Evaluation of methods for subtyping Campylobacter jejuni during an outbreak involving a food handler.

In October 1998, the Centers for Disease Control and Prevention (CDC) assisted in an investigation of an outbreak of campylobacteriosis at a school in Salina, Kansas. Twenty-two isolates were submitted from the Kansas state public health laboratory to CDC, 9 associated with the outbreak and 13 epidemiologically unrelated sporadic isolates. Pulsed-field gel electrophoresis (PFGE) using SmaI and SalI was initially used to validate the epidemiologic data. We then tested the ability of other subtyping techniques to distinguish the outbreak-associated isolates from unrelated sporadic isolates. The methods employed were somatic O serotyping, PCR-restriction fragment length polymorphism (RFLP) analysis of flaA, DNA sequence analysis of 582 bp of flaA that included the short variable region (SVR), and sequencing of the entire flaA gene. PFGE was the most discriminatory technique, yielding 11 SmaI and 10 SalI restriction profiles. All outbreak isolates were indistinguishable by PFGE, somatic O serotyping, and sequencing of the 582-bp region of the flaA gene. fla typing by PCR-RFLP grouped one sporadic isolate with the outbreak strain. Analysis of the DNA sequence of a 582-bp segment of flaA produced strain groupings similar to that generated by PCR-RFLP but further differentiated two flaA PCR-RFLP types (with a 1-bp difference in the 582-bp region). Two sporadic strains were distinct by flaA PCR-RFLP but differed only by a single base substitution in the 582-bp region. The entire flaA gene was sequenced from strains differing by a single base pair in the 582-bp region, and the data revealed that additional discrimination may in some cases be obtained by sequencing outside the SVR. PFGE was superior to all other typing methods tested for strain discrimination; it was crucial for understanding the Kansas outbreak and, when SmaI was used, provided adequate discrimination between unrelated isolates.

An outbreak of Campylobacter jejuni infections associated with food handler contamination: the use of pulsed-field gel electrophoresis.

In 1998, an outbreak of Campylobacter jejuni infections occurred in Kansas among persons attending a school luncheon; community cases were also reported. In a cohort study of luncheon attendees, 27 (17%) of 161 persons reported illness. Consuming gravy (relative risk [RR], 4.2; 95% confidence interval [CI], 1.5-11.7) or pineapple (RR, 2.4; 95% CI, 1.0-5.7) was associated with illness. Both foods were prepared in a kitchen that served 6 other schools where no illness was reported. A cafeteria worker at the luncheon had a diarrheal illness and was the likely source of the outbreak. The pulsed-field gel electrophoresis (PFGE) patterns of the isolates from the food handler and those of 8 lunch attendees were indistinguishable. Isolates from 4 community patients differed. This was the first use of PFGE in a Campylobacter outbreak in the United States; its use was critical in determining that community cases were not linked.

Human tumor and normal tissue reactivity of the anti-(breast cancer) monoclonal antibody BA-Br-3 and its similarity to the anti-(epithelial membrane antigen) monoclonal antibody E29.

A mouse monoclonal antibody (BA-Br-3) raised against the breast carcinoma cell line CAMA-1 was previously shown to react with a greater than or equal to 300-kDa globule-like glycoprotein from human milk fat also expressed in the cytoplasm and on the surface of human carcinoma cells of different histological types. In this report the reactivity of this mAb with a large number of normal and malignant human tissues was analyzed using immunoperoxidase techniques. When tested on sections of both fresh-frozen tissues and formalin-fixed, paraffin-embedded tissues, BA-Br-3 reacted with a formalin-resistant antigenic determinant expressed by normal and malignant epithelial cells. Preferential reactivity was observed at the apical portion of ductal epithelial cells in normal breast and in glandular epithelia distributed in several other organs. Reactivity with mucin-like secretions in the lumina of ducts was also found. BA-Br-3 reacted mostly in heterogenous staining patterns with 88% of 49 breast carcinoma specimens tested, regardless of their histological type or whether they were primary or secondary neoplasms. Testing of epithelial malignant tumors other than breast carcinomas with this antibody showed that 127 of 151 (84%) were also reactive. mAb BA-Br-3 and E29 (a commercially available anti-(epithelial membrane antigen) shared very similar staining patterns and distributions of reactivity with breast and other epithelial tumors. However, BA-Br-3 showed a significantly higher percentage of reactivity with melanoma (33% versus 6%, P = 0.003) and a trend toward a higher percentage of reactivity with sarcoma (55% versus 27%, P greater than 0.05). This antibody, therefore, defines a molecule that is a member of the mucin-like epithelial membrane antigen family. Further studies are warranted to determine its usefulness in antibody-directed cancer diagnosis, prognosis, and immunotherapy.

Binding and functional properties of a mouse-human chimeric monoclonal antibody of the human IgG1 subclass with specificity for human carcinomas.

Recombinant DNA techniques were utilized successfully to join the coding regions for the variable region of a mouse anti-tumor antibody (BA-Br-1) and the human IgG1 constant region for both the light and heavy chains. After insertion into a mouse myeloma host cell line, the chimeric genes were expressed successfully and the resulting antibody (ING-1) was purified. In this study, we describe biochemical, serological, immunohistochemical, and functional properties of the chimeric ING-1 antibody. Analysis of the synthesized antibody revealed that while it was similar in size to the mouse antibody, it had a different pI as determined by isoelectrofocusing. The flow cytometric binding profiles of the new molecule were found to be essentially identical to the parental mouse immunoglobulin. The specificity of the chimeric ING-1 and mouse BA-Br-1 antibodies were compared by extensive immunohistochemical analysis on human normal and tumor tissues. The chimeric antibody retained the same broad carcinoma binding activity, showing strong reactivity with greater than 90% of epithelial tumor tissues, as was previously observed for the mouse BA-Br-1 antibody. The chimeric and mouse antibodies also recognized the same selected normal tissues: primarily glandular epithelia, gastrointestinal mucosa, bile ducts, and thyroid follicles. Analysis of the biological function of the chimeric antibody revealed that it possessed ADCC activity against antigen-bearing tumor targets in vitro which was absent from the mouse form of the antibody. Competent effector cells could be either PBMCs from normal healthy donors, PBMCs from cancer patients receiving LAK/IL-2 therapy, or LAK cells prepared from cancer patients. Enhanced cytotoxicity even in the presence of LAK cell killing was noted with effector cells from the latter two sources. This contrasts sharply with the absence of activity in the same systems when the native murine antibody was used. The in vitro activation of cell-dependent cytolysis observed with the chimeric antibodies when effector cells from both normal and tumor-bearing donors were used strongly suggests that comparable activity would be observed in vivo. These results, along with the broad carcinoma binding activity and minimal normal tissue reactivity, suggest that the ING-1 chimeric antibody may be useful in cancer therapy. The application of the ING-1 chimeric antibody for treatment of tumors thus offers a promising avenue for future research.

Mechanism of suppression of lipopolysaccharide-driven B cell differentiation by anti-mu antibodies. Evidence for a trans-acting repressor of transcription.

Bivalent anti-mu antibodies suppress LPS-driven B cell differentiation by inhibiting the coordinate activation of a family of differentiation-related genes, including those encoding the heavy, light, and J chains of IgM. We have shown that the presence of inhibitors of RNA or protein synthesis during a pulse with anti-mu can interfere with induction of suppression. We suggest that suppression is mediated by a trans-acting repressor protein with specificity for common motifs in regulatory regions of each of these genes.

Anti-mu antibody blocks LPS driven B cell differentiation by suppressing specific mRNAs.

High concentrations of anti-mu antibodies inhibit differentiation and increase proliferation of adult mouse splenic B cells stimulated by LPS. Cells from treated cultures express Ia antigens and on removal of anti-mu reexpress membrane IgM. They do not develop into plasmablasts secreting IgM. To investigate the mechanism of suppression we used Northern blots hybridized with cDNA probes to mu, kappa, J, and I-A beta chains to analyze RNAs in anti-mu suppressed and control cultures. Messenger RNAs for mu, kappa, and J chains but not I-A beta chain are diminished in anti-mu treated cells as compared to controls. Cells treated with anti-kappa antibodies have decreased mRNA levels for mu as well as kappa chains. Suppression mediated by modulation of the IgM receptor is not immunologically specific, but selectively inhibits expression of a cluster of unlinked genes which are coordinately activated during differentiation.