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C Czerkinsky

Publications and source records attributed to C Czerkinsky.

15 recordsLinked to original sources
PubMed1992

Amplified ELISPOT assay for the detection of HIV-specific antibody-secreting cells in subhuman primates.

A novel immunoenzyme amplification technique has been evaluated in an ELISPOT assay for the detection of antigen-specific antibody-secreting cells (ASC) in monkeys. In this assay, mononuclear cells containing putative ASC are incubated for a few hours in antigen-coated wells. Following removal of the cells, zones of solid phase bound antibodies secreted by individual ASC are visualized in four consecutive steps. First, a primary biotinylated anti-immunoglobulin (Ig) reagent is added followed by enzyme-labelled avidin. The amplification procedure comprises the addition of biotinylated anti-enzyme antibodies in the third stage, followed by enzyme-conjugated avidin and substrate. When evaluated in a modified ELISPOT assay for the detection of simian B cells secreting antibodies to the envelope glycoprotein gp120 of the human immunodeficiency virus type 1 (HIV-1), this amplification procedure proved to be suitable even when using anti-human Ig antisera as primary antibody reagents. This development should be useful for other ELISPOT assays where species specific anti-Ig reagents are not always available and, most importantly, for enumerating cells producing immunoreactive substances in such minute amounts that they may escape detection by conventional ELISPOT assays. Furthermore, a functional simian HIV-specific ELISPOT assay could prove valuable for assessing the humoral immunogenicity of future candidate vaccines against the acquired immunodeficiency syndrome (AIDS).

Animals↗
PubMed1992

Secretory immunoglobulins in serum from human immunodeficiency virus (HIV)-infected patients.

Infection by the human immunodeficiency virus is associated with polyclonal B cell activation and increased levels of serum IgA. In order to characterize the molecular species of serum IgA, we have measured total IgA, IgA1, and IgA2 in sera from 60 HIV-1-infected patients and 40 healthy controls. In addition, secretory IgA (S-IgA), secretory IgM (S-IgM), free immunoreactive secretory component (SC), and the distribution of monomeric and polymeric IgA were determined. The data confirm the elevation of total serum IgA levels in HIV-1-infected patients, and both IgA1 and IgA2 concentrations are elevated. Furthermore, the data show a substantial increase in serum levels of both monomeric and polymeric IgA. Serum S-IgA levels were significantly increased in CDC group II patients versus controls and more frequently elevated in CDC group IV patients. The highest S-IgA levels were found among patients with the lowest blood CD4+ cell counts. Serum S-IgA levels were not correlated with serum levels of either total IgA or polymeric IgA. Serum S-IgM levels were also increased in HIV-1-infected patients and positively correlated with serum S-IgA levels. Conversely, serum levels of free SC were not altered. An increase in serum S-IgA was not related to human hepatitis B virus infection and/or to hepatic dysfunction or to diarrhea or overt intestinal infection. The data indicate that secretory Ig (S-IgM and S-IgA), which are likely to be produced at mucosal sites, increase in the serum of HIV-1-infected patients.

Adult↗
PubMed1992

Mucosal immunity: implications for vaccine development.

The mucosal surfaces in e.g. the gastrointestinal, respiratory and urogenital tracts represent a very large exposure area to exogenous agents including microorganisms. Not surprising, therefore, those mucosal tissues are defended by a local immune system with properties and functions that in many respects are separate from the systemic immune system. The intestine is the largest immunological organ in the body. It comprises 70-80% of all immunoglobulin-producing cells and produces more secretory IgA (SIgA) (50-100 mg/kg body weight/day) than the total production of IgG in the body (ca. 30 mg/kg/day). The local immune system of the gut has two main functions: to protect against enteric infections, and to protect against uptake of and/or harmful immune response to undergraded food antigens. The best known entity providing specific immune protection for the gut is the SIgA system. The resistance of SIgA against normal intestinal proteases makes antibodies of this isotype uniquely well suited to protect the intestinal mucosal surface. The main protective function of SIgA antibodies is the "immune exclusion" of bacterial and viral pathogens, bacterial toxins and other potentially harmful molecules. SIgA has also been described to mediate antibody-dependent T cell-mediated cytotoxicity (ADCC), and to interfere with the utilization of necessary growth factors for bacterial pathogens in the intestinal environment, such as iron. It is now almost axiomatic that in order to be efficacious, vaccines against enteric infection must be able to stimulate the local gut mucosal immune system, and that this goal is usually better achieved by administering the vaccines by the oral route rather than parenterally. Based on the concept of a common mucosal immune system through which activated lymphocytes from the gut can disseminate immunity also to other mucosal and glandular tissues there is currently also much interest in the possibility to develop oral vaccines against e.g. infections in the respiratory and urogenital tracts. It has previously been widely assumed that only live vaccines would efficiently stimulate a gut mucosal immune response. However, an oral cholera vaccine, composed of the nontoxic B subunit of cholera toxin in combination with killed whole cell (WC) cholera vibrios has been shown to stimulate a strong intestinal SIgA antibody response associated with long-lasting protection against cholera. We have used this new cholera subunit vaccine and developed ELISPOT methods for examining at the clonal B and T cell level the dynamics of intestinal and extra-intestinal immune responses in humans after enteric immunizations.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Cholera as a model for research on mucosal immunity and development of oral vaccines.

During the past year, the extensive investigational use of a recently developed oral vaccine against cholera has led to significant advances in our understanding of both immunity to cholera and related diarrhoeal diseases, and the mucosal immune response in general after oral immunization. The oral cholera vaccine has been shown to protect, through its cholera toxin B subunit component, against travellers' diarrhoea caused by enterotoxigenic Escherichia coli. The elaboration of sensitive new techniques has allowed detailed clonal analyses of the activation of specific B and T cells and immunologic memory in intestinal mucosa in humans after oral cholera vaccination. These techniques have also been used to demonstrate a transient appearance after immunization of specific gut-derived IgA antibody-producing cells in the circulation and also, a few days later, in a distant mucosal tissue such as the salivary glands.

Administration, Oral↗
PubMed1992

Antibody-secreting cells in human peripheral blood after oral immunization with an inactivated enterotoxigenic Escherichia coli vaccine.

Vaccine antigen-specific antibody-secreting cell (ASC) responses in peripheral blood of healthy adult volunteers were studied after oral immunization with a prototype enterotoxigenic Escherichia coli (ETEC) vaccine by means of the enzyme-linked immunospot technique. Three doses of vaccine consisting of formalin-killed ETEC bacteria expressing fimbrial colonization factor antigens I and II (CFA/I and CFA/II) in combination with purified cholera toxin B subunit (CTB) were given 2 weeks apart. The ASC responses were detected 7 days after each immunization. Immunoglobulin A (IgA) was the predominant isotype produced by CFA/I- as well as CFA/II-specific ASCs. Moderate CFA/I- and CFA/II-specific IgM-secreting ASC (IgM-ASC) responses were also seen, whereas IgG-ASC responses to either of the CFAs were negligible. The ASC responses to CTB, on the other hand, comprised both IgA- and IgG-ASCs, with few if any specific IgM-ASCs. Almost 90% of the volunteers developed CFA-specific ASC responses after vaccination. Maximal CFA-specific ASC responses were usually observed after a single dose or two doses of vaccine. A third dose of vaccine did not result in increased but rather resulted in decreased magnitudes of CFA-specific ASC responses. Furthermore, it was found that CTB did not function as a mucosal adjuvant, since CFA-specific ASC responses were not enhanced by the simultaneous administration of CTB. These results suggest that two oral doses of ETEC vaccine induce a strong mucosal immune response, as reflected by the presence of large numbers of antigen-specific mucosal B cell immunoblasts in the blood.

Administration, Oral↗
PubMed1992

Oral administration of immunomodulators and the mucosal immune system.

Orally administered antigens reach the lymphoid tissue in Peyer's patches in the gut where they initiate an immune response with clonal expansion of antigen-specific T and B cells. Activated T cells migrate through lymph and blood to intestinal epithelium (intra-epithelial leukocytes) whereas activated B cells migrate to the lamina propria, other mucosae and exocrine glands where they differentiate into plasma cells secreting polymeric IgA1 or IgA2. These antibodies are transported across the epithelial cells after binding to a poly-Ig receptor, then excreted in the lumen as secretory IgA. Reciprocal interactions have been demonstrated between lymphoid and epithelial cells in the mucosae. Oral administration of antigens in different experimental models may induce the production of secretory antibodies and/or systemic unresponsiveness with suppression of delayed-type hypersensitivity or specific IgG and IgE antibody production or both. New strategies are currently being explored for the development of oral vaccines using recombinant antigens or viral vectors (e.g. pox-viruses, vaccinia virus, cholera toxin B subunit etc.). Conversely, immunomodulating compounds or procedures which could enhance specific oral tolerance in association with antigen would have considerable therapeutic applications in auto-immune diseases and allergy.

Adjuvants, Immunologic↗
PubMed1991

Immunizations of monkeys with synthetic peptides disclose conserved areas on gp120 of human immunodeficiency virus type 1 associated with cross-neutralizing antibodies and T-cell recognition.

Site-directed immunization was employed to identify sites on the envelope glycoprotein gp120 for antibody-mediated neutralization of human immunodeficiency virus type 1 (HIV-1). Antisera were raised in monkeys (Macaca fascicularis) against a series of 40 overlapping synthetic peptides covering the entire amino acid sequence of gp120 from the HTLV-IIIB strain of HIV-1. Immune sera against 12 of these peptides were reactive with gp120 by immunoblotting analysis, and antisera raised against 5 peptides, corresponding to amino acids (aa) 152-176, 193-218, 206-230, 248-269, and 307-330, were highly efficient in neutralizing HIV-1 (HTLV-IIIB) infectivity in vitro. Admixture of individual neutralizing anti-peptide monkey sera resulted in increment in neutralizing antibody titer. Antisera with reactivity to the relatively conserved regions defined by aa 152-176, 193-230, and 248-269 also neutralized to different extents the infectivity of the five Swedish clinical isolates of HIV-1 tested. Only a few HIV-1-infected people were found to make antibodies to these three conserved domains of gp120 as judged by ELISA using synthetic peptides as antigens. Three of the peptides (aa 152-176, 248-269, and 307-330) that induced neutralization antibodies also induced interleukin 2 production and lymphocyte proliferation when added to cultures of peripheral blood mononuclear cells from monkeys immunized with the corresponding peptides, indicating that these domains accommodate T-cell recognition sites. The results have obvious implications for the rational design of subunit vaccines against HIV-1 infection.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Enumeration of human peripheral blood lymphocytes secreting immunoglobulins of major classes and subclasses in healthy children and adults.

The reverse enzyme-linked immunospot assay was modified to enumerate peripheral blood mononuclear cells (PBMC) secreting IgG1-4, IgA1-2, and IgM. Anti-human IgG F(ab')2 and mouse monoclonal antibodies specific to each of the isotypes were used as solid-phase capture antibodies and developing antibodies, respectively. Although attempts were also made to detect IgD- and IgE-secreting cells (SC), their numbers in the peripheral blood were too few to be reliably estimated. The assay was applied to study healthy subjects including 21 neonates within 3 days of birth, 44 1- to 48-month-old children, and 32 adults. Sixty percent of these neonates had IgM SC in small numbers (less than 20 per 10(6) PBMC), but only three neonates had IgSC of other isotypes. In contrast, by 1-2 months of age children had IgSC of all isotypes, including IgA2 and IgG4, often in higher numbers than adults. The relative frequencies of IgSC were IgA1 greater than IgG1 greater than IgM greater than IgG2 greater than IgG3 greater than IgG4 greater than IgA2 in the children and IgA1 greater than IgG1 greater than IgA2 greater than IgM greater than IgG4 greater than IgG2 greater than IgG3 in the adults. The order of the serum concentrations in the adults was IgG1 greater than IgG2 greater than IgA greater than IgM greater than IgG4 greater than IgG3. No correlation was found between the serum level and the IgSC number of individual isotypes (except for serum IgA and IgA1-SC). This new methodology should facilitate investigating the current status of immunoglobulin synthesis and the Ig repertoire in adults and children, in health and in disease.

Adult↗
PubMed1991

Contact sensitivity in the murine oral mucosa. I. An experimental model of delayed-type hypersensitivity reactions at mucosal surfaces.

We have examined in a murine model, the potential of the oral mucosa (OM) to serve as inductive and/or expression site(s) of delayed-type hypersensitivity (DTH) reactions. The expression of DTH reactions in the murine buccal mucosa was studied after topical application of oxazolone or picryl chloride onto the OM of animals previously sensitized with either hapten. Irrespective of the site of priming (skin or buccal mucosa), inflammatory cells appeared in the OM following buccal elicitation with the pertinent hapten. The density of infiltrating cells peaked at 24 h after hapten elicitation. Such inflammatory reactions, which comprised mainly mononuclear cells at 24 h, were preceded by an early inflammatory reaction that developed only in animals previously sensitized at skin sites. This early reaction, comprising mainly PMN neutrophils, peaked at 6-8 h, declined by 8-16 h, and was not observed in mice previously sensitized in the buccal mucosa. The 24 h reactions failed to develop in nude mice similarly treated, in intact unsensitized mice, as well as in animals sensitized with an irrelevant hapten. These reactions could be adoptively transferred to naive animals by LN cells but not by serum from sensitized syngeneic donors. Furthermore, LN cell suspensions depleted of T cells failed to transfer sensitization for subsequent OM DTH. Topical application of contact sensitizing haptens onto OM induced priming for subsequent DTH reactions elicited with recall antigen applied at a distant skin site or at a local buccal site. These results demonstrate that the OM has the capacity to serve both as an inductive and as an expression site for T cell-mediated inflammatory reactions, be these expressed or induced at local mucosal sites or at remote systemic (skin) sites. This animal model should be valuable for studying the regulation of T cell-mediated inflammatory responses at mucosal surfaces.

Administration, Buccal↗
PubMed1991

Antibody-producing cells in peripheral blood and salivary glands after oral cholera vaccination of humans.

We examined whether immunization with a newly developed oral cholera vaccine would elicit gut-derived antibody-producing cells in the blood and in distant mucosal tissues, such as the minor salivary glands, in 30 adult Swedish volunteers. The results of this study demonstrated that this vaccine indeed induced production of specific antibody-producing cells against the cholera toxin B subunit in both peripheral blood and salivary glands. The response in blood, which after primary and booster immunizations comprised both immunoglobulin A (IgA) and IgG antibody-forming cells, was highly transient and preceded the response in salivary glands; the latter response was restricted to the IgA isotype. The results provide further evidence of the existence of a common mucosal immune system in humans. Furthermore, these findings support previous observations that in animals, the cholera toxin B subunit may be a useful carrier protein for preparing enteric vaccines against pathogens encountered at intestinal and extraintestinal mucosal sites.

Administration, Oral↗
PubMed1991

Intestinal immune responses in humans. Oral cholera vaccination induces strong intestinal antibody responses and interferon-gamma production and evokes local immunological memory.

We have examined secretory antibody and cell-mediated immune responses to oral cholera vaccine in the human gastrointestinal mucosa. Freshly isolated peripheral blood lymphocytes and intestinal lymphocytes obtained by enzymatic dispersion of duodenal biopsies were assayed for numbers of total and vaccine specific immunoglobulin-secreting cells by enzyme-linked immunospot assay (ELISPOT) techniques; the frequency of cells secreting interferon-gamma (IFN-gamma) was also examined by a new modification of the ELISPOT technique. After booster immunizations with oral cholera vaccine, large numbers of cholera toxin-specific antibody-secreting cells (ASC) appeared in the small intestine. The responses were dominated by IgA ASC. A single immunization, performed 5 mo after the initial vaccinations, gave rise to an ASC response similar to that seen after the first booster immunization, with respect to both magnitude and isotype distribution. Each of the immunizations also evoked an ASC response in blood which was of lower magnitude than that seen in the small intestine, and comprised similar proportions of IgA and IgG ASC. A booster immunization also resulted in increased frequencies of IFN-gamma-secreting cells, but this increase was confined to the duodenal mucosa. This study establishes the feasibility of studying, at the single-cell level, intestinal immune reactivity in humans. Furthermore, it indicates that the small intestinal mucosa is an enriched source of IFN-gamma. It also demonstrates marked differences between intestinal and peripheral blood immune responses after enteric immunization, and confirms the notion that the mucosal immune system in humans displays immunological memory.

Administration, Oral↗