Role of gamma interferon inToxoplasma gondii infection

Toxoplasma gondii has emerged as an important pathogen in the ever increasing numbers of patients with disorders of the immune system. Better understanding of the mechanisms of resistance of the host against this protozoan is important for development of safe, effective alternative treatment regimens for toxoplasmosis. Gamma interferon is the cytokine that plays a central role in protection againstToxoplasma gondii. The purpose of this review is to highlight the current knowledge of the role of gamma interferon inToxoplasma gondii infection.     

Acute Bordetella pertussis infection in an adult.

Bordetella pertussis was transmitted from an immunized boy to his father and possibly to other family members. This case report demonstrates that although unusual, acute adult B. pertussis infection can occur. B. pertussis immunization may not prevent infection but can reduce the severity. B. pertussis was detected in sputum from the adult by direct-fluorescent antibody staining and was grown on Regan-Lowe medium. Serology tests confirmed the infection in the adult.     

Role of insulin in the hypoglycaemic effect of sublethal Bordetella pertussis infection in mice.

Mice were infected intranasally with a sub-lethal dose of Bordetella pertussis organisms (1.2 x 10(5) colony forming units per mouse), control animals receiving the vehicle intranasally. The experiments were performed 14 days later. Serum glucose and insulin concentrations were studied across a 24 h period in freely fed animals and the changes in serum glucose and insulin concentrations in response to feeding were examined in mice fasted for 18 h. The responsiveness of mice to injected insulin (0.5 and 5.0 units/kg i.v.) was also examined. Pertussis-infected mice developed hypoglycaemia and hyperinsulinaemia relative to the controls. These changes were present across a 24 h period although the magnitude of the differences between values seen in control and infected animals varied and the hyperinsulinaemia was not seen at all times. Infected mice showed a markedly diminished hyperglycaemia and an exaggerated hyperinsulinaemia following food ingestion, relative to normal controls. Pertussis-induced hypoglycaemia was abolished following destruction of the pancreatic islet B cells with alloxan (80 mg/kg i.v.). The serum glucose response to a low dose of insulin was significantly attenuated by B. pertussis infection although the hypoglycaemia produced by a high dose was prolonged. It was concluded that B. pertussis infection-induced hypoglycaemia was secondary to hyperinsulinaemia, possibly caused by an exaggerated insulin secretory response to food intake.     

Role of gamma interferon during infection with Plasmodium chabaudi chabaudi.

A role has been proposed for inflammatory mediators such as gamma interferon (IFN-gamma) and reactive oxygen intermediates in the control of the blood stages of Plasmodium organisms. It was previously shown that IFN-gamma can be detected in the plasma of mice with a primary infection by Plasmodium chabaudi chabaudi (AS). We found that susceptible and other resistant mouse strains produced IFN-gamma, suggesting that susceptibility is not due to a defect in IFN-gamma production. Administration of IFN-gamma to intact C57BL/6 mice slightly decreased and partially delayed parasitemia, whereas in vivo depletion of IFN-gamma through injection of a "cocktail" of monoclonal antibodies against IFN-gamma exacerbated infection. Since CD4+ T cells are essential for the development of a protective immune response to P. chabaudi chabaudi, we tested whether CD4+ T cells are responsible for IFN-gamma production in vivo and whether exogenous IFN-gamma can replace the protective function of the CD4+ T cells. Mice depleted of CD4+ T cells were unable to produce IFN-gamma, but factors in addition to IFN-gamma may be important in parasite clearance.     

Identification of Bordetella pertussis infection by shared-primer PCR.

A shared-primer PCR method for the detection of infection was developed by using primers derived from DNA sequences upstream of the structural genes for the porin proteins of Bordetella pertussis and Bordetella parapertussis. This method resulted in a 159-bp PCR product specific for B. pertussis and a 121-bp DNA fragment specific for B. parapertussis and allowed for the simultaneous detection of these pathogens. The PCR procedure was shown to be very specific since no PCR product was obtained from 36 non-Bordetella bacterial DNAs. Nasopharyngeal aspirates (NPAs) from children suspected of having pertussis were evaluated by the PCR method, culture, and the Chinese hamster ovary (CHO) cell assay, which detects pertussis toxin. B. pertussis was cultured from 119 of 205 NPAs assayed, and the presence of pertussis toxin was detected in 69 of the NPAs by the CHO cell assay. When ethidium bromide staining was used to detect PCR products, 100 NPAs gave positive results by shared-primer PCR; 94 of these NPAs were also positive by culture. The result indicated a sensitivity of 79% for PCR when culture was used as the standard. The sensitivity of PCR was increased to 95% when a digoxigenin immunoblot system was used. An additional 20 NPAs from patients with suspected pertussis that were culture negative also gave positive results by PCR. The specific and sensitive PCR method described here should be useful for both the clinical diagnosis of pertussis and case identification in vaccine trials.     

Mechanism of pertussis toxin B oligomer-mediated protection against Bordetella pertussis respiratory infection.

Immunization with the B oligomer of pertussis toxin protected neonatal mice from a lethal respiratory challenge with Bordetella pertussis. All mice immunized with 8 micrograms of B oligomer survived aerosol challenge and had peripheral leukocyte counts and weight gains similar to those of mice immunized with pertussis toxoid before challenge and to those of control mice that were not challenged. Unprotected mice challenged with an aerosol of B. pertussis had an increase in peripheral leukocyte count, failed to gain weight, and died within 21 days of challenge. Protection appeared to be dose dependent, since a dose of 1 microgram of B oligomer per mouse prevented death in 100% of the mice challenged with B. pertussis, whereas 0.4 micrograms of B oligomer protected 50% of the challenged mice. Mice immunized with the B oligomer had increases in immunoglobulin G (IgG) anti-B oligomer in sera and in IgG and IgA anti-B oligomer in bronchoalveolar lavage fluids 1 to 3 weeks after respiratory challenge. Specific anti-B oligomer antibodies could not be detected in unimmunized, infected mice at the same time after challenge. Intravenous administration of the monoclonal antibody 170C4, which binds to the S3 subunit of the B oligomer, protected neonatal mice from B. pertussis respiratory challenge, while administration of an IgG1 anti-tetanus toxin monoclonal antibody, 18.1.7, was not protective. We conclude that anti-B-oligomer-mediated neutralization of pertussis toxin is one mechanism of protection in the mouse model of B. pertussis aerosol challenge.     

Role of insulin in the hypoglycaemic effect of sublethal Bordetella pertussis infection in mice

Mice were infected intranasally with a sub-lethal dose of Bordetella pertussis organisms (1.2 x 10(5) colony forming units per mouse), control animals receiving the vehicle intranasally. The experiments were performed 14 days later. Serum glucose and insulin concentrations were studied across a 24 h period in freely fed animals and the changes in serum glucose and insulin concentrations in response to feeding were examined in mice fasted for 18 h. The responsiveness of mice to injected insulin (0.5 and 5.0 units/kg i.v.) was also examined. Pertussis-infected mice developed hypoglycaemia and hyperinsulinaemia relative to the controls. These changes were present across a 24 h period although the magnitude of the differences between values seen in control and infected animals varied and the hyperinsulinaemia was not seen at all times. Infected mice showed a markedly diminished hyperglycaemia and an exaggerated hyperinsulinaemia following food ingestion, relative to normal controls. Pertussis-induced hypoglycaemia was abolished following destruction of the pancreatic islet B cells with alloxan (80 mg/kg i.v.). The serum glucose response to a low dose of insulin was significantly attenuated by B. pertussis infection although the hypoglycaemia produced by a high dose was prolonged. It was concluded that B. pertussis infection-induced hypoglycaemia was secondary to hyperinsulinaemia, possibly caused by an exaggerated insulin secretory response to food intake.     

Aerosol infection of mice with Bordetella pertussis

Aerosol inhalation of Bordetella pertussis Tohama phase I resulted in a reproducible and uniform infection of mice (strain DDY or ICR). Mice in groups of 10 exposed for 30 min to aerosols generated from bacterial suspensions of 10(9) and 10(10) organisms per ml resulted in mean bacterial counts of 2.3 (+/- 0.3) X 10(4) and 1.0 (+/- 0.3) X 10(5) colony-forming units, respectively, in the lung of each animal. Subsequent studies using a 30-min aerosol inoculation of ICR mice with 2 X 10(9) bacterial cells per ml showed: (i) B. pertussis cells reached a maximum of about 10(7) colony-forming units per lung 14 days after inhalation. (ii) Deaths (10 to 100%, depending on mouse age) occurred 10 to 14 days after exposure. (iii) The lung weight and the leukocyte count increased from basal values of 100 mg and 10(4) leukocytes per mm3 to a plateau of 950 mg and 1.95 X 10(5) leukocytes per mm3, respectively, 14 days after challenge. (iv) There was a significantly reduced rate of body weight gain by infected mice compared to noninfected mice. (v) With mortality as the criterion for disease, susceptibility varied with the age of mice as follows: 10 days old greater than 18 greater than 28 greater than 49. (vi) Bacteria were associated with ciliated respiratory epithelial cells by scanning electron microscopy.     

Role of antibodies against Bordetella pertussis virulence factors in adherence of Bordetella pertussis and Bordetella parapertussis to human bronchial epithelial cells

Immunization with whole-cell pertussis vaccines (WCV) containing heat-killed Bordetella pertussis cells and with acellular vaccines containing genetically or chemically detoxified pertussis toxin (PT) in combination with filamentous hemagglutinin (FHA), pertactin (Prn), or fimbriae confers protection in humans and animals against B. pertussis infection. In an earlier study we demonstrated that FHA is involved in the adherence of these bacteria to human bronchial epithelial cells. In the present study we investigated whether mouse antibodies directed against B. pertussis FHA, PTg, Prn, and fimbriae, or against two other surface molecules, lipopolysaccharide (LPS) and the 40-kDa outer membrane porin protein (OMP), that are not involved in bacterial adherence, were able to block adherence of B. pertussis and B. parapertussis to human bronchial epithelial cells. All antibodies studied inhibited the adherence of B. pertussis to these epithelial cells and were equally effective in this respect. Only antibodies against LPS and 40-kDa OMP affected the adherence of B. parapertussis to epithelial cells. We conclude that antibodies which recognize surface structures on B. pertussis or on B. parapertussis can inhibit adherence of the bacteria to bronchial epithelial cells, irrespective whether these structures play a role in adherence of the bacteria to these cells.     

Bordetella pertussis infection in northern Taiwan, 1997-2001.

The clinical presentations of laboratory-confirmed Bordetella pertussis infection in Chang Gung Children's Hospital during 1997 and 2001 were analyzed. Of the 46 cases, 25 (54.3%) were male. The patients ages ranged from 24 days to 37 years, with a mean and median of 4.3 years and 10.5 years, respectively. Forty four patients had vaccination records, among them 23 patients (52.2%) had received > or = 3 doses of pertussis vaccine. Of the patients who were partially vaccinated (received 1 or 2 doses vaccine) or unvaccinated, 16 (69.6%) presented with whooping cough, 5 (22.2%) with post-tussive vomiting, and 13 (59.1%) with cyanosis. Leukocytosis (white blood cells > or = 15,000 cells/microL) and lymphocytosis (lymphocytes > or = 10,000 cells/microL) were observed in 17 (47.2%) and 16 (44.4%) of the patients, respectively. Fourteen patients (30.4%) developed complications, among which pneumonia was the most common (92.3%). Among infants < or = 1 year of age, 95.2% were partially vaccinated (20/21), compared with 5% (1/20) of the patients > 1 year of age (p<0.05). The overall complication rate was 37.5%, compared with 18.2% for patients > 1 year of age (p<0.05). One 2-month-old patient required ventilatory support after the development of cardiopulmonary failure. There was no mortality in this study. In summary, pertussis most commonly occurred in infants who were unvaccinated or partially vaccinated. These patients usually presented with atypical symptoms such as cyanosis or apnea. The importance of vaccination still cannot be overemphasized because immunized patients usually present with milder disease than those who are not immunized.     

Orally administered microencapsulated Bordetella pertussis fimbriae protect mice from B. pertussis respiratory infection.

Fimbriae from Bordetella pertussis have been encapsulated in poly(lactide-co-glycolide) microparticles of a size appropriate for uptake by the immune inductive tissues of the gastrointestinal tract. Mice were immunized by oral gavage with a single dose of 10 micrograms of microencapsulated fimbriae. The resulting immune responses were compared with those resulting from intraperitoneal injection of mice with equivalent amounts of fimbriae absorbed onto alhydrogel. The examination of serum and mucosal secretions, collected over a 6-week period, for specific antifimbrial antibodies clearly demonstrated that only orally immunized animals mounted measurable immune responses in external secretions. Six weeks after immunization, all immunized animals were protected against intranasal challenge with live B. pertussis.     

Immune suppression and induction of gamma interferon by pertussis toxin.

It has been suggested that pertussis toxin is a virulence factor of Bordetella pertussis. Although extracts enriched in pertussis toxin activity have been reported to enhance immune responsiveness, other studies have demonstrated a suppressive ability, suggesting that the toxin may contribute to the virulence of B. pertussis through mechanisms involving immune suppression. We report that purified pertussis toxin suppressed the in vitro immunoglobulin M antibody response of mouse splenocytes to sheep erythrocytes. At submitogenic doses, the toxin also suppressed [3H]thymidine incorporation by splenocytes, suggesting that it interfered with antibody formation by inhibiting lymphocyte proliferation. Antiviral activity was detected in culture supernatants obtained from pertussis toxin-suppressed splenocyte cultures by using a cytopathic effect inhibition assay. This antiviral activity was virus nonspecific, sensitive to pH 2.0 treatment, stable to heating at 56 degrees C, and neutralized by anti-gamma interferon antiserum. Finally, the fractionation of splenocytes by anti-immunoglobulin panning techniques suggested that Lyt2+ lymphocytes proliferated in response to pertussis toxin and produced interferon. Our results suggest that pertussis toxin may contribute to the virulence of B. pertussis through stimulation of Lyt2+ lymphocytes, resulting in the induction of gamma interferon and the subsequent inhibition of the primary antibody response.     

Isotype and antigen specificity of pertussis agglutinins following whole-cell pertussis vaccination and infection with Bordetella pertussis.

Elevated agglutinin titers have been shown to correlate with protection from disease following whole-cell pertussis vaccination, but the isotype and antigen specificity of human agglutinating antibodies is unknown. In 13 immunoassays, immunoglobulin G antifimbria antibodies had the strongest correlation with agglutinin titers following culture-proven infection with Bordetella pertussis (R' = 0.79; P < 0.0001) and following whole-cell pertussis vaccination (R' = 0.87, P < 0.0001).     

Role of carbohydrate recognition domains of pertussis toxin in adherence of Bordetella pertussis to human macrophages.

Pertussis toxin (PT) and filamentous hemagglutinin can each mediate the association of Bordetella pertussis with human macrophages. Adherence via filamentous hemagglutinin leads to integrin-mediated entry and survival of the bacteria within the human cell. We determined the contribution of PT to bacterial adherence to human macrophages. Plating macrophages on wells coated with recombinant PT subunit 2 (S2) or S3 decreased PT-dependent bacterial binding by greater than 60%; S1, S4, and S5 were ineffective. S3-dependent adherence was reduced 63% +/- 8% by sialic acid, while S2-dependent adherence was reduced 53% +/- 11% by galactose. Loss of the carbohydrate recognition properties of S2 by deletion of residues 40 to 54 or site-specific mutations at Asn-93, His-47, or Arg-50 eliminated the ability of the subunit protein to competitively inhibit bacterial binding. Peptides corresponding to residues 28 to 45 of S2 and S3 competitively inhibited adherence. Treatment of macrophages with antibodies to Le(a) or Le(x) but not CD14, CD15, CD18, or HLA interfered with PT-mediated binding. Exposure of the macrophages to the B oligomer, S2, or S3 increased binding to the CD11b/CD18 integrin. These results indicate that the carbohydrate recognition domains of both S2 and S3 participate in adherence of B. pertussis to human macrophages. The PT receptor(s), as yet unidentified, appears to carry the Le(a) or Le(x) determinants and is functionally capable of modulating integrin-mediated binding to the macrophage.     

Role of gamma interferon in induction of natural killer activity by Legionella pneumophila in vitro and in an experimental murine infection model.

Legionella pneumophila has been shown to induce gamma interferon (IFN-gamma) both in vitro and in vivo during experimental infections of mice. With complement-mediated serologic depletion of murine splenocytes, the cellular sources of IFN-gamma following in vitro stimulation with L. pneumophila antigens were Thy-1.2+, Lyt-2-, L3T4-, and asialo-GM1+, which is consistent with the natural killer (NK) cell phenotype. Additionally, Percoll density discontinuous centrifugation demonstrated that maximal production of IFN coincided with high NK activity in fractions which were enriched for large granular lymphocytes. Furthermore, 18- to 24-h incubation of splenocytes with L. pneumophila whole-cell vaccine resulted in augmented NK cytotoxic activity against YAC-1 tumor target cells in a 51Cr release assay. The addition of macrophages to purified large granular lymphocyte populations augmented both IFN-gamma production and NK activity, suggesting that antigen is required for optimal responses. In an experimental infection model using an intratracheal inoculation route, NK activity was enhanced in the spleen, peripheral blood, and lung cells of infected mice, with maximal stimulation in the lung leukocytes at the site of infection. The results of the present study indicate that NK cells respond in vivo and in vitro to stimulation by L. pneumophila by producing IFN-gamma and by increased cytolytic activity.     

Lethal infection by Bordetella pertussis mutants in the infant mouse model.

Different aspects of lethal infection of infant mice with Bordetella pertussis were examined. Mutants deficient in vir-regulated genes were tested for the ability to cause a lethal infection in the infant mouse model. Adenylate cyclase toxin-hemolysin and pertussis toxin were required to cause a lethal infection at low doses. Mixed infection caused by challenging the mice with an equal number of pertussis toxin and adenylate cyclase toxin-hemolysin mutants at a dose at which neither alone was lethal was also unable to cause a lethal infection. Production of the filamentous hemagglutinin and the dermonecrotic toxin was not required to cause a lethal infection. Nine other mutants in vir-regulated genes whose phenotypes have yet to be determined were also tested. Only two of these mutants were impaired in the ability to cause a lethal infection. Expression of fimbriae does not appear to affect the dose required to cause a lethal infection; however, fimbrial expression was correlated with the later stages of a nonlethal, persistent infection. Growth of the bacteria in MgSO4, a condition which reversibly suppresses expression of the genes required for virulence, did not alter the ability of the bacteria to cause a lethal infection. Auxotrophic mutants deficient in leucine biosynthesis were as virulent as the parental strain; however, mutants deficient in methionine biosynthesis were less virulent. A B. parapertussis strain was much less effective in promoting a lethal infection than any of the wild-type B. pertussis strains examined. A persistent infection in the lungs was observed for weeks after challenge for mice given a sublethal dose of B. pertussis, and transmission from infected infants to the mother was never observed.     

Resistance to adenovirus infection after administration of Bordetella pertussis vaccine in mice.

Treatment of mice with Bordetella pertussis vaccine rendered mice resistant to mouse adenovirus infection. The resistant state took at least 5 days to develop, and susceptibility returned to a portion of the test population 35 days after treatment. Transient resistance developed in congenitally athymic mice also. Treatment with a dose of 25 micrograms (dry weight) of B. pertussis vaccine protected approximately 50% of the test population. Vaccines prepared from several different strains of B. pertussis were capable of inducing resistance, and the induction of resistance was not dependent on the mouse strain used for testing. Cross-reacting antibodies capable of neutralizing the virus or protecting against a challenging infection were not induced by treatment with B. pertussis vaccine.