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Background: Reports on treatment patterns of biologic disease-modifying antirheumatic drugs (bDMARDs)/Janus kinase inhibitors (JAKi) for rheumatoid arthritis (RA) in clinical practice are still sparse in Japan, especially in combination with conventional synthetic DMARDs (csDMARDs)., Objectives: The aim of this study was to investigate treatment patterns of bDMARD/JAKi in the treatment of RA in real-world clinical practice in Japan., Method: A retrospective cohort study was conducted using the Japanese Medical Data Vision health claims database. The inclusion criteria required a recorded diagnosis of RA, defined by ICD-10 codes, in patients aged 18 years and older on the index date. We analyzed 39,903 RA patients treated with DMARDs from 2008 to 2020., Results: Among analyzed subjects, 10,196 patients (25.6%) were prescribed bDMARDs/JAKi in combination with csDMARDs, and 3067 patients (7.7%) were prescribed these drugs without csDMARDs. Among the bDMARDs/JAKi, tumor necrosis factor inhibitors (TNFi) were the most commonly prescribed DMARD overall, and also the most common first-line therapy, accounting for 60.0% or 45.5% of patients prescribed these drugs in combination with or without csDMARDs, respectively. Switching, temporary discontinuation (restarting with the same agents), and discontinuation of bDMARDs/JAKi were observed in 3150 (30.9%), 1379 (13.5%), and 2025 (19.9%) patients with csDMARDs, and in 849 (27.7%), 513 (16.7%), and 833 (27.2%) patients without csDMARDs, respectively., Conclusions: Real-world treatment trajectories of bDMARDs/JAKi with and without csDMARDs was analyzed in RA patients in Japan between 2008 and 2020. TNFi were the predominant first-line therapy, and likely to be switched to different classes. Understanding the current treatment patterns, including discontinuation, is important to find an optimal treatment strategy for RA patients., (© 2024. The Author(s).)

Introduction: The persistence of golimumab (GLM) treatment in Japanese patients with rheumatoid arthritis (RA) has been evaluated previously, but evidence of long-term real-world use is lacking. This study assessed the long-term persistence of GLM use, its influencing factors, and impact of prior medications in patients with RA in actual clinical practice in Japan., Methods: This is a retrospective cohort study of patients with RA using data from a hospital insurance claims database in Japan. The identified patients were stratified as only GLM treatment (naïve), had one biological disease-modifying anti-rheumatic drug (bDMARD)/Janus kinase (JAK) inhibitor treatment prior to GLM [switch (1)] and had at least two bDMARDs/JAK prior to GLM treatment [switch (≥ 2)]. Patient characteristics were evaluated using descriptive statistics. Kaplan-Meier survival and Cox regression methods were used to analyze GLM persistence at 1, 3, 5, and 7 years and the associated factors. Treatment differences were compared using a log-rank test., Results: GLM persistence rate in the naïve group was 58.8%, 32.1%, 21.4%, and 11.4% at 1, 3, 5, and 7 years, respectively. Overall persistence rates in the naïve group were higher than in switch groups. Higher GLM persistence was observed among patients aged 61-75 years and those concomitantly using methotrexate (MTX). Also, women were less likely to discontinue treatment compared to men. Higher Charlson Comorbidity Index score, initial GLM dose of 100 mg, and switch from bDMARDs/JAK inhibitor were related to a lower persistence rate. As a prior medication, infliximab showed the longest persistence for subsequent GLM, and using this as a reference, tocilizumab, sarilumab, and tofacitinib subgroups had significantly shorter persistence, respectively (p = 0.001, 0.025, 0.041)., Conclusion: This study presents the long-term real-world results for persistence of GLM and its potential determinants. These most recent and long-term observations demonstrated that GLM and other bDMARDs continue to benefit patients with RA in Japan., (© 2023. The Author(s).)

Melanocortin-1 receptor (MC1R) is primarily activated by α-melanocyte-stimulating hormone (α-MSH) and plays a crucial role, such as keeping homeostasis in the skin against melanogenesis and external stimuli, anti-inflammatory effects, and tissue fibrosis suppression. Afamelanotide, an α-MSH analog MC1R agonist, is clinically used for treating erythroblastic protoporphyria (EPP) by subcutaneous implantation administration. Therefore, we initiated an investigation aimed at orally available small molecule nonpeptide MC1R agonists. Optimization from the internal hit compound 6a finally resulted in the discovery of N-(1-benzyl-1H-imidazol-2-yl)amide derivative 9g bearing isonipecotinic acid moiety, which demonstrated good MC1R agonistic activity and metabolic stability., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Activation of melanocortin 1 receptor (MC1R) is known to exert broad anti-inflammatory and anti-fibrotic effects. The purpose of this study is to investigate the potential of dersimelagon, a novel oral MC1R agonist, as a therapeutic agent for systemic sclerosis (SSc)., Methods: The effects of dersimelagon phosphoric acid (MT-7117) on skin fibrosis and lung inflammation were evaluated in bleomycin (BLM)-induced SSc murine models that were optimized for prophylactic and therapeutic evaluation. Microarray-based gene expression analysis and serum protein profiling were performed in the BLM-induced SSc models. The effect of MT-7117 on transforming growth factor-β (TGF-β)-induced activation of human dermal fibroblasts was evaluated in vitro. Immunohistochemical analyses of MC1R expression in the skin of SSc patients were performed., Results: Prophylactic treatment with MT-7117 (≥ 0.3 mg/kg/day p.o.) significantly inhibited skin fibrosis and lung inflammation, and therapeutic treatment with MT-7117 (≥ 3 mg/kg/day p.o.) significantly suppressed the development of skin fibrosis in the BLM-induced SSc models. Gene array analysis demonstrated that MT-7117 exerts an anti-inflammatory effect via suppression of the activation of inflammatory cells and inflammation-related signals; additionally, vascular dysfunction was extracted as the pathology targeted by MT-7117. Serum protein profiling revealed that multiple SSc-related biomarkers including P-selectin, osteoprotegerin, cystatin C, growth and differentiation factor-15, and S100A9 were suppressed by MT-7117. MT-7117 inhibited the activation of human dermal fibroblasts by suppressing TGF-β-induced ACTA2 (encoding α-smooth muscle actin) mRNA elevation. MC1R was expressed by monocytes/macrophages, neutrophils, blood vessels (endothelial cells), fibroblasts, and epidermis (keratinocytes) in the skin of SSc patients, suggesting that these MC1R-positive cells could be targets for MT-7117., Conclusions: MT-7117 demonstrates disease-modifying effects in preclinical models of SSc. Investigations of its mechanism of action and target expression analyses indicate that MT-7117 exerts its positive effect by affecting inflammation, vascular dysfunction, and fibrosis, which are all key pathologies of SSc. The results of the present study suggest that MT-7117 is a potential therapeutic agent for SSc. A phase 2 clinical trial investigating the efficacy and tolerability of MT-7117 in patients with early, progressive diffuse cutaneous SSc is currently in progress., (© 2022. The Author(s).)

Rickettsiae are obligate intracellular bacteria that have small genomes as a result of reductive evolution. Many Rickettsia species of the spotted fever group (SFG) cause tick-borne diseases known as "spotted fevers". The life cycle of SFG rickettsiae is closely associated with that of the tick, which is generally thought to act as a bacterial vector and reservoir that maintains the bacterium through transstadial and transovarial transmission. Each SFG member is thought to have adapted to a specific tick species, thus restricting the bacterial distribution to a relatively limited geographic region. These unique features of SFG rickettsiae allow investigation of how the genomes of such biologically and ecologically specialized bacteria evolve after genome reduction and the types of population structures that are generated. Here, we performed a nationwide, high-resolution phylogenetic analysis of Rickettsia japonica, an etiological agent of Japanese spotted fever that is distributed in Japan and Korea. The comparison of complete or nearly complete sequences obtained from 31 R. japonica strains isolated from various sources in Japan over the past 30 years demonstrated an extremely low level of genomic diversity. In particular, only 34 single nucleotide polymorphisms were identified among the 27 strains of the major lineage containing all clinical isolates and tick isolates from the three tick species. Our data provide novel insights into the biology and genome evolution of R. japonica, including the possibilities of recent clonal expansion and a long generation time in nature due to the long dormant phase associated with tick life cycles., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Objectives: The complexity and heterogeneity of human bone, as well as ethical issues, frequently hinder the development of clinical trials. The purpose of this in vitro study was to determine the modulus of elasticity of a polyurethane isotropic experimental model via tension tests, comparing the results to those reported in the literature for mandibular bone, in order to validate the use of such a model in lieu of mandibular bone in biomechanical studies., Material and Methods: Forty-five polyurethane test specimens were divided into 3 groups of 15 specimens each, according to the ratio (A/B) of polyurethane reagents (PU-1: 1/0.5, PU-2: 1/1, PU-3: 1/1.5)., Results: Tension tests were performed in each experimental group and the modulus of elasticity values found were 192.98 MPa (SD=57.20) for PU-1, 347.90 MPa (SD=109.54) for PU-2 and 304.64 MPa (SD=25.48) for PU-3., Conclusion: The concentration of choice for building the experimental model was 1/1.

Malignancies of the gallbladder, including neuroendocrine tumors, are uncommon, mostly found incidentally after cholecystectomy and are frequently asymptomatic in the early stages, but highly fatal. Limited data is available on adrenocorticotropic hormone (ACTH)-producing neuroendocrine tumors specifically originating from the gallbladder. We report the clinical and radiographic findings, which included positron emission tomography and computed tomography, of a patient with a gallbladder mass who presented with Cushing's syndrome. Subsequently, a diagnosis of ACTH-producing large cell neuroendocrine carcinoma of the gallbladder was made. Despite being rare and having a poor prognosis, hormone-producing neuroendocrine tumors should be part of the differential diagnosis in the approach of patients with Cushing's syndrome.

The activation of mitogen-activated protein kinases (MAPKs) is critically involved in inflammatory events through mediation of the production of various inflammatory cytokines. The Tpl2 (tumor progression locus 2)-MEK (MAPK/ERK kinase)-ERK (extracellular signal-regulated kinase) signaling pathway plays an essential role in the production of tumor necrosis factor alpha (TNFalpha) in macrophages stimulated with lipopolysaccharide (LPS). Here, we studied the molecular mechanisms of Tpl2-mediated TNFalpha production using a potent Tpl2 kinase inhibitor, 1,7-naphtyridine-3-carbonitrile, and LPS-stimulated RAW264.7 cells. This inhibitor was effective in suppressing the in vitro Tpl2 kinase activity, and caused a significant reduction in TNFalpha production via specific suppression of the phosphorylation of MEK and ERK but not that of p38 and c-Jun N-terminal kinase (JNK). A p38 inhibitor, SB203580, also inhibited the TNFalpha production dose-dependently. Although the TNFalpha mRNA level was not altered by either inhibitor, the Tpl2 inhibitor increased the nuclear TNFalpha mRNA level, while decreasing that in the cytoplasm. Tip-associated protein (TAP), a key molecule in the nucleocytoplasmic transport of TNFalpha mRNA, was up-regulated by LPS, but this increase was impaired by the Tpl2 inhibitor. In all cases, SB203580 was without effect in the presence of LPS. These results suggest that the LPS-induced TNFalpha production via the Tpl2-MEK-ERK signaling pathway is regulated by changing the TAP level at the nucleocytoplasmic transport level. These results improve understanding of TNFalpha regulatory mechanisms and might provide a new therapeutic strategy against inflammatory diseases.

Background: We describe a case of bilateral linezolid-associated optic neuropathy in a patient with ocular sarcoidosis., Case: A 70-year-old woman with sarcoidosis noted foggy vision in both eyes. Best-corrected visual acuity was 0.5 in the right eye and 0.9 in the left. No abnormality other than slight optic disc hyperemia was visible in either eye. A central scotoma in both eyes and enlargement of the blind spot in the right eye were detected by Goldmann perimetry examination, and magnetic resonance imaging demonstrated an edematous optic nerve in the right eye. Therefore, retrobulbar optic neuritis resulting from sarcoidosis was initially suspected. Sub-Tenon's capsule injection of triamcinolone acetonide along with steroid pulse therapy was given; however, best-corrected visual acuity worsened to 0.06 in the right eye and 0.08 in the left. Pulse therapy was discontinued on day 1, and the possibility of linezolid-associated optic neuropathy was speculated because linezolid had been given for methicillin-resistant Staphylococcus aureus osteomyelitis 2 years before by an orthopedist. After discontinuation of linezolid, best-corrected visual acuity improved to 0.8 in the right eye and 0.9 in the left, and the optic disc hyperemia in both eyes disappeared., Conclusion: Our findings demonstrate that it is important for ophthalmologists as well as physicians and orthopedists to consider the possibility of optic neuropathy caused by long-term use of linezolid.

Background: We treated a patient with anterior ischemic optic neuropathy caused by peginterferon., Case: Upon medical examination of the eyes before starting interferon therapy for chronic hepatitis C at Saiseikai Izuo hospital, a 64-year-old man showed corrected visual acuity of 0.9 in the right eye and 1.0 in the left. No abnormality was visible in either eye except for mild cataracts. Six weeks after combination therapy with peginterferon alpha-2b and ribavirin was started, corrected visual acuity was found to have decreased in the right eye, and swelling of the optic nervehead in both eyes was evident. Bilateral anterior ischemic optic neuropathy caused by interferon therapy was diagnosed. Combination therapy with peginterferon alpha-2 b and ribavirin was discontinued, and administration of prednisolone was started at a dose of 60 mg. However, visual acuity declined in both eyes and the visual field defects worsend. At the most recent examination, visual acuity was 1.0 in the right eye and 0.01 in the left. The visual field included only the temporal periphery in the left eye, and part of the central and upper temporal periphery in the right., Conclusion: Since the outcome of visual acuity and visual fields in anterior ischemic optic neuropathy caused by interferon can be poor, an effective therapy for this complication needs to be developed.

Purpose: To determine the concentration of pigment epithelium-derived factor (PEDF) in the aqueous humor of highly myopic eyes., Design: Observational case series., Methods: The PEDF concentration in the aqueous humor of 23 eyes of 17 patients with high myopia (axial length >26 mm) who underwent cataract surgery was measured by enzyme-linked immunosorbent assay., Results: The mean concentration of PEDF in eyes with high myopia (0.54 +/- 0.12 microg/ml) was significantly lower than that in control eyes (0.86 +/- 0.04 microg/ml, P = .0022). The PEDF level in myopic eyes with chorioretinal atrophy (0.32 +/- 0.05 microg/ml) was lower than that in myopic eyes without chorioretinal atrophy (0.71 +/- 0.12 microg/ml; P = .041) and control eyes (P = .0003)., Conclusions: The significantly lower concentration of PEDF in eyes with chorioretinal atrophy-associated high myopia probably resulted from degeneration of the retinal pigment epithelial cells and/or the retinal ganglion cells that are the main sources of PEDF in the eye.

The sialyl Lewis X (sLe(x)) determinant on leukocytes serves as a ligand for selectin family cell adhesion molecules, and selectin-carbohydrate interaction is considered to play an important role in the process of leukocyte extravasation during inflammation. Among several alpha1-3 fucosyltransferases (FucTs), FucT-VII plays a critical role in the biosynthesis of sLe(x)-epitopes. Therefore, small molecules specifically designed to inhibit the FucT-VII enzyme may have potential as anti-inflammatory agents. Here, we have developed a versatile cell-based assay system to monitor sLe(x) biosynthesis using the GeneSwitch System. This system is a mifepristone (MFP)-inducible mammalian expression system, and human transfectant T lymphoblasts expressed the mRNA of FucT-VII and the sLe(x)-epitopes on the cell surface in a time-dependent manner in the presence of MFP, with very low background transcription. Furthermore, when the transfectants were treated with the FucT-VII inhibitor panosialin, sLe(x) expression on the induced cells was inhibited dose dependently without alteration at the mRNA level of FucT-VII. These results suggest that the FucT-VII may be a major regulator of the biosynthesis of the sLe(x)-epitopes on T lymphoblasts, and this cell-based assay may be utilized for a screening system of FucT-VII inhibitors.

The biosynthesis of the carbohydrate antigen sialyl Lewis X (sLe(x)) in human leukocytes is mediated by alpha1-3 fucosyltransferase-VII (FucT-VII), which catalyzes the transfer of fucose from GDP-beta-fucose to the 3-OH of alpha2-3 sialyl N-acetyllactosamine (SA-LN). We developed a simple method for quantitating the reaction product of FucT-VII involving Anion-Exchange Chromatography (AEC). The AEC assay involved the separation of a radio-labeled acceptor from the unreacted nucleotide sugars with 0-0.5 M NH(4)OAc (pH9.0) on QAE-Toyopearl 550C. Furthermore, this assay enabled the separation of the fucosylated products of sialylated and non-sialylated oligosaccharides with this column. Analysis of the FucT-VI reaction mixture showed that Lewis X (Le(x)) was eluted in the flow-through fraction and sLe(x) was eluted with 0.1 M NH(4)OAc, and these products were clearly separated from the fraction of unreacted GDP-[(3)H]fucose. Therefore, this method could be a powerful tool for the characterization of recombinant FucT-VII and for establishing a high-throughput screening system for FucT-VII inhibitors. Beside FucT-VII, this method will be applicable to the assaying of many different glycosyltransferases, including sialyltransferases and glucosaminyltransferases, which are reactive to alpha2-3 SA-LN or N-acetyllactosamine sequences.

The C-terminal two-thirds of nonstructural protein 3 (NS3) of hepatitis C virus (HCV) exhibits RNA-dependent NTPase/helicase activity. This enzyme is considered to be involved in viral replication and is expected to be one of the target molecules of anti-HCV drugs. In a search for NTPase inhibitors specific to HCV, we expressed and purified the truncated NS3 NTPase/helicase domain. Here, we report the characterization of its RNA-dependent ATPase activity. This enzyme preferred Mg(2+) and the optimal pH was 7.0. We further investigated the effects of heavy metal ions on the ATPase activity. The mercuric ion inhibited it significantly, the 50% inhibitory concentration being 49 nM. The fact that the inhibitory profile was competitive and that this inhibition was blocked in the presence of a large excess of cysteine or dithiothreitol, suggested that a cysteine residue in the DECH box was the main target site of mercury.

Hepatitis C virus (HCV) NS5B protein has been shown to have RNA-dependent RNA polymerase (RdRp) activity by itself and is a key enzyme involved in viral replication. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that human eukaryotic initiation factor 4AII (heIF4AII), which is a component of the eIF4F complex and RNA-dependent ATPase/helicase, interacted with NS5B protein. These two proteins were shown to be partially colocalized in the perinuclear region. The binding site in HCV NS5B protein was localized within amino acid residues 495 to 537 near the C terminus. Since eIF4A has a helicase activity and functions in a bidirectional manner, the binding of HCV NS5B protein to heIF4AII raises the possibility that heIF4AII facilitates the genomic RNA synthesis of NS5B protein by unwinding the secondary structure of the HCV genome and is a host component of viral replication complex.

Aim: To clarify the incidence of choroidal neovascularisation (CNV) and predisposing findings for development of CNV in the second eye of Japanese patients with unilateral exudative age related macular degeneration (AMD)., Methods: The second eyes of unilaterally affected patients with exudative (neovascular) AMD treated in our clinic during the past 10 years (1988-97) were carefully followed up for more than a year. Evidence of CNV was confirmed by fluorescein and indocyanine green angiography. Macular lesions in patients, in whom CNV developed in the second eye, were retrospectively evaluated from patient records., Results: 170 patients met the criteria. The average follow up period was 47 months (range 12-108 months). All patients were Japanese. CNV developed in the second eye in 12 (7%) of 170 patients, 30.3 months on average after the first examination. Cumulative incidence of developing CNV in the second eye using Kaplan-Meier life table analysis was: 0.6% by 1 year, 5.6% by 3 years, and 12.3% by 5 years, and was relatively low compared with that in white patients. CNV developed most frequently from serous pigment epithelial detachment (PED) in the macula (58%). Soft drusen were not prevalent and risk of developing CNV was not very high (18%)., Conclusion: It was confirmed that there were some differences in the incidence and predisposing findings for CNV developing in AMD among Japanese and other Asian patients compared with those in white people. It is important to recognise these differences between the two populations to understand the pathogenesis and epidemiology of AMD.

Purpose: To report a penetrating ocular injury resulting from inadvertent placement of a fetal scalp monitoring spiral electrode into the right eye of a preterm male with a face presentation., Methods: Case report and review of the literature., Results: A spiral electrode was screwed clockwise into the right eye, tearing the inferior retina and creating two inferior iridotomies. Severe myopic astigmatism resulted from gradual lens dislocation combined with elongation of the eye. Despite persistent occlusive therapy and aggressive optical correction, before and after lensectomy at age 3 years, visual acuity was only 20/200 at age 8 years., Conclusions: Although complications from spiral monitoring electrodes are uncommon, this case emphasizes that before inserting a spiral monitoring electrode during labor, face presentation must be excluded to prevent inadvertent ocular injury.

Purpose: To report a case of Lyme disease that presented with a single nummular unilateral interstitial keratitis., Methods: Case report and review of the literature., Results: A 57-year-old black man who had contact with freshly killed deer had a chief complaint of foreign-body sensation in his right eye (OD) that had been diagnosed and treated for herpes simplex stromal keratitis. The patient underwent a systemic workup for interstitial keratitis. All results including RPR and MHA-TP were negative except for Lyme antibody titer (enzyme-linked immunosorbent assay [ELISA]) 178 U/ml (normal, <159 U/ml)., Conclusion: Interstitial keratitis from Lyme disease has been regarded as a bilateral disease in the literature. We present this infrequent ocular manifestation of Lyme disease as a rare single nummular unilateral presentation.

The standard treatment for herniated "bags" of the lower eyelid is surgical removal of excess fat. Sachs and Bosniak in 1986 and de la Plaza and Arroyo in 1988 described a new technique for treatment of palpebral bags that consisted of returning the herniated fat to the orbital cavity and retaining it by continuous sutures of the capsulopalpebral fascia either to the dehiscent portion of the orbital septum or to the periosteum of the lower orbital rim. This article reports a prospective study of 26 patients who underwent standard blepharoplasty in one lower eyelid and capsulopalpebral fascia hernia repair in the other lower eyelid. All were evaluated at 6 weeks and at 6 months after surgery, and the outcomes were compared. The results of the two different techniques in the same patient have shown comparable aesthetic outcomes in the treatment of palpebral bags. However, results indicate that the capsulopalpebral fascia hernia repair technique carries less discomfort and pain during the operation and may be less prone to postoperative bleeding and hematoma formation. In addition, in contrast to standard lower blepharoplasty with fat resection, hollowing of the lower lid or potential sunken appearance of the globe may remain absent with capsulopalpebral fascia hernia repair beyond the 6-month period of this study.

Purpose: There is considerable evidence that vascular endothelial growth factor (VEGF) mediates ocular neovascularization in retinal vascular diseases. We investigated the time-dependent changes in the expression of VEGF and its receptor KDR/ Flk in a transient retinal ischemia-reperfusion injury model., Methods: Transient retinal ischemia was induced by increasing the intraocular pressure in albino rats eyes for 45 min. In situ hybridization was used to identify the retinal cells synthesizing VEGF mRNA and KDR mRNA at various times following reperfusion. Immunohistochemical analysis was also carried out to detect VEGF immunoreactivity., Results: In the control, non-ischemic retinas, signals for VEGF mRNA and KDR mRNA were observed in the cells of the ganglion cell layer. Immunoreactivity to VEGF was also found in the nerve fiber layer, the ganglion cell layer, and the retinal pigment epithelial (RPE) cell layer. Immediately and 6 h after reperfusion, VEGF and KDR mRNA expression was markedly decreased, but recovered by 24 h to the levels observed in normal retinas. Immunoreactivity for VEGF was also decreased immediately and 6 h after reperfusion, and was detected in the endothelial cells of the retinal vessels after 24 h. Immunoreactivity to VEGF recovered by 48 h after reperfusion., Conclusions: The hybridization pattern of VEGF and KDR mRNA in the ganglion cell layer strongly suggests that the ganglion cells are the major source of this growth factor. The decrease of VEGF mRNA, KDR/Flk mRNA and VEGF protein levels after ischemia and recovery after reperfusion suggest that transient hypoxia might mediate short-term down-regulation of VEGF and KDR mRNA.

The C-terminal two-thirds of the nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses RNA helicase activity. This enzyme is considered to be involved in the viral replication and is expected to be one of the target molecules of anti-HCV drugs. The conventional method for the measurement of RNA helicase activity includes the step of gel electrophoresis which makes the screening of multiple samples inconvenient. In this study, to establish a high-throughput screening system for HCV helicase inhibitors, we applied the scintillation proximity assay (SPA) system to the detection of this enzymatic activity. We could detect the helicase activity using the NS3 protein purified by an immunoaffinity column. The activity was dependent on the concentration of the enzyme and the reaction time. The RNA helicase activity measured by the SPA system was in a good correlation with that obtained by the conventional method. Furthermore, the SPA system showed better reproducibility and less deviation of the data than the conventional method, which makes the former suitable for quantitative analysis. Since any separation step is not required and microtiter plates can be used in this method, it has the advantage of dealing with multiple samples.

Background: The normal levels of immunoglobulin and IgG subclasses in African American and Hispanic populations are uncertain. To determine immunoglobulin and IgG subclass levels in this community, we measured serum IgG, IgM, and IgA levels along with IgG subclasses in 303 African American and Hispanic patients in a general medical clinic and an allergy/asthma clinic in East Harlem in New York City., Methods: Prospective measurement of immunoglobulins and IgG subclasses in a general medical clinic and an allergy/asthma clinic in East Harlem., Results: Ten (3.4%) patients had IgG levels below the lower limit of normal values, two (0.07%) patients had IgA levels below the lower limit of normal values, and two (0.07%) patients had an IgM level below the lower limit of normal values. Twenty-four (8.1%) patients had IgG subclass levels below the lower limit of normal values; 1 patient had low levels of IgG1 and IgG3, 5 patients had low levels of IgG2, and 18 patients had low levels of IgG3. Because low IgG subclasses and allergy/asthma appeared to be associated, we compared IgG subclass levels of the patients with and without allergy/asthma. The mean IgG2 level in the patients without allergy/asthma was 425.1 +/- 199.1 mg/dL (p = 0.05) compared with 345.5 +/- 133.1 mg/dL in the allergy/asthma group, the mean IgG3 level in the patients without allergy/asthma was 85.0 +/- 57.1 mg/dL compared with 64 +/- 34.1 mg/dL in the allergy/asthma group (p = 0.016) but there were no differences in IgG1 and IgG4 levels between the two groups., Conclusion: Altogether, our data indicate that humoral immunoglobulin and IgG subclass levels below the lowest normal values occur in the low socioeconomic African American and Hispanic populations, especially in patients with asthma in East Harlem.

Background: Scatter photocoagulation induces regression of retinal neovascularization, but the mechanism of its therapeutic effect is incompletely understood. To elucidate the mechanism of therapeutic effect of photocoagulation is the main focus of our research. We have already demonstrated basic fibroblast growth factor (bFGF) immunolocalization during retinal wound repair following laser photocoagulation. Transforming growth factor beta (TGF beta) reportedly inhibits endothelial cell growth and bFGF-induced cell proliferation in vitro. In the present study, we evaluated the immunohistochemical localization of TGF-beta 1 and -beta 2 during wound repair in the rat retina following laser photocoagulation., Methods: Krypton laser photocoagulation was performed on the eyes of pigmented rats. The eyes were then enucleated on day 1, 3, 7, 14, 28 or 56 following the photocoagulation and enrolled into the analysis of immunohistochemical localization of TGF-beta 1 and -beta 2., Results: Immunoreactivity for TGF-beta 1 and -beta 2 was present in the ganglion cell layer and photoreceptor outer segments of the normal adult rat retina. The cytoplasm of RPE cells at the photocoagulated lesion showed intense TGF-beta 1 and -beta 2 immunoreactivity on day 3 after laser photocoagulation. Macrophages that migrated into the lesion lacked positive staining for TGF-beta 1 and -beta 2. TGF-beta immunoreactivity in RPE cells continued to be upregulated for more than 1 month compared with that in normal RPE cells. Controls did not exhibit any positive staining., Conclusion: An elevated expression of TGF-beta immunoreactivity for a longer period of time than bFGF was observed in RPE cells at the photocoagulated lesion in vivo. In the late phase of retinal wound repair, TGF-beta may inhibit cell proliferation induced by mitogens, introduce an end stage of cellular events, and induce extracellular matrix induction.

Purpose: Midkine (MK), a 13-kDa heparin-binding growth factor, is known to exert neurotrophic activities on various nerve cells including retinal cells. To initiate studies toward determining the physiological role of endogenous MK, we investigated the spatial and temporal expression profile of MK before and after intraocular pressure-induced retinal ischemia., Methods: Retinal ischemia was induced in Wistar strain rats by increasing the intraocular pressure to 110 mm Hg for 45 min via cannulation into the anterior chamber. The localization and abundance of the MK protein and mRNA were determined by the use of immunohistochemistry and in situ hybridization in the normal retina, as well as the retina after reperfusion. The protein expression profile was confirmed by Western blot analysis., Results: Immunohistochemical analysis showed that MK protein was expressed in the ganglion cell layer, the inner portion of the inner nuclear layer, and in the retinal pigment epithelium of the normal rat. MK expression transiently decreased 3 h to 2 days after reperfusion, and then dramatically increased to a level higher than normal after 7 to 28 days. The temporal expression profile of the MK protein was confirmed by Western blot analysis. In situ hybridization analysis gave results comparable to those obtained with immunohistochemistry., Conclusions: MK was expressed in the neural cells of the retina in the normal state, but became more abundant after pressure-induced retinal ischemia. Thus, endogenous MK responds to ischemic treatment by an initial decrease in expression and then a period of expression above basal levels.

A selective inhibitor of cyclic nucleotide phosphodiesterase (PDE) 4, rolipram, markedly enhanced the forskolin-induced increase of intracellular dopamine and dihydroxyphenylacetate (DOPAC, a metabolite of dopamine) levels in primary cultured rat mesencephalic neurons and the forskolin-induced increase of dopamine and DOPAC in extracellular medium. Selective inhibitors of PDE2, PDE3, PDE5 and PDE6 did not have such a promoting effect, and the PDE1 inhibitor vinpocetine and W-7 caused dopamine depletion in the neurons. These findings suggested that PDE4 plays a major role in regulating the intracellular cAMP level to control the dopamine biosynthesis in mesencephalic neurons, whereas PDE1 regulates dopamine release instead.

Retinal ischemia promotes retinal neovascularization. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) are important growth factors for neovascularization. We did experimental retinal vein occlusion (RVO) and examined the expression of basic FGF and FGF receptor 1(one of the basic FGF receptors) by in situ hybridization. We used adult pigmented rats (Brown-Norway strain). Dye laser photocoagulation (577 nm) was applied to the retinal arteries and veins within two disc diameters of the optic nerve head to injure the retinal vessels. After one week, laser photocoagulation was applied to only the retinal veins to occlude them (RVO model). As a control, laser photocoagulation was applied to the posterior retina avoiding the retinal vessels. After treatment, the eyes were removed and 10 microns thick cryostat-cut chorioretinal section were used for in situ hybridization with probes as mentioned above. In the RVO model, expression of messenger RNA of basic FGF (b-FGF) and FGF receptor 1 increased in the inner nuclear layer and the inner segment of the photoreceptors, and appeared in the retinal vessel wall in the early stage. This shows that b-FGF and FGF receptor 1 increased in the ischemic retina, and were produced on the retinal vessel wall. This suggests that b-FGF may be involved in protection, regeneration, and proliferation of the retinal vascular endothelial cells in retinal circulatory disturbance.

Purpose: Transforming growth factor beta (TGF-beta) is a multifunctional cytokine that modulates biological events as diverse as wound healing and angiogenesis and which may be important in the pathogenesis of choroidal neovascularization. We investigated the mRNA expression of TGF-beta isoforms in a model of experimental choroidal neovascularization induced by krypton-laser photocoagulation., Methods: Rat TGF-beta 1, mouse TGF-beta 2 or TGF-beta 3 cDNAs was inserted into the pBluescript vector to prepare antisense and sense riboprobes. Intense laser burns were applied to the posterior poles of the eyes of pigmented rats according to a protocol described for producing choroidal neovascularization in these animals. At intervals up to 4 weeks after photocoagulation, the eyes were obtained and cut into thin sections. The sections were subjected to in situ hybridization with digoxigenin (DIG)-labeled single-strand riboprobes synthesized from each TGF-beta cDNA., Results: In normal adult rat retinas and choroids, TGF-beta 1 mRNA was found only in cells of the ganglion cell layer, TGF-beta 2 mRNA was found in cells of the ganglion cell layer and choriocapillaris endothelium, whereas TGF-beta 3 mRNA was not detected at all. During the process of neovascularization, TGF-beta 1 and TGF-beta 2 mRNAs (the latter being expressed more prominently) were detected in retinal pigment epithelial cells, fibroblast-like cells and the endothelium of the neovascular region. TGF-beta 2 was the predominant isoform of TGF-beta, and its expression was especially strong in the endothelium of the choroidal neovascularization at 2 weeks. However, TGF-beta mRNAs was decreased in cells 4 weeks after photocoagulation., Conclusions: Our findings suggest that TGF-beta may act in the retina as a neurotrophic agent, since TGF-beta 1 is normally transcribed in ganglion cells and TGF-beta 2 is also transcribed in ganglion cells and choriocapillaris endothelium. TGF-beta 1 and TGF-beta 2 mRNA expression were increased in photocoagulated lesions from 3 days to 2 weeks after laser treatment. Therefore, it is likely that TGF-beta acts as a mediator of the neovascularization process.

Background: Basic fibroblast growth factor (bFGF) stimulates the mitogenesis of various cells and plays a key role in wound repair. We studied the immunohistochemical localization of bFGF during wound repair in the rat retina after laser photocoagulation., Methods: Krypton laser photocoagulation was performed on the eyes of pigmented rats. The eyes were enucleated on days 1, 3, 7, 14 and 28 after the photocoagulation, and the immunohistochemical localization of bFGF was assessed. Two different monoclonal antibodies and one polyclonal antibody against bFGF as first antibodies were used., Results: Marked immunoreactivity for bFGF was found in the ganglion cell layer, and weak immunoreactivity for bFGF was found in the retinal pigment epithelial (RPE) cells of the normal adult rat retina. On day 3 after laser photocoagulation, the nuclei and cytoplasm of proliferating RPE cells at the center of the photocoagulated lesion showed intense bFGF immunoreactivity. The nuclei of RPE cells around the lesion showed intense bFGF immunoreactivity. Macrophages that migrated into the lesion showed positive staining for bFGF. These immunoreactivity decreased with time. Controls (0.05 M Tris-HCl buffer, normal serum, or these same antibodies preabsorbed with bFGF) did not show positive staining., Conclusion: The finding of an elevated expression of bFGF immunoreactivity in the photocoagulated lesion suggests that bFGF may play a role in wound repair in the rat retina after laser photocoagulation.

We investigated the expression of mRNA of basic fibroblast growth factor (bFGF) and FGF receptor 1 in rat retina after laser photocoagulation using in situ hybridization method. Pigmented rats (Brown Norway strain) received weak photocoagulation by krypton laser (500 microns, 0.05 sec, 60 mW) in the posterior retina. On 1, 3, 5, 7, 14 days after laser photocoagulation, the rats were fixed by perfusion with phosphate-buffered 4% paraformaldehyde and the eyes were enucleated. The eyes were further fixed by immersion in the same fixative, then quickly frozen in liquid nitrogen and finally sectioned with a cryostat. In situ hybridization was performed on frozen sections with digoxigenin (DIG) labeled riboprobes synthesized from rat bFGF cDNA and FGF receptor 1 cDNA. In normal chorioretinal tissue, the signals of bFGF and FGF receptor 1 mRNA were seen in the ganglion cell layer and inner nuclear layer. On day 3 after photocoagulation, we observed expression of bFGF and FGF receptor 1 mRNA in the proliferating retinal pigment epithelial (RPE) cells and endothelial cells of choriocapillaris at the photocoagulated lesion. We also observed expression of bFGF mRNA in some macrophage-like cells. On day 14 after photocoagulation, these expressions had disappeared. Our results suggest that bFGF may be involved in the process of retinal wound healing after laser photocoagulation.

Basic fibroblast growth factor (bFGF) stimulates the mitogenesis of various cells and plays a key role in wound repair. Using in situ hybridization, we studied mRNA expressions of bFGF and one of its receptors, FGF receptor 1 (FGFR1), during wound repair of the rat retina after laser photocoagulation. Gene expressions of bFGF and FGFR1 were detected in the ganglion cell and inner nuclear layers of the normal adult rat retina. On day 3 following laser photocoagulation, proliferating retinal pigment epithelial (RPE) cells of the lesion showed intense gene expressions of bFGF and FGFR1. Macrophage-like cells that migrated into the lesion also showed gene expression of bFGF. These gene expressions decreased over time. The finding of elevated gene expressions of bFGF and FGFR1 after laser photocoagulation suggests the bFGF may be a factor in retinal wound repair.