Mechanisms of apoptosis induced by purine nucleosides in astrocytes

Astrocytes release adenine-based and guanine-based purines under physiological and, particularly, pathological conditions. Thus, the aim of this study was to determine if adenosine induced apoptosis in cultured rat astrocytes. Further, if guanosine, which increases the extracellular concentration of adenosine, also induced apoptosis determined using the TUNEL and Annexin V assays. Adenosine induced apoptosis in a concentration-dependent manner up to 100 microM. Inosine, hypoxanthine, guanine, and guanosine did not. Guanosine or adenosine (100 microM) added to the culture medium was metabolized, with 35% or 15%, respectively, remaining after 2-3 h. Guanosine evoked the extracellular accumulation of adenosine, and particularly of adenine-based nucleotides. Cotreatment with EHNA and guanosine increased the extracellular accumulation of adenosine and induced apoptosis. Inhibition of the nucleoside transporters using NBTI (100 microM) or propentophylline (100 microM) significantly decreased but did not abolish the apoptosis induced by guanosine + EHNA or adenosine + EHNA, respectively. Apoptosis produced by either guanosine + EHNA or adenosine + EHNA was unaffected by A(1) or A(2) adenosine receptor antagonists, but was significantly reduced by MRS 1523, a selective A(3) adenosine receptor antagonist. Adenosine + EHNA, not guanosine + EHNA, significantly increased the intracellular concentration of S-adenosyl-L-homocysteine (SAH) and greatly reduced the ratio of S-adenosyl-L-methioine to SAH, which is associated with apoptosis. These data demonstrate that adenosine mediates apoptosis of astrocytes both, via activation of A(3) adenosine receptors and by modulating SAH hydrolase activity. Guanosine induces apoptosis by accumulating extracellular adenosine, which then acts solely via A(3) adenosine receptors.     

Identification and ranking of genetic and laboratory environment factors influencing a behavioral trait,thermal nociception,via computational analysis of a large data archive

Laboratory conditions in biobehavioral experiments are commonly assumed to be ‘controlled’, having little impact on the outcome. However, recent studies have illustrated that the laboratory environment has a robust effect on behavioral traits. Given that environmental factors can interact with trait-relevant genes, some have questioned the reliability and generalizability of behavior genetic research designed to identify those genes. This problem might be alleviated by the identification of the most relevant environmental factors, but the task is hindered by the large number of factors that typically vary between and within laboratories. We used a computational approach to retrospectively identify and rank sources of variability in nociceptive responses as they occurred in a typical research laboratory over several years. A machine-learning algorithm was applied to an archival data set of 8034 independent observations of baseline thermal nociceptive sensitivity. This analysis revealed that a factor even more important than mouse genotype was the experimenter performing the test, and that nociception can be affected by many additional laboratory factors including season/humidity, cage density, time of day, sex and within-cage order of testing. The results were confirmed by linear modeling in a subset of the data, and in confirmatory experiments, in which we were able to partition the variance of this complex trait among genetic (27%), environmental (42%) and genetic×environmental (18%) sources.     

RNASEL mutation screening and association study in Ashkenazi and non-Ashkenazi prostate cancer patients.

Epidemiologic and genetic studies support the considerable effect of heritable factors on prostate tumorigenesis, although to date, no unequivocal susceptibility gene has been identified. The extensive study of RNASEL in prostate cancer patients worldwide has yielded conflicting results. We reevaluated the role of the RNASEL 471delAAAG Ashkenazi founder mutation in 1,642 Ashkenazi patients with prostate, bladder, breast/ovarian, and colon cancers; Ashkenazi controls; and in non-Ashkenazi prostate cancer patients and controls. The entire RNASEL coding sequence was also screened using denaturing high-performance liquid chromatography and multiplex ligation-dependent probe amplification for possible sequence variations or copy number changes in a population of prostate cancer patients. The 471delAAAG mutation was detected in 2.4% of the Ashkenazi prostate cancer patients; in 1.9% of patients with bladder, breast/ovarian, and colon cancers; and in 2.0% of the Ashkenazi controls. Seven additional variants were detected in RNASEL, including a novel potentially pathogenic splice site mutation, IVS5+1delG, although none were associated with increased prostate cancer risk. Multiplex ligation-dependent probe amplification analysis showed two RNASEL gene copies in all 300 prostate cancer patients tested. We estimated that the RNASEL 471delAAAG founder mutation, which was detected in 2% of the Ashkenazi Jews, originated between the 2nd and 5th centuries A.D., compared with the less frequent (1%) BRCA1 185delAG founder mutation, which originated hundreds of years earlier. Taken together, our analysis does not support a role for the RNASEL 471delAAAG Ashkenazi mutation nor for the other alterations detected in RNASEL in prostate cancer risk in Jewish men.     

Mind the gap between policy imperatives and service provision: a qualitative study of the process of respiratory service development in England and Wales

Background  

Healthcare systems globally are reconfiguring to address the needs of people with long-term conditions such as respiratory disease. Primary Care Organisations (PCOs) in England and Wales are charged with the task of developing cost-effective patient-centred local models of care. We aimed to investigate how PCOs in England and Wales are reconfiguring their workforce to develop respiratory services, and the background factors influencing service redesign.     

Histopathology of failed osteoarticular shell allografts

Fresh cadaveric osteochondral fragment allografts were used to replace damaged articular surfaces of knee joints. Of the original 100 patients, 22 experienced graft failure necessitating graft removal. From these patients, a total of 44 osteochondral allografts were extirpated between 12 and 84 months after insertion. These were examined radiologically, histologically, and ultrastructurally. The bone and bone marrow were necrotic and had undergone variable replacement by host bone, which appeared to be independent of the duration of the graft. The articular cartilage showed degenerative changes ranging from fibrillation to erosion. Viable donor cartilage was present as late as seven years, proving that fresh graft cartilage can survive transplantation. Host bone interfaced with the cartilage, but in 14 grafts there was focal invasion of the cartilage. In some grafts, pannus formation with resorption of cartilage was evident. There was no histologic evidence of transplant rejection. This study is encouraging because hyaline cartilage has been shown to survive for as long as seven years and because bone can be replaced in a homogeneous fashion if the correct biomechanical conditions are met.     

Murine neuroblastoma attenuates dendritic cell cysteine cysteine receptor 7 (CCR7) expression

Background/Purpose

Dendritic cell (DC) migration from tumors to T-cell priming sites is critical in developing antitumor cytotoxicity. Cysteine cysteine receptor 7 (CCR7), a promigratory chemokine receptor, regulates DC recruitment to secondary lymphoid organs. Tumors may inhibit CCR7 expression to evade immunodetection. Previous work implicates impaired DC migration as a critical defect in immunity to neuroblastoma (NB). However, the mechanism has yet to be defined. We hypothesize that NB abrogates DC CCR7 expression and signaling, leading to decreased antitumor immunity.

Methods

A/J mice (N = 36) were injected with saline (control) or murine NB (TBJ) and bone marrow-derived DC were isolated at 7, 14, and 28 days. CCR7 expression was analyzed by polymerase chain reaction, Western blot, and flow cytometry. Cytometry data were analyzed using the paired Student's t test.

Results

Dendritic cells isolated from mice with NB had a 60% increase in CCR7 protein expression by flow cytometry compared with control mice at day 7. However, there was a 43% downregulation of CCR7 expression by DC from tumor-bearing mice compared with controls 2 weeks postinoculation (P < .005). These observations were confirmed by polymerase chain reaction and Western blot analysis.

Conclusion

Neuroblastoma initially upregulates CCR7 expression by DC. However, with tumor progression, this chemokine is downregulated, likely leading to impaired DC migration. Immunotherapeutic strategies to bypass or augment CCR7-dependent DC trafficking may improve survival for patients with aggressive disease.     

Principles of Good Practice for the Translation and Cultural Adaptation Process for Patient-Reported Outcomes (PRO) Measures: Report of the ISPOR Task Force for Translation and Cultural Adaptation

In 1999, ISPOR formed the Quality of Life Special Interest group (QoL-SIG)--Translation and Cultural Adaptation group (TCA group) to stimulate discussion on and create guidelines and standards for the translation and cultural adaptation of patient-reported outcome (PRO) measures. After identifying a general lack of consistency in current methods and published guidelines, the TCA group saw a need to develop a holistic perspective that synthesized the full spectrum of published methods. This process resulted in the development of Translation and Cultural Adaptation of Patient Reported Outcomes Measures--Principles of Good Practice (PGP), a report on current methods, and an appraisal of their strengths and weaknesses. The TCA Group undertook a review of evidence from current practice, a review of the literature and existing guidelines, and consideration of the issues facing the pharmaceutical industry, regulators, and the broader outcomes research community. Each approach to translation and cultural adaptation was considered systematically in terms of rationale, components, key actors, and the potential benefits and risks associated with each approach and step. The results of this review were subjected to discussion and challenge within the TCA group, as well as consultation with the outcomes research community at large. Through this review, a consensus emerged on a broad approach, along with a detailed critique of the strengths and weaknesses of the differing methodologies. The results of this review are set out as "Translation and Cultural Adaptation of Patient Reported Outcomes Measures--Principles of Good Practice" and are reported in this document.     

A novel intranasal Protollin-based measles vaccine induces mucosal and systemic neutralizing antibody responses and cell-mediated immunity in mice

Protollin-MV is a vaccine produced by mixing split measles virus (MV) antigen with the novel adjuvant Protollin (Neisseria meningitidis outer membrane proteins non-covalently complexed with Shigella flexneri 2a lipopolysaccharide). Intranasal immunization of mice with two or three doses of Protollin-MV induces both serum IgG and mucosal IgA with strong neutralizing activity. There is a dose-dependent shift towards lower IgG1:IgG2a ratios and MV-specific IFNgamma production in splenocytes. Intranasal Protollin-MV can therefore induce systemic and mucosal neutralizing antibody responses as well as elicit a balanced TH1/TH2-type response.     

Interleukin-6 and platelet protagonists in T lymphocyte and virological response

The present cross-sectional study evaluated the status and relationship of interleukin-6, a platelet growth factor, with platelet counts, viral load, CD4 counts, and antiretroviral treatment in 75 HIV-infected subjects with thrombocytopenia and 50 gender-, race-, age- and antiretroviral treatment-matched controls without thrombocytopenia. Mean IL-6 production was significantly higher in thrombocytopenic participants (13 432+/-8596) than in non-thrombocytopenic subjects (12 859+/-3538 pg/10(5) Lym). Univariate analyses indicated, however, that thrombocytopenic patients were more likely to have <3000 pg of IL-6 than non-thrombocytopenic patients (OR=7 95% CI 1.3-12; P=0.01). For additional analyses, participants were dichotomized above and below 3000 pg of IL-6. Despite similar age, gender, drug use and antiretroviral treatment, thrombocytopenic participants had lower CD4 counts (186.5+/-149 vs. 401+/-286, P=0.005) than non-thrombocytopenic subjects. Thrombocytopenic participants with elevated IL-6, with or without HAART, were more likely to have higher HIV-replication (496 273+/-210 416; 34 656+/-25 332) than thrombocytopenic individuals with low IL-6 levels (105 332+/-42 699; 19 015+/-14 296 P=0.05). Non-thrombocytopenic patients with high IL-6 levels exhibited the highest CD4s (466.7+/-333) and the lowest viral burden (63 094+/-53 300) of the groups. Two distinct categories of HIV-associated thrombocytopenia exist: one accompanied by low IL-6, and another with compensatory elevations of IL-6. In thrombocytopenic individuals, the latter was associated with the poorest immunological and virological responses.