Race-specific association of an IRGM risk allele with cytokine expression in human subjects.

Immunity-related GTPase family M (IRGM), located on human chromosome 5q33.1, encodes a protein that promotes autophagy and suppresses the innate immune response. The minor allele of rs13361189 (-4299T>C), a single nucleotide polymorphism in the IRGM promoter, has been associated with several diseases, including Crohn's disease and tuberculosis. Although patterns of linkage disequilibrium and minor allele frequency for this polymorphism differ dramatically between subjects of European and African descent, studies of rs13361189 have predominantly been conducted in Europeans and the mechanism of association is poorly understood. We recruited a cohort of 68 individuals (30 White, 34 African American, 4 other race) with varying rs13361189 genotypes and assessed a panel of immune response measures including whole blood cytokine induction following ex vivo stimulation with Toll-like Receptor ligands. Minor allele carriers were found to have increased serum immunoglobulin M, C-reactive protein, and circulating CD8+ T cells. No differences in whole blood cytokines were observed between minor allele carriers and non-carriers in the overall study population; however, minor allele status was associated with increased induction of a subset of cytokines among African American subjects, and decreased induction among White subjects. These findings underline the importance of broad racial inclusion in genetic studies of immunity.

Glucocorticoid Receptor Mutations and Hypersensitivity to Endogenous and Exogenous Glucocorticoids.

Background: The glucocorticoid receptor (GR) consists of two alternatively spliced isoforms: GRα, which activates gene transcription, and GRß, a dominant-negative receptor. Theoretically, inactivating variants of GRß could result in glucocorticoid hypersensitivity. Design: A 46-year-old woman presented for evaluation of adrenal insufficiency prompted by low plasma cortisol levels and multiple unexplained symptoms but without clinical evidence of glucocorticoid insufficiency. To explain these findings, extensive clinical, genetic, and molecular studies were performed. Methods: Standard clinical methods assessed the patient's hypothalamic-pituitary-adrenal axis. Validated molecular techniques were used for receptor sequencing, stable transfections, stimulation of candidate genes, cDNA arrays, Ingenuity Pathway Analysis, volcano analysis, and isolation and analysis of the patient's mononuclear cells. Results: Clinical studies excluded primary or secondary adrenal insufficiency, established consistently low basal cortisol levels, and demonstrated hypersensitivity to ultra-low-dose dexamethasone. Receptor sequencing identified two variants of GR9ß (A3669G and G3134T) as well as the known Bcl1 polymorphism. Reductionist studies using stable osteosarcoma cell lines transfected with the GRß variants demonstrated glucocorticoid hypersensitivity of transcribed genes on cDNA array analysis. The patient's monocytes responded to hydrocortisone with exaggerated stimulation of the candidate genes GILZ and FKBP5. Conclusion: Two variants of the dominant-negative GRß, in conjunction with a common Bcl1 intron variant, resulted in hypersensitivity to endogenous and exogenous glucocorticoids and, as a reflection of severity, low circulating cortisol levels without clinical evidence of glucocorticoid insufficiency. This prismatic case exemplifies the unique effects of variants of a dominant-negative receptor.

Generating diversity in human glucocorticoid signaling through a racially diverse polymorphism in the beta isoform of the glucocorticoid receptor.

Alternative splicing of the human glucocorticoid receptor gene generates two isoforms, hGRα and hGRß. hGRß functions as a dominant-negative regulator of hGRα activity and but also has inherent transcriptional activity, collectively altering glucocorticoid sensitivity. Single-nucleotide polymorphisms in the 3' UTR of hGRß have been associated with altered receptor protein expression, glucocorticoid sensitivity, and disease risk. Characterization of the hGRß G3134T polymorphism has been limited to a relatively small, homogenous population. The objective of this study was to determine the prevalence of hGRß G3134T in a diverse population and assess the association of hGRß G3134T in this population with physiological outcomes. In a prospective cohort study, 3730 genetically diverse participants were genotyped for hGRß G3134T and four common GR polymorphisms. A subset of these participants was evaluated for clinical and biochemical measurements. Immortalized human osteosarcoma cells (U-2 OS), stably transfected with wild-type or G3134T hGRß, were evaluated for receptor expression, stability, and genome-wide gene expression. Glucocorticoid-mediated gene expression profiles were investigated in primary macrophages isolated from participants. In a racially diverse population, the minor allele frequency was 74% (50.7% heterozygous carriers and 23.3% homozygous minor allele), with a higher prevalence in Caucasian non-Hispanic participants. After adjusting for confounding variable, carriers of hGRß G3134T were more likely to self-report allergies, have higher serum cortisol levels, and reduced cortisol suppression in response to low-dose dexamethasone. The presence of hGRß G3134T in U-2 OS cells increased hGR mRNA stability and protein expression. Microarray analysis revealed that the presence of the hGRß G3134T polymorphism uniquely altered gene expression profiles in U-2 OS cells and primary macrophages. hGRß G3134T is significantly present in the study population and associated with race, self-reported disease, and serum levels of glucocorticoids. Underlying these health differences may be changes in gene expression driven by altered receptor stability.

Healthy glucocorticoid receptor N363S carriers dysregulate gene expression associated with metabolic syndrome.

The glucocorticoid receptor single-nucleotide polymorphism (SNP) N363S has been reported to be associated with metabolic syndrome, type 2 diabetes, and cardiovascular disease. Our aim was to determine how the N363S SNP modifies glucocorticoid receptor signaling in a healthy population of individuals prior to the onset of disease. We examined the function of the N363S SNP in a cohort of subjects from the general population of North Carolina. Eighteen N363S heterozygous carriers and 36 noncarrier, control subjects were examined for clinical and biochemical parameters followed by a low-dose dexamethasone suppression test to evaluate glucocorticoid responsiveness. Serum insulin measurements revealed that N363S carriers have higher levels of insulin, although not statistically significant, compared with controls. Glucocorticoid receptor protein levels evaluated in peripheral blood mononuclear cells from each clinical subject showed no difference between N363S and control. However, investigation of gene expression profiles in macrophages isolated from controls and N363S carriers using microarray, quantitative RT-PCR, and NanoString analyses revealed that the N363S SNP had an altered profile compared with control. These changes in gene expression occurred in both the absence and the presence of glucocorticoids. Thus, our observed difference in gene regulation between normal N363S SNP carriers and noncarrier controls may underlie the emergence of metabolic syndrome, type 2 diabetes, and cardiovascular disease associated with the N363S polymorphism.

Randomized, double-blind, Phase 1 trial of an alphavirus replicon vaccine for cytomegalovirus in CMV seronegative adult volunteers.

Development of a cytomegalovirus (CMV) vaccine is a priority. We evaluated a two component alphavirus replicon particle vaccine expressing CMV gB or a pp65/IE1 fusion protein, previously shown to induce robust antibody and cellular immune responses in mice, in a randomized, double-blind Phase 1 clinical trial in CMV seronegative subjects. Forty subjects received a lower dose (LD) or higher dose (HD) of vaccine or placebo by intramuscular or subcutaneous injection at Weeks 0, 8 and 24. The vaccine was well tolerated, with mild to moderate local reactogenicity, minimal systemic reactogenicity, and no clinically important changes in laboratory parameters. All vaccine recipients developed ex vivo, direct IFN-gamma ELISPOT responses to CMV antigens (maximal mean spot-forming cells per 10(6) PBMC in LD and HD groups of 348 and 504 for pp65, 83 and 113 for IE1, and 138 and 114 for gB), and neutralizing antibodies (maximal geometric mean titer 110 with LD and 218 with HD). Polyfunctional CD4(+) and CD8(+) T cell responses were detected by polychromatic flow cytometry. This alphavirus replicon particle vaccine was safe and induced neutralizing antibody and multifunctional T cell responses against three CMV antigens that are important targets for protective immunity.

Novel lavendamycin analogues as potent HIV-reverse transcriptase inhibitors: synthesis and evaluation of anti-reverse transcriptase activity of amide and ester analogues of lavendamycin.

Novel lavendamycins including two water soluble derivatives were synthesized via short and efficient methods. Pictet-Spengler condensation of 7-N-acylamino-2-formylquinoline-5,8-diones with tryptophans produced lavendamycin esters or amides 11-17. Lavendamycins 18-21 were obtained, respectively, by further transformations of 13-15 and 17. Several lavendamycins were found to be potent HIV reverse transcriptase inhibitors with very low toxicity in vitro and in vivo. Several compounds also acted either additively or synergistically to inhibit enzyme activity together with AZT-triphosphate.