Neuroblastomas vary widely in their sensitivities to herpes simplex virotherapy unrelated to virus receptors and susceptibility.

Although most high-risk neuroblastomas are responsive to chemotherapy, relapse is common and long-term survival is < 40%, underscoring the need for more effective treatments. We evaluated the responsiveness of 12 neuroblastoma cell lines to the Δγ134.5 attenuated oncolytic herpes simplex virus (oHSV), Seprehvir (HSV1716), which is currently used in pediatric phase I trials. We found that entry of Seprehvir in neuroblastoma cells is independent of the expression of nectin-1 and the sum of all four known major HSV entry receptors. We observed varying levels of sensitivity and permissivity to Seprehvir, suggesting that the cellular anti-viral response, not virus entry, is the key determinant of efficacy with this virus. In vivo, we found significant anti-tumor efficacy following Seprehvir treatment, which ranged from 6/10 complete responses in the CHP-134 model to a mild prolonged median survival in the SK-N-AS model. Taken together, these data suggest that anti-tumor efficacy cannot be solely predicted based on in vitro response. Whether or not this discordance holds true for other viruses or tumor types is unknown. Our results also suggest that profiling the expression of known viral entry receptors on neuroblastoma cells may not be entirely predictive of their susceptibility to Seprehvir therapy.

In situ labeling of granule cells for apoptosis-associated DNA fragmentation reveals different mechanisms of cell loss in developing cerebellum.

We have examined the role apoptosis plays during postnatal development of the mouse cerebellum by a new method utilizing T7 DNA polymerase for the in situ detection of DNA fragmentation associated with cell death. Granule cell loss between the third and fifth postnatal weeks, hypothesized to affect the granule cell to Purkinje cell stoichiometry, is not associated with DNA fragmentation. However, cerebellar granule cells undergo extensive nuclear DNA fragmentation between postnatal days 5 and 9. Cell death prior to synaptogenesis may help regulate granule cell number. Our results suggest that different mechanisms of cell death within the same neuronal cell population occur during development.

Expression of Fas ligand by human cytotrophoblasts: implications in placentation and fetal survival.

Recent data indicated that local production of Fas ligand (FasL) by cells of the eye and testis may confer immune tolerance at these sites. In the present study, we examined the in vivo and in vitro patterns of expression of FasL in the human placenta to provide a potential mechanism through which the fetus is afforded protection against the cytolytic actions of lymphocytes present within maternal decidua across gestation. Immunohistochemical staining of first trimester human placental tissue sections revealed the presence of FasL in cytotrophoblasts in free floating villi, anchoring villi, and cytotrophoblastic islands. FasL staining was also pronounced in syncytiotrophoblasts of term placenta indicating that FasL expression is maintained across gestation. Multiple molecular forms of FasL, suggestive of altered patterns of glycosylation, were detected in extracts of term placenta, amnion and chorion by Western blotting. In addition, in vitro expression of FasL was demonstrated to increase 2 to 3-fold during differentiation of primary cultures of cytotrophoblasts isolated from human term placentas. Local production of FasL by human cytotrophoblasts provides a mechanism through which cytotrophoblasts may induce immune tolerance and self-regulate survival during invasion and subsequent placentation.

Apoptosis and Fas expression in human fetal membranes.

Apoptosis (i.e. programmed cell death) plays a key role in maintaining reproductive function in the ovary, mammary and prostate glands, uterus, and testis. The purpose of the present report was to determine, based on biochemical and morphological parameters, whether cells in human fetal membranes undergo apoptosis and express Fas (CD95), a cell surface receptor that mediates apoptosis. Using the terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling immunohistochemical technique, apoptotic nuclei were identified in amnion epithelial, chorionic trophoblast, and decidua parietalis cell layers of human fetal membranes at term. Electron microscopy of fetal membranes revealed ultrastructural characteristics in amnion epithelium and chorion trophoblast cell layers consistent with apoptosis, including condensation of chromatin along the periphery of the nucleus and nuclear shrinkage. The apoptotic index (percentage of terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling-positive nuclei of the total nuclei) ranged from 8-29% in amnion epithelial, chorionic trophoblast, and decidual cell layers from women at 23-30, 31-36, and 37-42 weeks gestation. The apoptotic index was statistically greater in the 37-42 week group than in the 23-30 week group in chorionic trophoblast (P < 0.05) and decidual cell (P < 0.01) layers. In contrast, the apoptotic index in the amnion epithelial cell layer was statistically greater (P < 0.05) in the 23-30 week group than in the 31-36 week group, suggesting that apoptosis may be independently regulated in amnion epithelial, chorionic trophoblast, and decidual cell types. Based on the importance of Fas in mediating apoptosis, we investigated whether Fas was expressed by human fetal membrane cells. Immunohistochemical staining of fetal membranes with anti-Fas antibody localized Fas in amnion epithelial, chorionic trophoblast, and decidua parietalis cell layers. A 266-bp band corresponding to the cytoplasmic domain of Fas was detected in samples of amnion, chorion, decidua, and placenta by RT-PCR. Northern blotting revealed a molecular weight of approximately 1.9 kilobases for Fas messenger ribonucleic acid in amniotic tissue. These data suggest that apoptosis and Fas signaling may play a role in remodeling of fetal membrane architecture across gestation.

Religious coping and depression among elderly, hospitalized medically ill men.

OBJECTIVE: The investigators examined the frequency of religious coping among older medical inpatients, the characteristics of those who use it, and the relation between this behavior and depression. METHOD: The subjects were 850 men aged 65 years and over, without psychiatric diagnoses, who were consecutively admitted to the medical or neurological services of a southern Veterans Administration medical center. Religious coping was assessed with a three-item index. Depressive symptoms were assessed by self-rating (the Geriatric Depression Scale) and observer rating (the Hamilton Rating Scale for Depression). RESULTS: One out of every five patients reported that religious thought and/or activity was the most important strategy used to cope with illness. Variables that were associated with religious coping included black race, older age, being retired, religious affiliation, high level of social support, infrequent alcohol use, a prior history of psychiatric problems, and higher cognitive functioning. Depressive symptoms were inversely related to religious coping, an association which persisted after other sociodemographic and health correlates were controlled. When 202 men were reevaluated during their subsequent hospital admissions an average of 6 months later, religious coping was the only baseline variable that predicted lower depression scores at follow-up. CONCLUSIONS: These findings suggest that religious coping is a common behavior that is inversely related to depression in hospitalized elderly men.

Induction of immediate-early gene expression in preoptic and hypothalamic neurons by the glucocorticoid receptor agonist, dexamethasone.

Glucocorticoid receptors (GR) exist in several preoptic and hypothalamic nuclei that participate in neuroendocrine control of anterior pituitary function. GR may mediate effects of endogenous steroids on hormone secretion, since intracerebral administration of exogenous ligands alters plasma levels of several pituitary hormones. The following studies utilized selective antisera for the transcriptional proteins, Fos and Jun, to examine whether these immediate-early gene products are upregulated in response to the GR agonist, dexamethasone (DEX). DEX was administered to groups of male rats by either a subcutaneous (s.c., 5.0 mg/kg) or intracerebroventricular route (i.c.v., 10.0 microg/rat); matched controls received vehicle only. Two hours later, the rats were sacrificed by transcardial perfusion, and serial 25 microm sections through the preoptic area and hypothalamus were processed by avidin-biotin immunocytochemistry for Fos- and Jun-like proteins. Animals treated with DEX i.c.v. exhibited Fos-like immunoreactivity (-li) in several sites in close proximity to the third ventricle, including the preoptic and anterior hypothalamic nuclei, and the periventricular zone of the paraventricular nucleus. In the same group, Jun-li was detected only in the arcuate and suprachiasmatic nuclei. Subcutaneous injection of DEX resulted in more widespread immunostaining for Fos, which occurred in lateral, as well as medial, loci in the preoptic area and hypothalamus, whereas Jun-li was restricted to only medial sites. These data show that discrete populations of preoptic/hypothalamic neurons express c fos and/or jun in response to GR activation. The differential distribution of Fos-li following s.c. vs. i.c.v. administration of DEX suggests that steroid induction and/or amplification of this cellular signaling cascade may depend upon resultant hormone concentrations in neural tissue. In addition, the wide pattern of immunolabeling for Fos in the systematically treated group may reflect both central and peripheral (indirect) steroid effects. Additional studies are in progress to characterize those neurons within sites of neuroendocrine significance that exhibit possible upregulation of these regulatory gene products in response to GR stimulation.

Heterogeneity and modulation of tumor-associated antigens in human glioblastoma cell lines.

Seven human glioblastoma cell lines established in vitro from primary tumor explants were studied. A marked heterogeneity of glial fibrillary acidic protein was observed whereas vimentin was uniformly expressed by all cell lines. Indirect immunofluorescence and flow cytofluorometry revealed a heterogeneous distribution of surface GE 2 and CG 12 tumor-associated antigens (TAA's): three cell lines were positive (greater than 69% TAA-positive cells) and three cell lines were negative (less than 9% TAA-positive cells). One cell line (Hu 228) was moderately positive at early culture passages and subsequently acquired a TAA-negative phenotype. The difference in the relative amounts of surface TAA's of the three positive cell lines was less than twofold. In spite of the heterogeneous distribution of surface TAA's, all cell lines exhibited considerable amounts of intracellular TAA. Treatment with phorbol esters and density-dependent growth arrest decreased the percentage of the TAA-positive cells and the amount of cell-surface TAA's in one cell line (Hu 195). Interferon-gamma treatment in vitro increased the percentage of CG 12-positive cells by 12% and the amount of cell-surface CG 12 antigens by 38% as compared to untreated cells. The percentage of TAA-positive cells among phorbol ester-treated cells of the Hu 195 cell line was lowest 48 hours after treatment, but returned to normal values within the next 48 hours. Reduction of 3H-thymidine incorporation preceded the decrease in number of TAA-positive cells by about 18 hours. Two-color fluorescence analysis performed in positive cell lines for simultaneous determination of surface TAA's and deoxyribonucleic acid content or reactivity with the proliferation-associated Ki67 intracellular marker indicated that GE 2 and CG 12 antigens are expressed preferentially by actively proliferating glioma cells. The results of this study indicate the existence of two different phenotypes in cultured human glioblastoma cells: surface TAA-positive/cytosol TAA-positive and surface TAA-negative/cytosol TAA-positive cell populations. In addition, modulation of TAA expression was dependent on the cell-cycle differentiation stage, culture conditions, and proliferative state of the cells.

Morphological heterogeneity and phenotype modifications during long term in vitro cultures of six new human glioblastoma cell lines.

Long term in vitro cultures of six human malignant gliomas were established to obtain permanent lines and to assess, under conditions of prolonged culture, changes in morphology and phenotype of neoplastic cells and the extent of these modifications. We analyzed expression of the following markers by immunocytochemistry: glioma-specific antigens (GE2 and CG12), fibronectin, intermediate filaments (GFAP, vimentin, neurofilaments), class I and II histocompatibility antigens (HLA-ABC and HLA-DR), growth factor and receptor (alpha TGF and EGF-receptor), proliferation-associated antigen (Ki-67). Strong and stable staining with the two antiglioma monoclonal antibodies (GE2 and CG12) was seen, with coexpression of GFAP and fibronectin in five of six cell lines (after 20 passages) and presence of vimentin and neurofilaments. HLA-DR expression was heterogeneous, with a peculiar intracellular compartmentation in four of six cell lines. Cells showed clear cytoplasmic positivity for alpha TGF and strong membrane staining for EGF-receptor. In previous studies we showed that these cell lines have increased copies of chromosome 7; therefore we speculate that an autocrine pathway of stimulation may maintain the neoplastic growth. The percentage of Ki-67 positive proliferating cells ranged from 40 to greater than 60%, depending on cell line and passage. A slight decrease in the positivity of some markers (GFAP, vimentin and HLA-DR in 2/6 cell lines) was observed after prolonged in vitro culture (greater than 12 months), but morphophenotypic modifications, established within a few passages after explanation, were maintained with time. A clonogenic assay showed values of plating efficiency (PE) higher than corresponding values of other similar cell lines with a tendency to increase in the late passages. PE and Ki-67 positivity were not associated with tumorigenicity into nude mice (except the Hu 197 cell line). These results indicate that, in culture, all six cell lines acquired stable morphology, a well defined antigenic phenotype and high growth rate. Further studies will be performed on these permanent cell lines to clarify differentiation steps of malignant gliomas.

Establishment and characterization of a new human melanoma cell line (HU 214) with a high growth potential and stable properties.

A human melanoma cell line (HU 214) with high growth potential was established from a lymph node metastasis of a patient with advanced cutaneous melanoma. The cells of this line were able to grow in monolayer (according to the Rosenblum technique) and in agar (according to the Courtenay-Mills method), and formed tumors when injected in nude mice. The line has been maintained in culture for more than 47 passages. The cell cultures were periodically characterized (every 6-8 passages) by immunohistochemistry using a panel of monoclonal antibodies (MoAbs) including MoAbs against tumor-associated antigens (antimelanoma, antiglioma and anti-LLA), against vimentin, and against major histocompatibility antigens, and including also Ki 67, a MoAb which reacts with a nuclear antigen associated with cell proliferation. The results of this characterization indicate that we have established a human melanoma cell line with a stable antigenic phenotype during subculturing, poorly differentiated cells, and a high growth potential.

Massive ovarian edema associated with ovarian serous cystadenoma: a case report and review of the literature.

Massive ovarian edema is a rare entity that can be confused with an ovarian neoplasm. A few ovarian lesions have been reported that are associated with massive ovarian edema. This article describes the first case of an ovarian serous cystadenoma associated with a massive ovarian edema. The patient was a 17-year-old female who was referred to the emergency room because of lower abdominal pain. Subsequent ultrasound and computed tomography scanning studies revealed an abdominopelvic cystic mass suggestive of an ovarian neoplasm. She underwent an exploratory laparoscopy, and a left salpingo-oophorectomy was performed. The specimen weighed 1610 g and consisted of a cystic mass measuring 17 x 15 x 8 cm attached to a solid mass measuring 13 x 11 x 4 cm. Microscopy revealed a cystic and a solid lesion. The cystic structure was composed of a flat or cuboidal single-layer lining showing ciliated epithelium and focal areas of papillary structures compatible with a diagnosis of ovarian serous cystadenoma. The solid mass had an intact capsule and diffuse interstitial edema, preserving the overall structure of the ovary and sparing the outer cortex. These findings are compatible with the diagnosis of ovarian massive edema. This report of an association of serous cystadenoma with massive ovarian edema broadens the histologic spectrum in which a massive ovarian edema may be encountered.

Immunohistochemical characterization of nurse cells in normal human thymus.

Lymphoepithelial complexes known as thymic "nurse" cells (TNC) have been isolated and described in the thymus of several animal species including man. Most of the investigations on TNC have been carried out in enzymatically digested thymuses in which TNC were isolated by differential sedimentation. In the present study we demonstrate TNC in immunohistochemically stained sections of human thymus as ring-shaped cells completely enclosing thymocytes and localized not only in the cortex, but also at the corticomedullary junction where they have not been previously described. TNC expressed epithelial markers [low and high molecular weight keratins identified by 35 beta H11 and 34 beta E12 monoclonal antibodies, a cortical antigen shared with neuroectodermal neoplasms recognized by the GE2 monoclonal antibody, and tissue polypeptide antigen (TPA:B1)], class II histocompatibility antigens (HLA-DR), and thymosin alpha 1. Double staining experiments with the nuclear proliferation-associated antigen Ki-67 and the cortical epithelium marker GE2 showed that most thymocytes enclosed in these cortical TNC were not proliferating. The antigens expressed by TNC indicate that not only cortical, but also medullary epithelial cells are part of the TNC system. The possible role of TNC in the education and maturation of thymocytes is discussed.

Programmed cell death in heterokaryons. A study of the transfer of apoptosis between nuclei.

Thymocytes undergoing apoptosis induced by dexamethasone showed nuclear refractivity changes under Nomarski optics that correlated precisely with internucleosomal DNA degradation and was prevented by cycloheximide. When heterokaryons between thymocytes and 9L or NIH3T3 cells were examined, 99.75% of the nuclei in heterokaryons followed the original and distinct fate characteristic of the parental cells. Thymocyte nuclei proceeded to undergo apoptotic cell death, whereas the 9L and NIH3T3 nuclei in the heterokaryons did not show the morphologic changes of apoptosis or any DNA cleavage on gels, and remained viable and mitotic. Cycloheximide prevented the induction of apoptosis in thymocyte nuclei in the heterokaryons. An excess of up to seven thymocyte nuclei undergoing programmed cell death in a heterokaryon did not detectably damage the 9L nucleus, and an excess of six 9L nuclei did not protect the thymocyte nucleus from apoptosis. The proposed model stating that programmed cell death results from de novo synthesis of death-causing gene products is difficult to reconcile with these findings. A cell-type-specific activity, present in thymocyte nuclei before induction of apoptosis with corticosteroids and unable to diffuse between nuclei, appears to be responsible for DNA fragmentation. These results also show that nuclear disintegration is the trigger of thymocyte death and not a consequence of cell death due to another mechanism.

Apoptosis and DNA degradation induced by 1-methyl-4-phenylpyridinium in neurons.

Apoptosis is a prominent mechanism of programmed cell death in lymphocytes and in cancer cells not previously found in neurons. We have identified apoptosis and internucleosomal DNA degradation in cultures of cerebellar granule neurons. 1-methyl-4-phenylpyridinium, a selective neurotoxin that destroys the dopaminergic nigrostriatal pathway and results in a parkinsonian syndrome, increases the rate of apoptosis and kills cerebellar granule cells in culture via induction of programmed cell death. Inhibition of gene expression in granule cells with cycloheximide prevents the MPP(+)-induced apoptosis and the DNA fragmentation. Our findings demonstrate a new pathway of neuron death and suggest the possibility that neurodegenerative diseases may result from the inappropriate activation of programmed cell death by apoptosis.

Human glioma cell lines: tumour associated antigens distribution and sensitivity to antibody-toxin or ligand-toxin conjugates. A preliminary report.

We have investigated the phenotype of seven human glioma cell lines established in vitro from primary tumour explants. Indirect immunofluorescence and flow cytofluorimetry revealed a heterogeneous distribution of surface GE 2 and CG 12 Tumour Associated Antigens (TAA). In one group of cell lines TAA were detected both at the cell surface and in the cytosol, whereas in a second group of glioma cell lines TAA were found only in the cytosol. We have also investigated the sensitivity of glioma-derived cell lines to antibody-toxin and ligand-toxin conjugates (Immunotoxins). Monoclonal antibodies anti GE 2 antigen linked to ricin toxin A subunit (RTA) showed poor cytotoxicity, which increased about 50 fold when the whole toxin was linked to anti GE 2 monoclonals. Treatment with human recombinant interferon gamma (IFN-gamma) greatly augmented the percentage of HLA-DR+ cells and the amount of HLA-DR antigens per cell. IFN-gamma treatment resulted in a net increase of sensitivity to anti HLA-DR Immunotoxins (IT). Human diferric transferrin linked to RTA exhibited a potent cytotoxic effect against human glioma-derived cells when used in the presence of the lysosomotropic carboxylic ionophore monensin.

Immunohistochemical evidence of active thymocyte proliferation in thymoma. Its possible role in the pathogenesis of autoimmune diseases.

Eight cases of human thymoma have been analyzed on cryostat sections with the monoclonal antibody Ki67, which reacts with cells in the proliferative phases of the cell cycle. The aim was to assess the proportion of proliferating thymocytes among lymphoid cells in the thymoma samples. In all cases a large number of cells (mean, 58.75%; range, 35-80%), recognized as thymocytes by morphology and lack of cytokeratin expression in a combined immunohistochemical assay, exhibited nuclear Ki67 staining. These findings differ from the reactivity pattern observed in age-matched nonneoplastic thymuses where lower growth activity of cortical thymocytes was observed (15-20% Ki67+ cells). Intensive thymocyte proliferation in thymomas may represent one of the factors which lead to autoimmunity in myasthenia gravis and thymomas.