Application of specific ELISAs for BMP15 and GDF9 to cumulus cell extracts from infertile women.

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific paracrine factors which regulate ovarian cumulus cell (CC) functions. This study aimed to investigate if BMP15 and GDF9 bound to CCs can be characterized, quantified, and show an association with IVF outcomes in infertile women. BMP15 and GDF9 ELISAs were validated and applied to discarded CC extracts. Pooled CCs from individual patients were collected from 120 (cohort 1; BMP15 only) and 81 infertility patients (cohort 2; BMP15 and GDF9) undergoing superovulation. BMP15 and GDF9 levels expressed per CC DNA were correlated with maternal age, clinical and embryology data. Total BMP15 and GDF9 were highly correlated with each other (r = 0.9, p < 0.001). The GDF9:BMP15 ratio was unrelated to oocyte number or age. BMP15/CC DNA and GDF9/CC DNA were unaffected by the type of superovulation and were not related to oocyte/embryo outcomes.

The differentiation pathway of T lymphocytes. Evidence for two differentiated cell types.

Two experimental models have been used to study T-cell differentiation. The first, a graft-vs.-host reaction, was induced by injecting thymocytes or cortisone-resistant thymocytes into lethally irradiated allogeneic mice. The second was tumor graft rejection in allogeneic hosts. Ultrastructural studies at various time intervals revealed two differentiated T-cell types. One of these (the "pale" cell) is probably high cytotoxic as measured in the chromium-release assay, the other (the "dark" cell) may be an "amplifier" cell, helping in the differentiation of cytotoxic cells.

Mouse thymus-independent and thymus-derived lymphoid cells. I. Immunofluorescent and functional studies.

The simultaneous use on mouse lymphoid suspensions of heterologous antisera directed against thymus-derived (T) cell mouse-specific lymphocyte antigen and brain-associated theta antigen (MSLA and BAtheta) or thymus-independent (B) cell mouse-specific bone marrow-derived lymphocyte antigen (MBLA) surface antigens allowed direct proof of the different specificity of these antisera by double immunofluorescence (IF) staining with selective visualization of fluorochromes. These antisera and antisera against mouse Ig and its different types of chains were then used with technique of either double IF staining or IF combined with radioautography, allowing the following conclusions: (a) Surface Ig (sIg) was found exclusively on B cells and never on T cells, but not all B cells had sIg. Cells containing detectable amounts of Ig were MBLA+, but had less sIg than other B cells or none at all. There was evidence for the existence of a significant number of MBLA+ lymphocytes, neither bearing nor containing detectable Ig. (b) micro-Chains were the most frequent but not the only heavy chains found on spleen cells; however, it could not be decided with the technique used, if a single cell can bear more than one type of heavy chain. No cell containing gamma-chains was found to bear surface micro-chains, although a very few cells containing both micro- and gamma-chains were observed. (c) The antigen-binding cells detected after immunization with bacteriophage T4, bovine serum albumin, Maia squinado hemocyanin, and sheep erythrocytes were analyzed for MSLA, MBLA or sIg using double IF, a combination of IF and radioautography, or inhibition of "rosette" formation. Practically all the antigen-binding cells detected were MSLA-, MBLA+, sIg+. (d) More B cells than T cells were found among short-lived lymphoid cells labeled by repeated in vivo injections of tritiated thymidine, but the results did not support a simplified concept equating T cells to long-lived and B cells to short-lived lymphocytes. (e) Cells dividing rapidly in the lymph nodes draining the sites of immunization with various antigens were predominantly T cells 2 days after immunization and in majority B cells a few days later. (f) Incubation of lymphoid cells at 37 degrees C with rabbit anti-mouse Ig or anti-kappa chains led to complete disappearance of sIg and to decrease of MBLA ("antigenic modulation"). In the same conditions, anti-MBLA gave partial modulation of MBLA and of sIg; MBLA, however, reappeared much faster than sIg. No modulation of T cell surface antigens by the appropriate antisera was observed. Cell treatment with Pronase could remove MBLA, sIg, MSLA, and BAtheta, which reappeared within a few hours. Neuraminidase treatment was without detectable effect on these antigens.

Mouse thymus-independent and thymus-derived lymphoid cells. II. Ultrastructural studies.

The ultrastructural features of B-, T-, and surface Ig(sIg)-bearing cells have been studied on cell suspensions from lymphoid organs of mice at different stages of immunization. The cells were identified by exposure to rabbit antibodies against mouse-specific lymphocyte antigens (MSLA) or brain-associated theta antigen (BAtheta) for T cells, mouse-specific bone marrow-derived lymphocyte antigens (MBLA) for B cells, and mouse Ig for sIg-bearing cells. The rabbit antibodies fixed on the cell surfaces were detected by peroxidase-labeled sheep anti-rabbit Ig antibodies or by a "bridge" technique using southern bean mosaic virus or bacteriophage T4 as the final markers. In some experiments, short-lived lymphoid cells were labeled in vivo with repeated tritiated thymidine and the ultrastructural detection of their surface antigens was combined with radioautography. MBLA+ lymphoid cells showed a whole range of ultrastructural patterns. Most were small and medium-sized lymphocytes with a clear cytoplasm containing mono- and polyribosomes, but they comprised also blasts and large cells with various amounts of endoplasmic reticulum, as well as plasma cells at different stages of maturation. sIg-bearing cells appeared to be identical with MBLA+ cells, except that sIg was less easily detectable on large blasts, and only very rarely observed on plasma cells. MSLA+ and BAtheta+ cells fell into three categories. One of them (T(1) cells) consisted of small to medium-sized lymphocytes with a clear cytoplasm and few organelles, mostly monoribosomes. A second consisted of large cells (T(2) cells) characterized by numerous polyribosomes often in a "rosette"-like pattern, occasional dark, membrane-bound granules, and a developing "filamentous network." The third, very characteristic type, (T(3) cells) was represented by dark small to medium-sized lymphocytes, usually containing large amounts of closely packed ribosomes and showing a striking accumulation of filamentous network, often condensed in large areas devoid of cell organelles. This filamentous network appeared to correspond to the cytochalasin B-sensitive system of microfilaments found in other cells and considered to be one of the contractile elements of the cell. The T(3) lymphocytes showed frequently vesicles suggestive of a strong pinocytic activity, and assumed a variety of shapes, including uropods. Evidence is presented that T(1) lymphocytes represent "virgin" T cells, T(2) "activated," and T(3) "differentiated" lymphocytes.

A morphometric study on the nexus of rat cardiac muscle.

A morphometric study of the nexus of rat cardiac muscle was carried out. The nexus surface of one intercalated disk of one 15 microm thick fiber is found to range between 47 microm(2) and 94 microm(2), the latter value taking into account the maximal underestimation caused by tangential sectioning. Dividing the lower, minimal value by the surface of the observed subunits (90 A periodicity), one obtains for one intercalated disk 6.7 x 10(5) subunits, each of them assumed to be permeated by a central pore. These pores are thought to be equivalent to intercellular channels in a recently proposed model. Taking our morphometric and recently reported physiological values, this model is examined for its consistency with a low resistance pathway between cardiac muscle cells.

Red sea drillings.

Recent drilling in the Red Sea has shown that much of the basin is underlain by evaporites of a similar age to that of evaporites found in the Mediterranean Sea. These evaporites and their structural positions indicate that other brine areas are present-and, indeed, several others have been discovered.

Interaction of retinoic acid and 3-methylcholanthrene on the fine structure of mouse prostate epithelium in vitro.

The effects of 3-methylcholanthrene (MCA) and retinoic acid (RA) on the fine structure of AKR mouse prostate epithelium in organ culture were correlated with changes in cell proliferation. In intact glands before explantation, the epithelial cytoplasm showed concentric flat or globular cisternae of endoplasmic reticulum in both supranuclear and basal areas, a well-developed Golgi complex, secretory vesicles, and numerous microvilli at the luminal surface. After explantation, the cytoplasmic organelles, particularly the endoplasmic reticulum, regressed and tonofilaments appeared. The regression was largely prevented by RA. MCA induced considerable epithelial hyperplasia and squamous metaplasia. The fine structure of the newly formed cells revealed a complete loss of endoplasmic reticulum, Golgi apparatus, secretory vesicles, and microvilli, with the appearance of bundles of tonofilaments and a striking increase in the number of desmosomes. Administration of RA to explants pretreated with the carcinogen partially reversed the hyperplasia and squamous metaplasia. The tonofilaments disappeared and the number of desmosomes greatly decreased, whereas endoplasmic reticulum, Golgi complex, secretory vesicles, and microvilli were largely reestablished. Planimetric measurements of the alveolar epithelium showed that the squamous transformation and its partial reversal by RA coincide with the rise and decline of epithelial hyperplasia. The data suggest that the restoration of secretory differentiation by RA was responsible for the initial breakdown of the hyperplastic epithelium, whereas the lowering of DNA synthesis by RA prevented further hyperplasia and kept cell replication within normal limits.

Retinoic acid-binding protein in human breast cancer and dysplasia.

Seventy-five specimens of human breast tissue were checked for the presence of cellular retinoic acid-binding protein (cRABP). Fifty-two percent of the primary carcinomas and 43% of the dysplastic breast lesions (stage MII) contained detectable amounts of crabp, whereas no cRABP was found in normal tissue. Sucrose gradient centrifugation and electrophoresis on agarose were used for analysis of the presence of cRABP. The cRABP of human origin (normal uterus and neoplastic mammary tissue) differed in its mobility in agarose electrophoresis from that of rat testis cRABP.

CGS 16949A, a new nonsteroidal aromatase inhibitor: effects on hormone-dependent and -independent tumors in vivo.

CGS 16949A is a very potent and highly selective inhibitor of the aromatase enzyme system in vitro and of estrogen biosynthesis in vivo. These characteristics are reflected in the marked efficacy with which it affects growth of estrogen-dependent 7,12-dimethylbenz(a)antracene-induced mammary carcinomas in intact female Sprague-Dawley rats. Daily p.o. treatment of tumor-bearing rats for 42 days with CGS 16949A at doses of 1.0 to 8.0 mg/kg caused almost complete regression of palpable tumors and almost totally suppressed the appearance of new tumors. A dose of about 0.1 mg/kg corresponded to the 50% effective dose, and a fully effective dose was estimated to be about 2.0 mg/kg. Eight to 10 days after cessation of treatment, tumor regrowth was observed. No unexpected side-effects were noted during the course of treatment. Tumors, which were allowed to regrow after a first treatment with CGS 16949A, were similarly efficaciously suppressed with a second treatment with CGS 16949A. Continuous long-term treatment with 2.0 mg/kg for 27 wk caused complete regression of tumors, suppressed the appearance of new tumors completely, and significantly prolonged the survival time of the tumor-bearing rats. This treatment schedule caused no major hematological or blood chemistry changes and was very well tolerated. CGS 16949A was ineffective against transplantable hormone-independent tumors such as R-3230AC mammary carcinoma, 11095 prostate carcinoma, leukemia L1210, and B16 melanoma.

New S-adenosylmethionine decarboxylase inhibitors with potent antitumor activity.

Methylglyoxal bis(guanylhydrazone) (MGBG) has been studied clinically as an antitumor and antileukemic agent and is recognized as a potent but nonspecific inhibitor of the key polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (SAMDC). A series of four SAMDC inhibitors with structural features similar to MGBG have been found to have improved potency and specificity toward the target enzyme, SAMDC. Relative to MGBG, the new derivatives were much more effective in inhibiting partially purified preparations of SAMDC (50% inhibitory concentration, 10 to 100 nM), much less effective at inhibiting diamine oxidase, and inactive toward ornithine decarboxylase. The inhibitors varied relative to MGBG in their ability to compete with spermidine for uptake, with two being similar and two being less effective. Against L1210 leukemic cells and T24 bladder carcinoma cells, the compounds were slightly less effective than MGBG at inhibiting cell growth, with 50% inhibitory concentration values of 1 to 10 microM as compared with 0.5 and 1.1 microM, respectively, for MGBG. Under 50% growth-inhibitory conditions, the inhibitors decreased SAMDC activity, increased ornithine decarboxylase activity and putrescine pools, and markedly depleted spermidine and spermine pools of L1210 cells. At the same time, mitochondrial integrity as assessed by whole-cell pyruvate oxidation and mitochondrial DNA content was not affected as it was with MGBG. At doses less than one tenth that of the maximally tolerated dose, all of the new inhibitors strongly suppressed the growth of B16 melanoma in vivo with minimal weight loss or toxicity. At doses less than one sixth the maximally tolerated dose, they effectively inhibited the growth of T24 human bladder carcinoma xenografts. In these same systems, MGBG showed only marginal antitumor activity. These studies identify two potent and efficacious inhibitors of SAMDC as potential antitumor agents and reaffirm the importance of SAMDC as a target in anticancer drug discovery.

Wnt addiction of genetically defined cancers reversed by PORCN inhibition.

Enhanced sensitivity to Wnts is an emerging hallmark of a subset of cancers, defined in part by mutations regulating the abundance of their receptors. Whether these mutations identify a clinical opportunity is an important question. Inhibition of Wnt secretion by blocking an essential post-translational modification, palmitoleation, provides a useful therapeutic intervention. We developed a novel potent, orally available PORCN inhibitor, ETC-1922159 (henceforth called ETC-159) that blocks the secretion and activity of all Wnts. ETC-159 is remarkably effective in treating RSPO-translocation bearing colorectal cancer (CRC) patient-derived xenografts. This is the first example of effective targeted therapy for this subset of CRC. Consistent with a central role of Wnt signaling in regulation of gene expression, inhibition of PORCN in RSPO3-translocated cancers causes a marked remodeling of the transcriptome, with loss of cell cycle, stem cell and proliferation genes, and an increase in differentiation markers. Inhibition of Wnt signaling by PORCN inhibition holds promise as differentiation therapy in genetically defined human cancers.

Markers of tumor angiogenesis and proteolysis independently define high- and low-risk subsets of node-negative breast cancer patients.

PURPOSE: To compare the prognostic impact of tumor angiogenesis factors (vascular endothelial growth factor [VEGF], angiogenin, and basic fibroblast growth factor [bFGF]), tumor proteolysis factors (urokinase-type plasminogen activator [uPA] and plasminogen activator inhibitor-1 [PAI-1]), and conventional tumor markers (stage, grade, and steroid receptors) in early breast cancer. PATIENTS AND METHODS: In the primary clinical study, tumor angiogenesis and other factors were detected in frozen biopsies from 305 primary breast tumors. VEGF expression was assessed by chemiluminescence immunosorbent assay (ICMA); angiogenin, bFGF, uPA, and PAI-1 by enzyme-linked immunosorbent assay (ELISA); and steroid receptors (estrogen receptor [ER] and progesterone receptor [PgR]) by enzyme immunoassay (EIA). In the validating clinical study, another set of 190 node-negative primary breast tumor samples were collected at a separate institution. RESULTS: Univariate analysis of the primary study showed that VEGF levels were positively correlated with recurrence (P < .001). Angiogenin levels were positively correlated with disease relapse (P < .005) for the overall collective group, but not within the node-negative subset. No significant correlations were found between tumor bFGF levels and patient survival. In multivariate regression analysis, the only independent predictors of relapse-free survival (RFS) were VEGF, uPA, and lymph node status. In the validation set, the distribution of VEGF and uPA values were similar to those in the primary study; low expression of both VEGF and uPA identified patients with a < or = 20% likelihood of recurrence within 7 years. CONCLUSION: Separate primary and validating clinical studies concur that tumor VEGF level is the most important prognostic parameter among several markers of tumor angiogenesis and proteolysis.

Culture conditions for induction of suppressor cells in vitro.

Culture conditions have been established for optimal generation of cytotoxic T lymphocytes (CTL) in one-way mixed lymphocyte cultures (MLC). The responder cell concentration was found to be most critical. In general the more active the proliferation was, the lower was the optimum for CTL generation. Optimal proliferation was usually found to occur at lower cell concentrations than optimal CTL generation. Above the optimal CTL generation proliferation also decreased, most likely due to overcrowding. These observations were made on both unpurified and nylon wool-passaged spleen cells, though the latter generally produced higher levels of cytotoxic activity per equal number of harvested cells. Cells prestimulated for 2 or 4 days in the absence or presence of antigen (M-locus differences) were unable to mount an effective response when restimulated with another antigen presenting an H-2 difference. This hyporesponsiveness was not due to the exhaustion of the culture medium or the presence of dead cells (since these were removed before restimulation). A high degree of hyporesponsiveness was also induced in the absence of antigen during prestimulation which rendered antigenic competition highly unlikely. Overcrowding could be excluded since the observed inhibition could not be diluted out by lowering the responder cell concentrations. A carry-over of CTL destroying newly added antigen (Fitch et al., 1975) could not account for our observations since in the system utilized (M-locus stimulation) there was no generation of CTL (Festerstein, 1973; Röllinghoff et al., 1975). The development of suppressor cells would seem the most likley explanation of the data.

Protection against murine ascites tumours by lymphoid cell populations with T memory or cytotoxicity.

It was possible to protect irradiated C3H mice against lethal doses of allogeneic ascitic tumor cells (RBL-3 or P-815 Y) by systemic administration of low doses of syngeneic sensitized lymphoid cells. Two types of cell populations were active at similar doses: (1) spleen cells harvested 2 months after a nonlethal inoculation of tumour cells, at which time no cytotoxic lymphocytes wer in vivo sensitization or in vitro sensitized spleen cells; sucll and dose dependent and target cell specific. The findings imply that T memory cells as well as cytotoxic cells are able to protect, although it was not possible to exclude the presence of memory lymphocytes in the cytotoxic cell population. Antiserum to cytotoxic T cells (CTL) was prepared in an attempt to distinguish memory and cytotoxic effector cells. The antiserum did not react with the majority of T cells in a normal spleen or thymus, but showed specificity for the CTL cell lineage. Antiserum treatment and complement abolished protection in all types of sensitized cell populations. Since memory and cytotoxic cells appeared to share differentiation antigens, we could not establish whether memory cells are precursors or products of cytotoxic cells.

Cytogenetics of breast cancer.

Chromosome counts were performed on 1,100 cells from 17 malignant breast carcinomas and on 168 cells of four normal tissue samples after amethopterin treatment and G-banding. Karyotypes were established from 216 cells of 11 tumor-derived cultures and from 47 cells of four nonmalignant tissue-derived cultures. Karyotypes of cells from nonmalignant samples showed a normal diploid chromosomal constitution with no consistent loss or gain of a specific chromosome. Structural chromosomal abnormalities were not observed. Tumor-derived cultures could be distinguished from normal cultures on the basis of a significantly increased incidence of numerical changes and structural chromosomal aberrations. In nine of 11 tumor-derived cultures, numerically normal cells were shown to be pseudodiploid, with frequencies ranging to 43% (mean, 13.2%) of the diploid cells. In agreement with previous reports, cytogenetic analyses showed predominantly diploid cells. Clonal numerical changes of chromosomes 17, 18, 20, and 21 could be detected in three tumor samples. Clonal structural abnormalities could be observed in two of 11 analyzed tumours. A t(6;12)(p21;p13) and an enlarged chromosome 7 (7q+) were found in a patient with invasive ductal carcinoma. An inversion of chromosome 7 [inv(7)(q11.2q32)] was observed in one case, also diagnosed as invasive ductal carcinoma. The significance of these findings in relation to clinical data is discussed.

CGP 6809, a sugar-containing nitrosourea derivative: pharmacological and physicochemical properties.

CGP 6809 is a water-soluble nitrosourea derivative with quite distinct chemical and biological properties as compared with the well-known representatives of this class of compounds. It is related to the antibiotic streptozotocin, from which it is distinguished in the structure of the sugar moiety and the position of the methylnitrosourea residue. CGP 6809 possesses practically the same alkylating potential as streptozotocin; however, its carbamoylating activity is comparable with that of CCNU. In contrast to other nitrosourea derivatives, CGP 6809 showed relatively little activity in murine leukemias but was markedly active in solid transplantable melanomas (Harding-Passay, B16), in the 11095 prostate carcinoma, and in a substrain of Yoshida hepatoma (AH 7974) resistant to BCNU and CCNU. In the Ehrlich and Yoshida ascitic tumors complete responses were seen with no toxic death. Dose-dependent activity was found in the human lung carcinoma MBA 9812 and almost complete growth inhibition was achieved in the human melanoma WM 47 by both the oral and parenteral routes of administration. However, mammary tumor lines (Ca 755, 2661/61, R-3230AC), the Guerin-T8 uterus epithelioma, and the Rous sarcoma/S-R proved to be relatively refractory to this drug. This was also the case for the Lewis lung carcinoma implanted i.m. or s.c. However, development of lung metastases was markedly inhibited. Combination therapy using CGP 6809 with cyclophosphamide, 5-fluorouracil, or chlorambucil in the same model led to partial responses of the primary tumor as well as almost total eradication of lung metastases.

Interactive image-guided resection of cerebral cavernous malformations.

Between July 1992, and February 1997, 15 patients with cavernous malformations underwent interactive image-guided resection of their lesions. There were eight women and seven men in the group, ranging in age from 6 years to 62 years (mean 34 years). Clinical presentations included seizures (n = 7), headache (n = 4), and hemorrhage (n = 4). Prior conventional subtotal resection had been performed in one patient, and a history of prior hemorrhage was found for two patients. Diagnosis was made using magnetic resonance imaging and digital substraction angiography. Locations of the lesions were temporal (n = 9), frontal (n = 3), thalamus (n = 1), basal ganglia (n = 1), and pons (n = 1). Size ranged from 9 to 20 mm (mean 12 mm). For those lesions located near or within eloquent areas (n = 7), an awake craniotomy with functional cortical and subcortical mapping was performed. An infrared system was used intraoperatively to confirm the location and the extent of the resection of these lesions in real time. In 1996 we started using a robotic microscope to aid in localization and resection. Clinical follow-up ranged from 2 to 54 months (mean 27 months). In all 15 patients, complete surgical resection was achieved as demonstrated by postoperative magnetic resonance imaging studies. Two patients had postoperative transient neurological deficits (13.3%) that cleared over a 6-month period. One of them had a lesion in the pons, with multiple cranial nerve deficits postoperatively that gradually improved. There was no associated mortality. Histological diagnosis was consistent with cavernous angioma in all cases. Clinical follow-up revealed that 13 patients experienced complete recovery from preoperative symptoms and two patients with seizures showed marked improvement. We conclude that interactive image-guided surgery for deep-seated cavernous malformations represents a very accurate and safe approach.

Interactive image-guided surgical resection of intracranial arteriovenous malformations.

Surgical excision is the only treatment method that immediately prevents increased morbidity or mortality as a result of hemorrhage from arteriovenous malformations (AVMs). For those lesions located deep within the cerebral hemispheres or near eloquent areas, conventional surgical resection may be associated with an unacceptable degree of morbidity and mortality. Herein we report our experience in the resection of these lesions using interactive image guidance. There were five women and five men in the patient group. Their age ranged from 16 to 73 years (mean = 41). Clinical presentation included hemorrhage (n = 7), headaches (n = 2), and seizures (n = 1). All lesions were classified using the Spetzler-Martin grading system as follows: grade I (n = 4), grade II (n = 5), and grade III (n = 1). The locations of the lesions were supratentorial (9) and infratentorial (1). Surgical planning was carried out using the Neurological Surgery Planning System software developed at Wayne State University. An infrared-based system was used to locate and define the lesion intraoperatively. For those lesions located near or within eloquent areas, an awake craniotomy with functional mapping was carried out. Clinical follow-up ranged from 3 to 62 months (mean = 34). Complete surgical excision was achieved in all patients, which was demonstrated postoperatively by digital substraction angiography. The preoperative neurological status remained unchanged in seven patients and improved in three. There was no associated morbidity and mortality with this technique. Image-guided surgical resection of arteriovenous malformations represents a valuable technique, especially in small deep-seated lesions and in those near eloquent areas.

Obesity among secondary school students in Bahrain.

The aim of this study is to estimate the prevalence of obesity and factors associated with it in Bahraini secondary school students. A cross-sectional study involving a sample of 825 students (417 boys and 408 girls) aged 15 to 21 years was obtained from secondary schools. Obesity was determined using body mass index (BMI = Wt/Ht2). The findings revealed that 15.6% of boys and 17.4% of girls were either overweight or obese (BMI > or = 25). Family size, parents education, and family history of obesity were significantly associated with obesity among boys, while family history was the only socio-economic factors statistically associated with obesity among girls. Meal patterns such as eating between meals, number of meals per day, and method of eating were not associated with obesity in students. Boys who ate alone were 3 times more likely to be obese than those who ate with family members (odd ratio = 3.4). Measures to prevent and control obesity among children are suggested.