TLR4 activation induces IL-1ss release via an IPAF dependent but caspase 1/11/8 independent pathway in the lung
Background: The IL-1 family of cytokines is known to play an important role in inflammation therefore understanding the mechanism by which they are produced is paramount. Despite the recent plethora of publications dedicated to the study of these cytokines, the mechanism by which they are produced in the airway following endotoxin, Lipopolysaccharide (LPS), exposure is currently unclear. The aim was to determine the mechanism by which the IL-1 cytokines are produced after LPS inhaled challenge.
Methods:Mice were challenged with aerosolised LPS, and lung tissue and bronchiolar lavage fluid (BALF) collected. Targets were measured at the mRNA and protein level; caspase activity was determined using specific assays.
Results: BALF IL-1b/IL-18, but not IL-1a, was dependent on Ice Protease-Activating Factor (IPAF), and to a lesser extent Apoptosis-associated Speck-like protein containing a CARD (ASC). Interestingly, although we measured an increase in mRNA expression for caspase 1 and 11, we could not detect an increase in lung enzyme activity or a role for them in IL-1a/b production. Further investigations showed that whilst we could detect an increase in caspase 8 activity at later points in the time course (during resolution of inflammation), it appeared to play no role in the production of IL-1 cytokines in this model system.
Conclusions: TLR4 activation increases levels of BALF IL-1b/IL-18 via an IPAF dependent and caspase 1/11/8 independent pathway. Furthermore, it would appear that the presence of IL-1a in the BALF is independent of these pathways. This novel data sheds light on innate signalling pathways in the lung that control the production of these key inflammatory cytokines.
BACKGROUND: One method to understand and evaluate an experiment that produces a large set of genes, such as a gene expression microarray analysis, is to identify overrepresentation or enrichment for biological pathways. Because pathways are able to functionally describe the set of genes, much effort has been made to collect curated biological pathways into publicly accessible databases. When combining disparate databases, highly related or redundant pathways exist, making their consolidation into pathway concepts essential. This will facilitate unbiased, comprehensive yet streamlined analysis of experiments that result in large gene sets.
METHODS: After gene set enrichment finds representative pathways for large gene sets, pathways are consolidated into representative pathway concepts. Three complementary, but different methods of pathway consolidation are explored. Enrichment Consolidation combines the set of the pathways enriched for the signature gene list through iterative combining of enriched pathways with other pathways with similar signature gene sets; Weighted Consolidation utilizes a Protein-Protein Interaction network based gene-weighting approach that finds clusters of both enriched and non-enriched pathways limited to the experiments' resultant gene list; and finally the de novo Consolidation method uses several measurements of pathway similarity, that finds static pathway clusters independent of any given experiment.
RESULTS: We demonstrate that the three consolidation methods provide unified yet different functional insights of a resultant gene set derived from a genome-wide profiling experiment. Results from the methods are presented, demonstrating their applications in biological studies and comparing with a pathway web-based framework that also combines several pathway databases. Additionally a web-based consolidation framework that encompasses all three methods discussed in this paper, Pathway Distiller (http://cbbiweb.uthscsa.edu/PathwayDistiller), is established to allow researchers access to the methods and example microarray data described in this manuscript, and the ability to analyze their own gene list by using our unique consolidation methods.
CONCLUSIONS: By combining several pathway systems, implementing different, but complementary pathway consolidation methods, and providing a user-friendly web-accessible tool, we have enabled users the ability to extract functional explanations of their genome wide experiments.
BACKGROUND: For treatment and prevention of metastatic disease, one of the premier challenges is the identification of pathways and proteins to target for clinical intervention. Micro RNAs (miRNAs) are short, non-coding RNAs, which regulate cellular activities by either mRNA degradation or translational inhibition. Our studies focused on the invasive properties of hsa-mir30c based on its high expression in MDA-MB-231 metastatic cells and our bioinformatic analysis of the Cancer Genome Atlas that identified aberrant hsa-mir-30c to be associated with poor survival.
METHODS: Contributions of hsa-mir-30c to breast cancer cell invasion were examined by Matrigel invasion transwell assays following modulation of hsa-mir-30c or hsa-mir-30c* levels in MDA-MB-231 cells. hsa-mir-30c in silico predicted targets linked to cell invasion were screened for targeting by hsa-mir-30c in metastatic breast cancer cells by RT-qPCR. The contribution to invasion by a target of hsa-mir-30c, Nephroblastoma overexpressed (NOV), was characterized by siRNA and invasion assays. Significant effects were determined using Student's T-tests with Welch's correction for unequal variance.
RESULTS: MCF-7 and MDA-MB-231 cells were used as models of poorly invasive and late-stage metastatic disease, respectively. By modulating the levels of hsa-mir-30c in these cells, we observed concomitant changes in breast cancer cell invasiveness. From predicted targets of hsa-mir-30c that were related to cellular migration and invasion, NOV/CCN3 was identified as a novel target of hsa-mir-30c. Depleting NOV by siRNA caused a significant increase in the invasiveness of MDA-MB-231 cells is a regulatory protein associated with the extracellular matrix.
CONCLUSIONS: NOV/CCN3 expression, which protects cells from invasion, is known in patient tumors to inversely correlate with advanced breast cancer and metastasis. This study has identified a novel target of hsa-mir-30c, NOV, which is an inhibitor of the invasiveness of metastatic breast cancer cells. Thus, hsa-mir-30c-mediated inhibition of NOV levels promotes the invasive phenotype of MDA-MB-231 cells and significantly, the miR-30/NOV pathways is independent of RUNX2, a known target of hsa-mir-30c that promotes osteolytic disease in metastatic breast cancer cells. Our findings allow for mechanistic insight into the clinical observation of poor survival of patients with elevated hsa-mir-30c levels, which can be considered for miRNA-based translational studies.
BACKGROUND: Telephone counseling Quitlines can support smoking cessation, but are under-utilized. We explored the use of smoker peer-referrals to increase use of a Quitline in Mississippi and Alabama.
FINDINGS: Collaborating with the Alabama and Mississippi Quitline, we piloted peer-referrals to Quitlines. Successful 'quitters' who had used the Quitline were contacted at routine follow-up and recruited to participate as a peer-referrer and refer their friends and family who smoked to the Quitline. Peer-referrers completed a training session, received a manual and a set of Quitline brochures a peer-referral forms. These peer-referral forms were then returned to the Quitline telephone counselors who proactively called the referred smokers. Of the initial potential pool of 96 who quit using the Quitline, 24 peer-referrers (75% Women, 29% African-American, and high school graduates/GED 67%) were recruited and initially agreed to participate as peer-referrers. Eleven of the 24 who initially agreed were trained, and of these 11, 4 (4%) actively referred 23 friends and family over 2 months. From these 23 new referrals, three intakes (100% Women, 66% African-American) were completed. Of the initial pool of 96, 4 (4%) actively participated in referring friends and family. Quitline staff and peer-referrers noted several barriers including: time-point in which potential peer-referrers were asked to participate, an 'overwhelming' referral form to use and limited ways to refer.
CONCLUSIONS: Though 'quitters' were willing to agree to peer-refer, we received a minority of referrals. However, we identified several areas to improve this new method for increasing awareness and access to support systems like the Quitline for smokers who want to quit.
Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA
BACKGROUND: The human peptidyl-prolyl isomerase Cyclophilin A (CypA) binds HIV-1 capsid (CA) and influences early steps in the HIV-1 replication cycle. The mechanism by which CypA regulates HIV-1 transduction efficiency is unknown. Disruption of CypA binding to CA, either by genetic means or by the competitive inhibitor cyclosporine A (CsA), reduces the efficiency of HIV-1 transduction in some cells but not in others. Transduction of certain cell types increases significantly when CypA binding to particular HIV-1 CA mutants, i.e., A92E, is prevented. Previous studies have suggested that this cell type-specific effect is due to a dominant-acting, CypA-dependent restriction factor.
RESULTS: Here we investigated the mechanism by which CypA regulates HIV-1 transduction efficiency using 27 different human cell lines, 32 HeLa subclones, and several previously characterized HIV-1 CA mutants. Disruption of CypA binding to wild-type CA, or to any of the mutant CAs, caused a decrease in HIV-1 reverse transcription in all the cell lines analyzed here. This block to reverse transcription, though, did not correlate with cell type-specific effects on transduction efficiency. The level of 2-LTR circles, a marker for nuclear transport of the viral cDNA that results from reverse transcription, correlated closely with effects on infectivity. No correlation was observed between the cell type-specific effects on infectivity and the steady-state CypA protein levels in these cells. Instead, as indicated by a fate-of-capsid assay, CsA released the HIV-1 CA core from an apparent state of hyperstabilization, in a cell type-specific manner.
CONCLUSION: These data demonstrate that, while CypA promotes reverse transcription under all conditions tested here, its effect on HIV-1 infectivity correlates more closely with effects on nuclear entry of the viral cDNA. The data also support the hypothesis that a cell-type specific CypA-dependent restriction factor blocks HIV-1 replication by delaying CA core uncoating and hindering nuclear entry.
A novel rabbit monoclonal antibody platform to dissect the diverse repertoire of antibody epitopes for HIV-1 Env immunogen design
The majority of available monoclonal antibodies (MAbs) in the current HIV vaccine field are generated from HIV-1-infected people. In contrast, preclinical immunogenicity studies have mainly focused on polyclonal antibody responses in experimental animals. Although rabbits have been widely used for antibody studies, there has been no report of using rabbit MAbs to dissect the specificity of antibody responses for AIDS vaccine development. Here we report on the production of a panel of 12 MAbs from a New Zealand White (NZW) rabbit that was immunized with an HIV-1 JR-FL gp120 DNA prime and protein boost vaccination regimen. These rabbit MAbs recognized a diverse repertoire of envelope (Env) epitopes ranging from the highly immunogenic V3 region to several previously underappreciated epitopes in the C1, C4, and C5 regions. Nine MAbs showed cross-reactivity to gp120s of clades other than clade B. Increased somatic mutation and extended CDR3 were observed with Ig genes of several molecularly cloned rabbit MAbs. Phylogenic tree analysis showed that the heavy chains of MAbs recognizing the same region on gp120 tend to segregate into an independent subtree. At least three rabbit MAbs showed neutralizing activities with various degrees of breadth and potency. The establishment of this rabbit MAb platform will significantly enhance our ability to test optimal designs of Env immunogens to gain a better understanding of the structural specificity and evolution process of Env-specific antibody responses elicited by candidate AIDS vaccines.
Estudio Parto: postpartum diabetes prevention program for hispanic women with abnormal glucose tolerance in pregnancy: a randomised controlled trial - study protocol
BACKGROUND: Diabetes and obesity have reached epidemic proportions in the U.S. with rates consistently higher among Hispanics as compared to non-Hispanic whites. Among Hispanic women diagnosed with gestational diabetes mellitus (GDM), 50% will go on to develop type 2 diabetes within 5 years of the index pregnancy. Although randomised controlled trials among adults with impaired glucose tolerance have shown that diet and physical activity reduce the risk of type 2 diabetes, such programs have not been tested in high-risk postpartum women. The overall goal of this randomised controlled trial is to test the efficacy of a culturally and linguistically modified, individually-tailored lifestyle intervention to reduce risk factors for type 2 diabetes and cardiovascular disease among postpartum Hispanic women with a history of abnormal glucose tolerance during pregnancy.
METHODS/DESIGN: Hispanic pregnant women who screen positive for GDM will be recruited and randomly assigned to a Lifestyle Intervention (n = 150) or a Health and Wellness (control) Intervention (n = 150). Multimodal contacts (i.e., in-person, telephone, and mailed materials) will be used to deliver the intervention from late pregnancy (29 weeks gestation) to 12 months postpartum. Targets of the intervention are to achieve Institute of Medicine Guidelines for postpartum weight loss; American Congress of Obstetrician and Gynecologist guidelines for physical activity; and American Diabetes Association guidelines for diet. The intervention draws from Social Cognitive Theory and the Transtheoretical Model and addresses the specific cultural and environmental challenges faced by low-income Hispanic women. Assessments will be conducted at enrollment, and at 6-weeks, 6-months, and 12-months postpartum by trained bicultural and bilingual personnel blinded to the intervention arm. Efficacy will be assessed via postpartum weight loss and biomarkers of insulin resistance and cardiovascular risk. Changes in physical activity and diet will be measured via 7-day actigraph data and three unannounced 24-hour dietary recalls at each assessment time period.
DISCUSSION: Hispanic women are the fastest growing minority group in the U.S. and have the highest rates of sedentary behavior and postpartum diabetes after a diagnosis of GDM. This randomised trial uses a high-reach, low-cost strategy that can readily be translated into clinical practice in underserved and minority populations.
TRIAL REGISTRATION: NCT01679210.
Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag-encoded RNA
BACKGROUND: All retroviruses synthesize essential proteins via alternatively spliced mRNAs. Retrovirus genera, though, exploit different mechanisms to coordinate the synthesis of proteins from alternatively spliced mRNAs. The best studied of these retroviral, post-transcriptional effectors are the trans-acting Rev protein of lentiviruses and the cis-acting constitutive transport element (CTE) of the betaretrovirus Mason-Pfizer monkey virus (MPMV). How members of the gammaretrovirus genus translate protein from unspliced RNA has not been elucidated.
RESULTS: The mechanism by which two gammaretroviruses, XMRV and MLV, synthesize the Gag polyprotein (Pr65Gag) from full-length, unspliced mRNA was investigated here. The yield of Pr65Gag from a gag-only expression plasmid was found to be at least 30-fold less than that from an otherwise isogenic gag-pol expression plasmid. A frameshift mutation disrupting the pol open reading frame within the gag-pol expression plasmid did not decrease Pr65Gag production and 398 silent nucleotide changes engineered into gag rendered Pr65Gag synthesis pol-independent. These results are consistent with pol-encoded RNA acting in cis to promote Pr65Gag translation. Two independently-acting pol fragments were identified by screening 17 pol deletion mutations. To determine the mechanism by which pol promoted Pr65Gag synthesis, gag RNA in total and cytoplasmic fractions was quantitated by northern blot and by RT-PCR. The pol sequences caused, maximally, three-fold increase in total or cytoplasmic gag mRNA. Instead, pol sequences increased gag mRNA association with polyribosomes ~100-fold, a magnitude sufficient to explain the increase in Pr65Gag translation efficiency. The MPMV CTE, an NXF1-binding element, substituted for pol in promoting Pr65Gag synthesis. A pol RNA stem-loop resembling the CTE promoted Pr65Gag synthesis. Over-expression of NXF1 and NXT, host factors that bind to the MPMV CTE, synergized with pol to promote gammaretroviral gag RNA loading onto polysomes and to increase Pr65Gag synthesis. Conversely, Gag polyprotein synthesis was decreased by NXF1 knockdown. Finally, overexpression of SRp20, a shuttling protein that binds to NXF1 and promotes NXF1 binding to RNA, also increased gag RNA loading onto polysomes and increased Pr65Gag synthesis.
CONCLUSION: These experiments demonstrate that gammaretroviral pol sequences act in cis to recruit NXF1 and SRp20 to promote polysome loading of gag RNA and, thereby license the synthesis of Pr65Gag from unspliced mRNA.
BACKGROUND: In addition to the core catalytic machinery, bacterial replicative DNA polymerases contain a Polymerase and Histidinol Phosphatase (PHP) domain whose function is not entirely understood. The PHP domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. In E. coli DNA polymerase III, however, the PHP domain has lost several metal-coordinating residues and is likely to be catalytically inactive.
RESULTS: Genomic searches show that the loss of metal-coordinating residues in polymerase PHP domains is likely to have coevolved with the presence of a separate proofreading exonuclease that works with the polymerase. Although the E. coli Pol III PHP domain has lost metal-coordinating residues, the structure of the domain has been conserved to a remarkable degree when compared to that of metal-binding PHP domains. This is demonstrated by our ability to restore metal binding with only three point mutations, as confirmed by the metal-bound crystal structure of this mutant determined at 2.9 A resolution. We also show that Pol III, a large multi-domain protein, unfolds cooperatively and that mutations in the degenerate metal-binding site of the PHP domain decrease the overall stability of Pol III and reduce its activity.
CONCLUSIONS: While the presence of a PHP domain in replicative bacterial polymerases is strictly conserved, its ability to coordinate metals and to perform proofreading exonuclease activity is not, suggesting additional non-enzymatic roles for the domain. Our results show that the PHP domain is a major structural element in Pol III and its integrity modulates both the stability and activity of the polymerase.
Background and Purpose: Hodgkin lymphoma (HL) treatment has evolved to reduce or avoid radiotherapy (RT) dose and volume and minimize the potential for late effects. Some older adolescents are treated on adult protocols. The purpose of this study is to examine the protocol assignment of older adolescents and its impact on radiation dose to relevant thoracic structures.
Materials and Methods: Cooperative group data were reviewed and 12 adolescents were randomly selected from a pediatric HL protocol. Treatment plans were generated per one pediatric and two adult protocols. Dose volume histograms for heart, lung, and breast allowed comparison of radiation dose to these sites across these three protocols.
Results: A total of 15.2% of adolescents were treated on adult HL protocols and received significantly higher radiation dosage to heart and lung compared to pediatric HL protocols. Adolescents treated on either pediatric or adult protocols received similar RT dose to breast.
Conclusion: Older adolescents treated on adult HL protocols received higher RT dose to thoracic structures except breast. Level of nodal involvement may impact overall RT dose to breast. The impact of varying field design and RT dose on survival, local, and late effects needs further study for this vulnerable age group.
For centuries the criminal justice system has struggled to define the methodology of and the justifications for social control of sexual behavior that does not conform to community mores. This poster compares and contrasts the historical and contemporary attempts in the United States, Canada, Belgium, the United Kingdom, and Germany to address the risk created by individuals who engage in behaviors broadly characterized as sexually deviant. Where available, we consider the rationale for sentencing, and the earliest attempts to bring “treatment” into the criminal dispositional formula for sexual based prosecution. We also consider the impact that the choice of societal response has on risk assessment and evaluation in the various systems, including where available, the assessment and commitment of juvenile offenders. The current practice of civil commitment for a person deemed to be a sexually violent predator (SVP) is discussed highlighting the U.S. Supreme Court decision in Kansas v. Hendricks. This practice will then be compared and contrasted with the approach of designating an offender as a Dangerous Offender (DO) or a Long-Term Offender (LTO) under the criminal law. We also highlight sex offender registries where applicable. This poster is intended as an overview of the law as it exists, and not as a defense or a critique of any specific model.
Association of Demographic Factors and Comorbid Diagnoses with Crime Typein an Arrest Cohort with Schizophrenia and/or Related Psychosis
The implications of the interface between the criminal justice system and individuals with schizophrenia persist despite decades of research on criminalization and risk of arrest. Research exploring the broader construct of criminality has predominantly focused on individuals with severe mental illness as a collective. This study diverges from others by examining diagnoses comorbid with schizophrenia and related psychoses and their relationships with risk of arrest across a spectrum of criminal categories ranging in severity.
Helping law enforcement, health care, and social service communities work together to develop a seamless continuum of care for persons with serious mental illness who are charged with non-serious crimes.
A recent survey of state statutes for outpatient commitment (Torrey and Kaplan, 1995) indicates that while thirty-five states and the District of Columbia have laws permitting outpatient commitment, Massachusetts is not one of them. Rather, Massachusetts uses a competency-based, substituted-decision-making model for the involuntary administration of medication in the community. To appreciate the Massachusetts model, it is important to understand how this court-ordered involuntary outpatient treatment fits into the overall scheme of outpatient commitment and how it is structured.
A review of involuntary outpatient treatment (IOT) literature indicates that it is prudent to distinguish between outpatient commitment, conditional release, and conservatorship-guardianship (Torrey and Kaplan, 1995). Two states whose IOT is based on the guardianship process and is described in the literature are California and New Mexico. Lamb and Weinberger (1992, 1993) have discussed California’s use of guardians for the gravely disabled psychiatric outpatient, and Schneider-Braus (1986) has presented a single case report from New Mexico.
Temporal Patterns of Arrest in a Cohort of Adults Receiving Mental Health Services: The Massachusetts Mental Health / Criminal Justice Cohort Study
Criminal Justice Involvement among Clientele is a Major Concern for State Mental Health Agencies. Mental health and criminal justice systems provide services at various points along the interface of these systems to reduce offending and re-offending, including:
- Diversion programs
- Mental Health Courts
Little information about scope of offending to guide service development. This study provides data on the prevalence, type and temporal patterns of arrest for a large sample of adults followed for roughly 9.5 years.
Serious Mental Illness and Chronic Crimial Justice Involvement: Findings from The Massachusetts Mental Healthy / Criminal Justice Cohort Study
Presents findings from 1990 through 2000 from The Massachusetts Mental Health/Criminal Justice Cohort Study.
Institutional Review Boards (IRBs) are the primary organizations designed to protect research subjects from harm and assure that they participate voluntarily. At the same time, many researchers feel that they intrude into the research process without making research safer.
• Identify which issues about applications are the focus of IRB attention; e.g., the scientific validity of a protocol, issues of risk, informed consent
• Clarify how, if at all, the occupants of different roles (chair, community member, attorney, scientific expert, etc.) differ in their discussion of applications
• Describe how IRB members identify problems in applications; what information resources they use and how they use them
• Identify how IRBs organize the work of application review through the use of staff, pre-meeting review, and formatl meetings
Presentation on how to receive a high survey response rate using the Dillon Method, Survey Monkey and various contact steps.
Regional differences in the reporting of mental health symptoms among juvenile justice (JJ) involved youth were examined using data from the 70,423 youths in the MAYSI-2 national norm study (Vincent et al., 2008). The percentage of youth scoring above Caution on MAYSI-2 scales was examined by race/ethnicity (white vs. minorities) and sex. Regional differences were assessed using Cochran’s Mantel-Haenszel (CMH) analyses. White youth were more likely to score above caution on all clinical scales except Depressed-Anxious. An interesting gender and race/ethnicity effect emerged such that White male youth in the Northeast and Midwest were more likely than Minority youth to score above caution; whereas White female youth in the West were more likely than Minority youth to score above caution.
This poster reports on factors that influenced the rapid adoption and implementation of the Massachusetts Youth Screening Instrument-version 2 (MAYSI-2) and the perceived consequences of routine MAYSI-2 mental health screening. Semi-structured interviews and focus groups were conducted with administrators, managers, and front-line staff in juvenile detention centers in three states (n=19). Results will allow us to better inform juvenile justice facilities regarding the conditions under which screening can more often result in increases in mental health services to youth entering the system and help guide future efforts to provide technology to juvenile justice programs in the interest of youths.