Immune responses induced by intramuscular or gene gun injection of protective deoxyribonucleic acid vaccines that express the circumsporozoite protein from Plasmodium berghei malaria parasites.

The circumsporozoite protein (CSP) is a target for effector Ab and cell mediated immunity against malaria parasites; DNA vaccination can induce both types of effector response. The immunogenicity and efficacy of two DNA plasmids expressing different amounts of Plasmodium berghei CSP were evaluated by immunizing BALB/c mice i.m. or epidermally and by varying the number of immunizations (one to three doses) and the interval between immunizations. Expanding the interval gave the strongest effect, increasing efficacy and antibody boosting, and, in the case of epidermal vaccination, promoting a switch in CSP-specific IgG isotypes from IgG1 to a balance with IgG2a. The strongest humoral immune response and the greatest level of protection were induced by vaccinating epidermally with high expresser plasmid, using a gene gun to administer three doses at 6-wk intervals. For this group, the mean, repeat-specific, prechallenge antibody titer among mice not infected after challenge was significantly higher than that in infected mice, but the mean prechallenge titers for antibody reactive with whole sporozoites were not significantly different. The interval-dependent induction of IgG2a antibodies by epidermal vaccination contradicts the widely held belief that antibody responses induced by this method are restricted to those that are Th2 dependent.

Attenuated vaccinia virus-circumsporozoite protein recombinants confer protection against rodent malaria.

NYVAC-based vaccinia virus recombinants expressing the circumsporozoite protein (CSP) were evaluated in the Plasmodium berghei rodent malaria model system. Immunization of mice with a NYVAC-based CSP recombinant elicited a high level of protection (60 to 100%). Protection did not correlate with CS repeat-specific antibody responses and was abrogated by in vivo CD8+ T-cell depletion. Protection was not enhanced by modification of the subcellular localization of CSP. These results suggest the potential of poxvirus-based vectors for the development of vaccine candidates for human malaria.

Co-localization of inducible-nitric oxide synthase and Plasmodium berghei in hepatocytes from rats immunized with irradiated sporozoites.

Both CD8+ T cells and IFN-gamma (IFN-gamma) are important components in the regulation of inducible-nitric oxide synthase (iNOS) which contribute to liver stage anti-malarial activity in rodents immunized with irradiated sporozoites. IFN-gamma, provided by malaria-specific CD8+ T cells, stimulates liver cells to produce nitric oxide (NO) for the destruction of infected hepatocytes or the parasite within these cells. To identify the cell source of iNOS in livers from Brown Norway rats challenged with Plasmodium berghei sporozoites, we probed tissue sections with antisera that recognize iNOS and the malarial exoerythrocytic stage parasite. Immunofluorescence analysis of parasitized livers demonstrate that 1) iNOS was found in infected hepatocytes, not Kupffer or endothelial cells; and 2) a higher proportion of infected hepatocytes express iNOS in immunized rats compared with naive animals after challenge. There was no immunoreactivity to the iNOS antisera in liver sections of immunized rats 15 h after sporozoite challenge, however, iNOS activity was present in 18% of the infected hepatocytes by 24 h and reached 81% by 31 h. In contrast, < 10% of the infected hepatocytes displayed iNOS activity in naive or immune animals 48 h after challenge. We also found a significant decrease in the ability of the immunized animals to express iNOS in response to sporozoite challenge by accelerating the removal of pre-existing irradiated-attenuated parasites from hepatocytes with the antimalarial drug, primaquine. Therefore, induction and maintenance of iNOS activity were dependent on intrahepatic persistence of the irradiated-attenuated parasite. These results suggest that liver-iNOS expression following sporozoite challenge is restricted to the infected hepatocyte and dependent on the presence of the irradiated-attenuated parasite in immune animals.

Nitric oxide: cytokine-regulation of nitric oxide in host resistance to intracellular pathogens.

To discover how nitric oxide (NO) synthesis is controlled in different tissues as cells within these tissues combat intracellular pathogens, we examined three distinctively different experimental murine models designed for studying parasite-host interactions: macrophage killing of Leishmania major; nonspecific protection against tularemia (Francisella tularensis) by Mycobacterium bovis (BCG); and specific vaccine-induced protection against hepatic malaria with Plasmodium berghei. Each model parasite and host system provides information on the source and role of NO during infection and the factors that induce or inhibit its production. The in vitro assay for macrophage antimicrobial activity against L. major identified cytokines involved in regulating NO-mediated killing of this intracellular protozoan. L. major induced the production of two competing cytokines in infected macrophages: (1) the parasite activated the gene for tumor necrosis factor (TNF), and production of TNF protein was enhanced by the presence of interferon-gamma (IFN-gamma). TNF then acted as a autocrine signal to amplify IFN-gamma-induced production of NO; and (2) the parasite upregulated production of transforming growth factor-beta (TGF-beta), which blocked IFN-gamma-induced production of NO. Whether parasite-induced TNF (parasite destruction) or TGF-beta (parasite survival) prevailed depended upon the presence and quantity of IFN-gamma at the time of infection. The relationship between NO production in vivo and host resistance to infection was demonstrated with M. bovis (BCG).(ABSTRACT TRUNCATED AT 250 WORDS)

L-carnosine (beta-alanyl-L-histidine) and carcinine (beta-alanylhistamine) act as natural antioxidants with hydroxyl-radical-scavenging and lipid-peroxidase activities.

Carnosine (beta-alanyl-L-histidine) and carcinine (beta-alanylhistamine) are natural imidazole-containing compounds found in the non-protein fraction of mammalian tissues. Carcinine was synthesized by an original procedure and characterized. Both carnosine and carcinine (10-25 mM) are capable of inhibiting the catalysis of linoleic acid and phosphatidylcholine liposomal peroxidation (LPO) by the O2(-.)-dependent iron-ascorbate and lipid-peroxyl-radical-generating linoleic acid 13-monohydroperoxide (LOOH)-activated haemoglobin systems, as measured by thiobarbituric-acid-reactive substance. Carcinine and carnosine are good scavengers of OH. radicals, as detected by iron-dependent radical damage to the sugar deoxyribose. This suggests that carnosine and carcinine are able to scavenge free radicals or donate hydrogen ions. The iodometric, conjugated diene and t.l.c. assessments of lipid hydroperoxides (13-monohydroperoxide linoleic acid and phosphatidylcholine hydroperoxide) showed their efficient reduction and deactivation by carnosine and carcinine (10-25 mM) in the liberated and bound-to-artificial-bilayer states. This suggests that the peroxidase activity exceeded that susceptible to direct reduction with glutathione peroxidase. Imidazole, solutions of beta-alanine, or their mixtures with peptide moieties did not show antioxidant potential. Free L-histidine and especially histamine stimulated iron (II) salt-dependent LPO. Due to the combination of weak metal chelating (abolished by EDTA), OH. and lipid peroxyl radicals scavenging, reducing activities to liberated fatty acid and phospholipid hydroperoxides, carnosine and carcinine appear to be physiological antioxidants able to efficiently protect the lipid phase of biological membranes and aqueous environments.

Induction of nitric oxide synthase protects against malaria in mice exposed to irradiated Plasmodium berghei infected mosquitoes: involvement of interferon gamma and CD8+ T cells.

Exposure of BALB/c mice to mosquitoes infected with irradiated Plasmodium berghei confers protective immunity against subsequent sporozoite challenge. Immunized mice challenged with viable sporozoites develop parasitemia when treated orally with substrate inhibitors of nitric oxide synthase (NOS). This suggests that the production of nitric oxide (NO) prevents the development of exoerythrocytic stages of malaria in liver. Liver tissue from immunized mice expressed maximal levels of mRNA for inducible NOS (iNOS) between 12 and 24 h after challenge with sporozoites. Intraperitoneal injection of neutralizing monoclonal antibody against interferon gamma (IFN-gamma) or in vivo depletion of CD8+ T cells, but not CD4+ T cells, at the time of challenge blocked expression of iNOS mRNA and ablated protection in immunized mice. These results show that both CD8+ T cells and IFN-gamma are important components in the regulation of iNOS in liver which contributes to the protective response of mice immunized with irradiated malaria sporozoites. IFN-gamma, likely provided by malaria-specific CD8+ T cells, induces liver cells, hepatocytes and/or Kupffer cells, to produce NO for the destruction of infected hepatocytes or the parasite within these cells.

Comparison of obese and non-obese patients with gestational diabetes.

The aim of this study was to assess the effect of obesity on the outcome of gestational diabetes (GD). The age of the subject (31 +/- 5 years) and duration of pregnancy (29 +/- 5 weeks) were similar at the time of diagnosis for the 19 obese (34 +/- 6 kg/m2) and 19 non-obese (23.0 +/- 2.1 kg/m2) subjects. Before treatment, the area under the curve of the glucose tolerance test and the mean capillary blood glucose levels were similar in both groups; however, the baseline blood glucose levels of the obese patients (6.0 +/- 0.8 mmol/l) were higher than those of the non-obese patients (5.3 +/- 0.8 mmol/l; P < 0.01). During pregnancy, the total weight gain of the obese patients was less (10.6 +/- 5.0 vs. 15.2 +/- 4.7 kg, P < 0.006). Fifteen of the obese patients required treatment with insulin, while only six of the non-obese women required insulin (P < 0.005). During insulin therapy, the mean capillary blood glucose, glycosylated haemoglobin and fructosamine levels were similar in both groups. In conclusion, our data suggest that GD in obese patients is characterized by lower weight gain and higher baseline glucose with the result that insulin therapy is more frequently instituted.

Interactions of Plasmodium berghei sporozoites and murine Kupffer cells in vitro.

Malaria sporozoites must leave the bloodstream and cross a layer of sinusoidal lining cells in order to infect hepatocytes and undergo exoerythrocytic schizogony. To determine whether Kupffer cells (KC) derived from this layer interact with sporozoites, murine KC were isolated from perfused livers of BALB/cJ mice and incubated in vitro with Plasmodium berghei sporozoites. Isolated KC had characteristic macrophage surface Ag and were phagocytic, ingesting both latex particles and Leishmania major amastigotes. In the absence of immune serum, sporozoites associated with fewer than 10% of these KC. By 30 min, 10% of the cell-associated sporozoites were completely ingested, 30% were in the process of being ingested, and 60% were attached to the surface of the cells. Opsonization of sporozoites with monoclonal or polyclonal antibodies directed against P. berghei circumsporozoite protein markedly enhanced sporozoite association with KC. Up to 40% of cells exposed to opsonized sporozoites had parasites inside or attached to their surfaces. Sporozoites attached to or ingested by KC were uniformly destroyed within 240 min in all cultures; there was no evidence of conversion of sporozoites to the exoerythrocytic stage within KC by light microscopy, and there was no evidence of residual sporozoites, either inside or outside of cells, by either light or electron microscopy. These data suggest that under nonimmune conditions, KC play a minor role in resistance to infection by malaria sporozoites. However, when sporozoites are opsonized by circumsporozoite antibodies, phagocytosis by KC may be an important immune mechanism that prevents parasitization of hepatocytes.

A rapid and safe procedure for preparing the murine toxins of the plague bacillus (Yersinia pestis).

The murine toxins A and B of Y. pestis were purified from a crude extract of the nonencapsulated strain M23NP of Y. pestis using HPLC gel filtration. Toxins A and B had molecular weights of 240,000 and 120,000, respectively. Both toxins had isoelectric points of 3.8. The mouse LD50 of toxin A was 2.5 micrograms, that of toxin B was 1.8 micrograms. Of the protein injected on the column, approximately 9% was toxin, including 2.9% toxin A and 6.1% toxin B. The crude M23NP extract was used periodically over 10 months to purify lots of the toxins. Each HPLC purification required about 30 min.

Epitopic analysis of antigenic determinants on the surface of dengue-2 virions using monoclonal antibodies.

The relative binding sites of dengue serotype-specific, dengue subcomplex-specific, dengue complex-specific, flavivirus subgroup-reactive, and flavivirus group-reactive monoclonal antibody preparations were identified by using competitive antibody binding assays. A dengue complex-specific epitope, capable of mediating infection enhancement, was identified on a 20,000 dalton protein found on intracellular virions. The other epitopes were assigned relative positions on the E glycoprotein by competitive antibody binding. These could be grouped into 3 linkage groups based on the ability of some monoclonal antibodies to block contiguous binding sites. Some antibodies were able to increase or "promote" the binding of antibodies from other linkage groups. These results suggest that a continuum of antigenic reactivities exist on the E glycoprotein of the dengue viruses, and that the conformation of this glycoprotein may be altered after antibody binding.

Rapid identification of dengue virus isolates by using monoclonal antibodies in an indirect immunofluorescence assay.

Type-specific monoclonal antibodies prepared against the four dengue (DEN) virus serotypes were evaluated for their ability to identify low-passage human and mosquito isolates from Jamaica and West Africa by an indirect immunofluorescence assay. Serotyped human isolates from Jamaican dengue fever patients included 12 DEN-1, two DEN-2, and five DEN-4 viruses. Viruses from West Africa included 84 DEN-2 mosquito strains as well as two DEN-1 and one DEN-2 from humans. Results obtained using the immunofluorescence assay were consistent with virus identifications obtained using the more classical but costly and time-consuming plaque-reduction neutralization test. More viral isolates and higher virus yields were obtained using the C6/36 clone of Aedes albopictus cells rather than LLC-MK2 (monkey kidney) cells. Dengue type-specific monoclonal antibodies detected prototype viral antigens 24-48 hours postinfection in C6/36 cells. This is the first time that monoclonal antibodies have been used to serotype low-passage flavivirus isolates.