An ovine hepatorenal fibrocystic model of a Meckel-like syndrome associated with dysmorphic primary cilia and TMEM67 mutations.

Meckel syndrome (MKS) is an inherited autosomal recessive hepatorenal fibrocystic syndrome, caused by mutations in TMEM67, characterized by occipital encephalocoele, renal cysts, hepatic fibrosis, and polydactyly. Here we describe an ovine model of MKS, with kidney and liver abnormalities, without polydactyly or occipital encephalocoele. Homozygous missense p.(Ile681Asn; Ile687Ser) mutations identified in ovine TMEM67 were pathogenic in zebrafish phenotype rescue assays. Meckelin protein was expressed in affected and unaffected kidney epithelial cells by immunoblotting, and in primary cilia of lamb kidney cyst epithelial cells by immunofluorescence. In contrast to primary cilia of relatively consistent length and morphology in unaffected kidney cells, those of affected cyst-lining cells displayed a range of short and extremely long cilia, as well as abnormal morphologies, such as bulbous regions along the axoneme. Putative cilia fragments were also consistently located within the cyst luminal contents. The abnormal ciliary phenotype was further confirmed in cultured interstitial fibroblasts from affected kidneys. These primary cilia dysmorphologies and length control defects were significantly greater in affected cells compared to unaffected controls. In conclusion, we describe abnormalities involving primary cilia length and morphology in the first reported example of a large animal model of MKS, in which we have identified TMEM67 mutations.

Health care costs attributable to overweight calculated in a standardized way for three European countries.

This article presents a tool to calculate health care costs attributable to overweight in a comparable and standardized way. The purpose is to describe the methodological principles of the tool and to put it into use by calculating and comparing the costs attributable to overweight for The Netherlands, Germany and Czech Republic. The tool uses a top-down and prevalence-based approach, consisting of five steps. Step one identifies overweight-related diseases and age- and gender-specific relative risks. Included diseases are ischemic heart disease, stroke, hypertension, type 2 diabetes mellitus, colorectal cancer, postmenopausal breast cancer, endometrial cancer, kidney cancer and osteoarthritis. Step two consists of collecting data on the age- and gender-specific prevalence of these diseases. Step three uses the population-attributable prevalence to determine the part of the prevalence of these diseases that is attributable to overweight. Step four calculates the health care costs associated with these diseases. Step five calculates the costs of these diseases that are attributable to overweight. Overweight is responsible for 20-26% of the direct costs of included diseases, with sensitivity analyses varying this percentage between 15-31%. Percentage of costs attributable to obesity and preobesity is about the same. Diseases with the highest percentage of costs due to overweight are diabetes, endometrial cancer and osteoarthritis. Disease costs attributable to overweight as a percentage of total health care expenditures range from 2 to 4%. Data are consistent for all three countries, resulting in roughly a quarter of costs of included diseases being attributable to overweight.

Bystander help within a polyepitope DNA vaccine improves immune responses to influenza antigens.

A polyepitope DNA vaccine has the potential to generate protective immune responses to a range of antigens in a single construct. We investigated whether it was possible to obtain responses to individual epitopes from different antigens, directly linked in a string, and whether the response to a given epitope was enhanced by adjacent epitopes within the construct. A polyepitope plasmid was created, which included three Th epitopes (influenza haemagglutinin, moth cytochrome c and ovalbumin), a Tc epitope (ovalbumin) and two B cell epitopes (haemagglutinin and ovalbumin). Mice were immunized with DNA by using a gene gun. Responses to the polyepitope DNA vaccine were compared with those to DNA vaccine comprising only the haemagglutinin Th and B epitopes (HAT(h)B) or with responses to the recombinant protein. These experiments showed that the polyepitope DNA vaccine induced greater antigen-specific responses to HAT(h)B peptide than the HAT(h)B DNA vaccine. Antigen-specific in vivo cytotoxic responses following polyepitope DNA vaccination were also clearly demonstrable. We conclude that a 'naked DNA' polyepitope vaccine generates specific responses to constituent epitopes and that adjacent irrelevant epitopes may enhance these responses.

IL-2 linked to a peptide from influenza hemagglutinin enhances T cell activation by affecting the antigen-presentation function of bone marrow-derived dendritic cells.

Chimeric proteins containing antigen linked to cytokines have shown some promise as vaccine candidates but little is known of their mechanism of action, particularly at the level of the antigen-presenting cell. We have investigated this using a chimeric protein in which an immunodominant T cell epitope from influenza hemagglutinin peptide (HA), recognized in the context of I-E(d), was fused to IL-2. Immature murine dendritic cells (DC) derived from bone marrow (BMDC) were used to present the chimeric protein to a T cell hybridoma with TCR specific for the HA peptide (A5 cell line). HA-IL-2 was found to induce significantly higher T cell activation than HA alone. Although the inclusion of IL-2 and HA separately did increase the response of A5 cells compared to HA alone, they were not as effective as the HA-IL-2 chimeric protein. When an antibody known to block IL-2 receptor alpha chain (CD25) was included, A5 activation was reduced, suggesting a role for the receptor in this process. Expression of CD25 on A5 cells was low during activation, implying that the effect was mediated by CD25(+) BMDC. Antigen uptake and processing of HA-IL-2 by BMDC was required since fixing BMDC, prior to antigen exposure, greatly reduced their ability to activate A5 cells. The function of CD25 on DC is currently unknown. Our results suggest this receptor may play a role in antigen uptake and subsequent T cell activation by receptor-mediated endocytosis of antigen attached to IL-2. This finding that may have implications for the development of a new generation of vaccines.

2,3,7,8-Tetrachlorodibenzo-p-dioxin and diindolylmethanes differentially induce cytochrome P450 1A1, 1B1, and 19 in H295R human adrenocortical carcinoma cells.

Diindolylmethane (DIM) is an acid-catalyzed condensation product of indole-3-carbinol, a constituent of cruciferous vegetables, and is formed in the stomach. DIM alters estrogen metabolism and inhibits carcinogen-induced mammary tumor growth in rodents. DIM is a weak agonist for the aryl hydrocarbon (Ah) receptor and blocks the effects of estrogens via inhibitory Ah receptor-estrogen receptor cross-talk. DIM and various structural analogs were examined in H295R cells for effects on 3 cytochrome P450 (CYP) enzymes involved in estrogen synthesis and/or metabolism: CYP1A1, CYP1B1, and CYP19 (aromatase). Aromatase activity was measured by conversion of 1 beta-(3)H-androstenedione to estrone and (3)H(2)O. H295R cells were exposed to the test chemicals dissolved in dimethyl sulfoxide for 24 h prior to analyses. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) (0--30 nM) and DIM (0--10 microM) induced ethoxyresorufin-O-deethylase (EROD) activity, as a measure of CYP1A1 and possibly 1B1 activity, with EC(50) values of about 0.3 nM and 3 microM, respectively. DIM, but not TCDD, induced aromatase activity with an apparently maximal 2-fold increase at 10 microM; higher concentrations of DIM and many of its analogs were cytotoxic. TCDD (30 nM) significantly increased CYP1A1 and 1B1 mRNA levels, but had no effect on mRNA for CYP19. DIM (3 microM) significantly increased mRNA levels for all three CYPS: DIM analogs with substitutions on the 5 and 5' position (3 microM) induced aromatase and EROD activity, together with mRNA levels of CYP1A1, 1B1, and 19; analogs that were substituted on the central carbon of the methane group showed little or no inductive activity toward the CYPS: In conclusion, DIM and several of its analogs appear to induce CYPs via multiple yet distinct pathways in H295R human adrenocortical carcinoma cells.

Targeting early events in T cell activation to construct improved vaccines.

Live, attenuated vaccines currently offer the best protection against virulent pathogens. Recent advances in Immunology and Molecular Biology provide an opportunity to design vaccines that will be more effective and safer than existing ones. Immunologists are rapidly developing the capacity to identify and construct the minimal immunogenic units from pathogens. The molecular signals required to fully activate antigen presenting cells (APCs) and responder T cells are becoming apparent. Improved vaccine delivery systems are being designed which will mimic the actions of pathogens in vivo. These vaccines will incorporate protective epitopes fused to immunoregulatory cytokines in chimeric proteins. They will be encapsulated in formulations which allow for the slow release of these chimeric proteins thereby inducing the memory T cells required for long-lived immunity. These vaccine formulations will target receptors present on the most active APCs. Here we discuss how these advances will allow us to rationally construct "virtual pathogens" which will provide improved protection against new and old microbial foes.

In vitro responses of cervine macrophages to bacterial stimulants.

The function of cervine (deer) mononuclear phagocytes is poorly defined. In the present study, the potential of cervine macrophages to generate phagocytic and immunoregulatory responses following stimulation with bacterial products was investigated. Blood-derived macrophages of red deer were cultured in vitro with particulate stimulants (Mycobacterium bovis BCG and Staphylococcus aureus SAC) or soluble stimulants (M. bovis PPD and Escherichia coli LPS), prior to assessment of phagocytic responses, prostaglandin secretion and cytokine production. Particulate stimulants induced vigorous phagocytic responses (superoxide anion generation, lysosomal enzyme release), secretion of prostaglandin E2 and transcription of mRNA specific for the cytokines IL-1 beta, IL-10 and TNF alpha, while soluble products invoked weaker responses. These results are discussed in relation to the role of cervine mononuclear phagocytes in regulating and participating in inflammatory and immune processes relevant to bacterial challenge.

Macrophage function in deer.

Macrophage inflammatory and immune functions were characterised in red deer (cervus elaphus), for use as a model for natural infection with bovine tuberculosis. Highly enriched populations of deer macrophages were obtained from 14 day cultures of plastic-adherent peripheral blood mononuclear cells. Cervine macrophages produced superoxide anion in response to respiratory burst stimuli (serum-opsonised zymosan and phorbol myristic acetate), but nitric oxide production could not be detected under the conditions tested. The lysosomal enzymes acid phosphatase and lysozyme were detected at the intercellular and extracellular level. Stimulation with bacterial lipopolysaccharide extract (Escherichia coli LPS) enhanced the production of superoxide and acid phosphatase with a peak increase in activity observed after 2h. Production of interleukin 1 (IL-1) and tumour necrosis factor (TNF), determined using cytokine-sensitive cell lines and mRNA analysis (Northern blotting), indicated maximal secretion of both cytokines after 24 h stimulation with LPS, preceded by a peak in message accumulation at 2-6 h post-stimulation. Cervine macrophages stimulated proliferative responses in T cell-enriched lymphocyte populations derived from the peripheral blood of autologous animals that had been primed to mycobacterial antigens (Mycobacterium bovis Bacille Calmette-Guerin, BCG). Macrophages were able to stimulate responses after pulsing with particulate (BCG) or soluble (purified protein derivative) mycobacterial antigens. These results indicate that macrophage inflammatory and immune responses in red deer are similar to those in other mammalian species, and that macrophages may play an important role in resistance to mycobacterial infection.