Seiho Nagafuchi

AIRE deficiency in thymus of 2 patients with Omenn syndrome

Omenn syndrome is a severe primary immunodeficiency with putative autoimmune manifestations of the skin and gastrointestinal tract. The disease is caused by hypomorphic mutations in recombination-activating genes that impair but do not abolish the process of VDJ recombination, leading to the generation of autoreactive T cells with a highly restricted receptor repertoire. Loss of central tolerance in genetically determined autoimmune diseases, e.g., autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, is associated with defective expression by medullary thymic epithelial cells of AIRE, the transcription activator that induces thymic expression of tissue-specific antigens. Analysis of AIRE expression in the thymi of 2 Omenn syndrome patients and 1 SCID patient, by real-time RT-PCR and immunohistochemistry, demonstrated a profound reduction in the levels of AIRE mRNA and protein in patients as compared with a normal control subject. Lack of AIRE was associated with normal or even increased levels of keratin and lymphotoxin-beta receptor mRNAs, while mRNAs of the self-antigens insulin, cytochrome P450 1a2, and fatty acid-binding protein were undetectable in thymi from immunodeficiency patients. These results demonstrate that deficiency of AIRE expression is observed in severe immunodeficiencies characterized by abnormal T cell development and suggest that in Omenn syndrome, the few residual T cell clones that develop may escape negative selection and thereafter expand in the periphery, causing massive autoimmune reactions.

Expression of AIRE gene in peripheral monocyte/dendritic cell lineage

The responsible gene for autoimmune polyglandular syndrome type 1, known as autoimmune regulator (AIRE), was identified by positional cloning. The AIRE gene was reported to be expressed in the thymus medulla and lymph nodes. However, an expression of the AIRE gene in peripheral blood cells has not yet been reported. In the present study, we found that the AIRE gene was restrictively expressed in peripheral CD14-positive monocytes but not in CD4-positive T cells nor polymorphonuclear cells, as assessed by RT-PCR. Moreover, immunocytochemical study revealed the expression of the AIRE protein not only in CD14-positive monocytes but also in differentiated dendritic cells, cultured in RPMI1640 medium containing 800 U/ml GM-CSF, 1000 U/ml IL-4 and 100 U/ml TNF-alpha. Thus, it was concluded that the AIRE gene is restrictively expressed in the peripheral monocyte/dendritic cell lineage.

Human cord blood--derived cells generate insulin-producing cells in vivo.

Here we report the capacity of human cord blood (CB)–derived cells to generate insulin‐producing cells. To investigate in vivo capacity of human CB–derived cells, T cell–depleted mononuclear cells were intravenously transplanted into nonobese diabetic/severe combined immunodeficient/β2‐microglobulinnull mice within 48 hours of birth. At 1–2 months post‐transplantation, immunofluorescence staining for insulin and fluorescence in situ hybridization (FISH) analysis using a human chromosome probe indicated that human CB–derived cells generated insulin‐producing cells at a frequency of 0.65% ± 0.64% in xenogeneic hosts. Reverse transcription–polymerase chain reaction analysis confirmed the transcription of human insulin in the pancreatic tissue of the recipient mice. To clarify the mechanism underlying CB‐derived insulin‐producing cells, double FISH analysis using species‐specific probes was performed. Almost equal proportions of human chromosome+ murine chromosome− insulin+ cells and human chromosome+ murine chromosome+ insulin+ cells were present in recipient pancreatic islets. Taken together, human CB contains progenitor cells, which can generate insulin‐producing cells in recipient pancreatic tissues across a xenogeneic histocompatibility barrier by fusion‐dependent and ‐independent mechanisms.

CLINICOPATHOLOGICAL STUDY OF SEVERE CHRONIC ACTIVE EPSTEIN-BARR VIRUS INFECTION THAT DEVELOPED IN ASSOCIATION WITH LYMPHOPROLIFERATIVE DISORDER AND/OR HEMOPHAGOCYTIC SYNDROME

Chronlc active Epstein‐Barr virus (CAEBV) infection has been prevlously reported to be sometimes associated with an aggresslve clinical course. However, the role of EBV in the CAEBV Is not well clarifled. A retrospective study was performed on nine adult and five child patients (eight males and six females). Histologlcally, at first admission, the presence of neoplastic lesions could not be confirmed. The lymph nodes In half of all cases revealed paracortical hyperplasla with transformed lymphocytes (hyperplastic type). Half of the cases showed nonsuppuratlve necrosis and an Increased number of histiocytes with phagocytosis (histiocytic type). Activated histiocytes with lymphokine positivity were frequently detected in the histiocytic type. In the phenotypical study, 10 of the examined 11 cases showed Increased numbers of natural killer (NK) cells and/or CD8 positive T lymphocytes. In situ hybridization (ISH) showed EBV‐Infected lymphoid cells, but the number of EBV‐infected cells varied. Double‐labeling irnmunochemistryASH demonstrated EBV‐infected T cells, including NK cells, but not B cells. In addition, three cases showed a monoclonal dissemination of EBV terminal repetitive sequence (TR), and two cases showed oligoclonal dissemination. From those findings, monoclonal, oligoclonal and polyclonal populations of EBV‐infected T or NK cells were considered to be present in CAEBV states. During the clinical course, 12 of the 14 cases died within 5 years. Six cases died from EBV‐associated hematopoietic tumors (histiocytlc tumor, T cell lymphoma, B cell lymphoma, plasmacytoma, and NK cell leukemia); one from non‐EBV‐associated acute myeloge‐nous leukemia, and five due to hemophagocytic syndrome. The examined EBV‐associated hematopoietic tumors showed monoclonal EBV terminal repetitive sequences. There is a possibility that the monoclonal dissemination of EBV‐infected cells develops from oligoclonal or polyclonal EBV‐infected cells. And active histlocytes with lymphokine positivity were frequently detected In the cases with histologically histiocytic type. These findings seem to be related with the causes of death due to hemophagocytic syndrome.

Identification of a novel type 1 diabetes susceptibility gene, T-bet

The gene encoding interferon (IFN)-γ, IFNG, is known as one of the candidate susceptibility genes for type 1 diabetes. In addition, cytokines, including IFN-γ, play important roles in the pathogenesis of type 1 diabetes. Therefore, we focused on the Th1-specific T-box transcription factor gene (T-bet), which contributes to the induction of the hallmark Th1 cytokine, IFN-γ. We first screened for polymorphisms in the T-bet gene and detected two microsatellite repeat polymorphisms located in intron 1 and the 3′- flanking region, and two single nucleotide polymorphisms, including a His33Gln substitution within the coding region. By association studies, the Gln-positive phenotype and (CA)14 allele in 3′-flanking region of T-bet were found to be associated with type 1 diabetes in the Japanese population. Furthermore, Gln33 T-bet showed a significantly higher transcriptional activity of the IFNG gene via a dual luciferase reporter assay. Our study suggests the first evidence of an association between type 1 diabetes and polymorphisms in the T-bet gene, and that variation in T-bet transcriptional activity may play a role in the development of type 1 diabetes, possibly through the effect on IFN-γ production in Th1 cells.

In vivo comparative therapeutic study of optimal administration of 5-fluorouracil and cisplatin using a newly established HST-1 human squamous-carcinoma cell line

SummaryThe efficacy and toxicity of 5-fluorouracil (5-FU) and cisplatin (CDDP) given in a sequential combination were evaluated in nude mice transplanted with HST-1, a newly established human squamous-carcinoma cell line. 5-FU and CDDP were given i.p. for 5 days and 1 day, respectively, either as single agents or in a sequential manner separated by a 24-h interval. The treatment was repeated every 30 days. Although inhibition of tumor growth was seen in all of the treated groups after two cycles, the sequence of 5-FU followed by CDDP significantly reduced the tumor burdens throughout all three courses and was more effective than the reverse sequence or either drug alone. Neither treatment-related death nor significant hematologic or nephrologic toxicities were seen, even following three cycles of therapy. Significant weight loss was observed only in mice treated with CDDP followed by 5-FU. This sequence dependence of the activity and toxicity of the 5-FU and CDDP combination should thus be incorporated into the design of a clinical trial.

The Role of Thymic Immunity and Insulitis in the Development of Streptozocin-induced Diabetes in Mice

This article is concerned with the role of thymic immunity in the development of diabetes experimentally induced by multiple injections of subdiabetogenic doses of streptozocin (STZ). Euthymic + / +, + /nu, and athymic nu/nu mice of CD-1 and BALB/cAJcl origin were studied. Daily intraperitoneal (i.p.) injections of 30 mg /kg body wt of STZ for 5 consecutive days in CD-1 + / + and + /nu mice resulted in hyperglycemia and mononuclear cell infiltrations of islets (insulitis). The CD-1 nu/nu mice developed neither insulitis nor hyperglycemia after the same treatment. In the nu/nu mice, when thymic immunity was restored by thymus grafting, both insulitis and hyperglycemia developed, thus demonstrating that thymic immunity was a prerequisite for the development of insulitis and hyperglycemia. There was a positive correlation among the degrees of thymic immunity, insulitis, and hyperglycemia in CD-1 + /nu,nu/nu with thymus grafts, and nu/nu mice, indicating that thymic immunity may amplify the diabetogenic effect of STZ by eliciting insulitis. In contrast, in BALB/cAJcl mice, a nonsusceptible strain to insulitis, no significant differences in plasma glucose levels were observed between the + /nu and nu/nu or between the nu/nu and thymus-grafted nu/nu mice. Furthermore, no significant difference was found in plasma testosterone levels between the + /nu and nu/nu mice of both CD-1 and BALB/cAJcI origin. In conclusion, our data indicate that thymic immunity enhances the diabetogenic effect of STZ by eliciting insulitis in susceptible mice.

Aire-Dependent Thymic Expression of Desmoglein 3, the Autoantigen in Pemphigus Vulgaris, and Its Role in T-Cell Tolerance

In the mechanism of thymus-induced central tolerance, the transcription factor Aire has been demonstrated to promote the expression of a wide range of peripheral organ-specific antigens (Ags) in the medullary thymic epithelial cells (mTECs), which serve as self-Ags in negative selection. We examined the expression of desmoglein 3 (Dsg3), the autoantigen in pemphigus vulgaris (PV), in mouse thymus and the involvement of Aire in tolerance to Dsg3. Immunofluorescence and in situ hybridization revealed Dsg3 in single cells or in clusters in ∼3% of mTECs near the cortico-medullary junction of the thymus in C57BL/6 mice. Dsg3-expressing mTECs also expressed some Ags of skin-unrelated peripheral organs simultaneously. In contrast, Dsg3-positive mTECs were not detected in the Aire(-/-) thymus. Adoptive transfer of splenocytes from Aire(-/-) mice immunized with Dsg3 did not induce anti-Dsg3 IgG production or PV phenotype in Rag2(-/-) recipient mice. However, Aire(-/-) CD4(+) T cells, but not Aire(+/+) CD4(+) T cells, induced low levels of anti-Dsg3 IgG production when transferred with Dsg3(-/-) B cells. These findings indicate that Aire has an important role in Dsg3 expression as well as in selection of T cells that help B cells to produce anti-Dsg3 IgG in thymus.

Role of Lyt-1 Positive Immune T Cells in Recovery from Herpes Simplex Virus Infection in Mice

It has been shown that T cell-mediated immunity plays a major role (4, 5, 710, 12) in control of and recovery from herpes simplex virus (HSV) infection. Nevertheless, the mechanism of immune T cell-mediated protection in vivo is complex and poorly understood. Recent studies have revealed that peripheral T cells express different Lytalloantigens on their surfaces and are divisible into three subpopulations with phenotypes Lyt-1.2.3, Lyt-l, and Lyt-2.3 (2). Lyt-1.2.3 cells are presumed to be precursors for Lyt-I or Lyt-2.3 cells (2). Lyt-l cells are programmed to help or amplify activities of other cells after stimulation by an antigen (2). They help B cells to produce antibody and activate macrophages in the generation of the delayed type hypersensitivity reaction (2). Lyt-2.3 cells are known to suppress antibody production and other immune responses, and to be cytotoxic cells (2). In this study, we have used Lyt-alloantisera for the examination of immune T lymphocytes responsible for providing protection in mice lethally infected with HSV. Sixto 10-week-old athymic nude (nu/nu) mice and their littermates (nu/+) with BALB/c genetic background were purchased from the Central Laboratories of Experimental Animals Co., Ltd., Japan. When athymic nude mice were injected intracutaneously with 2.5 X 104 plaque-forming units (PFU) of the virulent Hayashida strain of HSV-l per 0.05 ml, all of the mice died after development of severe zosteriform skin lesions, while all of the control nul + mice survived the infection, as reported previously (10), indicating that resistance of HSV infection is T-cell dependent. In order to determine the phenotypes of committed lymphocytes responsible for HSV resistance, immune spleen cells were transferred to numu mice that were lethally infected with HSV, following treatment with various antilymphocyte sera. Anti-lymphocyte alloantisera were obtained from Cederlane Laboratories, Canada. Anti-Thy-1.2 alloantisera were produced in AKR/J mice by injecting thymocytes

Genome-wide linkage analysis of type 2 diabetes mellitus reconfirms the susceptibility locus on 11p13–p12 in Japanese

AbstractType 2 diabetes mellitus is a heterogeneous disorder, and the development of type 2 diabetes mellitus is associated with both insulin secretion defect and insulin resistance. The primary metabolic defect leading to type 2 diabetes mellitus has been thought to be varied among populations, especially in Japanese and Caucasians. Here, we have done the genome-wide scan for type 2 diabetes mellitus using 102 affected Japanese sib-pairs to identify the genetic factors predisposing to type 2 diabetes mellitus. Nonparametric linkage analysis showed one suggestive evidence for linkage to 11p13-p12 [D11S905: two-point maximum LOD score (MLS) of 2.89 and multipoint MLS of 2.32] and one nominally significant evidence for linkage to 6q15-q16 (D6S462: two-point MLS of 2.02). Interestingly, the 11p13-p12 region was reported to be a susceptibility locus for Japanese type 2 diabetes mellitus with suggestive evidence of linkage, and D11S905 was within 5 cM to D11S935 with the highest MLS in the previous linkage analysis reported. The only overlapped susceptibility region with suggestive evidence of linkage for Japanese type 2 diabetes mellitus was D11S935-D11S905 among the three reports including this study. These results taken together suggest that a susceptibility gene for type 2 diabetes mellitus in Japanese will reside in 11p13-p12.