Mutations of carnitine palmitoyltransferase II (CPT II) in Japanese patients with CPT II deficiency.

Carnitine palmitoyltransferase II (CPT II) deficiency is an inherited disorder involving beta-oxidation of long-chain fatty acids. CPT II deficiency is a wide-spectrum disorder that includes a lethal neonatal form, an infantile form, and an adult-onset form. However, the ethnic characteristics and the relationship between genotype and clinical manifestation are not well understood. We investigated three non-consanguineous Japanese patients with CPT II deficiency and examined cell lines from 4 unrelated patients and 50 healthy donors. The CPT 2 gene was typed by direct DNA sequencing of polymerase chain reaction-amplified gene products. Case 1 (infantile form) was heterozygous for a phenylalanine to tyrosine substitution at position 383 (p.F383Y) and a novel valine to leucine substitution at 605 (p.V605L). Cases 2, 4, and 5 (infantile form) and case 3 (adult-onset form) were heterozygous for a single mutation at F383Y. Case 6 (adult-onset form) was compound heterozygous at the CPT 2 locus, with deletion of cytosine and thymine at residue 408, resulting in a stop signal at 420 (p.Y408fsX420), and an arginine to cysteine substitution at position 631 (p.R631C). Case 7 (adult-onset form) was homozygous for the p.F383Y mutation. In conclusion, we identified p.F383Y mutations in six of seven patients with CPT II deficiency and two novel variants of the coding gene: p.Y408fsX420 and p.V605L. These mutations differ from those in Caucasian patients, who commonly harbor p.S113L, p.P50H, and p.Q413fsX449 mutations; therefore, our data and those of other Japanese groups suggest that the p.F383Y mutation is significant in Japanese patients with CPT II deficiency.

Disseminated strongyloidiasis in nephrotic syndrome.

Strongyloides stercoralis is endemic in the southwestern islands Amami and Ryukyu in Japan. Systemic strongyloidiasis occurs in immunocompromised hosts. We report here on a 60-year-old patient with minimal-change nephrotic syndrome (MCNS) without eosinophilia or HTLV-I infection. She was treated with corticosteroid for MCNS and died of disseminated strongyloidiasis. The patient developed systemic purpura, ileus, respiratory distress, malabsorption, pancytopenia, pulmonary hemorrhage and sepsis due to Escherichia coli before death. Massive infestation with Strongyloides stercoralis was disclosed by autopsy, and the larvae was considered as a pathomechanism or exacerbating agent of nephrotic syndrome in endemic areas.

Suppression of experimental membranous glomerulonephritis in rats by an anti-MHC class II antibody.

BACKGROUND: We previously reported that idiopathic membranous nephropathy (IMN) strongly correlated with HLA-DRB1*1501-DRB5*0101-DQAI*0102-DQB1* 0602, a specific haplotype of human major histocompatibility complex (MHC), in Japanese patients. To investigate the role of MHC in the development of rat Heymann nephritis (HN), an animal model of membranous nephropathy, a monoclonal antibody (mAb) specific to rat MHC class II antigen (RT1B) was administered, and its effectiveness in inhibiting HN was assessed. METHODS: Active HN was induced in HN-sensitive Lewis rats by administering brush border proteins of rat proximal uriniferous tubules (FX1A). Rats were divided into four groups: rats treated with 1,000 micorg anti-rat MHC class II mAb, rats treated with 100 microg anti-rat MHC class II mAb, rats treated with murine myeloma IgG, and rats that did not receive either FX1A or any other mAb. We examined the differences in 24-hour urinary protein excretion and serum alloantibody titers against FX1A between groups at different time intervals, and the histologic features of kidneys at the end of the study. RESULTS: HN was induced in Lewis rats by inoculation with FX1A antigen. Administration of anti-MHC class II mAb successfully lowered urinary proteins, production of anti-FX1A alloantibodies, and the development of glomerular lesions in a dose-dependent manner. CONCLUSION: The present results demonstrated that the MHC class II molecule itself is directly involved in the pathogenesis of HN, and suggest that this therapy would be any better (or less toxic) than nonselective immunosuppressants in the treatment of IMN.

Relationship between fluctuation pattern of blood pressure during hemodialysis treatment and cardiovascular morphology: an autopsy study of 53 cases.

BACKGROUND: Cardiovascular morphological changes are often conspicuous in autopsy examination of chronic hemodialysis (HD) patients. On the other hand, the fluctuation pattern in blood pressure (BP) during HD treatment varies from one patient to another. Cardiovascular changes may correlate with clinical findings including BP fluctuation patterns during HD, although no autopsy studies have previously examined this issue. METHODS: In this study, 53 autopsies of patients who had been on chronic HD were reviewed. We determined the relationship between BP fluctuation during HD treatment along with stable and cardiovascular morphology, including heart weight, ventricular wall thickness, circumferences of the valves and the severity of aortic arteriosclerosis and coronary stenosis. Patients were divided into 4 groups according to the pattern of BP fluctuation during HD treatment at about 6 months before death: group 1 (n = 13), symptomatic hypotension and/or decline pattern during HD; group 2 (n = 11), continuously high BP during HD treatment; group 3 (n = 17), continuous normal BP during HD treatment, and group 4 (n = 12), continuously low BP without symptomatic hypotension during HD treatment. RESULTS: Heart weight and ventricular wall thickness were greatest in group 2. The scores for aortic arteriosclerosis in groups 1 and 2 were higher than in groups 3 and 4. The coronary stenosis index was significantly higher in group 1 than in the other groups, and that in group 2 was higher than in group 4. Multiple regression analysis showed that age, HD duration and pulse pressure were independent variables for the score of arteriosclerosis, and the decline pattern of BP fluctuation during HD and pulse pressure were independent variables for coronary stenosis index. CONCLUSIONS: Our results suggest that certain clinical parameters including BP during HD may reflect cardiovascular morphological changes in stable HD patients, although further examination, such as 24-hour blood pressure measurement is recommended to elucidate the pathophysiology of cardiovascular diseases in HD patients.

Proximal symphalangism with "coarse" facial appearance, mixed hearing loss, and chronic renal failure: new malformation syndrome?

A 25-year-old man is described with short stature, moderate mental retardation, an abnormal facial appearance, a webbed neck, skeletal abnormalities including proximal symphalangism of bilateral second through fifth fingers, mixed hearing loss, and slowly progressive, sclerosing nephropathy. He was large at birth with generalized edema, more pronounced around the jaw, neck and the upper part of the body, but became short with increasing age, and currently measures 143 cm (-4.9 SD). He had intermittent proteinuria and slowly progressive deterioration of the renal function. A biopsy of the left kidney showed global glomerular sclerosis with interstitial fibrosis. He was placed on maintenance peritoneal dialysis at age 17 years, and now on hemodialysis. His skeletal abnormalities included, in addition to proximal symphalangism, stenosis of the cervical canal, scoliosis, brachydactyly of the hands, hypoplastic hip joints, and pes valgus. Other abnormalities noted were a communicating defects of the diaphragm (surgically corrected), bilateral inguinal hernia and cryptorchidism. These clinical manifestations indicate a hitherto undescribed combination of manifestations and nephropathy.

Protein tyrosine phosphorylation: A possible common signaling pathway in human Th1 and Th2 cell clones.

BACKGROUND: It has been reported that protein tyrosine phosphorylation is essential for cytokine production in mouse Th1 cells but not in Th2 cells. However, little is known about the difference of signal transduction between human antigen-specific Th1 and Th2 cell clones. METHODS: Purified protein derivative-specific Th1 and Dermatophagoides farinae-specific Th2 cell clones were established. T cell clones were stimulated with anti-CD3 Abs and further treated with goat antimouse immunoglobulin Abs for cross-linking of TCR/CD3 complex. RESULTS: In contrast to murine Th2 clones, tyrosine phosphorylation including both ZAP-70 and phospholipase-C-Al was necessary for cell activation in both types of human T cell clones. This was further supported by the observation that genistein (protein tyrosine kinase inhibitor) inhibits IFN-A production for Th1 and IL-4 for Th2 in a dose-dependent manner. CONCLUSION: Protein tyrosine phosphorylation is thus an essential component in transducing the activation signal from TCR-CD3 complex both in human Th1 and Th2 cells.

Association between HLA-DRB1*15 and Japanese patients with rheumatoid arthritis complicated by renal involvement.

We performed serological phenotyping of HLA antigens in 175 patients with rheumatoid arthritis (RA) with (n = 41) and without (n = 134) renal involvement (RI), and DNA typing of HLA class II alleles in 75 patients. Among the patients with RA, the frequency of serologically determined HLA-DR4 was found to be significantly increased (odds ratio: 1.8, confidence interval: 1.3-2.5, p = 2.4x10(-4)). In the patients without RI, the frequency of serological DR4 significantly increased (odds ratio: 2.2, confidence interval: 1.6-3.3, p = 2. 6x10(-5)). On the other hand, among the patients with RI, a serological determinant, DR15, did significantly increase (odds ratio: 2.7, confidence interval: 0.9-8.4, p = 1.2x10(-3)) in comparison to the controls. At the DNA level, we found that the association of Japanese RA patients with serological HLA-DR4 was based on that with a genotype of HLA-DRB1*0405 (odds ratio: 2.4, confidence interval: 1.5-4.0, p = 4.4x10(-4)) and also found an association of HLA-DRB1*1501 (odds ratio: 2.8, confidence interval: 1.2-6.6, p = 0.017) with RA patients having RI. Our results confirmed the association of HLA-DRB1*04 with RA over the ethnic barrier at the DNA level. Our results also suggested a distinct genetic effect of HLA-DRB1*1501 in the aspect of the susceptibility of RI in RA.

[Superantigens and autoimmune diseases].

Superantigens are potent immunomodulators derived from microorganisms such as bacteria, viruses and mycoplasmas. Their effects on immune systems are obtained through their binding both to outer portion of binding grooves of an MHC on antigen presenting cells and to non-recognizing structure of hypervariable region of T cell antigen receptors. X-ray crystallography revealed precise structures of some superantigens, a beta barrel domain as a ligand to MHC class II alpha chain and a beta grasp domain to TCRV beta chain. Superantigens induce not only T/B cell activation but also immunological tolerance through oligoclonal deletion and/or anergy. Contribution of superantigens to the pathogenesis of autoimmune diseases has been discussed since a superantigen, mycoplasma arthritis T cell mitogen revealed to be arthritogenic to mice, and the murine arthritis resembled human rheumatoid arthritis in the pathological findings. Rheumatoid arthritis as well as Kawasaki Disease, Sjögren syndrome, and multiple sclerosis is now well studied through the oligoclonal expression of TCR beta specificities on infiltrating T cells. Application of superantigens to the treatment of autoimmune diseases is also discussed.

Autoreactive T cell clones from patients with systemic lupus erythematosus support polyclonal autoantibody production.

This work examines the functional properties and TCRbeta gene utilization of 15 autoreactive T cell clones derived from five patients with systemic lupus erythematosus. All these clones proliferated and secreted cytokine when stimulated in vitro by autologous (but not allogenic) B cells. Individual T cell clones used diverse TCRbeta genes and did not show skewing toward the preferential usage of anionically charged receptors. Autoreactive T cell clones supported polyclonal B cell activation, as characterized by the production of anti-DNA, anti-Sjögren syndrome A, and anti-tetanus toxoid (anti-TT) Abs. This T cell help was mediated through the production of immunostimulatory cytokines, especially IL-6. Although stimulation of the autoreactive clones was blocked by anti-HLA class II Abs, the T cell clones did not proliferate, nor did they support polyclonal IgG production by HLA class II-matched normal B cells. Unlike the autoreactive clones, TT-specific clones derived from the same patients provided help selectively to B cells secreting anti-TT Abs. These findings suggest that autoreactive T cells from systemic lupus erythematosus patients are triggered to provide help following cognate interactions with self-peptides presented in the context of HLA class II molecules expressed on autologous B cells regardless of their specificities.

Role of gammadelta T lymphocytes in the development of Behçet's disease.

Phenotypic and functional properties of gammadelta T cells, which play an important role in mucocutaneous immunity, were examined to elucidate whether immunological abnormality in Behcet's disease may be related to a specific T cell population. We found that CD45RA+ Vgamma9+ Vdelta2+ gammadelta T cells, which constitute a minor population of gammadelta T cells in healthy individuals, were increased in number in Behçet's disease irrespective of disease activity. This CD45RA+ subset of gammadelta T cells in the active, but not inactive, phase of this disease expressed IL-2Rbeta and HLA-DR, suggesting that they are activated in vivo in active Behçet's disease. In addition, the CD45RA+ gammadelta T cells produced extreme amounts of tumour necrosis factor and contained perforin granules. These data indicate that a phenotypically distinct subset of gammadelta T cells, CD45RA+ CD45RO- Vgamma9+ Vdelta2+, may contribute to immunological abnormalities which may lead to complexity of pathophysiology in Behçet's disease.

Excessive function of peripheral blood neutrophils from patients with Behçet's disease and from HLA-B51 transgenic mice.

OBJECTIVE: To elucidate the role played by HLA-B51 in the neutrophil hyperfunction of Behçet's disease, we determined the superoxide production by purified peripheral blood neutrophils from Behçet's disease patients, from HLA-B51 positive healthy individuals, and from HLA-B51 transgenic mice. METHODS: Neutrophil function was evaluated by flow cytometric analysis, detecting the conversion of 2',7'-dichlorofluorescin diacetate into dichloroflurescein, induced by superoxide in the neutrophils. RESULTS: A significant correlation between the neutrophil hyperfunction and the possession of HLA-B51 phenotype, regardless of the presence of the disease, was observed in humans. FMLP-stimulated neutrophils (without in vitro priming) from HLA-B51 transgenic mice, but not those from HLA-B35 transgenic mice or from nontransgenic mice, produced substantial amounts of superoxide. CONCLUSION: The HLA-B51 molecule itself may be responsible, at least in part, for neutrophil hyperfunction in Behçet's disease.

Induction of differentiation into monocyte/macrophage cell lineage of a human eosinophilic leukaemia cell line EoL-1 by simultaneous stimulation with tumour necrosis factor-alpha and interferon-gamma.

Human myeloid leukaemia cell lines have been shown to differentiate into distinct cell lineages in vitro in response to several differentiation-inducing agents. A human eosinophilic leukaemia cell line, EoL-1, has been shown to differentiate into mature eosinophilic granulocytes by treatment with the culture supernatant of a human T-cell line, HIL-3. In this study we have studied whether the EoL-1 cell line has potential to differentiate into cell lineage other than eosinophils. We found that EoL-1 cells cultured in the presence of tumour necrosis factor (TNF)-alpha (10 u/ml) and interferon (IFN)-gamma (1000 u/ml) for 2-4 d differentiated into macrophage-like cells in morphology, and expressed CD14 antigen on their cell surface. It is possible that the small subpopulation of EoL-1 cells which contains non-specific esterase (NSE) activity may be preferentially differentiated by TNF-alpha and IFN-gamma. To clarify this issue, we have cloned the EoL-1 cell line and obtained NSE negative and positive sublines. Both EoL-1 sublines differentiated into monocyte/macrophage-like cells, because: (a) EoL-1 sublines were induced to express CD14 antigen, and (b) they attached firmly to the plastic wells; (c) after differentiation they became strongly positive for NSE staining, and secreted TNF-alpha in response to the stimulation with lipopolysaccharide; and (d) they exhibited potent phagocytic activity. Therefore, we found that the EoL-1 cell line has the ability to differentiate not only into mature eosinophilic cells but also into monocyte/macrophage cell lineage, suggesting that EoL-1 cells represent immature cells with ability to differentiate into multiple cell lineages.

Fine antigen specificity of human gamma delta T cell lines (V gamma 9+) established by repetitive stimulation with a serotype (KTH-1) of a gram-positive bacterium, Streptococcus sanguis.

We have established human gamma delta T cell lines specific for Streptococcus sanguis (S. sanguis) KTH-1 present in normal oral cavity flora. The CD4-CD8-CD3+V gamma 9+V delta 1-CD45RO+ CD25+ T cell lines showed a proliferative response to the streptococcal antigen (Ag) in the presence of autologous antigen-presenting cells without apparent evidence of HLA restriction. The proliferative response of the gamma delta T cell lines was completely blocked by anti-TcR gamma delta monoclonal antibody (mAb) and anti-HLA class I mAb (W6/32), whereas anti-HLA classical class Ia mAb (B-H9; anti-HLA-A,B,C), anti-HLA class II mAb (anti-DR, anti-DQ, and anti-DP) and anti-CD4 mAb did not have any inhibitory effects. Surprisingly, the gamma delta T cell lines showed the proliferative response against the original bacterial Ag KTH-1 exclusively, and exhibited no cross-reactivity with nominal Ag such as purified protein derivative of tuberculin, tetanus toxoid and Mycobacterium tuberculosis, or the same species but different strain of S. sanguis, American Type Culture Collection (ATCC) standard strain (10556), or even with the same strain but different serotype of S. sanguis, KTH-3. Moreover, cytokine production of the gamma delta T cell lines was similar to the Th1 pattern [interferon-gamma, tumor necrosis factor (TNF)-alpha and TNF-beta]. They also produced interleukin-8 that functions as one of chemoattractants for polymorphonuclear cells. Using direct sequencing technique of the polymerase chain reaction products, we found that junctional diversity of the T cell receptor (TcR) used by the parental KTH-1 specific gamma delta T cell line and its subclones is rather limited. It is suggested that gamma delta T cells with canonical TcR could preferentially respond to KTH-1 Ag. Thus, in addition to a broad or cross-reactivity of gamma delta T cells against phylogenetically conserved stress/heat-shock protein, which is well characterized by others, some peripheral blood gamma delta T cells could recognize and kill exogenous agents with fine antigenic specificity to protect the body against them.

Human T cell clones reactive against U-small nuclear ribonucleoprotein autoantigens from connective tissue disease patients and healthy individuals.

SLE and mixed connective tissue disease (MCTD) are characterized by the presence of high titers of autoantibodies against uridine-rich RNA-small nuclear ribonucleoprotein (snRNP) Ag. Because the presence of such snRNP-reactive autoantibodies has recently been shown to be associated with polymorphisms of HLA, this study was undertaken to determine whether snRNP-reactive T cells could be identified and characterized from patients. PBMC were stimulated with affinity-purified snRNP Ag and cloned by limiting dilution in the presence of rIL-2 and rIL-4, snRNP-reactive human T cell clones were generated from three patients and two healthy blood donors who possessed disease-associated HLA genotypes. The cell surface phenotype of clones determined by flow cytometry was CD3+, CD4+, CD45RO+, TCR V alpha beta+. TCR V beta analysis, performed using V beta-specific primers and polymerase chain reaction, revealed that the T cell lines generated were clonal; a limited number of TCR V beta genes were expressed among the clones tested. All clones tested by mAb blocking of Ag-induced proliferation were restricted by HLA-DR. Several T cell clones were identified that were specific for B'/B or D polypeptides. These results demonstrate that snRNP-reactive T cells can be isolated from SLE and MCTD patients in vitro, and that Ag-driven expansion of such T cells could play a role in the immunopathogenesis of these diseases in vivo.

Effects of activin A on IgE synthesis and cytokine production by human peripheral mononuclear cells.

Activin A not only stimulates the synthesis and release of pituitary follicle-stimulating hormone, but exerts various effects on haematopoietic cells, embryos, and fibroblasts. In the present study we have examined effects of activin A on IgE synthesis and cytokine production by peripheral blood mononuclear cells (PBMC) in normal humans. When PBMC were cultured in the presence of IL-4, activin A significantly augmented IgE production induced by IL-4. Activin A did not affect, however, IgE production from highly purified B cells when they were stimulated with anti-CD40 MoAb and IL-4. The fact that in the latter condition IgE synthesis was T cell- and monocyte-independent indicated that activin A does not directly influence B cells for IgE synthesis. Rather, production as well as gene expression of IL-6, which is known to enhance IgE synthesis by purified monocytes, was induced by activin A alone. In addition, activin A induced other monokines such as IL-1 and tumour necrosis factor (TNF)-alpha from monocytes. In contrast, activin A neither induced nor augmented the production of TNF-beta or interferon-gamma (IFN-gamma), both of which are known to be exclusively generated by T cells. These data indicate that activin A plays a certain role in physiological functions for monocytes in normal humans.