Isolation of the most immature population of murine fetal thymocytes that includes progenitors capable of generating T, B, and myeloid cells.

Thymus cells of murine fetuses at day 12 of gestation are exclusively of the CD3-CD4-CD8-CD44+CD25- phenotype, which is known as a hallmark of the most immature subset of thymus cells. In the present study, we show that day 12 fetal thymus (FT) cells express Fc gamma RII/ III (FcR) at a broad range of levels on their surface. The FcR+ FT cells seem to represent T lineage cells, because a large majority of them express the T lineage specific transcription factors TCF-1 and GATA-3 as well as CD3 epsilon in the cytoplasm. Also shown is that the FcR- population contains progenitors capable of developing into not only T cells but also B and myeloid cells, whereas FcR+ progenitors are mostly committed to the T lineage. These findings indicate that thymic T lineage cells express FcR on their surface at the earliest stage of differentiation, and thus FcR is a useful marker in isolating the most immature population of murine FT cells.

Commitment of common T/Natural killer (NK) progenitors to unipotent T and NK progenitors in the murine fetal thymus revealed by a single progenitor assay.

We have established a new clonal assay system that can evenly support the development of T and natural killer (NK) cells. With this system, we show that all T cell progenitors in the earliest CD44(+)CD25(-)FcgammaRII/III(-) fetal thymus (FT) cell population retain NK potential, and that the NK lineage-committed progenitors (p-NK) also exist in this population. T cell lineage-committed progenitors (p-T), which are unable to generate NK cells, first appear at the CD44(+)CD25(-) FcgammaRII/III(+) stage in day 12 FT. The proportion of p-T markedly increases during the transition from the CD44(+)CD25(-) stage to the CD44(+)CD25(+) stage in day 14 FT. On the other hand, p-NK preferentially increase in number at the CD44(+)CD25(-) stage between days 12 and 14 of gestation. The production of p-NK continues up to the CD44(+)CD25(+) stage, but ceases before the rearrangement of T cell receptor beta chain genes. It was further shown that the CD44(+)CD25(-) CD122(+) population of day 14 FT exclusively contains p-NK. These results indicate that the earliest T cell progenitor migrating into the FT is T/NK bipotent, and strongly suggest that the bipotent progenitor continuously produces p-NK and p-T until the CD44(+)CD25(+) stage.

Involvement of transcription factors TCF-1 and GATA-3 in the initiation of the earliest step of T cell development in the thymus.

Flow cytometric and immunocytochemical analyses of murine fetal thymus (FT) cells with antibodies to various surface markers and transcription factors reveal that the synthesis of TCF-1 and GATA-3 protein begins simultaneously in a fraction of the most immature population of FT cells, which have the phenotype of CD4-CD8-CD44+CD25-. No TCF-1-producing cells is found in the fetal liver (FL). In CD44+CD25- FT cells, the production of TCF-1 is immediately followed by intracellular expression of CD3 epsilon. It is also found that the T cell development from FL, but not FT, progenitors in the FT organ culture system is severely inhibited by the addition of antisense oligonucleotides for either TCF-1 or GATA-3. These results strongly suggest that TCF-1 and GATA-3 play essential roles in the initiation of the earliest steps of T cell development in the thymus.

Micrometer-scale monolayer SnS growth by physical vapor deposition.

Recently, monolayer SnS, a two-dimensional group IV monochalcogenide, was grown on a mica substrate at the micrometer-size scale by the simple physical vapor deposition (PVD), resulting in the successful demonstration of its in-plane room temperature ferroelectricity. However, the reason behind the monolayer growth remains unclear because it had been considered that the SnS growth inevitably results in a multilayer thickness due to the strong interlayer interaction arising from lone pair electrons. Here, we investigate the PVD growth of monolayer SnS from two different feed powders, highly purified SnS and commercial phase-impure SnS. Contrary to expectations, it is suggested that the mica substrate surface is modified by sulfur evaporated from the Sn2S3 contaminant in the as-purchased powder and the lateral growth of monolayer SnS is facilitated due to the enhanced surface diffusion of SnS precursor molecules, unlike the growth from the highly purified powder. This insight provides a guide to identify further controllable growth conditions.

Interobserver agreement in endoscopic evaluation of reflux esophagitis using a modified Los Angeles classification incorporating grades N and M: a validation study in a cohort of Japanese endoscopists.

The Los Angeles classification system is the most widely employed criteria associated with the greatest interobserver agreement among endoscopists. In Japan, the Los Angeles classification system has been modified (modified LA system) to include minimal changes as a distinct grade of reflux esophagitis, rather than as auxiliary findings. This adds a further grading M defined as minimal changes to the mucosa, such as erythema and/or whitish turbidity. The modified LA system has come to be used widely in Japan. However, there have been few reports to date that have evaluated the interobserver agreement in diagnosis when using the modified LA classification system incorporating these minimal changes as an additional grade. A total of 100 endoscopists from university hospitals and community hospitals, as well as private practices in the Osaka-Kobe area participated in the study. A total of 30 video clips of 30-40 seconds duration, mostly showing the esophagocardiac junction, were created and shown to 100 endoscopists using a video projector. The participating endoscopists completed a questionnaire regarding their clinical experience and rated the reflux esophagitis as shown in the video clips using the modified LA classification system. Agreement was assessed employing kappa (kappa) statistics for multiple raters. The kappa-value for all 91 endoscopists was 0.094, with a standard error of 0.002, indicating poor interobserver agreement. The endoscopists showed the best agreement on diagnosing grade A esophagitis (0.167), and the poorest agreement when diagnosing grade M esophagitis (0.033). The kappa-values for the diagnoses of grades N, M, and A esophagitis on identical video pairs were 0.275-0.315, with a standard error of 0.083-0.091, indicating fair intraobserver reproducibility among the endoscopists. The study results consistently indicate poor agreement regarding diagnoses as well as fair reproducibility of these diagnoses by endoscopists using the modified LA classification system, regardless of age, type of practice, past endoscopic experience, or current workload. However, grade M reflux esophagitis may not necessarily be irrelevant, as it may suggest an early form of reflux disease or an entirely new form of reflux esophagitis. Further research is required to elucidate the pathophysiological basis of minimal change esophagitis.

Clinical characteristics of synchronous colorectal cancer are different according to tumour location.

AIMS: The purpose of this study was to investigate the clinical characteristics of synchronous cancer patients, with particular attention given to variations in tumour location. METHODS: A retrospective evaluation of 249 synchronous cancer cases out of 3061 consecutive colorectal cancer patients. RESULTS: Multivariate analysis of risk factors for synchronous cancer according to tumour location revealed that male gender was a significant risk for synchronous lesions in the left colon only (odds ratio=2.05, 95% confidence interval 1.34-3.13). Meanwhile, aging was a risk factor for synchronous cancer in the right colon only (odds ratio=1.05, 95% confidence interval 1.02-1.08), and in both sides of the colon (odds ratio=1.03, 95% confidence interval 1.01-1.05), but not in the left colon only (odds ratio=0.98, 95% confidence interval 0.97-1.00). In addition, patients with synchronous lesions in the right colon only tended to have adenomas in the right colon, while those with synchronous lesions in the left colon only tended to have adenomas in the left colon (each P value <0.05). CONCLUSION: The risk factors and status of concurrent adenomas of synchronous cancer cases varied according to tumour location, suggesting that the colonic site susceptible to neoplasia varies according to patient characteristics.

A case of combined laparoscopic reduction and open preperitoneal mesh repair for incarcerated small bowel in a retroperitoneal hernia between the external and internal iliac vessels.

We report a rare case of an incarcerated retroperitoneal hernia with or involving the small bowel through the orifice between the right external and internal iliac vessels. A 39-year-old woman was admitted to our hospital because of vomiting and abdominal pain. She had a history of right oophorocystectomy and appendectomy. Abdominal computed tomography revealed small bowel obstruction resulting from an incarcerated retroperitoneal hernia. The small bowel herniated into the retroperitoneal fossa through the orifice between the right external and internal iliac vessels. Laparoscopic reduction of the small bowel was performed, followed by ligation of the sac and placement of a mesh prosthesis through the preperitoneal approach, using a lower midline incision along the previous laparotomy scar. Her postoperative course was uneventful and no recurrence has been observed after surgery.

A multidisciplinary treatment strategy that includes high-dose chemotherapy for metastatic retinoblastoma without CNS involvement.

The prognosis of patients with metastatic retinoblastoma is poor with conventional chemotherapy and radiation. Since retinoblastoma is highly chemosensitive, dose-escalation of chemotherapeutic agents with stem cell support should be promising. We report our experience with high-dose chemotherapy (HDC) and autologous stem cell transplantation (SCT) in patients with metastatic retinoblastoma. Five patients with metastatic retinoblastoma underwent HDC with autologous SCT following conventional chemotherapy and local radiation therapy. Stem cells (bone marrow in four and peripheral blood stem cells in one) were collected after marrow involvement was cleared. Melphalan was a key drug in all patients, and was administered in combination with other agents such as cisplatin, cyclophosphamide, carboplatin or thiotepa. Three patients are currently alive disease-free at 113, 107 and 38 months, respectively, from the time of SCT. They had no central nervous system (CNS) involvement. The two patients who died of disease had CNS involvement. No long-term sequelae of HDC have been noted. Our treatment strategy using HDC appears to be effective for treating metastatic retinoblastoma without CNS involvement.

Effects of lithium ion on the inhibitory GTP-binding protein and its coupling response.

Addition of lithium ion to the inhibitory GTP-binding (Gi) protein resulted in a decrease of its ADP-ribosylation by islet-activating protein (pertussis toxin, IAP). The possibility that this decrease was due to dissociation of the Gi protein trimer was examined. Results showed that lithium ions had no appreciable effect on either the Gi protein trimer or its dissociation into its three subunits induced by Mg2+ and GTP gamma S. Next, the effect of lithium ions on Gi protein-mediated adenylate cyclase inhibition and alpha 2-adrenoceptor in human platelet membranes was examined. Lithium ion was found to impair adenylate cyclase inhibition of alpha 2-adrenoceptor stimulation of forskolin-stimulated enzyme activities. The monovalent ion also abolished guanine nucleotide modulation (GTP shift) of agonist binding, while it had no remarkable effects on antagonist binding in alpha 2-adrenoceptor of human platelet membranes. These results suggested that lithium ion caused functional change of the Gi protein without remarkable change of its dissociation, causing modulation in a coupling between alpha 2-adrenoceptor and Gi protein.

Evaluation of covered metallic stents in malignant biliary stenosis--prominent effectiveness in gallbladder carcinoma.

BACKGROUND/AIMS: The survival time of patients with unresectable malignant biliary stenosis and the patent period of metallic biliary stents are different in each disease. The efficacy of the covered metallic stent was analyzed according to the primary disease. METHODOLOGY: Seventy-three patients with bile duct carcinoma (12 cases), gallbladder carcinoma (22 cases), and pancreas carcinoma (39 cases) were retrospectively enrolled. Covered metallic stents were used in 42 patients and uncovered metallic stents in 31 patients. The patency of covered stents was compared with that of uncovered stents for each disease. RESULTS: The patent rate at 6 months after insertion was 80.6% (95% CI [72.6%, 88.6%]) for the covered stent, and 49.5% (95% CI [37.6%, 61.4%]) for the uncovered stent. The mean patent periods of the covered stent and the uncovered stent were 14.6 and 27.6 months for bile duct carcinoma (p=0.424), 12.7 and 3.0 months for gallbladder carcinoma (p=0.003), and 11.9 and 9.6 months for pancreas carcinoma (p=0.919), respectively. CONCLUSIONS: The covered metallic stent was the most effective in patients with gallbladder carcinoma.

High-dose chemotherapy for high-risk retinoblastoma: clinical course and outcome of 14 cases in the National Cancer Center, Japan.

The prognosis of high-risk retinoblastoma (RB) with extraocular disease, relapse, or invasion of the cut end of the optic nerve is extremely poor. Following the discontinuation of thiotepa production in Japan, BU- and melphalan (Mel)-based regimens have been used, followed by the standard treatment for neuroblastoma. This study retrospectively analyzed 14 high-risk RB patients who underwent high-dose chemotherapy (HDC) and hematopoietic SCT; 8 received a BU/Mel conditioning regimen and 6 received other regimens. The disease status at HDC was relapse in 8 patients and extraocular involvement in 5. All patients received peripheral blood stem cell infusion >1.5 × 10(6)/kg. Engraftment occurred within a median of 11 days (BU/Mel: 10-13, others: 9-13). Primary toxicities included mucositis (⩾grade 3) in 9 patients (4 with BU/Mel, 5 with others). Veno-occlusive disease (VOD) occurred in two 1-year-old patients in the BU/Mel group. There were no treatment-related deaths. Of 4 (2 with BU/Mel, 2 with others) patients with central nervous system (CNS) relapse after HDC, 3 died. In conclusion, the BU/Mel regimen may be feasible for high-risk RB under careful monitoring for VOD, particularly in younger patients. CNS relapse associated with a lethal prognosis occurred after all regimens; therefore, further evaluation of HDC efficacy for high-risk RB is required.

Human NELL-1 expressed in unilateral coronal synostosis.

Surgical correction of unilateral coronal synostosis offers a unique opportunity to examine the molecular differences between an abnormal and a normal cranial suture. We isolated and identified a cDNA fragment whose expression was up-regulated in the premature fusing and fused coronal sutures, as compared with normal coronal sutures. The nucleotide sequence of the full-length cDNA of this gene, human NELL-1, has approximately 61% homology with the chicken Nel gene. Both chicken Nel and human NELL-1 are comprised of six epidermal growth factor-like repeats. The human NELL-1 messages were localized primarily in the mesenchymal cells and osteoblasts at the osteogenic front, along the parasutural bone margins, and within the condensing mesenchymal cells of newly formed bone in sites of premature sutural fusion. Human multiorgan tissue mRNA blot showed that NELL-1 was specifically expressed in fetal brain but not in fetal kidney, liver, or lung. We also showed that Nell-1 was expressed in rat calvarial osteoprogenitor cells and was largely absent in rat tibiae and fibroblast cell cultures. In conclusion, our data suggest that the NELL-1 gene is preferentially expressed in cranial intramembranous bone and neural tissue (both of neural crest cell origin) and is up-regulated during unilateral premature closure of the coronal suture. The precise role of this gene is unknown.

Stepwise lineage restriction of progenitors in lympho-myelopoiesis.

It has long been controversial whether hematopoiesis progresses through ordered stages of determination as in embryonic development. This is due to the absence of a methodology capable of exactly determining the developmental potential of hematopoietic stem/progenitor cells. The multilineage progenitor (MLP) assay enabled us to discriminate among seven types of hematopoietic progenitors, which are multipotent progenitor p-MTB (capable of generating myeloid, T and B cells), bipotent progenitors p-MT, p-MB and p-TB, and unipotent progenitors p-M, p-T and p-B. Among these seven types, the p-TB type progenitor was found to be absent. These findings indicate that the process of lineage commitment proceeds through an ordered but not random process. By extending the area of investigation to include the erythroid lineage, more convincing evidence for the ordered process was obtained. Detailed and exact illustration of the process of hematopoiesis will provide an opportunity to revive hematopoiesis as one of the most fascinating targets of research in developmental biology.

Characterization of Norwalk virus GI specific monoclonal antibodies generated against Escherichia coli expressed capsid protein and the reactivity of two broadly reactive monoclonal antibodies generated against GII capsid towards GI recombinant fragments.

BACKGROUND: Norwalk virus causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. In a previous study, the capsid protein of recombinant Norwalk virus genogroup II was expressed in an E. coli system and monoclonal antibodies were generated against it. The analysis of the reactivity of those monoclonal antibodies suggested that the N-terminal domain might contain more antigenic epitopes than the C-terminal domain. In the same study, two broadly reactive monoclonal antibodies were observed to react with genogroup I recombinant protein. RESULTS: In the present study, we used the recombinant capsid protein of genogroup I and characterized the obtained 17 monoclonal antibodies by using 19 overlapping fragments. Sixteen monoclonal antibodies recognized sequential epitopes on three antigenic regions, and the only exceptional monoclonal antibody recognized a conformational epitope. As for the two broadly reactive monoclonal antibodies generated against genogroup II, we indicated that they recognized fragment 2 of genogroup I. Furthermore, genogroup I antigen from a patient's stool was detected by sandwich enzyme-linked immunosorbent assay using genogroup I specific monoclonal antibody and biotinated broadly reactive monoclonal antibody. CONCLUSION: The reactivity analysis of above monoclonal antibodies suggests that the N-terminal domain may contain more antigenic epitopes than the C-terminal domain as suggested in our previous study. The detection of genogroup I antigen from a patient's stool by our system suggested that the monoclonal antibodies generated against E. coli expressed capsid protein can be used to detect genogroup I antigens in clinical material.

Successful treatment of meningoencephalitis caused by methicillin-resistant Staphylococcus aureus with intrathecal vancomycin in an allogeneic peripheral blood stem cell transplant recipient.

Methicillin-resistant Staphylococcus aureus (MRSA) is a common infectious pathogen during stem cell trans-plantation. We report a case of meningoencephalitis with multiple abscess formation caused by MRSA, which occurred in a 4-year-old boy soon after allogeneic peripheral blood stem cell transplantation. We successfully cured the infection with a combination of intravenous and intrathecal vancomycin.

Seasonal changes in calcium homeostasis affect the incidence of postoperative tetany in patients with Graves' disease.

BACKGROUND: We have found that postoperative tetany occurs in patients with Graves' disease who have secondary hyperparathyroidism caused by a deficiency in calcium and vitamin D concomitant with transient hypoparathyroidism after surgery. There are seasonal variations in serum 25-hydroxyvitamin D (25[OH]D) concentrations. The purpose of this study was to investigate the effects of seasonal changes in calcium homeostasis on the incidence of postoperative tetany in patients with Graves' disease who undergo subtotal thyroidectomy. PATIENTS AND METHODS: A prospective study was carried out to investigate sequential changes in serum levels of intact parathyroid hormone (iPTH), calcium and other electrolytes, 25(OH)D, and 1,25-dihydroxyvitamin D (1,25[OH]2D) in female patients with Graves' disease who underwent subtotal thyroidectomy during the summer (n = 89) and during the winter (n = 89). RESULTS: The serum levels of calcium, magnesium, and 25(OH)D were significantly higher, but iPTH levels and 1,25(OH)2D levels were lower in summer than in winter. The percentage of vitamin D deficiency (25(OH)D < 25 nmol/L) was 23% in summer and 62% in winter (P < .001). iPTH was below the detection limit on the first postoperative day in 15 patients (13.8%) in summer and in 13 patients (11.4%) in winter. In summery, tetany developed in only 4 of 15 patients and in one patient whose iPTH level was below normal (incidence of tetany, 5.6%). In winter, however, tetany developed in 6 of 13 patients and in 4 patients whose iPTH level was below normal (incidence of tetany, 11.2%). CONCLUSIONS: Patients with Graves' disease are more susceptible to calcium and vitamin D deficiency during the winter than during the summer, resulting in the tendency toward a higher incidence of postoperative tetany in winter.

In vitro inhibitory effects of J-113397 on nociceptin/orphanin FQ-stimulated.

J-113397 (1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one) is a recently developed antagonist of the opioid receptor-like 1 (ORL1) receptor. We compared the in vitro functional profile J-113397 on [35S]guanosine 5'-O-(gamma-thio)triphosphate (GTPgammaS) binding to mouse brain with that of [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 and naloxone benzoylhydrazone (NalBzoH). J-113397 antagonized nociceptin/orphanin FQ-stimulated [35S]GTPgammaS binding to mouse brain with an IC50 value of 7.6 nM, but had no effect on basal [35S]GTPgammaS binding by itself. [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 partially antagonized nociceptin/orphanin FQ-stimulated [35S]GTPgammaS binding but showed agonistic activity on ORL1 by itself. NalBzoH showed antagonistic activity on ORL1 receptor but had significant agonistic activity on other opioid receptors at lower doses. Schild plot analysis demonstrated competitive antagonism of J-113397 on ORL1 receptor in mouse brain. A [35S]GTPgammaS binding study using ORL1 receptor-deficient mice confirmed the selective antagonism of J-113397 on ORL1 receptor. These data indicate that J-113397 is the most potent and selective antagonist of ORL1 receptor in mouse brain that has yet been reported, and therefore will be a useful tool for characterization of ORL1 receptors in the brain.

Papillary thyroid carcinoma: modified radical neck dissection improves prognosis.

OBJECTIVE: To ascertain whether modified radical neck dissection offers a survival advantage for some subsets of patients with papillary cancer of the thyroid. DESIGN: A retrospective cohort study of 2966 patients curatively treated at the Noguchi Thyroid Clinic and Hospital Foundation, Oita, Japan, between 1946 and 1991. SETTING: A center for the treatment of thyroid disease, where about 1400 thyroid operations are performed per year. PATIENTS: Between 1946 and 1991, patients with papillary cancer whose primary tumor was 1 cm or larger and who were curatively treated were studied. Of the 2859 patients, 72.1% underwent modified radical neck dissection, 8.5% underwent partial node excision, and 19.4% underwent no node excision. RESULTS: A univariate analysis revealed a subset of patients who benefited from modified radical neck dissection. A multivariate analysis revealed that sex (P<.001), age at the time of the operation (P<.001), size of the primary tumor (P<.001), extrathyroidal invasion (P<.001), and the presence of nodal metastasis (P<.01) are significant risk factors. CONCLUSION: Patients with nodal metastasis, patients in whom the primary tumor invades beyond the thyroid capsule, and women older than 60 years can benefit from modified radical neck dissection.

Ultrasonographic characteristics of thyroid nodules: prediction of malignancy.

BACKGROUND: High-resolution real-time ultrasonography (US) can detect characteristics of thyroid nodules, but the US differentiation between malignant nodules and benign nodules is not well described. HYPOTHESIS: Ultrasonography is useful for predicting malignancy of thyroid nodules. DESIGN: A retrospective study of 329 thyroid nodules (> or =5 mm) in 309 patients comparing US characteristics and pathological results. SETTING: A center for the treatment of thyroid diseases where about 1400 thyroid operations are performed per year. PATIENTS: Between January 1 and June 30, 1999, 309 patients were examined by US before thyroidectomy. MAIN OUTCOME MEASURE: The US characteristics to predict malignancy for both follicular and nonfollicular neoplasms by means of multiple logistic regression analysis. RESULTS: The sensitivity of preoperative US diagnosis was 86.5% for nonfollicular neoplasms and 18.2% for follicular neoplasms. The specificity was 92.3% and 88.7%, respectively. According to multiple logistic regression analysis, margin, shape, echo structure, echogenicity, and calcification were reliable indication of malignancy in nonfollicular neoplasms. According to a receiver operating characteristic curve constructed from this multiple logistic regression analysis, the best point not to overlook malignancy is the point at which sensitivity is 94% and specificity is 87%. The probability of malignancy at this point is greater than 0.2. For follicular neoplasms, ultrasonographic diagnosis was unreliable, even when multiple logistic regression analysis was applied. CONCLUSION: We can predict malignancy of nonfollicular neoplasms of the thyroid by using multiple logistic regression analysis based on only 5 features: margin, shape, echo structure, echogenicity, and calcification.