An analysis of osteoporosis in patients with hereditary multiple exostoses.

We analyzed osteoporosis in 20 HME patients. According to the T-score of BMD, 30% and 67.5% of the patients fell in the range of osteopenia in the lumbar spine and femoral neck. Our results indicate HME patients have low bone mass. They do not have abnormal bone metabolism. INTRODUCTION: There are few reports of osteoporosis in hereditary multiple exostoses (HME) patients. Therefore, the purpose of this study was to analyze osteoporosis in HME patients. METHODS: This retrospective cohort study included 20 patients diagnosed with HME. Patients underwent bone mineral density (BMD) measurement of the lumbar spine (n = 20) and femoral neck (n = 40). Bone metabolic parameters, including serum osteocalcin and urinary cross-linked N-telopeptide of type 1 collagen (NTx), were analyzed in all subjects. EXT1 and EXT2 genes were sequenced using genomic DNA. We also examined the correlation between genotype and BMD Z-score and T-score. RESULTS: The mean BMD values of the lumbar spine were 1.085 ± 0.116 g/cm2 (n = 11) in male and 1.108 ± 0.088 g/cm2 (n = 9) in female. The mean BMD values of the femoral neck area were 0.759 ± 0.125 g/cm2 (n = 22) in male and 0.749 ± 0.115 g/cm2 (n = 18) in female. Z-score of most HME patients show < 0, indicating that these patients tend to have low bone mass compared with the age-matched population. According to the T-score of BMD, 30% (6 of 20) and 67.5% (27 of 40) of the patients fell in the range of osteopenia in the lumbar spine and femoral neck areas, respectively. Serum osteocalcin and urinary NTx were in the normal range in most patients. There was no significant correlation between genotypes and Z-score. CONCLUSION: HME patients have low bone mass, especially in the femoral neck area. They do not have abnormal bone metabolism, and there was no correlation between genotypes and Z-score.

Sodium temperature/wind lidar based on laser-diode-pumped Nd:YAG lasers deployed at Tromsø, Norway (69.6°N, 19.2°E).

An Nd:YAG laser-based sodium temperature/wind lidar was developed for the measurement of the northern polar mesosphere and lower thermosphere at Tromsø (69.6N, 19.2E), Norway. Coherent light at 589 nm is produced by sum frequency generation of 1064 nm and 1319 nm from two diode laser end-pumped pulsed Nd:YAG lasers. The output power is as high as 4W, with 4 mJ/pulse at 1000 Hz repetition rate. Five tilting Cassegrain telescopes enable us to make five-direction (zenith, north, south, east, west) observation for temperature and wind simultaneously. This highly stable laser system is first of its kind to operate virtually maintenance-free during the observation season (from late September to March) since 2010.

Improper waste disposal of silver-mercury amalgam.

The objective of this work was to estimate the quantity of mercury residue present in dental amalgam that is generated and discarded in the city of Manaus (Amazon-Brazil). For this purpose, the locations of amalgam usage (10 public and 31 private dental clinics), the method by which the residue is discarded (14 clinics improper disposal), and the analysis of total mercury in the sediment of the controlled landfill (2.68-3 µgHg/g), were described. It was concluded that: there are dental clinics in the city that discard mercury residue into the common waste disposal system, which contravenes health safety standards.

Xenografting of testicular tissue from an infant human donor results in accelerated testicular maturation.

BACKGROUND: Grafting of testicular tissue into immunodeficient mice has been used to differentiate the neonatal testes from different animal species up to the level of complete spermatogenesis; however, this approach has not been successful for human testicular tissue. The aim of this study was to evaluate the capacity for differentiation of infant human testicular tissue grafts. METHODS AND RESULTS: Testicular tissue from a 3-month-old patient with testicular cancer was grafted into immunodeficient nude mice. At the time of grafting, A spermatogonia were the only germ cells present in the testicular tissue. B spermatogonia and first spermatocytes were observed at 7 months and 1 year after grafting, respectively. Positive immunostaining with antibodies against BOULE and CDC25A suggested that spermatocytes in the graft were not arrested but in meiosis. Furthermore, ultrastructural and immunohistochemical analyses showed that the onset of both Sertoli cell maturation and partial differentiation of Leydig cells preceded the appearance of spermatocytes. Differentiation of testicular cells was accelerated compared with in vivo development. CONCLUSIONS: Spermatogenesis in the xenograft of infant human testicular tissues proceeded successfully from the stage of spermatogonial stem cells until pachytene spermatocyte formation. The differentiation of Sertoli cells and Leydig cells was reproduced in a manner similar to that in normal testicular development. Grafting of infant human testicular tissue may be a powerful tool to examine the early period of human spermatogenesis and may pave the way for fertility preservation among infant patients.

100 ps time-resolved solution scattering utilizing a wide-bandwidth X-ray beam from multilayer optics.

100 ps time-resolved X-ray solution-scattering capabilities have been developed using multilayer optics at the beamline NW14A, Photon Factory Advanced Ring, KEK. X-ray pulses with an energy bandwidth of DeltaE/E = 1-5% are generated by reflecting X-ray pulses (DeltaE/E = 15%) through multilayer optics, made of W/B(4)C or depth-graded Ru/C on silicon substrate. This tailor-made wide-bandwidth X-ray pulse provides high-quality solution-scattering data for obtaining photo-induced molecular reaction dynamics. The time-resolved solution scattering of CH(2)I(2) in methanol is demonstrated as a typical example.

A ten-color tube with dried antibody reagents for the screening of hematological malignancies.

INTRODUCTION: The workflow in clinical flow cytometry laboratories must constantly be reviewed to develop technical procedures that improve quality and productivity and reduce costs. Using the Beckman Coulter dry coating technology, we customized a ten-color tube with dried antibody reagents, designated the Duraclone screening tube (DST), for screening hematological malignancies. Here, we compared the applicability, clinical and numerical equivalence, and cost and time required for the technical procedures between the liquid reagents and the DST. METHODS: The DST contains CD4 + Kappa-FITC, CD8 + Lambda-PE, CD3 + CD14-ECD, CD33-PE-Cy5.5, CD20 + CD56-PE-Cy7, CD34-APC, CD19-APC-AlexaFluor700, CD10-APC-AlexaFluor750, CD5-Pacific Blue, and CD45-Krome Orange. We evaluated 20 bone marrow samples, 13 peripheral blood samples, 6 lymph node biopsy samples, 5 fine-needle aspirate samples, 5 cerebrospinal fluid samples, and 1 pleural fluid sample. RESULTS: The DST was useful for more than 60% of our samples. It was able to enumerate the majority of the populations in all types of samples with a statistically acceptable correlation with the liquid reagents. The use of the DST translated into significant time and cost savings of 15.8% and 12.3%, respectively, compared with the use of the liquid reagent. The cost was reduced by $14.36 per sample. CONCLUSIONS: The DST is an efficient solution for screening hematological malignancies with improved quality, productivity, standardization, and sustainability. These improvements could benefit patients by providing faster diagnoses using a higher quality and lower cost reagent.

Estrogen deficiency stimulates B lymphopoiesis in mouse bone marrow.

We have found that an estrogen deficiency causes a marked increase in bone marrow cells. To examine the effect of estrogen on hemopoiesis, we characterized the increased population of bone marrow cells after ovariectomy (OVX). In OVX mice, the percentage of myeloid cells and granulocytes was decreased, whereas that of B220-positive B lymphocytes was selectively increased 2-4 wk after surgery. The total number of myeloid cells and granulocytes did not change appreciably, but that of B220-positive cells was greatly increased by OVX. When OVX mice were treated with estrogen, the increased B lymphopoiesis returned to normal. B220-positive cells were classified into two subpopulations, B220low and B220high. The majority of the B220low cells were negative for the IgM mu chain, whereas most of the B220high cells were mu-positive. OVX selectively increased the precursors of B lymphocytes identified by B220low. mu-negative phenotype, suggesting that an estrogen deficiency stimulates accumulation of B lymphocyte precursors. When bone marrow-derived stromal cells (ST2) were pretreated with estrogen then co-cultured with bone marrow cells in the presence of estrogen, the stromal cell-dependent B lymphopoiesis was greatly inhibited. The present study suggests that estrogen plays an important role in the regulation of B lymphocyte development in mouse bone marrow.

8-1A, a human monoclonal antibody that reacts with intact human chorionic gonadotropin.

The incidence of choriocarcinoma has decreased over time and therapeutic results have improved about 90% complete remission in patients without extensive metastasis. However, some choriocarcinomas metastasize to other organs and show resistance to chemotherapy, having a poor prognosis despite multidisciplinary treatment. Better methods of early diagnosis for recurrence or micrometastasis, and treatment against cases with intractable gestational trophoblastic neoplasia (GTN) are needed to improve the prognosis. Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of two dissimilar subunits and a tumor marker to make a diagnosis and monitor therapeutic effect in GTN. Even when hCG levels in the serum become too low to measure with the hCG beta-CTP system which is the most sensitive assay, there are estimated to be approximately 10,000 trophoblastic cells in the body. Residual trophoblast cells may cause symptoms such as bleeding or undergo malignant transformation to choriocarcinoma. Since most monoclonal antibodies developed so far are murine, administration creates human anti-mouse antibodies, resulting in clinical failure. More recent mouse/human chimeric antibodies or humanized antibodies still possess substantial immunogenicity that makes repeated administration difficult. In the present study, KM mice that can produce completely human monoclonal antibodies were used to prepare hCG-specific human monoclonal antibody. This yielded 8-1A, a human monoclonal antibody capable of reacting with intact hCG. In the future, new diagnostic techniques and treatments for chorionic diseases may be developed using this kind of human monoclonal antibody.

Effects of progesterone on human endometrial carcinoma cells in vivo and in vitro.

The effects of progesterone on the growth and differentiation of human endometrial adenocarcinoma cells (cell lines SNG-P and SNG-M derived from primary and metastatic tumors, respectively) were assessed in vitro and in vivo. Progesterone suppressed their growth and induced cell differentiation in vitro. The suppressive effect of progesterone was stronger in the primary tumor cells than in the metastatic ones. Progesterone produced morphologic changes such as multinucleation, multinucleolation, vacuolation, extensive Golgi apparatus, and papillary arrangement of cells. The cells were transplanted sc into nude BALB/c mice where they produced undifferentiated adenocarcinomas in untreated mice and well-differentiated adenocarcinomas in progesterone-treated ones. Progesterone reduced tumor growth and decreased transplantability in nude mice. This hormone produced no change in the distribution of the chromosome numbers or in the karyology.

Establishment and characterization of HUOT, a human ovarian malignant teratoma cell line producing alpha-fetoprotein.

A cell line designated HUOT was established from a recurrent tumor of human ovarian malignant teratoma. The cell line grew slowly and stably and serial passages were performed 50 times within 35 months. The cells, polygonal or spindle, with neoplastic and pleomorphic features grew in multiple layers and without contact inhibition. Population doubling time was 98 hours and the plating efficiency was less than 6%. The chromosome number varied from 43 to over 256, and the modal number was stable at the hyperdiploid range (52-56). The cultured cells produced anaplastic carcinomas by heterotransplantation into the subcutis of nude mice and were characterized as producing large amounts of alpha-fetoprotein, in vitro, at the stationary growth phase and as forming cystlike structures. Dibutyl cAMP suppressed the cellular proliferation and increased the production of alpha-fetoprotein. Therefore, this HUOT line is expected to have a wide application for various laboratory studies.

Effect of prenatal exposure to bisphenol A on the serum testosterone concentration of rats at birth.

In the rat, testosterone (T) in the neonatal period plays an important role in sexual differentiation and there is a serum T surge in male rats 2 hours after birth. Pregnant female rats were exposed to various doses of bisphenol A (BPA) from gestational day 1 (GD1) through 2 hours after parturition. About half of the BPA-exposed and control dams were subjected to cesarean section on GD22. The male fetuses on GD22 were immediately sacrificed and blood was collected. The other half of the BPA-treated and control dams delivered at GD23 (parturition day). The male pups were sacrificed 2 hours after birth. Serum T concentration was determined by radioimmunoassay (RIA). The BPA concentration in the fetal serum on GD22 increased inversely to the T levels in the serum. The T concentration in the pups' serum 2 hours after birth decreased inversely to the BPA concentration in the serum. However, there were no differences in the serum T concentration among the various doses of BPA. These results suggest that exposure to BPA in utero inhibits the T surge in the neonatal period and we speculate that exposure to BPA in utero disrupts the endocrine environment in the neonatal male.

Effects of 17beta-estradiol and progesterone on growth and morphology of human endometrial carcinoma cells in vitro.

The effects of 17beta-estradiol and progesterone on the rate of growth and the morphological changes of human endometrial adenocarcinoma cells were studied in in vitro culture. 17beta-Estradiol enhanced their growth and produced no cellular morphological changes at low concentrations of less than 1 microgram/ml, whereas it suppressed their growth and produced such cellular changes as enlargement of nuclei, karyorrhexis, and karyolysis at high concentrations of more than 5 microgram/ml. On the other hand, progesterone did not affect the cells at less than 1 microgram/ml, but it suppressed their growth and induced differentiation at more than 5 microgram/ml. Specific morphological changes produced by progesterone were characterized by multinucleation, multinucleolation, prominent Golgi apparatus, occurrence of vacuoles, and papillary-like arrangement of cells. These features suggested that progesterone acted directly on the endometrial carcinoma cells and induced their histological differentiation. These changes could not be detected by the adminstration of 17beta-estradiol.

Complementary DNA cloning for galactosyltransferase associated with tumor and determination of antigenic epitopes recognized by specific monoclonal antibodies.

The galactosyltransferase associated with tumor (GAT) was the name given to the isoenzyme that tends to polymerize resulting in slower moving in a nondenaturing polyacrylamide gel electrophoresis than normal (beta 1-4)galactosyltransferase (normal GalT). A complementary DNA (cDNA) library was constructed from a human ovarian cancer cell line, RMG-I, which secreted an amount of GAT into the culture supernatant and screened with monoclonal antibodies (MAbs) against GAT and normal GalT. One of six cDNA clones, UG86-1, encoded an epitope recognized by a GAT-specific MAb, 8513. Recombinant proteins expressed by UG86-1 in Escherichia coli also had antigenic epitopes recognized by the other MAbs against normal GalT. The 229-base pair nucleotide sequence encoded by UG86-1 was identical to the stem region sequence of HGT832 which encodes a full-length cDNA of human GalT. Using recombinant proteins directed by deletion mutant cDNAs, the antigenic epitopes recognized by each MAb were determined. The epitope of MAb8628, which reacts to both the GAT and normal GalT, was localized to the COOH-terminal side of proteolytic cleavage site where the membrane-bound form enzyme is cleaved to be converted to soluble forms, while MAb8513 epitope was at the NH2-terminal side from this cleavage site between the COOH-terminal end of the membrane-binding domain and the cleavage site. These results demonstrate that GAT is produced by aberrant proteolytic cleavage at the different site, closer to the membrane-binding domain, from the normal GalT.

Development and characterization of established cell lines from primary and metastatic regions of human endometrial adenocarcinoma.

Cell lines designated SNG-P and SNG-M were established from operation specimens of primary and metastatic regions of human endometrial adenocarcinoma. The cell lines grew well without interruption for over 13 months and were subcultivated more than 65 times. They continue to exhibit stable growth. The cultured cells appeared epithelial in shape, showing a pavement arrangement and multilayering without contact inhibition. The cytology revealed anaplastic and pleomorphic features. Upon electron microscopic observation, most of the cultured cells were characterized by highly indented nuclei with multiple large nucleoli and by desmosomal cell contact. The chromosomal number varied widely and showed aneuploidy, but the modal chromosomal number was stable at the diploid range. No marker chromosome could be identified. Both of these cell lines, SNG-P and SNG-M, were transplanted to an immune-depressed hamster cheek pouch and produced a tumor resembling the original.

Subcellular localization of blood group Leb carbohydrate antigen (MSN-1-reactive antigen) in endometrial cancer cells.

MSN-1 is a monoclonal antibody, which was raised by immunizing mice with a human endometrial cancer cell line, SNG-II. The MSN-1 specifically recognizes a blood group carbohydrate antigen, Leb. We investigated the subcellular and suborganellar localization of the MSN-1-reactive carbohydrate antigens in the endometrial adenocarcinomas and the normal endometria using immunofluorescence microscopy, confocal laser scanning microscopy, and immunoelectron microscopy. In normal endometrium, the apical plasma membranes of endometrial glandular cells were weekly positive. In contrast, in endometrial adenocarcinoma specimens, the apical plasma membranes, the lateral plasma membranes, intracytoplasmic vesicular structures, and the Golgi apparatus were strongly positive. We also found that there are differences in the manner of the distribution of the MSN-1 antigen within the Golgi apparatus between the normal and tumor samples; namely, in the endometrial adenocarcinoma cells the antigens are found abundantly throughout the Golgi apparatus or tend to be located not only at the trans side but also close to the cis side, while the localization of this antigen in normal counterpart is restricted to the trans-Golgi cisternae. These findings imply that aberrant glycosylation occurs in endometrial adenocarcinomas, presumably as a result of abnormal expression of some glycosyltransferases involving the expression of the Leb carbohydrate antigen in the Golgi apparatus.

Compensatory occurrence of IV2Fucalpha,II3NeuAcalpha-Gg4Cer and human fetal antigen Lc4Cer in small cell carcinomas of the human lung.

By means of a thin-layer chromatography immunostaining procedure involving a human monoclonal anti-Lc4Cer antibody, which was established by hybridizing murine myeloma cells and human lymphocytes from a cancer patient, Lc4Cer was proven to be a fetal antigen of human lung and to be a cancer-related antigen in small cell carcinomas of human lung, but not of other lung cancers, i.e., large cell carcinomas, adenocarcinomas, and squamous carcinomas. With the simultaneous detection of IV2Fuc alpha,II3NeuAc alpha-Gg4Cer with rabbit anti-IV2Fuc alpha,II3NeuAc alpha-Gg4Cer antiserum, the expression of Lc4Cer and IV2Fuc alpha,II3NeuAc alpha-Gg4Cer was found to be compensatory and, consequently, small cell lung carcinomas could be classified into Lc4Cer- and IV2Fuc alpha,II3NeuAc alpha-Gg4Cer-expressing types, L-SCLC and F-SCLC, respectively, which were detected in four and 27 of 31 patients' tissues and in one and three of four nude mouse-transplanted small cell lung carcinoma tissues, respectively. The compensatory expression of Lc4Cer and IV2Fuc alpha,II3NeuAc alpha-Gg4Cer in small cell carcinomas indicated that different metabolic pathways for glycosphingolipids were activated to give the distinct glycosphingolipid compositions in the two types of small cell lung carcinomas.

Mouse monoclonal antibodies which recognize a human (beta 1-4)galactosyl-transferase associated with tumor in body fluids.

Mouse monoclonal antibodies against human (beta 1-4)galactosyl-transferase (GalT) purified from human ovarian tumor effusion fluids were prepared and characterized. GalT purified from normal human plasma showed a single diffused band in nondenaturing polyacrylamide gel electrophoresis, but GalT purified from human ovarian tumor effusion fluids showed several oligomeric bands and a monomeric band in nondenaturing polyacrylamide gel electrophoresis. These oligomeric bands were dissociated into monomer by urea treatment and polymerized by a 2-mercaptoethanol treatment. Nine monoclonal antibodies (MAb) were prepared by immunization of purified GalT from human ovarian tumor effusion fluids and classified into three groups. Type I MAbs (MAb8611, MAb8913, and MAb8919) reacted only to the GalT monomer. Type II MAbs (MAb4880, MAb8507, and MAb8628) reacted to both the GalT monomer and the GalT polymer. Type III MAbs (MAb7907, MAb8513, and MAb8677) reacted only to the GalT polymer. These MAbs except MAb7907 could recover GalT enzyme activity from effusion fluids by immunoprecipitation. A fraction passed through MAb8513 affinity chromatography still showed reactivity to MAb8919, demonstrating that an epitope of MAb8513 resides on a minor part of GalT. A sandwich immunoassay (MAb8513-MAb8628HRP) was developed, and serum samples from ovarian cancer patients and benign ovarian patients were tested. The levels of sandwich immunoassay of serum samples from cancer were elevated significantly compared to those from benign and did not necessarily correlate to total GalT enzyme activity in serum samples. These results suggested that MAb8513 (Type III) might recognize a unique GalT associated with tumor (GAT).

Glycosphingolipids of various human ovarian tumors: a significantly high expression of I3SO3GalCer and Lewis antigen in mucinous cystadenocarcinoma.

Among several human ovarian tumors, which include mucinous cystadenocarcinoma, serous cystadenocarcinoma, and clear cell adenocarcinoma, the mucinous cystadenocarcinoma showed a unique glycosphingolipid composition. In particular, more than 90% of the acidic glycosphingolipids in the mucinous cystadenocarcinoma is comprised of sulfolipids, which are hardly detected in normal ovary and are contained in concentrations of less than 40% in the other type of ovarian tumors. By means of negative ion fast atom bombardment mass spectrometry and gas liquid chromatography, the major sulfolipid in mucinous cystadenocarcinoma is confirmed to be I3SO3-GalCer with N-cerebronoyl phytosphingosine, that which contrasts with I3SO3-GalCer with N-nonhydroxy fatty acyl sphingosine as the major molecular species in the other ovarian cancers. In mucinous cystadenocarcinoma, galactosylceramide is found in the relatively high concentration and is also composed of N-cerebronoyl phytosphingosine. In addition, the concentrations of glycolipids with Le(a) and Le(b) antigenicities are significantly higher in mucinous cystadenocarcinoma than those in normal ovary and the other ovarian tumors.

Newly established uterine cervical cancer cell line (SKG-III) with Regan isoenzyme, human chorionic gonadotropin beta-subunit, and pregnancy-specific beta 1-glycoprotein phenotypes.

The production of Regan isoenzyme (heat-stable, L-phenylalanine-sensitive term-placental alkaline phosphatase), human chorionic gonadotropin beta-subunit, and pregnancy-specific beta 1-glycoprotein by newly characterized human uterine cervical cancer cell lines, SKG-IIIa and SKG-IIIb, is reported. These cell lines were derived from a moderately differentiated epidermoid cancer partially mixed with epidermoid clear-cell components. At the end of the first 4 months in culture 2 sublines with different morphologies were identified. In nude mice, SKG-IIIa produce clear-cell epidermoid cancer with much glycogen, while SKG-IIIb grew as a moderately differentiated epidermoid cancer rich in tonofilaments. The presence of Regan isoenzyme was established by biochemistry, enzyme cytochemistry, immunocytochemistry, and immunoelectrophoresis. However, the copresence of small amounts of early placental alkaline phosphatase was also demonstrated. The alkaline phosphatase specific activities of SKG-IIIa cells and SKG-IIIb cells were 3.7 and 1.4 nmol per mg protein per min, respectively. The existence was proven by radioimmunoassay of human chorionic gonadotropin beta-subunit (SKG-IIIa, 5.0 mlU/mg protein; SKG-IIIb, 4.4 mlU/mg protein), pregnancy-specific beta 1-glycoprotein (SKG-IIIa, 0.7 ng/mg protein) in the culture media as a tumor cell product. The described cell lines may serve as a more representative model system for studies of regulation of oncodevelopmental genes in gynecological tumors in general and in epidermoid cervical cancer in particular.

Beta-catenin mutation in carcinoma of the uterine endometrium.

Beta-catenin forms complexes with Tcf and Lef-1 and functions as a transcriptional activator downstream of the Wnt signaling pathway. Activation of the pathway by stabilization of beta-catenin has been shown to be important in the development of colorectal carcinoma, which is mainly caused by inactivating mutations of the adenomatous polyposis coli tumor suppressor gene or by activating mutations in exon 3 of the beta-catenin gene. Here, we analyzed mutations in exon 3 of the beta-catenin gene in endometrial carcinoma cases in which loss of heterozygosity at the adenomatous polyposis coli tumor suppressor gene locus has been rarely reported. We found that 10 of 76 cases had beta-catenin gene mutations. All mutations identified were single-base missense mutations on serine/threonine residues (codons 33, 37, 41, and 45), altering the glycogen synthase kinase-3beta phosphorylation consensus motif, which participates in the degradation of beta-catenin. To determine whether these beta-catenin mutations actually led to stabilization of this protein, expression of beta-catenin was analyzed immunohistochemically, and 9 of 10 cases with the beta-catenin mutation and 20 of 66 cases without it showed accumulation of beta-catenin in the cytoplasm and/or nucleus. In total, 38% of cases showed accumulation of beta-catenin. These data indicate that stabilization of beta-catenin due to mutations in exon 3 of the beta-catenin gene and other mechanisms may have an important role in development of endometrial carcinomas.