Primary prevention of beta-cell autoimmunity and type 1 diabetes - The Global Platform for the Prevention of Autoimmune Diabetes (GPPAD) perspectives.

OBJECTIVE: Type 1 diabetes can be identified by the presence of beta-cell autoantibodies that often arise in the first few years of life. The purpose of this perspective is to present the case for primary prevention of beta-cell autoimmunity and to provide a study design for its implementation in Europe. METHODS: We examined and summarized recruitment strategies, enrollment rates, and outcomes in published TRIGR, FINDIA and BABYDIET primary prevention trials, and the TEDDY intensive observational study. A proposal for a recruitment and implementation strategy to perform a phase II/III primary prevention randomized controlled trial in infants with genetic risk for developing beta-cell autoimmunity is outlined. RESULTS: Infants with a family history of type 1 diabetes (TRIGR, BABYDIET, TEDDY) and infants younger than age 3 months from the general population (FINDIA, TEDDY) were enrolled into these studies. All studies used HLA genotyping as part of their eligibility criteria. Predicted beta-cell autoimmunity risk in the eligible infants ranged from 3% (FINDIA, TEDDY general population) up to 12% (TRIGR, BABYDIET). Amongst eligible infants, participation was between 38% (TEDDY general population) and 97% (FINDIA). Outcomes, defined as multiple beta-cell autoantibodies, were consistent with predicted risks. We subsequently modeled recruitment into a randomized controlled trial (RCT) that could assess the efficacy of oral insulin treatment as adapted from the Pre-POINT pilot trial. The RCT would recruit infants with and without a first-degree family history of type 1 diabetes and be based on general population genetic risk testing. HLA genotyping and, for the general population, genotyping at additional type 1 diabetes susceptibility SNPs would be used to identify children with around 10% risk of beta-cell autoimmunity. The proposed RCT would have 80% power to detect a 50% reduction in multiple beta-cell autoantibodies by age 4 years at a two-tailed alpha of 0.05, and would randomize around 1160 infants to oral insulin or placebo arms in order to fulfill this. It is estimated that recruitment would require testing of between 400,000 and 500,000 newborns or infants. CONCLUSION: It is timely and feasible to establish a platform for primary prevention trials for type 1 diabetes in Europe. This multi-site European infrastructure would perform RCTs, supply data coordination and biorepository, provide cohorts for mechanistic and observational studies, and increase awareness for autoimmune diabetes.

N-cadherin/FGFR promotes metastasis through epithelial-to-mesenchymal transition and stem/progenitor cell-like properties.

N-cadherin and HER2/neu were found to be co-expressed in invasive breast carcinomas. To test the contribution of N-cadherin and HER2 in mammary tumor metastasis, we targeted N-cadherin expression in the mammary epithelium of the MMTV-Neu mouse. In the context of ErbB2/Neu, N-cadherin stimulated carcinoma cell invasion, proliferation and metastasis. N-cadherin caused fibroblast growth factor receptor (FGFR) upmodulation, resulting in epithelial-to-mesenchymal transition (EMT) and stem/progenitor like properties, involving Snail and Slug upregulation, mammosphere formation and aldehyde dehydrogenase activity. N-cadherin potentiation of the FGFR stimulated extracellular signal regulated kinase (ERK) and protein kinase B (AKT) phosphorylation resulting in differential effects on metastasis. Although ERK inhibition suppressed cyclin D1 expression, cell proliferation and stem/progenitor cell properties, it did not affect invasion or EMT. Conversely, AKT inhibition suppressed invasion through Akt 2 attenuation, and EMT through Snail inhibition, but had no effect on cyclin D1 expression, cell proliferation or mammosphere formation. These findings suggest N-cadherin/FGFR has a pivotal role in promoting metastasis through differential regulation of ERK and AKT, and underscore the potential for targeting the FGFR in advanced ErbB2-amplified breast tumors.

Over-expression of CDKIs p15INK4b, p16INK4a and p21CIP1/WAF1 genes mediate growth arrest in human osteosarcoma cell lines.

Human somatic cells cultured in vitro exhibit a limited number of divisions. In contrast immortal cells have lost their growth regulatory mechanisms and, thus, continue to divide indefinitely. Cyclin-dependent kinase inhibitors (CDKIs) represent one of the most important regulatory factors in both mortality and immortality, as they are over-expressed in senescent cells and are down-regulated in a number of human cancers. In the present study we determined the effect of CDKIs on the proliferation ability of human osteosarcoma cell lines. Transient expression of various CDKIs (p15INK4b, p16INK4a and p21CIP1/WAF1) in KHOS cells resulted in growth arrest and the cells failed to enter the S-phase of the cell cycle as shown by a DNA synthesis inhibition assay. In addition, stable transfection of p21CIP1/WAF1 and p16INK4a genes in two osteosarcoma cell lines (KHOS and U2-OS cells) showed that p21CIP1/WAF1 was able to repress the immortal phenotype in both cell lines, whereas temporary over-expression of p16INK4a reversibly inhibited the cell growth. Therefore this study indicates that CDKIs mediate growth arrest in human osteosarcoma cell lines and provides further evidence of the existence of molecular links between cellular mortality and immortality.

Correlation of in vitro cytotoxicity and clinical response to chemotherapy in ovarian and breast cancer patients.

During the last years, a number of assays have been developed aiming at predicting the most effective chemotherapy regimen for each individual, avoiding possible toxicity of ineffective drugs. In the present study we have used an in vitro chemosensitivity/chemoresistance assay in order to evaluate cytotoxic treatment in ovarian and breast cancer patients. The assay was applied in 77 ovarian and breast cancer samples and the observed in vitro responses to various chemotherapeutic drugs or combinations of drugs were then correlated to the in vivo responses and the overall clinical data of the examined patients. Direct comparison was possible for 25 cases. The overall positive predictive value of the assay was 50% and the negative predictive value was 57%. However, it was observed that the positive predictive value for ovarian patients was 69% and that the negative predictive value for breast patients was 100%. Therefore this study indicates that although in vitro chemosensitivity/chemoresistance is a valuable assay, further analysis and implications of other factors are required for a general evaluation of cytotoxic treatment for patients with ovarian and breast cancer.

Evaluation of cytotoxic treatment of patients with osteosarcoma by an in vitro chemoresistance assay.

During the previous two decades several assays have been developed aiming to select the most effective chemotherapy regimen for each individual, avoiding the possible toxicity of ineffective drugs. In order to evaluate cytotoxic treatment for patients with osteosarcoma, we applied an in vitro chemoresistance assay by culturing tumour cells and determining their in vitro survival rates after exposure to various chemotherapeutic drugs. The conditions of the assay were optimised in two established osteosarcoma cell lines (KHOS and U2-OS), as compared with data derived after treatment of primary normal adult osteoblasts. Chemotherapeutic drugs (cisplatin, adriamycin or methotrexate or combinations) concentrations were chosen in a range that has been reported to induce tumour cell death in the plasma patients' The method applied successfully in 6 cell cultures originated from biopsies of 7 patients with osteosarcoma and the in vitro response to chemotherapeutic drugs was correlated with the clinical outcome. Such analysis revealed both positive and negative correlation of the in vitro data to the patients clinical responses. Therefore, this study indicated that, although in vitro chemoresistance is a valuable assay, additional analysis and implications of other factors are required for a general evaluation of cytotoxic treatment for patients with osteosarcoma.

Aging and longevity. A paradigm of complementation between homeostatic mechanisms and genetic control?

Aging is a universal and inevitable phenomenon that affects nearly all animal species. It can be considered the product of an interaction between genetic, environmental, and lifestyle factors, which in turn influence longevity that varies between and within species. It has been proposed not only that the aging process is under genetic control, but that it can also be considered a result of the failure of homeostasis due to the accumulation of damage. This review article discusses these issues, focusing on the function of genes that associate with aging and longevity, as well as on the molecular mechanisms that control cell survival and maintenance during aging.

Cloning and identification of genes that associate with mammalian replicative senescence.

Cellular senescence and limited proliferative capacity of normal diploid cells has a dominant phenotype over immortality of cancerous cells, suggesting its regulation by the expression of a set of genes. In order to isolate the genes that associate with senescence, we have employed a clonal system of conditional SV40 T antigen rat embryo fibroblast cell lines which undergo senescence upon T antigen inactivation. Construction of cDNA libraries from two conditional cell lines and application of differential screening and subtractive hybridization techniques have resulted in the cloning of eight senescence-induced genes (SGP-2/Apo J, alpha 1-procollagen, osteonectin, fibronectin, SM22, cytochrome C oxidase, GTP-alpha, and a novel gene) and a senescence-repressed gene (FRS-2). Three of these genes encode for extracellular matrix proteins, others are involved in the calcium-dependent signal transduction pathways, while the SGP-2/Apo J gene may have a cellular protective function. RNA analysis has shown that the senescence-associated genes are overexpressed in both normal rat embryonic fibroblasts and human osteoblasts cell cultures undergoing aging in vitro. In comparison, the expression of these genes in a rat fibroblast immortalized cell line (208F cells) was down-regulated after both its partial and its full transformation by ras oncogenes. Thus, cloning of senescence-associated genes opens up new ways to elucidate and/or to modulate aging and cancer.

High levels of anti-phospholipid antibodies in uncomplicated and severe Plasmodium falciparum and in P. vivax malaria.

The majority (75%) of adult patients with uncomplicated Plasmodium falciparum and P. vivax malaria are positive for anti-phospholipid antibodies (aPLA) as demonstrated by ELISA using a panel of anionic and cationic phospholipids. The highest IgG and IgM binding was to the anionic phospholipids, phosphatidylserine (PS), phosphatidic acid (PA) and cardiolipin (CL), but excluding phosphatidylinositol (PI) to which only low antibody levels were found. Comparison of the mean IgG and IgM aPLA showed a trend for anti-PA > CL > PS > PC > PE > PI. Anti-PI levels were compared in two groups of African children, one group with non-severe and the other with severe (cerebral) falciparum malaria. Children with cerebral disease had significantly lower IgM anti-PI. The results are discussed with the view that serum-derived aPLA may have a role in 'anti-disease' immune responses. Their possible role in the opsonization and phagocytosis of parasitized erythrocytes and in thrombocytopenia is also considered.