Dietary Protein Deficiency and Mycobacterium Bovis BCG Affect Interleukin-2 Activity in Experimental Pulmonary Tuberculosis

Inbred strain 2 guinea pigs were vaccinated with Mycobacterium bovis BCG or were left unvaccinated. They were maintained for 6 weeks on defined, isocaloric diets containing either 30% (control animals) or 10% (animals receiving low protein) ovalbumin as the sole protein source. Animals were challenged by the respiratory route with a low dose of virulent M. tuberculosis H37Rv and killed 4 weeks later. Protein-malnourished animals were not protected by previous vaccination with BCG. Lymphocytes isolated from various tissues were tested in vitro for proliferative responses to mitogen (concanavalin A) and antigen (purified protein derivative [PPD]), production of interleukin-2 (IL-2), and response to exogenous recombinant IL-2 (rIL-2). Protein-malnourished guinea pigs responded only weakly to PPD skin tests, and their blood and lymph node lymphocytes exhibited impaired proliferation when cultured with PPD in vitro. IL-2 levels were consistently low in cultures of stimulated blood and spleen lymphocytes from protein-deprived animals. BCG vaccination of nutritionally normal guinea pigs, on the other hand, induced significantly more IL-2 production by PPD- and concanavalin A-stimulated lymphocytes. The addition of exogenous mouse rIL-2 (40 and 80 U/ml) in vitro to PPD-stimulated blood and lymph node cells from nonvaccinated, protein-deprived guinea pigs resulted in no improvement of the proliferative response. Previous vaccination of malnourished guinea pigs did not consistently enhance the response of PPD-stimulated lymphocytes to added rIL-2. Dietary protein deficiency and BCG vaccination appear to modulate antigen-driven cellular immunity in animals with tuberculosis by altering the production of, and the response to, IL-2 by PPD-stimulated lymphocytes

Localizing Transcriptional Regulatory Elements at the Mouse Dlk1 Locus

Much effort has focused recently on determining the mechanisms that control the allele-specific expression of genes subject to genomic imprinting, yet imprinting regulation is only one aspect of configuring appropriate expression of these genes. Imprinting control mechanisms must interact with those regulating the tissue-specific expression pattern of each imprinted gene in a cluster. Proper expression of the imprinted Delta-like 1 (Dlk1) - Maternally expressed gene 3 (Meg3) gene pair is required for normal fetal development in mammals, yet the mechanisms that control tissue-specific expression of these genes are unknown. We have used a combination of in vivo and in vitro expression assays to localize cis-regulatory elements that may regulate Dlk1 expression in the mouse embryo. A bacterial artificial chromosome transgene encompassing the Dlk1 gene and 77 kb of flanking sequence conferred expression in most endogenous Dlk1-expressing tissues. In combination with previous transgenic data, these experiments localize the majority of Dlk1 cis-regulatory elements to a 41 kb region upstream of the gene. Cross-species sequence conservation was used to further define potential regulatory elements, several of which functioned as enhancers in a luciferase expression assay. Two of these elements were able to drive expression of a lacZ reporter transgene in Dlk1-expressing tissues in the mouse embryo. The sequence proximal to Dlk1 therefore contains at least two discrete regions that may regulate tissue-specificity of Dlk1 expression

Cytokine gene expression by cultures of human lymphocytes with autologous Mycobacterium tuberculosis-infected monocytes.

In order to better understand the immunoregulation following Mycobacterium tuberculosis infection, cytokine mRNA induction in response to in vitro infection of human monocytes with live virulent M. tuberculosis H37Rv cocultured with autologous lymphocytes was quantitated by reverse transcriptase-PCR. Induced levels of interleukin 1 beta (IL-1 beta), IL-2, tumor necrosis factor alpha, and gamma interferon (IFN-gamma) were compared among groups of individuals representing three phases of immunity to infection with M. tuberculosis: naive normal control subjects, purified protein derivative (PPD)-reactive normal donors, and individuals with active tuberculosis (TB [diseased]). Levels of IL-1 beta and tumor necrosis factor alpha mRNA in cocultured cells from TB patients were 51 and 45%, respectively, of those obtained in cells from sensitized healthy volunteers and were comparable to those from naive normal donors. Lymphoproliferative responses to M. tuberculosis and induction of the T-cell cytokine IL-2 were predictably high in the cells of PPD-sensitized donors, low in normal naive individuals, and variable among TB patients. In contrast, the induced level of another lymphokine, IFN-gamma, did not follow the pattern seen in IL-2 induction. Infection with live M. tuberculosis induced high levels of IFN-gamma mRNA in lymphocytes of both PPD-sensitized and normal naive donors compared with those of TB patients. Interestingly, polyclonal stimulation with the mitogen concanavalin A induced similar IFN-gamma levels in cells from all three donor groups. The high level of IFN-gamma induced by the infection of monocytes from naive normal donors suggests a role for natural killer (NK) cells in the production of IFN-gamma in this coculture system. This response appears independent of the role performed by T cells

Dietary vitamin D affects cell-mediated hypersensitivity but not resistance to experimental pulmonary tuberculosis in guinea pigs.

Outbred, Hartley strain guinea pigs were fed purified diets varying only in their levels of vitamin D. The amounts of vitamin D in the diets were adjusted to represent 0, 25, 50, 100, or 200% of the recommended level (1,180 IU/kg of body weight) for guinea pigs. In some experiments, half of the animals in each diet group were vaccinated with Mycobacterium bovis BCG vaccine at the time the diets were introduced. Six weeks later, all guinea pigs were infected by the respiratory route with a low dose of virulent M. tuberculosis H37Rv. Vitamin D-deficient animals exhibited marked reductions in levels of the major vitamin D metabolite, 25-hydroxyvitamin D3, in plasma. Altered vitamin D intake was accompanied by changes in antigen (purified protein derivative)-induced, cell-mediated immune responses both in vivo (tuberculin hypersensitivity) and in vitro (lymphoproliferation). Dermal tuberculin reactivity developed more slowly in vitamin D-deficient guinea pigs but eventually achieved normal levels. The proliferation of splenocytes cultured with purified protein derivative was suppressed by both deficiency and excess of dietary vitamin D. Vitamin D status did not affect the abilities of naive guinea pigs to control primary, pulmonary tuberculosis, nor did it influence the protective efficacy of BCG vaccination. We conclude that changes in dietary vitamin D are associated with alterations in some cellular immune functions but may not be an important determinant of disease outcome in pulmonary tuberculosis, as has been suggested previously

Clinical and Pathologic Changes in a Guinea Pig Aerosol Challenge Model of Acute Q Fever

Acute Q fever is a zoonotic disease caused by the obligate intracellular bacterium Coxiella burnetii and can manifest as a flu-like illness, pneumonia, or hepatitis. A need exists in Q fever research for animal models mimicking both the typical route of infection (inhalation) and the clinical illness seen in human cases of Q fever. A guinea pig aerosol challenge model was developed using C. burnetii Nine Mile phase I (RSA 493), administered using a specialized chamber designed to deliver droplet nuclei directly to the alveolar spaces. Guinea pigs were given 10(1) to 10(6) organisms and evaluated for 28 days postinfection. Clinical signs included fever, weight loss, respiratory difficulty, and death, with the degree and duration of response corresponding to the dose of organism delivered. Histopathologic evaluation of the lungs of animals infected with a high dose showed coalescing panleukocytic bronchointerstitial pneumonia at 7 days postinfection that resolved to multifocal lymphohistiocytic interstitial pneumonia by 28 days. Guinea pigs receiving a killed whole-cell vaccine prior to challenge with the highest dose of C. burnetii were protected against lethal infection and did not develop fever. Clinical signs and pathological changes noted for these guinea pigs were comparable to those seen in human acute Q fever, making this an accurate and valuable animal model of human disease

Predictors of Development of Diabetes in Patients With Chronic Heart Failure in the Candesartan in Heart Failure Assessment of Reduction in Mortality and Morbidity (CHARM) Program

OBJECTIVE: The purpose of this study was to identify predictors of incident diabetes during follow-up of nondiabetic patients with chronic heart failure (CHF) in the Candesartan in Heart Failure Assessment of Reduction in Mortality and Morbidity (CHARM) program.<p></p> RESEARCH DESIGN AND METHODS: A total of 1,620 nondiabetic patients had full baseline datasets. We compared baseline demographic, medication, and laboratory data for patients who did or did not develop diabetes and conducted logistic regression and receiver operator characteristic curve analyses.<p></p> RESULTS: Over a median period of 2.8 years, 126 of the 1,620 patients (7.8%) developed diabetes. In multiple logistic regression analysis, the following baseline characteristics were independently associated with incident diabetes in decreasing order of significance by stepwise selection: higher A1C (odds ratio [OR] 1.78 per 1 SD increase; P &#60; 0.0001), higher BMI (OR 1.64 per 1 SD increase; P &#60; 0.0001), lipid-lowering therapy (OR 2.05; P = 0.0005), lower serum creatinine concentration (OR 0.68 per 1 SD increase; P = 0.0018), diuretic therapy (OR 4.81; P = 0.003), digoxin therapy (OR 1.65; P = 0.022), higher serum alanine aminotransferase concentration (OR 1.15 per 1 SD increase; P = 0.027), and lower age (OR 0.81 per 1 SD increase; P = 0.048). Using receiver operating characteristic curve analysis, A1C and BMI yielded areas under the curve of 0.723 and 0.712, respectively, increasing to 0.788 when combined. Addition of other variables independently associated with diabetes risk minimally improved prediction of diabetes.<p></p> CONCLUSIONS: In nondiabetic patients with CHF in CHARM, A1C and BMI were the strongest predictors of the development of diabetes. Other minor predictors in part reflected CHF severity or drug-associated diabetes risk. Identifying patients with CHF at risk of diabetes through simple criteria appears possible and could enable targeted preventative measures