Local cholera immunity in mice: intestinal antitoxin-containing cells and their correlation with protective immunity

Abstract

Methods for light and electron microscopic identification and characterization of intestinal cholera antitoxin-containing cells (ACC) using peroxidase-labeled immunoreagent are described and used to study the ACC response in mice after immunizations with cholera toxin. Specific ACC appeared in significant numbers after two oral immunizations and increased six- to eightfold with two additional oral boosters, whereas further oral immunizations caused no additional stimulation. Intravenous immunizations had to be repeated seven times before ACC could be detected. After two oral immunizations, most of the ACC were found in the proximal part of the small intestine and no ACC were seen in the colon. This uneven distribution of ACC within the small intestine was eliminated after four oral immunizations, when ACC could also be detected in the colon. The ACC response after four oral immunizations demonstrated a peak at 4 days with far fewer cells present at 2 and 7 days. Electron microscopic studies showed that the ACC were mature plasma cells with the staining localized to the endoplasmic reticulum. Regression analysis of the relationship between the number of ACC and the magnitude of protective immunity against intestinal challenge with cholera toxin indicates a highly significant correlation (r = 0.91, P less than 0.001).

Antitoxic Cholera Immunity in Mice: Influence of Antigen Deposition on Antitoxin-Containing Cells and Protective Immunity in Different Parts of the Intestine

“…served as controls and were analyzed concurrently with the immunized animals. We used three or four initial immunizations for building up a local immunological memory in the intestine, and after the actual antibody response resulting from this priming had vanished (12,20), a single booster immunization was given for eliciting the local antibody response under study.…”

“…was conjugated to the immunoglobulin fractions of antisera specific for mouse IgA, IgG2B, and IgM (Meloy Laboratories, Springfield, Va.) (17). Conjugation of peroxidase to anti-cholera toxin immunglobulin and control studies of the binding specificities of the conjugates were done as described previously (12,17).…”

“…Stained cells were examined after incubation with 3'3'-diaminobenzidine and hydrogen peroxide. The optimal reagent concentrations were determined by chessboard titrations (12).…”

Induction of pinocytosis in cerebral vessels by acute hypertension and by hyperosmolar solutions

“…Choleratoxin, choleragen, is known to consist of two different types of polypeptide subunits, one large one that is toxic and another that is atoxic, binding specifically to the ganghoside G M l [5,9] . The conjugate used thus consisted of the binding part of the toxin ie, choleragenoidto which horseradish peroxidase was attached through glutaraldehyde [13]. The endothelial cells of all the examined types of cerebral and leptomeningeal blood vessels became labeled with the conjugate, reflecting that G M l is a widespread constituent of the outer parts of cell membranes (cf 51.…”

Protective Effect of Immunization with Heat-Labile Enterotoxin in Gnotobiotic Rats Monocontaminated with Enterotoxigenic Escherichia coli

“…The E. coli LT holotoxin and its A and B subunits have both shared and unique immunological determinants in relationship with cholera toxin and its subunits (3-5), and observations conducted to date have shown the same pattern of antibody response to immunization with either of these toxins when given by different routes of administration. Parenteral immunization with cholera toxin or toxoid yields elevated serum antitoxin levels (36), whereas peroral immunization arouses a local mucosal antibody response that is protective in the absence of elevated serum antitoxin titers (25,26,34). Parenteral priming also primes, in some unexplained manner, the local mucosal antibody response for subsequent boosting by the peroral route, and protection is afforded by this immunization regimen (the IP/PO route) in the absence of a marked increase in serum antitoxin titers (35,37).…”