Establishment and gene expression analysis of disease-derived induced pluripotent stem cells of scleroderma.

BACKGROUND: We recently generated induced pluripotent stem cells (iPSCs) from cultured dermal fibroblasts of systemic sclerosis (SSc-iPSC) to study the disease mechanisms. OBJECTIVE: In the present study, we have performed gene expression analysis using cultured SSc dermal fibroblasts, SSc-iPSC, and fibroblasts re-differentiated from SSc-iPSC (SSc-iPSC-FB). METHODS: mRNA and protein levels of collagen and integrins were analyzed using PCR array, PCR, immunoblotting, and immunofluorescence. RESULTS: We compared expression pattern of TGF-ß-related genes between normal iPSC (NS-iPSC) and SSc-iPSC by PCR array, and found constitutive and significant down-regulation of S100A8, Smad6, and TGF-ß2 in SSc-iPSC. The expression of these genes was not altered in cultured SSc fibroblasts or SSc-iPSC-FB compared to NS fibroblasts or NS-iPSC-FB, respectively. On the other hand, the expression of collagen, integrin α and ß was up-regulated in SSc fibroblasts, while SSc-iPSC-FB showed normalized levels of collagen and integrin ß. CONCLUSIONS: So far, there have been no reports investigating disease-derived iPSCs of SSc. Our results suggest that S100A8, Smad6, and TGF-ß2 may be the key molecules of this disease. On the other hand, the normalization of collagen and integrins by iPSC reprogramming suggests that epigenetic modifications of genes may play a role in the mechanism of collagen accumulation seen in SSc fibroblasts, and that gene reprogramming may become novel therapeutic approach. As the limitation of this study, we established only one iPSC line from each patient, which may not be enough to discuss disease-specific phenotypes. Larger studies including increased number of iPSC lines are needed in the future.

Cytokine expression profiles in the sera of cutaneous squamous cell carcinoma patients.

We focused on the interaction of cytokines in squamous cell carcinoma (SCC), and determined the expression profile of multiple cytokines in the serum of each patient with SCC in the present study. Serum samples were obtained from 12 SCC patients and 7 normal subjects. Four cytokines (IFN-γ, IL-6, GM-CSF, and TGF-ß) were selected because they are reported to be involved in keratinocyte proliferation and SCC progression. Serum levels were measured using ELISA. We found a statistically significant increase of serum IFN-γ levels in SCC patients compared to those in normal subjects, and areas under the curve (AUC) of 0.82 for the serum levels of IFN-γ were higher than those for other cytokine levels according to ROC curve analysis. Patients with increased IFN-γ levels had a significantly more severe cancer stage. Furthermore, the combination of IFN-γ levels and TGF-ß levels could improve the AUC to 0.84. We also found there was a significant correlation between IFN-γ levels and GM-CSF levels or between GM-CSF levels and TGF-ß levels only in SCC patients. Our results suggest that the combination of IFN-γ levels and TGF-ß levels is more effective to diagnose SCC, while serum levels of IFN-γ alone are useful to evaluate tumor progression. Furthermore, expression of these cytokines was not independent, but may be regulated by common upstream factors (e.g. cytokines or methylation) in SCC patients, and such factors may play some roles in the pathogenesis of SCC.

Long non-coding RNA TSIX is upregulated in scleroderma dermal fibroblasts and controls collagen mRNA stabilization.

Long non-coding RNAs (lncRNAs) are thought to have various functions other than RNA silencing. We tried to evaluate the expression of lncRNAs in patients with systemic sclerosis (SSc) and determined whether lncRNAs controls collagen expression in dermal fibroblasts. lncRNA expression was determined by real-time PCR and in situ hybridization. Protein and mRNA levels of collagen were analysed using immunoblotting and real-time PCR. We found TSIX, one of the lncRNAs, was overexpressed in SSc dermal fibroblasts both in vivo and in vitro, which was inhibited by the transfection of transforming growth factor (TGF)-ß1 siRNA. TSIX siRNA reduced the mRNA expression of type I collagen in normal and SSc dermal fibroblasts, but not the levels of major disease-related cytokines. In addition, TSIX siRNA significantly reduced type I collagen mRNA stability, but not protein half-lives. Furthermore, we first investigated serum lncRNA levels in patients with SSc, and serum TSIX levels were significantly increased in SSc patients. TSIX is a new regulator of collagen expression which stabilizes the collagen mRNA. The upregulation of TSIX seen in SSc fibroblasts may result from activated endogenous TGF-ß signalling and may play a role in the constitutive upregulation of collagen in these cells. Further studies on the regulatory mechanism of tissue fibrosis by lncRNAs in SSc skin lead to a better understanding of the pathogenesis, new diagnostic methods by their serum levels and new therapeutic approaches using siRNAs.

EBI3 Downregulation Contributes to Type I Collagen Overexpression in Scleroderma Skin.

IL-12 family cytokines are implicated in the pathogenesis of various autoimmune diseases, but their role in the regulation of extracellular matrix expression and its contribution to the phenotype of systemic sclerosis (SSc) remain to be elucidated. Among the IL-12 family members, IL-35 decreases type I collagen expression in cultured dermal fibroblasts. IL-35 consists of p35 and EBI3 subunits, and EBI3 alone could downregulate the protein and mRNA expression of type I or type III collagen in the presence or absence of TGF-ß costimulation. We found that collagen mRNA stability was reduced by EBI3 via the induction of miR-4500. The IL-35 levels in the sera or on the surface of T cells were not altered in SSc patients, while EBI3 expression was decreased in the keratinocytes of the epidermis and regulatory T cells of the dermis in SSc skin compared with normal skin, which may induce collagen synthesis in SSc dermal fibroblasts. We also found that gp130, the EBI3 receptor, was expressed in both normal and SSc fibroblasts. Moreover, we revealed that EBI3 supplementation by injection into the skin improves mice skin fibrosis. Decreased EBI3 in SSc skin may contribute to an increase in collagen accumulation and skin fibrosis. Clarifying the mechanism regulating the extracellular matrix expression by EBI3 in SSc skin may lead to better understanding of this disease and new therapeutic strategies using ointment or microinjection of the subunit.

Streptococcus pulmonary empyema after varicella infection in a serologically immunocompetent boy.

Varicella zoster virus (VZV) is the etiologic agent of varicella, and it remains common among children in Japan due to low vaccination rates. It can cause a variety of serious and life-threatening complications. Generally, the most frequent complication of varicella in healthy children is bacterial superinfection, but empyema after VZV infection is a rare condition. This case report describes a previously healthy 21-month-old boy who attended nursery school with a recent varicella and group A ß-hemolytic streptococcus (GABHS) pharyngitis outbreak and who presented with a 7 day history of vesicular rash along with progressive fever. Due to continued mild cough and prolonged fever, however, chest radiography was done, which showed a right pleural effusion. Further computed tomography showed a right pulmonary empyema, and purulent material was drained and eventually grew GABHS. This report hereby describes the development of pleural empyema caused by GABHS after VZV infection in a serologically immunocompetent patient.

Life-threatening QT prolongation in a boy with attention-deficit/hyperactivity disorder on atomoxetine.

As a noncentral nerve-stimulating agent blocking reuptake of noradrenalin, atomoxetine is used for treatment of attention-deficit/hyperactivity disorder (ADHD). Because it has less potential for addiction and abuse and improves core symptoms of ADHD, it is commonly prescribed in many children and adolescents internationally. Its common side effects include headache, abdominal pain, decreased appetite, and weight loss. In addition, cardiac effects such as tachycardia and hypertension have also been reported. In this case report, an 11-year-old Japanese boy with a past medical history of ADHD on atomoxetine for more than 2 years presented with a loss of consciousness. Initial electrocardiogram (ECG) showed significant QT prolongation, and 9 h later, it worsened, along with bradycardia, inversed T waves, and multiple premature ventricle contractions (PVCs). Transthoracic echocardiography showed akinesis with dilation and systolic ballooning of the left ventricle's (LV) apical segment (Takotsubo cardiomyopathy). At that point, bisoprolol and transcutaneous pacing were started. After 5 days, transcutaneous pacing was discontinued due to improvement in his cardiac rhythm. He continued to remain asymptomatic for the next year, while his QT interval returned to normal. Conclusion: This case report suggests a serious side effect of atomoxetine, and to avoid life-threatening cardiovascular events for children and adolescents with ADHD on atomoxetine, prior screening for cardiovascular conditions by ECG with close monitoring is necessary.

Impaired IL-17 signaling pathway contributes to the increased collagen expression in scleroderma fibroblasts.

Among IL-17 families, IL-17A and IL-17F share amino acid sequence similarity and bind to IL-17R type A. IL-17 signaling is implicated in the pathogenesis of various autoimmune diseases, but its role in the regulatory mechanism of extracellular matrix expression and its contribution to the phenotype of systemic sclerosis (SSc) both remain to be elucidated. This study revealed that IL-17A expression was significantly increased in the involved skin and sera of SSc patients, whereas the IL-17F levels did not increase. In contrast, the expression of IL-17R type A in SSc fibroblasts significantly decreased in comparison with that in normal fibroblasts, due to the intrinsic TGF-ß1 activation in these cell types. Moreover, IL-17A, not IL-17F, reduced the protein expression of α1(I) collagen and connective tissue growth factor. miR-129-5p, one of the downregulated microRNAs in SSc fibroblasts, increased due to IL-17A and mediated the α1(I) collagen reduction. These results suggest that IL-17A signaling, not IL-17F, has an antifibrogenic effect via the upregulation of miR-129-5p and the downregulation of connective tissue growth factor and α1(I) collagen. IL-17A signaling is suppressed due to the downregulation of the receptor by the intrinsic activation of TGF-ß1 in SSc fibroblasts, which may amplify the increased collagen accumulation and fibrosis characteristic of SSc. Increased IL-17A levels in the sera and involved skin of SSc may be due to negative feedback. Clarifying the novel regulatory mechanisms of fibrosis by the cytokine network consisting of TGF-ß and IL-17A may lead to a new therapeutic approach for this disease.

Down-regulation of mir-424 contributes to the abnormal angiogenesis via MEK1 and cyclin E1 in senile hemangioma: its implications to therapy.

BACKGROUND: Senile hemangioma, so-called cherry angioma, is known as the most common vascular anomalies specifically seen in the aged skin. The pathogenesis of its abnormal angiogenesis is still unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we found that senile hemangioma consisted of clusters of proliferated small vascular channels in upper dermis, indicating that this tumor is categorized as a vascular tumor. We then investigated the mechanism of endothelial proliferation in senile hemangioma, focusing on microRNA (miRNA). miRNA PCR array analysis revealed the mir-424 level in senile hemangioma was lower than in other vascular anomalies. Protein expression of MEK1 and cyclin E1, the predicted target genes of mir-424, was increased in senile hemangioma compared to normal skin or other anomalies, but their mRNA levels were not. The inhibition of mir-424 in normal human dermal microvascular ECs (HDMECs) using specific inhibitor in vitro resulted in the increase of protein expression of MEK1 or cyclin E1, while mRNA levels were not affected by the inhibitor. Specific inhibitor of mir-424 also induced the cell proliferation of HDMECs significantly, while the cell number was decreased by the transfection of siRNA for MEK1 or cyclin E1. CONCLUSIONS/SIGNIFICANCE: Taken together, decreased mir-424 expression and increased levels of MEK1 or cyclin E1 in senile hemangioma may cause abnormal cell proliferation in the tumor. Senile hemangioma may be the good model for cutaneous angiogenesis. Investigation of senile hemangioma and the regulatory mechanisms of angiogenesis by miRNA in the aged skin may lead to new treatments using miRNA by the transfection into senile hemangioma.

Pediatric kidney transplantation is safe and available for patients with urological anomalies as well as those with primary renal diseases.

The aim of the current study was to evaluate long-term outcomes of pediatric live kidney transplantation in patients with genitourinary anomalies relative to those with primary kidney diseases. The study included 35 pediatric patients who received a live kidney transplantation during the last 25 yr (28 males, six females). Median age at the time of transplantation was nine yr (range 1-15 yr), and the median follow-up period was 151 months (range 6-239 month). The patients were divided into two groups. The urological group (n = 14) included patients with primary obstructive/reflux nephropathy. The renal group (n = 20) included patients with primary renal disorders. Differences between groups in graft survival, clinical course, and final graft function were evaluated. Original diseases represented in the urological group included five cases with primary VUR and eight cases with secondary VUR. Diseases in the renal group included eight cases with bilateral hypo-dysplastic kidney, three cases with focal/segmental glomerular sclerosis, two cases with membranous proliferative glomerulonephritis, two cases with congenital nephrotic syndrome and five cases with other forms of chronic nephritis. Ten of 14 cases in the urological group, relative to six of 20 in the renal group, were preemptive. Median age at transplantation was 7.5 or 10 yr old, respectively, in the urological or renal group. Twelve kidney recipients in the urological group had also undergone other urinary surgeries, including upper urinary tract drainage, ureteroneocystostomy, augmentation cystoplasty, endoscopic incision of posterior-urethral valve, urethroplasty, etc. Cumulative post-operative complications occurred in nine or 16, respectively, in the urological or renal group. The acute rejection free and overall graft survival were similar in both groups. One patient in the urological group lost his graft while six patients in the renal group lost their grafts. Thus, the post-transplant clinical outcome of pediatric transplantation in patients with urological anomalies is comparable to that of recipients with primary renal disease. Appropriate urinary tract reconstruction and management is essential to reduce the risk of graft dysfunction because of urinary problems.

Elevation of serum Krebs von den Lunge-6 levels in patients with tubulointerstitial nephritis and uveitis syndrome.

BACKGROUND: Tubulointerstitial nephritis and uveitis (TINU) syndrome is characterized by a combination of idiopathic acute tubulointerstitial nephritis and uveitis. Krebs von den Lunge-6 (KL-6) is a human glycoprotein secreted by type II alveolar cells in the lung, and its serum levels increase in patients with pneumonia of various causes, as well as ocular sarcoidosis. The aim of the present study is to quantify serum KL-6 levels in patients with TINU syndrome, which has no pulmonary and sarcoid lesions, and elucidate the usefulness of KL-6 for the diagnosis and follow-up of this syndrome. METHODS: Serum and urinary samples were obtained from 17 patients with TINU syndrome and 36 age-matched patients with uveitis from other causes. Sarcoidosis was eliminated because serum KL-6 levels increased during their lung lesion. Serum KL-6 level was determined by using a human KL-6 electrochemiluminescence immunoassay. Formalin-fixed paraffin-embedded renal tissue sections were incubated with anti-KL-6 monoclonal antibody, then examined immunohistochemically. RESULTS: Mean serum KL-6 levels for patients with TINU syndrome and those with uveitis from other causes were 363.35 +/- 51.06 and 213.19 +/- 10.28 U/mL, respectively (P < 0.001). Urinary beta(2)-microglobulin levels of patients with TINU syndrome and uveitis from other causes were 4.06 +/- 1.31 and 0.16 +/- 0.20 mg/L, respectively (P < 0.001). All patients with TINU syndrome showed a simultaneous decrease in serum KL-6 and urinary beta(2)-microglobulin levels after the beginning of treatment. Strong immunoreactivity for KL-6 was observed in renal distal tubules in biopsy tissue of patients with TINU syndrome. CONCLUSION: We show a significant increase in serum KL-6 levels in patients with TINU syndrome, whereas levels were normal in patients with other causes of uveitis without nephritis. Renal distal tubules stained strongly with anti-KL-6 antibody, suggesting that high KL-6 levels may reflect the renal lesion of TINU syndrome. Serum KL-6 may be a potential laboratory parameter for the diagnosis and follow-up of patients with TINU syndrome that could complement urinary beta(2)-microglobulin measurements.

Renal failure due to tubulointerstitial nephropathy in an infant with cranioectodermal dysplasia.

Cranioectodermal dysplasia (CED) is a rare autosomal recessive disease with characteristic craniofacial, skeletal, and ectodermal-derived tissue abnormalities. In this disease, tubulointerstitial nephropathy (TIN) has been reported as one of the life-threatening combinations. Here we report a sporadic case of CED showing signs of renal failure during the perinatal period. Renal biopsy at the age of 6 months revealed TIN consisting of marked interstitial fibrosis with inflammatory cell infiltration accompanied by scattered tubular atrophy. Glomeruli were often sclerosed and others showed prominent immaturity; the findings are supportive of progressive deterioration of renal function in this infant. This case suggests that TIN in CED can occur during the fetal period and progress rapidly, leading to end-stage renal failure in infancy.

Glomerular localization of erythropoietin receptor mRNA and protein in neonatal and mature mouse kidney.

BACKGROUND/AIMS: Erythropoietin (EPO) possesses well-established hematopoietic properties as the primary stimulator of red blood cell formation by binding to its receptor (EPO-R). Recent evidence suggests pathophysiological roles of EPO in several non-hematopoietic tissues including kidney. Our aim was to further clarify the glomerular localization of EPO-R in normal kidney, as well as changes in its expression during glomerulogenesis. METHODS: We analyzed EPO-R mRNA and protein expression in neonatal and adult mouse kidney by in situ hybridization and immunohistochemistry. To confirm the precise localization and developmental changes of EPO-R expression in podocytes in mature and developing glomeruli, we examined co-expression with the podocyte markers WT-1 and synaptopodin. RESULTS: In addition to tubular expression as reported recently, EPO-R expression was observed in podocytes as well as endocapillary cells in the glomeruli from adult mice. In newborn kidney, EPO-R mRNA and protein expression was first observed in developing podocytes in S-shaped bodies with expression subsequently increasing in glomeruli at the capillary-loop and maturing stages. Immunoelectron microscopy also demonstrated cytoplasmic expression of EPO-R that was prominent at the basal sides of podocytes in glomeruli at the late capillary-loop and maturing stage. CONCLUSION: EPO-R is expressed in developing and mature podocytes in mouse kidney, suggesting a possible role for EPO in podocyte biology.

[Two cases of autosomal dominant polycystic kidney diseases who presented bilateral enlarged kidneys and severe hypertension in the neonatal period].

Autosomal dominant polycystic kidney disease(ADPKD) is rarely observed in the neonatal period. We report 2 cases of ADPKD who showed bilateral enlarged, hyperechoic kidneys and severe hypertension. It is difficult to differentiate ADPKD from autosomal recessive polycystic kidney disease (ARPKD) based on the initial clinical presentations in this period. In both cases, bilateral enlarged kidneys and severe hypertension were detected without oligohydramnion and respiratory distress. The mother of case 1 has polycystic kidneys. The father of case 2 was diagnosed as ADPKD. Case 2 had heart failure due to hypertension. Angiotensin converting enzyme inhibitor (ACE-I) was administered to both patients and resulted in good control of blood pressure. ADPKD in the neonatal and very early infantile period has diverse clinical courses. In general, although severe cases are rare, some cases have renal failure and/or hypertension as we reported. We emphasize that both the prompt diagnosis of ADPKD and the start of medication are of great importance in the neonatal and very early infantile period. We recommend that neonates and infants with a family history of ADPKD undergo screening including physical examinations, blood pressure measurements and urinalysis.