Hiroaki Nishizaka

Analysis of Fas ligand gene mutation in patients with systemic lupus erythematosus

OBJECTIVE To investigate the possible association of a Fas ligand (FasL) gene mutation(s) or polymorphism(s) with systemic lupus erythematosus (SLE). METHODS For amplification of the introns of the FasL gene, long polymerase chain reaction (PCR) using exon-based primers was utilized, followed by partial sequencing to construct exon-specific oligonucleotide primers for the analyses of FasL genomic DNA in SLE patients. Structural defects were studied by use of a composite analysis of reverse transcriptase-PCR/single-strand conformational polymorphism (SSCP) analysis of messenger RNA (mRNA) transcripts of the FasL gene in 35 SLE patients and PCR/SSCP analysis of FasL genomic DNA in 143 SLE patients. RESULTS The sizes of the introns were approximately 0.6 kb for intron 1, 4.3 kb for intron 2, and 1.3 kb for intron 3. By SSCP analysis, we did not identify any mutations or polymorphisms in the FasL mRNA transcripts or in any of the 4 exons or areas of the introns adjacent to the exons. CONCLUSION Using the same methods used in the present studies (PCR/SSCP), one group of investigators identified a structural defect of the FasL molecule in 1 of 75 SLE patients evaluated. Among the 143 SLE patients in the present study, however, we did not identify any mutations or polymorphisms of the FasL gene. Our results suggest that a FasL defect is not the major contributing factor in the pathogenesis of SLE.

Production of the third and fourth component of complement (C3, C4) by smooth muscle cells

Production of the third and fourth components of complement (C3, C4) by smooth muscle cells was investigated by using normal human aortic smooth muscle cells (AoSMC), human smooth muscle cell line (G402) and vascular smooth muscle cells obtained from human umbilical cord vein (UVSMC). AoSMC spontaneously produced both C3 and C4 at 15 ng/106 cells/72 hr and 22 ng/106 cells/72 hr, respectively, and both were enhanced by interferon‐γ (IFN‐γ). Although phorbol 12‐myristate 13‐acetate (PMA) and tumour necrosis factor‐α (TNF‐α) enhanced C3 production, C4 production was reduced by these agents. On the other hand, G402 produced C4 but not C3 in a dose‐dependent manner when cultured with IFN‐γ. UVSMC produced only a small amount of C3 and C4 compared with AoSMC or G402. C3 and C4 produced by AoSMC were confirmed to be identical with their human serum counterparts as determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and measurement of haemolytic activity. Northern blotting analysis showed that the expression of mRNA of C3 and C4 was enhanced by TNF‐α and IFN‐γ, respectively, in AoSMC. Our findings suggest the importance of smooth muscle cells as a source of components of complement in vascular diseases including vasculitis.

Analysis of CD40 ligand gene mutations in patients with primary biliary cirrhosis

An elevated immunoglobulin (Ig)M concentration in serum is a common and distinctive feature of primary biliary cirrhosis (PBC). Little is known, however, about the mechanism of hyper-IgM in PBC. CD40 ligand (CD40L) has a crucial role in immunoglobulin class switching in B cells. Mutations in the gene encoding CD40L are known to induce X-linked hyper-IgM syndrome. To identify mutations in the gene for CD40L in PBC patients, we analyzed CD40L gene mutations, using reverse transcription (RT)-PCR single-strand conformation polymorphism (SSCP) analysis. No mutations were detected in cDNA from any of 24 PBC patients by the RT-PCR-SSCP technique. These data suggest that other, unidentified mechanisms are involved in hyper-IgM in PBC patients.

Successful Treatment of Invasive Pulmonary Aspergillosis by Transbronchial Injection of Urinary Trypsin Inhibitor and Amphotericin B

Accessible online at: www.karger.com/aha Invasive pulmonary aspergillosis is one of the fatal complications in immunocompromised hosts. Although amphotericin B (AmB) is effective against invasive aspergillosis, its systemic use is often limited by severe drugrelated adverse events. Other new agents, such as AmB colloidal dispersion and voriconazole, have shown better results [1, 2], but sometimes it takes such a long time to achieve complete cure that patients under chemotherapy cannot receive the next course. We report dramatic improvement of invasive pulmonary aspergillosis in 4 patients with hematological malignancies who were treated with urinary trypsin inhibitor (UTI) in combination with AmB administered via bronchoscopic fiber. UTI is a Kunitz-type protease inhibitor which inhibits trypsin, plasmin and granulocyte elastase [3]. UTI also decreases the production of TNF-·, IL-6 and IL-8 from leukocytes [4]. In addition, we verified that aspergillus species cannot grow on a plate containing UTI. Based on these observa-

Molecular bases for human complement C7 polymorphisms, C7*3 and C7*4

Complement C7 is one of the components of membrane attack complex (MAC) generated by the terminal complement cascade. C7 protein is polymorphic and most of its polymorphisms have been identified using isoelectric focusing (IEF), which detects protein charge differences. To date, the molecular bases of the polymorphisms detected by IEF have not been determined. In this paper, we describe the structural bases of two C7 IEF-detected polymorphisms, C7*3 and C7*4, both of which are common in Asian populations. C7*3 resulted from substitution of cysteine (Cys) at amino acid residue 106 by charged arginine (Arg; C106R), while charged lysine (Lys) at amino acid residue 398 was replaced by neutral glutamine (Gln; K398Q) in C7*4. As C7*3 is hypomorphic, it is important to study its possible associations with diseases such as immunological disorders and infections. We present genetic bases for this C7 polymorphism, which we determined using polymerase chain reaction (PCR)-based genotyping, a simple and accurate method suitable for large-scale studies.

An NcoI polymorphism in the human complement component 7 (C7) gene.

AbstractA novel polymorphic site has been found in the 3′ untranslated region (UTR) of the human complement component 7 (C7) gene. The polymorphic site at 14-bp downstream from the TAG stop codon was either C or A (NcoI-digested), with allele frequencies of 0.660 and 0.340. This NcoI polymorphism would be useful to perform a DNA marker haplotype study in patients with deficiencies of the complement genes, such as C6, C7, C9, which are located closely on chromosome 5p13.

An inflammatory pseudotumor of the liver in a patient with reactive arthritis

A rare case of an inflammatory pseudotumor (IPT) of the liver with reactive arthritis (ReA) is herein described. A 34-year-old woman presented with post-infectious ReA in the bilateral knees and foot joints. As the primary infected site for ReA could not be determined, a systemic survey revealed an IPT in her liver, which was diagnosed histologically. The arthritis and the tumor regressed simultaneously after being treated by indomethacin farnesil 400 mg/day. Although the exact etiology of IPT or ReA still remains unknown, it is hypothesized that the same pathogenetic mechanisms may be involved in the development of IPT and ReA.