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Infection and Immunity, August 2003, p. 4808-4814, Vol. 71, No. 8
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.8.4808-4814.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Antigen-Specific Immunoglobulin A Antibodies Secreted from Circulating B Cells Are an Effective Marker for Recent Local Immune Responses in Patients with Cholera: Comparison to Antibody-Secreting Cell Responses and Other Immunological Markers

Firdausi Qadri,1* Edward T. Ryan,2,3,4 A. S. G. Faruque,1 Firoz Ahmed,1 Ashraful Islam Khan,1 M. Monirul Islam,1 Syed M. Akramuzzaman,1 David. A. Sack,1 and Stephen B. Calderwood3,5

International Centre for Diarrhoeal Disease Research, Bangladesh, Centre for Health and Population Research, Mohakhali, Dhaka 1212, Bangladesh,1 Tropical & Geographic Medicine Center,2 Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts 02114,3 Department of Immunology and Infectious Diseases, Harvard School of Public Health,4 Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 021155

Received 5 February 2003/ Returned for modification 21 April 2003/ Accepted 24 May 2003

Gut-derived lymphocytes transiently migrate through the peripheral circulation before homing back to mucosal sites and can be detected using an ELISPOT-based antibody secreting cell (ASC) assay. Alternatively, transiently circulating lymphocytes may be cultured in vitro, and culture supernatants may be assayed for antigen-specific responses (antibody in lymphocyte supernatant [ALS] assay). The ALS assay has not been validated extensively in natural mucosal infection, nor has the ALS response been compared to the ASC assay and other cholera-specific immunological responses. Accordingly, we examined immune responses in 30 adult patients with acute cholera in Bangladesh, compared with 10 healthy controls, measuring ALS-immunoglobulin A (IgA), ASC-IgA, and serum and fecal IgA responses to two potent Vibrio cholerae immunogens, the nontoxic B subunit of cholera toxin (CtxB) and lipopolysaccharide (LPS) and a weaker V. cholerae immunogen, the mannose-sensitive hemagglutinin (MSHA). We found significant increases of anti-CtxB, anti-LPS, and anti-MSHA IgA in supernatants of lymphocytes cultured 7 days after onset of cholera using the ALS assay. We found that ALS and ASC responses correlated extremely well; both had comparable sensitivities as the vibriocidal responses, and both procedures were more sensitive than fecal IgA measurements. An advantage of the ALS assay for studying mucosal immune responses is the ability to freeze antibodies in supernatants for subsequent evaluation; like the ASC assay, the ALS assay can distinguish recent from remote mucosal infection, a distinction that may be difficult to make in endemic settings using other procedures.

* Corresponding author. Mailing address: Laboratory Sciences Division, ICDDR,B, GPO Box 128, Dhaka 1000, Bangladesh. Phone: 880 2 8811751 to 8811760, ext. 2431. Fax: 8802 8823116 or 8802 8826050. E-mail: fqadri{at}icddrb.org.

Editor: W. A. Petri, Jr.

Infection and Immunity, August 2003, p. 4808-4814, Vol. 71, No. 8
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.8.4808-4814.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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