Population-based genetic alterations in Ewing's tumors from Japanese and European Caucasian patients.

BACKGROUND: The incidence of Ewing's tumors (ETs) is lower in Asians or African-Americans than in Caucasians. PATIENTS AND METHODS: Japanese ETs were available for analysis of chromosomal aberrations by comparative genomic hybridization (n = 16) and for expression of chimeric EWS transcripts by reverse-transcriptase polymerase chain reaction (n = 11). These results in Japanese patients were compared with those of 62 ETs in European Caucasian patients registered in the European Intergroup Cooperative Ewing's Sarcoma Study. RESULTS: Japanese patients with ET had lower overall survival (P = 0.0446) and relapse-free survival (P = 0.0371) compared with European Caucasian patients. Ten of 11 Japanese ETs and 31 of 62 European Caucasian ETs had type I (EWS exon 7 to FLI1 exon 6) fusion transcripts. In Japanese ETs, the median numbers of chromosomal aberrations were 2.0 and 6.0 in 11 primary tumors and five relapsed tumors, respectively. In European Caucasian ETs, the median number of changes were 2.5 and 5.0 in 52 primary and 10 relapsed tumors, respectively. Frequent gains were 8q (38%), 8p (31%) and 12q (25%) in Japanese ETs and 8q (52%), 8p (48%) and 12q (19%) in European Caucasian ETs. Frequent losses were 19q (44%), 19p (38%) and 17p (25%) in Japanese ETs and 16q (21%), 19q (18%) and 17p (15%) in European Caucasian ETs. The incidence of losses of 19p (P = 0.0215) and 19q (P = 0.0277) were significantly higher in Japanese ETs than in European Caucasian ETs. An amplification (1p33-p34) was observed in only one Japanese ET. CONCLUSIONS: Japanese patients with ET in this study had a worse prognosis than European Caucasian patients. In molecular genetic analyses, Japanese ETs had a higher frequency of loss of chromosome 19 than European Caucasian ETs. Different genetic aberrations may explain the different incidences and prognoses of ET between Caucasian and Japanese patients.

Development of acute myeloid leukemia after chemotherapy for osteosarcoma.

The current study describes two patients with osteosarcoma who had acute myeloid leukemia develop after treatment with multiagent chemotherapy. The incidence density for the chemotherapy protocol was 129.8 per 10,000 person-year of followup. Karyotype analysis of 16 reported patients (including the current two patients) indicated that most leukemias after treatment of osteosarcoma correlated with the use of topoisomerase II inhibitors, such as doxorubicin. The deoxyribonucleic acid-damaging activity of doxorubicin reinforced by the use of alkylating agents is highly suspected as a causative event in the development of leukemia after treatment of osteosarcoma. As the next step in the development of treatment for patients with osteosarcoma, the type and intensity of treatment must be evaluated to minimize possible leukemogenic effects without compromising the potential for cure.

A reverse transcriptase-polymerase chain reaction assay in the diagnosis of soft tissue sarcomas.

BACKGROUND: Many types of sarcomas are characterized by specific chromosomal translocations that result in the production of novel chimeric genes. Detection of these fusion genes could be a sensitive molecular diagnostic assay. However, to the authors' knowledge there have been few systemic comparisons between the current histopathologic diagnosis and the presence or absence of particular fusion genes in patients with adult soft tissue sarcomas (STSs). METHODS: Total RNA was extracted from 75 cases of STS and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the detection of a variety of fusion transcripts. The results of the molecular assay were compared with standard histopathologic diagnoses. RESULTS: Of the 18 tumors diagnosed as synovial sarcoma, 17 (94%) expressed SYT-SSX chimeric transcripts. All nine myxoid liposarcomas were positive for FUS-CHOP fusion transcripts. Of the four cases of Ewing sarcoma, two had an EWS-FLI1 fusion transcript and one had an EWS-ERG fusion transcript. A clear cell sarcoma had a EWS-ATF1 fusion transcript. None of 19 cases of malignant fibrous histiocytoma nor 3 leiomyosarcomas contained a fusion transcript. Three cases with an initial diagnosis other than synovial sarcoma expressed a SYT-SSX fusion transcript. A review of the slides and additional examination showed that a diagnosis of synovial sarcoma was appropriate for these cases. There was a trend for biphasic synovial sarcoma to contain the SYT-SSX1 fusion. CONCLUSIONS: The authors believe RT-PCR assay for the detection of a specific fusion gene provides a useful tool for confirmation of the diagnosis of adult STS in diagnostically difficult cases and in retrospective studies.

Synovial sarcoma with a large hematoma in the inguinal region.

We report the case of a patient with synovial sarcoma and a large hematoma of the inguinal region. The patient underwent tumor resection of the lower 2/3 of the acetabulum after preoperative chemotherapy. Twenty-four months after surgery, she is alive without any relapse and can walk without support.

Reconstruction of the hip abductors after resection of the proximal femur.

Three patients with malignant bone tumors of the proximal femur underwent implantation of an endoprosthesis with reconstruction of the joint capsule and hip abductors using artificial mesh.

The 20-kilodalton (kDa) human growth hormone (hGH) differs from the 22-kDa hGH in the effect on the human prolactin receptor.

Previously we have demonstrated that 20-kDa human GH (20K-hGH) is a full agonist for hGH receptor (hGHR) even though its complex formation with hGHR and hGH-binding protein differs from that of 22-kDa human GH (22K-hGH). In this study, we focused on the effect of 20K-hGH on human PRL receptor (hPRLR). To elucidate the effects of 20K-hGH on hPRLR and compare them with those of 22K-hGH, we prepared two cells stably expressing full-length hPRLR, Ba/F3-hPRLR and CHO-hPRLR. In the proliferation of Ba/F3-hPRLR cells, which can grow in a dose-response to lactogenic hormones, both 20K- and 22K-hGH exhibited bell-shaped curves in the absence of exogenous zinc ion (Zn2+); however, the curve of 20K-hGH was shifted to a 10-fold higher concentration than that of 22K-hGH in view of EC50 value (the EC50 of 20K- and 22K-hGH were 15 nM and 1.5 nM, respectively). Addition of Zn2+ up to 25 microM increased the activities of both 20K- and 22K-hGH; however, the enhancement by Zn2+ was greater in 20K-hGH than in 22K-hGH, thereby the activities of both hGH isoforms reached the same level at 25 microM Zn2+. Nevertheless, in the presence of 0.25-1 microM free Zn2+, which is equal in human serum, the activity of 20K-hGH was still lower than that of 22K-hGH. The modest effect of 20K-hGH on activating hPRLR in the absence of Zn2+ was confirmed in the rat serine protease inhibitor 2.1 (Spi2.1) gene promoter activation and JAK2/Stat5 tyrosine phosphorylation in CHO-hPRLR. In addition, in human breast cancer cell T-47D, 20K-hGH was proved to stimulate Stat5 tyrosine phosphorylation to much lower degree than 22K-hGH via not hGHR but hPRLR. Taken together, our data suggest that 20K-hGH may be a weaker agonist for hPRLR than 22K-hGH in the human body; therefore 20K-hGH may alleviate the hPRLR-mediated side-effects such as breast cancer when administered to human body.

Vancomycin-impregnated polymethylmethacrylate beads for methicillin-resistant Staphylococcus aureus (MRSA) infection: report of two cases.

Two patients with methicillin-resistant Staphylococcus aureus (MRSA) infection were treated with vancomycin (VCM)-impregnated polymethylmethacrylate (PMMA) beads. One patient, who had a history of polycystic kidney and diabetes mellitus, who was receiving hemodialysis due because of non-functional kidney, underwent resection of an intermediate grade chondrosarcoma in the pelvis. MRSA infection developed and curettage of the lesion was performed, but MRSA infection recurred. During the second revision surgery, VCM-impregnated PMMA beads were implanted. MRSA infection has not recurred for 16 months since the implantation of the VCM beads. The second patient had a history of total hip arthroplasty (THA) performed because of coxarthrosis. After the initial surgery, MRSA infection developed, recurring after the second revision surgery for THA. After curettage following removal of the prosthesis, VCM beads were implanted with a spacer composed of VCM-PMMA and a Luque rod. Infection did not recur and THA revision was performed 3 months after the VCM beads implantation. Fifteen months after the last revision surgery, infection has not recurred.

High complication rate of reconstruction using Ilizarov bone transport method in patients with bone sarcomas.

We performed five reconstructions by bone transport in patients with bone sarcoma: three osteosarcomas, one Ewing's sarcoma and one chondrosarcoma. Four sarcomas were in the distal femur and one in the shaft of the tibia. Four patients received multidrug chemotherapy. The average length of the skeletal defect after tumour resection was 17 (range 10-25) cm. All patients underwent double elongation from both proximal and distal sites of the bone defect. The average follow-up period was 48 (range 40-66) months, and the average duration of external fixation was 32 months (range 579-1340 days). In one case, bone formation was satisfactory, but in the other cases, it was poor and slow. The average treatment index was 95 (range 53-191) days per 1 cm of elongation. In one case, the bone fragment disappeared during the bone transport. In one case, the end of the bone protruded from the skin, and osteomyelitis set in. This patient underwent above-knee amputation due to failure of infection control. Three patients suffered talipes equinus. One patient died of pulmonary metastasis. Two patients had fair and three had poor function. This method is not recommended for patients with bone sarcoma who may have a poor prognosis, as it has an unacceptably high complication rate.

Secretion of authentic 20-kDa human growth hormone (20K hGH) in Escherichia coli and properties of the purified product.

Using Bacillus amyloliquefaciens neutral protease gene (npr), we have constructed a secretion system of 20-kDA human growth hormone (20K hGH) in E. coli. The secretion-signal region from npr was modified inserting a fragment coding a 2Lys-5Leu cluster. In this system we found that co-expression of glutathione reductase remarkably increased accumulation level of 20K hGH in periplasm and confirmed that secreted 20K hGH was correctly processed. The recombinant 20K hGH was highly purified and subjected to analyses of physicochemical properties and biological activities which are still unclear and controversial due to difficulty in preparing the sample with authentic structure. The secreted recombinant product had authentic disulfide linkages and showed molecular weight of 20,270.5 +/- 3.7 (theoretical value, 20,269.9). The results suggest that the recombinant 20K hGH is a full agonist on rat growth promotion and lipolysis stimulation in isolated rat adipose tissues. In particular, the lipolysis-stimulating activity of 20K hGH was distinct as compared with that of 22K hGH under physiological concentration. Cell proliferation activity via prolactin-receptor in Nb-2 lymphoma was obviously low as compared with that of 22K hGH.

Morphological changes of Candida albicans induced by BMY-28864, a highly water-soluble pradimicin derivative.

The time course of BMY-28864-dependent morphological changes in Candida albicans A9540 was studied by electron microscopy. Scanning electron microscopy revealed that BMY-28864 often induced cell surface deformations such as abnormal swelling and bulging around budding sites and bud scars. The cell membrane damage was visualized by freeze-fracturing technique as deep pit-like invaginations. Transmission electron microscopy with thin-sectioned specimens also demonstrated that BMY-28864 induced cell membrane invaginations together with cell membrane detachment from the cell wall, nuclear membrane fragmentation and mitochondrial aberration. Statistical sequence analysis of the prominent BMY-28864-dependent morphological changes of the yeast cells led to the conclusion that BMY-28864 first attacked the cell membrane and then caused disintegration of other intracytoplasmic organelles, resulting in the lethal effect.

Anti-thyroglobulin autoantibodies in sera from patients with chronic thyroiditis and from healthy subjects: differences in cross-reactivity with thyroid peroxidase.

A significant portion (about 12.7%) of healthy subjects was found to contain anti-thyroglobulin (anti-Tg) antibodies in their sera. We compared the binding activities of these antibodies and of anti-Tg autoantibodies from sera of patients with chronic thyroiditis with human thyroid peroxidase (TPO). The results obtained by ELISA indicated that out of 10 healthy subjects with anti-Tg antibodies, only four had anti-Tg antibodies capable of binding to TPO, whereas anti-Tg autoantibodies from almost all patients with chronic thyroiditis possessed high binding activities to TPO. By use of the immunoprecipitation method, it was also shown that although all anti-Tg autoantibodies from patients precipitated TPO, a majority of anti-Tg antibodies from healthy subjects could not precipitate TPO. Such findings cannot be ascribed to the differences in levels of anti-Tg autoantibodies and anti-TPO autoantibodies in sera and the differences in avidities of anti-Tg antibodies in sera between healthy subjects and patients with chronic thyroiditis. Thus, it can be concluded that anti-Tg antibodies from healthy subjects differ from those of patients with chronic thyroiditis with respect to TPO binding, probably due to difference in fine specificities of these anti-Tg antibodies.

Salmonid pituitary gonadotrophs. II. Ontogeny of GTH I and GTH II cells in the rainbow trout (Salmo gairdneri irideus).

Immunocytochemistry of rainbow trout pituitary gonadotrophs (GTH I- and GTH II-producing cells) during gametogenesis was investigated. GTH I and GTH II were found in distinctly different cells in all stages of reproductive development that were examined. Only GTH I cells were present in trout prior to puberty. GTH II appeared in addition to GTH I coincident with the onset of vitellogenesis and spermatogenesis. Both GTH I and GTH II cells were found in trout at the time of final reproductive maturation, although the number of GTH II cells was greater than that of GTH I cells. These data indicate that GTH I and GTH II are localized in separate cells in the trout pituitary throughout gametogenesis, and that synthesis of GTH I and GTH II varies during reproductive development.

Anti-thyroid peroxidase antibody activity in sera of patients with systemic lupus erythematosus.

Although patients with SLE have autoantibodies to thyroid peroxidase (TPO), IgG from sera of SLE patients does not inhibit TPO activities, in contrast with IgG from sera of patients with thyroid disorders. This finding suggests that the specificities of anti-TPO autoantibodies in SLE are different from those in cases of thyroid disorders. These autoantibodies to TPO should be considered when searching for associations between SLE and autoimmune thyroid disorders.

The duality of teleost gonadotropins.

The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I ß is more similar to bovine FSHß than bovine LHß and salmon GTH II ß shows higher homology to bovine LHß than to bovine FSHß. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.

Thyroglobulin and thyroid peroxidase share common epitopes recognized by autoantibodies in patients with chronic autoimmune thyroiditis.

Monoclonal antibodies specific for human thyroid peroxidase (TPO) were prepared by the hybridoma technique using hyperimmune spleen cells from mice immunized with TPO purified from thyroid glands from patients with Graves' disease. Use of the microenzyme-linked immunosorbent assay method revealed that some of the monoclonal antibodies cross-reacted strongly with human thyroglobulin (Tg). Conversely, monoclonal anti-Tg antibodies cross-reacted with TPO, albeit to a lesser degree. Some anti-Tg autoantibodies in serum from patients with chronic autoimmune thyroiditis purified by Tg affinity chromatography bound TPO, and such binding was completely inhibited by Tg. Western blotting experiments revealed that thyroid microsomal 103K proteins recognized by mouse monoclonal and polyclonal anti-TPO antibodies were recognized by some monoclonal anti-Tg antibodies and anti-Tg autoantibodies, and conversely, that 19S Tg was recognized by some monoclonal anti-TPO antibodies. TPO was immunoprecipitated by anti-Tg autoantibodies isolated by Tg affinity chromatography. On the other hand, the specificity for TPO of the anti-Tg autoantibodies was not identical with that of anti-TPO autoantibodies. These cross-reactivities were not due to contamination of TPO with Tg or vice versa, or to contamination of the anti-Tg autoantibody preparations with anti-TPO autoantibodies. Taken together, these data indicate that Tg and TPO share common antigenic determinants and that some of those determinants are recognized by autoantibodies in the serum of patients with chronic autoimmune thyroiditis.

Enhanced thyroid iodine metabolism in patients with triiodothyronine-predominant Graves' disease.

Some patients with hyperthyroid Graves' disease have increased serum T3 and normal or even low serum T4 levels during treatment with antithyroid drugs. These patients with elevated serum T3 to T4 ratios rarely have a remission of their hyperthyroidism. The aim of this study was to investigate thyroid iodine metabolism in such patients, whom we termed T3-predominant Graves' disease. Mean thyroid radioactive iodine uptake was 51.0 +/- 18.1% ( +/- SD) at 3 h, and it decreased to 38.9 +/- 20.1% at 24 h in 31 patients with T3-predominant Graves' disease during treatment. It was 20.0 +/- 11.4% at 3 h and increased to 31.9 +/- 16.0% at 24 h in 17 other patients with hyperthyroid Graves' disease who had normal serum T3 and T4 levels and a normal serum T3 to T4 ratio during treatment (control Graves' disease). The activity of serum TSH receptor antibodies was significantly higher in the patients with T3-predominant Graves' disease than in control Graves' disease patients (60.5 +/- 19.2% vs. 20.4 +/- 18.2%; P less than 0.001). From in vitro studies of thyroid tissue obtained at surgery, both thyroglobulin content and iodine content in thyroglobulin were significantly lower in patients with T3-predominant Graves' disease than in the control Graves' disease patients. Thyroid peroxidase (TPO) activity determined by a guaiacol assay was 0.411 +/- 0.212 g.u./mg protein in the T3-predominant Graves' disease patients, significantly higher than that in the control Graves' disease patients (0.129 +/- 0.112 g.u./mg protein; P less than 0.01). Serum TPO autoantibody levels determined by immunoprecipitation also were greater in T3-predominant Graves' disease patients than in control Graves' disease patients (52.6 +/- 27.7% vs. 32.4 +/- 11.4%; P less than 0.05). Binding of this antibody to TPO slightly inhibited the enzyme activity of TPO, but this effect of the antibody was similar in the two groups of patients. The data suggest enhanced iodine metabolism in the thyroid gland of patients with T3-predominant Graves' disease, which may relate to the discordant T3 overproduction in patients with this type of Graves' disease.

Immunocytochemical identification of the prolactin-secreting cells in the teleost pituitary with an antiserum to chum salmon prolactin.

An antiserum raised to highly purified chum salmon (Oncorhynchus keta) prolactin (sPRL) was used to identify prolactin-producing cells in the adenohypophysis of 15 species of teleosts by the immunocytochemical peroxidase-antiperoxidase method. In the chum salmon, the only pituitary cells that reacted with sPRL antibody were the PRL cells organized as follicular structures in the rostral pars distalis. When the antiserum was absorbed with sPRL, on the other hand, no immunoreactive cell was observed in the pituitary, indicating the specificity of the antiserum. Furthermore, the antibody to sPRL reacted only with PRL cells in the pituitaries of three species of salmonids, a plecoglossid, eel, carp, goldfish, killifish, tilapia, and five species of marine fishes, thus showing no species specificity of the antibody among the teleosts tested. The PRL cells of the eel decreased in number and also in immunoreactivity after adaptation to seawater for 1 month. On the other hand, highly immunoreactive PRL cells were observed in the pituitaries of marine fishes, although the cells were much fewer in number than in eels and in other fishes in fresh water.

Isolation and properties of chum salmon prolactin.

A highly purified prolactin (PRL) was isolated from the chum salmon pituitary by extraction with acid acetone, gel filtration on Sephadex G-25 and ion-exchange chromatography on CM-Sephadex C-25 with a yield of 1 mg/g of wet tissue. It was 10-15 times more potent than ovine PRL in sodium-retaining activity for juvenile rainbow trout adapted to 50% seawater. The salmon PRL emerged as a single and symmetrical peak on Sephadex G-100 with Ve/Vo = 2.0. Polyacrylamide gel electrophoresis revealed only one band at pH 4.3, whereas no band was seen at pH 7.5. The isoelectric point was estimated to be 10.3 by gel electric focusing. The circular dichroism spectrum of the salmon PRL was similar to that of tilapia PRL, showing an alpha-helix content of 50%. The salmon PRL had a molecular weight of 23,400 daltons by gel filtration and 22,300 daltons by sodium dodecyl sulfate gel electrophoresis, with a single NH2-terminal residue, isoleucine, and a single COOH-terminal residue, half-cystine. In the sequence comparison with those of mammalian PRLs and growth hormones, the clusters of invariant residues were found in both terminal regions, although the disulfide at NH2-terminal of mammalian PRLs was missing. Specific salmon PRL antisera were prepared in rabbits giving a precipitin reaction against the salmon PRL and a pituitary extract of tilapia in agar diffusion but no cross reaction with purified mammalian PRLs. The antibody was localized specifically in PRL cells of the chum salmon pituitary.

Pancreatic arteriovenous malformation in a patient with gastrointestinal hemorrhage.

A 39-year old woman, with a pancreatic arteriovenous malformation (AVM) which ruptured into the pancreatic duct, was treated successfully with distal pancreatectomy and splenectomy. Histological examinations revealed an AVM with extravasation into the pancreatic ducts. A review of the cases reported to date emphasizes the importance of physical signs, individual histories and angiographic studies for early diagnosis and surgery.