Production of recombinant SERA proteins of Plasmodium falciparum in Escherichia coli by using synthetic genes

We expressed two regions of the serine repeat antigen (SERA) protein of Plasmodium falciparum in Escherichia coli by synthesizing the genes with a changed codon usage. One of the synthetic gene sequences encodes amino acid residues 17-382 (SE47') and the other encodes amino acid residues 586-802 (SE50A). The products produced by the synthetic gene sequences in E. coli accounted for 15-30% of the total bacterial protein. Antisera against both the purified gene products prepared in rats inhibited malaria parasite growth in vitro. The anti-SE47' serum was significantly more inhibitory than the anti-SE50A serum. The described methods provide a large scale preparation of recombinant antigens for improving and producing malaria vaccine.     

Laboratory and preliminary clinical characterization of Vi capsular polysaccharide-protein conjugate vaccines

To improve its immunogenicity for children and adults and to make it suitable for routine immunization of infants against typhoid fever, the capsular polysaccharide of Salmonella typhi (Vi) was bound to the B subunit of the heat-labile toxin (LT-B) of Escherichia coli or the recombinant exoprotein A (rEPA) of Pseudomonas aeruginosa. The conjugates elicited higher levels of antibodies (micrograms per milliliter of serum) in mice and in guinea pigs than did Vi and, unlike Vi alone, elicited booster antibody responses in both species. In adult volunteers, Vi-LT-B and Vi-rEPA, respectively, elicited higher levels of antibodies than Vi alone after the first injection (4.74 versus 1.77 and 4.91 versus 1.77; P < 0.005) and 26 weeks later (2.32 and 2.69 versus 0.54; P < 0.04); a second injection of the conjugates did not elicit a booster response of Vi antibodies. None of the 51 vaccinees had fever or significant local reactions. Vi-rEPA elicited slightly higher levels of Vi antibodies than did Vi-LT-B at all intervals after injection, but these differences were not significant. Each conjugate elicited antibodies to its carrier protein. The antibody responses elicited in adults by Vi bound to LT-B and rEPA are similar to those of other polysaccharide-protein conjugates. These conjugates promise to be an improved Vi vaccine. Studies of Vi conjugates with adults and infants in areas where typhoid is endemic are planned.     

Susceptibility of three strains of conventional adult mice to intestinal colonization by an isolate of Escherichia coli O157:H7

Three mouse strains were assessed for their susceptibility to intestinal colonization by a strain of the enteric bacterial pathogen Escherichia coli O157:H7. Following intragastric inoculation of E. coli O157:H7, the intestines of young adult female CD1, BALB/c, and C57BL/6 mice became colonized, as evidenced by faecal shedding of the pathogen for periods of up to 5 weeks. None of the three mouse strains examined developed overt disease in response to colonization by the organism. Following clearance of the primary inoculum, BALB/c mice, but not CD1 or C57BL/6 mice, appeared to acquire enhanced resistance to recolonization by E. coli O157:H7, as evidenced by a decreased faecal shedding period. This enhanced resistance correlated with the presence and persistence of immunoglobulin A, but not immunoglobulin G, in the serum and faeces directed against the O157 antigen. The implications of these findings to vaccine development against E. coli O157:H7 are discussed.     

Inhibition of bacterial respiration by a low-molecular weight lectin, scyllin, from Scylla serrata crab hemolymph

Interaction of plant and/or invertebrate lectins with mammalian cells and different microorganisms is well known. In the present study, we have demonstrated that scyllin, a low molecular weight (MW 4000) lectin from the edible crab Scylla serrata hemolymph, purified by GalNAc-Sepharon affinity column followed by Mono-Q ion exchanger in FPLC exhibits antimicrobial activity against Bacillus cereus and Escherichia coli by inhibiting endogenous respiration as well as exogenous glucose oxidation. In both the cases oxygen consumption has been measured in an oxygraph. Scyllin has produced 50% inhibition of endogenous respiration at a concentration of 110 micrograms/ml and 125 micrograms/ml in B. cereus and E. coli respectively. It also reduced the exogenous glucose oxidation by 50% at a concentration of 12 micrograms/ml and 80 micrograms/ml respectively in B. cereus and E. coli. From the above study the mechanism of bacterial growth inhibitory property of scyllin is suggested though the other studies such as inhibition of nucleic acid biosynthesis, cell wall biosynthesis etc. to evaluate its total mode of inhibitory action are not yet obtained.     

Effects of dietary retinoic acid on skin papilloma and carcinoma formation in female SENCAR mice

Previously we have shown that dietary retinoids are essential for papilloma formation induced by either an initiation-promotion or a complete skin carcinogenesis protocol. The present study was conducted to further determine the effect of dietary retinoic acid (RA) on papilloma formation and the conversion of papillomas to carcinomas. Skin tumors were induced in 3 week old female SENCAR mice by an initiation-promotion protocol with one application of 20 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA), followed by 20 weekly applications of 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA). Mice were fed RA at one of the three doses: 0.3 (nutritionally marginal dose), 3 (near physiological) and 30 (pharmacological) micrograms/g of diet. Mice fed 30 micrograms of RA/g of diet had the same survival rate as the other two groups despite a lower body weight and all three groups had similar papilloma incidence, which reached 100% at age 18 weeks. Mice fed 3 micrograms of RA/g of diet had the highest papilloma yield (approximately 14 papillomas/mouse) of all groups and it peaked between weeks 18 and 38 of age. These papillomas later regressed such that mice from all three groups had about the same papilloma yield at week 44 of age. Mice fed 30 micrograms of RA/g of diet failed to develop any visible carcinoma, while mice fed 0.3 or 3 micrograms/g showed 1.9% conversion of papillomas to carcinomas. Therefore, dietary RA at 30 micrograms/g of diet inhibited the conversion of papillomas to carcinomas without affecting papilloma incidence. In addition, dietary RA at 30 and 0.3 micrograms/g of diet lowered papilloma yield.     

Translocation of Escherichia coli from the gastrointestinal tract to the mesenteric lymph nodes in gnotobiotic mice receiving Escherichia coli vaccines before colonization

Germfree mice were immunized orally or intraperitoneally for 6 weeks with heat-killed vaccines of indigenous Escherichia coli or nonindigenous E. coli O 127: B8 before colonization with these strains. The mice exhibited increases in specific serum antibodies and intestinal immunoglobulin A reacting with the E coli antigens. Prior immunization did not reduce the gastrointestinal population levels of the E. coli strains attained 3 and 7 days after colonization. Neither oral nor intraperitoneal immunization with the E. coli strains before colonization decreased the incidence of bacterial translocation to the mesenteric lymph nodes or reduced the number of viable E. coli cells per mesenteric lymph node. There also was no relation in individual mice between serum antibody titers and the numbers of viable E. coli cells translocating to the mesenteric lymph nodes. Thus, prior vaccination with E. coli in this study did not decrease the incidence or reduce the numbers of viable E. coli translocating to the mesenteric lymph nodes in gnotobiotic mice monoassociated with E. coli.     

Effect of in vivo hyperoxia on the glutathione system in neonatal rat lung

A DNA clone encoding a cathepsin D inhibitor CathInh was isolated from a potato genomic library using a CathInh cDNA as hybridization probe. The amino acid sequence of the coding region is nearly identical with a CathInh cDNA and CathInh proteins previously isolated from a tuber-specific cDNA library and from tubers, respectively. Analysis of GUS activity resulting from expression of chimeric CathInh promoter-GUS genes in transgenic potato plants revealed expression exclusively confined to potato tubers. No GUS activity could be detected in any other organ of the transgenic plants either constitutively or after wounding or treatment with abscisic and jasmonic acid (JA). Interestingly, part of the promoter region of the CathInh gene, essential for GUS activity in tubers, shows striking similarity to promoter regions of tuber-specific class I patatin genes.     

DNA vaccines expressing either the GP or NP genes of Ebola virus protect mice from lethal challenge

DNA vaccines expressing the envelope glycoprotein (GP) or nucleocapsid protein (NP) genes of Ebola virus were evaluated in adult, immunocompetent mice. The vaccines were delivered into the skin by particle bombardment of DNA-coated gold beads with the Powderject-XR gene gun. Both vaccines elicited antibody responses as measured by ELISA and elicited cytotoxic T cell responses as measured by chromium release assays. From one to four vaccinations with 0.5 microgram of the GP DNA vaccine resulted in a dose-dependent protection from Ebola virus challenge. Maximal protection (78% survival) was achieved after four vaccinations. Mice were completely protected with a priming dose of 0.5 microgram of GP DNA followed by three or four subsequent vaccinations with 1.5 micrograms of DNA. Partial protection could be observed for at least 9 months after three immunizations with 0.5 microgram of the GP DNA vaccine. Comparing the GP and NP vaccines indicated that approximately the same level of protection could be achieved with either vaccine.     

Protection of C3H/He mice from experimental Borrelia burgdorferi infection by immunization with a 110-kilodalton fusion protein

A 110-kDa Borrelia burgdorferi fusion protein, Escherichia coli expressing the fusion protein, transformed E. coli lacking the fusion protein insert, and lyophilized whole B. burgdorferi bacteria were compared for immunogenicity in C3H/He mice. Immunized mice were challenged with a variety of isolates from the United States or the European isolate P/Gau 3 weeks following the last inoculation. An average of 76.7% of the mice immunized with 25 micrograms of lyophilized whole B. burgdorferi cells were protected from infection, while 60% of the mice immunized with the 110-kDa fusion protein were protected. Whole E. coli bacteria expressing the fusion protein protected 57.7% of immunized mice against experimental challenge. Lower levels of protection occurred in mice challenged with the European isolate than in those challenged with isolates originating from the United States. These results demonstrate the potential of the 110-kDa fusion protein for use as a component of a subunit vaccine for prevention of Lyme borreliosis.     

Localization of a domain in the FimH adhesin of Escherichia coli type 1 fimbriae capable of receptor recognition and use of a domain-specific antibody to confer protection against experimental urinary tract infection

The FimH subunit of type 1-fimbriated Escherichia coli has been implicated as an important determinant of bacterial adherence and colonization of the urinary tract. Here, we sought to localize the functionally important domain(s) within the FimH molecule and to determine if antibodies against this domain would block adherence of type 1-fimbriated E. coli to the bladder mucosa in situ and in vivo in an established mouse model of cystitis. We generated translational fusion proteins of disparate regions of the FimH molecule with an affinity tag MalE, and tested each of the fusion products in vitro for functional activity. The minimum region responsible for binding mouse bladder epithelial cells and a soluble mannoprotein, horseradish peroxidase, was contained within residues 1-100 of the FimH molecule. We validated and extended these findings by demonstrating that antibodies directed at the putative binding region of FimH or at synthetic peptides corresponding to epitopes within the binding domain could specifically block type 1 fimbriae-mediated bacterial adherence to bladder epithelial cells in situ and yeast cells in vitro. Next, we compared the ability of mice passively immunized intraperitoneally with antisera raised against residues 1-25 and 253-264 of FimH or 1-13 of FimA to resist bladder colonization in vivo after intravesicular challenge with type 1-fimbriated E. coli. Only the antibody directed at the putative binding region of FimH (anti- s-FimH1-25) significantly reduced E. coli bladder infections in the experimental mouse model of urinary tract infections. Similar results were obtained when the mice were actively immunized with synthetic peptides corresponding to residues 1-25 and 253-264 of FimH or 1-13 of FimA. The mechanism of protection was attributed, at least in part, to inhibition of bacterial adherence to the bladder surface by s-FimH1-25-specific antibody molecules that had filtered through the kidneys into the urine. The level of FimH antibodies entering the bladder from the circulatory system of the immunized mice was found to be markedly enhanced upon bacterial challenge. The potential broad spectrum activity of the protective FimH antibody was indicated from its serologic cross-reactivity with various urinary tract bacterial isolates bearing type 1 fimbriae. These findings could be relevant in the design of an efficacious and broadly reactive FimH vaccine against urinary tract infections.     

The origin of congenital cholesteatoma

Transgenic potatoes were engineered to synthesize a cholera toxin B subunit (CTB) pentamer with affinity for GMI-ganglioside. Both serum and intestinal CTB-specific antibodies were induced in orally immunized mice. Mucosal antibody titers declined gradually after the last immunization but were restored following an oral booster of transgenic potato. The cytopathic effect of cholera holotoxin (CT) on Vero cells was neutralized by serum from mice immunized with transgenic potato tissues. Following intraileal injection with CT, the plant-immunized mice showed up to a 60% reduction in diarrheal fluid accumulation in the small intestine. Protection against CT was based on inhibition of enterotoxin binding to the cell-surface receptor GMI-ganglioside. These results demonstrate the ability of transgenic food plants to generate protective immunity in mice against a bacterial enterotoxin.     

Impairment of bacterial clearance induced by norepinephrine infusion in rabbits

OBJECTIVE: Purpose of the study was to investigate the potential influence of norepinephrine (NE) on immune functions in terms of systemic and organ-specific bacterial clearance in rabbits. DESIGN: To enable quantification of the clearance process, defined numbers of exogenous Escherichia coli (1.3 x 10(8) CFU) were injected intravenously 60 min after starting the NE infusion at a low dose (1 microgram/kg per min, n = 6), causing an increase (30 mmHg) in mean arterial pressure without affecting the oxygen uptake, and at a higher dose (7.5 micrograms/kg per min, n = 6), resulting in a marked decrease (20%) in oxygen uptake, after infusion of NaCl solution (control, n = 6). In additional experiments (n = 6) NE (1 microgram/kg per min) was tested in endotoxemia induced by simultaneous infusion of endotoxin (40 micrograms/kg per h). Parameters monitored were arterial pressure, oxygen uptake, and rates of bacterial elimination from the blood. At 180 min after E. coli injection, the animals were sacrificed, and tissue samples of liver, kidney, spleen, and lung were collected for bacterial counts. RESULTS: NE infusion resulted in a dose-dependent prolonged elimination of the injected E. coli from the blood and in significantly higher (p < 0.05) numbers of CFU in liver and lung compared to the controls. Significant impairment of bacterial clearance was found after shock-producing endotoxemia, whereas simultaneous infusion of NE and endotoxin caused only a slightly delayed blood clearance of the injected bacteria. CONCLUSION: NE dose dependently affected bacterial clearance, which might be due to ischemia-derived hypoxic impairment of the phagocytosis and lysis function of the reticuloendothelial system, whereas NE improved elimination of bacteria in a state of endotoxic shock.     

Therapy with cefoperazone plus sulbactam against disseminated infection due to cefoperazone-resistant Pseudomonas aeruginosa and Escherichia coli in granulocytopenic mice

Using a granulocytopenic murine model, we evaluated the efficacy of cefoperazone plus sulbactam against disseminated infection due to isolates of beta-lactamase-producing, cefoperazone-resistant (MIC, > or = 50 micrograms/ml) Escherichia coli and Pseudomonas aeruginosa. Both isolates were susceptible in vitro to cefoperazone plus sulbactam (MIC, < or = 6.3 micrograms/ml). Mice rendered granulocytopenic with cyclophosphamide were divided into three groups: group A--infected, untreated mice (controls); group B--infected, cefoperazone-treated mice (700 mg/kg of body weight); and group C--infected, cefoperazone-plus-sulbactam-treated mice (700 mg plus 350 mg). In the E. coli experiment, survival rates in groups A, B, and C were 25, 46, and 73%, respectively. In the experiment with P. aeruginosa, survival rates in groups A, B, and C were 0, 10, and 50%, respectively (P < 0.001). Highly significant differences also were noted for colony counts in the blood, liver, and spleen of group C mice versus group A or B mice in both experiments. Thus, cefoperazone plus sulbactam appears to be a promising combination for the treatment of infections due to certain cefoperazone-resistant gram-negative bacilli, including P. aeruginosa.     

Inactivation of the sapA to sapF locus of Erwinia chrysanthemi reveals common features in plant and animal bacterial pathogenesis

We investigated the role in pathogenesis of bacterial resistance to plant antimicrobial peptides. The sapA to sapF (for sensitive to antimicrobial peptides) operon from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It has five open reading frames that are closely related (71% overall amino acid identity) and are in the same order as those of the sapA to sapF operon from Salmonella typhimurium. An E. chrysanthemi sap mutant strain was constructed by marker exchange. This mutant was more sensitive than was the wild type to wheat alpha-thionin and to snakin-1, which is the most abundant antimicrobial peptide from potato tubers. This mutant was also less virulent than was the wild-type strain in potato tubers: lesion area was 37% that of the control, and growth rate was two orders of magnitude lower. These results indicate that the interaction of antimicrobial peptides from the host with the sapA to sapF operon from the pathogen plays a similar role in animal and in plant bacterial pathogenesis.     

Effect of different combinations of dietary additives on bacterial translocation and survival in gut-derived sepsis

BACKGROUND: Dietary arginine, glutamine, and fish oil each have been shown to improve resistance to infection. The purpose of this study was to assess the potential benefit of different combinations and amounts of these components on bacterial translocation and related mortality during gut-derived sepsis. METHODS: Balb/c mice were fed for 10 days with an AIN-76A diet supplemented with different combinations and percentages of arginine, glutamine, glycine, fish oil, and medium-chain triglycerides. Controls were fed a complete AIN-76A diet or chow. After 10 days of feeding, all animals were transfused. On day 15, the animals were gavaged with 10(10) 111In-radiolabeled or unlabeled Escherichia coli and given a 30% burn injury. Animals gavaged with unlabeled bacteria were observed for survival (n = 317). Groups that showed the best survival as well as control groups were gavaged with labeled bacteria and killed 4 hours postburn (n = 60) for harvest of mesenteric lymph nodes, liver and spleen. RESULTS: Mice fed diets enriched with 5% fish oil + 2% arginine, 2% arginine + 2% glutamine, or 5% fish oil + 2% glutamine had higher survival than control groups. The animals fed fish oil+glutamine had significantly reduced translocation to the liver and spleen. Animals fed arginine+glutamine had an enhanced ability to kill translocated organisms in the liver compared with other groups. Fish oil+arginine improved both barrier function and microbial killing. CONCLUSIONS: Feeding with arginine+glutamine, fish oil+arginine, or fish oil+glutamine supplemented diets positively affects the outcome in a gut-derived sepsis model.     

Immunogenicity of recombinant Escherichia coli expressing the omp31 gene of Brucella melitensis in BALB/c mice

BALB/c mice were immunized with recombinant Escherichia coli expressing the omp31 gene of Brucella melitensis, a gene coding for a major outer membrane protein. Immunization resulted in the production of specific antibodies to B. melitensis in the serum, the production of which was considerably increased after boosting with a dose ten times lower than the first. A significant specific proliferative response of immune spleen cells to B. melitensis was observed 5 weeks after the first immunization but this response did not persist. Despite the induction of systemic humoral and cellular immune responses by recombinant E. coli expressing the B. melitensis omp31 gene, no significant protection against a challenge with smooth B. melitensis H38S was observed in immunized mice. These results demonstrate that despite the strong antibody response induced in mice, immunization with the recombinant Omp31 of B. melitensis does not confer any protective effect against a virulent smooth B. melitensis. However, its potential protective effect for protection against rough Brucella would be worth testing.     

IFN-gamma decreases translocation and improves survival following transfusion and thermal injury

The effects of recombinant murine interferon-gamma (rmIFN-gamma) on survival and host defense were studied during gut-derived sepsis that included transfusion-induced immunosuppression. Balb/c mice (n = 153) were transfused with allogeneic blood and then treated with different doses of rmIFN-gamma: 10, 100, 1000, 10,000 U, or sterile saline as control once daily for 3 days. Five days after transfusion they were gavaged with 10(10) Escherichia coli and given a 20% TBSA burn injury. Survival was significantly higher in groups receiving 10 U compared to control and the group receiving 10,000 U (P < 0.0001 and P = 0.02, respectively). Groups receiving 100 or 1000 U also showed an improvement of survival compared to nontreated control animals (P = 0.02). The effect of rmIFN-gamma on the degree of translocation and the host's ability to kill translocated organisms was also investigated. Mice were treated as described above, except they were gavaged with 111In oxine-labeled E. coli and then subjected to a 20% TBSA burn. Mesenteric lymph nodes (MLN), liver, and spleen were harvested aseptically. Less translocation to the liver was observed compared to the nontreated group (P = 0.002) to the MLNs and spleen of the group treated with 100 U rmIFN-gamma compared to controls and the group treated with 10 U (P < 0.005). Animals receiving 1000 U showed fewer bacteria in the liver and spleen compared to the control group (P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)     

Tacrolimus therapy for persistent or recurrent acute rejection after lung transplantation

Purified native F1 antigen from Yersinia pestis was used to assess controlled-release vaccine delivery systems in poly(lactide-co-glycolide) (PLG) microparticles and liposomes. Antigen encapsulated in PLG microparticles induced high serum titres when injected i.p. in mice: mucosal IgA was also detected. Mice immunized with F1 in Alhydrogel or PLGs were protected against subcutaneous challenge with Y. pestis. F1 antigen surface-labelled onto liposome vesicles stimulated high serum titres in Balb/c mice and also induced a mucosal response: F1-labelled liposomes protected mice against challenge with up to 1 x 10(5) organisms. These findings indicate that a significant immune response is induced by immunizing with F1 formulated in PLGs and liposomes and that protection was achieved after only one dose.