Virus-like particles for antigen delivery at mucosal surfaces.

Virus-like particles are a new type of vaccine platform that presents an attractive alternative to more traditional live-attenuated or inactivated/subunit vaccines. Virus-like particles (VLP) are composed of viral structural proteins that assemble spontaneously in cells, mimicking the live virus without the possibility of replication. They are readily recognized by the immune system, inducing both humoral and cellular immune responses. Here we review the development of VLP as vaccine delivery systems at mucosal surfaces. We first summarize the current status of VLP vaccines in general, and then discuss their use in mucosal vaccination approaches for several viruses that enter the host via the urogenital, respiratory or gastrointestinal tract.

The use of feathers in monitoring bioaccumulation of metals and metalloids in the South African endangered African grass-owl (Tyto capensis).

Few studies have quantified metals in South African species and no published data on residues specifically in South African owl feathers exist. Tyto capensis is listed as vulnerable within South Africa, making it preferable to use a non-invasive technique to determine metal bioaccumulation for this species. Comparisons are made with the cosmopolitan T. alba to determine whether this species could be used as a surrogate. Concentrations of various metals were thus determined in feathers of the two species and compared with liver and muscle samples. Samples were taken from 119 owls collected as road kill along a national road. A comparison of concentrations in feathers revealed similarly higher concentrations of aluminium, antimony, lead, nickel, and strontium, whereas concentrations of chromium, copper, iron, manganese, selenium, titanium and zinc were similarly higher in internal tissues for both species. Metal concentrations of owls were comparable to those reported in literature and below toxic levels, suggesting that these metals were not likely to impact the owls. Further regressions between feathers and corresponding livers were examined to determine if feathers were indicative of internal metal burdens. Significant positive relationships were found for aluminium, copper, lead, nickel and vanadium in T. alba and nickel, manganese and vanadium in T. capensis. Preliminary results support the feasibility of using feathers as non-destructive indicators of environmental contamination in T. capensis although caution needs to be taken when interpreting the results.

Delivery of interferon-beta to the monkey nervous system following intranasal administration.

We determined the nervous system targeting of interferon-beta1b (IFN-beta1b), a 20 kDa protein used to treat the relapsing-remitting form of multiple sclerosis, following intranasal administration in anesthetized, adult cynomolgus monkeys. Five animals received an intranasal bolus of [(125)I]-labeled IFN-beta1b, applied bilaterally to the upper nasal passages. Serial blood samples were collected for 45 min, after which the animals were euthanized by transcardial perfusion-fixation. High resolution phosphor imaging of tissue sections and gamma counting of microdissected tissue were used to obtain the distribution and concentration profiles of [(125)I]-IFN-beta1b in central and peripheral tissues. Intranasal administration resulted in rapid, widespread targeting of nervous tissue. The olfactory bulbs and trigeminal nerve exhibited [(125)I]-IFN-beta1b levels significantly greater than in peripheral organs and at least one order of magnitude higher than any other nervous tissue area sampled. The basal ganglia exhibited highest [(125)I]-IFN-beta1b levels among CNS regions other than the olfactory bulbs. Preferential IFN-beta1b distribution to the primate basal ganglia is a new finding of possible clinical importance. Our study suggests both IFN-beta and IFN-alpha, which share the same receptor, may be bound with relatively high affinity in these structures, possibly offering new insight into a neurovegetative syndrome induced by IFN-alpha therapy and suspected to involve altered dopamine neurotransmission in the basal ganglia. Most importantly, our results suggest intranasally applied macromolecules may bypass the blood-brain barrier and rapidly enter the primate CNS along olfactory- and trigeminal-associated extracellular pathways, as shown previously in the rat. This is the first study to finely detail the central distribution of a labeled protein after intranasal administration in non-human primates.

Inhibition of HIV-1 progeny virion release by cell-surface CD4 is relieved by expression of the viral Nef protein.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) Nef protein is required for efficient virus replication in vivo and displays a number of distinct and apparently unrelated biological activities in vitro. Of these, one of the most readily demonstrated is the efficient internalization and degradation of cell-surface CD4, the receptor for the HIV-1 envelope protein. The biological purpose of this internalization has, however, remained unclear. RESULTS: Using human 293T cells expressing high levels of cell-surface CD4 or CD8, we demonstrate that CD4, but not CD8, can dramatically reduce the release of infectious virions bearing the HIV-1 envelope protein and induce a concomitant increase in the accumulation of cell-associated HIV-1 structural proteins. In contrast, CD4 had no effect on the release of HIV-1 bearing a heterologous envelope protein unable to bind CD4. Nef expression totally reversed CD4-mediated inhibition but only if the CD4 used remained susceptible to Nef-induced internalization. CONCLUSIONS: These results support the hypothesis that cell-surface CD4 can interact with the envelope protein present on budding HIV-1 virions to inhibit their release. The internalization and degradation of cell-surface CD4 induced by the viral Nef protein can fully reverse this inhibition and is, therefore, likely to facilitate the spread of virus in vivo.

Using death to one's advantage: HIV modulation of apoptosis.

Infection by human immunodeficiency virus (HIV) is associated with an early immune dysfunction and progressive destruction of CD4+ T lymphocytes. This progressive disappearance of T cells leads to a lack of immune control of HIV replication and to the development of immune deficiency resulting in the increased occurrence of opportunistic infections associated with acquired immune deficiency syndrome (AIDS). The HIV-induced, premature destruction of lymphocytes is associated with the continuous production of HIV viral proteins that modulate apoptotic pathways. The viral proteins, such as Tat, Env, and Nef, are associated with chronic immune activation and the continuous induction of apoptotic factors. Viral protein expression predisposes lymphocytes, particularly CD4+ T cells, CD8+ T cells, and antigen-presenting cells, to evolve into effectors of apoptosis and as a result, to lead to the destruction of healthy, non-infected T cells. Tat and Nef, along with Vpu, can also protect HIV-infected cells from apoptosis by increasing anti-apoptotic proteins and down-regulating cell surface receptors recognized by immune system cells. This review will discuss the validity of the apoptosis hypothesis in HIV disease and the potential mechanism(s) that HIV proteins perform in the progressive T cell depletion observed in AIDS pathogenesis.

Drugs which stimulate or facilitate central cholinergic transmission interact synergistically with delta-9-tetrahydrocannabinol to produce marked catalepsy in mice.

In experiments in which mice were placed with their forepaws over a 4 cm high horizontal bar, delta-9-tetrahydrocannabinol (THC; 10 mg/kg i.p.) delayed descent from the bar. This effect on descent latency was markedly enhanced by physostigmine (0.05 or 0.25 mg/kg s.c.) and oxotremorine (0.04 or 0.08 mg/kg s.c.), administered immediately before THC. These interactions were attenuated by atropine (2.0 mg/kg s.c.) and (-)-scopolamine (1.9 mg/kg s.c.) but not by atropine methyl nitrate (2.11 mg/kg s.c.), which does not readily cross the blood-brain barrier. However, atropine methyl nitrate did prevent salivation induced by oxotremorine in the presence of THC. No synergism was detected between THC and neostigmine (0.047 mg/kg s.c.). Atropine and (-)-scopolamine also decreased the ability of chlordiazepoxide (10 mg/kg s.c.) to enhance the effect of THC on descent latency. The interaction was not antagonized by atropine methyl nitrate or mecamylamine (1.17 or 2.34 mg/kg s.c.). These results point to an involvement of central acetylcholine-releasing pathways in the cataleptic response of mice to THC.

Intranasal administration of interferon beta bypasses the blood-brain barrier to target the central nervous system and cervical lymph nodes: a non-invasive treatment strategy for multiple sclerosis.

Intranasal (i.n.) administration of IFN beta-1b was examined as a route for targeted delivery to the rat central nervous system (CNS). Intranasal administration resulted in significant delivery throughout the CNS and cervical lymph nodes with low delivery to peripheral organs. At similar blood levels, intravenous (i.v.) administration of IFN beta-1b yielded 88-98% lower CNS levels and 100-1650% greater peripheral organ levels compared to intranasal. Autoradiography confirmed much greater delivery to the CNS with intranasal administration. Intranasally administered IFN beta-1b reached the brain intact and produced tyrosine phosphorylation of IFN receptor in the CNS. Intranasal administration offers a non-invasive method of drug delivery for multiple sclerosis (MS) that bypasses the blood-brain barrier (BBB) and directly targets the CNS and lymph nodes.

Enhanced avidity maturation of antibody to human immunodeficiency virus envelope: DNA vaccination with gp120-C3d fusion proteins.

DNA vaccination can elicit both humoral and cellular immune responses and can confer protection against several pathogens. However, DNA vaccines expressing the envelope (Env) protein of human immunodeficiency virus (HIV) have been relatively ineffective at generating high titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, we report that fusion of Env and the complement component, C3d, in a DNA vaccine, enhances the titers of antibody to Env. Plasmids were generated that expressed a secreted form of Env (sgp120) from three isolates of HIV and these same forms fused to three tandem copies of the murine homologue of C3d (sgp120-3C3d). Analyses of titers and avidity maturation of the raised antibody indicated that immunizations with each of the sgp120-3C3d-expressing DNAs accelerated both the onset and the avidity maturation of antibody to Env.

Long-term ethanol-exposure markedly changes the cellular composition of cerebral glial cultures.

The vulnerability of morphologically distinct glial subpopulations to ethanol toxicity was surveyed in tissue culture. Secondary cultures of rat glia were examined at intervals during 56 days of ethanol treatment for changes in growth and cellular characteristics. Beginning at 6 days in vitro (DIV), the experimental cultures were treated with either 0.2% or 0.5% (w/v) ethanol in the medium; control cultures received ethanol-free medium. Relative to control cultures, the ethanol-treated cultures exhibited a consistent and dose-dependent suppression in cell number. The development of these cultures was documented with sequential phase-contrast photomicrography. Prior to treatment day 5 (11 DIV), the preponderance of cells were epithelioid in configuration; the astrocytic character of these cells was verified by the immunocytochemical localization of glial fibrillary acidic protein. In control cultures, a subpopulation of process-bearing cells was acquired gradually during the first 3 weeks in culture. The majority of these process-bearing cells were considered to be oligodendrocytes due to their position above the astrocytic carpet and by the immunocytochemical localization of galactocerebroside. Exposure to 0.5% ethanol markedly suppressed the acquisition of process-bearing cells. This ethanol-related suppression of process-bearing cells was apparent in the photomicrographic records of culture development and was confirmed by differential cell counts after 50 days of treatment. These results suggest a possible differential sensitivity of oligodendrocytes of their precursors to ethanol toxicity at elevated (0.5% w/v) concentrations.

Glutathione S-transferase-mediated induction of GC-->AT transitions by halomethanes in Salmonella.

Halomethanes are among the most common mutagenic and carcinogenic disinfection by-products present in the volatile/semivolatile fraction of chlorinated drinking water. Recent studies have demonstrated that the mutagenicity of dichloromethane (CH2Cl2) and bromodichloromethane (BrCHCl2) can be mediated by a theta-class glutathione S-transferase (GSTT1-1). These studies used strain RSJ100 of Salmonella, which is a derivative of the base-substitution strain TA1535 (hisG46, rfa, delta uvrB), into which has been cloned the GSTT1-1 gene from rat. In the present report, we have extended these studies by demonstrating that the mutagenicity of two additional brominated trihalomethanes, bromoform (CHBr3) and chlorodibromomethane (CICHBr2), are also mediated by GSTT1-1 in RSJ100. Using a Tedlar bag vaporization technique, the mutagenic potencies (revertants/ppm) for these two compounds as well as the compounds tested previously rank as follows: CHBr3 approximately CICHBr2 > BrCHCl2 approximately CH2Cl2. To explore the mutational mechanism, we determined the mutation spectra of all four halomethanes at the hisG46 allele by performing colony probe hybridizations of approximately 100 revertants induced by each compound. The majority (96-100%) of the mutations were GC-->AT transitions, and 87-100% of these were at the second position of the CCC/GGG target. In contrast, only 15% of mutants induced by CH2Cl2 were GC-->AT transitions in the absence of the GSTT1-1 gene in strain TA100 (a homologue of TA1535 containing the plasmid pKM101). The ability of GSTT1-1 to mediate the mutagenicity of these di- and trihalomethanes and the induction of almost exclusively GC-->AT transitions by these compounds suggest that these halomethanes are activated by similar pathways in RSJ100, possibly through similar reactive intermediates. The implications of these findings are discussed in relation to previous experimental work on the GST-mediated bioactivation of dihalomethanes, which includes the possible formation of GSH intermediates and/or GSH-DNA adducts.

Physiologically based estimation of in vivo rates of bromodichloromethane metabolism.

Bromodichloromethane (BDCM) is a rodent carcinogen formed by chlorination of drinking water containing bromide and organic precursors. BDCM is a member of the class of disinfection by-products known as trihalomethanes (THMs), compounds that have been shown to be carcinogenic in rodents. A physiologically-based pharmacokinetic (PBPK) model has been developed and applied to provide estimates of the rates of metabolism of BDCM in vivo in rats. The model consists of five compartments (liver, kidney, fat and slowly and rapidly perfused tissues). Tissue partition coefficients were determined using a modified vial equilibration technique and rates of metabolism were estimated by fitting data obtained from stable metabolite (bromide ion, (Br-)) analysis following 4 h constant concentration BDCM inhalation exposure (50-3200 ppm) and closed chamber gas uptake experiments. Metabolism was described using a single saturable pathway representing a high capacity, high affinity process (Vmaxc = 12.8 mg/h/kg; Km = 0.5 mg/l). Rate constants obtained from Br- data adequately described data from gas uptake experiments and literature data on exhalation of 14CO and 14CO2 produced following oral gavage with 14C-BDCM. Pretreatment with trans-dichloroethylene (t-DCE), an inhibitor of CYP2E1, increased the apparent Km from 0.5 to 225 mg/l indicating that CYP2E1 is the major P450 isoform involved in the bioactivation of BDCM to reactive intermediates.

An investigation of the involvement of GABA in certain pharmacological effects of delta-9-tetrahydrocannabinol.

Experiments were performed with mice to determine whether doses of the benzodiazepine, flurazepam, or the GABA uptake inhibitor, NO-328, known to potentiate catalepsy induced by delta-9-tetrahydrocannabinol (THC), would also interact synergistically with THC in the production of certain other effects. No synergism was detected either in the production of antinociception (tail flick test) or in a test in which the ability of flurazepam to delay onset of clonic convulsions induced by intravenous infusion of pentylenetetrazole was compared in the presence and absence of THC or cannabidiol. The hypothermic effect of THC was unaffected by NO-328 but enhanced by flurazepam, albeit only at doses higher than those needed to potentiate THC-induced catalepsy. In vitro experiments with guinea pig ileum showed that the ability of THC to inhibit electrically evoked contractions was unaffected by delta-amino-n-valeric acid, a GABA(B) receptor antagonist, and that preparations rendered tolerant to GABA responded normally to THC. Contractions induced by GABA in unstimulated ileal longitudinal muscle were attenuated by THC. We conclude that there is little evidence from our data that any of the THC effects studied were GABA mediated.

The ability of HIV type 1 to use CCR-3 as a coreceptor is controlled by envelope V1/V2 sequences acting in conjunction with a CCR-5 tropic V3 loop.

Although infection by primary HIV type 1 (HIV-1) isolates normally requires the functional interaction of the viral envelope protein with both CD4 and the CCR-5 coreceptor, a subset of such isolates also are able to use the distinct CCR-3 receptor. By analyzing the ability of a series of wild-type and chimeric HIV-1 envelope proteins to mediate CCR-3-dependent infection, we have determined that CCR-3 tropism maps to the V1 and V2 variable region of envelope. Although substitution of the V1/V2 region of a CCR-3 tropic envelope into the context of a CCR-5 tropic envelope is both necessary and sufficient to confer CCR-3 tropism, this same substitution has no phenotypic effect when inserted into a CXCR-4 tropic HIV-1 envelope context. However, this latter chimera acquires both CCR-3 and CCR-5 tropism when a CCR-5 tropic V3 loop sequence also is introduced. These data demonstrate that the V1/2 region of envelope can, like the V3 loop region, encode a particular coreceptor requirement and suggest that a functional envelope:CCR-3 interaction may depend on the cooperative interaction of CCR-3 with both the V1/V2 and the V3 region of envelope.

Gracilis muscle repair of rectovaginal fistula after restorative proctocolectomy. Report of two cases.

Gracilis muscle interposition flaps have been used to treat two patients with rectovaginal fistulas. The fistulas occurred following restorative proctocolectomy with a J-shaped ileal reservoir and ileoanal anastomosis. Attempts at local repair of the fistulas had failed. A diverting loop ileostomy was constructed simultaneously. Anterior sphincteroplasty was performed in one patient for associated incontinence. Excellent results were achieved in both patients. The fistulas have healed, and intestinal continuity has been re-established. This procedure can be useful to salvage a pelvic pouch complicated by a rectovaginal fistula.

Trihalomethane comparative toxicity: acute renal and hepatic toxicity of chloroform and bromodichloromethane following aqueous gavage.

Bromodichloromethane (BDCM) and chloroform (CHCl3) are by-products of drinking water chlorination and are the two most prevalent trihalomethanes (THMs) in finished drinking water. To date, no comprehensive comparison of the acute renal and hepatic effects of BDCM and CHCl3 following oral gavage in an aqueous dosing vehicle has been conducted. To characterize BDCM- and CHCl3-induced nephro- and hepatotoxicity following aqueous gavage and compare directly the responses between these THMs, 95-day-old male F-344 rats were given single oral doses of 0.0, 0.75, 1.0, 1.5, 2.0, or 3.0 mmol BDCM or CHCl3/kg body wt in an aqueous 10% Emulphor solution. Compound-related hepatic and renal damage was evaluated by quantitating clinical toxicity markers in the serum and urine, respectively. Both THMs appear to be equally hepatotoxic after 24 h, but BDCM caused significantly greater elevations in serum hepatotoxicity markers than CHCl3 at 48 h following exposure to 2.0 and 3.0 mmol/kg. In addition to causing more persistent liver toxicity than CHCl3, BDCM also appears to be slightly more toxic to the kidney at lower doses. Potency differences between the two THMs may be due to pharmacokinetic dissimilarities such as greater metabolism of BDCM to reactive metabolites or more extensive partitioning of BDCM into kidneys and fat depots, resulting in prolonged target tissue exposure.

Multiple residues contribute to the inability of murine CCR-5 to function as a coreceptor for macrophage-tropic human immunodeficiency virus type 1 isolates.

Infection of CD4-positive cells by human immunodeficiency virus type 1 (HIV-1) requires functional interaction of the viral envelope protein with a coreceptor belonging to the chemokine receptor family of seven-membrane-spanning receptors. For the majority of macrophage-tropic HIV-1 isolates, the physiologically relevant coreceptor is the human CCR-5 (hCCR-5) receptor. Although the murine homolog of CCR-5 (mCCR-5) is unable to mediate HIV-1 infection, chimeric hCCR-5/mCCR-5 molecules containing single extracellular domains derived from hCCR-5 are effective coreceptors for certain macrophage-tropic HIV-1 isolates. Here, we have sought to identify residues in hCCR-5 critical for HIV-1 infection by substitution of mCCR-5-derived residues into the context of functional chimeric hCCR-5/mCCR-5 receptor molecules. Using this strategy, we demonstrate that residues 7, 13, and 15 in the first extracellular domain and residue 180 in the third extracellular domain of CCR-5 are important for HIV-1 envelope-mediated membrane fusion. Of interest, certain substitutions, for example, at residues 184 and 185 in the third extracellular domain, have no phenotype when introduced individually but strongly inhibit hCCR-5 coreceptor function when present together. We hypothesize that these changes, which do not preclude chemokine receptor function, may inhibit a conformational transition in hCCR-5 that contributes to HIV-1 infection. Finally, we report that substitution of glycine for valine at residue 5 in CCR-5 can significantly enhance the level of envelope-dependent cell fusion by expressing cells. The diversity of the mutant phenotypes observed in this mutational analysis, combined with their wide distribution across the extracellular regions of CCR-5, emphasizes the complexity of the interaction between HIV-1 envelope and coreceptor.