Does point-of-care ultrasonography improve diagnostic accuracy in emergency department patients with undifferentiated hypotension? An international randomized controlled trial from the SHOC-ED investigators.

PURPOSE: Point-of-care ultrasonography (POCUS) is an established tool in the management of hypotensive patients in the emergency department (ED). We compared the diagnostic accuracy of a POCUS protocol versus standard assessment without POCUS in patients with undifferentiated hypotension. METHODS: This was an international, multicenter randomized controlled trial included three EDs in North America and three in South Africa from September 2012 to December 2016. Hypotensive patients were randomized to early POCUS protocol plus standard care (POCUS group) or standard care without POCUS (control group). Initial and secondary diagnoses were recorded at 0 and 60 min. The main outcome was measures of diagnostic accuracy of a POCUS protocol in differentiating between cardiogenic and non-cardiogenic shock. Secondary outcomes were diagnostic performance for shock sub-types, as well as changes in perceived category of shock and overall diagnosis. RESULTS: Follow-up was completed for 270 of 273 patients. For cardiogenic shock, the POCUS-based diagnostic approach (POCUS) performed similarly to the non-POCUS approach (control) for specificity [95.5% (89.9-98.5) vs.93.8% (87.7-97.5)]; positive likelihood ratio (17.92 vs 14.80); negative likelihood ratio (0.21 vs 0.09) and diagnostic odds ratio (85.6 vs 166.57), with a similar overall diagnostic accuracy between the two approaches [93.7% (88-97.2) vs 93.6% (87.8-97.2)]. Diagnostic performance measures were similar across sub-categories of shock. CONCLUSION: This is the first randomized controlled trial to compare diagnostic performance of a POCUS protocol to standard care without POCUS in undifferentiated hypotensive ED patients. POCUS performed well diagnostically in undifferentiated hypotensive patients, especially as a rule-in test; however, performance did not differ meaningfully from standard assessment.

RéSUMé: OBJECTIF: L'échographie au point d'intervention (POCUS) est un outil bien établi dans la gestion des patients hypotendus dans le service des urgences. Nous avons comparé la précision diagnostique d'un protocole POCUS par rapport à une évaluation standard sans POCUS chez des patients présentant une hypotension indifférenciée. MéTHODES: Il s'agissait d'un essai contrôlé randomisé international multicentrique incluant 3 services d'urgence en Amérique du Nord et 3 en Afrique du Sud de septembre 2012 à décembre 2016. Les patients hypotenseurs ont été répartis par randomisation selon le protocole POCUS précoce plus les soins standard (groupe POCUS) ou les soins standard sans POCUS (groupe témoin). Les diagnostics initiaux et secondaires ont été enregistrés à 0 et 60 minutes. Le principal résultat était la mesure de la précision diagnostique d'un protocole POCUS pour différencier le choc cardiogénique du choc non cardiogénique. Les résultats secondaires étaient la performance diagnostique pour les sous-types de chocs, ainsi que les changements dans la perception de la catégorie de choc et du diagnostic global. RéSULTATS: Le suivi a été complété pour 270 des 273 patients. Pour le choc cardiogénique, l'approche diagnostique basée sur le POCUS (POCUS) a donné des résultats similaires à l'approche non-POCUS (Contrôle) pour la spécificité (95,5 % (89,9­98,5) vs 93,8 % (87,7­97,5)) ; Rapport de vraisemblance positif (17,92 vs 14,80) ; Le rapport de vraisemblance négatif (0,21 vs 0,09) et le rapport de cotes diagnostiques (85,6 vs 166,57), avec une précision diagnostique globale similaire entre les deux approches (93,7 % (88­97,2) vs 93,6 % (87,8­97,2). Les mesures de performance diagnostique étaient similaires dans toutes les sous-catégories de choc. CONCLUSION: Il s'agit du premier essai contrôlé randomisé visant à comparer la performance diagnostique d'un protocole POCUS aux soins standard sans POCUS chez des patients hypotendus indifférenciés aux urgences. La POCUS a donné de bons résultats diagnostiques chez les patients hypotendus indifférenciés, surtout en tant que test de référence ; cependant, les performances ne diffèrent pas de manière significative de l'évaluation standard.

Canine astrocytic tumors: a comparative review.

Tumors of astrocytic lineage are among the most common primary brain neoplasms in people and dogs. Current understanding of the pathogenesis of astrocytic tumors is limited in dogs compared with humans. In dogs, critical biological data concerning the natural history of disease progression, tumor imaging features, and response to therapeutic intervention are lacking. This review outlines the clinical, genetic, immunologic, and histopathologic characteristics of astrocytic tumors in dogs with special focus on comparative neuro-oncology. Common problems associated with the diagnosis of these neoplasms are summarized. Traditional veterinary histologic typing and grading of astrocytic tumors must be updated and supplemented with molecular data so that future studies directed toward therapeutic intervention and outcome can be optimized.

[Preventive and emergency embolization of angiomyolipomas: our experience]. / Embolisation préventive et en urgence des angiomyolipomes: notre expérience.

AIM: To present our experience with emergency or programmed embolization of angiomyolipomas. PATIENTS AND METHODS: The retrospective study 1999-2000 included a total of 20 patients with AML, five of whom had hypothyroidism. Group I emergency embolization: 11 patients age being 61.4 ± 15.6 years and the size of AML 8.2 ± 2.8 cm presented retroperitoneal hemorrhage from spontaneous rupture. Two had a hemorrhagic shock. A transfusion of 3.4 blood units per patient was performed for five patients. A clinical and radiological follow-up was done by scanning during the first week and in one month. Group II preventive embolization: nine patients, with age between 58.3 ± 15.2 years and tumor size 5.2 ± 2.2 cm, all asymptomatic. All successfully received a unilateral preventive embolization. A scan was performed one month later. RESULTS: Group I: the embolization was effective in 100% of patients. No intraoperative incident was reported. After one month, the reduction in tumor volume was 40%. At eight months, a patient underwent nephrectomy because of a new fracture, and another a second embolization after 14 months. The technical result was maintained in 83% of cases after 18 months. Two patients developed HTA after embolization controlled by a single treatment, and five had limited renal ischemic sequels. Group II: no intraoperative incidents and no postoperatively complications have been reported. One month after embolization, the reduction in tumor volume was 23%. After 24 months, patients remained completely asymptomatic, no spontaneous bleeding has been reported, no surgery has been performed, and no HTA has been described. Only one re-embolization was done at 20 months (artery duplicity). Limited renal ischemic sequels were reported for one patient but no renal failure. CONCLUSIONS: The required embolization became the method of choice in emergency with excellent results and few complications at distance. Programmed embolization effectively prevented the risk of bleeding, without impact on the renal function, with a low economic cost compared to hospitalization and emergency care. The significance of the observed AML--hypothyroidism association in our series requires a confrontation with more important cohorts.

Monosodium luminol upregulates the expression of Bcl-2 and VEGF in retrovirus-infected mice through downregulation of corresponding miRNAs.

The retrovirus ts1 is a mutant of Moloney murine leukemia virus (MoMuLV) that causes neurodegeneration (ND) in susceptible mice. Our previous studies showed that the antioxidant drug monosodium luminol (GVT) prevented the development of ND in ts1-infected mice. In this study, we analyzed effect of GVT on the expression of B-cell lymphoma-2 protein (Bcl-2) and vascular endothelial growth factor (VEGF) in central nervous system (CNS) tissues of these animals. Our data showed that GVT treatment of ts1-infected mice significantly increased their expression of Bcl-2 and VEGF in brainstem compared with ts1-infected untreated mice. We also studied the expression of specific microRNAs (miRNAs) such as miRNA-15 and -16 (targeting Bcl-2), and miRNA-20 (targeting VEGF). We found that the expression of miRNAs inversely correlated with the upregulation of their target proteins in ts1-infected untreated as well as in GVT-treated-ts1-infected mice. The data showed that GVT treatment prevented ts1-induced ND at least in part by upregulating Bcl-2 and VEGF expression, what likely occurred as a consequence of downregulation of their corresponding miRNAs.

Recruitment of insulin receptor substrate-1 and activation of NF-kappaB essential for midkine growth signaling through anaplastic lymphoma kinase.

Anaplastic lymphoma kinase (ALK) is a transmembrane receptor tyrosine kinase in the insulin receptor superfamily. We recently demonstrated that the growth factors pleiotrophin (PTN) and midkine (MK) are ligands for ALK and that upon ALK activation, insulin receptor substrate-1 (IRS-1) and other substrates are phosphorylated. Here, the role of IRS-1 in ligand-mediated ALK signaling is investigated in interleukin-3 (IL-3)-dependent 32D murine myeloid cells. These cells do not express ALK and IRS family members, and do not respond to exogenously added PTN or MK. We show that expression of ALK plus IRS-1 renders these cells independent of IL-3 owing to the activation of ALK by endogenous MK. Mutational analysis reveals that this transformed phenotype of 32D cells requires kinase-active ALK as well as the interaction of ALK with IRS-1. Furthermore, 32D/IRS-1/ALK cells display an enhanced activation of mitogen-activated protein kinase and PI3-kinase pathways, and a selective transcriptional activation of nuclear factor (NF)-kappaB. Small interfering RNA-mediated knockdown of the endogenous MK or p65/NF-kappaB revealed that both these are rate limiting for the transformed phenotype induced by ALK plus IRS-1. We conclude that the recruitment of IRS-1 to activated ALK and the activation of NF-kappaB are essential for the autocrine growth and survival signaling of MK.

Osteoraticular Tuberculosis-Brief Review of Clinical Morphological and Therapeutic Profiles.

Osteoarticular tuberculosis (OATB) is a rare form of tuberculosis (TB) whose incidence rose significantly nowadays especially in the underdeveloped countries. The main risk factors predisposing to this new challenge for the medical system are the Human Immunodeficiency Virus (HIV) epidemic, the migration from TB endemic areas and the development of drug and multidrug-resistant strains of Mycobacterium tuberculosis (Mt). The disease affects both genders and any age group although the distribution depending on gender is controversial and that depending on age has a bimodal pattern. In most cases the initial focus is elsewhere in the organism and the most frequent pathway of dissemination is lympho-haematogenous. The clinical picture includes local symptoms as pain, tenderness and limitation of motion, with some particularities depending on the segment of the osteoarticular system involved, sometimes accompanying systemic symptoms specific for TB and other specific clinical signs as cold abscesses and sinuses. The radiographic features are not specific, CT demonstrates abnormalities earlier than plain radiography and MRI is superior to plain radiographs in showing the extent of extraskeletal involvement. Both CT and MRI can be used in patient follow-up to evaluate responses to therapy. TBhas been reported in all bones of the body, the various sites including the spine (most often involved) and extraspinal sites (arthritis, osteomyelitis and tenosynovitis and bursitis). Two basic types of disease patterns could be present: the granular type (most often in adults) and the caseous exudative type (most often in children) one of which being predominant. The algorithm of diagnosis includes several steps of which detection of Mt is the gold standard. The actual treatment is primarily medical, consisting of antituberculosis chemotherapy (ATT), surgical interventions being warranted only for selected cases. It is essential that clinicians know and refresh their knowledge about manifestations of OATB.

Rat ovarian granulosa cell culture: a model system for the study of cell-cell communication during multistep transformation.

A spontaneously immortalized clonal granulosa cell line (SIGC) derived from primary rat ovarian granulosa cell cultures was developed as a model system to explore the process of transformation using an epithelial cell type. SIGC has an epithelial morphology and grows in culture without undergoing luteinization. The cell line is thought to represent an intermediate step in carcinogenesis because it seems to grow indefinitely in culture but does not form clones in soft agar or tumors in nude mice. Indirect immunofluorescence and Western blot analysis verified the constitutive expression of the recessive oncogene product p53 in the cell line, thereby suggesting a possible mechanism of immortalization. Ultrastructural studies indicated that SIGC cells are characterized by an undifferentiated phenotype with prominent intermediate filaments, desmosomes, and gap junctions. The identification of cytokeratin by indirect immunofluorescence and Western blot analysis suggests that SIGC functions as an epithelial cell type. Functional studies of cell-cell communication by a dye transfer technique (fluorescence recovery after photobleaching) showed reduced communication compared to normal primary granulosa cells in culture. SIGC cells were transfected with early region genes of SV40 virus in an attempt to generate fully transformed cell lines. The resulting cell line SV-SIGC expressed T-antigen, was anchorage independent, formed tumors in nude mice, and had reduced intercellular communication as compared to SIGC cells. Explants from the tumors in nude mice were used to generate another cell line (T-SV-SIGC), which exhibited further reduction in both the incidence and the rate of communication. These results clearly demonstrated a progressive loss of functional communication during multistep transformation of an ovarian cell type. These data demonstrate that this assay system based on an epithelioid cell type can be used to study the relationship between intercellular communication and the multistep process of carcinogenesis.

Modulation of tumor angiogenesis by stem cell factor.

Mast cells accumulate within solid tumors and can release many angiogenic factors, suggesting that they may modulate vascularization of tumors. Stem cell factor (SCF) stimulates mast cell migration, proliferation, and degranulation and therefore may influence mast cell behavior within tumors. We investigated the contribution of SCF to tumor angiogenesis by manipulating its level in mammary tumors. Sense or antisense cDNA fragments of rat SCF were ligated into an episomal expression vector. Ethylnitrosourea-induced rat mammary tumor cell lines were transfected with vector containing either control (no insert, C-P), sense (S-P), or antisense (AS-P) SCF DNA. The functional nature of the transfectants was confirmed by measuring SCF in cell lysates and conditioned media. Immunohistochemical analysis of the tumors induced in Berlin-Druckrey rats by these transfected cells demonstrated that mast cell number and microvascular density were significantly higher in S-P tumors and significantly lower in AS-P tumors, compared with C-P tumors. The expression of von Willebrand factor, an endothelial cell marker, showed a similar pattern. AS-P tumors were significantly smaller than either C-P or S-P tumors. These data suggest that SCF modulates tumor growth and angiogenesis via the involvement of mast cells.

Differential suppression of mammary and prostate cancer metastasis by human chromosomes 17 and 11.

Metastasis suppressor activities have previously been mapped to human chromosomes 17 and 11. Decreased expression of the metastasis suppressor gene NM23, which is located on chromosome 17, has been correlated with increased metastatic potential in mammary cancers. A region on human chromosome 11, from 11p11.2-p13, has been shown to suppress metastasis in rat prostatic carcinoma cells. In both cases the metastasis suppressor activity had no effect on tumorigenicity or tumor growth rate, demonstrating that the encoded activities are distinct from effects of tumor suppression. To determine whether these human chromosomes encode general or tissue-specific metastasis suppressor activities, a truncated human chromosome 17 (i.e., pter-q23) and a full-length human chromosome 11 were separately transferred into highly metastatic rat mammary and prostate cancer cell lines and tested for their ability to suppress spontaneous metastasis in vivo. These studies demonstrated that when the pter-q23 region of human chromosome 17 is retained by the microcell hybrids, the metastatic ability of both mammary and prostatic cancer cells is suppressed. In contrast, when the pter-q14 region of human chromosome 11 is retained, only the metastatic ability of prostatic cancer cells is suppressed. Additional studies demonstrated that the metastasis suppressor activity encoded by the chromosome 17 pter-q23 region is p53-independent and not due to enhanced expression of NM23 protein.

The BCR-ABL tyrosine kinase inhibits apoptosis by activating a Ras-dependent signaling pathway.

BCR-ABL is a deregulated tyrosine kinase that is expressed in Philadelphia chromosome (Ph1) positive human leukemias. When expressed in hematopoietic cells, BCR-ABL causes cytokine independent proliferation, induces tumorigenic growth and prevents apoptosis in response to cytokine deprivation or DNA damage. One mechanism by which BCR-ABL signals in cells is by activating the small guanine nucleotide binding protein Ras. BCR-ABL-transformed cells have constitutively high levels of active, GTP-bound Ras. Here we use 32D cells that inducibly express a dominant negative Ras protein to define the Ras requirements in BCR-ABL-transformed cells. Dominant negative Ras inhibits BCR-ABL-mediated Ras activation, and induces cell death by an apoptotic mechanism. Therefore, BCR-ABL inhibits apoptosis through activation of a Ras-dependent signaling pathway.

Murine leukemia virus induced central nervous system diseases.

The ts1 mutant of Moloney murine leukemia virus TB (MoMuLV-TB) causes a degenerative neurologic and immunologic disease in mice characterized by development of spongiform encephalomyelopathy that results in hind-limb paralysis, marked thymic atrophy associated with immunodeficiency, and generalized body wasting. T cells, particularly CD4+ helper T cells, play a key role in the pathogenesis of the disease induced by ts1. Therefore, ts1 is unique among the described murine retroviruses in its ability to afflict both the central nervous system (CNS) and the T-cell compartment of the immune system in the same host. This particular ability to cause degenerative diseases involving both the CNS and immune system is shared by the lentiviruses responsible for development of the acquired immunodeficiency syndromes of humans and macaques. Our goal has been to elucidate the specific cellular and molecular mechanisms that underlie this neuro- and immunopathogenicity of ts1. We have previously reported that the primary neuropathogenic determinant of ts1 maps to a single amino acid substitution, Val-25-Ile, in the precursor envelope protein gPr80env. Further, at the restrictive temperature, the Val-25-Ile substitution did not prevent oligomerization of the gPr80env proteins; however, the structure of the oligomer was incompetent for transport from the ER to the Golgi. These findings suggest that the cytopathic effect of ts1 in neural cells might be due to accumulation of the gPr80env oligomers in the ER. Since glial cells are targets of ts1 infection in vivo, primary astrocytic cultures were established and the cytopathic effect of ts1 and MoMuLV-TB on these cells assessed. Both viruses replicate well in astrocytes and their replication is cytopathic, albeit to different degrees. The ts1 mutant appears to produce greater cell killing than the wild-type virus. Furthermore, it was found that the rate of processing of gPr80env of ts1 in astrocytes is slower than that of MoMuLV-TB. Therefore, the inefficient transport and processing of gPr80env of ts1 appears to correlate with its cytopathic effect in these cells. Electron microscopic studies of the ts1-infected astrocytes revealed large numbers of aberrant particles in the ER. The in vitro cytopathic effect of ts1 on astrocytes may reflect what happens in vivo. An indirect mechanism of neuronal-cell killing by ts1 is proposed.

Effect of the bisphosphonate risedronate on bone metastases in a rat mammary adenocarcinoma model system.

Risedronate (NE-58095) is a third-generation bisphosphonate with very potent antiresorptive activity but few toxic effects. The purpose of this work was to evaluate the effect of risedronate treatment on bone metastases produced in a rat breast cancer model. Berlin Druckrey IV rats inoculated with ENU1564 mammary adenocarcinoma cells were treated daily with risedronate or a saline placebo. Survival times, dictated by extraskeletal metastases (lung, heart, and brain), were not affected by risedronate treatment. Risedronate-treated animals had skeletal changes associated with decreased remodeling of bones undergoing endochondral ossification, most prominently affecting the appendicular skeleton. Despite the skeletal alterations induced by the treatment, the distribution of bone metastases throughout the surveyed skeletal sites was similar for treated and untreated animals. Bone metastases were enumerated in histologic sections of distal femur, spine, and skull. Tumor size was estimated from area measurements obtained from histologic lesions in distal femoral metaphyses and vertebral bodies. A greater number of treated rats had no bone metastases in any of the examined sections (30 versus 16.1% of untreated rats). Multiple bone metastases were observed less frequently in treated rats (33.3 versus 71% of untreated rats). Treated rats had fewer observed bone metastases in each examined site than untreated rats (p < or = 0.025). Mean tumor areas in femora and vertebrae were smaller in treated rats (p < or = 0.05), due to the less frequent presence of very large lesions. In untreated animals, osteoclasts appeared to be active at the tumor/bone interface and osseous structures were often completely replaced by expanding tumors. In contrast, metastases in treated animals caused less disruption of skeletal histoarchitecture. The apparent lack of osteoclastic activity and retention of bone within lesions suggested a decreased contribution of osteoclasts to the bone resorptive process. An in vivo immunohistochemical cell proliferation assay failed to reveal differences in the percentage of dividing tumor cells in bone metastatic sites in treated versus untreated animals. The results demonstrate significant effects of risedronate treatment on the incidence and size of observed skeletal metastases in this model.

A role for Akt in mediating the estrogenic functions of epidermal growth factor and insulin-like growth factor I.

This study examines whether the serine/threonine protein kinase, Akt, is involved in the cross-talk between epidermal growth factor (EGF) and insulin-related growth factor I (IGF-I) receptors and ER-alpha. Treatment of MCF-7 cells with either EGF or IGF-I resulted in a rapid phosphorylation of Akt and a 14- to 16-fold increase in Akt activity, respectively. Akt activation was blocked by inhibitors of phosphatidylinositol 3-kinase, but not by an inhibitor of the ribosomal protein kinase p70S6K. Stable transfection of cells with a dominant negative Akt mutant blocked the effects of EGF and IGF-I on ER-alpha expression and activity, whereas stable transfection of cells with a constitutively active Akt mutant mimicked the effects of EGF and IGF-I. In the latter cells, there was a decrease in the amount of ER-alpha protein and messenger RNA (70-80%) and an increase in the amount of progesterone receptor protein, messenger RNA (4- to 9- and by 3.5- to 7-fold, respectively) and pS2 (3- to 5-fold). Coexpression of wild-type ER-alpha and the dominant negative Akt mutant in COS-1 cells also blocked the growth factor-stimulated activation of ER-alpha, but coexpression of the wild-type receptor with the constitutively active Akt mutant increased ER-alpha activity. Receptor activation was blocked by an antiestrogen. Studies using mutants of ER-alpha demonstrated that Akt increased estrogen receptor activity through the amino-terminal activation function-1 (AF-1). Serines S104 S106, S118, and S167 appear to play a role in the activation of ER-alpha by Akt.

Characterization of brain and bone-metastasizing clones selected from an ethylnitrosourea-induced rat mammary carcinoma.

An animal model for breast cancer, brain and bone metastasis was developed using ENU1564, a cell line established from a metastatic mammary adenocarcinoma induced by N-ethyl-N-nitrosourea in a female Berlin-Druckrey IV rat. The original tumor isolate (designated FP1) spontaneously metastasizes to regional lymph nodes and lung following orthotopic inoculation into mammary fat pad (mfp) and metastasizes widely following left cardiac ventricle (LV) inoculation. From FP1, two sublines were selected from brain metastases (designated Br7-C5) or from slowly growing colonies in vitro (FP2-A11), then cloned and compared in assays of spontaneous and experimental metastasis. After inoculation of 10(5) cells into mfp, Br7-C5 formed large tumors at the inoculation site (9.4 +/- 3.3 g) and spontaneously metastasized to lung and lymph node by 55 days post-inoculation (dpi). In contrast, FP2-A11 produced much smaller tumors within mfp (0.6 +/- 0.3 g) and failed to metastasize by 55 dpi. Rats inoculated via the LV with 10(4) Br7-C5 cells developed signs of weight loss, head tilt, and dyspnea by 24 +/- 1.4 dpi with consistent colonization of brain, bone, lung, heart, kidney, and stomach. Rats inoculated similarly with FP2-A11 showed no signs until 53 +/- 12.3 dpi, when all developed rear limb paresis. There was significant colonization of only brain and bone, with only minor lung involvement. These ENU1564 sublines thus differ in their apparent rates of tumor growth and lesion development in vivo, their capacity to metastasize from orthotopic implantation sites, and in the spectrum of tissues colonized in experimental metastasis assays. Both clones provide reproducible models of breast cancer metastasis in syngeneic hosts, particularly to brain and bone.

Relationship between Moloney murine sarcoma-virus tissue tropism and tumor-development.

Previous work from our laboratory has demonstrated that a clone of Moloney murine sarcoma virus (MoMuSV-349) inoculated intraperitoneally into newborn BALB/c mice induces multiorgan disseminated angiosarcomatous tumors. These tumors develop in two stages: sarcomatous and angiosarcomatous. The present report investigates the relationship between tumor stage development and viral replication within the target cells using the technique of in situ hybridization. A nonradioactive RNA probe (riboprobe) labeled with digoxigenin-linked uridine triphosphate (DIG-UTP) was used to detect the presence of MoMuSV-349 in frozen tissue sections. Infected and control mice were euthanized at various times post-inoculation and tissues were collected for in situ hybridization. The riboprobe hybridized to macrophages, splenic reticular cells, mesothelial cells, megakaryocytes, Kupffer cells and neoplastic (spindled and endothelial) cells in mice infected with MoMuSV-349. The riboprobe failed to hybridize to any tissues in the non-infected mice. Cells hybridizing with the riboprobe were detected as early as 3 days post-inoculation. As the neoplasms progressed from the sarcomatous to angiosarcomatous type there was a shift in cellular target hybridized by the riboprobe from spindled cells to endothelial cells. Positive labeling of endothelial cells during the angiosarcomatous stage of tumor development suggests-that viral replication correlates with cellular proliferation. This demonstrates that in situ hybridization utilizing a non-radioactive riboprobe was useful for a pathogenesis study involving MoMuSV-349.

Expression of a 65 kda oncofetal phosphoprotein in the altered hepatic foci of rats fed 2-acetylaminofluorene followed by phenobarbital.

The altered hepatic foci (AHF) developed in livers of Sprague-Dawley male rats,by acetylaminofluorene/phenobarbital protocol, were analyzed on paraffin sections for gamma-glutamyl-transpeptidase, glutathione S-transferase P and 65 kDa oncofetal protein (p65). 92% percent of the foci were GST-P-positive, 89% of those were also positive for GGT but only 10-12% were positive for p65. Comparison of tissues from different sources such as fetal and normal liver, foci, nodules, surrounding parenchyma and hepatocellular carcinomas revealed the presence of p65 protein in all tissues except normal rat liver and liver parenchyma surrounding the focal lesions.

The role of fibroblast growth factor (FGF) in neoplasms induced by MoMuSV-349.

Previous results from our laboratory have demonstrated that intraperitoneal inoculation of newborn BALB/c mice with MoMuSV-349 virus induced multiorgan disseminated angiomatous tumors. Changes in histological pattern, from sarcomatous to angiosarcomatous, stimulated our interest in investigating the possible role of angiogenic growth factors released by the spindled (sarcomatous) cells in angiosarcoma development in this model. Cell lines were obtained from various tumors and assayed for production of acidic (a) and basic (b) fibroblast growth factors (FGFs). All tumor cell lines released detectable amounts of growth factor(s) into the cultured media that induced proliferation of endothelial cells and mitogenesis of mouse fibroblasts. This growth factor(s) bound to heparin Sepharose (HS) beads and its effects on cell proliferation were partially blocked by a neutralizing bFGF antibody. Proteins released by tumor cells into the conditioned medium were detected by bFGF antibodies on Western blots. In addition, proteins that reacted with both bFGF and aFGF antibodies were detected in various conditioned media by ELISA. This protein(s) from the FGF family detected in the conditioned media from various tumor cell lines might be responsible for the angiomatous proliferation observed in the late (angiosarcomatous) stage of MoMuSV-induced tumors.

In vitro malignant transformation of in vivo ENU-induced rat ovarian Sertoli cell tumor (adenoma).

An N-ethyl-N-nitrosourea-induced rat ovarian Sertoli cell tumor was grown in tissue culture in Dulbecco's modified Eagle's medium (DMEM) supplemented with 25% horse serum (HS) and a hormone combination of 20 ng/ml each of hydrocortisone, insulin, and prolactin. This tissue culture derived from a nonsteroid hormone-producing tumor. Cytofluorometry and karyotyping of the nonhormone-producing tumor cell line (SCTL-1) revealed a diploid pattern for the early passage (P1), which became hyperdiploid (P10), and then aneuploid (P20). These cells had an epitheloid pattern, grew in a monolayer at early passages. After P10 the cells were transplanted into newborn rats and nude mice and resulted in high incidences of tumors (up to 100%). The cell line (SCTL-1) continued to grow in DMEM, 10% HS, and no hormone supplementation after P10. This study revealed that a benign rat ovarian Sertoli cell tumor after multiple passages in vitro underwent sequential genotypic and phenotypic changes and became highly malignant.

Detection of Moloney murine sarcoma virus in tissues and cultured cells by the polymerase chain reaction.

The polymerase chain reaction was used for Moloney murine sarcoma virus (MoMuSV) detection in frozen and formalin-fixed, paraffin-embedded tissue sections and cultured cells isolated from MoMuSV-induced tumors. Rapid DNA extraction by proteinase K digestion, followed by CHROMA SPIN + TE-100 column purification proved to be satisfactory. Two pairs of overlapping primers, flanking 1026 base pair (bp) to 221 bp, allowed to choose among four different length of DNA-amplified segments. Although net amplification was obtained for frozen tissue and tumor cultured cells in all combinations of primers, the maximum specificity and sensitivity resulted with 602 bp fragment. This product was fully and adequately digestible using Apa I and Sau3A I restriction endonucleases. DNA extracted from paraffin-embedded sections yielded an amplification product only when the primer pair which delineated a 221-bp segment was used. This reproducible method could be useful for diagnostic and for pathogenetic investigations of MoMuSV infections.

Neurodegeneration induced by MoMuLV-ts1 and increased expression of Fas and TNF-alpha in the central nervous system.

Infection of neonatal mice with ts1, the neuropathogenic mutant of the Moloney murine leukemia virus, results in motor neuronal death in the brainstem and the spinal cord, with gliosis and demyelination, but no inflammatory cell infiltration into the CNS. To evaluate the possible mechanism(s) of ts1-induced neuropathogenesis, we measured CNS expression of cytokines and cell death-related genes in ts1-infected mice with neurological signs and compared with control uninfected mice. In the brainstem, the expression of Fas and tumor necrosis factor alpha (TNF-alpha) was increased in the ts1-infected mice. Both TNF-alpha and Fas were detected in astrocytes, and Fas was also detected in neurons in the brainstem. Some TNF-alpha-immunolabeled cells also appeared to be microglial cells. Most Fas-positive cells, including astrocytes and neurons, showed cytoplasmic vacuolization and other degenerative changes. In addition, Fas ligand-immunolabeled cells were also detected in sites where spongiform degeneration occurred. This study suggests that neural cell death in ts1-induced neurodegeneration is likely due to Fas- and TNF-alpha-mediated cell death mechanisms.