Functional Analysis of PTH1R Variants Found in Primary Failure of Eruption.

Although many variants of the parathyroid hormone 1 receptor (PTH1R) gene are known to be associated with primary failure of eruption (PFE), the mechanisms underlying the link remains poorly understood. We here performed functional analyses of PTH1R variants reported in PFE patients-namely, 356C>T (P119L), 395C>T (P132L), 439C>T (R147C), and 1148G>A (R383Q)-using HeLa cells with a lentiviral vector-mediated genetic modification. Two particular variants, P119L and P132L, had severe reduction in a level of N-linked glycosylation when compared with wild-type PTH1R, whereas the other 2 showed modest alteration. PTH1R having P119L or P132L showed marked decrease in the affinity to PTH1-34, which likely led to severely impaired cAMP accumulation upon stimulation in cells expressing these mutants, highlighting the importance of these 2 amino acid residues for ligand-mediated proper functioning of PTH1R. To further gain insights into PTH1R functions, we established the induced pluripotent stem cell (iPSC) lines from a patient with PFE and the heterozygous P132L mutation. When differentiated into osteoblastic-lineage cells, PFE-iPSCs showed no abnormality in mineralization. The mRNA expression of RUNX2, SP7, and BGLAP, the osteoblastic differentiation-related genes, and that of PTH1R were augmented in both PFE-iPSC-derived cells and control iPSC-derived cells in the presence of bone morphogenetic protein 2. Also, active vitamin D3 induced the expression of RANKL, a major key factor for osteoclastogenesis, equally in osteoblastic cells derived from control and PFE-iPSCs. In sharp contrast, exposure to PTH1-34 resulted in no induction of RANKL mRNA expression in the cells expressing P132L variant PTH1R, consistent with the idea that a type of heterozygous PTH1R gene mutation would spoil PTH-dependent response in osteoblasts. Collectively, this study demonstrates a link between PFE-associated genetic alteration and causative functional impairment of PTH1R, as well as a utility of iPSC-based disease modeling for future elucidation of pathogenesis in genetic disorders, including PFE.

Long-term Survival in a Kidney Transplantation Patient With Progressive Multifocal Leukoencephalopathy: A Case Report.

Post-kidney transplantation progressive multifocal leukoencephalopathy (PML) is a rare disease on which there are very few published reports on record. PML is a demyelinating disease caused by a destructive infection of the oligodendrocytes by the JC polyomavirus. No effective therapeutic protocol has been established other than measures to revive the immune function by reducing or discontinuing the administration of immunosuppressive agents. Most cases are progressive and show a poor prognosis. We herein report a case in which renal function has been maintained for 2 years following the onset of PML, which was initially diagnosed 3 years after kidney transplantation.

Detection of the MYD88 mutation by the combination of the allele-specific PCR and quenching probe methods.

INTRODUCTION: The MYD88 missense mutation c.794T>C, p.Leu265Pro, is found in patients with Waldenstörm's macroglobulinemia and lymphoma. Direct sequencing, allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and high-resolution melting analysis (HRM) are currently used to detect the mutation; however, they are either time-consuming or have low detection sensitivity. Here, we developed a novel highly sensitive and rapid detection method based on the quenching probe (QP) technique and AS-PCR. METHOD: A lymphoma cell line heterozygous for the MYD88 mutation, two wild-type cell lines, and two samples from Waldenstörm's macroglobulinemia patients were analyzed by AS-PCR, PCR-RFLP, HRM, and QP, and their detection sensitivity was examined using the mixtures of the mutant and wild-type DNA. RESULTS: For mutation-carrying heterozygous samples, the QP method produced W-shaped melting profiles presenting curves derived from the wild-type and mutant alleles. The QP analysis was performed in 2 h and demonstrated the detection limit of 5%, which was similar to that of the other methods. However, the combination of AS-PCR and QP (AS-QP) improved the sensitivity to 0.62% of the mutant allele. CONCLUSION: The AS-QP analysis is rapid and minimally improves detection sensitivity compared to the AS-PCR.

Reprogramming human B cells into induced pluripotent stem cells and its enhancement by C/EBPα.

B cells have been shown to be refractory to reprogramming and B-cell-derived induced pluripotent stem cells (iPSC) have only been generated from murine B cells engineered to carry doxycycline-inducible Oct4, Sox2, Klf4 and Myc (OSKM) cassette in every tissue and from EBV/SV40LT-immortalized lymphoblastoid cell lines. Here, we show for the first time that freshly isolated non-cultured human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20+ B cells can be reprogrammed to iPSCs carrying complete VDJH immunoglobulin (Ig) gene monoclonal rearrangements using non-integrative tetracistronic, but not monocistronic, OSKM-expressing Sendai Virus. Co-expression of C/EBPα with OSKM facilitates iPSC generation from both CB- and PB-derived B cells. We also demonstrate that myeloid cells are much easier to reprogram than B and T lymphocytes. Differentiation potential back into the cell type of their origin of B-cell-, T-cell-, myeloid- and fibroblast-iPSCs is not skewed, suggesting that their differentiation does not seem influenced by 'epigenetic memory'. Our data reflect the actual cell-autonomous reprogramming capacity of human primary B cells because biased reprogramming was avoided by using freshly isolated primary cells, not exposed to cytokine cocktails favoring proliferation, differentiation or survival. The ability to reprogram CB/PB-derived primary human B cells offers an unprecedented opportunity for studying developmental B lymphopoiesis and modeling B-cell malignancies.

Appendiceal mucocele: sonographic findings.

BACKGROUND: Appendiceal mucocele (AM) is a relatively rare disease, and its sonograms (US) have not been sufficiently analyzed. METHODS: We studied the US findings of five patients with AM, with special attention to AM size, shape, internal echoes, and the mode of back echoes. RESULTS: All five cases showed an elongated mass in the lower right abdomen. Internal echoes were present in all cases and M-mode US confirmed the movement of those echoes. The echogenecity of the lesion changed according to the frequency of the transducer used. Only one case showed posterior echo enhancement, and no case showed lateral shadowing. CONCLUSION: AM appears as an elongated echo-poor mass without posterior echo enhancement. The cyst wall is less distinct than what one would expect for a cyst. When encountering such a mass in the lower right abdomen, one should strongly suspect an AM. In such cases, appropriate diagnostic and therapeutic strategies are especially necessary to prevent rupture that results in development of pseudomyxoma peritonei.

Case of ulcerative colitis associated with oesophageal ulcer.

A case of ulcerative colitis complicated by oesophageal ulcers is reported. A woman was admitted to our hospital because of exacerbations of ulcerative colitis both in 1992 (aged 15 years) and 1995 (aged 18 years). When she was admitted in 1995 she complained of bloody diarrhoea, sore throat and pain on swallowing. Oesophagogastro-duodenoscopy revealed oesophageal ulcers. Oesophageal pH monitoring (24-h) showed no evidence of gastro-oesophageal reflux disease. After the patient was treated she with oral prednisolone showed considerable improvement clinically and endoscopically. Initial dosage was 60 mg/day, and 1 week later, the dosage was gradually dropped since the patient responded favourably. The improvement of the oesophageal lesions coincided with the remission of ulcerative colitis. The oesophageal ulcers are, therefore, thought to be an extracolonic manifestation of ulcerative colitis.

A new mitochondrial DNA mutation at 14577 T/C is probably a major pathogenic mutation for maternally inherited type 2 diabetes.

From a family of 16 diabetic patients with typical maternal inheritance, we investigated a 69-year-old woman with type 2 diabetes. The proband showed no major deletions in the mitochondrial DNA (mtDNA). Direct sequencing revealed 7 missense and 5 ribosomal RNA homoplasmic nucleotide substitutions when compared with the Cambridge Sequence and its recent revision. When compared with the control cybrid cells, the proband cybrid cells showed 6 nucleotide substitutions. Among these, 14577 T/C, which turned out to be 98.9% heteroplasmic, is a new missense substitution in the NADH dehydrogenase 6 gene. We also observed 2 other patients with 14577 T/C substitution from another group of 252 unrelated diabetic patients, whereas no individual from a group of 529 control subjects had 14577 T/C substitution. Furthermore, these 6 substitutions were in linkage disequilibrium. Mitochondrial respiratory chain complex I activity and O2 consumption rates of the proband cybrid cells, which were obtained by the fusion of mtDNA-deleted (rho0) HeLa cells and mtDNA from the proband, showed 64.5 and 61.5% reductions, respectively, compared with control cybrid cells. The present study strongly indicates that the new mtDNA mutation at 14577 T/C is probably a major pathogenic mutation for type 2 diabetes in this family.

A novel cell adhesion inhibitor, K-7174, reduces the endothelial VCAM-1 induction by inflammatory cytokines, acting through the regulation of GATA.

A novel inhibitor for the adhesion of monocytes to cytokine-stimulated endothelial cells, K-7174, was selected by an assay system using the cultured human monocytic cells and human endothelial cells. K-7174 inhibited the expression of vascular cell adhesion molecule-1 (VCAM-1) induced by either tumor necrosis factor alpha or interleukin-1beta, without affecting the induction of intercellular adhesion molecule-1 or E-selectin. K-7174 had no effect on the stability of VCAM-1 mRNA. Electrophoretic mobility shift assay revealed that its inhibitory effect on VCAM-1 induction was mediated by an effect on the binding to the GATA motifs in the VCAM-1 gene promoter region. K-7174 did not influence the binding to any of the following binding motifs: octamer binding protein, AP-1, SP-1, ets, NFkappaB, or interferon regulatory factor. These results suggest that the regulation of GATA binding may become a new target for anti-inflammatory drug development, acting through a mechanism independent from NFkappaB activity.

Endoscopic urease sensor system for detecting Helicobacter pylori on gastric mucosa.

BACKGROUND: It is now accepted that the curing of Helicobacter pylori infection will result in healing of chronic active gastritis and will change the natural history of gastroduodenal ulcer disease. Both endoscopic observation and evaluation of H. pylori status of the stomach are necessary for diagnosis and treatment of such patients. We carried out a clinical evaluation of an endoscopic tube type urease sensor system for the detection of H. pylori on the gastric mucosa. The differential output of two pH-sensitive field effect transistors at the tip of the endoscopic tube reflects the pH change in a urea solution depending on the existence of urease. METHODS: In vitro experiments and clinical evaluation of the system were performed. Fifty-one patients who were suspected to have a gastroduodenal disorder were examined for H. pylori infection with this system, using the combination of histologic and bacteriologic examinations and rapid urease test as the references. RESULTS: Clinical sensitivity and specificity of this system were 26 of 28 (92.9%) and 22 of 23 (95.7%), respectively. A measurement at 1 site is completed in about 1 minute. Repetition of the procedure provides multi-site measurements. CONCLUSIONS: The present system makes possible quick on-site detection of H. pylori under endoscopic observation, with satisfactory sensitivity and specificity.

Transient isolated thyrotropin deficiency associated with cavernous sinus syndrome.

The cause and course of isolated thyrotropin (TSH) deficiency are not well understood. We report a 65-year-old man with a transient, probable isolated TSH deficiency associated with cavernous sinus syndrome secondary to tympanitis. On his admission, serum TSH and triiodothyronine levels were very low. No TSH response to thyrotropin releasing hormone (TRH) was observed. However, 6 years later, TSH response to TRH was restored. The present case showed that inflammation in the cavernous sinus could be one of the causes of TSH deficiency. Further, it demonstrated that TSH deficiency is not always permanent and the reevaluation of pituitary function is necessary.

[(Neurological CPC-59). A 65-year-old man with a history of gastric cancer who presented progressive loss of vision, memory loss and consciousness disturbance].

We report a 65-year-old man with progressive loss of vision and consciousness disturbance. The patient was well until his age of 63 when he was found to have a gastric cancer. He was treated by the tumor resection and chemotherapy; he was apparently well, but hepatic metastases were found in the next year (1996). In June, 1996, he noted an onset of blurred vision more on the left. He was admitted to the ophthalmology service of our hospital on July 14, 1996. His vision was 0.8 on the right and 0.15 on the left. He was treated with oral prednisolone with slight improvement. He was also found to have IgM kappa-type monoclonal gammopathy; Bence-Jones protein was positive and a bone marrow aspiration revealed that approximately 10% of bone marrow cells were atypical plasma cells. His vision had progressively got worse and he became blind by the end of October 1996. A chest X-ray and cranial CT scan revealed multiple abnormal nodular densities. In the middle of November 1996, he became confused, disoriented and agitated. His mental symptoms had progressively became worse, and a neurologic consultation was asked on December 10, 1996. Neurologic examination revealed that he was somnolent with decreased attention to his surroundings. He showed marked disorientation and memory loss. Higher cerebral functions appeared intact. He was able to recognize only light and dark. Pupils were moderately dilated with very sluggish light reflex remained. Vertical gaze was moderately restricted and horizontal nystagmus was noted upon left and right lateral gaze. The remaining of the neurologic examination were unremarkable. General physical examination revealed hepatosplenomegaly; the liver was palpable by 3 cm below the right costal margin. Laboratory examination revealed anemia (Hb10.1 g/dl) and thrombocytopenia (43,000/microliter). A cranial CT scan and MRI revealed a mass lesion in involving the chiasmatic and bilateral hypothalamic areas. The tumor showed intense homogeneous enhancement after Gd-DTPA infusion. The patient was treated with dexamethasone and radiation. After 9 Gray radiation, he showed deterioration in the sensorium; a cranial CT scan revealed a hydrocephalus of the right ventricle with the midline shift towards left. The radiation was discontinued. The subsequent clinical course was complicated by aspiration pneumonia and thrombocytopenia. He expired on January 4, 1997. The patient was discussed in a neurological CPC and the chief discussant arrived at the conclusion that the patient had systemic malignant lymphoma with metastasis to the brain judging from the characteristics of MRI and CT findings. Opinions were divided between malignant lymphoma and metastatic brain tumor. Post-mortem examination revealed plasmacytoid lymphocytic infiltration in the bone marrow. Immunologically, these cells were positive for IgM and kappa-type light chain. These plasmacytic infiltrations were seen in the lungs and lymph nodes. These findings were consistent with the diagnosis of Waldenström's macroglobulinemia. In the liver metastatic cancer tissues were seen; microscopic pictures were essentially similar to those of resected gastric cancer. No local recidive was noted in the stomach. In the central nervous system, a necrotic tissue was involving the hypothalamic area bilaterally; no clear neoplastic cells were found, however, lymphocytic and plasmacytic infiltrations were seen in the perivascular space. In the optic nerves, loss of myelin and axons were seen. These findings most likely mass formation from macroglobulinemia which underwent necrotic change after radiation. Mass formation in the brain is rare for Waldenström's macroglobulinemia, although it has been reported. The relation between gastric cancer and macroglobulinemia in this patient is unclear.

Epidemiological approach to the prevention of imported infectious diseases in the age of globalization.

As globalization progresses, over 10% of Japanese citizens now travel abroad. With this, there are many cases in which people catch a variety of infectious and contagious diseases abroad. In addition, new infectious diseases also have been emerging in the world. Therefore, protection of travelers' health and prevention of the import of infectious diseases are of ever increasing importance. From this point of view, at the Narita Airport Quarantine Station, a Health Information Corner for travelers going abroad was established in 1992, along with Health Consultation Rooms for arriving passengers. In order to overcome imported infectious diseases, the promotion of domestic and international cooperation among the health and medical organizations concerned is required for primary prevention, and early detection or treatment of the diseases.

Heparin-induced thrombocytopenia in hemodialysis patients.

This study was conducted to determine the incidence of heparin-induced thrombocytopenia (HIT), an important complication in heparin therapy, in 154 hemodialysis patients, with characterization of the subtypes using an enzyme-linked immunosorbent assay. The etiology of the immunologic type of HIT is suggested to involve the binding of a specific antibody for platelet factor 4-heparin complex to platelets and their consequent activation. The 154 consecutive patients were newly treated with hemodialysis due to chronic or acute renal failure between January 1993 and July 1995. Heparin-induced thrombocytopenia was suspected in six patients (3.9%), and its presence was confirmed by platelet aggregation testing. Five of the patients with HIT had anti-platelet factor 4-heparin complex antibody detected by the enzyme-linked immunosorbent assay and were diagnosed with immunologic HIT. The patient who did not have the antibody was thought to have another type of HIT.

A case of glycogen storage disease type III (glycogen debranching enzyme deficiency) with liver cirrhosis and hypertrophic cardiomyopathy.

We present a 26-year-old woman with glycogen storage disease type III (debranching enzyme deficiency) complicated with liver cirrhosis and hypertrophic cardiomyopathy. Glycogen debranching enzyme has two catalytic sites, oligo-1,4,-1,4- glucantransferase (EC 2.4.1.25) and amylo-1,6-glucosidase (EC 3.2.1.33). Variability in the clinical phenotype could be a function of the involvement of one or other catalytic site, or differences in tissue expression of the defective enzyme, or both. We hypothesize that some subtypes of glycogen storage disease (GSD) type III may cause liver cirrhosis as seen in GSD type IV due to the accumulation of glycogen of abnormal structure.

[Studies on Vibrio cholerae non-O1 isolated from diarrheal patients arrived from overseas].

Between the period January 1991 to June 1993, there were 23,976,238 travellers who arrived from overseas to Narita Airport, of which 20,501 stool specimens were collected from diarrheal patients for bacteriological examination, and infectious agents were detected from 2,751 cases (13.4%) including 250 cases (1.2%) of Vibrio cholerae non-O1. Countries suspected of infection of these patients were Thailand, the most in number, and followed by Indonesia, India and so on these mostly distributed in South-east or South Asia. About fifty percent of the patients were associated with abdominal pain and some with vomiting or fever. Diarrhea was mostly mild except in 16 patients who had severe diarrhea of more than ten times a day. 237 of the 250 isolated V. cholerae non-O1 strains were classified into 48 serogroups. There were 2 rough strains and 11 other strains which were out of the confirmed serogroups. Positive rate of these strains to haemolysin, cholera toxin (CT) and NAG-ST tests were 87.2, 0.4 and 0.8% respectively.

Desensitization to somatostatin analogue (Octreotide) observed in a patient with acromegaly.

We encountered a 33-year-old female patient with a pituitary growth hormone (GH)-secreting macroadenoma. The patient was treated with somatostatin analogue (Octreotide) in combination with bromocriptine for 2 months before a transsphenoidal adenomectomy was carried out. Octreotide (300-800 micrograms/day) in combination with bromocriptine was effective in reducing the size of the adenoma by 36%, but produced only a marginal decrease in serum GH. After the operation, bromocriptine alone (15 mg/day) did not lower the level of GH which was produced by residual adenoma tissue. When octreotide (200 micrograms/day) was resumed along with the bromocriptine one year after the operation, it effectively lowered serum GH for 6 months. Thereafter, octreotide therapy became ineffective with a concomitant rise in serum GH and somatomedin C, which was not accompanied by an increase in tumor size. This was a rare case of acromegaly that showed desensitization to octreotide after long-term treatment.

Variant forms of glucokinase gene in Japanese patients with late-onset type 2 diabetes.

We have applied the technique of single-strand conformation polymorphism analysis to detect mutations of the glucokinase gene in 50 Japanese patients with late-onset type 2 diabetes and in 50 normal Japanese subjects. Out of the 50 patients with late-onset type 2 diabetes, we observed three kinds of variant patterns: one in exon 1b, one in exon 4, and one in exon 5. The incidence of these patterns was one in exon 1b, two in exon 4 and one in exon 5. Direct sequencing of exon 1b and exon 5 revealed mutations in intron areas at the 12th nucleotide downstream from the 5' splice points in two cases. Direct sequencing of exon 4 revealed a heterozygous silent mutation, CCC[Pro]-->CCG[Pro] at codon 145. In contrast, 50 normal Japanese subjects showed no variant patterns in any exons. Our results showed that although 8% (4 out of 50) of Japanese patients with late-onset type 2 diabetes have variant forms of the glucokinase gene, none is expected to cause apparent qualitative changes in glucokinase. We think that the frequency of mutations of the glucokinase gene which could cause qualitative change is very low in Japanese patients with late-onset type 2 diabetes.

The effect of prostaglandin E1.alpha CD on vibratory threshold determined with the SMV-5 vibrometer in patients with diabetic neuropathy.

We studied the effect of prostaglandin E1.alpha CD (PGE1) on diabetic peripheral neuropathy by evaluating subjective symptoms and vibration sensation using a new vibrometer (SMV-5). Patients with diabetic neuropathy (n = 38) were divided into three groups; group A received no drugs (control), group B was treated with 1500 micrograms/day of oral methyl vitamin B12 (VB12) for four weeks, and group C received 1.2 micrograms/kg/day PGE1 intravenously for four weeks. There was a close relationship between symptom scores and vibratory threshold (VT). The effect of PGE1 on subjective symptoms and VT were compared with those in groups A and B. Patients who received PGE1 showed a significant improvement rate in pain and hypesthesia compared to patients in groups A and B, and in numbness compared to group A. During the study period, there was no significant change in VT in groups A and B, whereas VT was significantly improved at styloid process (P < 0.05) and at medial malleolus (P < 0.001) in group C. Our results confirmed that PGE1 significantly improved both subjective symptoms and VT, indicating that PGE1 therapy may be useful in diabetic neuropathy.