Point mutation of neu oncogene in animal peripheral nerve sheath tumors.

Thirty-four peripheral nerve sheath tumors of four domesticated animal species were characterized and assayed for point mutation of the neu oncogene. Based on their morphoimmunophenotype, 32 tumors were classified as schwannomas. Schwannoma morphology was characterized by the presence of Antoni type A and B pattern and immunoreactivity for S-100 protein and vimentin. Two anaplastic and metastatic tumors originating from spinal cord root, immunonegative for S-100 protein and positive for vimentin, were classified as malignant peripheral nerve sheath tumors (MPNSTs). Four malignant schwannomas and two MPNSTs expressed a point mutation of the neu oncogene by the polymerase chain reaction-restriction fragment length polymorphism method. The finding of neu oncogene mutation could be a useful diagnostic genetic marker in the malignant form of peripheral nerve sheath tumors in animals.

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.

Motor neuronal loss and neurofilament-ubiquitin alteration in MoMuLV-ts1 encephalopathy.

A temperature-sensitive mutant of Moloney murine leukemia virus (MoMuLV-ts1) induces immunosuppression and spongiform encephalopathy in susceptible newborn mice. The associated neuronal degeneration primarily involves the motor neurons in specific target areas of the central nervous system (CNS). Neuronal loss occurs in the absence of direct viral infection of neurons and is the most dramatic pathological change in the CNS of infected mice. To quantitatively demonstrate neuronal loss, an unbiased morphometric stereological study was undertaken using the optical disector method. Using highly susceptible FVB/N mice, neuronal loss was quantitated in the tissue sections of brain stem from infected and noninfected mice at 20 and 35 days post inoculation (dpi). Results indicated that there was no significant neuronal loss at 20 dpi, but significant (P < 0.05) at 35 dpi. In addition, histology, transmission electron microscopy and immunohistochemistry revealed Lewy body-like inclusions consisting of aggregates of neurofilaments and cellular organelles. Degenerated neurons and glial cells were heavily ubiquitinated. Together, these results suggest that significant neuronal loss occurs at the end of the disease process and that Lewy body-like formation and protein ubiquitination are part of the pathogenic process in ts1-induced encephalopathy.

Molecular phylogeny and in situ detection of the etiologic agent of necrotizing hepatopancreatitis in shrimp.

Necrotizing hepatopancreatitis (NHP) is a severe disease of farm-raised Penaeus vannamei that has been associated with mortality losses ranging from 20 to 95%. NHP was first recognized in Texas in 1985 (S. K. Johnson, p. 16, in Handbook of Shrimp Diseases, 1989) and is an economically important disease that has limited the ability to culture shrimp in Texas. The putative cause of NHP is a gram-negative, pleomorphic, intracellular, rickettsia-like bacterium that remains uncultured in part because of the absence of established shrimp cell lines. The inability to culture the NHP bacterium necessitated the use of molecular methods for phylogenetic placement of the NHP bacterium. The gene encoding the 16S rRNA (16S rDNA) of this shrimp pathogen was amplified by PCR, cloned, and sequenced. Sequence analysis of the cloned 16S rDNA indicates that the NHP bacterium is a member of the alpha subclass of the Proteobacteria. Within the alpha subclass, the NHP bacterium is shown to be most closely related to bacterial endosymbionts of protozoa, Caedibacter caryophila and Holospora obtusa. Also, the NHP bacterium is distinct from but related to members of the typhus group (Rickettsia typhi and R. prowazekii) and spotted fever group (R. rickettsii) of the family Rickettsiaceae. Fluorescently labeled oligonucleotide DNA probes that bind to variable regions (V2, V6, and V8) of 16S rRNA of the NHP bacterium were used to detect the bacterium in infected shrimp by in situ hybridization. This technique provided direct visual evidence that the 16S rDNA that was amplified, cloned, and sequenced was derived from the intracellular bacterium that infects the hepatopancreas of farm-raised P. vannamei shrimp.

Immunohistochemical localization of proliferating cells, basic fibroblast growth factor and Fos protein following iliac artery balloon angioplasty in hypercholesterolemic rabbits.

The restenotic process in iliac arteries of hyperlipidemic rabbits following balloon angioplasty was evaluated in a temporal sequence by monitoring smooth muscle cell (SMC) proliferation (proliferating cell nuclear antigen -PCNA marker), growth factor (basic fibroblast growth factor -bFGF) production, and Fos oncogene protein expression using immunohistochemical methods. The percutaneous transluminal angioplasty was performed in iliac artery of rabbits fed a 1% cholesterol diet, with a balloon catheter inflated from 4 to 8 atm for 2 min under cineangiographic control until an acceptable (at least 50% stenosis reduction) result was obtained. The morphometric analysis indicated that the number of PCNA-positive cells in contralateral control arteries was low, but was increased in balloon-injury. The bFGF-positive labeled cells were localized in the media and in recently-migrated SMCs within the intima. Two days following angioplasty these cells were more abundant and scattered in the entire neo-intima. After 14 days their number was still higher than that of the controls. Cellular Fos protein expression was detected in the intima and, to a lesser extent, in the media 2 hours after angioplasty. These data suggest that early expression of Fos protein and bFGF in iliac arteries of hyperlipidemic rabbits following balloon angioplasty could be involved in development of restenosis in this animal model.

Temporal central and peripheral nervous system changes induced by a paralytogenic mutant of Moloney murine leukemia virus TB.

BACKGROUND: The temporal localization of cellular targets for viral replication and the morphopathogenesis of neurodegeneration in the central nervous system (CNS) and peripheral nervous system induced by ts1, a neuropathogenic and lymphocytopathic mutant of Moloney murine leukemia virus-TB, were studied in the highly susceptible FVB/N mouse strain in order to better understand the mechanisms of this neurodegenerative disease. EXPERIMENTAL DESIGN: Newborn FVB/N mice were inoculated intraperitoneally with 0.1 ml of viral suspension containing 10(6) to 10(7) infectious units/ml. The mice were observed daily for clinical signs of disease and killed at specific time points. Their nervous system tissues were collected and processed for light and electron microscopy and for immunohistochemical viral-antigen detection. RESULTS: ts1-Infected FVB/N mice developed a rapidly progressive wasting disease that culminated in hindleg paralysis or paraplegia 30 to 35 days postinoculation (pi). CONCLUSIONS: Clear evidence of CNS lesions involving the cerebellar ventricular system, the grey and white matter of the brain stem and the spinal cord were seen as early as 5 to 10 days pi. These lesions, which began as mild perivascular and paraventricular neuropil spongiform changes and cytoplasmic vacuolation of neuronal and glial cell processes, progressed in severity with time and culminated in almost complete destruction of the white and gray matter in the brain stem and the cervical and lumbar spinal cord. Viruses were detected as early as 5 to 10 days pi in the fourth ventricle choroid plexus and ventricular lumen and budding from endothelial cells within the brain stem and cerebellum. Endothelial, ependymal, microglial, astroglial, and oligodendroglial cells were positive for gp70env. Astroglial and microglial cell proliferation with microglial syncytia formation was detected only within the areas showing spongiform degeneration. Viral replication was consistently high in the capillary endothelial cells of those areas showing spongiform degeneration, whereas in the glial cells, relatively few budding viruses were present. Neurodegeneration was accompanied by demyelinization within the CNS and peripheral nervous system and by hindleg muscle degeneration and necrosis. Multiple cellular targets for ts1 viral infection and replication were detected within the nervous system. The presence of budding virus and the immunodetection of viral antigen in the choroid plexus and ependymal cells of the fourth ventricle and the central canal of the spinal cord demonstrated that cerebrospinal fluid as well as blood can disseminate virus within the CNS. Pathologic and functional changes within the blood-brain barrier and glial system probably account for the neuronal necrosis and spongiform changes that result in paralysis induced by ts1 infection.

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.

Culture of pericytes isolated from rat adipose microvasculature and characterization of their prostanoid production.

In order to investigate the role of pericytes (PCs) at microvascular level, a PC line from rat epididymal fat pads was isolated and its prostanoid production in culture was further examined. PC cultures were characterized morphologically by phase contrast and electron microscopy. PC prostanoid production was compared with that of a smooth muscle cell (SMC) line isolated from bovine aorta. The same PGI2 production magnitude was assayed in PC and SMC cultures, but TXA2 and PGF2 alpha synthesis was 8-10 times higher in the PC line. At 4-day postconfluence, when PC layers started retraction, prostanoid synthesis was significantly lower than at confluence. Histamine and bradykinin (both 100 nM) acted similarly, increasing the PGI2 basal production of PC cultures. The results argue for a possible contractile role of PCs at microvascular level.

Ultrastructural autoradiographic localization of exogenous arachidonic acid in cultured endothelial and smooth muscle cells.

The uptake and intracellular localization of exogenous arachidonic acid (AA) were investigated in cultured endothelial (EC) and smooth muscle cells (SMC) isolated from bovine aorta. The [14C]AA uptake was assessed biochemically and by light and electron microscopic autoradiography. The highest values of silver grain surface density were associated with the mitochondria, lysosomes, and the Golgi apparatus of the EC. The grain linear density was greater on the nuclear envelope than on plasmalemma. On SMC, the grain density was highest on lipid droplets whereas the linear densities of the nuclear envelope and plasmalemma were similar. The share of each subcellular compartment in the AA distribution was estimated as the percentage of the individual silver grain count out of the total cell-associated radioactivity. The results showed that cytoplasm (including endoplasmic reticulum, ribosomes, and small vesicles) made the main contribution followed by the nucleus and at lower values by other organelles. These subcompartments may represent the intracellular sites from which AA could be mobilized for prostanoid synthesis by EC and SMC.