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Background: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare but life-threatening disorders characterized by widespread epidermal necrosis of the skin and mucosa. The severity-of-illness scoring system for TEN (SCORTEN) was widely used since 2000 as a standard prognostic tool consisting of seven clinical values., Methods: To evaluate the prognosis using current treatments and risk factors for mortality, we retrospectively analyzed 59 cases of TEN, including SJS/TEN overlap treated in two university hospitals from January 2000 to March 2020., Results: The mortality rate of TEN was 13.6% (8/59). All patients treated with high-dose steroid administration in combination with plasma exchange and/or immunoglobulin therapy recovered. Logistic regression analysis showed nine clinical composite scores, namely: heart rate (≧120 bpm), malignancy present, percentage of body surface area with epidermal detachment (>10%), blood urea nitrogen (>28 mg/dL), serum bicarbonate level (<20 mEq/L), serum glucose level (>252 mg/dL), age (≧71 years), the interval between disease onset and treatment initiation at the specialty hospital (≧8 days), and respiratory disorder within 48 h after admission. The receiver operating characteristic curves confirmed a high potential for predicting the prognosis of TEN., Conclusions: Recent developments in treatment strategies have contributed to the improved prognosis of TEN patients. A modified severity scoring model composed of nine scores may be helpful in the prediction of TEN prognosis in recent patients. Further large-scale studies are needed to confirm mortality findings to improve prognostication in patients with TEN., (Copyright © 2020 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

Background: Recent studies have indicated that serum levels of squamous cell carcinoma antigen (SCCA) 1 and 2 induced by type 2 cytokines such as IL-4 and IL-13, are increased in patients with atopic dermatitis (AD). However, no clinical studies have analyzed serum levels of SCCA2 in larger series of AD patients or their association with various clinical characteristics. This study was performed to clarify whether serum levels of SCCA2 are associated with disease severity and clinical phenotypes of adult AD patients., Methods: An enzyme-linked immunosorbent assay was performed to examine serum SCCA2 levels in 240 adult patients with AD and 25 healthy controls in this study. Serum SCCA2 levels were analyzed with clinical characteristics and laboratory parameters including thymus and activation-regulated chemokine (TARC), lactate dehydrogenase (LDH), blood eosinophils, total IgE, and specific IgE (Japanese cedar pollen, Dermatophagoides farina, Candida, malassezia, Staphylococcal enterotoxin B). Expression of SCCA2 in AD eruption was examined by immunohistochemistry. The effect of treatment on serum SCCA2 was also assessed., Results: Serum SCCA2 level showed a positive correlation with disease severity, levels of TARC, LDH, eosinophil counts, and IgE levels. Robust expression of SCCA2 was detected in the supra basal keratinocytes in the epidermis of AD patients. Serial measurements of serum SCCA2 revealed decreased levels of SCCA2 after treatment for AD., Conclusions: Serum SCCA2 levels reflected disease severity and clinical type of AD. Serum SCCA2 may thus be a relevant biomarker for AD., (Copyright © 2017 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

Background: Patients with steroid-resistant bullous pemphigoid (BP) require an appropriate treatment option., Objective: A multicenter, randomized, placebo-controlled, double-blind trial was conducted to investigate the therapeutic effect of high-dose intravenous immunoglobulin (IVIG; 400mg/kg/day for 5days) in BP patients who showed no symptomatic improvement with prednisolone (≥0.4mg/kg/day) administered., Methods: We evaluated the efficacy using the disease activity score on day15 (DAS15) as a primary endpoint, and changes in the DAS over time, the anti-BP180 antibody titer, and safety for a period of 57days as secondary endpoints., Results: We enrolled 56 patients in this study. The DAS15 was 12.5 points lower in the IVIG group than in the placebo group (p=0.089). The mean DAS of the IVIG group was constantly lower than that of the placebo group throughout the course of observation, and a post hoc analysis of covariance revealed a significant difference (p=0.041). Furthermore, when analyzed only in severe cases (DAS≥40), the DAS15 differed significantly (p=0.046). The anti-BP180 antibody titers showed no difference between the two groups., Conclusion: IVIG provides a beneficial therapeutic outcome for patients with BP who are resistant to steroid therapy., (Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Background: MicroRNAs are non-coding small RNAs that regulate expression of target genes by binding to 3' untranslated regions. In this study, we used bronchial epithelial cells to investigate in vitro the role of the microRNA miR-155 in the expression of chemokines associated with airway inflammation. miR-155 has previously been reported to regulate allergic inflammation., Methods: BEAS-2B bronchial epithelial cells were cultured and transfected with mimic or inhibitor oligonucleotides to overexpress or downregulate miR-155, as confirmed by real-time PCR. Cells were then stimulated with tumor necrosis factor-alpha, interleukin-13 (IL-13), and a double stranded RNA that binds Toll-like receptor 3. Expression and secretion of the chemokines CCL5, CCL11, CCL26, CXCL8, and CXCL10 were then quantified by real-time PCR and ELISA, respectively. Phosphorylation of signal transducer and activator of transcription 6 (STAT6), a target of the IL-13 receptor, was analyzed by ELISA., Results: miR-155 overexpression significantly suppressed IL-13-induced secretion of CCL11 and CCL26. These effects were specific, and were not observed for other chemokines, nor in cells with downregulated miR-155. miR-155 overexpression also suppressed CCL11 and CCL26 mRNA, but did not affect expression of the IL-13 receptor or phosphorylation of STAT6., Conclusions: miR-155 specifically inhibits IL-13-induced expression of eosinophilic chemokines CCL11 and CCL26 in bronchial epithelial cells, even though the 3'-untranslated region of these genes do not contain a consensus binding site for miR-155., (Copyright © 2016 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

Background: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare but severe adverse drug reactions with high mortality., Methods: To present the clinical characteristics of SJS and TEN in Japan and evaluate the efficacy of treatments, we retrospectively analyzed cases of SJS and TEN treated in 2 university hospitals during 2000-2013., Results: Fifty-two cases of SJS (21 males and 31 females; average age, 55.1 years) and 35 cases of TEN (17 males and 18 females; average age, 56.6 years) were included in this study. Twenty-eight cases of SJS (53.8%) and all cases of TEN were caused by drugs. Hepatitis was the most common organ involvement in both SJS and TEN. Renal dysfunction, intestinal disorder, and respiratory disorder were also involved in some cases. The major complication was pneumonia and sepsis. All cases except for 3 cases were treated systemically with corticosteroids. Steroid pulse therapy was performed in 88.6% of TEN. Plasmapheresis and/or immunoglobulin therapy was combined with steroid therapy mainly in TEN after 2007. The mortality rate was 6.9% and the rates for SJS and TEN were 1.9% and 14.3%, respectively. These were much lower than predicted mortality according to a severity-of-illness scoring system for TEN prognosis (SCORTEN) score. When comparing the mortality rate between 2000-2006 and 2007-2013, it was decreased from 4.5% to 0.0% in SJS and from 22.2% to 5.3% in TEN., Conclusions: Treatment with steroid pulse therapy in combination with plasmapheresis and/or immunoglobulin therapy seems to have contributed to prognostic improvement in SJS/TEN., (Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Pneumonia is a leading cause of death among elderly patients. Although aspiration pneumonia (AP) commonly occurs with aging, its clinical features and outcomes are still uncertain. The aims of this study were to describe the clinical features and outcomes of AP and to assess whether presence of AP affects clinical outcomes in patients with community-acquired pneumonia (CAP) and healthcare-associated pneumonia (HCAP). We retrospectively analyzed patients with CAP and HCAP hospitalized in our institution in Japan from October 2010 to March 2012. We compared clinical features and outcomes between AP and non-AP, and investigated risk factors for recurrence of pneumonia and death. Of 214 consecutive patients, 100 (46.7%) were diagnosed as having aspiration pneumonia. These patients were older and had lower body mass index, more comorbidities, and poorer Eastern Cooperative Oncology Group performance status (ECOG PS) than the patients with non-AP. Patients with AP had more severe disease, required longer hospital stays, and had a frequent recurrence rate of pneumonia and higher mortality. In multivariate analyses, AP, age, and ECOG PS were related to recurrence of pneumonia, and the prognostic factors were CURB-65 score and ECOG PS. AP was not a significant indicator for prognosis but was the strongest risk factor for recurrence of pneumonia. Clinical background and outcomes including recurrence and mortality of AP were obviously different from those of non-AP; therefore AP should be considered as a distinct subtype of pneumonia, and it is important to prevent the recurrence of pneumonia in the patients with AP., (Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

IL-17F is known to be involved in many inflammatory diseases, but its role in skin diseases has not been fully examined. Because IL-8 is involved in many skin diseases such as psoriasis, we investigated the production of IL-8 in normal human epidermal keratinocytes (NHEKs) stimulated by IL-17F, tumor necrosis factor-alpha (TNF-alpha), IL-17A, and control using real-time PCR and ELISA. The results showed that IL-17F induced production of IL-8 in NHEKs in a time-dependent manner. Interestingly, the amounts of IL-8 stimulated by IL-17F were much higher than those stimulated by TNF-alpha or IL-17A. Next, we confirmed that selective mitogen-activated protein kinase kinase inhibitors significantly inhibited IL-17F-induced IL-8 production. Moreover, mouse skin intradermally injected with IL-17F expressed high level of IL-8 mRNA and induced ERK1/2 phosphorylation. Histological examination of mouse skin that was injected with IL-17F revealed marked neutrophilia in dermis and the infiltration was significantly inhibited by anti-IL-8 antibody. Finally, IL-17F expression in skin biopsy samples from psoriasis patients were examined by western blotting and ELISA. IL-17F was upregulated in lesional psoriatic skin compared with nonlesional skin. These results indicate that IL-17F may be involved in psoriasis via, in part, the activation of ERK1/2 and the induction of IL-8 in keratinocytes.

Background: Allergic reactions after ingestion of fermented soybeans have rarely been reported. Fermented soybeans were recently reported to be a causative food of IgE-mediated, late-onset anaphylaxis without early phase responses. The objectives of our study are to clarify the clinical and laboratory features and to characterize the allergens in allergy due to fermented soybeans., Methods: Seven patients with suspected hypersensitivity to fermented soybeans, from whom informed consent had been obtained, underwent skin prick-prick tests with fermented soybeans and challenge test with fermented soybeans. Additionally, specific IgE against fermented soybeans and the allergens of fermented soybeans were detected by ELISA and IgE-immunoblotting, respectively., Results: Seven male patients, aged 26 to 42 years (mean age, 33.1 years), participated. All patients reported generalized urticaria and dyspnea; 5, loss of consciousness; 2, collapse; 2, vomiting; and 2, diarrhea after fermented soybean ingestion. The interval between fermented soybean ingestion and onset of symptoms was 5 to 14 hours (mean, 9.6 hours). All patients were positive on skin prick-prick tests with fermented soybeans. In 2 patients, oral challenge with fermented soybeans was positive 5.5 and 13 hours after ingestion. In ELISA, all 5 patients tested showed elevated IgE levels to the fermented soybean extract. Furthermore, IgE-immunoblotting using 5 patients' sera showed six bands, of which three bands at 38, 28, and 26-kd were bound to sera from 4 patients., Conclusions: Cases with hypersensitivity after ingestion of fermented soybeans most frequently correspond to IgE-mediated, late-onset anaphylactic reactions due to fermented soybeans.

Angiogenesis is a crucial component of bone remodeling under both normal and pathophysiological conditions. Among the various mediators that regulate the angiogenic process is the angiopoietin (Ang) family of growth factors. Ang-1 stabilizes new blood vessels by recruiting surrounding mesenchymal cells and promoting their differentiation into vascular smooth muscle cells, whereas Ang-2 is a natural antagonist of Ang-1 and can inhibit angiogenesis. The expression of Ang-1 and Ang-2 in human osteoblasts (hOBs) isolated from rheumatoid arthritis (RA) and osteoarthritis (OA) patients and from healthy individuals has been examined. After incubation in the presence or absence of tumor necrosis factor-alpha (TNF-alpha) and/or interferon-gamma (IFN-gamma), the culture supernatants were assayed for Ang using an enzyme-linked immunosorbent assay. In addition, expression of Ang protein and mRNA was examined using immunohistochemical techniques and quantitative real-time polymerase chain reaction, respectively. It was found that hOBs expressed Ang-1 but not Ang-2 protein, and cultured hOBs from RA and OA patients and from healthy individuals all spontaneously secreted significant amounts of Ang-1 in the absence of any stimulation. Although stimulation with TNF-alpha or IFN-gamma had little or no effect on Ang-1 secretion, costimulation with IFN-gamma plus TNF-alpha dose- and time-dependently diminished secretion of Ang-1 from hOBs. This inhibitory effect was mediated in part by nuclear factor-kappa B via upregulated expression of inducible nitric oxide synthase and enhanced synthesis of nitric oxide. Taken together, these findings suggest that OBs are an important cellular source of Ang-1 and may modulate bone remodeling through regulation of angiogenesis.

Ghrelin is a novel brain-gut peptide that stimulates food intake and body weight gain. We studied the anabolic effect of ghrelin in a cancer cachexia mouse model. SEKI, a human melanoma cell line, was inoculated into nude mice to examine the effects of ghrelin on food intake and body weight. The intraperitoneal administration of ghrelin twice a day (6 nmol/mice/day) for 6 days suppressed weight loss in SEKI-inoculated mice and increased the rate of weight gain in vehicle-treated nude mice. Ghrelin administration also increased food intake in both SEKI- and vehicle-treated mice. Both the weight of white adipose tissue and the plasma leptin concentration were reduced in tumor-inoculated mice compared with vehicle-treated mice; these factors increased following ghrelin administration. The levels of both ghrelin peptide and mRNA in the stomach were upregulated in tumor-inoculated mice. The anabolic effect of ghrelin efficiently reverses the cachexia in mice bearing SEKI human melanoma. Ghrelin therefore may have a therapeutic ability to ameliorate cancer cachexia.

We examined the characteristic clinical features of one family of familial amyotrophic lateral sclerosis (FALS) with a His46Arg mutation in the enzyme Cu/Zn superoxide dismutase-1 (SOD1). The disease duration for this family was 18.1 +/- 13.2 (mean +/- S.D.) years, with the age at onset being 39.7 +/- 10.5 years old (mean +/- S.D.). The initial sign was distal weakness of the unilateral lower limb, extending to the lower limb of the other side. A wheel chair became necessary at 9.8 +/- 3.2 years after the onset. Upper limb weakness started at 15.5 +/- 8.9 years following from the onset. An autopsy was performed on a 71-year-old woman of the family with the mutation. Her disease duration was 47 years, and she died of pneumonia. She had no clear upper motor neuron involvement. Bulbar sign and respiratory muscle weakness had developed 2 years before her death. Neuropathological findings showed degeneration of corticospinal tracts, anterior/posterior spinocerebellar tracts, posterior columns, and Clarke's columns. There were few anterior horn cells in the lumbar spinal cord and no Lewy body-like hyaline inclusion bodies in these remaining anterior horn neurons. This is the first autopsy report of FALS with a His46Arg mutation in the SOD1 enzyme.

Study Objectives: To investigate the role of neutrophil peptides named alpha-defensins in patients with pulmonary tuberculosis (TB)., Patients: Thirty-seven patients with TB and 25 healthy subjects., Measurements and Results: Concentrations of alpha-defensins (human neutrophil peptide [HNP]-1, HNP-2, and HNP-3) were measured by radioimmunoassay in plasma and BAL fluid (BALF). Concentrations of alpha-defensins were significantly higher in plasma and BALF of patients with TB than in healthy subjects. In BALF of patients with TB, the concentration of alpha-defensins correlated positively with the levels of interleukin 8, and higher concentrations of alpha-defensins in BALF were also detected in patients with cavitary lesions. There was an inverse relationship between plasma alpha-defensins and FEV(1)/FVC ratio before treatment, and between plasma concentrations of alpha-defensins before treatment and the improvement in percentage of vital capacity after treatment. Plasma alpha-defensin concentrations returned to the normal range after treatment., Conclusion: Our data suggest that alpha-defensins released from neutrophils may play an important role in the pathogenesis of TB, and that plasma alpha-defensin concentration may be a useful marker of disease severity and deterioration of pulmonary function.

Neuromedin U (NMU) is a hypothalamic peptide that has been recently found to reduce food intake, but few is known about its other functions in the central nervous system. We here studied behavioral activities induced by an intracerebroventricular (ICV) administration of NMU in rats and mice. NMU increased gross locomotor activity, face washing behavior, and grooming. NMU-induced stress response was significantly abolished by pretreatment with an antagonist of corticotropin-releasing hormone (CRH), alpha-helical CRH (9-41) (alpha-hCRH), or anti-CRH IgG. NMU did not induce locomotor activity in CRH knockout mice. NMU that interacts anatomically and/or functionally with the CRH system is a novel physiological regulator of stress response., (Copyright 2001 Academic Press.)

Study Objective: Recently, the number of new cases of Paragonimus westermani in humans has gradually increased, and paragonimiasis is a re-emerging public health issue in Kyusyu, Japan. We review our recent experience with pleuropulmonary Paragonimus westermani., Patients: Pulmonary paragonimiasis was diagnosed in 13 patients at the Third Department of Internal Medicine, Miyazaki Medical College between 1993 and 1999., Results: Both sputum and bronchoscopic examinations revealed ova in four of nine patients; bronchoscopy yielded ova in two additional patients. Twelve patients (92%) had respiratory symptoms, including cough (92%), sputum and/or hemoptysis (92%), and chest pain (46%). Chest radiography and CT showed pleural lesions (62%) and parenchymal lesions (92%). Of note was the high frequency of solitary nodular lesions (62%), mimicking lung cancer, tuberculosis, or fungal diseases. Immunodiagnosis and bronchoscopic examination were also useful for diagnosis. Praziquantel treatment was very effective and had minimal side effects. One patient required surgical decortication for empyema in spite of treatment with praziquantel. Eosinophilia was noted in peripheral blood and body fluids, which was probably due to increased levels of interleukin-5., Conclusions: Our findings indicate that our patients with Paragonimus westermani presented with a wide variety of radiographic findings, which were different from the classic presentations reported earlier. Bronchoscopic examination and serologic tests are very useful for accurate diagnosis. As dietary habits change and international transportation increases, it appears likely that paragonimiasis will also increase in frequency in various parts of the world.

Study Objectives: To investigate the roles of human alpha-defensin (HAD), human beta-defensin (HBD)-1, and HBD-2, novel antimicrobial peptides, in patients with Mycobacterium avium-intracellulare infection (MAI)., Patients: The study included 25 patients (10 men) with MAI who visited our hospital between June 1998 and August 1999., Measurements and Results: In patients with pulmonary MAI, we measured HAD and HBD-1, and HBD-2 levels in plasma and in BAL fluid (BALF) by radioimmunoassay. Plasma concentrations of HAD and HBD-2 in those patients were higher than those in control subjects, whereas HBD-1 levels were similar to those in the control subjects. High levels of HAD and HBD-2, but not HBD-1, also were observed in the BALF of MAI patients. There was a positive correlation between HAD and interleukin (IL)-8 concentrations in the BALF of patients with MAI. BALF HBD-2 concentrations also correlated positively with those of plasma HBD-2 and BALF IL-1 beta in MAI patients. Patients with cavity formation on the chest roentgenogram had higher HAD and HBD-2 levels in their BALF than those of patients without cavity formation. Treatment with clarithromycin combined with two or three other antibiotics, including ethambutol, rifampicin, ofloxacin, or ciprofloxacin, for at least 6 months resulted in a significant fall in plasma HBD-2 concentrations in responders, but not in nonresponders., Conclusion: Our findings suggest that HAD and HBD-2 may participate in host defense and local remodeling of the respiratory tract in patients with MAI and that plasma HBD-2 levels may be a useful marker of disease activity in patients with pulmonary MAI.

Ghrelin is a novel gut-brain peptide that binds to the growth hormone secretagogue receptor (GHS-R), thereby functioning in the regulation of growth hormone (GH) release and food intake. Ghrelin-producing cells are most abundant in the oxyntic glands of the stomach. The regulatory mechanism that governs the biosynthesis and secretion of ghrelin has not been clarified. We report that ghrelin mRNA expression in the gastric fundus was increased, but that ghrelin peptide content decreased after a 48-h fast. Both values returned to control levels after refeeding. The ghrelin plasma concentration in the gastric vein and systemic venous blood increased after 24- and 48-h fasts. Furthermore, des-octanoylated ghrelin and n-octanoylated ghrelin were found in rat stomach, with the ratio of des-octanoylated ghrelin to n-octanoylated ghrelin markedly increased after fasting. The ghrelin mRNA level in the stomach also increased after administration of insulin and leptin. Conversely, db/db mice, which are deficient in the leptin receptor, had lower ghrelin mRNA levels than control mice. These findings suggest that this novel gastrointestinal hormone plays a role in the regulation of energy balance., (Copyright 2001 Academic Press.)

Ghrelin is a novel acylated peptide that functions in the regulation of growth hormone release and energy metabolism. It was isolated from rat stomach as an endogenous ligand for growth hormone secretagogue receptor. Ghrelin is also localized in the arcuate nucleus of rat hypothalamus. Intracerebroventricular (ICV) administration increases food intake and body weight. We examined the effect of ghrelin on gastric acid secretion in urethane-anesthetized rats and found that ICV administration of ghrelin increased gastric acid output in a dose-dependent manner. Vagotomy and administration of atropine abolished the gastric acid secretion induced by ghrelin. ICV administration of ghrelin also induced c-fos expression in the neurons of the nucleus of the solitary tract and the dorsomotor nucleus of the vagus, which are key sites in the central nervous system for regulation of gastric acid secretion. Our results suggest that ghrelin participates in the central regulation of gastric acid secretion by activating the vagus system., (Copyright 2001 Academic Press.)

Neuromedin U (NMU) is a brain-gut peptide whose peripheral activities are well-understood but whose central actions have yet to be clarified. The recent identification of two NMU receptors in rat brain has provided a springboard for further investigation into its role in the central nervous system. Intracerebroventricular administration of NMU to free-feeding rats decreased food intake and body weight. Conversely, NMU increased gross locomotor activity, body temperature, and heat production. NMU, a potent endogenous anorectic peptide, serves as a catabolic signaling molecule in the brain. Further investigation of the biochemical and physiological functions of NMU will help our better understanding of the mechanisms of energy homeostasis., (Copyright 2000 Academic Press.)

Ghrelin, a novel growth-hormone-releasing acylated peptide, was recently isolated from rat stomach by the search of an endogenous ligand to an "orphan" G-protein-coupled-receptor. Ghrelin neuron is present in the arcuate nucleus of rat hypothalamus, but its central effect on growth hormone (GH) release has yet to be clarified. We determined the plasma GH concentration and GH mRNA level in the pituitary in response to central administration of ghrelin. A single intracerebroventricular (ICV) administration of ghrelin to rats increased the plasma GH concentration dose-dependently. A continuous ICV administration of ghrelin via osmotic pump for 12 days increased the plasma GH concentration on day 6, but did not keep the high GH concentration on day 12. The GH mRNA levels in both groups of single and continuous administration of ghrelin were not significantly different from those of controls. A single administration of growth-hormone secretagogue also did not stimulate GH synthesis. Central ghrelin stimulated GH release but did not augment GH synthesis. In addition to gastric ghrelin, hypothalamic ghrelin functions to regulate GH release., (Copyright 2000 Academic Press.)

We identified two novel missense mutations in exon 1 of adrenoleukodystrophy (ALD) gene in two unrelated Japanese families. The first, G(874)C transition results in Arg(163)Pro substitution in the cytoplasmic domain of the ALD protein in adrenomyeloneuropathy family. The second, C(679)G results in Ser(98)Trp substitution in the first transmembrane loop in childhood onset cerebral ALD family. Both mutations cause the substitution of polar amino acid (arginine and serine) with non-polar amino acid (proline and tryptophan). Bone marrow transplantation (BMT) from his non-affected his younger sister was performed on a boy with childhood onset cerebral ALD who showed neurological deficit and brain MRI abnormalities. We evaluated the effect of BMT over a 6-year period in terms of neurological deficit, the level of very-long-chain fatty acids (VLCFA) in plasma and fibroblasts, and brain MRI. After BMT, patient's peripheral white blood cells were replaced by donor's XX ones carrying a normal ALD gene confirmed by in situ hybridization using satellite DNA of the centromere of X and Y chromosomes as probes and the level of VLCFA in lymphocytes was within normal limit. However, his neurological state progressively deteriorated. BMT was not beneficial to him.

Agouti protein and agouti-related protein (AGRP) antagonize alpha-melanocyte-stimulating hormone that binds to and activates the melanocortin-4 receptor (MC4-R) in the hypothalamus, thereby stimulating food intake. Melanin-concentrating hormone (MCH) and orexin are orexigenic peptides that specifically are synthesized in the lateral hypothalamus. MCH gene expression was augmented in A(y)/a (agouti) mice which overexpress agouti protein, but orexin mRNA was not. AGRP administered intracerebroventricularly into wild-type rats augmented MCH but not orexin gene expression. Also, SHU9119, a peptidergic antagonist of MC4-R, increased only MCH mRNA. These findings indicate that interruption of signaling at MC4-R activates the MCH but not the orexin gene. The biosyntheses of MCH and orexin are regulated through different pathways., (Copyright 2000 Academic Press.)

Orexins A and B, novel hypothalamic peptides encoded by a single mRNA transcript, stimulate food intake. Two antisera specific for the individual rat orexins were prepared and sensitive RIAs developed. Orexin-A and -B are abundant in the rat hypothalamus, medulla-pons, and midbrain-thalamus, and moderately abundant in the cerebral cortex. No orexins were found in the adipose tissues or visceral organs studied. The major endogenous molecule of orexin-A is a 33-amino-acid peptide, and that of orexin-B a 28-amino-acid peptide. After a 48 h fast, the orexin-A and -B contents of the lateral hypothalamus exhibited a trend to increase, but the contents of other brain tissues significantly decreased as compared with the fed control rats. No circadian variations in the orexin contents were found in the brain. The extensive and abundant distribution of orexins in the brain and changes in their contents upon fasting suggest that they serve as neuromodulators and/or neurotransmitters that regulate feeding behavior through interaction with diverse neural networks., (Copyright 1999 Academic Press.)

Human beta-defensin-2 (hBD-2), a novel antimicrobial peptide, was originally isolated from human skin. We found that synthetic hBD-2 has high bactericidal activity against Escherichia coli under conditions nearly the same as in human bronchial airway surface liquid. We prepared an antiserum against hBD-2 and established a highly sensitive radioimmunoassay (RIA). Reverse-phase high-performance liquid chromatography coupled with the RIA showed that hBD-2 in patients with human lung, bronchoalveolar lavage flid, and plasma. The plasma concentration of hBD-2 in patients with bacterial pneumonia was 32.1 +/- 3.7 fmol/ml (mean +/- SE), 3.9-fold that of normal individuals. By reverse transcription-polymerase chain reaction, the hBD-2 gene transcript was detected in the respiratory epithelial surface of human lung. Human beta-defensin-2 seems to function in airway mucosal defense. Our findings provide a clue to elucidate its pathophysiological significance in respiratory infection.

Background: Defensins, also known as human neutrophil peptides, are antimicrobial peptides present in the azurophil granules of neutrophils. We measured their level in pleural effusion in various pulmonary diseases to investigate whether they could be used as a diagnostic marker in the differential diagnosis of specific pleural diseases., Patients and Participants: We analyzed pleural effusion samples collected from 61 patients, including 50 exudates (11 with empyema, 3 parapneumonic, 15 tuberculous, 18 neoplastic, 3 miscellaneous) and 11 transudates as controls., Measurements: Defensins were measured by radioimmunoassay and also analyzed by reverse-phase high-performance liquid chromatography. The concentrations of interleukin (IL)-8 and granulocyte colony-stimulating factor (G-CSF) in pleural effusion fluid were measured by enzyme-linked immunosorbent assay to examine the correlation between these cytokines and defensins., Results: The concentration of defensins in all samples of empyema was >5,100 ng/mL and the mean concentration (13,265.8+/-1,895.2 ng/mL) in these samples was the highest among other groups. The concentration in the other 50 pleural effusion samples tested was <2,800 ng/mL. Defensins were mostly of the mature type in empyema. Pleural effusion levels of IL-8 and G-CSF in patients with empyema were also significantly higher than those in other samples. There was a significant correlation between defensins and IL-8 or G-CSF in pleural effusion fluid (r=0.762, and 0.827, respectively)., Conclusions: Our results suggest that the high effusion concentrations of defensins in pleural effusion may constitute an important component of the host defense system or may have a cytotoxic role in empyema. Our results also indicate that the high levels of IL-8 and G-CSF in empyema may indirectly explain the elevated levels of defensins by increasing the number of neutrophils in the pleural space.

Study Objective: Adhesion molecules have been implicated in the pathogenesis of inflammatory diseases. This study was designed to determine whether soluble adhesion molecules in serum reflect the disease activity in diffuse panbronchiolitis (DPB)., Patients and Methods: Using an enzyme-linked immunosorbent assay, we measured the serum levels of soluble L-, E-, and P-selectin (sL-, sE-, and sP-selectin), intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in 27 patients with DPB, 13 with bronchiectasis, and 15 normal adults. BAL was also performed, and the levels of interleukin (IL)-8 and IL-1 beta in BAL fluid (BALF) were measured., Results: The serum levels of these molecules were significantly elevated in DPB patients compared with the control subjects. DPB patients also had significant high levels of circulating sE- and sP-selectin compared with patients with bronchiectasis. There was a significant correlation between serum sE-selectin and the percentage of neutrophils in BALF in all patients. There was a significant inverse correlation between serum sE-selectin and percent vital capacity in DPB patients. In the same patients, the relationships between serum sE-selectin and BALF concentrations of IL-1 beta as well as between serum sL-selectin and BALF IL-8 were also significant. Treatment of DPB patients with macrolides significantly reduced the serum levels of these soluble adhesion molecules and BALF concentrations of IL-1 beta and IL-8., Conclusions: Our results suggest that these soluble adhesion molecules, particularly selectins, may reflect the disease activity of DPB, and that their levels may be regulated by cytokines produced in the lungs.

We identified three different missense mutations of the transthyretin (TTR) gene in three Japanese patients with familial amyloidotic polyneuropathy by analysis of their DNAs extracted from formalin-fixed and paraffin-embedded tissues. Patient 1 carried the TTR methionine-30 (Met) mutation (G to A transition at position 1679). DNA sequencing analysis of the TTR gene from patient 2 showed a G to T transversion at position 3830 in exon 3, resulting in an amino acid replacement of serine-50 (Ser) with isoleucine (Ile). Patient 3 had the novel mutation (G to T transversion at position 7314) in exon 4, resulting in an amino acid replacement of alanine-109 (Ala) with Ser. We established DNA diagnostic methods for detecting TTR Ile50 by polymerase chain reaction (PCR)-induced mutation restriction analysis and for TTR Ser109 by PCR-restriction fragment length polymorphism. Gene analysis of archival paraffin-embedded tissues is useful for the precise diagnosis of FAP and for clarifying its molecular pathogenesis in patients for whom fresh genomic DNA is not available.

Uroguanylin, a 16-amino acid peptide, is an endogenous activator of intestinal and possibly renal guanylate cyclase C (GC-C). Using two synthetic topological isoforms of human uroguanylin, one bioactive, the other inactive, we prepared two antisera specific for the individual isoforms and developed sensitive radioimmunoassay (RIAs). The respective plasma concentrations of the bioactive and inactive uroguanylins in the normal individuals tested were 5.0 +/- 0.3 fmol/ml (mean +/- SE) and 1.6 +/- 0.1 fmol/ml. These concentrations increased in chronic renal failure (CRF). The major endogenous uroguanylin molecule in normal human urine was 16 amino acids long, whereas in the plasma and urine of CRF patients the major molecule was 10-kDa prouroguanylin. The RlAs established are promising tools for clarifying the physiological functions and pathophysiological implications of uroguanylin in water and electrolyte homeostasis.

Uroguanylin, a member of the guanylin peptide family, is an endogenous activator of intestinal guanylate cyclase (GC-C). A cDNA encoding a precursor for human uroguanylin was cloned from a human colon cDNA library and sequenced. The precursor was 112 amino acids long and included a signal peptide at the N-terminus and the human uroguanylin sequence at the C-terminus. RNA blot analysis and the reverse transcription-polymerase chain reaction (RT-PCR) showed that human uroguanylin mRNA is expressed in the stomach and intestine. Uroguanylin, as well as guanylin, may be a potent physiological regulator of intestinal fluid and electrolyte transport.

Guanylin, a 15-amino acid peptide homologue of bacterial heat-stable enterotoxins, is an endogenous activator of guanylate cyclase C (GC-C). We isolated two novel guanylin molecules from rat intestinal mucosa. They contained guanylin-15 at their C-termini and were identified as guanylin-94 and guanylin-16 by amino acid sequencing and mass spectrometry. Guanylin-94 and guanylin-16 in total account for 85% of guanylin molecules in both the small and large intestine, guanylin-15 being a minor component. Rat guanylin-94 and guanylin-16 did not increase cyclic GMP production in T84 cells. Identification of the post-translational processing products of guanylin should provide a better understanding of the biosynthesis of the peptide.

To study the role of T cells in bronchiolitis obliterans organizing pneumonia (BOOP) and chronic eosinophilic pneumonia (CEP) and to examine the influence of differing racial background, T-cell subsets in bronchoalveolar lavage (BAL) fluid (BALF) and in peripheral blood of 8 Japanese patients with idiopathic BOOP and 5 with CEP were compared with those of 15 normal subjects. The BALF pattern in BOOP was characterized by a significantly high number and percentage of lymphocyte and by a low CD4 to CD8 ratio compared with patients with CEP and healthy volunteers. Patients with CEP showed a significantly higher percentage of BALF eosinophils compared with other groups. There was no significant difference in BALF CD4 to CD8 ratio between patients with CEP and volunteers. Two-color analysis of T-cell subsets revealed that CD3+HLA-DR+ cells (activated T cell) in BALF of patients with BOOP and CEP increased significantly compared with volunteers, while BALF CD3+CD25+ cells (interleukin 2 receptor+ T-cell) did not. In addition, BALF CD8+HLA-DR+ cells (activated suppressor/cytotoxic T cell) in patients with BOOP and CD4+HLA-DR+ cells (activated helper T cell) in patients with CEP were significantly higher than levels detected in healthy subjects. The percentage of CD8+CD57+ cells and the number of CD8+CD11b- cells (cytotoxic T cell) in BALF were significantly higher in patients with BOOP compared with patients with CEP and healthy volunteers. There were no significant differences in the expression of peripheral blood T-lymphocyte surface antigens among the groups. These findings indicate that cytotoxic T cells in Japanese patients with idiopathic BOOP and helper T cells in CEP appear in the lungs is consistent with a previous report in Caucasians, supporting the hypothesis that T cells may play an important role in the pathogenesis of these diseases.

Human neutrophil peptides (HNPs) are 29 to 30 amino acid antimicrobial peptides localized in the azulophil granules in neutrophils. We isolated endogenous molecular forms of HNPs from normal human bone marrow, plasma and peripheral blood neutrophils and determined their primary structures to investigate their post-translational processing. HNPs initially are synthesized as 94 amino acid precursors that produce 75 amino acid pro-HNPs by cleavage of a signal peptide. The majority of pro-HNPs was processed to 56 amino acid intermediates by preaspartate proteolytic cleavage in the bone marrow then to mature HNPs in peripheral blood neutrophils. Part of pro-HNPs was released into the plasma, in which they constituted 25-30% of the HNP molecules. Pro-HNPs and their mRNAs were detected both in peripheral blood neutrophils and bone marrow. Identification of the post-translational processing products of HNPs should provide a better understanding of the biosynthesis of the peptides.

Guanylin is a 15-amino acid peptide that acts on intestinal guanylate cyclase, thereby regulating intestinal fluid and electrolyte transport through the second messenger, cyclic GMP. Using synthetic rat guanylin, we prepared an antiserum that recognizes human and rat guanylin equally on a molar basis and developed a sensitive radioimmunoassay (RIA). The major endogenous guanylin molecule in human intestine and plasma is 10-kDa proguanylin, 15-amino acid guanylin being a minor component. Human guanylin is distributed widely from the duodenum to colon, the highest contents being in the ileum and proximal colon. The plasma concentration of immunoreactive guanylin in the normal individuals tested was 31.2 +/- 3.0 fmol/ml (mean +/- SE) and that in patients with chronic renal failure who were undergoing hemodialysis 7,924 +/- 2,140 fmol/ml. The RIA we established is a promising tool for clarifying the physiological functions and pathophysiological significance of guanylin in water and electrolyte homeostasis.

We investigated therapeutic effects of 4-methylcatechol (4-MC), a non-amine catechol compound, on streptozotocin (STZ)-induced diabetic neuropathy in rats. 4-MC is one of the potent stimulators of endogenous nerve growth factor (NGF) synthesis both in vitro and in vivo. Diabetic rats showed a statistically significant reduction in motor nerve conduction velocity (MNCV), mean myelinated axon diameter, and NGF content in the sciatic nerve during the experimental period of 8 weeks. The 4-MC treatment started 4 weeks after the STZ injection resulted in significantly greater NGF content, faster MNCV, and larger mean myelinated nerve fiber diameter and axon diameter than in untreated diabetic rats. These findings suggest that a decreased NGF level in the diabetic sciatic nerves may be involved in the pathogenesis of diabetic neuropathy and that 4-MC treatment could be useful for diabetic neuropathy.

Neutrophil peptide, also called defensin, is an antimicrobial peptide localized in azurophil granules in neutrophils. Using a synthetic human neutrophil peptide-1 (HNP-1), we prepared an antiserum against the peptide and established a highly sensitive radioimmunoassay (RIA). This RIA equally recognized HNP-1, -2, and -3 on a molar basis. Analyses of leukocyte and blood extracts obtained from normal individuals and patients with bacterial infection by reverse-phase high-performance liquid chromatography coupled with the RIA showed that the antiserum specifically detected HNP molecules. Concentrations of HNP 1-3 in total in normal human blood and plasma are 13.2 +/- 1.4 (mean +/- SEM) ng/mu 1 and 403.8 +/- 38.9 pg/mu 1, respectively. The values increased in infection and returned to within the normal levels after the recovery of the disorder. Granulocyte colony-stimulating factor (G-CSF) increased the plasma and blood concentrations of HNP 1-3. The RIA established here provides a useful tool to elucidate the pathophysiological significance of HNP and to evaluate the neutrophil functions in infection and inflammation.

A new mutation of transthyretin (TTR) has been identified in a patient with late-onset familial amyloidotic polyneuropathy (FAP) of Japanese origin. Peptide mapping by reverse-phase high performance liquid chromatography to compare the patient's TTR with normal TTR showed the presence of an abnormal peptide. Amino acid sequence analysis of the peptide (residues 49-61) showed a lysine-for-glutamic acid substitution at position 61. This substitution, verified by direct DNA sequencing, was the result of a guanine to adenine change on exon 3 of the TTR gene. A polymerase chain reaction-induced mutation restriction analysis (PCR-IMRA) system was established to rapidly detect the missense mutation. TTR-Lys61 is the first variant TTR with a replacement of the acidic with basic amino acid to be found in the amyloid precursor proteins of FAP.

Using a synthetic N-terminal hexadecapeptide of islet amyloid polypeptide (IAPP), we prepared an antiserum specific for IAPP[1-16] and established an extremely sensitive radioimmunoassay (RIA) for the peptide with a minimum detection level of 0.26 fmol/tube. Since the N-terminal sequence of IAPP is 100% conserved in many mammalian species, the RIA is widely applicable in quantifying their IAPP. Analyses of pancreatic extracts of human and hamster using reverse-phase high performance liquid chromatography coupled with the RIA revealed that almost all pancreatic IAPP consisted of IAPP[1-37]. On the other hand, rat and mouse pancreata contained substantial amounts of IAPP[1-16] and IAPP[1-17] in addition to IAPP[1-37] as a major molecular form. In human plasma, IAPP[1-37] is the major molecular form secreted into the circulation in response to glucose administration. The RIA established in this study is promising in elucidating the physiological functions and the pathophysiological significance of IAPP in diabetes mellitus.

We identified and determined the content and molecular form of islet amyloid polypeptide (IAPP/amylin) in the gastrointestinal (GI) tract of human, rat, mouse and cat. IAPP was isolated by anti- IAPP- IgG immunoaffinity chromatography and reverse-phase high performance liquid chromatography coupled with radioimmunoassays for human and rat/mouse IAPPs. Human IAPP [1-37], [17-37] and [24-37] were identified in human stomach with IAPP [1-37] being the major molecular form. In the GI tract of rat, mouse and cat, IAPP [1-37] and IAPP [19-37] were identified with the latter being the major molecular form. IAPP is present from stomach to colon with the highest concentration being observed in pyloric antrum of stomach. IAPP content in rat antrum fell to 69% of control after 4 days of fasting, with the molar ratio of IAPP [19-37] to IAPP [1-37] increasing from 1.4 in controls to 2.9 in fasted rats. Identification of IAPP and characteristic morphology of IAPP- cells in the GI tract indicate a possible biological function of IAPP as a gastrointestinal peptide.

C-type natriuretic peptide (CNP) is a new member of the natriuretic peptide family recently identified in porcine brain (1). We raised an antiserum against porcine CNP and set up a radioimmunoassay (RIA) for CNP. Using this RIA system, distribution of immunoreactive (ir-) CNP in porcine tissue was measured and compared with that of ir-atrial natriuretic peptide (ANP) and ir-brain natriuretic peptide (BNP). Tissue concentration of ir-CNP in brain was the highest of the three natriuretic peptides at about 0.79 pmol/g wet wt. CNP was present in medulla-pons in high concentration, with a significant concentration detected in cerebellum. In contrast, ir-CNP was not detected in peripheral tissue, including heart, in a significant concentration. These data demonstrated sharp contrasts in the distribution of the three natriuretic peptides, suggesting that CNP is a natriuretic peptide functioning in the central nervous system.

Skeletal muscle fibers from muscular dystrophic mice (C57BL/10-mdx) 1-4 months of age show elevated free Ca2+ concentrations both at resting and stimulated states, although contractility of adult (2-12 months old) mouse is similar to that of normal mouse. To evaluate the sensitivity of the contractile system of adult mdx mouse muscle to elevated free Ca2+ concentration, Mg2(+)-adenosine triphosphatase (ATPase) activity was examined using myosin, myosin B, and reconstituted actomyosin. Myosin Mg2(+)-ATPase activity of the mdx mouse was significantly higher than that of the normal mouse. Myosin B ATPase activity of the mdx mouse was also higher than that of normal mouse in free Ca2+ concentrations between 10(-9) and 10(-5) M, though there was no difference in the Ca2+ concentration required for half maximal activation of ATPase activity, 2 x 10(-7) M. Polymerized actin (FA) isolated from normal and mdx mice activated rabbit myosin Mg2(+)-ATPase identically, while activation of Mg2(+)-ATPase in mdx myosin by rabbit FA was significantly lower than that in normal mouse myosin. Rapid Pi liberation by Mg2(+)-ATPase in mdx mouse myosin was about half that of normal mouse myosin, being consistent with low activation of Mg2(+)-ATPase activity by rabbit FA. Polyacrylamide gel electrophoresis in the presence of pyrophosphate showed that myosin molecules of mdx and normal mice were both composed of three isozymes, although the fast migrating myosin isozyme (M1) was decreased while the slow migrating band (M3) was increased in mdx myosin. Subunit composition of myosin analyzed by polyacrylamide gel electrophoresis in the presence of SDS showed that the content of the smallest light chain (LC3) in mdx myosin was lower than that of normal mouse myosin, which agreed with findings that mdx myosin contained less M1 isozyme than normal myosin. These results indicated that the lowered response of mdx muscle fibers to elevated Ca2+ concentration can be attributed to the isozyme composition of myosin in mdx mouse.

Using a highly sensitive and specific radioimmunoassay (RIA) for rat islet amyloid polypeptide (IAPP), we clarified regional distribution and molecular forms of rat IAPP. IAPP[1-37] and IAPP[19-37] were identified in normal rat pancreas by sequence analyses IAPP[19-37], accounting for 57% of IAPP-immunoreactivity in rat pancreas, is a major molecular form of rat IAPP moiety. In human, however, IAPP[1-37] is the major component, with IAPP[17-37] composing as little as 2-6% of IAPP-immunoreactivity in pancreas. This indicates that processing of IAPP in pancreas differs in species. A large amount of IAPP (328.5 +/- 25.0 pmol/g wet weight) was found in rat pancreas and the peptide was also detected in pyloric antrum of the stomach, duodenum, jejunum, ileum, and colon at 0.1-0.8% of the level of pancreas. It was not detected in central nervous system. The content of rat IAPP in pancreas fell to 54% of control after 4 day fasting. The distribution of IAPP suggests its possible endocrine or paracrine function in pancreas and gastrointestinal tract.

Islet amyloid polypeptide (IAPP) was identified in human plasma using immunoaffinity chromatography, gel filtration and reverse- phase high performance liquid chromatography coupled with radioimmunoassay specific for the peptide. IAPP[1-37], IAPP[17-37], and other two IAPP- related peptides which were putative pro-IAPPs or different processing products of IAPP, were isolated. All of these IAPPs were also found in human pancreatic extract, indicating that they were secreted from B cell secretory granules into the circulation. IAPP[1-37] is a major molecular form of IAPP in the pancreas, but it accounted for 31% of immunoreactive IAPP in the plasma. Plasma concentration of IAPP in normal individuals increased to 3.0 times the basal level in response to oral administration of 75 g glucose. This study indicated that IAPP is a circulating hormone secreted under glucose stimulation.

Rat islet amyloid polypeptide (IAPP) was isolated from the pancreata of normal rats by utilizing cross-reactivity of a radioimmunoassay system for human IAPP with rat IAPP. Rat IAPP was a 37-amino acid polypeptide with tyrosine amide at the C-terminus, as was the case with human IAPP. Amino acid sequences of rat and human IAPPs were 84% identical, and the most highly conserved sequences were found in the N- and C-terminal regions. Rat IAPP sequence was also 51% identical to those of alpha and beta rat calcitonin gene-related peptide sequences.

A 29-year-old single woman had recurrent stroke-like episodes. She developed loss of consciousness, myoclonic seizures, and lactic acidosis. She died at the age of 30. A muscle biopsy study revealed mitochondrial myopathy, and the postmortem biochemical analysis demonstrated decreased cytochrome c oxidase activity in the skeletal muscles by 20% of normal control. The brain had multiple ischemic lesions in the cerebral cortex without major vascular occlusions. We present this case as an autopsy case of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) with a partial deficiency of cytochrome c oxidase. The analytical electron microscopic study of the calcified small vessels in the globus pallidus revealed increased calcium, phosphorus and iron. No accumulation of chromium, nickel or zinc was noted in this case, which was different from the previously reported cases of basal ganglia calcification.

Using a synthetic C- terminal tetradecapeptide of human islet amyloid polypeptide (IAPP), we prepared an antiserum for human IAPP [24-37] and established a highly sensitive radioimmunoassay (RIA) for human IAPP. Analyses of human pancreatic extract using reverse-phase high performance liquid chromatography coupled with the RIA revealed that the antiserum specifically detects human IAPP. The content of IAPP in the pancreas of two non-diabetic patients was 604.0 and 1447.7 pg/mg wet weight, and a small amount of IAPP-immunoreactivity was detected in the stomach, duodenum, and jejunum. The mean plasma concentration of IAPP in 10 normal individuals was 13.5 +/- 4.8 (SD) pg/ml. The RIA established in this study provides a useful tool to elucidate the physiological function of IAPP and its pathophysiological significance in non-insulin-dependent diabetes mellitus (NIDDM).

Striated muscle cells and storage granules observed in the atria were found in main branches of the pulmonary veins and superior and inferior venae cavae of the rat, pig, and ox. The presence of atrial natriuretic polypeptide (ANP) in these veins was examined by reverse-phase high-performance liquid chromatography coupled with a radioimmunoassay for ANP. The veins contained 0.6 to 8.0 ng ANP/mg wet tissue with the major molecular form being gamma-ANP. ANP was detected in the peripheral lung tissue in a small quantity, but was not detected in the pulmonary artery. The identification of gamma-ANP and storage granules stained with an anti-ANP antiserum in the pulmonary vein and vena cava suggest that the veins may participate in regulating volume status, blood pressure, and cardiovascular homeostasis through the release of ANP.