Family PArtners in Lifestyle Support (PALS): Family-based weight loss for African American adults with type 2 diabetes.

OBJECTIVE: To develop and test a family-centered behavioral weight loss intervention for African American adults with type 2 diabetes. METHODS: In this randomized trial, dyads consisting of an African American adult with overweight or obesity and type 2 diabetes (index participant) paired with a family partner with overweight or obesity but not diagnosed with diabetes were assigned in a 2:1 ratio to a 20-week special intervention (SI) or delayed intervention (DI) control group. The primary outcome was weight loss among index participants at the 20-week follow-up. RESULTS: One hundred eight participants (54 dyads-36 (SI) and 18 (DI) dyads) were enrolled: 81% females; mean age, 51 years; mean weight,103 kg; and mean BMI, 37 kg/m2 . At post-intervention, 96 participants (89%) returned for follow-up measures. Among index participants, mean difference in weight loss between groups was -5.0 kg, P <0.0001 (-3.6 kg loss among SI; 1.4 kg gain in DI). SI index participants showed significantly greater improvements in hemoglobin A1c, depressive symptoms, family interactions, and dietary, physical activity, and diabetes self-care behaviors. SI family partners also had significant weight loss (-3.9 kg (SI) vs. -1.0 kg (DI), P = 0.02). CONCLUSIONS: A family-centered, behavioral weight loss intervention led to clinically significant short-term weight loss among family dyads.

Defining spoken language benchmarks and selecting measures of expressive language development for young children with autism spectrum disorders.

PURPOSE: The aims of this article are twofold: (a) to offer a set of recommended measures that can be used for evaluating the efficacy of interventions that target spoken language acquisition as part of treatment research studies or for use in applied settings and (b) to propose and define a common terminology for describing levels of spoken language ability in the expressive modality and to set benchmarks for determining a child's language level in order to establish a framework for comparing outcomes across intervention studies. METHOD: The National Institute on Deafness and Other Communication Disorders assembled a group of researchers with interests and experience in the study of language development and disorders in young children with autism spectrum disorders. The group worked for 18 months through a series of conference calls and correspondence, culminating in a meeting held in December 2007 to achieve consensus on these aims. RESULTS: The authors recommend moving away from using the term functional speech, replacing it with a developmental framework. Rather, they recommend multiple sources of information to define language phases, including natural language samples, parent report, and standardized measures. They also provide guidelines and objective criteria for defining children's spoken language expression in three major phases that correspond to developmental levels between 12 and 48 months of age.

Are international differences in the outcomes of acute coronary syndromes apparent or real? A multilevel analysis.

STUDY OBJECTIVE: International variation in the outcomes of patients with acute coronary syndromes (ACS) has been well reported. The relative contributions of patient, hospital, and country level factors on clinical outcomes, however, remain unclear, and thus, was the objective of this study. DESIGN: Multilevel logistic regression models were developed for death/(re)infarction (MI) at 30 days and death in one year, with patients (1st level) nested in hospitals (2nd level) and hospitals in countries (3rd level). SETTINGS: The GUSTO IV ACS clinical trial was carried out at 458 hospital sites in 24 countries. PATIENTS: 7800 non-ST segment elevation (NSTE) ACS patients. MAIN RESULTS: There were substantial variations among countries in the processes and outcomes of care at 30 days, ranging from 5.4% to 50.0% for percutaneous coronary intervention, 4.3% to 21.2% for coronary artery bypass graft surgery, 5.0% to 13.9% for 30 day death/(re)MI, and 4.9% to 14.8% for one year mortality. However, the residual inter-country variations in 30 day death/(re)MI and one year mortality became non-significant and nearly disappeared (p > 0.500 for both) after adjusting for key baseline patient characteristics and hospital factors, which became significant (p < 0.01 for both). Patient level factors accounted for 96%-99% of total variation in these end points, leaving the remaining 1% and 4% of variance attributable to hospital level factors. CONCLUSION: The international differences in clinical outcomes in this study of NSTE ACS are primarily accounted for by the patient level factors, with hospital level factors playing a minor part, and the country level factors a negligible one. These findings have significant policy and research implications involving international collaboration and comparisons.

Molecular screening and association analyses of the interleukin 6 receptor gene variants with type 2 diabetes, diabetic nephropathy, and insulin sensitivity.

IL-6 levels and polymorphisms have been implicated in type 2 diabetes mellitus (T2DM) and insulin resistance. The IL-6 receptor (IL-6R) comprises two subunits, IL-6R and gp130, of which IL-6R confers specificity to IL-6 action and is located in a region of replicated linkage to T2DM on chromosome 1q21. We screened this gene for variation in Northern European Caucasian and African-American ethnic groups. We identified 11 variants with a minor allele frequency over 5%, including two amino acid changes (D358A and V385I) and four variants in the 3' untranslated region. No variant was associated with obesity or measures of insulin sensitivity, but two single nucleotide polymorphisms in the 3' untranslated region showed a trend to an association with T2DM in all Caucasians, and three single nucleotide polymorphisms, including D358A, showed a trend (P < 0.06) to an association with T2DM among the subset of Northern European Caucasians. Variant V385I was unique to African-Americans and was significantly associated with diabetes and diabetic nephropathy (P < 0.05). Among individuals heterozygous for the four variants in the transcribed sequence, one allele was significantly overrepresented, thus suggesting the existence of a regulatory variant controlling mRNA stability or expression. IL-6R is not likely to explain the linkage to diabetes in this region, but our work supports a minor role of variants in T2DM risk and suggests that sequence variants may alter IL-6R mRNA levels and possibly levels of soluble IL-6R.

Molecular screening of the human glutamine-fructose-6-phosphate amidotransferase 1 (GFPT1) gene and association studies with diabetes and diabetic nephropathy.

Increased glucose metabolism through the hexosamine pathway may result in insulin resistance, impaired insulin secretion, and diabetic nephropathy. We hypothesized that variants of GFPT1 encoding glutamine-fructose-6-phosphate amidotransferase, the rate limiting enzyme in this pathway, could increase GFPT1 gene expression and thus susceptibility to diabetes and diabetic nephropathy. To test this hypothesis, we screened for variation in the GFPT1 and flanking regions in Caucasian and African-American individuals. We tested each variant with over 5% allele frequency for an association with type 2 diabetes in Caucasian and African-American populations, and for an association with diabetic nephropathy in African-American subjects. We measured allele specific levels of GFPT1 mRNA and we compared mRNA levels across diagnostic categories for each ethnic group using RNA derived from transformed lymphocytes. None of the 8 variants detected altered the coding sequence or was present in a known regulatory region. We found a marginal association (p = 0.044) of 1/6 variants with diabetes in Caucasian subjects, and marginal associations of 2/7 variants with diabetic nephropathy among African-American subjects (p = 0.025, p = 0.041). Alleles marked by a variant in the 3' untranslated region were equally expressed, but in a small sample, GFPT1 mRNA levels were increased by 60% in Caucasians with diabetic nephropathy compared to diabetic individuals without nephropathy. Variants in the GFPT1 gene show suggestive evidence of an association with diabetic nephropathy among African-American individuals, and increased GFPT1 gene expression may characterize Caucasian subjects with diabetic nephropathy. Both findings need to be confirmed in other populations.

Standardized quantitative in situ hybridization using radioactive oligonucleotide probes for detecting relative levels of mRNA transcripts verified by real-time PCR.

In situ hybridization (ISH) is an essential technique for mapping gene expression in the brain. Although many ISH protocols provide for quantitative analysis of individual mRNAs in different brain regions or across experimental conditions, this technique has lacked the necessary standardization for quantitative comparisons between different mRNA transcripts. We have developed a standardized quantitative ISH (SQuISH) protocol that utilizes multiple radioactive oligonucleotide probes, providing for increased sensitivity, decreased background and accurate comparison of relative mRNA levels. We evaluated the SQuISH protocol against a riboprobe-based ISH procedure by comparing the mRNA expression levels in the brain for two transcripts, insulin receptor substrate p53 (IRSp53) and Calsenilin. The results of these two methods were then validated by real-time quantitative PCR. Both protocols exhibited identical mRNA expression patterns for IRSp53 and Calsenilin. In three brain regions analyzed, the levels of IRSp53 mRNA expression were approximately 1.5-fold higher with the riboprobe-based ISH than with the SQuISH procedure, although the relative abundance in regional expression levels was similar between the two methods. In contrast, the levels of Calsenilin mRNA expression were 10-17-fold higher with the riboprobe-based ISH than with the SQuISH procedure and the relative abundance in regional expression levels was different. When compared to the real-time PCR results, the SQuISH trade mark method showed almost identical relative levels of IRSp53 to Calsenilin mRNA in all three brain regions analyzed, while the riboprobe-based procedure showed a completely opposite trend. These results support the accuracy of the SQuISH protocol for determining relative mRNA levels in the brain.

Common variants in glutamine:fructose-6-phosphate amidotransferase 2 (GFPT2) gene are associated with type 2 diabetes, diabetic nephropathy, and increased GFPT2 mRNA levels.

Increased flux of glucose through the hexosamine biosynthetic pathway has been implicated in insulin resistance, altered insulin secretion, and diabetic nephropathy. Glutamine:fructose-6-phosphate amidotransferase (GFPT), the rate limiting enzyme in hexosamine biosynthesis, is encoded by the unlinked but highly homologous genes GFPT1 and GFPT2. We tested the hypothesis that GFPT2 sequence variation contributed to the susceptibility to type 2 diabetes mellitus (T2DM) and diabetic nephropathy in Caucasian and African-American individuals. We identified 11 single nucleotide polymorphisms (SNPs), of which seven were common. A single variant in exon 14, I471V, altered the amino acid sequence, is conserved between human and mouse genes, and was associated with T2DM among Caucasians (P = 0.05). A trend to an association was noted with diabetic nephropathy among African-American individuals (P = 0.15). Several variants in the 3' untranslated region (UTR) and exon 18 were also associated with T2DM in Caucasian individuals (P < 0.05), and the SNP in the 3' UTR was associated with diabetic nephropathy in African-American subjects (P = 0.047). GFPT2 mRNA levels in transformed lymphocytes from study subjects were significantly increased among African-American subjects compared with Caucasian individuals, regardless of diagnosis. Furthermore, the associated allele of the 3' UTR SNP was approximately 2-fold overexpressed. We propose that the 3' UTR variant results in increased GFPT2 mRNA levels with resultant increased hexosamine flux. The I471V variant may contribute to altered protein function or may simply be in linkage disequilibrium with the 3' UTR.

Cell culture modeling of specialized tissue: identification of genes expressed specifically by follicle-associated epithelium of Peyer's patch by expression profiling of Caco-2/Raji co-cultures.

Peyer's patch follicle-associated epithelium (FAE) regulates intestinal antigen access to the immune system in part through the action of microfold (M) cells which mediate transcytosis of antigens and microorganisms. Studies on M cells have been limited by the difficulties in isolating purified cells, so we applied TOGA mRNA expression profiling to identify genes associated with the in vitro induction of M cell-like features in Caco-2 cells and tested them against normal Peyer's patch tissue for their expression in FAE. Among the genes identified by this method, laminin beta3, a matrix metalloproteinase and a tetraspan family member, showed enriched expression in FAE of mouse Peyer's patches. Moreover, the C. perfringens enterotoxin receptor (CPE-R) appeared to be expressed more strongly by UEA-1(+) M cells relative to neighboring FAE. Expression of the tetraspan TM4SF3 gene and CPE-R was also confirmed in human Peyer's patch FAE. Our results suggest that while the Caco-2 differentiation model is associated with some functional features of M cells, the genes induced may instead reflect the acquisition of a more general FAE phenotype, sharing only select features with the M cell subset.

Heterogeneous expression of the triggering receptor expressed on myeloid cells-2 on adult murine microglia.

Microglial activation is an early and common feature of almost all neuropathologies, including multiple sclerosis, Alzheimer's disease and mechanical injury. To better understand the relative contributions microglia make toward neurodegeneration and neuroprotection, we used TOGA(R) to identify molecules expressed by microglia and regulated by inflammatory signals. Triggering receptor expressed on myeloid cells-2 (TREM-2) was among the mRNAs identified as being expressed by unactivated microglia, but down-regulated by lipopolysaccharide/interferon gamma. In the healthy CNS, not all microglia expressed TREM-2. Microglial expression of TREM-2 varied not only between brain regions but also within each brain region. Brain regions with an incomplete blood-brain barrier had the lowest percentages of TREM-2- expressing microglia, whereas the lateral entorhinal and cingulate cortex had the highest percentages. A novel form of TREM-2b that lacked a transmembrane domain was detected, perhaps indicating a soluble form of the protein. Taken together, these data suggest that (1) subsets of microglia are specialized to respond to defined extracellular signals; and (2) regional variations in TREM-2 expression may contribute to the varying sensitivities of different brain regions to similar pathological signals.