Evaluation of an amplicon-based custom gene panel for the diagnosis of hereditary tumors.

Genetic testing based on next-generation sequencing (NGS) analysis has recently been used to diagnose hereditary diseases. In this study, we explored the usefulness of our custom amplicon panel that targeted 23 genes related to hereditary tumors given in the American College of Medical Genetics and Genomics recommendations. We applied our custom NGS panel to samples from 12 patients previously diagnosed by Sanger sequencing as having the diseases or diagnosed clinically by meeting the diagnostic criteria in this study. Our gene panel not only successfully identified all variants detected by Sanger sequencing but also identified previously unrecognized variants that resulted in confirmation of the disease, or even in the revision of the diagnosis. For instance, a patient identified with an SDHD gene mutation actually had von Hippel-Lindau (VHL) syndrome, as determined by the presence of a pathogenic VHL gene variant. We also identified false-positive results that were generated by amplification of genome regions that are not intended to be investigated. In conclusion, NGS-based amplicon sequencing is a highly effective method to detect germline variants, as long as they are also carefully reviewed by manual inspection.

Dissociation dynamics of ethylene molecules on a Ni cluster using ab initio molecular dynamics simulations.

The atomistic mechanism of dissociative adsorption of ethylene molecules on a Ni cluster is investigated by ab initio molecular-dynamics simulations. The activation free energy to dehydrogenate an ethylene molecule on the Ni cluster and the corresponding reaction rate is estimated. A remarkable finding is that the adsorption energy of ethylene molecules on the Ni cluster is considerably larger than the activation free energy, which explains why the actual reaction rate is faster than the value estimated based on only the activation free energy. It is also found from the dynamic simulations that hydrogen molecules and an ethane molecule are formed from the dissociated hydrogen atoms, whereas some exist as single atoms on the surface or in the interior of the Ni cluster. On the other hand, the dissociation of the C-C bonds of ethylene molecules is not observed. On the basis of these simulation results, the nature of the initial stage of carbon nanotube growth is discussed.

Amplification of plant genomic DNA by Phi29 DNA polymerase for use in physical mapping of the hypermethylated genomic region.

Plant genomes contain a heavily methylated region in which cytosines are methylated in both the symmetrical and asymmetrical sequences. The physical mapping of such a hypermethylated region is difficult because many restriction enzymes are sensitive to methylated cytosine residues in their recognition sites. The Phi29 DNA polymerase provides an efficient and representative amplification of the genomic DNA that is methylation-free. Using this amplified genomic DNA, we were able to show that a heavily methylated genomic DNA region becomes amenable to physical mapping with any restriction enzymes. This protocol will be especially useful for analysis of the heavily methylated region of plant genomes.

[Mitral valve stenosis due to primary cardiac granulocytic sarcoma relapsing 8 years after complete remission: a case report].

A 28-year-old man was admitted because of dyspnea on effort. His tricuspid valve had been affected by granulocytic sarcoma and manifested tricuspid valve stenosis 8 years previously. After chemotherapy and radiation therapy, the tumor had disappeared and the tricuspid valve stenosis was relieved. Echocardiography showed that the posterior leaflet of the mitral valve was affected by the tumor, and Doppler ultrasonography revealed mild mitral valve stenosis. Biopsy of the anterior chest wall detected granulocytic sarcoma. Chemotherapy was started. The tumor size was reduced and the mitral valve stenosis became slight. Primary cardiac granulocytic sarcoma is very rare and stenosis of the atrioventricular valve by relapse of this tumor after complete remission is extremely unusual.

Size of the population of CD4+ natural killer T cells in the liver is maintained without supply by the thymus during adult life.

Given that there are few natural killer T (NKT) cells in the liver of athymic nude mice and in neonatally thymectomized mice, it is still controversial whether all NKT cells existing in the liver are supplied by the thymus or if some such cells develop in the liver. To determine whether or not NKT cells are consistently supplied from the thymus during adult life, thymectomy was conducted in mice at the age of 8 weeks. Interestingly, the proportion and number of CD4+ NKT cells increased or remained unchanged in the liver after adult thymectomy and this phenomenon continued for up to 6 months after thymectomy. The administration of alpha-galactosylceramide induced severe cytopenia (due to apoptosis) of CD4+ NKT cells in the liver on day 1, but subsequent expansion of these NKT cells occurred in thymectomized mice similar to the case in normal mice. However, in thymectomized mice given lethal irradiation (9.5 Gy) and subsequent bone marrow transfer, the population of CD4+ NKT cells no longer expanded in the liver, although that of CD8+ NKT cells did. These results suggest that thymic CD4+ NKT cells, or their progenitors, may migrate to the liver at a neonatal stage but are not supplied from the thymus in the adult stage under usual conditions. CD8+ NKT cells can be generated in the liver.

Infiltration of cervical lymph nodes by B- and T-cell non-Hodgkin's lymphoma and Hodgkin's lymphoma: preliminary ultrasonic findings.

Ultrasonography has been used in the evaluation of enlarged lymph nodes in patients with malignant lymphoma, which comprises a variety of histopathological subtypes. We assessed ultrasonic findings of infiltration in cervical lymph nodes of 47 patients with previously untreated malignant lymphoma and correlated them with histopathological subtypes. Common findings among various histopathological subtypes were hypoechoic masses, spot and/or line echoes, involvement of multiple nodes, and posterior enhancement. In addition, each histopathological subtype showed characteristic findings: Hodgkin's disease had a contour with a lobular pattern (9/10 cases), septum-like echo (9/10 cases), and tubular structure (9/10 cases) in the internal echo, reflecting histopathologically fibrous connective tissue surrounding lymph nodes and small vessels. In B-cell lymphoma, there was characteristically a tendency for lymph node fusion (19/28 cases) and spot and/or line echoes (17/28 cases), reflecting histopathological replacement and destruction of the structure of lymph node and capsule by proliferation of lymphocytes. These findings were strengthened in diffuse large B-cell lymphoma. T-cell lymphoma was distinguished from other types of lymphoma by the presence of a hilum-like echo (6/9 cases), reflecting histopathologically prominent vascularization in lymph nodes accompanied by a dilated hilum area. In summary, this preliminary study suggests that ultrasonic findings of infiltration of cervical lymph nodes in major subtypes of malignant lymphoma could reflect characteristic histopathological structures, therefore providing information helpful for differentiating them.

Ultrasonographic differentiation between tuberculous lymphadenitis and malignant lymph nodes.

OBJECTIVE: To assess the usefulness of ultrasonography in the differential diagnosis of cervical tuberculous lymphadenitis versus malignant lymph nodes. METHODS: Ultrasonography of cervical lymph nodes was performed with a real-time linear scanner using a 7.5- or 11-MHz probe or both. Ultrasonographic findings were retrospectively reviewed in 73 patients: 49 with malignant lymphoma, 15 with tuberculous lymphadenitis, and 9 with metastatic lymph nodes. RESULTS: Ultrasonographic features specific to cervical tuberculous lymphadenitis were strong echoes (33.3%) and an echogenic thin layer (86.7%). When the lymph nodes had at least 1 of these 2 features, tuberculous lymphadenitis was diagnosed with a sensitivity of 100% and a specificity of 100%. CONCLUSIONS: Ultrasonographic evaluation of cervical lymph nodes can be useful in the diagnosis of cervical tuberculous lymphadenitis.

Fate map of the avian anterior forebrain at the four-somite stage, based on the analysis of quail-chick chimeras.

To better understand the topological organization of the primordia within the anterior forebrain, we made a fate map of the rostral neural plate in the chick. Homotopic grafts at the four-somite stage were allowed to survive for up to 9 days to enable an analysis of definitive brain structures. In some cases, the topography of the grafted neuroepithelia was compared with gene expression patterns. The midpoint of the anterior neural ridge maps upon the anterior commissure in the closed neural tube, continuing concentrically into the preoptic area and optic field. Non-neural epithelium just in front of this median ridge gives rise to the adenohypophysis. Areas for the presumptive pallial commissure, septum, and prosencephalic choroidal tissue lie progressively more posteriorly along the ridge, peripheral to the telencephalic entopeduncular and striatopallidal primordia (the subpallium), and the pallium (olfactory bulb, dorsal ventricular ridge, and cortical domains). Subpallial structures lie topologically anterior to the pallial formations, and both are concentric to the septum. Within the pallium, the major cortical domains (Wulst and caudolateral, parahippocampal, and hippocampal cortices) appear posterior to the dorsal ventricular ridge. The amygdaloid region appears concentrically across both the subpallial and pallial regions. This fate map shows that the arrangement of the prospective primordia in the neural plate is basically a flattened representation of topological relationships present in the mature brain, though marked phenomena of differential growth and selective tangential migration of some cell populations complicate the histogenetic constitution of the mature telencephalon.

Key factors influencing bowel function after ileal W-pouch anal anastomosis: a spectral analysis of W-pouch motor activity.

Restorative proctocolectomy with ileal pouch anal anastomosis (IPAA) has become the standard surgical procedure for ulcerative colitis (UC). The purpose of this study was to determine which factors are important to achieve good anal continence after IPAA in terms of the motor activity and pressure-volume relationship. A total of 17 patients with UC who underwent IPAA were evaluated. The internal ileal pouch pressure was transanally measured with and without volume-loading of the pouch which induces the urge to evacuate. The maximum tolerable volume (MTV), first urge volume (FUV), and ileal pouch compliance were calculated and the internal ileal pouch pressure records were subjected to spectral analysis for intensive evaluation of the intraluminal pressure waves. The FUV, correlation of the compliance of the FUV with MTV, and the remaining volume up to the MTV (RVMTV) were analyzed. Compliance of the FUV was significantly correlated with the RVMTV (r = 0.736, P < 0.01). The frequency of the phasic waves in the pouch decreased with length of follow up, reflecting improved function (r = -0.588, P < 0.05). The findings of this intensive analysis of manometric measurement indicate that the key factors in postoperative pouch function are RVMTV and the frequency of phasic waves in the W-pouch.

Transfer of SV40 temperature-sensitive early gene into human epidermal keratinocytes by the recombinant adenovirus vector.

We constructed a recombinant adenovirus vector that contained the origin-defective SV40 early gene, coding temperature-sensitive T antigen. This vector transferred the SV40 early gene into human epidermal keratinocytes with high efficiency. T antigen conferred the ability of keratinocytes to grow with limited differentiation in the presence of serum and high calcium concentration at the permissive temperature (34 degrees C), although normal keratinocytes were induced to differentiate and stop growing under the same conditions. The serum/Ca++-resistant cells did not proliferate at the nonpermissive temperature (40 degrees C), indicating that they depended on T antigen for their proliferation. The temperature-sensitive T antigen dissociated from the tumor suppressor gene products, p53, at 40 degrees C. The serum/Ca++-resistant cells still had the ability to proceed to terminal differentiation when injected into SCID mice as cultured keratinocytes. However, they did not form an apparent basal layer. This indicated that the tissue remodeling process in the serum/Ca++-resistant keratinocytes was abnormal. All of these epidermoid cysts disappeared within 8 wk and no tumor developed for 6 mo. We consider that deltaE1/SVtsT is a useful tool to examine multistep carcinogenesis of human epithelial cells in vitro.

Inverse relationship between circulating oxidized low density lipoprotein (oxLDL) and anti-oxLDL antibody levels in healthy subjects.

Oxidized low density lipoprotein (oxLDL) has been implicated in the pathogenesis of atherosclerosis. Recent studies have shown that immunization of animals with oxLDL results in suppression of atherogenesis. Antibody against oxLDL (oxLDL Ab) is detectable in human sera, although its biological significance is not well established. We examined the relationship between oxLDL Ab titer and circulating oxLDL level in 130 healthy Japanese subjects. OxLDL was measured as apolipoprotein (apo) B-containing lipoproteins carrying oxidized phosphatidylcholines by a sensitive ELISA. IgG class oxLDL Ab titer was measured by ELISA. Plasma oxLDL concentration was very low and it corresponded on average to one to two out of 1000 apoB-containing lipoproteins in plasma. Plasma oxLDL correlated positively with LDL cholesterol and inversely with oxLDL Ab titer. These associations remained significant and independent in multiple regression analysis including age, gender, smoking, and high-density lipoprotein cholesterol. These data indicate that healthy subjects have a very low concentration of oxLDL in the circulation, and that oxLDL Ab titer is in an inverse relationship with plasma oxLDL concentration in this population. Although these results suggest that oxLDL Ab may play a role in maintaining the low level of plasma oxLDL, its role in atherogenesis awaits further studies.

Effects of cyclopiazonic acid on contraction and intracellular Ca2+ in oesophageal striated muscle of normotensive and spontaneously hypertensive rats.

1. The effects of cyclopiazonic acid (CPA), a selective inhibitor of sarcoplasmic reticulum (SR) Ca2+-ATPase, on twitch contraction and on the resting state of tension and intracellular Ca2+ level ([Ca2+]i) of the oesophageal striated muscle of stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar Kyoto rats (WKY) were compared. 2. CPA (10 micronM) augmented the twitch contraction of oesophageal striated muscle preparations from both SHRSP and WKY, reducing the rate of relaxation (-dT/dt), and thus resulting in the prolongation of the time to 80% relaxation. The effect was significantly smaller in the SHRSP preparations. 3. In the resting state, CPA caused a sustained elevation of [Ca2+]i. The elevation was greater in the WKY preparations. Tension development accompanied by the elevation was observed in WKY preparations, but not in SHRSP preparations. 4. The sustained elevation of [Ca2+]i induced by CPA was eliminated by the removal of extracellular Ca2+. Both the elevated [Ca2+]i and tension in the preparations from WKY were reduced by flufenamic acid (100 micronM), mefenamic acid (100 micronM), lanthanum (La3+, 100 micronM), gadolinium (Gd3+, 100 micronM) and SK&F 96365 (100 micronM) but not by verapamil (10 micronM). 5. Thapsigargin (3 micronM), another SR Ca2+-ATPase inhibitor, produced similar effects on basal tension to those of CPA, although it reduced the amplitude of twitch contraction. 6. These results suggest that in the rat oesophageal striated muscle, (1) CPA extends the sequestrating time of Ca2+ into the SR, (2) CPA induces a Ca2+ influx mediated through verapamil-insensitive pathways, possibly nonselective cation channels, and (3) the mechanism of [Ca2+](i) modulation due to CPA-sensitive SR Ca2+-ATPase is deteriorated in the oesophageal striated muscle from SHRSP as compared with WKY preparations.

Human acute myeloblastic leukemia-ascites model using the human GM-CSF- and IL-3-releasing transgenic SCID mice.

To generate an appropriate model for human acute myeloblastic leukemia (AML), we have successfully established a human hematopoietic growth factor-dependent AML cell line (TF-1 and UT-7/GM)-ascites model using human granulocyte-macrophage colony-stimulating factor (hGM-CSF)- and human interleukin 3 (hIL-3)-releasing transgenic (Tg)-SCID mice. When 1 x 10(7) cells of TF-1, a human erythroleukemia cell line, were transplanted into the peritoneum of irradiated Tg-SCID mice (TF-1 ip/Tg-SCID mice), TF-1 cells grew in both the single cell suspension form (asTF-1) and solid form in ascites and invaded various tissues: lungs, liver, pancreas, and genitals, 3-6 weeks following transplantation. Subsequently, 0.5-1 x 10(7) cells of UT-7/GM, a subline of the UT-7 human megakaryoblastic leukemia cell line, grown in the back of hGM-CSF Tg-SCID mice after subcutaneous inoculation, were transplanted into the peritoneum of other irradiated hGM-CSF Tg-SCID mice. After 4 weeks, UT-7/GM cells (asUT-7/GM) also grew in the same manner as TF-1 cells in hGM-CSF Tg-SCID mice. Analysis of the cells from the peritoneum and tissues by PCR amplifying ALU and human GM-CSF receptor beta sequences and by immunohistochemical staining using anti-human CD45 revealed that they possessed the original characteristics of the parental cells. To confirm the usefulness of this human AML-ascites model, experimental treatment of AML cells grown in these mice was carried out with a differentiation inducer, delta-aminolevulinic acid (deltaALA), which induces hemoglobin synthesis for TF-1 in vitro and is thus regarded as an anti-leukemia drug candidate. Unexpectedly, growth promotion of TF-1 cells was observed in the treated TF-1 ip/hIL-3 Tg-SCID mice without differentiation to erythroid cells after treatment with delta-ALA (5 mM) for 7 days. These results indicate that Tg-SCID mice can support the growth of human hematopoietic growth factor-dependent AML cell lines which are usually rejected by SCID mice, without modification of the parental cell characteristics. In addition, this Tg-SCID leukemia-ascites model may become a useful preclinical tool for estimation of drug efficacy in vivo, since the drug candidate which was promising in vitro did not act in the same manner in vivo.

An in vivo model of human skin acute graft-versus-host disease: transplantation of cultured human epidermal cells and dermal fibroblasts with human lymphocytes into SCID mice.

The ability of mixed epidermal cell-lymphocyte reactions to detect allogeneic reactivities in an in vivo model was investigated by developing an in vivo model of acute graft-versus-host disease (GVHD), using SCID mice with a C.B-17 background in which human skin structures were generated by transplantation of cultured human epidermal cells (HEC) with dermal fibroblasts (HDFC). Suspensions containing cultured HEC and HDFC from a single donor were mixed with autologous peripheral blood mononuclear cells (PBMNC) or with PBMNC from unrelated individuals, and were injected into the flanks of C.B-17-SCID mice. Ten and 21 days after injection, subcutaneous nodules generated in the mice were examined histologically and immunohistochemically. Cystic structures developing after injection of HEC and HDFC without human PBMNC showed normal epidermislike tissue. Human skin generated in SCID mice injected with HEC and HDFC with auto-PBMNC showed no graft-versus-host reaction (GVHR) histologically, whereas those mice injected with PBMNC from siblings that shared an HLA haplotype showed mild GVHR. Human skin in SCID mice injected with HEC and HDFC with histoincompatible unrelated PBMNC showed moderate to severe GVHR. The severity of GVHR paralleled the dose of unrelated PBMNC, and GVHR was prevented by peroral treatment with cyclosporine A. Immunohistochemically, inflammatory cells infiltrating human cutaneous tissue formed in the SCID mice were stained by an anti-human CD45RO antibody that reacts with human T cells but not with murine lymphocytes, and most T cells were stained by an anti-human CD8 antibody recognizing HLA class I antigens. These findings are similar to those in clinical skin graft-versus host disease (GVHD) observed in patients undergoing allogeneic bone marrow transplantation. This experimental system should be useful as an in vivo model of human skin GVHD.

Long-term effects of dietary alpha-linolenic acid from perilla oil on serum fatty acids composition and on the risk factors of coronary heart disease in Japanese elderly subjects.

Although important roles of dietary n-3 fatty acids in the prevention of coronary heart disease (CHD) have been suggested, long-term effects of dietary alpha-linolenic acid (ALA, 18:3n-3) have not yet been established under controlled conditions. We tested whether a moderate increase of dietary ALA affects fatty acids composition in serum and the risk factors of CHD. Oxidized LDL (OxLDL) was directly measured by ELISA using antibody specific to OxLDL. By merely replacing soybean cooking oil (SO) with perilla oil (PO) (i.e., increasing 3 g/d of ALA), the n-6/n-3 ratio in the diet was changed from 4:1 to 1:1. Twenty Japanese elderly subjects were initially given a SO diet for at least 6 mo (baseline period), a PO diet for 10 mo (intervention period), and then returned to the previous SO diet (washout period). ALA in the total serum lipid increased from 0.8 to 1.6% after 3 mo on the PO diet, but EPA and DHA increased in a later time, at 10 mo after the PO diet, from 2.5 to 3.6% and 5.3 to 6.4%, respectively (p<0.05), and then returned to baseline in the washout period. In spite of increases of serum n-3 fatty acids, the OxLDL concentration did not change significantly when given the PO diet. Body weight, total serum cholesterol, triacylglycerol, glucose, insulin and HbA1c concentrations, platelet count and aggregation function, prothrombin time, partial thromboplastin time, fibrinogen and PAI-1 concentration, and other routine blood analysis did not change significantly when given the PO diet. These data indicate that, even in elderly subjects, a 3 g/d increase of dietary ALA could increase serum EPA and DHA in 10 mo without any major adverse effects.

A case of gastrointestinal stromal tumor of the stomach.

In a 55-year-old man, a tumor about 3 cm in diameter was detected in the upper abdomen by abdominal ultrasound screening during follow-up of chronic hepatitis C discovered in 1990. There were no symptoms and no abnormalities on physical examination. Tests for tumor markers were negative. By barium meal and gastroscopy, submucosal tumor was found on the lesser curvature of the stomach, with bridging fold in the absence of central ulceration. Biopsy revealed no tumor tissue. Under the diagnosis of submucosal tumor of the stomach, either a leiomyoma or leiomyosarcoma, partial resection of stomach was performed. Direct invasion of the surrounding organs, lymph node metastasis or distant metastasis was not observed grossly in the operation. Histologic examination of the resected specimen revealed proliferation of spindle cells and oval cells in an interlacing pattern. Immunohistochemistry for CD34, vimentin and c-kit protein was strongly positive, while smooth muscle actin, S-100 protein, desmin and p53 protein were negative. The proliferating cell nuclear antigen index was about 50%, while the MIB-1 index was < or = 1%. From these findings, this tumor was diagnosed as a gastrointestinal stromal tumor of the uncommitted type.