Association of T-cell receptor Vbeta haplotypes with dry skin in DS-Nh mice.

BACKGROUND: Although dry skin and T cell-dependent disease exacerbation are characteristic features of atopic dermatitis (AD), the involvement of T cells in the development of dry skin remains unclear. AIMS: We aimed to elucidate the role of T cells in the development of dry skin in DS-Nh mice as a model for AD, and to evaluate this skin condition pharmacologically. METHODS: We prepared DS-Nh mice harbouring a T-cell receptor (TCR)Vbeta(a) haplotype with a central deletion in the TCRBV gene segments, and mice harbouring a TCRVbeta(b) haplotype without any deletion. We analysed the TCRVbeta chain usage and cytokine response to antimouse CD3 monoclonal antibodies in the splenocytes from the two mouse substrains. Transepidermal water loss (TEWL) was measured, and histochemical examination of these mice was carried out. Finally, a pharmacological analysis using loratadine was also performed to evaluate the features of spontaneous dry skin in DS-Nh mice as a model of AD. RESULTS: Although the deletion of TCRBV gene segments in the TCRVbeta(a) haplotype yielded different representations of each TCRVbeta mRNA, this deletion did not evoke distinct cytokine profiles in the splenocytes compared with those of mice with the TCRVbeta(b) haplotype. Furthermore, our results indicated that the onset of dry skin occurred earlier in mice with TCRVbeta(b) than in those with TCRVbeta(a). Pharmacologically, AD-like dry skin in DS-Nh with TCRVbeta(b) mice is susceptible to an H1 blocker. CONCLUSIONS: A specific lymphocyte subpopulation bearing T-cell receptors may be responsible for loratadine-responsive dermatitis in DS-Nh mice.

MAGE-A protein and MAGE-A10 gene expressions in liver metastasis in patients with stomach cancer.

Tumour samples from 71 patients with stomach cancer, 41 patients with liver metastasis (group A) and 15 patients each in stages II-IV (group B) and stage I (group C) without liver metastasis were analysed. MAGE-A protein expression was evaluated by immunohistochemistry using a 6C1 monoclonal antibody and MAGE-A10 mRNA expression was detected by highly sensitive in situ hybridisation using a cRNA probe. Expressions of MAGE-A protein and MAGE-A10 mRNA in group A were detected in 65.9 and 80.5%, respectively. Both protein and gene showed significantly higher expression in group A than those in groups B (6.7, 26.7%) and C (0, 0%) (P=0.0003, P=<0.0001, respectively). MAGE-A10 mRNA expression in liver metastasis was found in eight (88.9%) out of nine patients. The concordant rate between MAGE-A family protein expression and MAGE-A10 mRNA expression in the primary sites was 81.7% (P<0.0001). MAGE-A10 gene expression was associated with reduced survival duration. The results of this study suggest that MAGE-A10 is a possible target in active immunotherapy for advanced stomach cancer.

Spontaneous scratching behaviour in DS-Nh mice as a possible model for pruritus in atopic dermatitis.

Itching is one of the major clinical symptoms in atopic dermatitis (AD) and complicates the management of this pathological condition. An animal model of AD-like pruritus would contribute to a better understanding of AD and could lead to the development of safe and effective antipruritic agents. DS non-hair (DS-Nh) mice raised under conventional conditions spontaneously develop pruritus, which is associated with a dermatitis similar to human AD. There is a significant positive correlation between disease severity and the period of scratching behaviour in DS-Nh mice. In the present study, we found that levels of histamine and nerve growth factor (NGF) in serum and/or skin tissue were higher in DS-Nh mice with AD-like dermatitis than in age-matched mice without dermatitis. The histopathological data indicated that nerve fibres extend into and mast cells infiltrate the surrounding area of the skin lesion. NGF production by XB-2 cells, which was derived from mouse keratinocytes, was enhanced by histamine via the H1 receptor. We also found that prolonged treatment with an H1-antagonist was effective against pruritus through depression of the production of NGF, which is thought to be generated by keratinocytes. We conclude that DS-Nh mice can serve as a suitable model for gaining a better understanding of pruritus in AD, and that prolonged treatment with an H1-antagonist may be beneficial in patients with AD-associated pruritus.

Alteration of T-cell receptor repertoires during thymic T-cell development.

The majority of thymocytes die in the thymus, whereas small populations of T cells that are able to specifically recognize an antigen are considered to survive. Although the thymic selection is thought to have a profound effect on T-cell receptor (TCR) repertoire, little is known how TCR repertoire is formed during the thymocyte developmental process. We examined TCRalpha- and beta-chain variable regions (TCRAV and TCRBV) repertoire in thymic T-cell subpopulations from mice bearing different major histocompatibility (MHC) haplotypes. In Balb/c mice, but not C57BL/6, remarkable alterations of the TCR repertoire were observed in mature T-cell subpopulations as previously reported. In contrast, there were no significant differences of TCRBV repertoire between DN3 (CD25(+)CD44(-)) and DN4 (CD25(-)CD44(-)), and between DN4 and DP. These results suggest that (1) TCR repertoire of mature T cells was formed mainly under the influence of endogenous superantigens, while MHC haplotypes played the least role; (2) the 'beta-selection' process during immature stages had little impact on TCRBV repertoire formation; and (3) TCR repertoire in immature T-cell subpopulations was extremely similar between different strains of mice. We thus consider that pre-selection TCR repertoire in immature T cells could be determined by some genetic factors conserved among different strains.

Limited VH gene usage in B-cell clones established with nurse-like cells from patients with rheumatoid arthritis.

OBJECTIVES: Nurse-like stromal cells (NLC) in synovia and bone marrow of patients with rheumatoid arthritis (RA) can support pseudoemperipolesis, protect from apoptosis and enhance immunoglobulin production of peripheral blood B cells isolated from healthy individuals, suggesting the profound contribution of hyperactivation of B cells in RA. In the course of establishing RA-NLC from RA patients, we observed the growth of B cells in the presence of RA-NLC. METHODS: We cloned B cells from the synovium or bone marrow of RA patients using the limiting dilution technique. For established clones, nucleotide sequences of immunoglobulin and surface antigens were investigated. To investigate the dependence of these clones on NLC, differences in the proliferation and the amount of immunoglobulin produced in the presence or absence of NLC were compared. Immunocytochemical staining of various cells was performed using the antibody these clones produced. RESULTS: Nine B-cell clones established from RA patients showed RA-NLC-dependent growth. These B-cell clones expressed CD19, CD20, CD38, CD39 and CD40, suggesting that the cloned cells were mature and activated. All clones secreted immunoglobulins in culture media, which were specific for intracellular components of various cell lines, including RA-NLC. Interestingly, we found limited usage of immunoglobulin heavy-chain variable regions (VH) among B-cell clones from RA patients. These repertoires were reported to be detected preferentially in fetal livers. CONCLUSION: The present study provides a novel insight into the involvement of RA-NLC in the immunopathogenesis of RA via an autoreactive B cell development and/or activation mechanism.

The presence and longevity of peripherally expanded donor-derived TCRalphabeta+ mature T lymphocyte clones after allogeneic bone marrow transplantation for adult myeloid leukemias.

There are two major pathways for T-cell regeneration after allogeneic bone marrow transplantation; thymus-dependent T-cell differentiation of T-cell progenitors, and peripheral expansion of mature T cells in the graft. In order to learn to what extent the peripheral expansion of donor-derived mature T lymphocytes contributes to reconstitution of the TCRalphabeta+ T-cell repertoire after allogeneic bone marrow transplantation for adult myeloid leukemias, we pursued the fate of donor-derived T-cell clones using the amino-acid sequences of the complementarity-determining region 3 (CDR3) of the TCR-beta chain as a clonal marker. Clonal expansion of TCRalphabeta+ T lymphocytes with specific TCRBV subfamilies was identified in donor blood. Identical T-cell clones were not found in blood from recipients before transplantation. The donor-derived T-cell clones were identified in the circulating blood from recipients a few months after allogeneic bone marrow transplantation, and they remained in the blood for 18 months after transplant in two recipients, and for 56 months in one. These results suggest that the peripheral expansion of mature T lymphocytes in the graft makes a significant contribution to post-transplant T-cell regeneration during the early period of transplantation in humans, and that mature T cells can survive in recipients for several years. Further investigation will be required to explore which antigens drive the expansion of T-cell clones in donors and recipients, and the mechanisms of maintaining homeostatic balance between the thymus-dependent pathway and the peripheral expansion of mature T cells in post-transplant T-cell regeneration.

DS-Nh as an experimental model of atopic dermatitis induced by Staphylococcus aureus producing staphylococcal enterotoxin C.

DS-Nh mice raised under conventional conditions spontaneously develop dermatitis similar to human atopic dermatitis (AD), which is associated with staphylococcal infection. In the present study, we show that Staphylococcus aureus producing staphylococcus exotoxin C (SEC) was recovered from the culture of the skin lesions of DS-Nh mice with AD-like dermatitis and that the serum levels of anti-SEC antibodies from these mice were elevated. We describe here how to promote experimental AD by epicutaneous injection with SEC-producing S. aureus to DS-Nh mice. In order to assess the role of SEC in the pathogenesis of AD, the mitogenic activity, TCRBV repertoire analysis and the production of IL-4 and IFN-gamma from spleen mononuclear cells (MNC) from DS-Nh stimulated by SEC were compared with those due to SEA, SEB and TSST. The weakest was the mitogenic activity of SEC, and higher IL-4 responses and lower IFN-gamma responses to SEC showed correlation with TCRBV8S2-positive T cells, which were selectively stimulated by SEC. We also demonstrate that SEC-producing S. aureus was able to survive in DS-Nh after intradermal injection. These results suggest a possible role for SEC in the pathogenesis of AD through host-S. aureus relationships.

Distinct TCRAV and TCRBV repertoire and CDR3 sequence of T lymphocytes clonally expanded in blood and GVHD lesions after human allogeneic bone marrow transplantation.

Acute graft-versus-host disease (GVHD) is a disorder involving the skin, gut and liver that is caused by mismatches of major and/or minor histocompatibility antigens between the HLA-identical donor and recipient. If T lymphocytes infiltrating GVHD lesions recognize antigens expressed in these organs, T cell clones should expand in inflammatory tissues. We previously reported that recipients of allogeneic bone marrow grafts have clonally expanded TCRalphabeta(+) T lymphocytes soon after transplantation, which leads to a skew of TCR repertoires. To establish whether or not the same antigens cause clonal expansion of T lymphocytes in both blood and GVHD tissues, we examined the usage of TCR alpha and beta chain variable regions (TCRAV and TCRBV) and determined the complementarity-determining region 3 (CDR3) of T lymphocytes clonally expanded in circulating blood and GVHD lesions. We found that the repertoires and CDR3 diversity of TCRAV and TCRBV differed between the GVHD lesions and circulating blood, suggesting the selective recruitment of antigen-specific T cells into GVHD tissues. We also found that the usage of TCRAV and TCRBV by the clonally expanded T lymphocytes and their CDR3 sequences differed between the GVHD tissues and blood. These results suggest that the antigen specificity of TCRalphabeta(+) T lymphocytes clonally expanded in blood and GVHD lesions is different.

Liver abscesses associated with stromal tumour of the stomach in a young woman.

A 23-year-old Japanese woman was admitted to hospital because of pyrexia and anaemia. She was found to have liver abscesses and a gastric submucosal mass by computed tomography and ultrasonography. Gastroscopy and a barium swallow revealed a round submucosal mass with a giant ulceration in the body of the stomach. The liver abscesses were successfully treated by percutaneous transhepatic drainage and intravenous administration of antibiotics. Cultures of the fluid from a liver abscess and gastric juice yielded alpha-haemolytic streptococci. Three weeks after the drainage, partial gastrectomy was performed. The tumour was diagnosed as a stromal tumour of the stomach (leiomyosarcoma) in the final histological report. The patient was discharged on postoperative day 17 without receiving adjuvant radio-chemotherapy. There have been no signs of recurrence two years after surgery. This is a rare case of a liver abscess associated with a stromal tumour of the stomach in a young patient. The bacteriological examinations suggested a possible association between these diseases.

Oligoclonal expansion of CD4(+)CD28(-) T lymphocytes in recipients of allogeneic hematopoietic cell grafts and identification of the same T cell clones within both CD4(+)CD28(+) and CD4(+)CD28(-) T cell subsets.

Recipients of allogeneic bone marrow grafts have clonally expanded CD8(+)CD28(-) T lymphocytes during the early period after transplantation, which leads to skewing of T cell receptor (TCR) repertoires. Here, we have addressed the question of whether clonal expansion of CD28(-) T cells is also observed in CD4(+) T lymphocytes after human allogeneic hematopoietic cell transplantation. We found that the fraction of T cells lacking CD28 expression in the CD4(+) subset was increased after transplantation, and expanded CD4(+)CD28(-) T lymphocytes carrying certain TCRBV subfamilies showed limited TCR diversity. In order to further study the ontogeny of CD4(+)CD28(-) T cells, we analyzed the complementarity-determining region 3 (CDR3) of the TCR-beta chain of CD4(+)CD28(+) and CD4(+)CD28(-) cells. We identified the same T cell clones within both CD4(+)CD28(-) and CD4(+)CD28(+) T cell subsets. These results suggest that both subsets are phenotypic variants of the same T cell lineage.

A clinical evaluation of lymphangioma of the large intestine: a case presentation of lymphangioma of the descending colon and a review of 279 Japanese cases.

With the development and widespread use of colonoscopy, lymphangioma of the large intestine has recently been reported frequently. This paper presents some findings from a review of 279 cases of this disease in Japan, including a typical case that we encountered. A 69-year-old female was diagnosed as having lymphangioma of the descending colon based on the findings of a barium enema and a colonoscopy, and the lesion was successfully removed by an endoscopic resection. In the published reports, the etiology of this disease is not clear yet but the age at onset range shows a tendency toward a higher incidence in comparatively older patients and the male-to-female ratio indicates a higher incidence in males. If there is no complication, endoscopic treatment seems to be the preferable procedure for this disease.

Identification of the T cell clones expanding within both CD8(+)CD28(+) and CD8(+)CD28(-) T cell subsets in recipients of allogeneic hematopoietic cell grafts and its implication in post-transplant skewing of T cell receptor repertoire.

We have previously reported that skewed repertoires of T cell receptor-beta chain variable region (TCRBV) and TCR-alpha chain variable region (TCRAV) are observed at an early period after allogeneic hematopoietic cell transplantation. Furthermore, we found that T lymphocytes using TCRBV24S1 were increased in 28% of the recipients of allogeneic grafts and an increase of TCRBV24S1 usage was shown to result from clonal expansions. Interestingly, the arginine residue was frequently present at the 3' terminal of BV24S1 segment and was followed by an acidic amino acid residue within the CDR3 region. These results suggest that these clonally expanded T cells are not randomly selected, but are expanded by stimulation with specific antigens. This study was undertaken to elucidate the mechanisms of the post-transplant skewing of TCR repertoires. Since the CD8(+)CD28(-)CD57(+) T cell subset has been reported to expand in the peripheral blood of patients receiving allogeneic hematopoietic cell grafts, we examined the TCRAV and TCRBV repertoires of the CD8(+)CD28(-) T cell and CD8(+)CD28(+) T cell subsets, and also determined the clonality of both T cell populations. In all three recipients examined, the CD8(+)CD28(-) T cell subset appeared to define the post-transplant TCR repertoire of circulating blood T cells. Moreover, the CDR3 length of TCRBV imposed constraints in both CD8(+)CD28(-) T cell and CD8(+)CD28(+) T cell subsets. The DNA sequences of the CDR3 region were determined, and the same clones were identified within both CD8(+)CD28(-) and CD8(+)CD28(+) T cell subsets in the same individuals. These results suggest that the clonally expanded CD8(+)CD28(-) T cells after allogeneic hematopoietic cell transplantation derive from the CD8(+)CD28(+) T cell subset, possibly by an antigen-driven mechanism, resulting in the skewed TCR repertoire.

Extensive clonal expansion of T lymphocytes causes contracted diversity of complementarity-determining region 3 and skewed T cell receptor repertoires after allogeneic hematopoietic cell transplantation.

We previously described skewed repertoires of the T cell receptor-beta chain variable region (TCRBV) and the TCR-alpha chain variable region (TCRAV) soon after allogeneic hematopoietic cell transplantation. To determine the characteristics of skewed TCRBV after transplantation, we examined the clonality of T lymphocytes carrying skewed TCRBV subfamilies and determined the CDR3 sequences of expanded T cell clones. In all 11 recipients examined, TCR repertoires were skewed, with an increase of certain TCRBV subfamilies that differed among individuals. In nine of 11 patients, clonal/oligoclonal T cell expansion was observed, although the expanded T cells were not necessarily oligoclonal. The extent of expansion after transplantation appeared to predict clonality. The arginine (R)-X-X-glycine (G) sequence was identified in clonally expanded T cells from four of five recipients examined, and glutamic acid (E), aspartic acid (D) and alanine (A) were frequently inserted between R and G. These results suggest that T lymphocyte expansion may result from the response to antigens widely existing in humans, and that the extensive clonal expansion of a limited number of T cells leads to contracted CDR3 diversity and post-transplant skewed TCR repertoires.

Primary malignant melanoma of the esophagus treated by esophagectomy and systemic chemotherapy.

We describe herein a case of asymptomatic primary malignant melanoma of the esophagus. A 65-year-old man presented with a 4-cm filling defect in the middle third of the esophagus on a routine barium swallow. Subtotal esophagectomy accompanied by lymph node dissection was performed through a right thoracotomy. Postoperatively, the patient received five cycles of systemic chemotherapy with dacarbazine (DTIC), nimustine hydrochloride (ACNU), and vincristine (VCR) (DAV therapy), but ultimately died of generalized metastatic disease 15 months after surgery. Malignant melanoma of the esophagus has an extremely poor prognosis despite various therapeutic efforts.

Inhibited serum phospholipase A2 activity in hyperbilirubinemia.

BACKGROUND/AIMS: Phospholipase A2 activity is reported to be related to the physiology of various disorders, and the activity is modified by deoxycholic acid. METHODOLOGY: Serum phospholipase A2 activity was measured by counting the radioactivity of 14C-palmitic acid released from L-3-phosphatidylcholine (1-palmitoyl-2-14C-palmitoyl) in 40 patients with hyperbilirubinemia due to malignant neoplasms and benign cholestasis. Phospholipase A2 activity in serum from healthy subjects was also measured after incubation with 5-30 mg of bilirubin per dL or 0.1-5 mM of cholic acid for 60 min at 37 degrees C. RESULTS: Serum phospholipase A2 activity was significantly lower in patients with hyperbilirubinemia. There were negative correlations between serum phospholipase A2 activity and concentration of total bilirubin (r = 0.668; P < 0.0001) or total bile acids (r = 0.423; P < 0.05) in patients with hyperbilirubinemia. In 12 patients who underwent percutaneous transhepatic biliary drainage, the low serum phospholipase A2 activity was reversed while bilirubin concentrations decreased. Incubation of sera from healthy subjects with more than 3 mM cholic acid significantly reduced phospholipase A2 activity (P < 0.01), whereas incubation with bilirubin had no effect. CONCLUSIONS: Inhibition of serum phospholipase A2 activity in patients with hyperbilirubinemia may be caused by increased serum cholic acid concentration.

A new method for quantitative analysis of the mouse T-cell receptor V region repertoires: comparison of repertoires among strains.

We developed an adaptor ligation PCR-based microplate hybridization assay (MHA) to analyze the repertoires of mouse T-cell receptor (TCR) alpha- and beta-chain variable regions (TCRAV and TCRBV). RNA is transcribed to cDNA and an adaptor is ligated to the 5' end of the cDNA, which is then used as a template for PCR with an adaptor-specific 3' primer and a constant region-specific 5' primer. After hybridization of PCR products with TCRAV-and TCRBV-specific probes on the microplate, quantitative ELISA was carried out. The entire TCRAV or TCRBV repertoires could be analyzed using a single 96-well plate in triplicate and completed in less than 4 h. The assay results demonstrated the high level of specificity and reproducibility of this method. Furthermore, MHA results correlated well with those of fluorescence-activated cell sorting. This method may provide important information about various T-cell-associated diseases including autoimmune disease. The influence of the MHC on mouse TCR repertoires was next studied using the newly developed mouse TCRAV and TCRBV repertoire assay. The analysis in six strains showed no significant correlation between MHC haplotypes and TCRAV and TCRBV repertoires. However, large differences among strains was observed in TCRBV, but not in TCRAV repertoires. There were also large differences within same strain in TCRBV, but not in TCRAV repertoires, indicating differences in individuals independent of genetic factors. These data suggest that TCRBV repertoires are more susceptible than TCRAV repertoires not only to genetic factors but also some environmental factors.

Restricted usage of T-cell receptor alpha-chain variable region (TCRAV) and T-cell receptor beta-chain variable region (TCRBV) repertoires after human allogeneic haematopoietic transplantation.

We analysed T-cell receptor alpha-chain variable region (TCRAV) and T-cell receptor beta-chain variable region (TCRBV) repertoires in peripheral blood mononuclear cells (PBMCs) from 34 recipients of allogeneic bone marrow transplantation (allo-BMT), seven of allogeneic peripheral blood stem cell transplantation and 19 of autologous peripheral blood stem cell transplantation using the quantitative microplate hybridization assay. TCR usage skewed at an early period (6-7 weeks) after BMT. The change was more apparent in allogeneic recipients than in autologous recipients. In particular, a predominant increase was detected in the frequency of VA1-4 (26%, 11 of 41 recipients), VA3-1 (32%) and VB24-1 (28%). Interestingly, acidic amino acid residues frequently followed the arginine residue in complementarity-determining region 3 of BV24S1. We further examined the extent of skew using samples obtained at serial time points after transplantation. The normalization of skewed repertoires occurred over a long period of time (> 8 years). There was a significant difference in the rate of normalization of skewed TCR repertoires between adult and child recipients (P < 0.05). The results suggest that these T cells may have expanded in response to allogeneic antigens, such as miHA (minor histocompatibility antigen), and that altered repertoires are eventually normalized by T-cell regeneration via a thymic-dependent pathway in children.

Delayed recovery of CDR3 complexity of the T-cell receptor-beta chain in recipients of allogeneic bone marrow transplants who had virus-associated interstitial pneumonia: monitor of T-cell function by CDR3 spectratyping.

BACKGROUND: In the T-cell receptor (TCR)-beta chain, complementary-determining region 3 (CDR3) contains specific peptide sequences essential for recognition. Diversity of this region is considered to contribute to immunocompetence in humans. OBJECTIVE: The purpose of this study was to define the process of reconstitution of CDR3 complexity of the TCR-beta chain after allogeneic bone marrow transplantation and to investigate the association between host immunocompetence and CDR3 complexity. METHODS: Diversity of the CDR3 region of the TCR-beta chain was examined by CDR3 size distribution analysis with the use of an automated DNA sequencer. RESULTS: Reconstitution of the alphabeta T-cell repertoire and CDR3 diversity was incomplete for at least 2 months after bone marrow transplantation. Delayed reconstitution of T-cell diversity was more marked in immunocompromised hosts. Unlike the situation in patients who received allogeneic bone marrow grafts, the recovery of CDR3 complexity was almost perfect by 2 months after transplantation in patients who received allogeneic blood stem cells. Clonal expansion of alphabeta T cells after allogeneic bone marrow transplantation was readily detected by CDR3 size spectratyping analysis. CONCLUSION: PCR-based CDR3 size spectratyping may be a useful tool for clinically monitoring immune reconstitution after allogeneic bone marrow transplantation.

Detection of T cell apoptosis after major operations.

OBJECTIVE: To detect T cell apoptosis in reduced peripheral lymphocyte counts in patients having major operations. DESIGN: Prospective study. SETTING: University hospital, Japan. SUBJECTS: 11 patients having oesophagectomy and 5 having laparoscopic cholecystectomy. INTERVENTIONS: To investigate T cell apoptosis we detected DNA fragmentation using electrophoresis, and T-cell receptor-gamma (TCR-gamma) gene amplification using polymerase chain reaction (PCR) in serum. MAIN OUTCOME MEASURES: Peripheral lymphocyte count and DNA extracted from the serum preoperatively and on postoperative days 1, 3, 5, and 7. RESULTS: The lymphocyte count decreased significantly until day 5 and then increased in the patients who had had oesophagectomy. DNA fragmentation and PCR products for the TCRgamma variable region gene were found in the serum DNA of 10 patients until day 5. No DNA fragmentation or PCR products were found in the serum of patients who had had laparoscopic cholecystectomy. CONCLUSION: These results suggest that transient T cell apoptosis occurs after major operations.