Biomedical subjects

S Habu

Publications and source records attributed to S Habu.

At least 91 records · Page 5Linked to original sources

PubMed1994

Naive T cells can mediate delayed-type hypersensitivity response in T cell receptor transgenic mice.

We produced transgenic mice expressing T cell receptor-alpha beta chain genes, derived from the chicken ovalbumin (OVA)-specific I-Ad-restricted CD4+CD8- T helper cell clone 7-3-7. In transgenic mice with H-2d genetic background (Tg-d mice), delayed-type hypersensitivity (DTH) was induced in the hind footpad by one inoculation with OVA without any previous sensitization, suggesting that naive T cells have the potential to be involved in DTH response. Spleen cells from nonimmunized Tg-d mice showed a strong T cell proliferative response to in vitro stimulation with OVA. Furthermore, these spleen cells produce cytokines including interleukin(IL)-2, IL-3, interferon-gamma, granulocyte/macrophage colony-stimulating factor, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, which may play an important role in the attraction of mononuclear cells to an antigen-challenging site.

Animals↗

PubMed1994

Lack of involvement of the cholinergic mechanism in vasoactive intestinal peptide- and peptide-histidine methionine-induced growth hormone (GH) responses in acromegaly: comparison with the GH responses to thyrotropin-releasing hormone and GH-releasing hormone.

We examined whether the cholinergic mechanism is involved in the paradoxical GH responses to vasoactive intestinal peptide (VIP) and peptide histidine methionine (PHM) in acromegaly. 28 patients with active acromegaly underwent i.v. bolus injections of thyrotropin-releasing hormone (TRH, 500 micrograms), GH-releasing hormone (GHRH, 100 micrograms), VIP (100 micrograms), and PHM (100 micrograms) with or without a prior atropine treatment (1 mg, i.m., 30 min before). Blood samples were collected before and at intervals up to 120 min after the injection, and plasma GH levels were measured. In response to TRH, GHRH, VIP and PHM, 23 (82%), 24 (86%), 13 (46%) and 7 (25%) patients, respectively, responded with a significant GH increase (> 50% and 6 micrograms/l above the basal level). The effect of atropine pretreatment was examined in only these responders to the respective peptides. When the GH responses were estimated by the area under the response curve, the atropine pretreatment was able to significantly suppress the GH response to GHRH, but not to TRH, VIP, or PHM. Although the lack of cholinergic involvement in the TRH-induced GH release in acromegaly is confirmatory to previous reports, the same results with the VIP- and PHM-induced GH release are novel. The present study may suggest that in acromegaly the physiological GH response is mediated by the cholinergic mechanism, but the paradoxical ones are not.

Acetylcholine↗

PubMed1994

Acceleration of diabetes in young NOD mice with peritoneal macrophages.

To elucidate the roles of macrophages in the pathogenesis of NOD murine diabetes, peritoneal macrophages from NOD mice were injected into young NOD mice. We used 12 to 20 week-old NOD mice of both sexes as donors, and sex-matched 2-week-old NOD mice as recipients. Cyclophosphamide (CY), 200 mg/kg, was intraperitoneally injected into the donors. Two weeks later, peritoneal exudate cells (PEC) were collected from the diabetic donors. Macrophage-rich fractions (MRF) were collected by adherence. Then PEC(5-8 x 10(6)) or MRF(3-7 x 10(6)) were transferred, intraperitoneally, to the recipients. Two weeks later, some of the recipients were killed in order to perform immunofluorescent analysis of splenocytes and to assess pancreatic histology. Mac 1 positive splenocytes were increased in PEC- and in MRF-injected recipient mice. Insulitis was seen in PEC- and MRF-injected mice, but not in controls. Some of the recipients were injected with CY, 200 mg/kg, intraperitoneally, at two weeks post cell transfer. Two weeks after CY injection, the animals were examined for the presence of diabetes. The incidences of diabetes were 67% in PEC-injected mice, 40% in the MRF-injected group, and 3% in the controls. These results suggest that peritoneal macrophages accelerate the disease process in NOD mice.

Animals↗

PubMed1994

Implication of the common gamma chain of the IL-7 receptor in intrathymic development of pro-T cells.

The effects of IL-7 on the growth and differentiation of thymocytes were analyzed using murine fetal thymus organ cultures (FTOC) in the presence of mAbs specific for the conventional IL-7 receptor (IL-7R) and for the common gamma (gamma c) chain. In FTOC, the development of CD4-CD8- double-negative thymocytes to CD4+CD8+ double-positive (DP) and CD4+ or CD8+ single-positive (SP) cells was not completely blocked by adding these mAbs, although cell growth was reduced by the treatment. To define a developing stage sensitive to the mAbs, most immature thymocytes, Pgp-1+ c-kit+ cells, were cultured in 2-deoxyguanosine treated fetal thymus. In the presence of both mAbs in the culture, neither DP nor SP thymocytes developed whereas either of the mAbs partially blocked their development. These results indicate that the gamma c chain is involved in early T cell development as an indispensable subunit of the functional IL-7 receptor complex.

Animals↗

PubMed1994

Expression of tumor-associated glycoantigen, sialyl Lewis(a), in human head and neck squamous cell carcinoma and its application to tumor immunotherapy.

The glycoantigen sialyl Lewis(a) (sLe(a)) is widely expressed on a variety of gastrointestinal tumor cells. Here, we immunohistochemically demonstrated the expression of sLe(a) antigen in 54% (7 out of 13) of human head and neck squamous cell carcinoma (H-NSCC) samples. Frequent expression of sLe(a antigen was also demonstrated on a variety of H-NSCC cell lines using flow cytometry. Both CD4+ and CD8+ T cells, which were activated with immobilized OKT3 monoclonal antibody plus interleukin-2, showed augmented cytotoxicity against sLe(a)-positive H-NSCC, including autologous tumor cells, on targeting with anti-CD3 x anti-sLe(a) bispecific antibody, suggesting that sLe(a) antigen is a good target molecule for bispecific antibody-dependent adoptive tumor immunotherapy of human head and neck cancer.

Adult↗

PubMed1994

[An efficient methods for the induction of human antitumor effector CD4+ and CD8+ T cells: their application to tumor immunotherapy].

It is an important issues to investigate an efficient methods to induce antitumor effector T cells from peripheral blood lymphocytes of tumor patients for the development of a novel tumor immunotherapy. We established a large scale culture system of human CD4+ helper/killer T cells which have both helper and killer functions. Targeting of CD4+ helper/killer T cells to tumor using anti-CD3 x anti-c-erbB-2 mAb caused the lysis of tumor and triggering of IL-2 production. It was also demonstrated that culture of human CD4+ T cells with staphylococcal enterotoxin A (SEA) or IL-12 caused a selective induction of Th1 type of CD4+ helper/killer T cells. IL-12 also revealed a novel effect on CD8+CTL functions. Culture of CD8+ T cells with IL-12 resulted in the augmentation of IFN-gamma production and cytotoxicity. Moreover, culture of tumor-infiltrating lymphocytes with IL-12 caused a marked enhancement of CD8+CTL against autologous tumor cells. These findings suggest that IL-12 will become a useful cytokine for the tumor immunotherapy. In this paper, we will discuss the key role of CD4+ T cells for the induction of antitumor immunity in tumor-bearing host.

CD4-Positive T-Lymphocytes↗

PubMed1993

Inhibition of inflammatory liver injury by a monoclonal antibody against lymphocyte function-associated antigen-1.

When mice were given an i.v. injection of LPS 7 days after an i.v. injection of Propionibacterium acnes, liver injury and a rapid increase of serum alanine aminotransferase and asparagine acid aminotransferase occurred. The in vivo administration of mAb against LFA-1 on days 1, 2, and 3 after the i.v. injection of P. acnes resulted in a potent inhibition of all these dysfunctions. Using P. acnes and the LPS model, we found that anti-LFA-1 mAb protected the mice from P. acnes and LPS-induced lethal shock. During the course of P. acnes and LPS-induced liver injury, inflammatory cells infiltrated the liver and caused a massive hepatic cell necrosis. Flow cytometry revealed that the liver-infiltrating cells were mainly leukocytes expressing a higher level of LFA-1 antigen than that seen in the normal liver. These results suggested that the LFA-1 molecule on liver-infiltrating leukocytes may play an important role in the induction of inflammatory liver injury.

Alanine Transaminase↗

PubMed1993

Expression of sialyl Lewis(x) antigen on human T cells.

Sialyl Lewis(x) (sLe(x)) antigen, which has been introduced as tumor-associated cell surface carbohydrates, was demonstrated to be expressed on a subpopulation of human CD4+ and CD8+ T cells. It was also demonstrated that the majority of gamma delta T cells as well as natural killer cells expressed a high level of sLe(x) antigen. The sLe(x) expression on T cells were up-regulated by activation with various T cell stimulants. The sLe(x)+ CD4+ T cells were enriched in CD4+CD45RO+ memory type of T cells but not in CD4+CD45RA+ T cells. Moreover, sLe(x)+ CD4+ T cells were demonstrated to show higher proliferative responses to T cell stimulants such as CD4+CD45RO+ T cells. These results initially clarified the expression of sLe(x) carbohydrate on human T cells and indicated the important role of sLe(x)-expressing T cells in immune responses.

Antibodies, Monoclonal↗

PubMed1993

Lymphocyte infiltration of the skin in transgenic mice carrying the human interleukin-2 gene.

Inflammatory lesions of the skin such as erythema, depigmentation and hair loss were observed in C57/BL6(B6) transgenic mice that carried an intact human genomic interleukin-2 gene (gIL-2 transgenic mice). Accumulation of T lymphocytes in the perivascular and periadnexal areas of the dermis was the first change, followed by dermal papillary oedema, which occurred before the development of macroscopic skin lesions. In 3- or 4-week-old transgenic mice with slight erythema and depigmentation of the skin, there was an increase in the number of perivascular lymphocytes accompanied by the diffuse infiltration of neutrophils and monocytes in the damaged skin. These morphological skin changes were not observed in non-transgenic mice, which were bred together with transgenic litter mates. These findings suggest that lymphocyte infiltration of the perivascular space of the skin is a primary event of exogenously introduced human interleukin-2 gene, resulting in secondary cutaneous changes in gIL-2 transgenic mice.

Animals↗

PubMed1993

The role of cytotoxic macrophages in non-obese diabetic mice: cytotoxicity against murine mastocytoma and beta-cell lines.

The cytotoxicity of macrophages from non-obese diabetic (NOD) mice against murine mastocytoma (P-815), and murine beta-cell lines having the NOD gene background (MIN6N-9a), were examined. Peritoneal exudate cells from 20-week-old mice showed higher cytotoxicity, measured as inhibition of thymidine uptake into P-815, than those from 12-week-old mice (p < 0.01). In cyclophosphamide-injected mice, cytotoxicity of peritoneal exudate cells had increased at 8 days post-injection, at which time the mice were not diabetic. To confirm macrophage cytotoxicity against pancreatic cells and examine its cytolytic mechanism, the cytotoxicity of peritoneal exudate cells from cyclophosphamide-injected NOD mice against MIN6N-9a cells was measured by the chromium release assay. These peritoneal exudate cells showed higher cytotoxicity as compared to those of saline-injected mice (p < 0.001). Macrophages were demonstrated to be the major component of peritoneal exudate cells (50%) by flowcytometric analyses. Cytotoxicity increased with macrophage enrichment by adhesion (p < 0.01). Furthermore, a macrophage toxin, silica, completely blocked the cytotoxicity (p < 0.001). Cytokines (interleukin 1 and tumour necrosis factor) and a nitric-oxide-producing vasodilator, sodium nitroprusside, were cytotoxic to MIN6N-9a cells but only sodium nitroprusside showed cytotoxicity when incubated for the same period as peritoneal exudate cells. Thus, macrophages play an important role in beta-cell destruction and soluble factors other than cytokines (e.g. nitric oxide) may be mediators of this early cytolytic process.

Aging↗

PubMed1993

CD4+CD8+ cells lacking self-Mls reactive T cells are induced in mesenteric lymph nodes of Salmonella enteritidis-infected mice.

The percentage of mesenteric lymph node (MLN) cells that co-expressed both CD4 and CD8 was found to be from 5 to 7% in BALB/c and AKR/N mice bred under conventional conditions. In mice maintained under specific pathogen-free (SPF) conditions, the percentage fell below 2%. When mice were infected with an attenuated strain of Salmonella enteritidis (SER), the percentage of CD4+CD8+ cells in MLN rose to 20-30% transiently. In these mice, the total cell number and the percentage of CD8+ cells were not changed, but the CD4+ cell percentage was decreased. The expression intensity of TCR-alpha beta on CD4+CD8+ cells in the infected mice was higher in the MLN than in the thymus, but was similar to that of mature peripheral T cells. Among the CD4+CD8+ cell population in MLN, TCR-V beta 3+ cells were deleted but V beta 6+ cells were present in BALB/c mice which possess endogenous superantigen Mls-2a, but lack Mls-la. In AKR mice with the inverse of the occurrence of the superantigens, TCR-V beta 3+ cells were present and V beta 6+ cells were absent. These data suggest that CD4+CD8+ cells in the MLN of SER-infected mice may belong to thymus-derived mature T cells undergoing negative selection and that they may appear following exogenous stimulation.

Animals↗

PubMed1993

Characterization of the T cells in aged rat bone marrow.

In this study, we determined the characteristics of CD3-positive (CD3+) T cells existing in rat bone marrow (BM). In contrast to splenic T cells, BM CD3+ T cells are composed of a higher proportion of CD8+ T cells, and the number of both cell types increased with age. Such CD3+ T cells in aged rats showed a similar usage of TCR V beta as splenic T cells, suggesting that BM CD3+ T cells are thymus-dependent and composed of an ordinary population in view of the expression of the TCR beta-chain. Purified T cells obtained from aged rat BM showed a markedly proliferative response by stimulation with immobilized anti-CD3 mAb, as did splenic T cells. However, the addition of BM non-T cells completely inhibited the response of both BM and splenic T cells in vitro. These results suggest that T cells in rat BM are negatively regulated by BM non-T cells in their response to the TCR-mediated signal not to disrupt the microenvironment of the BM.

Aging↗

PubMed1993

Thymic nurse cell clone supports the differentiation of CD4-8- thymocytes into CD4+8+ thymocytes in vitro.

A previously reported thymic nurse cell clone, TNC-R3.1 could form a unique complex with isolated adult mouse CD4-8- (DN) thymocytes and greatly sustained the cell viability of DN thymocytes in suspension culture. In addition, the TNC-R3.1 clone supported the differentiation of DN thymocytes into CD4+8+ (DP) thymocytes in a short-term culture. Addition of IL-7 into the coculture markedly enhanced DN thymocyte-TNC interaction and induced the proliferation and differentiation of DN thymocytes, though IL-7 alone did not induce the differentiation of DN thymocytes. Separation of DN thymocytes from TNC-R3.1 monolayer using a Millicell caused a great inhibition of the DN thymocyte differentiation, suggesting that direct contact between TNC-R3.1 cells and immature thymocytes was required for the differentiation of DN thymocytes. The kinetics study demonstrated that DN thymocytes started to differentiate into DP thymocytes through CD3-CD4+J11d+ intermediate cells 8-12 h after the initiation of the culture with TNC-R3.1 plus IL-7. The generation of DP thymocytes became maximal 20 h after coculture and gradually decreased thereafter. Furthermore, we demonstrated that TNC-R3.1 could support the differentiation of CD3+CD4+CD8- or CD3+CD4-CD8+ thymocytes from CD3-CD4-CD8- thymocytes in the presence of IL-7 and IL-2. These data indicate that our established in vitro culture system mimics the early stage of the intrathymic T cell developing pathway.

Animals↗

PubMed1993

Potentiation of therapeutic effect of recombinant tumor necrosis factor against B16 mouse melanoma by combination with recombinant interleukin 2.

Treatment of B16 melanoma-bearing mice with recombinant tumour necrosis factor (rTNF) caused a marked inhibition of tumour growth but did not result in the complete cure of the tumour-bearing mice. In contrast, combination therapy of B16-bearing mice with r-TNF and recombinant interleukin 2 (rIL-2) potentiated the therapeutic effect of rTNF and 30% of the mice were totally cured from tumour. Spleen cells obtained from B16-bearing mice showed markedly decreased immune responses including IL-2 production, IL-2 responsiveness and mixed lymphocyte reaction owing to the existence of suppressor macrophages. However, spleen cells obtained from mice cured with rTNF plus rIL-2 showed the same level of T cell responsiveness as that from normal mice. The decreased induction of alloantigen-specific cytotoxic T lymphocytes (CTL) in B16-bearing mice was also recovered after treatment with rTNF plus rIL-2. Moreover, B16-specific CTL, which could not be induced in normal or B16-bearing mice, was effectively induced from the spleen cells of B16-cured mice by rTNF and rIL-2. These results demonstrated that local therapy of melanoma with rTNF and rIL-2 was effective and induced systemic antitumour immunity in vivo.

Animals↗

PubMed1993

Specific targeting of in vitro-activated human antitumour effector cells using anti-CD3 x anti-c-erbB-2 bispecific antibody.

Bispecific antibody (BSAb) consisting of anti-CD3 plus anti-c-erbB-2 Fab fragments for the application to adoptive tumour immunotherapy was prepared. This bifunctional hetero-F(ab')2 antibody reacted with both human CD3+ T cells and c-erbB-2 positive human tumour cells. Human CD8+ T cells activated with immobilized anti-CD3 plus interleukin 2 showed marginal cytotoxicity against tumour cells. However, addition of the prepared BSAb into the culture resulted in a marked augmentation of the cytotoxicity by the activated CD8+ T cells in a dose-dependent manner. The enhanced cytotoxicity of CD8+ T cells in the presence of BSAb was specific for c-erbB-2 positive tumour cells. Moreover, it was demonstrated that anti-CD3 x anti-c-erbB-2 BSAb was also effective for the specific targeting of various kinds of in vitro-activated antitumour effector cells such as lymphokine-activated killer cells, CD4+ helper/killer cells, gamma delta T cells and activated tumour-infiltrating CD8+ T cells. These results indicated that BSAb consisted of anti-CD3 and anti-c-erbB-2 will become a useful tool for the adoptive tumour immunotherapy of human cancer expressing c-erbB-2 oncogene products.

Animals↗

PubMed1993

Intestinal intraepithelial lymphocytes preferentially repopulate the intestinal epithelium.

We have used C.B-17 severe combined immune deficiency (SCID) mice to study the repopulation of intestinal intraepithelial cells in these mice. We have found that intestinal intraepithelial lymphocytes (IELs) injected into SCID mice preferentially repopulate the intestinal epithelium. About 5 weeks after injection we can detect significant numbers of IEL in repopulated SCID mice. Repopulation occurs in approximately 70% of the injected mice and the amount of recovered cells per mouse is variable. The recovered cells are of donor-type origin and exhibit a typical IEL phenotype. The donor-type T lymphocytes that can sometimes be found in other organs of IEL-repopulated SCID mice are generally of low number. They are not stained with antibodies against IEL-specific markers and their phenotypes appear to be more typical for T cells normally found in these sites. In contrast, the intestinal epithelium of SCID mice cannot be efficiently repopulated with lymphocytes using cells of other organs including thymocytes, Peyer's patch lymphocytes, and bone marrow cells. From our data we conclude that intestinal IELs are confined to the intestinal epithelium and possibly contain a precursor-type cell that preferentially regenerates cells of its own population.

Animals↗

PubMed1993

Production of a human monoclonal antibody to a synthetic peptide by active in vivo immunization using a SCID mouse grafted with human lymphocytes.

In order to meet the present increased demands, we have tried to improve the methods to produce human monoclonal antibodies by using a SCID mouse grafted with human mononuclear cells. Initially, we gave an anti-asialo GM1 antibody to a SCID mouse to suppress the NK activity, as a pretreatment. Then, fifty million human mononuclear cells (MNC) from a healthy volunteer were injected intraperitoneally to the SCID mouse so as to construct a human immune system in the mouse (PBL-SCID mouse). We immunized the mouse with a synthetic peptide (pep 190) conjugated with KLH four times. The spleen cells taken from the immunized PBL-SCID mouse were fused with (mouse x human) heteromyeloma cells. The hybridoma cells were selected in GIT medium containing HAT and IL-6. Among 68 hybridoma-growing wells, we obtained one hybridoma clone (#41-1-4) which secreted a specific antibody to pep 190. The reactivity of this monoclonal antibody was tested by ELISA and the specificity of this antibody was confirmed by an absorption test with different kinds of proteins. This paper is the first report of the successful production of peptide-specific human monoclonal antibody by active in vivo immunization using a PBL-SCID mouse. By this active in vivo immunization system using a PBL-SCID mouse, human monoclonal antibodies for any sort of peptide antigens may easily be made available.

Amino Acid Sequence↗

PubMed1992

Cellular and peptide requirements for in vitro clonal deletion of immature thymocytes.

Thymocytes from DO10 T-cell-receptor transgenic mice undergo apoptosis, or programmed cell death, when chicken ovalbumin-(323-339) peptide is administered in vivo. Using DO10 mice thymocytes, we have now developed a simple in vitro model system that recapitulates the in vivo clonal-deletion process. When transgenic thymocytes were cocultured with fibroblasts, B cells, or thymic nurse cell lines (all bearing I-Ad) in the presence of chicken ovalbumin-(323-339), deletion of the transgenic TCR+CD4+CD8+ thymocytes was seen within 8-20 hr. Thymocytes designed to bear I-Ad on their surface could mediate the deletion themselves. Thus, thymocyte clonal deletion entirely depends on the stage at which the thymocytes are vulnerable to the onset of apoptosis, rather than on the nature of the peptide antigen-presenting cells. Furthermore, thymic nurse cell line TNC-R3.1 could cause deletion, strongly suggesting that some thymic epithelial/stromal components are potentially capable of participating in negative selection. In all cases examined, little deletion could be induced at a peptide concentration less than 10 nM, thus defining the minimum amount of peptide antigen required for negative selection. The peptide-dependent in vitro negative-selection system will allow further dissection of the molecular and cellular processes involved in clonal deletion due to apoptosis in the thymus.

Amino Acid Sequence↗