The enzyme GK (glucokinase), which phosphorylates glucose to form glucose 6-phosphate, serves as the glucose sensor of insulin-producing beta-cells. GK has thermodynamic, kinetic, regulatory and molecular genetic characteristics that are ideal for its glucose sensor function and allow it to control glycolytic flux of the beta-cells as indicated by control-, elasticity- and response-coefficients close to or larger than 1.0. GK operates in tandem with the K(+) and Ca(2+) channels of the beta-cell membrane, resulting in a threshold for glucose-stimulated insulin release of approx. 5 mM, which is the set point of glucose homoeostasis for most laboratory animals and humans. Point mutations of GK cause 'glucokinase disease' in humans, which includes hypo- and hyper-glycaemia syndromes resulting from activating or inactivating mutations respectively. GK is allosterically activated by pharmacological agents (called GK activators), which lower blood glucose in normal animals and animal models of T2DM. On the basis of crystallographic studies that identified a ligand-free 'super-open' and a liganded closed structure of GK, on thermostability studies using glucose or mannoheptulose as ligands and studies showing that mannoheptulose alone or combined with GK activators induces expression of GK in pancreatic islets and partially preserves insulin secretory competency, a new hypothesis was developed that GK may function as a metabolic switch per se without involvement of enhanced glucose metabolism. Current research has the goal to find molecular targets of this putative 'GK-switch'. The case of GK research illustrates how basic science may culminate in therapeutic advances of human medicine.

Most HIV infections result from heterosexual transmission to women. Because cellular immunity plays a key role in the control of the infection, we sought to strengthen cellular immune responses in vaginal tissue. We explored a novel prime-boost protocol that used two live mucosal agents that trigger different pathways of innate immunity and induce strong cellular immunity. Adenovirus serotype 5 (Ad5) has frequently been used as a boost for DNA vaccines. In this study we used attenuated, recombinant L. monocytogenes-gag (rLm-gag) to prime mice by various mucosal routes—oral, intrarectal, and intravaginally (ivag)—followed by a systemic or mucosal boost with replication-defective rAd5-gag. Mice primed with a single administration of rLm-gag by any route and then boosted with rAd5-gag intramuscularly exhibited abundant Gag-specific CD8 T cells in spleen and vaginal lamina propria. Conversely, when boosted with rAd5-gag ivag, the immune response was reoriented toward the vagina with strikingly higher CD8 T cell responses in that tissue, particularly after ivag immunization by both vectors (ivag/ivag). Five weeks to 5 mo later, ivag/ivag-immunized mice continued to show high levels of effector memory CD8 T cells in vagina, while the pool of memory T cells in spleen assumed a progressively more central memory T cell phenotype. The memory mice showed high in vivo CTL activity in vagina, a strong recall response, and robust protection after ivag vaccinia-gag challenge, suggesting that this prime-boost strategy can induce strong cellular immunity, especially in vaginal tissues, and might be able to block the heterosexual transmission of HIV-1 at the vaginal mucosa.

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Vaccinology is the most cost effective and efficacious of medical interventions for the eradication or management of many infectious diseases. The generation of a cellular immune response is key to survival against a variety of viral and intracellular bacterial pathogens and cancer. Therefore, the development of safe vaccines capable of inducing strong cellular immunity continues to be a pressing challenge for medicine. We have explored the use of an attenuated strain of Listeria monocytogenes as a novel live vaccine vector for this purpose. L. monocytogenes is a gram-positive intracellular bacterial pathogen whose genome has been fully sequenced (22), greatly facilitating detailed studies of its biochemistry, genetics, and physiology and contributing to the existing wealth of knowledge of its adjuvant properties and immunogenicity.Since the early work of Mackaness in the 1960s (30), L. monocytogenes has been studied extensively as a paradigm for understanding innate and cell-mediated immunity. The organism can enter phagocytic cells through Fc receptors or type I macrophage scavenger receptors (12, 48) and can invade nonphagocytic cells by the interaction of the bacterial surface proteins InlA (16) and InlB (18) with their cognate host cell receptors. Following internalization by phagocytic cells, the majority of engulfed organisms are degraded in the phagosome (11). However, listeriolysin O (LLO), a pore-forming cytolysin, and PI-PLC, a phosphatidylinositol-specific phospholipase C, mediate permeabilization of the phagosomal membrane, enabling a fraction of the organisms to enter the host cell cytosol (9,31,38,45). Subsequently, proteins secreted by the organism can be delivered directly into the major histocompatibility complex (MHC) class I pathway of antigen processing and presentation (2, 5). Mutants of L. monocytogenes unable to enter the cytosol are avirulent. These bacteria fail to stimulate protective immunity in mice, and cells infected by such mutants are not recognized by L. monocytogenes-immune CD8 ϩ T cells (7, 34). Conversely, mice infected with sublethal doses of wildtype bacteria develop long-lasting immunity, mediated predominantly by CD8 ϩ T cells (13,29). This unique property of L. monocytogenes makes it attractive as a potential live vaccine vector, and recombinant strains expressing foreign antigens have successfully protected mice against lymphocytic choriomeningitis (23, 44), papillomavirus (26) and influenza virus (25) infections, and tumor challenge (24,36,37).Because of the potential use of this organism as a vaccine vector for protection against infectious diseases and cancer, the safety of L. monocytogenes is a critical issue. While infections by L. monocytogenes are fairly rare and can readily be controlled by a number of antibiotics, the organism can cause meningitis and death, particularly in immunocompromised or pregnant patients. A vaccine strain of L. monocytogenes should therefore be avirulent but immunogenic. We previously sought to generate an attenuated strain that could e...

Exposure to the endocrine disruptor bisphenol A (BPA) is ubiquitous and associated with health abnormalities that persist in subsequent generations. However, transgenerational effects of BPA on metabolic health are not widely studied. In a maternal C57BL/6J mice (F0) exposure model using BPA doses that are relevant to human exposure levels (10 μg/kg/day, LowerB; 10 mg/kg/day, UpperB), we showed male- and dose-specific effects on pancreatic islets of the first (F1) and second generation (F2) offspring relative to controls (7% corn oil diet; Control). In this study, we determined the transgenerational effects (F3) of BPA on metabolic health and pancreatic islets in our model. Adult F3 LowerB and UpperB male offspring had increased body weight relative to Controls, however glucose tolerance was similar in the three groups. F3 LowerB, but not UpperB, males had reduced ß-cell mass and smaller islets which was associated with increased glucose stimulated insulin secretion. Similar to F1 and F2 BPA male offspring, staining for markers of T-cells and macrophages (CD3 and F4/80) was increased in pancreas of F3 LowerB and UpperB male offspring, which was associated with changes in cytokine levels. In contrast to F3 BPA males, LowerB and UpperB female offspring had comparable body weight, glucose tolerance and insulin secretion as Controls. Thus, maternal BPA exposure resulted in fewer metabolic defects in F3 than F1 and F2 offspring, and these were sex- and dose-specific.

Listeria monocytogenes is a bacterial pathogen that elicits a strong cellular immune response and thus has potential use as a vaccine vector. An attenuated strain, L. monocytogenes dal dat, produced by deletion of two genes (dal and dat) used for D-alanine synthesis, induces cytotoxic T lymphocytes and protective immunity in mice following infection in the presence of D-alanine. In order to obviate the dependence of L. monocytogenes dal dat on supplemental D-alanine yet retain its attenuation and immunogenicity, we explored mechanisms to allow transient endogenous synthesis of the amino acid. Here, we report on a derivative strain, L. monocytogenes dal dat/pRRR, that expresses a dal gene and synthesizes D-alanine under highly selective conditions. We constructed the suicide plasmid pRRR carrying a dal gene surrounded by two res1 sites and a resolvase gene, tnpR, which acts at the res1 sites. The resolvase gene is regulated by a promoter activated upon exposure to host cell cytosol. L. monocytogenes dal dat/pRRR was thus able to grow in liquid culture and to infect host cells without D-alanine supplementation. However, after infection of these cells, resolvase-mediated excision of the dal gene resulted in strong down-regulation of racemase expression. As a result, this system allowed only transient growth of L. monocytogenes dal dat/pRRR in infected cells and survival in animals for only 2 to 3 days. Nevertheless, mice immunized with L. monocytogenes dal dat/pRRR generated listeriolysin O-specific effector and memory CD8؉ T cells and were protected against lethal challenge by wild-type Listeria. This vector may be an attractive vaccine candidate for the induction of protective cellular immune responses.Listeria monocytogenes is a gram-positive, facultative intracellular, food-borne bacterium that elicits strong cell-mediated immunity (16,23). It has shown promise as a live vaccine vector against model cancers (6,20,34,36,38), Mycobacterium tuberculosis (31), influenza virus (22), and lymphocytic choriomeningitis virus (19,48) and has been suggested for use as an AIDS vaccine vector (17, 28). However, L. monocytogenes is itself a pathogen that can cause fatal infections, particularly in immune-compromised or pregnant individuals (14,21,59). Therefore, the safety of L. monocytogenes as a vaccine vector is a critical issue that could block the potential utility of this organism.An ideal vaccine strain of any living organism must be highly attenuated but fully immunogenic. To achieve this, various mutations in virulence-associated determinants of L. monocytogenes have been exploited for potential use as vaccine vectors (1,2,6,8,19,24,29,30,55,57), but many tend to have the limitation of either weak immunogenicity or insufficient avirulence. We developed a highly attenuated strain, L. monocytogenes dal dat, by inactivating two genes (dal and dat) essential for D-alanine biosynthesis (58). This strain requires the unusual amino acid D-alanine, not synthesized by vertebrates, for viability and infection. It showed no re...

Natural transmission of human immunodeficiency virus (HIV) occurs at mucosal surfaces. During acute infection, intestinal and other mucosae are preferential sites of virus replication and rapidly become depleted of CD4؉ T cells. Therefore, mucosal immunity may be critical to control both initial infection and the massive early spread of virus. An attenuated D-alanine-requiring strain of the oral intracellular microorganism Listeria monocytogenes expressing HIV type 1 gag was shown to induce protective cell-mediated immunity in mice against viruses that express HIV gag when immunization occurs in the presence of a transient supply of D-alanine. In this study, we examined the efficacy of new attenuated strains that are able to synthesize D-alanine from a heterologous dal gene tightly regulated by an actA-promoted resolvase recombination system. In the absence of D-alanine, Gag-specific cytotoxic T lymphocytes (CTLs) were induced systemically after intravenous immunization, and one strain, Lmdd-gag/pARS, induced strong dose-dependent Gag-specific CTLs after oral immunization. A significant level of Gag-specific CD8 ؉ T cells was induced in the mucosal-associated lymphoid tissues (MALTs). Upon intravaginal challenge of these orally immunized mice with recombinant vaccinia virus (rVV) expressing HIV gag, gamma interferon-and tumor necrosis factor alpha-secreting Gag-specific CD8 ؉ T cells were dramatically increased in the spleen and MALTs. Oral immunization with Lmdd-gag/pARS led to complete protection against vaginal challenge by a homologous clade B gag-expressing rVV. In addition, strong cross-clade protection was seen against clades A and C and partial protection against clade G gag-expressing rVV. These results suggest that Lmdd-gag/pARS may be considered as a novel vaccine candidate for use against HIV/AIDS.

Listeria monocytogenes (Lm) is a Gram-positive intracellular pathogen that can elicit strong cellular immunity. An attenuated strain (Lmdd) with deletions in two genes (dal and dat) required for D-alanine synthesis and viability has been shown to induce long-lived protective systemic and mucosal immune responses in mice when administered in the presence of the required amino acid. To bypass the necessity for exogenous D-alanine without compromising the safety of the original strain, the defect of Lmdd was complemented with a heterologous Bacillus subtilis dal gene, and the effects of truncating the upstream region of the gene on its transcription efficiency and of modifying its protein product with an ssrA tag at the 39-terminus were examined. The strains with 551 bp and 80 bp upstream regions showed high levels of transcription and grew without D-alanine. The strains with the shortest upstream regions, 48 bp and 18 bp, showed greatly decreased levels of transcription and failed to grow in the absence of D-alanine. Addition of an ssrA tag to the longer genes resulted in a somewhat altered growth pattern in media and a reduced plaque size on L2 fibroblasts. These bacteria contained low levels of racemase protein and reduced free pools of D-alanine. One of the strains tested further, Lmdd/pA80S, was rapidly cleared from the spleens of infected mice but nevertheless induced a strong immune response that protected mice against challenge by wild-type L. monocytogenes. These bacteria can thus induce immune responses in mice comparable to the original Lmdd strain, but without the need for exogenous D-alanine, and may have use as a live vaccine vector against infectious diseases and cancers.