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C Czerkinsky

Publications and source records attributed to C Czerkinsky.

At least 37 records · Page 2Linked to original sources
PubMed1998

Antibodies and antibody-secreting cells in the female genital tract after vaginal or intranasal immunization with cholera toxin B subunit or conjugates.

We studied the antibody response including antibody-secreting cells (ASC) in the female genital tract of mice after mucosal immunizations with the recombinant B subunit of cholera toxin (rCTB) perorally, intraperitoneally, vaginally, and intranasally (i.n.). The strongest genital antibody responses as measured with a novel perfusion-extraction method were induced after vaginal and i.n. immunizations, and these routes also gave rise to specific immunoglobulin A (IgA) and IgG ASC in the genital mucosa. Specific ASC in the iliac lymph nodes, which drain the female genital tract, were seen only after vaginal immunization. Progesterone treatment increased the ASC response in the genital tissue after all mucosal immunizations but most markedly after vaginal immunization. We also tested rCTB as a carrier for human gamma globulin (HGG) and the effect of adding cholera toxin (CT) as an adjuvant for the induction of systemic and genital antibody responses to HGG after vaginal and i.n. immunizations. Vaginal immunizations with HGG conjugated to rCTB resulted in high levels of genital anti-HGG antibodies whether or not CT was added, while after i.n. immunization the strongest antibody response was seen with the conjugate together with CT. In summary, vaginal and i.n. immunization give rise to a specific mucosal immune response including ASC in the genital tissue, and vaginal immunization also elicits ASC in the iliac lymph nodes. We have also shown that rCTB can act as an efficient carrier for a conjugated antigen for induction of a specific antibody response in the genital tract of mice after vaginal or i.n. immunization.

Administration, Intranasal↗
PubMed1998

Induction and expression of intestinal humoral immunity in HIV-infected individuals: prospects for vaccination against secondary enteric infections.

In view of the high incidence and severity of AIDS-related mucosal complications and secondary mucosal infections, we have evaluated the impact of HIV infection on the mucosal humoral immune status. By measuring the number of total and specific antibody-secreting cells in duodenal biopsies, we could show that HIV infection induces a pronounced polyclonal B-cell activation already in the early stages of the disease. Furthermore, we could document normal mucosal B-cell responses following oral vaccination, also in the late stages of the disease, and despite a lack of concomitant systemic responses. The relevance of these observations will be discussed with respect to the possibility of vaccinating HIV-infected individuals against secondary opportunistic infections.

AIDS Vaccines↗
PubMed1997

A cholera toxoid-insulin conjugate as an oral vaccine against spontaneous autoimmune diabetes.

Mucosally induced immunological tolerance is an attractive strategy for preventing or treating illnesses resulting from untoward inflammatory immune reactions against self- or non-self-antigens. Oral administration of relevant autoantigens and allergens has been reported to delay or suppress onset of clinical disease in a number of experimental autoimmune and allergic disorders. However, the approach often requires repeated feeding of large amounts of tolerogens over long periods and is only partly effective in animals already systemically sensitized to the ingested antigen such as in animals already harboring autoreactive T cells, and thus presumably also in humans with an autoimmune disease. We have recently shown that oral administration of microgram amounts of antigen coupled to cholera toxin B subunit (CTB), can effectively suppress systemic T cell reactivity in naive as well as in immune animals. We now report that feeding small amounts (2-20 microg) of human insulin conjugated to CTB can effectively suppress beta cell destruction and clinical diabetes in adult nonobese diabetic (NOD) mice. The protective effect could be transferred by T cells from CTB-insulin-treated animals and was associated with reduced lesions of insulitis. Furthermore, adoptive co-transfer experiments involving injection of Thy-1,2 recipients with diabetogenic T cells from syngeneic mice and T cells from congenic Thy-1,1 mice fed with CTB-insulin demonstrated a selective recruitment of Thy-1,1 donor cells in the peripancreatic lymph nodes concomitant with reduced islet cell infiltration. These results suggest that protection against autoimmune diabetes can be achieved by feeding minute amounts of a pancreas islet cell autoantigen linked to CTB and appears to involve the selective migration and retention of protective T cells into lymphoid tissues draining the site of organ injury.

Administration, Oral↗
PubMed1997

Combined immunomagnetic cell sorting and ELISPOT assay for the phenotypic characterization of specific antibody-forming cells.

A combination of immunomagnetic cell sorting and ELISPOT techniques has been evaluated to permit enrichment and characterization of antibody-secreting cells (ASC). Cell suspensions containing putative ASC were first incubated with magnetic microbeads coated with antibodies specific for a given cell surface marker. After separation of bead-cell clusters and free cells, the resulting cell populations were examined for the presence of ASC by an ELISPOT assay. As a model system, the expression of selected cell differentiation markers by human circulating ASC has been evaluated after parenteral tetanus vaccination and during the course of a Leishmania infection. Prior treatment of blood MNC with beads coated with antibodies to CD38, HLA-DR or CD19 permitted the isolation of virtually all blood ASC. Further, prior immunomagnetic removal of T (CD2+) cells from blood MNC, followed by isolation of CD38+ cells facilitated the detection of Leishmania major-specific ASC in all six patients examined, whereas parasite-specific ASC among unfractionated blood mononuclear cells could only be detected in 3 out of these six patients. Simple and rapid, this approach provides not only accurate estimates of the frequency of ASC within a given B cell population or subpopulation, but can also efficiently enrich functional ASC from complex cell suspensions and thus should be particularly useful in situations where ASC are present at low frequencies.

Adult↗
PubMed1997

Differential expression of tissue-specific adhesion molecules on human circulating antibody-forming cells after systemic, enteric, and nasal immunizations. A molecular basis for the compartmentalization of effector B cell responses.

Expression of the adhesion molecules CD44, L-selectin (CD62L), and integrin alpha 4 beta 7 by antibody-secreting cells (ASC) was examined in human volunteers after oral, rectal, intranasal, or systemic immunization with cholera toxin B subunit. Almost all blood ASC, irrespective of immunization route, isotype (IgG and IgA), and immunogen, expressed CD44. On the other hand, marked differences were observed between systemically and intestinally induced ASC with respect to expression of integrin alpha 4 beta 7 and L-selectin, adhesion molecules conferring tissue specificity for mucosal tissues and peripheral lymph nodes, respectively. Thus, most ASC induced at systemic sites expressed L-selectin, whereas only a smaller proportion of ASC expressed alpha 4 beta 7. In contrast, virtually all IgA- and even IgG-ASC detected after peroral and rectal immunizations expressed alpha 4 beta 7, with only a minor fraction of these cells expressing L-selectin. Circulating ASC induced by intranasal immunization displayed a more promiscuous pattern of adhesion molecules, with a large majority of ASC coexpressing L-selectin and alpha 4 beta 7. These results demonstrate that circulating ASC induced by mucosal and systemic immunization express different sets of adhesion molecules. Furthermore, these findings provide for the first time evidence for differential expression of adhesion molecules on circulating ASC originating from different mucosal sites. Collectively, these results may explain the anatomical division of mucosal and systemic immune responses in humans as well as the compartmentalization of mucosal immune responses initiated in the upper vs. the lower aerodigestive tract.

Administration, Inhalation↗
PubMed1997

"PERFEXT": a direct method for quantitative assessment of cytokine production in vivo at the local level.

A method termed "PERFEXT", based on sequential perfusion and detergent extraction of lymphoid and non-lymphoid organs, has been developed for the quantitative measurement of cytokines produced at a local level in a given tissue. In vivo treatment of mice with Staphylococcus enterotoxin B (SEB) or lipopolysaccharide (LPS) served as the model systems. Interleukin-2 (IL2) and interferon-gamma (IFN gamma) levels were monitored by ELISA analysis of extracted samples. After local footpad (FP) injection with SEB, spleen and serum IL2 levels peaked at 2-4 h, while IL2 levels peaked at around 4-8 h in both FP and popliteal lymph nodes. SEB injection resulted in increased IFN gamma levels both in the FP and the draining lymph node. The detection of cytokines in the intestine allows for the application of the method at mucosal sites as well, provided enzyme inhibitors are present during the extraction procedure. After FP injection with LPS, IFN gamma production was significantly increased in the draining lymph node and was detectable in the FP, whereas IL2 was undetectable in any organ examined. IL2 and IFN gamma could also be detected at the site of elicitation of a delayed-type hypersensitivity reaction following local FP challenge. Local cytokine production correlated with the swelling response, whereas cytokine production in the spleen did not. IL2 peaked early, followed by a late increase in IFN gamma production, corresponding to the maximum swelling. This simple method should prove useful for analysing the production of other cytokines in vivo in distinct anatomical compartments.

Animals↗
PubMed1997

Suppression of delayed-type contact sensitivity in the murine oral mucosa by prior intragastric administration of hapten.

The effect of intragastric administration of antigen on the development of a mucosal cell-mediated immune response has been examined in a mouse model. Feeding mice with picryl sulfonic acid (PSA) abrogated development of buccal delayed-type contact hypersensitivity (CS) responses upon buccal challenge with picryl chloride (PCl) in animals that had previously been sensitized in the oral mucosa. In contrast, such feeding had no effect on buccal CS reactivity in animals similarly challenged but sensitized at skin sites. Conversely, gastric administration of PSA suppressed PCl-induced skin CS reactivity in skin-sensitized animals but had no effect on skin CS reactivity in buccally sensitized mice. Enterically induced suppression of buccal mucosa CS reactivity was associated with marked reductions in the numbers of infiltrating CD 11b+ macrophages and CD4+ cells, and a virtual disappearance of CD8+ cells in buccal tissue specimens examined 24 h after local challenge. Thus, depending on the initial site of immune sensitization, oral antigens may also suppress mucosal T cell reactivity.

Animals↗
PubMed1996

Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit.

Oral administration of autoantigens can prevent and partially suppress autoimmune diseases in a number of experimental models, Depending on the dose of antigen fed, this approach appears to involve distinct yet reversible and short-lasting mechanisms (anergy/deletion and suppression) and usually requires repeated feeding of large (suppression) to massive (anergy/deletion) amounts of autoantigens to be effective. Most importantly, this approach is relatively less effective in animals already systemically sensitized to the fed antigen, such as in animals already harboring autoreactive T cells and, thus, presumably also in humans suffering from an autoimmune disorder. We have previously shown that feeding a single dose of minute amounts of antigens conjugated to cholera toxin B subunit (CTB) can effectively suppress delayed-type hypersensitivity reactions in systemically immune animals. We now report that feeding small amounts of myelin basic protein (MBP) conjugated to CTB either before or after disease induction protected rats from experimental autoimmune encephalomyelitis. Such treatment was as effective in suppressing interleukin 2 production and proliferative responses of lymph node cells to MBP as treatment involving repeated feeding with much larger (50- to 100-fold) doses of free MBP. Different from the latter treatment, which led to decreased production of interferon-gamma in lymph nodes, low-dose oral CTB-MBP treatment was associated with increased interferon-gamma production. Most importantly, low-dose oral CTB-MBP treatment greatly reduced the level of leukocyte infiltration into spinal cord tissue compared with treatment with repeated feeding of large doses of MBP. These results suggest that the protection from experimental autoimmune encephalomyelitis achieved by feeding CTB-conjugated myelin autoantigen involves immunomodulating mechanisms that are distinct from those implicated by conventional protocols of oral tolerance induction.

Animals↗
PubMed1996

Experimental T cell-mediated inflammatory reactions in the murine oral mucosa. II. Immunohistochemical characterization of resident and infiltrating cells.

The nature and phenotype of infiltrating cells in DTH-like reactions elicited in the murine oral mucosa have been examined by routine histological and immunohistochemical procedures. During the first few hours that followed buccal challenge with the contact sensitizer oxazolone, a discrete lymphocytic reaction was disclosed in the oral mucosa of animals previously sensitized at skin sites, but was absent in animals that had been sensitized at buccal sites. The early lymphocytic reaction in the oral mucosa of skin-sensitized animals preceded the emergence of CD11+ polymorphonuclear cells (PMN) which was most prominent 8 h after hapten challenge and invaded the whole thickness of the oral epithelium. The PMN rapidly disappeared by 24 h. In contrast, early PMN infiltration was virtually absent in specimens from animals similarly challenged but that had been sensitized at local buccal sites. Irrespective of site of initial sensitization, inflammatory reactions developed in the oral mucosa, being maximal by 24 h. At that stage, CD11+ macrophages were the predominant cell type. Both CD4 and CD8 T lymphocytes were scattered in both lamina propria and epithelium, and their numbers were raised throughout the time period studied (2-168 h). IL-2 receptor expression was maximal 16 h post-challenge, and was paralleled by increased DNA synthesis in CD4+ and CD8+ cells, as demonstrated by paired immunohistoautoradiography. Focal accumulations of mononuclear cells containing IL-2 producing cells were readily detected as early as 2-3 h following local challenge with hapten in animals primed at skin but not at buccal sites. Maximal IL-2 staining was detected at 24 h irrespective of initial sensitization site. Interferon-gamma-producing cells were detected at 8 h post-challenge and remained increased during the first 24 h. MHC class II expression was detected on few oral mucosa cells during the first 4 h following hapten challenge, being mainly confined to dendritic-like cells. Consistent with increased numbers of macrophages, MHC class II expression was most intense in specimens obtained 8-24 h after hapten challenge. Thereafter, MHC class II expression was still observed in specimens obtained as late as 72 h, but was essentially associated with patches of basal keratinocytes. Taken together, these observations support the notion that the murine oral mucosa can serve as the site of expression of locally or remotely induced DTH reactions, but also indicate that the site of initial sensitization can profoundly affect the cellular composition of inflammatory reactions subsequent to local buccal challenge.

Animals↗
PubMed1996

Histaminergic regulation of interferon-gamma (IFN-gamma) production by human natural killer (NK) cells.

Monocytes, recovered from human peripheral blood by counter-current centrifugal elutriation, effectively inhibit the production of IFN-gamma by CD3-/56+ NK cells in response to IL-2. This study aimed at defining the nature of the inhibitory signal, particularly the importance of monocyte-derived reactive metabolites of oxygen. It was found that monocytes recovered from patients with chronic granulomatous disease (CGD), a condition characterized by deficient NADPH-oxidase activity of phagocytes, did not inhibit IFN-gamma production by NK cells. Further, catalase, a scavenger of hydrogen peroxide, completely reversed the inhibitory signal whereas scavengers of the superoxide anion, hypohalous acids, the hydroxyl radical, or nitric oxide synthesis inhibitors such as L-NMMA were ineffective. Inhibition of IFN-gamma production was operating on a pretranslational level, as indicated by the inability of enriched NK cells to accumulate IFN-gamma mRNA in the presence of elutriated monocytes. Hydrogen peroxide, at micromolar concentrations, reconstituted the inhibition of IFN-gamma production when added to enriched NK cells. Histamine, a biogenic amine which inhibits the generation of reactive oxygen metabolites in monocytes, abrogated the inhibition of IFN-gamma production in NK cells; by this mechanism, histamine strongly synergized with IL-2 to induce IFN-gamma in mixtures of NK cells and monocytes. The synergizing effect of histamine was specifically mediated by H2-type histamine receptors. We conclude that: (i) the induction of IFN-gamma mRNA in NK cells is effectively down-regulated by products of the oxidative metabolism of monocytes; and (ii) histamine effectively enhances IFN-gamma production by preventing monocyte-induced oxidative damage to NK cells.

Base Sequence↗
PubMed1996

Antigen presentation in the murine oral epithelium.

We have previously reported that the buccal mucosa can support delayed type hypersensitivity (DTH) reactions to contact sensitizers. In the present study, we show that cells isolated from the buccal epithelium are able to present soluble exogenous antigens to specific T cells. Single cell suspensions obtained by enzymatic dispersion of buccal epithelial sheets could present the native protein antigen hen-egg lysozyme (HEL) to the I-Ak-restricted CD4+ T-cell hybridoma specific for a.a 46-61 on HEL. T-cell activation resulted in interleukin-2 (IL-2) production which could be inhibited by anti-major histocompatibility complex (MHC) class-II antibodies of pertinent specificity. Immunohistochemical staining of whole buccal epithelial sheets revealed that all MHC II positive cells had a dendritic morphology and expressed ATPase activity, indicating that these cells represent a major antigen-presenting cell (APC) population in this tissue. Furthermore, single cell suspensions isolated from buccal epithelium (BEC) after local in vivo administration of either a native soluble protein, a synthetic dodecapeptide, or a contact sensitizer were able to activate antigen-specific T cells ex vivo. Kinetic analyses indicated that maximal APC activity in the oral epithelium occurred within 1 hr after local antigen administration, and had essentially vanished after 24 hr. Conversely, APC activity was undetectable in draining cervico-mandibular lymph node cell suspensions recovered 1 hr after local antigen injection but became manifest after 3-24 hr. These observations suggest that dendritic cells can acquire antigens in the buccal epithelium and migrate to draining lymph nodes where they present processed antigen to MHC class II-restricted T cells. This APC population may thus be a critical element in the initiation of Th1-driven DTH responses in the oral mucosa.

Animals↗
PubMed1995

Human circulating specific antibody-forming cells after systemic and mucosal immunizations: differential homing commitments and cell surface differentiation markers.

Circulating spontaneous antibody-secreting cells (ASC) induced by mucosal and systemic immunizations in human volunteers have been characterized with respect to differentiation stage and homing commitments. Irrespective of the immunization route, the large majority of ASC co-expressed CD19 and HLA-DR, which are normally lost during the transition of plasmablasts to plasmocytes, as well as CD38, a marker of activated B cell blasts, expressed also by plasmocytes. However, these cells expressed neither CD28, a molecule acquired by plasmocytes, nor CD22 and CD37, which are lost during the transition of plasmablasts to plasmocytes. Therefore, the large majority of ASC found in peripheral blood after oral and parenteral immunizations are terminally differentiated B cells, but not fully differentiated plasmocytes. As a whole, the mucosally derived ASC population seemed to be more homogenously differentiated. CD25 was detected on few ASC, whereas ASC expressing CD71 were more numerous, especially among systemically derived ASC. Almost all ASC expressed the adhesion molecules CD44 and alpha 4-integrins, irrespective of immunization route. However, virtually all systemically derived ASC expressed L-selectin, recognizing the peripheral lymph node addressin, whereas only a minority of mucosally induced blood ASC expressed L-selectin. These studies are the first to demonstrate in humans that circulating precursors of mucosal B cell immunoblasts utilize organ-specific recognition mechanisms distinct from those of corresponding systemic B cells and appear to be more advanced in the B lineage maturation pathway. Specialization of receptor expression could explain both the unification of immune responses in diverse mucosal sites and the physiologic segregation of mucosal from non-mucosal immune mechanisms in humans.

Adult↗
PubMed1995

Virus-specific antibody production and polyclonal B-cell activation in the intestinal mucosa of HIV-infected individuals.

OBJECTIVE: To examine possible changes in mucosal B-cell activation status. DESIGN: To examine the frequency and isotype distribution of total and HIV-specific antibody-secreting cells (ASC) in the intestinal mucosa of HIV-infected individuals. METHODS: Mucosal lymphocytes were obtained by enzymatic treatment of duodenal pinch biopsies and the numbers of ASC were assayed with the enzyme-linked immunospot technique. RESULTS: High numbers of HIV-specific ASC were found in the intestine of all HIV-infected individuals despite low levels of HIV-specific blood ASC. All HIV-infected individuals had large numbers of intestinal immunoglobulin (Ig) A-ASC against the HIV envelope glycoprotein gp160. Eight out of nine patients also had HIV gp160-specific intestinal IgG-ASC. These HIV-specific ASC were detected irrespective of disease stage, route of infection, or levels of circulating CD4+ T cells. HIV-specific ASC were found in peripheral blood from patients with CD4+ T cells > or = 100 x 10(6)/l blood, but in none of three patients with low CD4+ T-cell counts. The frequencies of virus-specific ASC in the blood were on average 100-fold lower than that observed within the intestinal mucosa. Mucosal polyclonal B-cell activation was evident in HIV-infected individuals, as documented by significantly elevated numbers of Ig-secreting cells (ISC) in all three major Ig classes; on average, seven-, five- and 20-fold numbers of IgA, IgG and IgM-ISC compared with healthy controls. Furthermore, substantial numbers of ASC reacting with unrelated antigens such as dog albumin and keyhole limpet haemocyanin were detected in HIV-infected patients. Interestingly, patients with CD4+ T cells < 100 x 10(6)/l blood displayed large numbers of HIV-specific intestinal ASC even though total numbers of ISC, including ASC reactive to unrelated antigens, were decreased. CONCLUSIONS: The large numbers of virus-specific ASC found in the intestine of HIV-infected individuals may be a consequence of local replication of HIV-1 resulting in a continuous antigen stimulation. The persistence of strong intestinal anti-HIV responses even at late stages of disease suggest that the mucosal B-cell responses are functionally intact throughout the disease. Furthermore, these results suggest that there is no correlation between HIV-specific ASC numbers and polyclonal B-cell activation. These observations indicate that intestinal B-cell activation is profoundly disregulated in HIV-infected individuals.

Antibodies, Viral↗
PubMed1995

Induction of compartmentalized B-cell responses in human tonsils.

The capacity of tonsillar and nasal mucosal lymphoid tissues to serve as induction sites of local and/or distant B-cell responses in humans has been examined. The frequencies of vaccine-specific antibody-secreting cells (ASC) in cell suspensions from palatine tonsils (PT) and adenoids were determined after local (intra-tonsillar [i.t.]) and regional (intranasal [i.n.]) immunizations as well as peroral and parenteral immunizations with cholera and tetanus toxoids. While peroral and parenteral immunizations evoked negligible ASC responses in PT, i.t. vaccination induced a substantial ASC response which consisted of immunoglobulin G (IgG) and IgA ASC. Responses were highly restricted to immunized tonsils. Primary immunization in one PT followed by a second immunization of both PT evoked a larger ASC response in the primed tonsil. The latter ASC response was associated with higher frequencies of ASC precursors in primed tonsils. Furthermore, two i.n. immunizations induced only modest ASC responses in PT, although such immunizations evoked high ASC responses in adenoids. However, both i.t. and i.n. routes of immunization induced specific peripheral blood ASC responses, suggesting that a fraction of B cells activated in tonsils or in nasal mucosa may enter the circulation and disseminate to distant organs. These blood ASC responses preceded increases in both IgA and IgG antibody titers in nasal washes and serum samples. However, vaccine-specific ASC were not detected in duodenal cell suspensions from volunteers who had received i.t. or i.n. immunizations. Collectively, these results indicate that tonsils can serve as expression sites of locally induced antibody responses and support the development of immunological memory. Furthermore, tonsils may serve as powerful inductive sites for immune responses expressed in the upper aerodigestive tract.

Adenoids↗
PubMed1995

Induction of specific immunity at mucosal surfaces: prospects for vaccine development.

We have demonstrated the feasibility of studying antigen-specific immune responses in a variety of mucosal tissues in humans after vaccination and during infection. In this respect, we have documented the usefulness of both oral cholera and ETEC vaccines for assessing in functional terms specific subpopulations of B- and T-cell immunocytes during an immune response initiated and/or expressed in human mucosal tissues. Circulating specific IgA antibody-secreting cells in blood appear to reflect recent or ongoing antigen exposure of mucosal surfaces. This implies that the detection of such cells in blood, the most accessible lymphoid compartment in humans, represents the simplest way to assess the immunogenicity of mucosal vaccines and to supplement the diagnostic and monitoring of active mucosal infections. Our studies indicate that while the concept of an integrated mucosal immune network is clearly operational in humans (at least in regards to induction of secretory antibody responses), its generalization appears somewhat simplistic as illustrated by the compartmentalization of immune responses initiated in certain mucosal organs such as the small intestine and the tonsils. Finally, the potential of the cholera toxin B subunit as a carrier for delivery of chemically or genetically linked foreign epitopes for induction of disseminated mucosal immune responses raises hope for the development of broadly applicable vaccines to control mucosal infections.

Animals↗