Thermoneutrality induces vascular dysfunction and impaired metabolic function in male Wistar rats: a new model of vascular disease.

OBJECTIVE: Cardiovascular disease is of paramount importance, yet there are few relevant rat models to investigate its pathology and explore potential therapeutics. Housing at thermoneutral temperature (30 °C) is being employed to humanize metabolic derangements in rodents. We hypothesized that housing rats in thermoneutral conditions would potentiate a high-fat diet, resulting in diabetes and dysmetabolism, and deleteriously impact vascular function, in comparison to traditional room temperature housing (22 °C). METHODS: Male Wistar rats were housed at either room temperature or thermoneutral temperatures for 16 weeks on either a low or high-fat diet. Glucose and insulin tolerance tests were conducted at the beginning and end of the study. At the study's conclusion, vasoreactivity and mitochondrial respiration of aorta and carotid were conducted. RESULTS: We observed diminished vasodilation in vessels from thermoneutral rats ( P  < 0.05), whereas high-fat diet had no effect. This effect was also observed in endothelium-denuded aorta in thermoneutral rats ( P  < 0.05). Vasoconstriction was significantly elevated in aorta of thermoneutral rats ( P  < 0.05). Diminished nitric oxide synthase activity and nitrotyrosine, and elevated glutathione activity were observed in aorta from rats housed under thermoneutral conditions, indicating a climate of lower nitric oxide and excess reactive oxygen species in aorta. Thermoneutral rat aorta also demonstrated less mitochondrial respiration with lipid substrates compared with the controls ( P  < 0.05). CONCLUSION: Our data support that thermoneutrality causes dysfunctional vasoreactivity, decreased lipid mitochondrial metabolism, and modified cellular signaling. These are critical observations as thermoneutrality is becoming prevalent for translational research models. This new model of vascular dysfunction may be useful for dissection of targetable aspects of cardiovascular disease and is a novel and necessary model of disease.

Alpha-1-antitrypsin inhibits human immunodeficiency virus type 1.

Several observations suggest the existence of potent endogenous suppressors of human immunodeficiency virus type 1 (HIV-1) production, and inhibitors of serine proteases may participate in this effect. Alpha-1-antitrypsin (AAT) is the most abundant circulating serine protease inhibitor. Physiological AAT concentrations inhibited HIV-1 production in chronically infected U1 monocytic cells, reduced virus replication in freshly infected peripheral blood mononuclear cells, and blocked infection of permissive HeLa cells. In U1 cells, AAT suppressed activation of the HIV-1-inducing transcription factor NF-kappaB. Similar results were obtained using CE-2072, a synthetic inhibitor of host serine proteases. HIV-1 did not replicate in blood obtained from healthy volunteers, but marked replication was observed in blood from individuals with hereditary AAT deficiency. These results identify AAT as a candidate circulating HIV-1 inhibitor in vivo. Two different mechanisms of AAT-induced HIV-1 inhibition were identified, including reduced HIV-1 infectivity and blockade of HIV-1 production. A novel host-pathogen interaction is suggested, and an alternative strategy to treat HIV-1-related disease may be possible.

Effect of cryopreservation on measurement of cytomegalovirus-specific cellular immune responses in HIV-infected patients.

To determine the feasibility of cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) studies using cryopreserved cells, we compared lymphocyte proliferation assays (LPA), responder cell frequency (RCF), interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production using fresh and cryopreserved peripheral blood mononuclear cells (PBMCs) from 53 HIV-infected patients and 15 uninfected controls. Qualitative CMV LPA results were concordant in >/=84% of the specimens from either HIV-infected patients or controls. Proliferation-based RCF, IL-2, and IFN-gamma comparisons showed that cryopreservation reduces the number of CMV-specific responders and decreases cytokine secretion, without changing the rank order of the results (p <.01). In contrast, the number of flow cytometry-enumerated IFN-gamma-producing CD4+ cells was not significantly changed by cryopreservation. In HIV-infected patients, the differences between fresh and frozen cell assays were not influenced by CD4 cell numbers or HIV viral load. These data indicate that cryopreserved cells are suitable for longitudinal studies of the CMV-specific immune response in HIV-infected patients and uninfected controls.

The isolation of FOS-1, a gene encoding a putative two-component histidine kinase from Aspergillus fumigatus.

In fungi, two-component histidine kinases are involved in response mechanisms to extracellular changes in osmolarity, resistance to dicarboximide fungicides, and cell-wall assembly. In the human opportunistic fungus, Candida albicans, each of the three histidine kinases plays a role in virulence. Here, we identify, for the first time, a gene, FOS-1, from the human pathogenic fungus Aspergillus fumigatus that predicts a protein with homology to two-component histidine kinases. The predicted FOS-1 protein is highly homologous to bacterial and other fungal histidine kinases in several functional domains, but is divergent at the amino- and carboxy-termini. A mutant lacking the FOS-1 locus, DeltaFOS-1, did not exhibit a detectable defect in either hyphal growth or morphology when grown on solid or liquid medium. However, in liquid medium, conidiophore development of the DeltaFOS-1 mutant was delayed. Compared to wild type, the DeltaFOS-1 strain was neither osmotically sensitive nor sensitive or resistant to a number of nondicarboximide antifungal drugs, but was highly resistant to dicarboximide fungicides and resistant to novozym 234, suggesting that FOS-1p may play a role in the regulation of cell-wall assembly.

Promoter-directed expression of recombinant fire-fly luciferase in the salivary glands of Hermes-transformed Aedes aegypti.

Molecular genetic analyses of biological properties characteristic of insect vectors of disease, such as hematophagy and competence for pathogens, require the ability to isolate and characterize genes involved in these processes. We have been working to develop molecular approaches for studying the promoter function of genes that are expressed specifically in the adult salivary glands of the yellow fever mosquito, Aedes aegypti. Genomic DNA fragments containing cis-acting promoter elements from the Maltase-like I (MalI) and Apyrase (Apy) genes were cloned so as to direct the expression of the reporter gene, luciferase (luc). The function of the promoters was assayed transiently in cultured insect cells and by germ-line transformation of Ae. aegypti. MalI and Apy DNA fragments consisting of at least 650 nucleotides (nt) of DNA immediately adjacent to the 5'-end of the initiation codon of the mosquito genes directed constitutive expression of the luc reporter gene in cultured cells. When introduced into Ae. aegypti chromosomes, approximately 1.5 kilobases (kb) of each promoter were able to direct the predicted developmental-, sex- and tissue-specific expression of the reporter gene in patterns identical to those determined for the respective endogenous genes.

Transient expression of a promoter-reporter construct in differentiated adult salivary glands and embryos of the mosquito Aedes aegypti.

Vector-borne pathogens develop in close association with specific tissues in their insect hosts. Efforts are being made to characterize insect genes that are expressed in tissues that have important roles in pathogen propagation. Successful transfection and expression of exogenous genes in terminally differentiated tissues of insects has previously proven difficult. Here we report a method that should allow the analysis of genes that are expressed in adult tissues and organs. Transient expression assays have been developed using the salivary glands of the mosquito, Aedes aegypti, which can now be used to analyze salivary gland-specific promoter sequences. A liposome-based transfection reagent was used to transfect cultured adult salivary glands with a DNA construct carrying the luciferase reporter gene under the control of the Drosophila melanogaster heat shock 70 promoter. Luciferase activity was detected in glands 18-20 hr post-transfection. This assay can now be used to determine the regulatory activity of other putative promoter sequences from salivary gland-specific genes. Alternatively, the assay may be used to study the effect of recombinant gene expression on parasite invasion and development. In addition, transient expression of gene constructs in embryos is shown to be a powerful tool for analyzing genes that are expressed at this stage of the mosquito life cycle.