Histological features of localized scleroderma 'en coup de sabre': a study of 16 cases.

BACKGROUND: Early lesions of localized scleroderma are histologically characterized by perivascular lymphocytic infiltrate in the reticular dermis and swollen endothelial cells. However, there have been few information regarding histological features other than these findings in localized scleroderma. OBJECTIVE: Since en coup de sabre (ECDS) is a certain subset of localized scleroderma with a relatively uniform clinical manifestation, we focused on this disease subset and evaluated its histopathological features. METHODS: A total of 16 patients with ECDS were retrospectively evaluated on the basis of clinical and histological findings. RESULTS: Regardless of clinical manifestations, vacuolar degeneration was found in all of the ECDS patients. Importantly, keratinocyte necroses were restricted to early and active ECDS lesions. In early ECDS patients (disease duration of <3 years), moderate to severe perivascular and/or periappendageal lymphocytic infiltrate and vacuolar changes in follicular epithelium were more prominent, whereas epidermal atrophy was less frequently observed, than in late ECDS patients (disease duration of ≥6 years). CONCLUSION: Vacuolar degeneration at the dermoepidermal junction is a common histological feature in ECDS and perivascular and/or periappendageal lymphocytic infiltrate and vacuolar degeneration of follicular epithelium are characteristic especially in early ECDS, further supporting a canonical idea that the elimination of mutated epidermal cells by immune surveillance contributes to tissue damage and resultant fibrosis in localized scleroderma.

Effects of bosentan on nondigital ulcers in patients with systemic sclerosis.

BACKGROUND: Bosentan is an oral dual endothelin receptor antagonist, which has been shown to be efficacious for preventing new digital ulcers in patients with systemic sclerosis (SSc) in two high-quality randomized controlled trials. However, its efficacy for nondigital ulcers in SSc remains unknown. OBJECTIVES: To evaluate the efficacy of bosentan on nondigital ulcers in patients with SSc. METHODS: Bosentan was administered to five patients with SSc with pulmonary arterial hypertension, who also had nondigital ulcers refractory to conventional treatments. The efficacy of bosentan on nondigital ulcers and its association with clinical features of ulcers were analysed. RESULTS: The nondigital ulcers refractory to conventional treatments were significantly improved by the administration of bosentan in cases surrounded with severe cyanosis. In contrast, nondigital ulcers without cyanosis were still refractory to bosentan therapy. CONCLUSIONS: Bosentan may be efficacious for accelerating the healing of nondigital ulcers with severe cyanosis, suggesting that nondigital ulcers caused by severely impaired peripheral circulation are highly responsive to this treatment.

An unstable beta 2-microglobulin: major histocompatibility complex class I heavy chain intermediate dissociates from calnexin and then is stabilized by binding peptide.

Proper assembly of the class I heavy chain (HC), beta 2-microglobulin (beta 2m), and peptide must occur in the endoplasmic reticulum (ER) in order for MHC class I molecules to be expressed on the cell surface. Newly synthesized class I HC bind calnexin, an ER resident chaperone. These calnexin-associated class I HC appeared to lack the stable association with beta 2m in peptide transporter-deficient T2 cells since beta 2m-unassociated class I HC-specific HC10 antibody, but not beta 2m-associated class I HC-specific W6/32 antibody, coimmunoprecipitated calnexin. To determine the precursor-product relationship of the pool of HC that bind peptide, class I-restricted peptides were added to lysates of T2 cells in vitro. These peptides stabilized preexisting beta 2m-associated HC complexes (beta 2m+:HC:pep-), but had no significant effect on the preexisting pool of calnexin-associated HC that lack beta 2m. Release of HC from calnexin appeared to be controlled by the binding of beta 2m, since beta 2m-deficient FO-1 cells showed a prolonged association of class I HC with calnexin, while beta 2m-transfected FO-1 cells displayed a more rapid dissociation of class I HC from calnexin. Consistent with this result, the dissociation of class I HC from calnexin did not appear to be dependent on peptide binding since the dissociation rates were similar in peptide transporter-deficient T2 cells and in wild-type T1 cells. From these observations, we speculate that in the stepwise assembly of class I molecules, calnexin may mediate dimerization of class I HC with beta 2m, and that the unstable beta 2m+:HC:pep- complexes, after dissociation from calnexin, subsequently bind peptide to complete the assembly.

Expression of different combinations of interleukins by human T cell leukemic cell lines that are clonally related.

We have analyzed expression patterns of 7 lymphokine mRNAs by Northern blot analyses in 19 different human T cell clones derived from patients with adult T cell leukemia. However, we were not able to reveal particular combinations of lymphokine production that allowed classification of human T cells. Especially, four clonally related leukemic lines that were established independently from the same patient with adult T cell leukemia expressed different combinations of lymphokine mRNAs, indicating that the expression of various lymphokines is not fixed but rather variable even among progenies of a single T cell clone.

Clonal V alpha 12.1+ T cell expansions in the peripheral blood of rheumatoid arthritis patients.

Rheumatoid arthritis (RA) represents a heterogenous disease characterized by chronic polyarthritis. Most patients with adult RA inherit HLA-DR4 or -DR1 major histocompatibility complex (MHC) genes. While the molecular basis for this genetic predisposition is unknown, the major function of these MHC-encoded molecules is to present peptides to T lymphocytes. It is hypothesized that an endogenous or environmental antigen initiates a MHC-restricted immune response mediated by T lymphocytes, which is followed by a chronic inflammatory reaction involving many cell types. In chronic RA, previous or ongoing antigenic activation might result in detectable skewing of the peripheral alpha/beta T cell receptor (TCR) repertoire. Here we demonstrate a marked expansion of V alpha 12.1-bearing CD8+ T cells in the peripheral blood (mean, 22%; range, 10-43%) of > 15% of RA patients. A major proportion of these patients shared HLA-DQ2 in addition to the expected high frequency DR1 and DR4 alleles. Detailed molecular analysis in three of the RA patients with elevated V alpha 12.1+ T cells identified repeated TCR alpha chain sequences consistent with clonal V alpha 12.1+,CD8+ T cell expansion. In addition to shared TCR V alpha 12.1 germline gene usage among unrelated subjects, a conserved J alpha motif was also detected. Together, these results suggest an antigen-driven mechanism of T cell expansion in these patients and may offer a new approach in examining specific antigen that stimulate T cells in RA.

Self-recognition of CD1 by gamma/delta T cells: implications for innate immunity.

The specificity of immunoglobulins and alpha/beta T cell receptors (TCRs) provides a framework for the molecular basis of antigen recognition. Yet, evolution has preserved a separate lineage of gamma/delta antigen receptors that share characteristics of both immunoglobulins and alpha/beta TCRs but whose antigens remain poorly understood. We now show that T cells of the major tissue gamma/delta T cell subset recognize nonpolymorphic CD1c molecules. These T cells proliferated in response to CD1+ presenter cells, lysed CD1c+ targets, and released T helper type 1 (Th1) cytokines. The CD1c-reactive gamma/delta T cells were cytotoxic and used both perforin- and Fas-mediated cytotoxicity. Moreover, they produced granulysin, an important antimicrobial protein. Recognition of CD1c was TCR mediated, as recognition was transferred by transfection of the gamma/delta TCR. Importantly, all CD1c-reactive gamma/delta T cells express V delta 1 TCRs, the TCR expressed by most tissue gamma/delta T cells. Recognition by this tissue pool of gamma/delta T cells provides the human immune system with the capacity to respond rapidly to nonpolymorphic molecules on professional antigen presenting cells (APCs) in the absence of foreign antigens that may activate or eliminate the APCs. The presence of bactericidal granulysin suggests these cells may directly mediate host defense even before foreign antigen-specific T cells have differentiated.

Ceramidase deficiency in Farber's disease (lipogranulomatosis).

Ceramidase activity could not be demonstrated in the kidney and cerebellum from a deceased patient with Farber's disease, whereas the activities of six control acid hydrolase enzymes appeared normal. This enzyme defect presumably accounts for the accumulation that has been described in two patients and may represent the biochemical basis of this disorder.

Cytoplasmic tail-dependent localization of CD1b antigen-presenting molecules to MIICs.

CD1 proteins have been implicated as antigen-presenting molecules for T cell-mediated immune responses, but their intracellular localization and trafficking remain uncharacterized. CD1b, a member of this family that presents microbial lipid antigens of exogenous origin, was found to localize to endocytic compartments that included the same specialized subset of endosomes in which major histocompatibility complex (MHC) class II molecules are proposed to bind endocytosed antigens. Unlike MHC class II molecules, which traffic to antigen-loading endosomal compartments [MHC class II compartments (MIICs)] primarily as a consequence of their association with the invariant chain, localization of CD1b to these compartments was dependent on a tyrosine-based motif in its own cytoplasmic tail.

The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression.

The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single-copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory-chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ;two out of three' and ;U:N wobble' mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.

EGFR regulation by microRNA in lung cancer: correlation with clinical response and survival to gefitinib and EGFR expression in cell lines.

BACKGROUND: Allelic loss in chromosome 3p is one of the most frequent and earliest genetic events in lung carcinogenesis. We investigated if the loss of microRNA-128b, a microRNA located on chromosome 3p and a putative regulator of epidermal growth factor receptor (EGFR), correlated with response to targeted EGFR inhibition. Loss of microRNA-128b would be equivalent to losing a tumor suppressor gene because it would allow increased expression of EGFR. PATIENTS AND METHODS: We initially showed that microRNA-128b is a regulator of EGFR in non-small-cell lung cancer (NSCLC) cell lines. We tested microRNA-128b expression levels by quantitative RT-PCR, genomic copy number by quantitative PCR, and mutations in the mature microRNA-128b by sequencing. We determined whether microRNA-128b loss of heterozygosity (LOH) in 58 NSCLC patient samples correlated with response to gefitinib and evaluated EGFR expression and mutation status. RESULTS: We determined that microRNA-128b directly regulates EGFR. MicroRNA-128b LOH was frequent in tumor samples and correlated significantly with clinical response and survival following gefitinib. EGFR expression and mutation status did not correlate with survival outcome. CONCLUSION: Identifying microRNA regulators of oncogenes could have far-reaching implications for lung cancer patients including improving patient selection for targeted agents, development of novel therapeutics, or development as early biomarkers of disease.

Pathways of CD1 and lipid antigen delivery, trafficking, processing, loading, and presentation.

Specific T cell responses to a variety of self and microbial lipids depend on proper assembly and intracellular trafficking of CD 1 molecules that intersect with and load processed lipid antigens. These pathways involve unique membrane trafficking and chaperones that are distinct from those utilized for major histocompatibility complex (MHC)-mediated presentation of peptide antigens, and thus define unique lipid antigen presentation pathways. Furthermore, recent studies have identified components of lipid metabolism that participate in lipid delivery, uptake, processing and loading onto CD1 molecules. Defects in these pathways result in impaired T cell development and function, underscoring their critical role in the lipid-specific T cell immune responses.

Nosocomial outbreak of epidemic keratoconjunctivitis accompanying environmental contamination with adenoviruses.

An outbreak of acute keratoconjunctivitis involving 27 patients occurred in the Department of Ophthalmology, Kurume University Hospital. Adenoviral DNA was detected in four inpatients, one outpatient and one healthcare worker. Sequence-based typing of adenoviral DNA indicated serotype 3 from one inpatient, the rest being serotype 37. At a later stage of the outbreak adenoviral DNA types 37 and/or 3 were also detected from almost all environmental instruments and commonly used eye drops, despite thorough disinfection of the environment and enforcement of various infection control measures. The detection rate of adenoviral DNA in environmental swabs was 81%. A further second disinfection of the environment reduced the detection rate of adenoviral DNA to 38%. The outbreak ceased after closing the ophthalmology ward and outpatient consulting room, accompanied by enhanced cleaning of environmental instruments and the introduction of disposable eye drops for individual patients.

RNA editing in the anticodon of tRNA Leu (CAA) occurs before group I intron splicing in plastids of a moss Takakia lepidozioides S. Hatt. & Inoue.

RNA editing of cytidine (C) to uridine (U) transitions occurs in plastids and mitochondria of most land plants. In this study, we amplified and sequenced the group I intron-containing tRNA Leu gene, trnL-CAA, from Takakia lepidozioides, a moss. DNA sequence analysis revealed that the T. lepidozioides tRNA Leu gene consisted of a 35-bp 5' exon, a 469-bp group I intron and a 50-bp 3' exon. The intron was inserted between the first and second position of the tRNA Leu anticodon. In general, plastid tRNA Leu genes with a group I intron code for a TAA anticodon in most land plants. This strongly suggests that the first nucleotide of the CAA anticodon could be edited in T. lepidozioides plastids. To investigate this possibility, we analysed cDNAs derived from the trnL-CAA transcripts. We demonstrated that the first nucleotide C of the anticodon was edited to create a canonical UAA anticodon in T. lepidozioides plastids. cDNA sequencing analyses of the spliced or unspliced tRNA Leu transcripts revealed that, while the spliced tRNA was completely edited, editing in the unspliced tRNAs were only partial. This is the first experimental evidence that the anticodon editing of tRNA occurs before RNA splicing in plastids. This suggests that this editing is a prerequisite to splicing of pre-tRNA Leu.

Cyclic GMP-specific phosphodiesterase inhibition and intracarotid bradykinin infusion enhances permeability into brain tumors.

Intracarotid infusion of bradykinin selectively increases the delivery of compounds into brain tumors. This study sought to determine the role of cyclic GMP in increased permeability across the blood-tumor barrier (BTB) after infusion of bradykinin. In permeability studies, 186 Wistar rats with RG2 gliomas and C6 gliomas were used. Transport across the BTB was quantified by autoradiography and reported as a unidirectional transport, Ki, for [14C]dextran (Mr 70,000) and [14C]aminoisobutyric acid (Mr 103,000), with or without inhibition of cyclic GMP-specific phosphodiesterase or soluble guanylate cyclase. We also determined cyclic GMP levels in tumors and normal brain, with or without intracarotid bradykinin infusion, using RIA. Intracarotid infusion of bradykinin selectively increased permeability in RG2 tumors and C6 tumors for both tracers. Simultaneous infusion of bradykinin and a cyclic GMP-specific phosphodiesterase inhibitor, zaprinast (20 mg/kg), resulted in significantly increased permeability across the BTB, compared to intracarotid bradykinin infusion alone. Zaprinast also significantly prolonged the permeability effects of bradykinin. Pretreatment using i.v. infusion of the soluble guanylate cyclase inhibitor, LY-83583 (125 microg/kg), significantly attenuated the bradykinin effect of opening the BTB. Cyclic GMP levels in RG2 and C6 tumors were significantly increased after intracarotid bradykinin infusion (2.8- and 2.2-fold, respectively). Cyclic GMP levels in normal brain were not increased by bradykinin infusion. These results show that increasing cyclic GMP in tumor microvessels can increase permeability in response to bradykinin.

Establishment of a human ovarian cancer cell line capable of forming ascites in nude mice and effects of tranexamic acid on cell proliferation and ascites formation.

The present study was designed to obtain an experimental tumor model as similar as possible to human ovarian cancer which often had a large amount of ascites and to assess the therapeutic value of tranexamic acid. Human tumor cell lines which form ascites in nude mice were established from ascites of patient with serous cystadenocarcinoma of the ovary. Two cloned cell lines designated HRA and HR-1 were obtained from the parent cell line designated HR. All of these cultured cell lines had about 2.5-3.5 times higher lactate dehydrogenase activities than the original tumor. The original tumor and the tumor grown in nude mice had all 5 bands of lactate dehydrogenase isoenzymes, while all cultured cell lines had only a marked lactate dehydrogenase-3 in addition to a faint lactate dehydrogenase-2. Modal chromosome numbers of HR cells ranged from 50-76, while that of HRA cells ranged widely from 40-140. The DNA histograms of HR and HRA cells were similar to each other, showing predominant G1 and S phases. Although these cell lines had ability to produce ascites in the nude mice when the cells were inoculated i.p., the HRA cell inoculation made ascites most rapidly and brought about the shortest median survival (39 days). The proliferations of all three cell lines were dose-dependently inhibited by tranexamic acid. However, the concentration of this drug required for 50% inhibition of the proliferation of HRA cells was about one-half of that of HR and HR-1 cells. In addition, i.p. injections of tranexamic acid to nude mice treated with cisplatin resulted in a significant inhibition of the ascites formation and prolongation of 50% survival.

[Surgical approach for lung cancer with multiple pulmonary nodules].

A computed tomography (CT) and high-resolution CT (HRCT) have provided us an increasing opportunity to find multiple small pulmonary nodules, which sometimes appear ground glass opacity (GGO). Recently, fluorodeoxyglucose-positron emission tomography (FDG-PET) had a great contribution to our assessment for these small pulmonary nodules. However, since it is yet difficult to establish a diagnosis for these nodules during preoperative workup, a surgical lung biopsy is often required for an accurate diagnosis. We have experienced 9 patients who had undergone lung resection for primary lung cancer accompanied by multiple pulmonary lesions. Since the multiple lesions were consisted of malignant and benign lesions, it is still uncertain whether excessive lung resection should be performed in such patients. In this brief article, we summarized the characteristics of the pulmonary lesions in those patients and discussed difficulty of preoperative diagnosis, viability of pulmonary resection and problems underlining a surgical treatment.

Cloning and characterization of the cDNA for a plastid sigma factor from the moss Physcomitrella patens.

We isolated a cDNA PpSig1 encoding a plastid sigma factor from the moss Physcomitrella patens. The PpSIG1 protein is composed of the conserved subdomains for recognition of -10 and -35 promoter elements, core complex binding and DNA melting. Southern blot analysis showed that the moss sig1 gene is likely a member of a small gene family. Transient expression assay using green fluorescent protein demonstrated that the N-terminal region of PpSIG1 functions as a chloroplast-targeting signal peptide. These observations suggest that multiple nuclear-encoded sigma factors regulate chloroplast gene expression in P. patens.

Identification of 10Sa RNA (tmRNA) homologues from the cyanobacterium Synechococcus sp. strain PCC6301 and related organisms.

We have isolated the 10Sa RNA (tmRNA) from the unicellular cyanobacterium Synechococcus sp. strain PCC6301. It comprises of 394 nucleotides (nt) and has 55% homology to Escherichia coli tmRNA. The cloning and sequencing of the corresponding gene have revealed that, like in many tRNA genes, the terminal CCA sequence reported in all the tmRNA species characterized so far is not encoded in the DNA. Hybridization analysis has shown that the tmRNA gene is present as a single copy. Fairly high levels of tmRNA accumulate throughout the cell cycle; however, a slight increase in its level is observed during late-log to stationary phase. This suggests that tmRNA is functional not only when cells divide actively but also when cell growth stops.

Identification of beta-D-mannosylceramide in hepatopancreas of the fresh-water bivalve, Hyriopsis schlegelii.

A mannosylceramide was isolated by preparative thin-layer chromatography on a 3% borate-impregnated silica gel plate from a monohexosylceramide fraction of the hepatopancreas of the fresh-water bivalve, Hyriopsis schlegelii. It contained only mannose as the sugar component and the ceramide moiety contained mainly sphingosine and palmitic acid. Anomeric configuration of the sugar moiety was determined by enzymatic hydrolysis with beta-D-mannosidase. The concentration of this glycolipid was 5% of the total monohexosylceramide fraction of the hepatopancreas.

Di- and trimannosylceramides in hepatopancreas of a fresh-water bivalve, Hyriopsis schlegelii.

An investigation of di- and triglycosylceramides of hepatopancreas of a fresh-water bivalve, Hyriopsis schlegelii, gave the following results: (1) The total amount of neutral glycosphingolipid was 7.2 mg/g of the dry weight of the hepatopancreas. (2) The diglycosylceramide fraction accounted for 4.8% of the total neutral glycolipid, and consisted of Man(beta 1 leads to 2)Man(beta 1 leads to 1)Cer, Man (beta 1 leads to 4)Glc leads to Cer, Gal(beta 1 leads to 4)Glc leads to Cer, and Gal(beta 1 leads to 4)Gal(beta 1 leads to 1)Cer. The presence of so many different types of diglycosylceramides in a single tissue appears to be the exception rather than the rule. (3) The triglycosylceramide fraction amounted to 12.2% of the total neutral glycolipid and contained a mixture of Man(alpha 1 leads to 3)Man(beta 1 leads to 2)Man(beta 1 leads to 1)Cer and Man(alpha 1 leads to 3)Man(beta 1 leads to 4)Glc leads to Cer. (4) The spectrum of di- and triglycosylceramides of the hepatopancreas was more complex than that of spermatozoa of this bivalve. (5) It is an interesting finding that a series of glycolipids containing one to three mannose residues occurs in nature. (6) The ceramide moieties of the above two fractions contained the normal saturated fatty acids in the range of C14-C24, and derivatives of 4-sphingenine. The structural analysis involved (1) gas-liquid chromatography of the component sugars, fatty acids and long-chain bases; (2) methylation studies coupled with gas-liquid chromatography and mass spectrometry to locate the bonds between the hexose units; and (3) enzymatic degradation to establish the sugar sequence and the anomeric configuration of the glycosidic links.