCR-CD3, after a short latency, the T cell developed a timed sequence of pushing and pulling forces against its target. These processes were defined by their initial constant growth velocity and loading rate (force increase per unit of time). LFA-1 engagement together with TCR-CD3 reduced the growing speed during the pushing phase without triggering the same mechanical behavior when engaged alone. Intracellular Ca2+ concentration ([Ca2+]i) was monitored simultaneously to verify the cell commitment in the activation process. [Ca2+]i increased a few tens of seconds after the beginning of the pushing phase although no strong correlation appeared between the two events. The pushing phase was driven by actin polymerization. Tuning the BFP mechanical properties, we could show that the loading rate during the pulling phase increased with the target stiffness. This indicated that a mechanosensing mechanism is implemented in the early steps of the activation process. We provide here the first quantified description of force generation sequence upon local bidimensional engagement of TCR-CD3 and discuss its potential role in a T cell mechanically-regulated activation process

Multi-Scale Modeling of HIV Infection in vitro and APOBEC3G-Based Anti-Retroviral Therapy

The human APOBEC3G is an innate restriction factor that, in the absence of Vif, restricts HIV-1 replication by inducing excessive deamination of cytidine residues in nascent reverse transcripts and inhibiting reverse transcription and integration. To shed light on impact of A3G-Vif interactions on HIV replication, we developed a multi-scale computational system consisting of intracellular (single-cell), cellular and extracellular (multicellular) events by using ordinary differential equations. The single-cell model describes molecular-level events within individual cells (such as production and degradation of host and viral proteins, and assembly and release of new virions), whereas the multicellular model describes the viral dynamics and multiple cycles of infection within a population of cells. We estimated the model parameters either directly from previously published experimental data or by running simulations to find the optimum values. We validated our integrated model by reproducing the results of in vitro T cell culture experiments. Crucially, both downstream effects of A3G (hypermutation and reduction of viral burst size) were necessary to replicate the experimental results in silico. We also used the model to study anti-HIV capability of several possible therapeutic strategies including: an antibody to Vif; upregulation of A3G; and mutated forms of A3G. According to our simulations, A3G with a mutated Vif binding site is predicted to be significantly more effective than other molecules at the same dose. Ultimately, we performed sensitivity analysis to identify important model parameters. The results showed that the timing of particle formation and virus release had the highest impacts on HIV replication. The model also predicted that the degradation of A3G by Vif is not a crucial step in HIV pathogenesis