Characterization of BrkA Expression in Bordetella bronchiseptica

BrkA confers resistance to killing by complement in Bordetella pertussis. Complement resistance in Bordetella bronchiseptica was examined. Four B. bronchiseptica strains possessed the brkA gene; however, only three expressed the protein. Only the strain lacking BrkA was susceptible to complement. Introduction of the B. pertussis brkA gene restored BrkA expression to this strain but did not confer resistance. brkA was mutated in the strains that naturally expressed BrkA, and loss of BrkA did not confer sensitivity to complement. As a species, B. bronchiseptica is more resistant to complement than B. pertussis, and BrkA does not mediate resistance.     

Bb2Bb3 regulation of murine Lyme arthritis is distinct from Ncf1 and independent of the phagocyte nicotinamide adenine dinucleotide phosphate oxidase

Several quantitative trait loci regulating murine Lyme arthritis severity have been mapped, including a highly significant linkage found on chromosome 5, termed Bb2Bb3. Within this region, the Ncf1 gene of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has recently been identified as a major regulator of arthritis severity in rodent models of rheumatoid arthritis, an effect attributed to protective properties of reactive oxygen species. To assess the role of Ncf1 in Lyme arthritis, we introgressed Bb2Bb3 from severely arthritic C3H/He mice onto mildly arthritic C57BL/6 mice. This increased Lyme arthritis severity, whereas the reciprocal transfer conferred protection from disease. A single nucleotide polymorphism was identified in the Ncf1 gene that did not influence the protein sequence or expression of Ncf1. Although polymorphonuclear leukocytes from C57BL/6 mice generated a greater oxidative burst than polymorphonuclear leukocytes from C3H/He mice, studies with the Bb2Bb3 congenic mice demonstrated this difference was not linked to Ncf1 alleles. Furthermore, Lyme arthritis severity was not altered in mice lacking either the Ncf1 or Gp91phox subunits of the NADPH oxidase complex. Together, these results argue that Ncf1 is not a candidate gene for regulation of Lyme arthritis and reveal Lyme arthritis to be independent of NADPH oxidase activity, distinguishing it from other models of rheumatoid arthritis.     

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.     

Determinants of activation by complement of group II phospholipase A2 acting against Escherichia coli.

Prompt killing of many strains of Escherichia coli during phagocytosis in vitro by isolated polymorphonuclear leukocytes (PMN) requires the presence of nonlethal doses of nonimmune serum (B. A. Mannion, J. Weiss, and P. Elsbach, J. Clin. Invest. 86:631-641, 1990). Because this requirement is bypassed in a phospholipase A (PLA)-rich mutant (pldA ) of E. coli, we have examined the effect of serum on bacteria] phospholipid (PL) degradation during phagocytosis of wild-type (pldA+) and PLA-deficient (pldA) E. coli. In parallel with increased killing, nonlethal doses of serum increased the degradation of prelabeled bacterial PL during phagocytosis by two- to fivefold, to nearly the same levels (ca. 50 to 60%) as those produced during phagocytosis of E. coli pldA in the absence of serum. The effects on the E. coli pldA mutant imply that there is a serum-mediated enhancement of granule-associated group II PMN PLA2 activity. At the same doses, serum promoted action against E. coli in the presence of purified rabbit and human group II PLA2 but did not activate bacterial PLA. Related PLA2s that lack specific structural determinants needed for optimal activity against E. coli treated with the bactericidal/permeability-increasing protein (BPI) of PMN are also less active than wild-type group II PLA2 against serum-treated E. coli. Treatment of E. coli with C7- or C9-depleted serum did not enhance bacterial killing or PL degradation during phagocytosis or the action of purified PLA2. In summary, these findings suggest that (i) nonlethal assemblies of the membrane attack complex promote intracellular killing and destruction of E. coli ingested by PMN, in part by promoting the action of granule-associated PLA2 against ingested bacteria, and (ii) structural determinants first implicated in PLA2 action against BPI-treated E. coli are also important in PLA2 action in concert with other host defense systems, such as complement.     

Tumours of histiocytes and accessory dendritic cells: an immunohistochemical approach to classification from the International Lymphoma Study Group based on 61 cases

Neoplasms of histiocytes and dendritic cells are rare, and their phenotypic and biological definition is incomplete. Seeking to identify antigens detectable in paraffin-embedded sections that might allow a more complete, rational immunophenotypic classification of histiocytic/dendritic cell neoplasms, the International Lymphoma Study Group (ILSG) stained 61 tumours of suspected histiocytic/dendritic cell type with a panel of 15 antibodies including those reactive with histiocytes (CD68, lysozyme (LYS)), Langerhans cells (CD1a), follicular dendritic cells (FDC: CD21, CD35) and S100 protein. This analysis revealed that 57 cases (93%) fit into four major immunophenotypic groups (one histiocytic and three dendritic cell types) utilizing six markers: CD68, LYS, CD1a, S100, CD21, and CD35. The four (7%) unclassified cases were further classifiable into the above four groups using additional morphological and ultrastructural features. The four groups then included: (i) histiocytic sarcoma (n=18) with the following phenotype: CD68 (100%), LYS (94%), CD1a (0%), S100 (33%), CD21/35 (0%). The median age was 46 years. Presentation was predominantly extranodal (72%) with high mortality (58% dead of disease (DOD)). Three had systemic involvement consistent with 'malignant histiocytosis'; (ii) Langerhans cell tumour (LCT) (n=26) which expressed: CD68 (96%), LYS (42%), CD1a (100%), S100 (100%), CD21/35 (0%). There were two morphological variants: cytologically typical (n=17) designated LCT; and cytologically malignant (n=9) designated Langerhans cell sarcoma (LCS). The LCS were often not easily recognized morphologically as LC-derived, but were diagnosed based on CD1a staining. LCT and LCS differed in median age (33 versus 41 years), male:female ratio (3.7:1 versus 1:2), and death rate (31% versus 50% DOD). Four LCT patients had systemic involvement typical of Letterer-Siwe disease; (iii) follicular dendritic cell tumour/sarcoma (FDCT) (n=13) which expressed: CD68 (54%), LYS (8%), CD1a (0%), S100 (16%), FDC markers CD21/35 (100%), EMA (40%). These patients were adults (median age 65 years) with predominantly localized nodal disease (75%) and low mortality (9% DOD); (iv) interdigitating dendritic cell tumour/sarcoma (IDCT) (n=4) which expressed: CD68 (50%), LYS (25%), CD1a (0%), S100 (100%), CD21/35 (0%). The patients were adults (median 71 years) with localized nodal disease (75%) without mortality (0% DOD). In conclusion, definitive immunophenotypic classification of histiocytic and accessory cell neoplasms into four categories was possible in 93% of the cases using six antigens detected in paraffin-embedded sections. Exceptional cases (7%) were resolvable when added morphological and ultrastructural features were considered. We propose a classification combining immunophenotype and morphology with five categories, including Langerhans cell sarcoma. This simplified scheme is practical for everyday diagnostic use and should provide a framework for additional investigation of these unusual neoplasms.     

No direct role for Epstein-Barr virus in American hepatocellular carcinoma

Epstein-Barr virus (EBV) was recently linked to hepatocellular carcinogenesis in Japanese patients. It is not clear whether EBV infection is also associated with hepatocellular carcinoma (HCC) occurring in American patients. We studied 41 cases of HCC from the Los Angeles area for evidence of EBV infection by in situ hybridization, immunohistochemistry, and polymerase chain reaction methods. Of 41 cases, 16 were seropositive for hepatitis B virus surface antigen (39%), 9 of 29 tested were seropositive for hepatitis C virus antibody (31%); in total, 22 cases were seropositive for hepatitis B virus and/or hepatitis C virus (53%). Of 41 cases, 1 was positive for EBV-encoded small nonpolyadenylated RNA (EBER)-1 (2%) by in situ hybridization. By immunohistochemistry, two cases were positive for EBV nuclear antigen (EBNA)-1 (5%), one was positive for the transactivating immediate early BZLF1 (ZEBRA) (2%), and none was positive for latent membrane protein-1. None of the 41 cases was positive for latent membrane protein-1 and EBV nuclear antigen (EBNA)-4 DNAs by polymerase chain reaction assay. All four positive cases showed rare EBER-1-, ZEBRA-, or EBNA-1- positive cells (<0.1%); in none of these cases was there expression of any other EBV viral genes. In the one case each that was positive for EBER-1 and ZEBRA, both of which occurred in patients of non-Asian ethnicity, the staining was limited to infiltrating small lymphocytes, and tumor cells were negative. In the two cases that were positive for EBNA-1, both of which occurred in patients of Asian ethnicity, the staining was limited to tumor cells, and infiltrating small lymphocytes were negative. Our study indicates that rare cases of American HCC may contain EBV-infected cells, but it is unlikely that EBV plays a major role in the carcinogenesis of HCC.     

Association of defensin beta-1 gene polymorphisms with asthma

BACKGROUND: Defensins are antimicrobial peptides that may take part in airway inflammation and hyperresponsiveness. OBJECTIVE: We characterized the genetic diversity in the defensin beta-1 (DEFB1) locus and tested for an association between common genetic variants and asthma diagnosis. METHODS: To identify single nucleotide polymorphisms (SNPs), we resequenced this gene in 23 self-defined European Americans and 24 African Americans. To test whether DEFB1 genetic variants are associated with asthma, we genotyped 4 haplotype-tag SNPs in 517 asthmatic and 519 control samples from the Nurses' Health Study (NHS) and performed a case-control association analysis. To replicate these findings, we evaluated the DEFB1 polymorphisms in a second cohort from the Childhood Asthma Management Program. RESULTS: Within the NHS, single SNP testing suggested an association between asthma diagnosis and a 5' genomic SNP (g.-1816 T>C; P = .025) and intronic SNP (IVS+692 G>A; P = .054). A significant association between haplotype (Adenine, Cytosine, Thymine, Adenine [ACTA]) and asthma ( P = .024) was also identified. Associations between asthma diagnosis and both DEFB1 polymorphisms were observed in Childhood Asthma Management Program, a second cohort: g.-1816 T>C and IVS+692 G>A demonstrated significant transmission distortion ( P = .05 and .007, respectively). Transmission distortion was not observed in male subjects. The rare alleles (-1816C and +692A) were undertransmitted to offspring with asthma, suggesting a protective effect, contrary to the findings in the NHS cohort. Similar effects were evident at the haplotype level: ACTA was undertransmitted ( P = .04) and was more prominent in female subjects ( P = .007). CONCLUSION: Variation in DEFB1 contributes to asthma diagnosis, with apparent gender-specific effects.     

Pseudotypes of vesicular stomatitis virus with envelope antigens provided by murine mammary tumor virus.

Infection of two mouse mammary carcinoma cell lines with vesicular stomatitis virus (VSV) resulted in the formation of at least two types of particles containing the VSV genome but expressing different envelope characteristics (VSV pseudotypes). One of these VSV pseudotypes was infectious for a cell line derived from normal mouse mammary epithelial cells and mouse embryo cells but noninfectious for 3T3 cells, mink lung cells, and Vero cells. If mouse mammary tumor cells were treated with dexamethason some days prior to infection with VSV, the titer of this pseudotype was significantly increased. In contrast, the second pseudotype was infectious for mink cells, but not for the other cell lines tested, and the titer of this second pseudotype was unaffected by the presence of dexamethasone. The first pseudotype was found to be almost completely neutralized by anti-murine mammary tumor virus (MuMTV) serum whereas the second pseudotype was only partially neutralized at a higher antiserum concentration. Neither pseudotype showed the neutralization, host range, or interference properties of either ecotropic or xenotropic murine C-type viruses. These results suggest that the first pseudotype is VSV(MuMTV). The other pseudotype is less well defined but conceivably may represent a xenotropic MuMTV. In the course of these studies, a filterable agent was observed in GR mammary carcinoma cultures that reactivated the infectivity of VSV neutralized by antiserum. This agent was transmissible to mink cells.