Iron-cofactored superoxide dismutase inhibits host responses to Mycobacterium tuberculosis.

Superoxide dismutase (SOD) is a ubiquitous metalloenzyme in aerobic organisms that catalyzes the conversion of superoxide anion to hydrogen peroxide. Mycobacterium tuberculosis is unusual in that it secretes large quantities of iron-cofactored SOD. To determine the role of SOD in pathogenesis, we constructed mutants of M. tuberculosis H37Rv with reduced SOD production. Compared with controls, SOD-diminished isolates were more susceptible to killing by hydrogen peroxide. The isolates were markedly attenuated, exhibiting nearly 100,000-fold fewer bacilli than virulent control strains in the lungs and spleens of C57BL/6 mice 4 wk after intravenous inoculation. In the lung, SOD-attenuated M. tuberculosis induced robust interstitial mononuclear cell infiltration within 24 h and many cells were apoptotic by TUNEL staining, whereas virulent H37Rv exhibited minimal early inflammatory response and only rare interstitial mononuclear cell apoptosis. During prolonged infections, C57BL/6 mice tolerated SOD-attenuated M. tuberculosis better than BCG, exhibiting 68% greater weight gain, quicker eradication of bacilli from the spleen, and less alveolar lung infiltration. These results establish the importance of SOD in the pathogenesis of tuberculosis. Its effect appears to be mediated in part by inhibiting innate host immune responses, including early mononuclear cell infiltration of infected tissues and apoptosis.

Expression of an antisense hla fragment in Staphylococcus aureus reduces alpha-toxin production in vitro and attenuates lethal activity in a murine model.

Isogeneic bacterial strains that differ only in the production of a single microbial factor have been invaluable in studying the pathogenesis of bacterial infections. The targeted, intentional inactivation of a gene encoding a potential virulence determinant generally requires homologous recombination to replace the gene with an inactivated allele. To determine whether the insertion and expression of a fragment of a bacterial gene in an antisense orientation could be used as a rapid alternative to allelic inactivation for producing paired isogeneic isolates, we inverted a 600-bp fragment of the Staphylococcus aureus gene encoding alpha-toxin, hla, behind its native promoter on an Escherichia coli-S. aureus shuttle vector. A transformant of an S. aureus strain carrying the antisense hla fragment produced antisense hla RNA and made 16-fold less alpha-toxin than either its parent or an isogeneic transformant containing vector DNA without hla. Also, intraperitoneal injection of 1.5 x 10(9) CFU of the antisense hla-containing transformant was significantly less lethal in a murine model than that of the parent (1 of 10 versus 7 of 10 mice expired [P < 0.02]) or the transformant without hla (1 of 10 versus 7 of 7 mice expired [P < 0.001]). We conclude that the expression of a fragment of hla in an antisense orientation in S. aureus on a plasmid vector reduces alpha-toxin production and the lethal activity of the strain in a murine model. The antisense strategy for creating isogeneic strains of bacteria may facilitate molecular investigations into the pathogenesis of infection. It also may be useful in creating novel live-attenuated strains of bacteria for use as vaccine candidates.

Booster response to acellular pertussis vaccine in children primed with acellular or whole cell vaccines.

Few data are available on the effect of a booster dose of acellular pertussis vaccine in children primed as infants with acellular vaccine. We administered acellular pertussis vaccine (ACV) at 19 months to children immunized in infancy with ACV or whole cell vaccine. Forty-one infants had been randomly assigned to receive either ACV or whole cell vaccine at 2, 4 and 6 months of age. Antibody titers to pertussis toxin and filamentous hemagglutinin were significantly higher in ACV than whole cell vaccine recipients at 7 months; at 15 months antibody to filamentous hemagglutinin (but not pertussis toxin) remained significantly higher among those receiving ACV. At 19 months all 41 children received an ACV booster. Local and systemic reactions were few and minor and were equally distributed between the two groups. All children responded to booster with significant increases in antibody; these increases tended to be greater for those having been primed with ACV. ACV booster immunization appears safe and immunogenic, regardless of the vaccine given for primary immunization.

An antimelanoma-barley ribosome inactivating protein conjugate is cytotoxic to melanoma cells in vitro.

The ribosome inactivating protein (BRIP) from barley is a single polypeptide chain (Mr = 32,000 Dalton) and is nontoxic to intact cells. The BRIP has been purified to homogeneity by modifications of the methods of Roberts and Selitrennikoff and crosslinked to monoclonal antibodies by Succinimidyl-3 (2- Pyridyldithio) -Propionate (SPDP) and by the cystamine-EDAC methods. The resulting hybrids were purified from the free BRIP by gel filtration on a Sephadex G-75 column. The model suicide transport agents were assayed against melanoma cells; K-562 cells were used as control. The hybrids were found to be selectively toxic to melanoma cells in a dose dependent manner.

A possible role for protein kinase C activity but not cyclic nucleotide-dependent protein kinases in human natural killer cell lytic activity.

Recent evidence suggests that a Ca++, phospholipid, diacylglycerol-dependent protein kinase, protein kinase C, plays a role in the activation of cytotoxic T lymphocytes by target cells. In this investigation we have examined the role of protein kinase C in human NK cell-mediated cytolysis of K-562 cells. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) inhibited human NK cell-mediated cytolysis in a dose dependent manner. On the other hand, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a specific inhibitor of cyclic nucleotide dependent protein kinases had no effect on human NK cell-mediated cytolysis of K562 cells. There is little or no effect on protein synthesis or N-glycosylation activity in human NK cells by H-7. The relative inhibitory ability of the two inhibitors suggest that protein kinase C, acting synergistically with Ca++ mobilization, plays a role in the early stages of human NK cell-mediated cytolysis of K562 target cells.

Evaluation of a new highly purified pertussis vaccine in infants and children.

Purified acellular pertussis vaccine (12.5 micrograms of lymphocytosis promoting factor [LPF] and 12.5 micrograms of filamentous hemagglutinin [FHA]) was compared with conventional pertussis vaccine in a randomized double-blind study involving 40 children aged 4-6 y, 40 children aged 18-24 mo, and 50 infants. Increases in antibody were significantly greater among recipients of acellular vaccine than among recipients of conventional vaccine for antibodies to LPF in all age groups and for antibodies to FHA in infants and children aged 4-6 y; the increase in FHA antibody was also greater with acellular vaccine among children aged 18-24 mo but not significantly so. Compared with conventional vaccine, acellular vaccine was significantly associated with reduced frequency of leg pain and fretfulness at all ages and less frequent fever and anorexia at some ages. The reduced reaction rates and comparable or enhanced immunogenicity of the acellular vaccine make it an attractive candidate for larger field trials, particularly among infants.