A modular approach to assembly of totally synthetic self-adjuvanting lipopeptide-based vaccines allows conformational epitope building

The technology described here allows the chemical synthesis of vaccines requiring correctly folded epitopes and that contain difficult or long peptide sequences. The final self-adjuvanting product promotes strong humoral and/or cell-mediated immunity. A module containing common components of the vaccine (T helper cell epitope and the adjuvanting lipid moiety S-[2,3-bis(palmitoyloxy)propyl]cysteine) was assembled to enable a plug and play approach to vaccine assembly. The inclusion within the module of a chemical group with chemical properties complementary and orthogonal to a chemical group present in the target epitope allowed chemoselective ligation of the two vaccine components. The heat-stable enterotoxin of enterotoxigenic Escherichia coli that requires strict conformational integrity for biological activity and the reproductive hormone luteinizing hormone-releasing hormone were used as the target epitopes for the antibody vaccines. An epitope from the acid polymerase of influenza virus was used to assemble a CD8(+) T cell vaccine. Evaluation of each vaccine candidate in animals demonstrated the feasibility of the approach and that the type of immune response required, viz. antibody or cytotoxic T lymphocyte, dictates the nature of the chemical linkage between the module and target epitope. The use of a thioether bond between the module and target epitope had little or no adverse effect on antibody responses, whereas the use of a disulfide bond between the module and target epitope almost completely abrogated the antibody response. In contrast, better cytotoxic T lymphocyte responses were obtained when a disulfide bond was used.     

Acylphloroglucinol and xanthones from Hypericum ellipticum

An acylphloroglucinol, elliptophenone A, and two xanthones, elliptoxanthone A and elliptoxanthone B, were isolated from the aerial portions of Hypericum ellipticum together with three known xanthones, 1,3,7-trihydroxy-8-(3-methyl-2-butenyl)-9H-xanthen-9-one, 1,6-dihydroxy-4-methoxy-9H-xanthen-9-one, and 1,4,5-trihydroxy-9H-xanthen-9-one. Their structures were determined by spectroscopic analyses. The acylphloroglucinol and xanthones were evaluated for cytotoxicity using three human colon cancer cell lines cell lines (HT-29, HCT-116 and Caco-2) and a normal human colon cell line (CCD-18Co).     

Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition

Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptor-mediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling.     

Intracerebroventricular administration of vasoactive intestinal peptide inhibits food intake

Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide expressed throughout the peripheral and central nervous systems. VIP and the VIP receptor VPAC(2)R are expressed in hypothalamic nuclei involved in the regulation of energy homeostasis. VIP has been shown to be involved in the regulation of energy balance in a number of non-mammalian vertebrates. We therefore examined the effects of intracerebroventricular (ICV) administration of VIP on food intake, energy expenditure and activity in adult male Wistar rats. VIP administration caused a potent short lived decrease in food intake and an increase in activity and energy expenditure. The pathways potentially involved in the anorexigenic effects of VIP were investigated by measuring the release of neuropeptides involved in the regulation of food intake from hypothalamic explants treated with VIP. VIP significantly stimulated the release of the anorexigenic peptide alpha-melanocyte stimulating hormone (αMSH). These studies suggest that VIP may have an endogenous role in the hypothalamic control of energy homeostasis.     

Restoring HSP70 deficiencies improves glucose tolerance in diabetic monkeys

We evaluated heat shock protein 70 (HSP70) changes in diabetes mellitus (DM) in a nonhuman primate model. To this end, two studies were conducted in DM vervet monkeys. 1) Normal control and streptozotocin-induced DM monkeys (Stz-DM) that were differentiated into moderately or poorly controlled DM by judicious insulin administration were evaluated. Liver was collected at 4, 8, 12, 16, and 20 wk after streptozotocin, exposed to ex vivo heat shock at 42°C, and immunoblotted for heat shock factor 1 (HSF1), HSP70, and phosphorylated HSF1. 2) Spontaneous DM monkeys that were not pharmacologically induced were included in a crossover study of the HSP70-inducing drug geranylgeranylacetone (GGA). GGA at 20 mg/kg was given for 14 days with a 6-wk washout period. Glucose tolerance testing and plasma and muscle HSP70 were the primary outcome measurements. In Stz-DM, hyperglycemia reduced hepatic HSP70 in a dose-dependent fashion. HSF1 was increased in livers of monkeys with Stz-DM, but responses to ex vivo heat shock were impaired vs. normal monkeys. Activation of HSF1 appears to be important, because the phosphorylation change with heat stress was nearly perfectly correlated with HSP70 increases. Impaired HSF1 activation was also seen in Stz-DM after chronic hyperglycemia (>12 wk). In naturally occurring DM, increased circulating HSP70 resulted in significantly improved glucose tolerance and significant, positive trends in other measurements of insulin resistance. No change in muscle HSP70 content was observed. We conclude that increasing HSP70, potentially through targeting hyperglycemia-related deficits in HSF1 induction and activation in the liver, is a potent and viable strategy to improve glucose tolerance.     

High cell-free virus load and robust autologous humoral immune responses in breast milk of simian immunodeficiency virus-infected african green monkeys

The design of immunologic interventions to prevent postnatal transmission of human immunodeficiency virus (HIV) will require identification of protective immune responses in this setting. Simian immunodeficiency virus (SIV)-infected rhesus monkeys (RMs), a species that develops an AIDS-like illness following experimental infection, transmit the virus at a high rate during breastfeeding. In contrast, postnatal transmission of SIV occurs rarely or not at all in natural, asymptomatic primate hosts of SIV. These contrasting transmission patterns provide a unique opportunity to study mechanisms that evolved to protect suckling infants from SIV infection. We compared the virologic and immunologic properties of milk of SIV-infected and uninfected natural hosts of SIV, African green monkeys (AGMs), to that of RMs. Interestingly, despite a low number of milk CD4(+) T lymphocytes in uninfected AGMs, milk virus RNA load in SIV-infected AGMs was comparable to that of SIV-infected RMs and that in AGM plasma. This observation is in contrast to the relatively low virus load in milk compared to that in plasma of SIV-infected RMs and HIV-infected women. Milk of SIV-infected AGMs also displayed robust virus-specific cellular immune responses. Importantly, an autologous challenge virus-specific neutralization response was detected in milk of five of six SIV-infected AGMs that was comparable in magnitude to that in plasma. In contrast, autologous challenge virus neutralization was not detectable in milk of SIV-infected RMs. The autologous virus-specific adaptive immune responses in breast milk of AGMs may contribute to impedance of virus transmission in the infant oral/gastrointestinal tract and the rarity of postnatal virus transmission in natural hosts of SIV.     

Durable mucosal simian immunodeficiency virus-specific effector memory T lymphocyte responses elicited by recombinant adenovirus vectors in rhesus monkeys

The induction of potent and durable cellular immune responses in both peripheral and mucosal tissues may be important for the development of effective vaccines against human immunodeficiency virus type 1 and other pathogens. In particular, effector responses at mucosal surfaces may be critical to respond rapidly to incoming mucosal pathogens. Here we report that intramuscular injection of nonreplicating recombinant adenovirus (rAd) vectors into rhesus monkeys induced remarkably durable simian immunodeficiency virus (SIV)-specific T lymphocyte responses that persisted for over 2 years in both peripheral blood and multiple mucosal tissues, including colorectal, duodenal, and vaginal biopsy specimens, as well as bronchoalveolar lavage fluid. In peripheral blood, SIV-specific T lymphocytes underwent the expected phenotypic evolution from effector memory T cells (T(EM)) to central memory T cells (TCM) following vaccination. In contrast, mucosal SIV-specific T lymphocytes exhibited a persistent and durable T(EM) phenotype that did not evolve over time. These data demonstrate that nonreplicating rAd vectors induce durable and widely distributed effector memory mucosal T lymphocyte responses that are phenotypically distinct from peripheral T lymphocyte responses. Vaccine-elicited T(EM) responses at mucosal surfaces may prove critical for affording protection against invading pathogens at the mucosal portals of entry.     

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) mediates vitamin K-dependent intracellular antioxidant function

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1), expressed in HEK 293T cells and localized exclusively to membranes of the endoplasmic reticulum, was found to support both vitamin K 2,3-epoxide reductase (VKOR) and vitamin K reductase enzymatic activities. Michaelis-Menten kinetic parameters for dithiothreitol-driven VKOR activity were: K(m) (μM) = 4.15 (vitamin K(1) epoxide) and 11.24 (vitamin K(2) epoxide); V(max) (nmol·mg(-1)·hr(-1)) = 2.57 (vitamin K(1) epoxide) and 13.46 (vitamin K(2) epoxide). Oxidative stress induced by H(2)O(2) applied to cultured cells up-regulated VKORC1L1 expression and VKOR activity. Cell viability under conditions of no induced oxidative stress was increased by the presence of vitamins K(1) and K(2) but not ubinquinone-10 and was specifically dependent on VKORC1L1 expression. Intracellular reactive oxygen species levels in cells treated with 2,3-dimethoxy-1,4-naphthoquinone were mitigated in a VKORC1L1 expression-dependent manner. Intracellular oxidative damage to membrane intrinsic proteins was inversely dependent on VKORC1L1 expression and the presence of vitamin K(1). Taken together, our results suggest that VKORC1L1 is responsible for driving vitamin K-mediated intracellular antioxidation pathways critical to cell survival.     

Seascape-scale trophic links for fish on inshore coral reefs

It is increasingly accepted that coastal habitats such as inshore coral reefs do not function in isolation but rather as part of a larger habitat network. In the Caribbean, trophic subsidies from habitats adjacent to coral reefs support the diet of reef fishes, but it is not known whether similar trophic links occur on reefs in the Indo-Pacific. Here, we test whether reef fishes in inshore coral, mangrove, and seagrass habitats are supported by trophic links. We used carbon stable isotopes and mathematical mixing models to determine the minimum proportion of resources from mangrove or seagrass habitats in the diet of five fish species from coral reefs at varying distances (0–2,200 m) from these habitats in Moreton Bay, Queensland, eastern Australia. Of the fish species that are more abundant on reefs near to mangroves, Lutjanus russelli and Acanthopagrus australis showed no minimum use of diet sources from mangrove habitat. Siganus fuscescens utilized a minimum of 25–44 % mangrove sources and this contribution increased with the proximity of reefs to mangroves (R ² = 0.91). Seagrass or reef flat sources contributed a minimum of 14–78 % to the diet of Diagramma labiosum, a species found in higher abundance on reefs near seagrass beds, but variation in diet among reefs was unrelated to seascape structure. Seagrass or reef flat sources also contributed a minimum of 8–55 % to a fish species found only on reefs (Pseudolabrus guentheri), indicating that detrital subsidies from these habitats may subsidize fish diet on reefs. These results suggest that carbon sources from multiple habitats contribute to the functioning of inshore coral reef ecosystems and that trophic connectivity between reefs and mangroves may enhance production of a functionally important herbivore.