Comparing the safety and efficacy of nintedanib starting dose in patients with connective tissue disease-associated interstitial lung diseases.

OBJECTIVE: This study aimed to analyse whether initiating nintedanib treatment at a reduced dose could improve the treatment continuation rate while maintaining efficacy in patients with connective tissue disease (CTD)-associated interstitial lung disease. METHOD: In total, 51 patients (age 61.6 ± 13.2 years; 38 women, 13 men) were retrospectively analysed. The primary endpoint was the cumulative discontinuation rate due to adverse events. Secondary endpoints included changes in drug dosage, efficacy evaluated based on annual changes in forced vital capacity (FVC), and safety assessed based on the frequency of adverse events. RESULTS: Eighteen patients who started treatment at the standard dose of 300 mg (standard dosage group) were compared with 33 patients who started treatment at a reduced dose (reduced dosage group). Systemic sclerosis was the most common CTD (n = 32), followed by idiopathic inflammatory myopathies and, rarely, rheumatoid arthritis. Both groups exhibited comparable cumulative discontinuation rates due to adverse events and similar frequencies of adverse events. No significant differences were observed in maintenance doses between the two groups; however, patients in the reduced dosage group had a lower cumulative dose for up to 52 weeks than those in the standard dosage group. No significant differences were observed in changes in FVC between the two groups. CONCLUSION: There was no evidence for a difference between the two groups in terms of discontinuation rates, efficacy, and safety. To provide further evidence, future studies using more precise dose-escalation protocols are warranted.

Contrast-enhanced CT rim sign may predict vestibular schwannoma adhesion and postoperative complications.

AIM: To investigate the clinical and radiological features to predict adhesion between vestibular schwannoma (VS) and brain tissue which is a critical risk factor for postoperative infarction and residual tumour. MATERIAL AND METHODS: One hundred and seven consecutive VS surgeries were analysed. After excluding cases without contrast-enhanced (CE) computed tomography (CT), Koos grades 1 and 2, and cases with incomplete clinical data, 44 patients were finally included in the study. Enhancement of the tumour capsule on the brainstem side on CE-CT was defined as the CE-CT rim sign, which was analysed along with clinical characteristics, including tumour adhesion and postoperative complications. RESULTS: Eight patients exhibited CE-CT rim signs; 17 had tumour adhesions. Four patients had postoperative infarction at the ipsilateral middle cerebellar peduncle; 18 exhibited postoperative infarction and/or residual tumour at the middle cerebellar peduncle. The CE-CT rim sign significantly correlated with tumour adhesion, postoperative infarction, and postoperative infarction and/or residual tumour in the cerebellar peduncle. Univariate regression analysis revealed that the CE-CT rim sign significantly correlated with tumour adhesion (odds ratio [OR] 6.81, 95% confidence interval [CI] 1.18-39.25, p=0.032) and postoperative infarction and/or residual tumour at the cerebellar peduncle (OR 6.00, 95% CI 1.04-34.31, p=0.044). CONCLUSION: The CE-CT rim sign was identified in 18.2% of patients with VS and significantly correlated with tumour adhesion and postoperative complications, such as postoperative infarction and residual tumour. This study highlights the importance of the preoperative CE-CT rim sign in VS, which is predictive of tumour adhesion and postoperative complications.

Generation of a novel CD30+ B cell subset producing GM-CSF and its possible link to the pathogenesis of systemic sclerosis.

Systemic sclerosis (SSc) is a T helper type 2 (Th2)-associated autoimmune disease characterized by vasculopathy and fibrosis. Efficacy of B cell depletion therapy underscores antibody-independent functions of B cells in SSc. A recent study showed that the Th2 cytokine interleukin (IL)-4 induces granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing effector B cells (GM-Beffs ) in humans. In this study, we sought to elucidate the generation mechanism of GM-Beffs and also determine a role of this subset in SSc. Among Th-associated cytokines, IL-4 most significantly facilitated the generation of GM-Beffs within memory B cells in healthy controls (HCs). In addition, the profibrotic cytokine transforming growth factor (TGF)-ß further potentiated IL-4- and IL-13-induced GM-Beffs . Of note, tofacitinib, a Janus kinase (JAK) inhibitor, inhibited the expression of GM-CSF mRNA and protein in memory B cells induced by IL-4, but not by TGF-ß. GM-Beffs were enriched within CD20+ CD30+ CD38-/low cells, a distinct population from plasmablasts, suggesting that GM-Beffs exert antibody-independent functions. GM-Beffs were also enriched in a CD30+ fraction of freshly isolated B cells. GM-Beffs generated under Th2 conditions facilitated the differentiation from CD14+ monocytes to DC-SIGN+ CD1a+ CD14- CD86+ cells, which significantly promoted the proliferation of naive T cells. CD30+ GM-Beffs were more pronounced in patients with SSc than in HCs. A subpopulation of SSc patients with the diffuse type and concomitant interstitial lung disease exhibited high numbers of GM-Beffs . Together, these findings suggest that human GM-Beffs are enriched in a CD30+ B cell subset and play a role in the pathogenesis of SSc.

Functional analysis of a novel G87V TNFRSF1A mutation in patients with TNF receptor-associated periodic syndrome.

Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autoinflammatory disease that is caused by heterozygous mutations in the TNFRSF1A gene. Although more than 150 TNFRSF1A mutations have been reported to be associated with TRAPS phenotypes only a few, such as p.Thr79Met (T79M) and cysteine mutations, have been functionally analyzed. We identified two TRAPS patients in one family harboring a novel p.Gly87Val (G87V) mutation in addition to a p.Thr90Ile (T90I) mutation in TNFRSF1A. In this study, we examined the functional features of this novel G87V mutation. In-vitro analyses using mutant TNF receptor 1 (TNF-R1)-over-expressing cells demonstrated that this mutation alters the expression and function of TNF-R1 similar to that with the previously identified pathogenic T79M mutation. Specifically, cell surface expression of the mutant TNF-R1 in transfected cells was inhibited with both G87V and T79M mutations, whereas the T90I mutation did not affect this. Moreover, peripheral blood mononuclear cells (PBMCs) from TRAPS patients harboring the G87V and T90I mutations showed increased mitochondrial reactive oxygen species (ROS). Furthermore, the effect of various Toll-like receptor (TLR) ligands on inflammatory responses was explored, revealing that PBMCs from TRAPS patients are hyper-responsive to TLR-2 and TLR-4 ligands and that interleukin (IL)-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) are likely to be involved in the pathogenesis of TRAPS. These findings suggest that the newly identified G87V mutation is one of the causative mutations of TRAPS. Our findings based on unique TRAPS-associated mutations provide novel insight for clearer understanding of inflammatory responses, which would be basic findings of developing a new therapeutic and prophylactic approach to TRAPS.

Randomized clinical trial comparing long-term quality of life for Billroth I versus Roux-en-Y reconstruction after distal gastrectomy for gastric cancer.

BACKGROUND: Patients' quality of life (QoL) deteriorates remarkably after gastrectomy. Billroth I reconstruction following distal gastrectomy has the physiological advantage of allowing food to pass through the duodenum. It was hypothesized that Billroth I reconstruction would be superior to Roux-en-Y reconstruction in terms of long-term QoL after distal gastrectomy. This study compared two reconstructions in a multicentre prospective randomized clinical trial to identify the optimal reconstruction procedure. METHODS: Between January 2009 and September 2010, patients who underwent gastrectomy for gastric cancer were randomized during surgery to Billroth I or Roux-en-Y reconstruction. The primary endpoint was assessment of QoL using the Functional Assessment of Cancer Therapy - Gastric (FACT-Ga) questionnaire 36 months after surgery. RESULTS: A total of 122 patients were enrolled in the study, 60 to Billroth I and 62 to Roux-en-Y reconstruction. There were no differences between the two groups in terms of postoperative complications or mortality, and no significant differences in FACT-Ga total score (P = 0·496). Symptom scales such as epigastric fullness (heaviness), diarrhoea and fatigue were significantly better in the Billroth I group at 36 months after gastrectomy (heaviness, P = 0·040; diarrhoea, P = 0·046; fatigue, P = 0·029). The rate of weight loss in the third year was lower for patients in the Billroth I group (P = 0·046). CONCLUSION: The choice of anastomotic reconstruction after distal gastrectomy resulted in no difference in long-term QoL in patients with gastric cancer. REGISTRATION NUMBER: NCT01065688 (http://www.clinicaltrials.gov).

Evaluation of the extent of ground-glass opacity on high-resolution CT in patients with interstitial pneumonia associated with systemic sclerosis: comparison between quantitative and qualitative analysis.

AIM: To verify whether quantitative analysis of the extent of ground-glass opacity (GGO) on high-resolution computed tomography (HRCT) could show a stronger correlation with the therapeutic response of interstitial pneumonia (IP) associated with systemic sclerosis (SSc) compared with qualitative analysis. MATERIALS AND METHODS: Seventeen patients with IP associated with SSc received autologous peripheral blood stem cell transplantation (auto-PBSCT) and were followed up using HRCT and pulmonary function tests. Two thoracic radiologists assessed the extent of GGO on HRCT using a workstation. Therapeutic effect was assessed using the change of vital capacity (VC) and diffusing capacity of the lung for carbon monoxide (DLco) before and 12 months after PBSCT. Interobserver agreement was assessed using Spearman's rank correlation coefficient and the Bland-Altman method. Correlation with the therapeutic response between quantitative and qualitative analysis was assessed with Pearson's correlation coefficients. RESULTS: Spearman's rank correlation coefficient showed good agreement, but Bland-Altman plots showed that proportional error could be suspected. Quantitative analysis showed stronger correlation than the qualitative analysis based on the relationships between the change in extent of GGO and VC, and change in extent of GGO and DLco. CONCLUSION: Quantitative analysis of the change in extent of GGO showed stronger correlation with the therapeutic response of IP with SSc after auto-PBSCT than with the qualitative analysis.

Mutation of DNASE1 in people with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a highly prevalent human autoimmune diseases that causes progressive glomerulonephritis, arthritis and an erythematoid rash. Mice deficient in deoxyribonuclease I (Dnase1) develop an SLE-like syndrome. Here we describe two patients with a heterozygous nonsense mutation in exon 2 of DNASE1, decreased DNASE1 activity and an extremely high immunoglobulin G titer against nucleosomal antigens. These data are consistent with the hypothesis that a direct connection exists between low activity of DNASE1 and progression of human SLE.

Association between habitual hot spring bathing and depression in Japanese older adults: A retrospective study in Beppu.

OBJECTIVES: Thermal therapy is used to manage various psychological diseases, such as depression. We investigated the relationship between hot spring bathing and depression in older adults using questionnaire responses. DESIGN AND SETTING: We comprehensively evaluated the preventive effects of long-term hot spring bathing in 10429 adults aged ≥ 65 years in Beppu, Japan, by conducting a questionnaire study on the prevalence of depression (n = 219). MAIN OUTCOME MEASURES: Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using a multivariable logistic regression model for history of depression. RESULTS: A separate multivariable logistic regression model for inference showed that female sex (OR, 1.56; 95 % CI, 1.17-2.08; p = 0.002), arrhythmia (OR, 1.73; 95 % CI, 1.18-2.52; p = 0.004), hyperlipidemia (OR, 1.63; 95 % CI, 1.14-2.32; p = 0.006), renal disease (OR, 2.26; 95 % CI, 1.36-3.75; p = 0.001), collagen disease (OR, 2.72; 95 % CI, 1.48-5.02; p = 0.001), allergy (OR, 1.97; 95 % CI, 1.27-3.04; p = 0.002), and habitual daily hot spring bathing (OR, 0.63; 95 % CI, 0.41-0.94; p = 0.027) were independently significantly associated with a history of depression. CONCLUSIONS: We found an inverse relationship between habitual daily hot spring bathing and history of depression. Prospective randomized controlled trials on habitual daily hot spring bathing as a treatment for depression are warranted to investigate whether the use of hot springs can provide relief to those with psychiatric and mental health disorders.

Risk modification by CYP1A1 and GSTM1 polymorphisms in the association of cigarette smoking and systemic lupus erythematosus in a Japanese population.

OBJECTIVES: Exposure to reactive oxygen species (ROS) through cigarette smoking is thought to contribute to the development of systemic lupus erythematosus (SLE). Metabolic enzymes are involved in ROS production. The aim of this study was to evaluate the modifying effect of metabolic polymorphisms on the association of cigarette smoking with SLE risk in a Japanese population. METHODS: We investigated the relationship of the cytochrome P450 (CYP) 1A1 rs4646903 and glutathione S-transferase (GST) M1 deletion polymorphisms to SLE risk with attention to interaction with cigarette smoking among 151 SLE cases and 421 controls in female Japanese subjects. Unconditional logistic regression was used to compute the odds ratios (ORs) and their 95% confidence intervals (CIs), with adjustments for several covariates. RESULTS: Smokers with the CC genotype of CYP1A1 rs4646903 were significantly associated with increased risk of SLE (OR 9.72, 95% CI 2.73-34.6). Similarly, smokers with the combined CYP1A1 rs4646903/GSTM1 'at-risk' genotype were significantly associated with increased risk of SLE (OR 17.5, 95% CI 3.20-95.9). More than 60% of the excess risk for SLE in smokers with the CC genotype and smokers with the combined 'at-risk' genotype was due to an additive interaction. A lack of association of the GSTM1 genotypes with smoking was observed. CONCLUSIONS: Our results suggest that a combination of smoking and either the CYP1A1 rs4646903 genotype or the combined metabolic genotype plays an important role in SLE susceptibility in our Japanese population. Additional studies are warranted to confirm the metabolic polymorphism-smoking interaction suggested in the present study.

Association study of TRAF1-C5 polymorphisms with susceptibility to rheumatoid arthritis and systemic lupus erythematosus in Japanese.

OBJECTIVE: The primary aim of this study was to investigate the association of polymorphisms of TRAF1-C5, a newly identified rheumatoid arthritis (RA) risk locus in Caucasians, with susceptibility to RA and systemic lupus erythematosus (SLE) in Japanese populations. Gene expression levels of TRAF1 and C5 to assess the functional significance of genotypes were also analysed. METHODS: A multicentre association study consisting of 4 RA case-control series (4397 cases and 2857 controls) and 3 SLE case-control series (591 cases and 2199 shared controls) was conducted. Genotyping was performed using TaqMan genotyping assay for two single nucleotide polymorphisms (SNPs) that showed the best evidence of association in the previous Caucasian studies. Quantifications of TRAF1 and C5 expression were performed with TaqMan expression assay. RESULTS: Significant differences in allele frequency for both SNPs were observed between RA and control subjects (combined odds ratio = 1.09), while no significant difference was detected between patients with SLE and controls. Interestingly, alleles rs3761847 A and rs10818488 G had increased the risk for RA in the present study, while they decreased the risk in the original studies. A significant difference was found between risk allele carriers and non-carriers of rs10818488 for the expression level of TRAF1 in phorbol myristate acetate-stimulated lymphoblastoid cell lines (p = 0.04). CONCLUSION: Association of TRAF1-C5 locus with RA susceptibility was detected in the Japanese populations with modest magnitude, while no significant association was observed for SLE. Significant positive effect of genotype on the expression of TRAF1 might support the genetic association between TRAF1 and RA.

Tacrolimus is effective for lupus nephritis patients with persistent proteinuria.

OBJECTIVES: To evaluate the safety and potential efficacy of tacrolimus for the treatment of patients with lupus nephritis and persistent proteinuria. METHODS: A total of 23 Japanese patients with lupus nephritis (21 females/2 males) were enrolled in this study. Patients were administered tacrolimus at a dose of 2-3 mg once daily after the evening meal for 6 months. The dose of tacrolimus was unchanged throughout the study period. Concomitant prednisolone therapy was unchanged or gradually tapered, while other immunosuppressants were stopped at the start of tacrolimus treatment. RESULTS: Tacrolimus was well tolerated, and none of the patients developed adverse drug reactions that required discontinuation of the study. Daily urinary protein loss, the U-prot/U-creat ratio, and serum albumin were significantly improved after 4 months, 3 months, and 1 month of treatment with tacrolimus (p<0.05), respectively, and the improvement persisted until 6 months. The serum complement hemolytic activity (CH50), complement C3 level, and CRP level were also significantly improved after treatment with tacrolimus (p<0.05). Improvement of the U-prot/U-creat ratio was most prominent for patients who were in WHO class IV. CONCLUSIONS: Tacrolimus is safe and effective as maintenance therapy for patients with lupus nephritis, at least for 6 months. A larger randomised, controlled trial over a longer period is needed to confirm these results.

Infectious complications in patients receiving autologous CD34-selected hematopoietic stem cell transplantation for severe autoimmune diseases.

Long-term analysis of infectious complication after high-dose immunosuppressive therapy with CD34-selected autologous hematopoietic stem cell transplantation for patients with severe autoimmune diseases (AD) was performed. Theoretically, CD34 selection can reduce the risk of reinfusion of autoreactive lymphocytes. However, it is also associated with a significant reduction in T cells, natural killer cells, and monocytes, which in turn may compromise immune reconstitution, thereby increasing the risk of infection. Moreover, AD compromises host immunity and causes organ damage resulting in dysfunction of the cutaneous or mucosal barrier. In this study, the incidence rate of infections is reported in 14 patients who underwent high-dose (200 mg/kg) cyclophosphamide therapy followed by reinfusion of CD34-selected autologous peripheral blood stem cells. Bacterial complication occurred in 3 of 14 (21%) patients. Cytomegalovirus reactivation and adenovirus hemorrhagic cystitis were observed in 9 (64%) and 2 (14%) patients, respectively. As for late infectious complications, 7 patients (50%) developed dermatomal varicella zoster virus infection. No infection-related mortality was seen in this case series. Because the risk for infections approaches that seen in allogeneic transplant recipients, infection surveillance, diagnostic workup, and prophylactic strategies similar to those applicable to allogeneic recipients are warranted.

A dose-escalation study of rituximab for treatment of systemic lupus erythematosus and Evans' syndrome: immunological analysis of B cells, T cells and cytokines.

OBJECTIVE: Accumulating evidence suggests that B-cell depletion therapy by rituximab may be effective for autoimmune disorders. However, an optimal dose of rituximab and a mechanism of its action remain to be established. We performed a dose-escalation study for treatment of Japanese patients with autoimmune diseases including eight with SLE and one with Evans' syndrome. METHODS: Rituximab was infused intravenously, weekly 4 times in a dose-escalating fashion at three different doses of 100, 250 or 375 mg/m(2) to three patients each. Immunological parameters were monitored at certain points until 12 months after the treatment. RESULTS: Rituximab was well tolerated and safe in these patients. Seven out of eight SLE patients and one with Evans' syndrome clinically responded completely or partially to the treatment. Four patients achieved long-term remission (18-30 months) without any additional treatment. In these patients, a significant decrease in circulating B cells continued for 6 months after the treatment. The mean fluorescence intensities of CD19, CD21, CD40 and BR3 on the residual B cells as well as the percentage of CD69+ CD4+ T cells decreased significantly. Serum TNF-alpha levels decreased significantly on day 2. The Th1/Th2 balance of CD4+ T cells gradually shifted towards a Th1 type by 6 months. CONCLUSION: In addition to B-cell depletion, modification of B-cell and T-cell phenotypes as well as cytokine profiles may be involved in the action of rituximab.

Association of polymorphisms in complement component C3 gene with susceptibility to systemic lupus erythematosus.

OBJECTIVE: Identification of the genes responsible for systemic lupus erythematosus (SLE). METHODS: All the exons and putative promoter regions of 53 candidate genes (TNFRSF6/Fas, TNFSF6/FasL, Fli1, TNFSF10/TRAIL, TNFSF12/TWEAK, Bcl-2, PTEN, FADD, TRADD, CDKN1A, TNFRSF1A/TNFR1, TNFRSF4/OX40, TNFSF4/OX40L, TNFSF5/CD40L, TNFSF13B/BAFF, ICOS, CTLA4, CD28, FYN, G2A, CR2, PTPRC/CD45, CD22, CD19, Lyn, PDCD1, PTPN6, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, TGFBR3, CD3Z, DNASE1, APCS, MERTK, C3, C1QA, C1QB, C1QG, C2, MBL2, IGHM, IL-2, IL-4, IL-10, IFNG, TNFA, MAN2A1, TNFRSF11A/RANK, TNFRSF11B/OPG, TNFSF11/OPGL) were screened for single nucleotide polymorphisms (SNPs) and their association with SLE was assessed by case-control studies. A total of 509 cases and 964 controls of Japanese descent were enrolled. RESULTS: A total of 316 SNPs was identified. When analysed in the Japanese population, the allele frequencies of T at rs7951 and G at rs2230201 of the C3 gene were 0.110 and 0.626, respectively, in SLE patients; significantly higher than the frequencies of 0.081 and 0.584, respectively, in controls [odds ratio (OR) = 1.40, 95% confidence interval (CI) = 1.05-1.86, P = 0.016 and OR=1.19, 95% CI = 1.01-1.41, P = 0.038, respectively]. The mean serum C3 level of carriers of the rs7951 T allele was significantly lower than that of non-carriers of the T allele in 87 SLE patients whose medical records were available (P = 0.0018). CONCLUSION: rs7951 T allele of the C3 gene was significantly associated with SLE, and decreased serum level of C3 seems to be correlated with this allele.

Increase of RP105-lacking activated B cells in the peripheral blood and salivary glands in patients with Sjögren's syndrome.

OBJECTIVE: To quantify the activated B cells in the peripheral blood and salivary glands of patients with Sjögren's syndrome (SS) by analyzing the expression of RP105 molecule on the B cells. METHODS: The expression of RP105 on the peripheral blood B cells of patients with SS (19 cases) was analyzed by flow cytometry. RP105-positive and negative B cells were sorted and cultured in vitro and the amount of immunoglobulins (IgG and IgM) produced in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA). Salivary gland biopsy samples from 9 SS patients were histologically evaluated and the sequential frozen sections were separately immunostained by anti-RP105 and anti-CD20 monoclonal antibodies. RESULTS: A significantly higher proportion of peripheral blood RP105-negative B cells was found in SS patients than in healthy individuals. RP105-negative, but not positive, B cells from SS patients were capable of producing IgG and IgM spontaneously in vitro, which was enhanced by the addition of Staphylococcus aureus Cowan I strain (SAC) or IL-6. Salivary glands from 2 of 9 SS patients were found to have lymphoid follicles whose germinal centers consisted of RP105-negative B cells. Moreover, a larger proportion of B cells extensively infiltrating the area other than lymphoid follicles was also RP105-negative. CONCLUSION: RP105-negative B cells, a subset of highly activated and well differentiated B cells, which are increased in number in the peripheral blood and extensively infiltrate salivary glands, may be responsible for the production of class-switched immunoglobulin in SS. In addition, those cells might be associated with the inflammation and tissue damage of the salivary glands.

Erythrocyte migration and gap formation in rabbit blood clots in vitro.

Thrombolytic agents must be carried by the blood circulation to thrombi to exert their functions. Structural gaps exist between blood vessels and thrombi or in the area surrounding thrombi. Therefore, information about fundamental gap formation at thrombotic areas is critically important for thrombolytic therapy. We previously reported that t-PA accelerates the activities of bovine erythrocytes and hemoglobin (Hb) towards bovine plasminogen activation. Here, we examined gap generation by observing morphological changes during thrombolytic processes in rabbit blood clots deformation of erythrocytes from blood clots and Hb transfer from erythrocytes to serum in vitro. Rabbit venous blood samples (1 ml) were stored under sterile conditions in glass tubes at 37 degrees C for 2, 24, 48 h, 1, and 2 weeks. We examined clot diameter, erythrocyte diameter and number as well as Hb volume in the serum, as well as histological changes in the clots. The diameter of blood clots did not change until 2 weeks after sampling. Erythrocyte diameter decreased within 48 h and at 2 weeks after sampling at the clot surface (p < 0.001) and interior (p < 0.001). The number of erythrocytes in the serum started to increase starting from 24 h after sampling (p < 0.01). Serum Hb volume also gradually increased from 24 h until 2 weeks after sampling (p < 0.01). The erythrocyte envelope became disrupted and cytoplasm started to flow through pores into the serum at 24 h. The results indicated that blood clots are reduced due to clot retraction, erythrocyte dissociation and cytoplasm leakage without a distinct fibrinolytic reaction. These results indicated that gaps start to form between 2 and 24 h after blood clotting.

Correction: Myeloproliferative leukemia protein activation directly induces fibrocyte differentiation to cause myelofibrosis.

Owing to the insufficient specificity of the anti-myeloproliferative leukemia protein (MPL) antibody in the original version of this Article, Figure 6 and parts of Figures 2a, 4e, and 5a do not represent the correct information. The corrected version of Figure 6 is in this correction and those of Figures 2a, 4e, and 5a are shown in the supplemental information.

Proprotein-processing endoprotease furin controls the growth and differentiation of gastric surface mucous cells.

Gastric surface mucous cells originate from progenitor cells at the isthmus of the gastric gland, from where the cells migrate to the luminal surface. With migration they form secretory granules and express TGF alpha. We found that proprotein-processing endoprotease furin-positive cells were layered around the upper one fourth of the gastric glands of adult rats, whereas they were distributed along an outer epithelial layer in fetal rats. Because the furin-positive cell layer was localized from the upper cell proliferating zone to the less proliferating pit-cell region in the gastric gland unit, we examined the role of furin in the growth and differentiation of surface mucous cells by using the cell line, GSM06. This cell line is derived from the gastric surface mucous cells of transgenic mice harboring the temperature-sensitive simian virus 40 T antigen. At T antigen-active temperature (33 degrees C), the cells grew to confluency, whereas at T antigen-inactive temperature (39 degrees C), the cells ceased growing. At 33 degrees C, the cells exhibited a high level of furin expression with a negligible level of periodic acid Schiff (PAS)-positive materials and a low level of TGF alpha. In contrast, at 39 degrees C the cells produced a high level of PAS-positive materials, TGF alpha, and secretory granules, with a negligible level of furin expression. To further examine the role of furin, we established a GSM06 cell line introduced with either a sense or an antisense furin cDNA. The cells with sense furin expression produced fewer PAS-positive materials and a low level of TGF alpha even at 39 degrees C, whereas the cells with antisense furin expression exhibited more PAS-positive materials and TGF alpha even at 33 degrees C. When furin expression was suppressed by its antisense oligonucleotide, the cell growth was retarded with enhanced expression of the differentiated characteristics. Thus, we conclude that furin is instrumental in controlling the growth of the surface mucous cells.

Identification of DNA cis elements essential for expansion of ribosomal DNA repeats in Saccharomyces cerevisiae.

Saccharomyces cerevisiae carries approximately 150 ribosomal DNA (rDNA) copies in tandem repeats. Each repeat consists of the 35S rRNA gene, the NTS1 spacer, the 5S rRNA gene, and the NTS2 spacer. The FOB1 gene was previously shown to be required for replication fork block (RFB) activity at the RFB site in NTS1, for recombination hot spot (HOT1) activity, and for rDNA repeat expansion and contraction. We have constructed a strain in which the majority of rDNA repeats are deleted, leaving two copies of rDNA covering the 5S-NTS2-35S region and a single intact NTS1, and whose growth is supported by a helper plasmid carrying, in addition to the 5S rRNA gene, the 35S rRNA coding region fused to the GAL7 promoter. This strain carries a fob1 mutation, and an extensive expansion of chromosomal rDNA repeats was demonstrated by introducing the missing FOB1 gene by transformation. Mutational analysis using this system showed that not only the RFB site but also the adjacent approximately 400-bp region in NTS1 (together called the EXP region) are required for the FOB1-dependent repeat expansion. This approximately 400-bp DNA element is not required for the RFB activity or the HOT1 activity and therefore defines a function unique to rDNA repeat expansion (and presumably contraction) separate from HOT1 and RFB activities.