Tumour-derived exosomes and their role in cancer-associated T-cell signalling defects

Dendritic and lymphoid ‘exosomes' regulate immune activation. Tumours release membranous material mimicking these ‘exosomes,' resulting in deletion of reactive lymphocytes. Tumour-derived ‘exosomes' have recently been explored as vaccines, without analysis of their immunologic consequences. This investigation examines the composition of tumour-derived ‘exosomes' and their effects on T lymphocytes. Membranous materials were isolated from ascites of ovarian cancer patients (n=6) and Western immunoblotting was performed for markers associated with ‘exosomes.' Using cultured T cells, ‘exosomes' were evaluated for suppression of CD3-ζ and JAK 3 expressions and induction of apoptosis, measured by DNA fragmentation. ‘Exosome' components mediating suppression of CD3-ζ were isolated by continuous eluting electrophoresis and examined by Western immunoblotting. ‘Exosomes' were shown to be identical with previously characterised shed membrane vesicles by protein staining and TSG101 expression. ‘Exosomes' expressed class I MHC, placental alkaline phosphatase, B23/nucleophosmin, and FasL. ‘Exosomes' suppressed expression of T-cell activation signalling components, CD3-ζ and JAK 3 and induced apoptosis. CD3-ζ suppression was mediated by two components: 26 and 42 kDa. Only the 42 kDa component reacted with anti-FasL antibody. These results indicate that, while ‘exosomes' express tumour antigens, leading to their proposed utility as tumour vaccines, they also can suppress T-cell signalling molecules and induce apoptosis

Dio-sensimedia: a novel culture medium for rapid detection of extended spectrum β-lactamases

BACKGROUND: Resistance to contemporary broad-spectrum β-lactams, mediated by extended-spectrum β-lactamases (ESBL), is an increasing problem worldwide. Many of the emerging antimicrobial resistance problems of this decade have been characterized by difficulty in the recognition of resistance in the laboratory, particularly by rapid susceptibility test methods. The plasmid-encoded ESBL represent such a resistance phenomenon that is difficult to recognize. We compared Dio-Sensimedia-ES (DSM-ES; Diomed, Istanbul, Turkey) and Mueller-Hinton (MH) agar in the double-disk synergy test (DDST) as a novel rapid system for detecting ESBL directly from bacterial culture. METHODS: Sixty ESBL-producing Klebsiella pneumoniae isolates cultured from blood (30), endotracheal aspirates (20), urine (5) and pus (5), as well as 40 Escherichia coli isolates cultured from endotracheal aspirates (15), urine (10), blood (8) and pus (7) were studied. Isolates positive for ESBL by the combined disk tests were tested with the DDST using MH and DSM-ES agar to detect ESBL-mediated resistance in K. pneumoniae and E. coli. DSM-ES agar was also used to determine the susceptibility of Enterobacteriaceae and staphylococci. RESULTS: Among 60 ESBL-producing K. pneumoniae isolates, 59 (98.3%) were identified as ESBL-positive by the DDST using MH, and 58 (96.6%), using DSM-ES agar. Of 40 ESBL-producing E. coli isolates, 38 (95%) were ESBL-positive by the DDST on MH agar, and 37 (92.5%), on DSM-ES agar. The average incubation period required for ESBL detection by the DDST on DSM-ES agar was 4 hours. CONCLUSIONS: Since the DDST results were available within 4 hours when DSM-ES agar was used, the use of this media may significantly lower the length of hospital stay, the total cost for patient care and even the mortality rate by fascilitating early treatment against ESBL-producing organisms

Let-7 MicroRNA Family Is Selectively Secreted into the Extracellular Environment via Exosomes in a Metastatic Gastric Cancer Cell Line

Background: Exosomes play a major role in cell-to-cell communication, targeting cells to transfer exosomal molecules including proteins, mRNAs, and microRNAs (miRNAs) by an endocytosis-like pathway. miRNAs are small noncoding RNA molecules on average 22 nucleotides in length that regulate numerous biological processes including cancer pathogenesis and mediate gene downregulation by targeting mRNAs to induce RNA degradation and/or interfering with translation. Recent reports imply that miRNAs can be stably detected in circulating plasma and serum since miRNAs are packaged by exosomes to be protected from RNA degradation. Thus, profiling exosomal miRNAs are in need to clarify intercellular signaling and discover a novel disease marker as well. Methodology/Principal Findings: Exosomes were isolated from cultured cancer cell lines and their quality was validated by analyses of transmission electron microscopy and western blotting. One of the cell lines tested, a metastatic gastric cancer cell line, AZ-P7a, showed the highest RNA yield in the released exosomes and distinctive shape in morphology. In addition, RNAs were isolated from cells and culture media, and profiles of these three miRNA fractions were obtained using microarray analysis. By comparing signal intensities of microarray data and the following validation using RT-PCR analysis, we found that let-7 miRNA family was abundant in both the intracellular and extracellular fractions from AZ-P7a cells, while low metastatic AZ-521, the parental cell line of AZ-P7a, as well as other cancer cell lines showed no such propensity. Conclusions/Significance: The enrichment of let-7 miRNA family in the extracellular fractions, particularly, in the exosome

Intestinal Damage Determines the Inflammatory Response and Early Complications in Patients Receiving Conditioning for a Stem Cell Transplantation

Contains fulltext : 87954.pdf (publisher's version ) (Open Access)BACKGROUND: Stem cell transplantation (SCT) is still complicated by the occurrence of fever and inflammatory complications attributed to neutropenia and subsequent infectious complications. The role of mucosal barrier injury (MBI) of the intestinal tract therein has received little attention. METHODS: We performed a retrospective analysis in 163 SCT recipients of which data had been collected prospectively on intestinal damage (citrulline), inflammation (C-reactive protein), and neutrophil count. Six different conditioning regimens were studied; 5 myeloablative (MA) and 1 non-myeloablative (NMA). Linear mixed model multivariate and AUC analyses were used to define the role of intestinal damage in post-SCT inflammation. We also studied the relationship between the degree of intestinal damage and the occurrence of early post-SCT complications. RESULTS: In the 5 MA regimen there was a striking pattern of inflammatory response that coincided with the occurrence of severe intestinal damage. This contrasted with a modest inflammatory response seen in the NMA regimen in which intestinal damage was limited. With linear mixed model analysis the degree of intestinal damage was shown the most important determinant of the inflammatory response, and both neutropenia and bacteremia had only a minor impact. AUC analysis revealed a strong correlation between citrulline and CRP (Pearson correlation r = 0.96). Intestinal damage was associated with the occurrence of bacteremia and acute lung injury, and influenced the kinetics of acute graft-versus-host disease. CONCLUSION: The degree of intestinal damage after myeloablative conditioning appeared to be the most important determined the inflammatory response following SCT, and was associated with inflammatory complications. Studies should explore ways to ameliorate cytotoxic therapy-induced intestinal damage in order to reduce complications associated with myeloablative conditioning therapy

Early Production of IL-22 but Not IL-17 by Peripheral Blood Mononuclear Cells Exposed to live Borrelia burgdorferi: The Role of Monocytes and Interleukin-1

If insufficiently treated, Lyme borreliosis can evolve into an inflammatory disorder affecting skin, joints, and the CNS. Early innate immunity may determine host responses targeting infection. Thus, we sought to characterize the immediate cytokine storm associated with exposure of PBMC to moderate levels of live Borrelia burgdorferi. Since Th17 cytokines are connected to host defense against extracellular bacteria, we focused on interleukin (IL)-17 and IL-22. Here, we report that, despite induction of inflammatory cytokines including IL-23, IL-17 remained barely detectable in response to B. burgdorferi. In contrast, T cell-dependent expression of IL-22 became evident within 10 h of exposure to the spirochetes. This dichotomy was unrelated to interferon-γ but to a large part dependent on caspase-1 and IL-1 bioactivity derived from monocytes. In fact, IL-1β as a single stimulus induced IL-22 but not IL-17. Neutrophils display antibacterial activity against B. burgdorferi, particularly when opsonized by antibodies. Since neutrophilic inflammation, indicative of IL-17 bioactivity, is scarcely observed in Erythema migrans, a manifestation of skin inflammation after infection, protective and antibacterial properties of IL-22 may close this gap and serve essential functions in the initial phase of spirochete infection