A systematic review of community-based participatory research interventions in pediatric and adolescent populations

Background. Community-based participatory research (CBPR) is a collaborative approach to research actively involving community members in all aspects of the research process. CBPR is not a new research method, but an approach that has gained increased attention in the field of public health over the last several years. Recognition of the inequalities in health status associated with social and environmental factors have led to calls for a renewed focus on ecological approaches to research. Ecological approaches acknowledge that the health of the community is dependent on an interaction between behavioral and environmental factors affecting the entire population. While many published studies document the benefits of CBPR in difficult-to-reach populations and describe successful implementation of this approach in adult populations, relatively few studies have been conducted in child and adolescent populations. Given that children and adolescents are particularly sensitive to the effects of their physical environments and may also be distrustful of outsiders, ecological approaches involving the community as partners, such as CBPR, may be especially useful in this population. ^ Objective. This thesis reviews published studies using a community-based participatory research approach in children and adolescents to assess the appropriateness of this approach in this population. ^ Method. Studies using CBPR in youth populations were identified using Medline and other Internet searches through both MeSH heading and text-word searches. ^ Results. A total of 16 studies were identified and analyzed for this review. Nine of the sixteen studies were experimental or quasi-experimental design, with Asthma being the most commonly studied disease. ^ Conclusions. While many studies using CBPR were not conducted with the level of scientific rigor typically found in clinical trial research, the studies reviewed each contributed to a greater understanding of the problems they investigated. Furthermore, interventional studies provided lasting benefits to communities under study above what would be found in studies using more traditional research approaches. While CBPR may not be appropriate for all research situations due to the time and resources required, we conclude that is a useful approach and should be considered when conducting community-based research for pediatric and adolescent populations.^

Intercellular adhesion and membrane polarity in rat hepatocytes: Localization of cell -CAM 105 and other domain-specific membrane proteins {\it in situ\/} and {\it in vitro\/} by electron microscopy

Cell-CAM 105 has been identified as a cell adhesion molecule (CAM) based on the ability of monospecific and monovalent anti-cell-CAM 105 antibodies to inhibit the reaggregation of rat hepatocytes. Although one would expect to find CAMs concentrated in the lateral membrane domain where adhesive interactions predominate, immunofluorescence analysis of rat liver frozen sections revealed that cell-CAM 105 was present exclusively in the bile canalicular (BC) domain of the hepatocyte. To more precisely define the in situ localization of cell-CAM 105, immunoperoxidase and electron microscopy were used to analyze intact and mechanically dissociated fixed liver tissue. Results indicate that although cell-CAM 105 is apparently restricted to the BC domain in situ, it can be detected in the pericanalicular region of the lateral membranes when accessibility to lateral membranes is provided by mechanical dissociation. In contrast, when hepatocytes were labeled following incubation in vitro under conditions used during adhesion assays, cell-CAM 105 had redistributed to all areas of the plasma membrane. Immunofluorescence analysis of primary hepatocyte cultures revealed that cell-CAM 105 and two other BC proteins were localized in discrete domains reminscent of BC while cell-CAM 105 was also present in regions of intercellular contact. These results indicate that the distribution of cell-CAM 105 under the experimental conditions used for cell adhesion assays differs from that in situ and raises the possibility that its adhesive function may be modulated by its cell surface distribution. The implications of these and other findings are discussed with regard to a model for BC formation.^ Analysis of molecular events involved in BC formation would be accelerated if an in vitro model system were available. Although BC formation in culture has previously been observed, repolarization of cell-CAM 105 and two other domain-specific membrane proteins was incomplete. Since DMSO had been used by Isom et al. to maintain liver-specific gene expression in vitro, the effect of this differentiation system on the polarity of these membrane proteins was examined. Based on findings presented here, DMSO apparently prolongs the expression and facilitates polarization of hepatocyte membrane proteins in vitro. ^

The p53 tumor suppressor pathway in culture and {\it in vivo\/}

p53 functions as a tumor suppressor through its ability to initiate either growth arrest or apoptosis in cells which have sustained DNA damage. p53 elicits these cellular phenotypes through its biochemical function as a transcriptional activator. By inducing the expression of a battery of target genes, p53 is able to prevent the propagation of cells with damaged DNA. However, the genes transcriptionally induced by p53 which have been identified to date do not fully explain p53 function. p53 has been demonstrated to activate genes involved in cell cycle inhibition, apoptosis and cell proliferation. The reasons for simultaneous activation of p53 targets with disparate, opposing functions are not clear, but may be due to the use of transformed cell lines in previous experiments. In the studies presented in this thesis, the pathway of p53 tumor suppression has been studied in detail in two systems chosen for their relevance to the natural cell environment. One utilizes a normal, unaltered cultured cell system; the other the whole mouse. In order to better understand the role of the known p53 targets in effecting p53 function in normal cells, early rat embryo fibroblasts were irradiated with ultraviolet light to induce DNA damage. It was discovered that p53 protein levels increased in response to irradiation. The known targets of p53, namely, $p21\sp{WAF1/CIP1},\ mdm2,\ cyclin\ G,$ and bax, were shown for the first time to have a differential temporal induction. The growth suppressor $p21\sp{WAF1/CIP1}$ was induced first, followed by cyclin G then mdm2, which is involved in proliferation through its inactivation of p53, and finally, the apoptosis promoter, bax. These findings indicated that p53 activates its target genes in a manner to allow maximum effectiveness of target function. The rat embryo fibroblasts were shown to undergo apoptosis 24 h after irradiation. Additionally, investigation of these cells for cell cycle alterations demonstrated a brief arrest in G1. In the second study, thymocytes from mice with wild type p53 were shown to undergo apoptosis and activate p53 target genes upon ionizing radiation treatment, while thymocytes from mice deficient in p53 could not. The p53 target genes mdm2 and fas were tested in vivo for their ability to mediate p53-regulated apoptosis, and were found dispensible for that cellular function. Therefore, the p53 targets identified to date do not fully explain the ability of p53 to function as a tumor suppressor. Potentially, functional redundancy between the known targets would account for the data seen in these experiments. Additionally, identification of additional target genes should add further understanding of the p53 pathway of tumor suppression. ^

Characterization of Jak, STAT, and Src interactions in Head and Neck Squamous Cell Carcinoma

Recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is common; thus, it is essential to improve the effectiveness and reduce toxicity of current treatments. Proteins in the Src/Jak/STAT pathway represent potential therapeutic targets, as this pathway is hyperactive in HNSCC and it has roles in cell migration, metastasis, proliferation, survival, and angiogenesis. During short-term Src inhibition, Janus kinase (Jak) 2, and signal transducer and activator of transcription (STAT) 3 and STAT5 are dephosphorylated and inactivated. Following sustained Src inhibition, STAT5 remains inactive, but Jak2 and STAT3 are reactivated following their early inhibition. To further characterize the mechanism of this novel feedback pathway we performed several experiments to look at the interactions between Src, Jak2, STAT5 and STAT3. We attempted to develop a non-radioactive kinase assay using purified recombinant Jak2 and Src proteins, but found that phospho-tyrosine antibodies were non-specifically binding to purified recombinant proteins. We then performed in vitro kinase assays (IVKAs) using purified recombinant Jak2, Src, STAT3, and STAT5 proteins with and without Src and Jak2 pharmacologic inhibitors. We also examined the interactions of these proteins in intact HNSCC cells. We found that recombinant Jak2, STAT3, and STAT5 are direct substrates of Src and that recombinant Src, STAT3, and STAT5 are direct substrates of Jak2 in the IVKA. To our knowledge, the finding that Src is a Jak substrate is novel and has not been shown before. In intact HNSCC cells we find that STAT3 can be reactivated despite continuous Src inhibition and that STAT5 continues to be inhibited despite Jak2 reactivation. Also, Jak2 inhibition did not affect Src or STAT5 activity but it did cause STAT3 inhibition. We hypothesized that the differences between the intact cells and the IVKA assays were due to a potential need for binding partners in intact HNSCC cells. One potential binding partner that we examined is the epidermal growth factor receptor (EGFR). We found that EGFR activation caused increased activation of Src and STAT5 but not Jak2. Our results demonstrate that although STAT3 and STAT5 are capable of being Src and Jak2 substrates, in intact HNSCC cells Src predominantly regulates STAT5 and Jak2 regulates STAT3. Regulation of STAT5 by Src may involve interactions between Src and EGFR. This knowledge along with future studies will better define the mechanisms of STAT regulation in HNSCC cells and ultimately result in an ideal combination of therapeutic agents for HNSCC.

Molecular analysis of TNF sensitivity in normal and Ha-{\it ras\/} transformed murine fibroblasts

Tumor necrosis factor (TNF) is known to have antiproliferative effects on a wide variety of tumor cells but proliferative effects on normal cells. However, the molecular basis for such differences in the action of TNF are unknown. The overall objectives of my research are to investigate the role of oncogenes in TNF sensitivity and delineate some of the molecular mechanisms involved in TNF sensitivity and resistance. To accomplish these objectives, I transfected TNF-resistant C3H mouse embryo fibroblasts (10T1/2) with an activated Ha-ras oncogene and determined whether these cells exhibit altered sensitivity to TNF. The results indicated that 10T1/2 cells transfected with an activated Ha-ras oncogene (10T-EJ) not only produced tumors in nude mice but also exhibited extreme sensitivity to cytolysis by TNF. In contrast, 10T1/2 cells transfected with the pSV2-neo gene alone were resistant to the cytotoxic effects of TNF. I also found that TNF-induced cell death was mediated through apoptosis. The differential sensitivity of 10T1/2 and 10T-EJ cell lines to TNF was not due to differences in the number of TNF receptors on their cell surface. In addition, TNF-resistant revertants isolated from Ha-ras-transformed, TNF-sensitive cells still expressed the same amount of p21 as TNF-sensitive cells and were still tumorigenic, suggesting that Ha-ras-induced transformation and TNF sensitivity may follow different pathways. Interestingly, TNF-resistant but not sensitive cells expressed higher levels of bcl-2, c-myc, and manganese superoxide dismutase (MnSOD) mRNA following exposure to TNF. However, TNF treatment resulted in a marginal induction of p53 mRNA in both TNF-sensitive and resistant cells. Based on these results I can conclude that (i) Ha-ras oncogene induces both transformation and TNF sensitivity, (ii) TNF-induced cytotoxicity involves apoptosis, and (iii) TNF-induced upregulation of bcl-2, c-myc, and MnSOD genes is associated with TNF resistance in C3H mouse embryo fibroblasts. ^