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Biomedical subjects

H Kiyono

Publications and source records attributed to H Kiyono.

At least 109 records · Page 6Linked to original sources
PubMed1997

A novel alkaline phosphatase-based isolation method allows characterization of intraepithelial lymphocytes from villi tip and crypt regions of murine small intestine.

The isolation of intestinal intraepithelial lymphocytes (IEL) is a major prerequisite for the investigation of cellular and molecular cross-talk in the intestinal mucosa. Since intestinal epithelial cells exhibit distinct functional features at the villi tip and crypt levels, such differences could extend to IEL. We developed a mechanical procedure for isolation of IEL from these distinct epithelial sites to test our hypothesis. Cells isolated from the intestinal epithelium by sequential incubations under stirring were segregated based upon their alkaline phosphatase (AP) activity since villi tip and crypt fractions expressed high and low AP activity, respectively. IEL preparations obtained after a further purification step in Percoll gradient contained > 90% Integrin alpha IEL chain+, CD3+ T cells, and no Ig+ cells. Villi tip IEL preparations possessed increased numbers of low density IEL when compared to crypt IEL, suggesting that distinct IEL-epithelial cell interactions occur at the intestinal villi tip and crypt levels.

Alkaline Phosphatase↗
PubMed1997

Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene.

Replication-deficient adenovirus (Ad) vectors are effective to specifically target the respiratory epithelium for either corrective gene therapy such as cystic fibrosis or for mucosal immunization. As a consequence of transducing the lower respiratory tract with an E1/E3 deleted Ad5 vector, host responses have been characterized by the duration of transgene expression and by the induction of CTL responses. However, limited emphasis has been devoted to understanding the contribution of CD4+ T cell responses to the Ad vector. Both CD4+ and CD8+ T cells migrate into the lung following sequential intratracheal Ad5 transgene instillations. Isolated CD3+ T lymphocytes from the lungs were predominantly of the Th2 type, and after cell sorting, the IL-4-producing T cells were largely CD4+, while IFN-gamma expression was associated with both CD4+ and CD8+ T cells. Ab responses to the Ad5 vector and to the expressed transgene beta-galactosidase (beta gal) revealed elevated bronchial and serum IgA and IgG Abs with low neutralization titers. Analysis of serum IgG subclass responses showed IgG1 and IgG2b with lower IgG2a Abs to Ad5 and IgG2a and IgG2b Ab responses to beta gal. Ad5-specifc CD4+ T cells produced both Th1 (IFN-gamma and IL-2)- and Th2 (IL-4, IL-5, IL-6)-type cytokines, while beta gal-specific CD4+ T cells secreted IFN-gamma and IL-6. This study provides direct evidence for the concomitant induction of Th2- with Th1-type responses in both the pulmonary systemic and mucosal immune compartments to the Ad5 vector as well as a Th1-dominant response to the transgene.

Adenoviridae↗
PubMed1997

Oral immunization with simian immunodeficiency virus p55gag and cholera toxin elicits both mucosal IgA and systemic IgG immune responses in nonhuman primates.

Rhesus macaques were orally immunized with a mucosal vaccine consisting of two different concentrations (1 mg vs 250 microg) of recombinant SIV p55gag (p55) with or without cholera toxin (CT, 50 microg) as a mucosal adjuvant. The plasma from macaques receiving the higher dose of p55 (1 mg) and CT had higher p55-specific IgG and IgA Ab titers compared with macaques that received the lower dose of p55 (250 microg) and CT. Further, high levels of p55-specific IgG and IgA Abs were present in external secretions from both groups. The level of p55-induced T cell responses was elevated in PBMCs isolated from the high dose group compared with the low dose group. When culture supernatants from these p55-stimulated PBMCs were examined for Th1 (IFN-gamma) and Th2 (IL-4 and IL-10) cytokines, both IFN-gamma and IL-10 were present, but IL-4 was absent. CD4+ T cells isolated from these p55-stimulated PBMCs contained IFN-gamma spot-forming cells (SFCs) but not IL-4 SFCs. These results were further confirmed by cytokine-specific reverse transcriptase PCR analysis, where p55-specific CD4+ T cells expressed mRNA for IFN-gamma, IL-6, and IL-10, but not IL-4. These findings suggest that oral immunization of nonhuman primates induced both IFN-gamma-secreting Th1 and select Th2 cytokine (e.g., IL-6 and IL-10)-producing CD4+ Th cells, which accounted for the generation of p55-specific systemic and mucosal Ab responses.

Administration, Oral↗
PubMed1997

A nontoxic mutant of cholera toxin elicits Th2-type responses for enhanced mucosal immunity.

We have characterized a nontoxic mutant of cholera toxin (CT) as a mucosal adjuvant in mice. The mutant CT was made by substitution of serine with phenylalanine at position 61 of the A subunit (S61F), which resulted in loss of ADP ribosyltransferase activity and toxicity. Mice were intranasally immunized with ovalbumin, tetanus toxoid, or influenza virus either alone or together with mutant CT S61F, native CT, or recombinant CT-B. Mice immunized with these proteins plus S61F showed high serum titers of protein-specific IgG and IgA antibodies that were comparable to those induced by native CT. Further, high protein-specific IgA antibody responses were observed in nasal and vaginal washes, saliva, and fecal extracts as well as increased numbers of IgG and IgA antibody forming cells in cervical lymph nodes and lung tissues of mice intranasally immunized with these proteins and S61F or native CT, but not with recombinant CT-B or protein alone. Both S61F and native CT enhanced the induction of ovalbumin-specific CD4(+) T cells in lung and splenic tissues, and these T cells produced a Th2-type cytokine pattern of interleukin 4 (IL-4), IL-5, IL-6, and IL-10 as determined by analysis of secreted proteins and by quantitation of cytokine-specific mRNA. These results have shown that mutant CT S61F is an effective mucosal adjuvant when administrated intranasally and induces mucosal and systemic antibody responses which are mediated by CD4(+) Th2-type cells.

Animals↗
PubMed1997

Mutants in the ADP-ribosyltransferase cleft of cholera toxin lack diarrheagenicity but retain adjuvanticity.

Cholera toxin (CT), the most commonly used mucosal adjuvant in experimental animals, is unsuitable for humans because of potent diarrhea-inducing properties. We have constructed two CT-A subunit mutants, e.g., serine-->phenylalanine at position 61 (S61F), and glutamic acid-->lysine at 112 (E112K) by site-directed mutagenesis. Neither mutant CT (mCT), in contrast to native CT (nCT), induced adenosine diphosphate-ribosylation, cyclic adenosine monophosphate formation, or fluid accumulation in ligated mouse ileal loops. Both mCTs retained adjuvant properties, since mice given ovalbumin (OVA) subcutaneously with mCTs or nCT, but not OVA alone developed high-titered serum anti-OVA immunoglobulin G (IgG) antibodies (Abs) which were largely of IgG1 and IgG2b subclasses. Although nCT induced brisk IgE Ab responses, both mCTs elicited lower anti-OVA IgE Abs. OVA-specific CD4+ T cells were induced by nCT and by mCTs, and quantitative analysis of secreted cytokines and mRNA revealed a T helper cell 2 (Th2)-type response. These results now show that the toxic properties of CT can be separated from adjuvanticity, and the mCTs induce Ab responses via a Th2 cell pathway.

Adjuvants, Immunologic↗
PubMed1997

Oral but not parenteral interleukin (IL)-12 redirects T helper 2 (Th2)-type responses to an oral vaccine without altering mucosal IgA responses.

Our past studies have shown that the mucosal adjuvant cholera toxin (CT) induces T helper type 2 (Th2) responses with systemic IgG1, IgE and mucosal secretory IgA (S-IgA) antibodies (Abs). In this study, recombinant murine IL-12 (rmIL-12) was given either parenterally or orally to mice orally immunized with tetanus toxoid (TT) and CT to determine whether this cytokine could redirect the CT-induced Th2-type responses and what effect this shift would have on S-IgA Ab responses. Intraperitoneal administration of rmIL-12 shifted TT-specific responses toward Th1-type and resulted in CD4+ T cells producing IFN-gamma and IL-2 with markedly reduced levels of Th2-type cytokines. This cytokine profile was accompanied by increased delayed-type hypersensitivity (DTH) and shifts in serum IgG1 to IgG2a and IgG3 anti-TT Ab responses. Further, serum IgE and S-IgA Ab responses were markedly reduced by parenteral IL-12. When IL-12 complexed to liposomes was given orally both shifts to IgG2a and IgG3 and low IgE Abs again occurred concomitant with enhanced serum IFN-gamma and DTH responses. Interestingly, oral rmIL-12 did not result in significant levels of serum IL-12 nor altered S-IgA Ab responses and resulted in higher levels of some Th2-type cytokines both in vitro and in vivo when compared with parenteral IL-12. Our results show that the shifts in systemic immune responses with intact S-IgA Abs which occur after oral delivery of IL-12-liposomes are due to cytokine effects in the Peyer's patches and suggest new strategies for the targeted manipulation of Th1- and Th2-type responses to mucosal vaccines.

Administration, Oral↗
PubMed1997

An interleukin-7 internet for intestinal intraepithelial T cell development: knockout of ligand or receptor reveal differences in the immunodeficient state.

Both interleukin-7 (IL-7) and IL-7 receptor (R) gene knockout (IL-7-/- and IL-7R-/-) mice were employed in order to directly investigate the importance of the IL-7 and IL-7R signaling pathway for the development of intestinal intraepithelial lymphocytes (IEL). Loss of the IL-7R-specific gene resulted in complete deficiency of the gamma delta T cell lineage with lack of V gamma 4- and V gamma 7-specific messages in the epithelium of the gastrointestinal (GI) tract in comparison to control mice of the same genetic background (approximately 40%). Disruption of the IL-7-specific gene resulted in marked, but not complete depletion of gamma delta T cells (2-3%) in IEL. Furthermore, mRNA for both V gamma 4 and V gamma 7 genes were detected in the gamma delta IEL subset of IL-7-/- mice. The subtle differences between IL-7-/- and IL-7R-/- mice suggest that although IL-7 controls most of the expansion and/or development of gamma delta IEL, another ligand binding to the IL-7R also plays a discernable role. Furthermore, alpha beta IEL developed more slowly in IL-7R-/- mice when compared with ligand knockouts; however, the frequency of IEL T cells subsequently increased with age and normal levels of CD3+ T cells expressing the alpha beta TCR were detected by 2 and 3 months of age in IL-7-/- and IL-7R-/- mice, respectively. The direct comparison of IL-7-/- and IL-7R-/- mice clearly supports the hypothesis that both IL-7 and another IL-7R binding molecule can influence the development of gamma delta T cells in the intestinal epithelium.

Animals↗
PubMed1997

Bacterial invasion induces interleukin-7 receptor expression in colonic epithelial cell line, T84.

The intestinal epithelial layer forms the interface between the external and the internal environments of a host. Since the interleukin-7 (IL-7) and IL-7 receptor (IL-7R) signaling pathway has been shown to play an important role in the mucosal immune system, we studied the expression of IL-7R in T84, a colonic epithelial cell line, after cells were infected with several types of enteropathogenic bacteria, including Salmonella typhimurium, enteropathogenic Escherichia coli and enteroinvasive Escherichia coli. Bacterial invasion induced IL-7R expression in T84 assessed by a semi-quantitative reverse transcription-polymerase chain reaction technique and flow cytometry. The inhibition of bacterial invasion by cytochalasin D, a specific inhibitor of actin polymerization, led to a reduction in the expression of IL-7R. These data indicate that bacterial invasion into intestinal epithelial cells is likely to be an essential process in the induction of IL-7R. The communication between the epithelium and mucosal lymphocytes which is mediated via IL-7 and IL-7R may be involved in the modulation of the mucosal inflammation which occurs in bacterial infection.

Antigens, CD↗
PubMed1997

CD4+ T-cell population mediates development of inflammatory bowel disease in T-cell receptor alpha chain-deficient mice.

BACKGROUND & AIMS: Increase of T cells expressing CD4 and T-cell receptor (TCR) alpha- beta+ (beta[dim]) was observed in the mucosal and peripheral lymphoid tissues of TCR alpha-/- mice with inflammatory bowel disease (IBD). The aim of this study was to characterize the CD4+ TCR alpha-beta+ T cells. METHODS: Cytokine production, TCR V beta usage, and helper function for Peyer's patch B cells by the CD4+ TCR alpha-beta+ T cells were assessed. RESULTS: The CD4+ TCR alpha-beta+ T cells purified from mesenteric lymph nodes and lamina propria of the intestine of IBD mice exclusively produced interleukin 4, used selected subsets (V beta6, V beta8, V beta14, and V beta15) of TCR, and massively proliferated after stimulation with staphylococcal enterotoxin B. Addition of the CD4+ TCR alpha-beta+ T cells to Peyer's patch B-cell cultures markedly enhanced immunoglobulin (Ig) A, IgG, and IgM antibody responses. Furthermore, depletion of the TCR alpha-beta+ T cells with monoclonal antibody against TCR beta chain completely suppressed the onset of IBD and polyclonal B-cell activation in the TCR alpha-/- mice. CONCLUSIONS: These findings suggest the CD4+ TCR alpha-beta+ T cells-mediated development of IBD in TCR alpha-/- mice.

Animals↗
PubMed1997

Molecular and cellular mechanisms for periodontal diseases: role of Th1 and Th2 type cytokines in induction of mucosal inflammation.

An accumulation of elevated numbers of macrophages (M phi) and Ig producing cells is associated with localized and chronically inflamed gingiva of patients with adult periodontitis. When gingival lymphocytes were isolated from inflamed tissues and examined by flow cytometry, approximately 20-30% of lymphocytes were CD4+ T cells. For the analysis of Th1 and Th2 cytokine expression by these CD4+ T cells, RNA was extracted and reverse transcriptase polymerase chain reaction (RT-PCR) was performed by using specific 5' and 3' primers for IFN-gamma and IL-2 (Th1), IL-4, IL-5, IL-6, IL-10 and IL-13, (Th2) and beta-actin (housekeeping gene). Two distinct cytokine profiles were noted based on the expression of selected Th1 and Th2 cytokines. Thus, one pattern was represented by the expression of mRNA for IFN-gamma, IL-6, IL-10 and IL-13, while the other case consisted of mRNA for IFN-gamma, IL-6, and IL-13. Except for a few cases, messages for IL-2, IL-4 and IL-5 were not detected by cytokine-specific RT-PCR. The predominant expression of Th2 cytokines (e.g. IL-6, IL-10 and IL-13) may contribute to the induction of high B cell responses in local disease sites. On the other hand, lack of IL-4 may be responsible for the accumulation of M phi in diseased periodontium. We also investigated whether a relationship exists between IL-4 receptor (IL-4R) expression and M phi persistence in the absence of exogenous IL-4. Gingival M phi, when compared with monocytes (MN)/M phi from peripheral blood mononuclear cells (PBMC), expressed high levels of IL-4R mRNA. When gingival M phi were incubated with recombinant IL-4 (rIL-4), the cell viability was dramatically reduced by apoptosis. These findings clearly show that the lack of IL-4 may contribute to the persistent occurrence of M phi at the disease site and addition of exogenous rIL-4 to gingival M phi cultures leads to cell death by apoptosis.

Adult↗
PubMed1997

Oral immunization with PspA elicits protective humoral immunity against Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a major respiratory mucosal pathogen affecting infants and children. Although a polysaccharide-based vaccine has been useful in adult populations, it does not elicit protective immunity in infants and young children. Pneumococcal surface protein A (PspA) is a highly immunogenic surface protein produced by all strains of Streptococcus pneumoniae. Previous studies have shown that systemic immunization of mice with PspA can elicit protective immunity against fatal pneumococcal infection. In this study, we demonstrated that oral immunization with PspA could elicit protective immune responses against pneumococcal infection. When mice were orally immunized with PspA alone, low levels of PspA-specific immunoglobulin G (IgG) responses were induced in serum; none was induced in secretion. On the other hand, when PspA was given orally with the mucosal adjuvant cholera toxin (CT), significant levels of IgG and IgA anti-PspA responses were induced in serum. The major IgG subclass was IgG1, followed by IgG2b, a profile of antibody response supported by Th2-type cells. In addition, all mice orally immunized with PspA and CT were protected from the lethal challenge with capsular serotype 3 S. pneumoniae A66. These results suggested that an oral PspA vaccine may be a useful means of preventing pneumococcal disease.

Adjuvants, Immunologic↗
PubMed1997

Spontaneously hypertensive rat: cholera toxin converts suppression to immunity through a Th2 cell-IL-4 pathway.

The spontaneously hypertensive rat (SHR) exhibits a number of T cell dysfunctions that develop concurrently with elevated blood pressure. Studies have shown a mitogen-induced lymphocyte suppression mediated in part by the production of interferon-gamma (IFN-gamma), which stimulated NO production by macrophages. To assess whether this immune suppression is reversible, SHR were immunized with diphtheria toxoid (DT) with or without cholera toxin (CT) as adjuvant. SHR immunized with DT only displayed weak serum immunoglobulin G (IgG) anti-DT titers, tenfold less than similarly treated normotensive Wistar-Kyoto rats (WKYR). SHR CD4+ T cells failed to proliferate upon in vitro stimulation with DT. In contrast, SHR coimmunized with DT and CT showed serum IgG antibody titers similar to WKYR and Brown Norway rats. Coimmunization with CT rescued SHR CD4+ T cells from suppression and supported DT- or B subunit of CT-specific proliferative responses, and these cells produced more interleukin-4 (IL-4) than IFN-gamma, and anti-IFN-gamma antibody treatment enhanced IL-4 production. Exogenous IL-4 increased the proliferation of antigen-specific CD4+ T cells, whereas IFN-gamma was inhibitory. This study shows that the adjuvant CT induces T helper 2-type responses, reversing the T cell dysfunction in the SHR.

Animals↗
PubMed1997

[Mucosal immune system and mucosal vaccine].

In the recent years, mucosal immune system is recognized as the new world in the area of immunology. The host is continuously exposed to the numerous numbers of environmental antigens via the mucosa and the skin. A total surface area of the mucosa is approximately 200 times larger than that of the skin, and the former surface area contains a large numbers of lymphoid cells (> 10(11)). In order to provide an effective defence for the host by vaccine, it is logical to consider the mucosal immune system. According to the new informations obtained by the modern cellular and molecular immunobiological knowledges and approaches, the concept of the mucosal immune system has been rapidly proceeded to apply for the development of mucosal vaccine. In this report, we have reviewed and discussed the recent progress in the characterization of mucosal immune system and the development of mucosal vaccine.

Animals↗
PubMed1997

Functional gamma delta T-lymphocyte defect associated with human immunodeficiency virus infections.

BACKGROUND: Antiviral cellular immune responses may influence immunological homeostasis in HIV-infected persons. Recent data indicate that V gamma 9/V delta 2 T lymphocytes display potent cytotoxic activities against human cells infected with certain viruses including HIV. Understanding the role of gamma delta T cells in the course of HIV infection may be helpful for designing novel treatment strategies for HIV-associated disorders. MATERIALS AND METHODS: The constitutive recognition of Daudi cells and monoethyl pyrophosphate (Etpp) by peripheral blood V gamma 9/V delta 2 T cells was assessed using a proliferation assay. The cytotoxicity of Daudi-stimulated lymphocyte populations was measured by chromium release assays. The HIV infectivity for gamma delta T cell clones was determined by measuring the levels of HIV p24 in cell supernatants. The effect of in vitro HIV-infection on cytokine mRNA production by gamma delta T cell clones was assessed by PCR. RESULTS: The constitutive proliferative responses of peripheral blood V gamma 9/V delta 2 T cells and the lytic functions of Daudi-expanded lymphoid cells from HIV+ persons were substantially diminished in comparison with those of HIV-seronegative persons. These alterations were present in asymptomatic HIV+ persons prior to substantial alpha beta CD4+ T cell loss. Productive HIV infection of gamma delta T cells in vitro had no measurable effect either on their proliferative response to Daudi stimuli or on the expression of cytokine mRNAs for IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-13. CONCLUSIONS: The constitutive responsiveness of V gamma 9/V delta 2 T lymphocytes to Daudi and Etpp is severely altered in HIV+ persons. HIV infection of gamma delta T cells in vitro does not substantially change their cytokine expression or antigenic response.

Cell Division↗
PubMed1997

[The mechanisms of oral tolerance].

Oral or nasal administration of a single high dose or repeated mucosal delivery of low doses of proteins have been shown to induce systemic unresponsiveness in the presence of mucosal IgA responses. The induction of oral tolerance(or mucosally-induced tolerance) is mediated by T cells involved in the generation of active suppression, clonal anergy or clonal deletion. Studies of T helper(Th) cytokine responses have suggested that Th1- and Th2-type cells are involved in the induction of oral tolerance. Further, gamma delta T cells appear to be an important T cell subset for the regulation of oral tolerance.

Administration, Oral↗