The presence and longevity of peripherally expanded donor-derived TCRalphabeta+ mature T lymphocyte clones after allogeneic bone marrow transplantation for adult myeloid leukemias.

There are two major pathways for T-cell regeneration after allogeneic bone marrow transplantation; thymus-dependent T-cell differentiation of T-cell progenitors, and peripheral expansion of mature T cells in the graft. In order to learn to what extent the peripheral expansion of donor-derived mature T lymphocytes contributes to reconstitution of the TCRalphabeta+ T-cell repertoire after allogeneic bone marrow transplantation for adult myeloid leukemias, we pursued the fate of donor-derived T-cell clones using the amino-acid sequences of the complementarity-determining region 3 (CDR3) of the TCR-beta chain as a clonal marker. Clonal expansion of TCRalphabeta+ T lymphocytes with specific TCRBV subfamilies was identified in donor blood. Identical T-cell clones were not found in blood from recipients before transplantation. The donor-derived T-cell clones were identified in the circulating blood from recipients a few months after allogeneic bone marrow transplantation, and they remained in the blood for 18 months after transplant in two recipients, and for 56 months in one. These results suggest that the peripheral expansion of mature T lymphocytes in the graft makes a significant contribution to post-transplant T-cell regeneration during the early period of transplantation in humans, and that mature T cells can survive in recipients for several years. Further investigation will be required to explore which antigens drive the expansion of T-cell clones in donors and recipients, and the mechanisms of maintaining homeostatic balance between the thymus-dependent pathway and the peripheral expansion of mature T cells in post-transplant T-cell regeneration.

Proliferative activity of mixed hyperplastic adenomatous polyp/serrated adenoma in the large intestine, measured by PCNA (proliferating cell nuclear antigen).

The proliferative activity of serrated adenomas of the large intestine was determined by examining proliferating cell nuclear antigen (PCNA). The PCNA labeling index, determined by visual inspection, and the PCNA area rate, determined with the newly developed image processor for analytical pathology (IPAP), of serrated adenoma were found to be similar to the values for tubular adenoma, and indicated the presence of high proliferative activity in the bottoms of crypts. Determination of the pattern of distribution of PCNA-positive cells indicated the presence of a proliferative zone in the lower region or bottom of the serrated adenoma. However, 5 of the 20 serrated adenomas exhibited an irregular on widely extended proliferative zone, and 2 were complicated by cancer. These findings indicated that serrated adenoma is also a highly proliferative tumor and that it may be complicated by cancer if atypia is increased and disturbance of the proliferative zone is present.

Molecular analysis of insulin receptor gene in Werner's syndrome.

Werner's syndrome is characterized by premature aging and frequent impaired glucose tolerance or overt diabetes. Insulin resistance may play an important role and may be caused by a post-receptor defect or dysfunctional insulin receptor. The present study was undertaken to investigate the insulin receptor gene mutation in Werner's syndrome. The genomic DNAs were obtained from four patients with Werner's syndrome. Exons 2-22 of the insulin receptor gene except exon 1 were amplified from genomic DNA by the polymerase chain reaction and screened for nucleotide variation by examining for single-stranded conformational polymorphisms. There were no nucleotide variations in exons 2, 4-->7, 9 and 12-->22. Variants were thus found in exons 3, 8, 10 and 11 and each were sequenced. The variant in exon 8 was due to a silent polymorphism (GAT-->GAC/T, Asp519) and other variants in exons 3, 10 and 11 were caused by nucleotide substitutions in introns. These results suggest that the patients with Werner's syndrome express normal insulin receptors and that the primary genetic lesion for insulin resistance is not in the insulin receptor gene. Insulin resistance in Werner's syndrome is thus likely by a post-receptor defect.

Generation of 28 sequence-tagged sites (STSs) from yeast artificial chromosome (YAC) clones anchored at human chromosome 21q22.1 region.

Twenty-eight sequence-tagged sites (STSs) were newly generated from the DNA sequences of vector-insert junctions from yeast artificial chromosomes (YACs) anchored at chromosome 21q22.1 region. The insert DNAs adjacent to vector arms were specifically amplified through inverse PCR method to clone into pUC19 vector for sequencing. Sixty DNA junctions from 44 CEPH YAC clones were cloned and sequenced. Of these DNA sequences of junctions between vector-arms and DNA inserts, twenty-eight STSs were finally obtained to show the accurate amplification, which is specific for human chromosome 21. The sets of 28 STSs were useful to build fine YAC contigs by STS-mediated YAC walking at the 21q22.1 region.

1.8-megabases fine physical map encompassing IFNAR and AML1 loci on human chromosome 21q22.1.

A long-range restriction map of the 1.8-megabases (mb) region encompassing the area between the interferon-alpha receptor and the acute myelogenous leukemia loci on human chromosome 21q22.1 was constructed after analysis of both the contiguous yeast artificial chromosome (YAC) clones and genomic DNA. Analysis of pulsed-field gel electrophoresis of lymphoblastoid DNA digested with three rare-cutting enzymes, Not I, Mlu I, and Nru I, revealed the positions of 17 markers on each restriction map. The 1.8-mb YAC contig that covers this region was obtained through YAC walking mediated by sequence-tagged sites (STSs), with 29 STSs including 12 newly generated YAC end-specific STSs. The consensus restriction map from 15 overlapping YACs and the positioning of the STS markers on each clone allowed 24 markers including 4 Not I-linking STSs to be ordered and mapped physically. Comparison of the maps revealed that the proximal region contains more unmethylated CpG islands than the distal region, which suggests that many expressed genes are in the proximal region. This fine consensus physical map will be informative and useful for construction of contigs of cosmid, P1, or BAC clones for further large-scale sequencing in this gene-rich region.

Non-hormonal adrenocortical adenoma with oncocytoma-like appearances.

We report a case of non-hormonal adrenocortical adenoma. The tumor was removed en block with the adrenal gland. The specimen was 5.0 X 4.5 X 3.0 cm, weighed 30 g and was solid. Histologically, this tumor had an oncocytoma-like appearance. However, as there is no concept of oncocytoma in connection with adrenocortical adenoma, this case was diagnosed as adrenocortical adenoma. A case with such histological findings has never been reported.

[Subdiaphragmatic bronchogenic cyst in the left crus of diaphragm: report of a case].

A 39-year-old man who had a subdiaphragmatic bronchogenic cyst in the left crus of diaphragm received surgical treatment. The cyst was located in the retroperitoneum just below the diaphragm and was adhered to the left crus of diaphragm and unconnected with any other structures. The surgically resected cyst was 50 x 25 x 22 mm diameter and the wall was thin and contained white turbid mucus. Histologically, the cyst consisted of ciliated epithelium, mucus glands, smooth muscle, cartilage and this evidence established the final diagnosis of bronchogenic cyst. The post operative course was uneventful and the patient was discharged 10 days after operation. This is the 4th reported case of a subdiaphragmatic bronchogenic cyst in the Japanese literature.

[Three cases of emergency video-assisted thoracoscopic surgery for spontaneous hemopneumothorax].

We experienced 3 cases of video-assisted thoracoscopic surgery for spontaneous hemopneumothorax. All the patients had received emergent operations because of massive intrathoracic bleeding. At the operation, a 3 cm-minithoracotomy and 2 trocar ports were fashioned. In the head up position, massive blood clots in the apex in the thoracic cavity was removed by using grasping forceps and the source of bleeding point was detected easily. The bleeding was successfully stopped. It was difficult to remove massive blood clots from trocar port by suction, however it was easy to remove massive blood clots from a 3 cm-minithoracotomy window by using a large grasping forceps. Post operative course was satisfactory and the all patients discharged within 2 weeks after admission. We concluded that the spontaneous hemopneumothorax may be a good indication for video-assisted thoracoscopic surgery.

[Pulmonary hyalinizing granuloma with massive infiltration of lymphocytes].

A 39-year-old man was admitted to our hospital for examination of multiple nodules and infiltrates on a chest radiograph. His chest HRCT revealed multiple nodules with or without thick- or thin-walled cavities. Specimens obtained by video-assisted thoracoscopic biopsy showed bundles of hyalinized collagen fibers, some of which contained accumulated plasma cells in the center. The nodules were surrounded by massive lymphoid cells which formed germinal centers. These findings are compatible with pulmonary hyalinizing granuloma. The lymphoid cells looked uniform in some areas and had infiltrated along the bronchioles and small vessels and into the intralobular septa in a manner resembling pulmonary lymphoma or pseudolymphoma. The findings suggested that pulmonary hyalinizing granuloma may overlap pulmonary lymphoma. The disease has shown no progression for four years although no treatment has been given.

[A case of fibrous histiocytoma of the appendix with twisted stalk].

A 74-year-old man consulted this hospital with the chief complaint of lower right abdominal pain on February 13, 1998. He was hospitalized, subjected to abdominalechography and CT, and diagnosed as having subileus caused by an intraperitoneal tumor. Surgery was performed on February 25, 1998. When the abdomen was incised, a chicken egg-sized tumor at the end of theappendix were found. In addition, the stalk of the appendix was twisted. Appendectomy was therefore performed. Upon histopathological examination, it was found that the submucosal tumor originated at the end of the appendix, and proliferation of spindle-shaped fibroblast-like cells and histocytic oval cells was observed in the tumor. Since various histiocyte markers were positive upon immunohistological examination, the tumor was considered to be of histiocytic origin. However, the tumordid not exhibit polymorphism, heteromorphism, or mitotic figures which would confirm a diagnosis of malignant fibrous histiocytoma. It was thus diagnosed as a fibrous histiocytic tumor on the borderline between malignant and benign. We report the present case because the occurrence of a primary fibrous histiocytoma in an appendix of which the stalk is twisted is very rare.

A simple, sensitive high-performance liquid chromatography -ultraviolet method for the quantification of concentration and steady-state pharmacokinetics of itraconazole and hydroxyitraconazole.

BACKGROUND: A steady-state trough plasma itraconazole concentration greater than 500 ng/mL is a therapeutic target for itraconazole. A simple, rapid and sensitive high-performance liquid chromatography-based method was developed for quantitation of itraconazole and hydroxyitraconazole in human plasma. METHODS: Itraconazole and hydroxyitraconazole were separated using a mobile phase of 0.5% KH2PO4 (pH 6.0)-acetonitrile (30:70, v/v) on a CAPCELLPAK C18 MGII column at a flow rate of 0.5 mL/min and ultraviolet absorbance at 260 nm. RESULTS: The analysis required 200 microL of plasma and involved a rapid, simple solid-phase extraction with an Oasis HLB cartridge, which resulted in recoveries of 87-92% for itraconazole and 91-94% for hydroxyitraconazole. The lower limit of quantification for itraconazole and hydroxyitraconazole was 5 ng/mL each. Intra- and interday coefficients of variation for itraconazole and hydroxyitraconazole were less than 11.3% and 12.2%, respectively, and accuracies were within 11.7% and 4.5% over the linear range, respectively. Although the steady-state plasma concentrations of itraconazole and hydroxyitraconazole ranged from 506 to 2482 ng/mL and from 766 to 2444 ng/mL, respectively, after a two-day loading dose of 400 mg/day intravenous itraconazole followed by the administration of 200 mg/day itraconazole oral solution, calibration curves of itraconazole and hydroxyitraconazole showed positive linearity in a concentration range of 5-2500 and 50-2500 ng/mL, respectively. CONCLUSIONS: Our results indicate that this method is applicable for the monitoring of plasma levels of itraconazole and hydroxyitraconazole in a clinical setting. Furthermore, the regimen presented here might also be effective in preventing infection, but further studies with large sample sizes are necessary to investigate this avenue.

Skewed T cell receptor repertoire of Vdelta1(+) gammadelta T lymphocytes after human allogeneic haematopoietic stem cell transplantation and the potential role for Epstein-Barr virus-infected B cells in clonal restriction.

The proliferation of Vdelta1(+) gammadelta T lymphocytes has been described in various infections including human immunodeficiency virus (HIV), cytomegalovirus (CMV) and malaria. However, the antigen specificity and functions of the human Vdelta1(+) T cells remain obscure. We sought to explore the biological role for this T cell subset by investigating the reconstitution of T cell receptor (TCR) repertoires of Vdelta1(+) gammadelta T lymphocytes after human allogeneic haematopoietic stem cell transplantation (HSCT). We observed skewed TCR repertoires of the Vdelta1(+) T cells in 27 of 44 post-transplant patients. Only one patient developed EBV-associated post-transplant lymphoproliferative disorder in the present patient cohort. The -WGI- amino acid motif was observed in CDR3 of clonally expanded Vdelta1(+) T cells in half the patients. A skew was also detected in certain healthy donors, and the Vdelta1(+) T cell clone derived from the donor mature T cell pool persisted in the recipient's blood even 10 years after transplant. This T cell clone expanded in vitro against stimulation with autologous EBV-lymphoblastoid cell lines (LCL), and the Vdelta1(+) T cell line expanded in vitro from the same patient showed cytotoxicity against autologous EBV-LCL. EBV-infected cells could also induce in vitro oligoclonal expansions of autologous Vdelta1(+) T cells from healthy EBV-seropositive individuals. These results suggest that human Vdelta1(+) T cells have a TCR repertoire against EBV-infected B cells and may play a role in protecting recipients of allogeneic HSCT from EBV-associated disease.

Purification and some properties of dipeptidyl carboxypeptidase from Bacillus pumilus.

An intracellular protease from a bacterium, Bacillus pumilus HL721, was purified about 5000-fold by chromatography with a Q-Sepharose Fast Flow column, TSK-gel HA-1000 glass column, and TSK-gel G3000SWXL column using Bz-Gly-Ala-Pro as a substrate. The enzyme was the most active at pH around 7.5 and stable from 4.5 and 8.0. The enzyme activity was inhibited by Cu2+, EDTA, N-ethylmaleimide, o-phenanthroline, and p-chloromercuribenzoic acid. The molecular weight of the enzyme was 155,000 by gel filtration. The enzyme removed dipeptide from the carboxyl end of some peptides used as substrates. From these results the enzyme seems to be a dipeptidyl carboxypeptidase.

Enzymatic properties of dipeptidyl carboxypeptidase from Bacillus pumilus.

Enzymatic properties of dipeptidyl carboxypeptidase (DCP) from Bacillus pumilus were investigated. The enzyme was more active on tri- and tetrapeptides than angiotensin-converting enzyme (ACE) from rabbit lung. The presence of chloride ion is essential for the hydrolysis. The Km value of angiotensin I for the enzyme was 0.119 x 10(-3) M. The enzyme was not inhibited by the mammalian ACE inhibitors lisinopril and enalaprilat. The enzyme is readily inhibited by EDTA but restored by Co2+, Mn2+, and Zn2+. Therefore, it seems to be a zinc-metallo protease.

Experimental infection of equine herpesvirus 9 in dogs.

Equine herpesvirus 9 (EHV-9), a new neurotropic equine herpesvirus, was inoculated intranasally at 107 plaque-forming units in five dogs to assess its pathogenicity. Dogs showed weight loss, pyrexia, anorexia, and neurologic signs on the fourth day. The EHV-9 virus was recovered from the examined brains. Histologically, dogs had a fulminant nonsuppurative encephalitis characterized by severe neuronal degeneration and loss, with intranuclear inclusions, slight glial reactions, perivascular cuffing, and multifocal hemorrhage. The olfactory bulb and the frontal and temporal lobes were predominantly affected. Immunohistochemistry revealed reactivity for EHV-9 antigen in neurons. All dogs had mild bronchopneumonia and various degrees of lymphoid necrosis. These findings indicate that dogs are fully susceptible to EHV-9 and that EHV-9 can cause fulminant encephalitis with high mortality in dogs, as in gazelles and goats.

Intraarterial lymphocytes injection for treatment of lymphedema.

We noted a marked improvement of lymphedema in a patient with recurrence of cancer 3 years after amputatio recti and with post-thrombotic syndrome of the leg, as the result of injection of a suspension of lymphocytes administrated as therapy against advanced cancer. Subsequently lymphocytes-injections were given 39 times to 7 patients with lymphedema of one limb, of various causes. A suspension of lymphocytes was injected repeatedly into the proximal artery of the affected limb. In one patient, lymphocytes were injected 4 times at intervals of 1 or 3 weeks. In most cases, this injection resulted in a marked reduction in the swelling of the affected limb and improvement was seen for several weeks or months. In all cases, there was a marked, continuous softening of the tissue.

Construction and evaluation of a porcine bacterial artificial chromosome library.

A porcine bacterial artificial chromosome (BAC) library consisting of 103,488 clones has been constructed. The average insert size in the BAC vector was calculated to be 133 kb based on the examination of 189 randomly selected clones, indicating that the library contained 4.4 genome equivalents. The library can be screened by two-step PCR. The first screening step is performed on 22 superpools, each containing 4704 clones (49 x 96 well plates). In the second screening step, 49 plates comprising a superpool are arrayed in a 7 x 7 matrix and 4D-PCR is performed. Screening of the library superpools by PCR for 125 marker sequences selected from different regions of swine genome revealed 123 sequences, indicating that the library is not biased. Subsequent screenings (4D-PCR) were successfully applied for identification of clones containing each marker sequence. This porcine BAC library and the PCR screening system are useful for isolation of genomic DNA fragments containing desired sequences.

[Choriocarcinoma of the stomach].

A 55-year-old Japanese man was admitted to our hospital in January 1985 complaining of epigastralgia. Radiologic and endoscopic examinations showed gastric carcinoma, but biopsy specimens taken by surgery showed the histologic pattern of choriocarcinoma. On autopsy, multiple metastatic sites wer observed, and histological examination of the primary and metastatic sites revealed choriocarcinoma. Primary gastric choriocarcinoma may develop from gastric cancer by retrodifferentiation.

[The pathogenesis of adenocarcinoma of the stomach after successful radiotherapy of squamous cell carcinoma of the lower intrathoracic esophagus--case report].

A case of a gastric cancer developed 3 years and 11 months after radiotherapy for esophageal cancer is reported. A 76-year-old man with a squamous cell carcinoma of the lower intrathoracic esophagus had received 50 Gy of irradiation as treatment. Thereafter, signs of the esophageal cancer disappeared radiologically and endoscopically, and a pathological biopsy of specimens taken from the site revealed no further cancer cells. The patient remained well for 3 years and 11 months after radiotherapy, at which time he again was admitted to hospital, having been diagnosed as having a gastric cancer. On admission, an upper G-I series showed a shadow defect along the lesser curvature of the upper-middle stomach but no evidence of any stenosis in the lower intrathoracic esophagus. Endoscopically, the mucosal surface of the esophagus was normal, and biopsy specimens taken from the site in the esophagus that had been treated with irradiation 3 years 11 months ago revealed no recurrence of his esophageal cancer. Endoscopical examination of the stomach showed an infiltrative tumor with ulceration, and a subsequent histological examination revealed a poorly differentiated adenocarcinoma. Upon a laparotomy, a metastasis was detected in the perigastric and paraaortic lymph nodes and the cancer had invaded the retroperitoneum. The stomach could not be removed and he died 3 months after the laparotomy.

A long-range physical map of human chromosome 21q22.1 band from the YAC continuum.

The human Chromosome (Chr) 21q22.1 region contains several genes for cytokines and neurotransmitters and the gene for superoxide dismutase (mutant forms of which can cause familial amyotrophic lateral sclerosis). A region of approximately 5.8 Mb encompassing D21S82 and the glycinamide ribonucleotide transformylase (GART) loci was covered by overlapping YAC clones, which were contiguously ordered by clone walking with sequence-tagged site (STSs). A total of 76 markers, including 29 YAC end-specific STSs, were unambiguously ordered in this 5.8-Mb region, and the average interval between markers was 76 kb. Restriction maps of the YAC clones with rare-cutting enzymes were simultaneously prepared, and the restriction sites were aligned to obtain a consensus restriction map of the proximal region of the 21q22.1 band. The restriction map made from 44 overlapping YACs contains 54 physically assigned STSs. By integrating the consensus map of the adjacent 1.8-Mb region, we obtained a fine physical map spanning 6.5 Mb of human Chr 21q22.1. This map contains 24 precisely positioned end-specific STSs and 12 NotI-linking markers. More than 39 potential CpG islands were identified in this region and were found to be unevenly distributed. This physical map and the YACs should be useful as a reference map and as a resource for further structural analysis of the Giemsa-negative band (R-band) of Chr 21q22.1.