Recombinant expression and neutralizing activity of an MHC class II binding epitope of toxic shock syndrome toxin-1

Toxic shock syndrome (TSS) is caused by the staphylococcal superantigen, TSST-1. The MHC class II binding domain of TSST-1 containing a conserved sequence with other related staphylococcal enterotoxins, comprising TSST-1 residues 47-64 [(T(47-64)], was expressed as a fusion protein with either glutathione-S-transferase (GST(47-64)), filamentous phage coat protein (pIII(47-64)), or E. coli outer membrane porin protein (OprF(47-64)), or synthesized as a peptide conjugated to bovine serum albumin, BSA(47-64). GST(47-64), OprF(47-64) and BSA(47-64), but not pIII(47-64), all induced high-titer T(47-64)-specific antibodies in Balb/c mice. However, only anti-GST(47-64) antibodies inhibited (125)I-TSST-1 binding to MHC class II and abrogated TSST-1-induced T cell mitogenesis and TNFalpha secretion in human peripheral blood mononuclear cells. Purified GST(47-64) also inhibited (125)I-TSST-1 binding in a dose-dependent manner. These findings suggest that GST(47-64) may have potential as a recombinant peptide vaccine or TSST-1 receptor inhibitor against TSS.     

Purification and characterization of Streptococcus pneumoniae palmitoylated pneumococcal surface adhesin A expressed in Escherichia coli

All Streptococcus pneumoniae isolates tested to date express a species-common lipoprotein designated as pneumococcal surface adhesin A (PsaA). This protein is cell-associated, hydrophobic, immunogenic, and genetically conserved. It is currently under investigation as a potential component in third-generation pneumococcal vaccine formulations. To overcome the problem of low-level expression of native hydrophobic PsaA in S. pneumoniae, and also of the recombinant PsaA (rPsaA) in Escherichia coli, we generated a stable E. coli construct expressing functional palmitoylated rPsaA ( approximately 10 mg/l of fermentation culture) using Borrelia burgdorferi outer surface protein A (OspA, a hydrophobic lipoprotein) signal peptide. By Western blot analysis, the chimeric rPsaA ( approximately 34 kDa) was detected in the cell lysate using anti-PsaA antibodies. It was partially purified by extracting the cell pellet with PBS/Triton X(R)-114 buffers, followed by anion exchange filter chromatography. A trypsin digestion profile of rPsaA closely resembled that of the native protein, as revealed by SDS-PAGE/silver staining. Lipidation of rPsaA was confirmed by labeling recombinant E. coli cells with [(3)H] palmitic acid and analyzing the labeled E. coli cells by Western blotting coupled with autoradiography. Further, analysis of purified rPsaA by mass spectrometry (MALDI-TOF) revealed a heterogenous spectrum with a major peak (M+H)(+1) of mass 33,384 Da (theoretical mass of palmitoylated rPsaA=33,361 Da). Purified rPsaA was immunogenic in CBA/NCAHN-XID female mice following intranasal immunization with or without adjuvant, as determined by measurement of anti-PsaA serum IgG levels. These anti-PsaA antibodies reacted with both native and rPsaA polypeptides. Our data strongly suggest that E. coli-expressed rPsaA is palmitoylated and closely resembles the native protein in structure and immunogenicity. It was also observed to elicit measurable protection against nasopharyngeal carriage with S. pneumoniae.     

Pseudomonas cepacia colonization in patients with cystic fibrosis: risk factors and clinical outcome

During the period of 1979 to 1983, 38 patients with cystic fibrosis (CF) at the CF center of St. Christopher's Hospital for Children in Pennsylvania developed respiratory tract colonization with Pseudomonas cepacia. Seventeen (45%) of the patients with colonization died. Yearly incidence rates of P. cepacia colonization fluctuated between 1.3% and 6.1%, suggesting an endemic phenomenon. Case-control studies showed that severe underlying CF, use of aminoglycosides, and having a sibling with CF and P. cepacia colonization were significant risk factors for P. cepacia colonization. Once colonized with P. cepacia, patients with CF were likely to be hospitalized longer (P = 0.008) and to die sooner (P = 0.0001) than control patients with CF. Environmental and microbiologic studies did not identify a common source or mode of transmission of P. cepacia among patients. The results of this investigation suggest that P. cepacia colonization of patients with CF was endemic in the hospital, occurred more frequently in those with severe disease, and was associated with adverse clinical outcome.     

A prospective randomized comparison of cefotaxime vs ampicillin and chloramphenicol for bacterial meningitis in children

Fifty children with bacterial meningitis were prospectively randomized to receive cefotaxime (50 mg/kg/dose every 6 hours) or ampicillin and chloramphenicol in standard doses. Twenty-three patients received cefotaxime and 27 received standard therapy. Bacterial isolates included: Haemophilus influenzae (29), Streptococcus pneumoniae (eight), Neisseria meningitidis (eight), group B streptococci (three), and Salmonella enteritidis (two). Ten (34%) of the H. influenzae isolates were resistant to ampicillin, nine on the basis of beta-lactamase production. All strains were susceptible to cefotaxime. Clinical cure rates for the cefotaxime (100%) and standard therapy (96%) groups were similar; survival without detectable sequelae was similar, at 78% and 77%, respectively. The duration of therapy, 11.1 +/- 2.4 days (range 10 to 21 days) vs 11.9 +/- 3.9 days (range 10 to 21 days), and days to defervescence, 4.7 +/- 2.6 days (range 1 to 14 days) vs 5.6 +/- 2.9 days (range 2 to 17 days), were similar in the cefotaxime and standard therapy groups, respectively. No adverse drug reactions or side effects were noted in either group. Cefotaxime was found to be as safe and effective as standard therapy for the treatment of bacterial meningitis in children.     

Hairy Cell Leukemia in Association with Thrombotic Thrombocytopenic Purpura and Factor VIII Antibodies

A unique patient is reported with longstanding hairy cell leukemia who manifested two distinct abnormalities of factor VIII; factor VIII antibodies and recurrent thrombotic thrombocytopenic purpura (TTP). The patient presented in 1977 with splenomegaly and pancytopenia and was diagnosed with hairy cell leukemia and was treated with splenectomy. In 1989 he received interferon-a because of a relapse which resulted in a hematologic remission. Hospitalization on two occasions for gross hema-turia was caused by the development of a factor VIII antibody. He was successfully treated on both occasions with cyclophosphamide, prednisone and active prothrombin complex (FEIBA). In October 1991 he presented with microangiopathic hemolytic anemia and thrombocytopenia. A diagnosis of thrombotic thrombocytopenic purpura (TTP) was made. Repeat bone marrow biopsy showed hairy cell leukemia. The patient responded to treatment with plasmapheresis, fresh frozen plasma replacement and prednisone. He had two subsequent relapses with the last being refractory and subsequently fatal. During the initial manifestation of TTP and in follow-up evaluation unusually large von Willebrand factor multimers were demonstrated.     

Molecular epidemiology of Salmonella typhimurium isolates from human sporadic and outbreak cases

The molecular epidemiology of a representative collection of sporadic foreign and domestically acquired Salmonella Typhimurium (S. Typhimurium) isolates from Norwegian patients in 1996-9 was studied by numerical analysis of pulsed-field gel electrophoresis (PFGE) profiles. Three subclusters (E5, F1 and G1) comprised 47% of the 102 sporadic isolates investigated and 45% of the domestically acquired isolates fell in subclusters E5 and F1. Distinct seasonal and geographic variations were evident for these strains which have been responsible for both local outbreaks (E5) and a national epidemic (F1) where salmonella-infected hedgehogs and birds constituted the suggested primary source of infection. Subcluster G1 was dominated by imported multi-resistant definitive type (DT) 104 isolates. All multi-resistant isolates contained integron-associated gene cassette-structures. This study presents valuable information on the relative significance, geographic distribution and antibiotic resistance features of distinct S. Typhimurium clones causing human salmonellosis among Norwegians.     

Detection of West Nile virus in oral and cloacal swabs collected from bird carcasses

We evaluated if postmortem cloacal and oral swabs could replace brain tissue as a specimen for West Nile virus (WNV) detection. WNV was detected in all three specimen types from 20 dead crows and jays with an average of >10(5) WNV PFU in each. These findings suggest that testing cloacal or oral swabs might be a low-resource approach to detect WNV in dead birds.