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Enumeration of Gut-Homing beta 7-Positive, Pathogen-Specific Antibody-Secreting Cells in Whole Blood from Enterotoxigenic Escherichia coli- and Vibrio cholerae-Infected Patients, Determined Using an Enzyme-Linked Immunosorbent Spot Assay Technique

Journal article
Authors T. R. Bhuiyan
M. R. Hoq
N. S. Nishat
D. Al Mahbuba
R. Rashu
K. Islam
L. Hossain
A. Dey
J. B. Harris
E. T. Ryan
S. B. Calderwood
Ann-Mari Svennerholm
F. Qadri
Published in Clinical and Vaccine Immunology
Volume 23
Issue 1
Pages 27-36
ISSN 1556-6811
Publication year 2016
Published at Institute of Biomedicine, Department of Microbiology and Immunology
Pages 27-36
Language en
Links dx.doi.org/10.1128/cvi.00526-15
Keywords human peripheral-blood, immune-responses, colonization factors, production invitro, bangladeshi children, immunological memory, enteric, infections, oral immunization, o1 infection, vaccine, Immunology, Infectious Diseases, Microbiology
Subject categories Infectious Medicine, Immunology in the medical area, Microbiology in the medical area

Abstract

Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) are noninvasive mucosal pathogens that cause acute watery diarrhea in people in developing countries. Direct assessment of the mucosal immune responses to these pathogens is problematic. Surrogate markers of local mucosal responses in blood are increasingly being studied to determine the mucosal immune responses after infection. However, the volume of blood available in children and infants has limited this approach. We assessed whether an approach that first isolates beta 7-positive cells from a small volume of blood would allow measurement of the antigen-specific immune responses in patients with cholera and ETEC infection. beta 7 is a cell surface marker associated with mucosal homing. We isolated beta 7-expressing cells from blood on days 2, 7, and 30 and used an enzyme-linked immunosorbent spot (ELISPOT) assay to assess the gut-homing antibody-secreting cells (ASCs) specific to pathogen antigens. Patients with ETEC diarrhea showed a significant increase in toxin-specific gut-homing ASCs at day 7 compared to the levels at days 2 and 30 after onset of illness and to the levels in healthy controls. Similar elevations of responses to the ETEC colonization factors (CFs) CS6 and CFA/I were observed in patients infected with CS6- and CFA/I-positive ETEC strains. Antigen-specific gut-homing ASCs to the B subunit of cholera toxin and cholera-specific lipopolysaccharides (LPS) were also observed on day 7 after the onset of cholera using this approach. This study demonstrates that a simple ELISPOT assay can be used to study the mucosal immunity to specific antigens using a cell-sorting protocol to isolate mucosal homing cells, facilitating measurement of mucosal responses in children following infection or vaccination.

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