Comprehensive screening for monogenic diabetes in 89 Japanese children with insulin-requiring antibody-negative type 1 diabetes.

BACKGROUND: Mutations in causative genes for neonatal diabetes or maturity-onset diabetes of the young have been identified in multiple patients with autoantibody-negative type 1 diabetes (T1D). OBJECTIVES: We aimed to clarify the prevalence and phenotypic characteristics of monogenic abnormalities among 89 children with autoantibody-negative insulin-requiring T1D. METHODS: Mutations in 30 genes were screened using next-generation sequencing, and copy-number alterations of 4 major causative genes were examined using multiplex-ligation-dependent probe amplification. We compared the clinical characteristics between mutation carriers and non-carriers. RESULTS: We identified 11 probable pathogenic substitutions (6 in INS , 2 in HNF1A , 2 in HNF4A , and 1 in HNF1B ) in 11 cases, but no copy-number abnormalities. Only 2 mutation carriers had affected parents. De novo occurrence was confirmed for 3 mutations. The non-carrier group, but not the carrier group, was enriched with susceptible HLA alleles. Mutation carriers exhibited comparable phenotypes to those of non-carriers, except for a relatively normal body mass index (BMI) at diagnosis. CONCLUSIONS: This study demonstrated significant genetic overlap between autoantibody-negative T1D and monogenic diabetes. Mutations in INS and HNF genes, but not those in GCK and other monogenic diabetes genes, likely play critical roles in children with insulin-requiring T1D. This study also suggests the relatively high de novo rates of INS and HNF mutations, and the etiological link between autoimmune abnormalities and T1D in the non-carrier group. Carriers of monogenic mutations show non-specific phenotypes among all T1D cases, although they are more likely to have a normal BMI at diagnosis than non-carriers.

Regioselective Diels-Alder reaction to open-cage ketolactam derivatives of C60.

Open-cage ketolactam fullerenes reacted with dienes on the rim of the orifice both regio- and stereoselectively. Unequivocal evidence for the structure of the Diels-Alder adduct was provided by 2D INADEQUATE 13C NMR studies on 13C enriched material, as well as via DFT-GIAO calculations. The theoretical calculations successfully model the regioselective and the endo stereoselective reaction, predicting molecular orbital control along with a repulsive steric interaction between the substituents on the nitrogen atom and those on the diene.

Controlled formation of ordered coordination polymeric networks using silsesquioxane building blocks.

In this report, we synthesized ordered coordination polymers using polyhedral oligomeric silsesquioxanes (POSS) as a building block. A POSS with eight carboxylic terminals was coordinated with copper ions at various temperatures, forming polymeric networks. This novel coordination polymer has a long-range ordered structure.

[Retrospective Risk Factor Analysis of Spinal Cord Ischemia after Thoracic Endovascular Aneurysm Repair].

BACKGROUND: Thoracic endovascular aneurysm repair carries a risk of spinal cord ischemia(SCI), similar to open repair. We conducted a retrospective study of the incidence and outcome of SCI after thoracic endovascular aneurysm repair (TEVAR). METHODS: From March 2007 to September 2012, 96 patients underwent TEVAR at Saitama Cardiovascular and Respiratory Center. A loss of lower extremity motor evoked potentials (MEP) or lower extremity strength was treated emergently to maintain a mean arterial blood pressure ≥ 70-80 mmHg. For the protection of spinal cord, combined use of naloxone and cerebrospinal fluid drainage (≤ 13 mmHg) was employed. RESULTS: 4 (4.2%) of the 96 patients had paraplegia. One had lower extremity strength loss after extubation and 3 developed delayed-onset paraparesis/paraplegia. Afterward 2 (50%) of them were dead. SCI patients were more likely to be elderly (78 ± 5.5 vs 70 ± 7.1 years; P = 0.0476). A trend toward an increase in SCI was noted in women (25.0% vs 2.2%; P = 0.0338), and in length of aortic coverage (25.9 ± 2.6 cm vs 16.2 ± 5.6 cm; P = 0.008). CONCLUSIONS: Early detection and intervention to improve spinal cord perfusion may benefit patients at risk for SCI.

A Novel Interaction between FLICE-Associated Huge Protein (FLASH) and E2A Regulates Cell Proliferation and Cellular Senescence via Tumor Necrosis Factor (TNF)-Alpha-p21WAF1/CIP1 Axis.

Dysregulation of the cell proliferation has been implicated in the pathophysiology of a number of diseases. Cellular senescence limits proliferation of cancer cells, preventing tumorigenesis and restricting tissue damage. However, the role of cellular senescence in proliferative nephritis has not been determined. The proliferative peak in experimental rat nephritis coincided with a peak in E2A expression in the glomeruli. Meanwhile, E12 (an E2A-encoded transcription factor) did not promote proliferation of Mesangial cells (MCs) by itself. We identified caspase-8-binding protein FLICE-associated huge protein (FLASH) as a novel E2A-binding partner by using a yeast two-hybrid screening. Knockdown of FLASH suppressed proliferation of MCs. This inhibitory effect was partially reversed by the knockdown of E2A. In addition, the knockdown of FLASH induced cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) expression, but did not affect p53 expression. Furthermore, overexpression of E12 and E47 induced p21, but not p53 in MCs, in the absence of FLASH. We also demonstrated that E2A and p21 expression at the peak of proliferation was followed by significant induction of FLASH in mesangial areas in rat proliferative glomerulonephritis. Moreover, we revealed that FLASH negatively regulates cellular senescence via the interaction with E12. We also demonstrated that FLASH is involved in the TNF-α-induced p21 expressions. These results suggest that the functional interaction of E2A and FLASH play an important role in cell proliferation and cellular senescence via regulation of p21 expression in experimental glomerulonephritis.

Effects of raloxifene on bone metabolism in postmenopausal women on chronic hemodialysis.

BACKGROUND: Postmenopausal women with end-stage renal failure are at an increased risk of fracture because of the effects of secondary hyperparathyroidism and postmenopausal osteoporosis. In the present study, we investigated the feasibility of using raloxifene to prevent fractures in postmenopausal women with end-stage renal failure on hemodialysis. METHODS: This study was conducted using a multicenter, single-arm, prospective design. Raloxifene was administered to postmenopausal women aged ≥50 years who were on maintenance hemodialysis and met any of the following criteria after a 24-week run-in period: an alkaline phosphatase level (bone formation marker) of ≥6.18 µkat/L (≥370 U/L), a bone-specific alkaline phosphatase (BAP; bone formation marker) level of ≥0.59 µkat/L (≥35.4 U/L), or a bone-derived tartrate-resistant acid phosphatase (TRACP-5b; bone resorption marker) level of ≥4.2 U/L. RESULTS: A total of 48 individuals were eligible for study inclusion. Of them, 30 individuals participated in this study. The BAP levels were significantly decreased at week 4, but returned to the baseline levels at week 24. Similarly, the TRACP-5b levels were significantly decreased at week 4, but returned to the baseline levels at week 24. The serum calcium value decreased consistently after the start of raloxifene therapy. The intact parathyroid hormone (iPTH) levels were likely increased at week 4. The ratio of BAP to iPTH levels and the ratio of TRACP-5b to iPTH levels both showed significant decreases over time. During the raloxifene therapy, no thrombosis or other drug-related adverse events developed. CONCLUSION: The study results indicated that raloxifene can transiently reduce the levels of bone metabolism markers and might be useful for preventing fractures in postmenopausal women with end-stage renal failure, although raloxifene use over the long term may not have adequate efficacy in the absence of appropriate concomitant active vitamin D therapy.

[Successful management of sigmoidectomy with sildenafil citrate in a patient with acute exacerbation of chronic thromboembolic pulmonary hypertension].

An 84-year-old woman with pulmonary hypertension (PH) secondary to chronic pulmonary thromboembolism suffered from continuous warfarin dependent bleeding from sigmoid colon cancer. Sigmoidectomy was scheduled to control continuous bleeding. Six hours after discontinuation of anticoagulant therapy for elective sigmoidectomy, the patient showed hypoxia, pulmonary thromboembolism and pulmonary hypertension with right ventricular systolic pressure (RVSP) of 81 mmHg. The operation was postponed and heparin was infused. Since two-day heparinization therapy did not improve PH, oral administration of sildenafil citrate 60 mg daily was initiated. Seven days after initiation of sildenafil administration, RVSP decreased to 49 mmHg without improvement of hypoxia. Sigmoidectomy was performed under general anesthesia. The patient showed severe hypotension managed with noradrenaline and dopamine infusion during and after surgery, resulting from interaction between sildenafil and vasodilators. The patient was discharged 36 days after the operation without complications.

Suppression of feline calicivirus replication using small interfering RNA targeted to its polymerase gene.

Feline calicivirus (FCV) is a pathogenic microorganism that causes upper respiratory diseases in cats. Recently, an FCV infection with a high mortality rate has been confirmed, and there is need to develop a treatment for cases of acute infection. We evaluated whether the replication of FCV could be prevented by RNA interference. For this study, we designed an siRNA targeted to the polymerase region of the strain FCV-B isolated from a cat that died after exhibiting neurological symptoms. Cells transfected with siR-pol dose-dependently suppressed the replication of FCV-B. siR-pol suppressed its replication by suppressing the target viral RNA.

HLA-class II and class I genotypes among Japanese children with Type 1A diabetes and their families.

OBJECTIVE: To determine the HLA-DRB1, DQB1, DPB1, A, C, and B genotypes among Japanese children with autoimmune type 1 diabetes. METHODS: Four hundred and thirty patients who were GADAb and/or IA-2Ab-positive (Type 1A) were recruited from 37 medical centers as part of a nationwide multicenter collaborative study. DNA samples from 83 siblings of the children with Type 1A diabetes and 149 parent-child trios were also analyzed. A case-control study and a transmission disequilibrium test (TDT) were then performed. RESULTS: The susceptible and protective DRB1 and DQB1 alleles and haplotypes were confirmed. DPB1 alleles unique to the Japanese population and those common to multiple ethnic groups were also present. A linkage disequilibrium (LD) analysis showed both susceptible and protective haplotypes. The TDT did not reveal any alleles that were transmitted preferentially from the mother or father to children with Type 1A. Homozygosity for DRB1-09:01-DQB1-03:03 and heterozygosity for DRB1-04:05-DQB1-04:01 and DRB1-08:02-DQB1-03:02 were associated with an extremely high risk of Type 1A. A comparison of children with Type 1A and their parents and siblings suggested a dose effect of susceptible DRB1-DQB1 haplotypes and an effect of protective alleles on immunological pathogenesis. DRB1-09:01 appeared to be strongly associated with an early onset in preschool children with Type 1A diabetes. CONCLUSIONS: This study demonstrated the characteristic association of HLA-class II and class I genes with Type 1A diabetes among Japanese children. A TDT did not reveal the genomic imprinting of HLA-class II and class I genes in Type 1A diabetes.

Detection and genotyping of canine coronavirus RNA in diarrheic dogs in Japan.

To clarify the prevalence of canine coronavirus (CCoV) infection in Japan, faecal samples from 109 dogs with diarrhoea were examined for CCoV RNA together with canine parvovirus type 2 (CPV-2) DNA. The detection rates of CCoV and CPV-2 for dogs aged less than 1 year were 66.3% and 43.8%, while those for dogs aged 1 year or older were 6.9% and 10.3%, respectively, which were significantly different (p<0.0001 and p=0.0003, respectively), indicating not CPV-2 but CCoV is an important diarrhoea-causing organism in juvenile dogs. Among the CCoV-positive dogs, 65.5% and 72.7% showed to be positive for CCoV types I and II, respectively, and simultaneous detection rate of both types was high at 40.0%. Furthermore, transmissible gastroenteritis virus (TGEV)-like CCoV RNA was detected from 8 dogs. These findings indicate that CCoV type I and TGEV-like CCoV are already circulating in Japan, though no reports have been presented to date.

Trophoblast glycoprotein: possible candidate mediating podocyte injuries in glomerulonephritis.

BACKGROUND: trophoblast glycoprotein (Tpbg), a 72-kDa transmembrane glycoprotein, is known to regulate the phenotypes of epithelial cells by modifying actin organization and cell motility. Recently, a microarray study showed that Tpbg is upregulated in Thy1 glomerulonephritis (Thy1 GN). We hypothesized that Tpbg regulates cytoskeletal rearrangement and modulates phenotypic alteration in podocytes under pathological conditions. METHODS: we examined Tpbg expression in Thy1 GN and Tpbg function in mouse podocytes. RESULTS: we demonstrated that Tpbg is upregulated in the injured podocytes of Thy1 GN. In vitro, immunofluorescence studies revealed that Tpbg colocalized with the focal adhesion protein, vinculin, in parallel with stress fiber formation. This colocalization was observed even when actin filaments were depolymerized with cytochalasin D. Tpbg localization at focal adhesions was induced by dominant-active RhoA and suppressed by the ROCK1 inhibitor Y-26732. In addition, transforming growth factor-ß increased Tpbg expression at focal adhesions concurrently with rearrangement of stress fibers. Stress fiber formation was suppressed in differentiated podocytes transfected with full-length Tpbg. Furthermore, knockdown of Tpbg using small interfering RNA decreased podocyte motility. CONCLUSION: our findings suggest a novel role of Tpbg in the phenotypic alteration of injured podocytes, and we accordingly propose a new mechanism of glomerular injury in glomerulonephritis.

Angio-embolization of renal artery pseudoaneurysm after renal biopsy: a case report.

Renal artery pseudoaneurysm is a rare clinical entity that has been reported after renal biopsy, percutaneous renal surgery, penetrating trauma, and rarely blunt renal trauma. We present the case of a 37-year-old man with ruptured renal artery pseudoaneurysm accompanied by massive gross hematuria, urinary clot retention, and bladder tamponade, which were the presenting signs seven hours after renal biopsy. Abdominal CT scan showed a large perinephric, intracapsular hematoma of left kidney. His angiogram revealed a left renal segmental artery pseudoaneurysm that measured 1 cm x 1 cm. He was successfully treated by selective embolization of the arterial branch supplying the pseudoaneurysm.

Urinary Smad1 is a novel marker to predict later onset of mesangial matrix expansion in diabetic nephropathy.

OBJECTIVE: We reported that Smad1 is a key transcriptional factor for mesangial matrix expansion in diabetic nephropathy. In this study, we examined whether urinary Smad1 in an early phase of diabetes can predict later development of glomerulosclerosis in diabetic nephropathy and how an angiotensin II type 1 receptor blocker (ARB) can modulate structural changes and urinary markers. RESEARCH DESIGN AND METHODS: Smad1 and albumin in the urine were examined 4 weeks after injection of streptozotocin in 48 rats or 6 weeks of diabetes in db/db mice. Their renal pathology was analyzed after 20 weeks in rats or 12 weeks in mice. Among 48 diabetic rats 7 rats were treated with olmesartan for 20 weeks. RESULTS: Urinary Smad1 of diabetic rats at 4 weeks was nicely correlated with mesangial matrix expansion at 24 weeks (r = 0.70, P < 0.001), while albuminuria showed a weaker association (r = 0.31, P = 0.043). Olmesartan treatment significantly ameliorated glomerulosclerosis and dramatically decreased urinary Smad1 (from 3.9 +/- 2.9 to 0.3 +/- 0.3 ng/mg creatinine, P < 0.05). In db/db mice, urinary Smad1 at 6 weeks was also significantly correlated with mesangial expansion at 18 weeks. In contrast, there was no change in urinary Smad1 in control diabetic rats or mice. CONCLUSIONS: The increase of urinary Smad1 in the early stages of diabetes is correlated with later development of glomerulosclerosis in two rodent models. These data indicate that urinary Smad1 could be a novel predictor for later onset of morphological changes and can be used to monitor the effect of ARBs in diabetic nephropathy.

Sensitivity of FCV to recombinant feline interferon (rFeIFN).

Feline calicivirus cause feline respiratory diseases, and inactivated and attenuated vaccines are available for its prevention. Moreover, the presence of vaccine breakdown strains (VBS) is problematic. In Japan, feline recombinant interferon (rFeIFN) has been used for its treatment. However the method of compare with each strains has not established. To examine the relationship between the breakdown vaccine strain and rFeIFN sensitivity, the sensitivity of 47 field isolates to rFeIFN was determined. The Log PDD(50) values were normally distributed within the range 1.1-3.7, with a mean value of 2.3 +/- 0.64. Since 68.3% of the PDD values fell in the range of the mean +/- standard deviation, the values in the range 1.7-2.9, the lower values, and the higher values were defined as representing moderate, low, and high sensitivity, respectively. Among the 15 vaccine breakdown strains, strain Fukuoka9 showed a low sensitivity, but strains ML89, T58, and N74 were highly sensitive, showing no association with vaccine breakdown. The amino acid sequence changes specific to the low rFeIFN-sensitive Fukuoka-9 strain were found, suggesting that these sites are involved in rFeIFN sensitivity.

FCV-VBS isolated from cats with typical symptoms caused VSD in experimental cats.

Commercially available vaccines have been used widely to prevent feline calicivirus infection (FCI). However, with their widespread use, field strains, which are weakly cross-reactive with the live-virus vaccine strain F9, have posed the problem of vaccine breakdown. Recently the existence of FCV--associated virulent systemic disease (VSD) has been published. But their molecular diversity, antigenic mutations and physicochemical property have not been sufficiently clarified. Thus, we experimentally gave the vaccine breakdown strain (VBS) H10 to cats that had been inoculated with an F9 live vaccine. After the administration of strain H10, vaccinated cats (1 through 4) had no respiratory symptoms, whereas the non-vaccinated cat 5 showed clinical symptoms such as a fever of over 40 degrees C, loss of vitality, decreased appetite, diarrhea, and nasal discharge after receiving strain H10, and died. Lethal FCV is rare, and may be a virulent systemic disease (VSD)--inducing strain. This is the initial report on VSD in Japan. It has been reported that symptoms of VSD were similar in vaccinated and nonvaccinated cats on experimental infection. However, no VSD-like symptoms developed, and the incidence of the disease varied depending on the presence or absence of vaccination, suggesting that there are two mechanisms of vaccine breakdown: one is associated with the vaccine immunity level, and the other is not. The characteristics of the VBS revealed were: (1) the duration of virus excretion was short when the originally carried antibody titer before virus challenge was high, (2) the excreted viral molecular species varied daily, not being limited to a specific species with time, and (3) the acquired physicochemical properties did not persist, and altered daily. FCV-VBS alters the molecular species and physicochemical properties daily due to the reduction of host immunity, which may lead to VSD.

Inhibition of MCP-1/CCR2 pathway ameliorates the development of diabetic nephropathy.

Monocyte chemoattractant protein (MCP-1) is an important mediator for macrophage recruitment in atherosclerosis and various glomerulonephritis. However, the role of MCP-1 and its receptor CCR2 in the progression of diabetic nephropathy remains unknown. Using a type 1 diabetic nephropathy model that shows noticeable glomerulosclerosis, we examined the role of MCP-1/CCR2 by propagermanium (Pro; CCR2 antagonist) treatment, and confirmed it by transfection of plasmids carrying the 7ND (a mutant of MCP-1) gene. We measured the mesangial matrix expansion, type IV collagen (Col4), transforming growth factor (TGF)-beta1 positive area, and macrophage infiltration in glomeruli after 12 weeks. Mesangial matrix expansion and macrophage infiltration were increased in diabetic mice and inhibited by Pro or 7ND-treatment. Increased glomerular expression of Col4 and TGF-beta1 in diabetic mice was also ameliorated. Thus blocking the MCP-1/CCR2 pathway ameliorated glomerulosclerosis, indicating that the MCP-1/CCR2 pathway plays a crucial role in the progression of diabetic nephropathy.

Evaluation of atrial and brain natriuretic polypeptides in association with angiotensin-converting enzyme gene polymorphism in Japanese non-diabetic hemodialysis patients.

OBJECTIVE: Insertion (I)/deletion (D) polymorphisms in the angiotensin-converting enzyme (ACE) gene have the potential to serve as a marker for an increased risk of cardiovascular events. Increased plasma levels of human atrial natriuretic polypeptide (ANP) and brain natriuretic polypeptide (BNP) are important indexes of cardiac function. The aim of this study was to examine possible relationships between I/D polymorphisms and the myocardial release of ANP and BNP in Japanese hemodialysis (HD) patients (n=131). MATERIAL AND METHODS: We studied 131 non-diabetic hemodialysis patients. The genotype of ACE gene was determined by polymerase chain reaction with a set of specific timers. ANP and BNP levels were measured before HD. RESULTS: The plasma levels of ANP and BNP were significantly lower in the DD genotype group compared to those in the II group. Corresponding levels in the ID genotype group were intermediate between those in the DD and II groups. ACE polymorphism was associated with neither ejection fraction nor left ventricular mass/height index (LVMI), as evidenced by echocardiographic findings (n=107). Plasma levels of ANP were significantly correlated with left atrial diameter (LAD) in patients as a whole, but this correlation was only observed in the II genotype group, and not in the DD or ID groups. Plasma levels of BNP were significantly correlated with LAD, left ventricular end systolic diameter and LVMI in patients as a whole, but these correlations were seen only in the II genotype group. CONCLUSION: The results suggest that the plasma levels of these natriuretic peptides should be evaluated on the basis of ACE polymorphism for assessing cardiac diseases due to volume overload in Japanese HD patients.

Characterization of cDNA and genomic sequences encoding a canine chemokine receptor, CXCR4 and its ligand CXCL12.

The interaction of chemokine receptor CXCR4 and its functional ligand CXCL12 plays a key role in bone marrow hematopoiesis, neuronal and cardiovascular development, and organization of the immune system. Despite the importance of the CXCL12-CXCR4 axis for regulating hematopoiesis, information on the canine CXCR4 and CXCL12 genes is insufficient. In this present study, we identified the canine counterparts of the CXCR4 and CXCL12 cDNAs and genes. The amino acid sequence encoding canine CXCR4 showed the structural characteristics of seven transmembrane domain G protein-coupled receptors and high homology with those of humans and other animals. Two isoforms, CXCL12 alpha and CXCL12 beta, were identified in dogs, as described in human and other animals. The gene structures for canine CXCR4 and CXCL12 were similar to those of other animals. The canine CXCL12 gene structure indicated that the transcripts of the isoforms arose from alternative mRNA splicing. A single nucleotide polymorphism (SNP) with synonymous substitution was observed in the exon of the canine CXCL12 gene. mRNAs encoding canine CXCR4 and CXCL12 were expressed widely and constitutively. Molecular homology and constitutive expression of CXCR4 and CXCL12 mRNAs in canine normal tissues suggests critical roles in hematopoiesis and trafficking of leukocytes, as shown in other animals.