Caudal lumbar vertebral fractures in California Quarter Horse and Thoroughbred racehorses.

REASONS FOR PERFORMING STUDY: To gain insight into the pathophysiology of equine lumbar vertebral fractures in racehorses. OBJECTIVES: To characterise equine lumbar vertebral fractures in California racehorses. STUDY DESIGN: Retrospective case series and prospective case-control study. METHODS: Racehorse post mortem reports and jockey injury reports were retrospectively reviewed. Vertebral specimens from 6 racehorses affected with lumbar vertebral fractures and 4 control racehorses subjected to euthanasia for nonspinal fracture were assessed using visual, radiographic, computed tomography and histological examinations. RESULTS: Lumbar vertebral fractures occurred in 38 Quarter Horse and 29 Thoroughbred racehorses over a 22 year period, primarily involving the 5th and/or 6th lumbar vertebrae (L5-L6; 87% of Quarter Horses and 48% of Thoroughbreds). Lumbar vertebral fractures were the third most common musculoskeletal cause of death in Quarter Horses and frequently involved a jockey injury. Lumbar vertebral specimens contained anatomical variations in the number of vertebrae, dorsal spinous processes and intertransverse articulations. Lumbar vertebral fractures examined in 6 racehorse specimens (5 Quarter Horses and one Thoroughbred) coursed obliquely in a cranioventral to caudodorsal direction across the adjacent L5-L6 vertebral endplates and intervertebral disc, although one case involved only one endplate. All cases had evidence of abnormalities on the ventral aspect of the vertebral bodies consistent with pre-existing, maladaptive pathology. CONCLUSIONS: Lumbar vertebral fractures occur in racehorses with pre-existing pathology at the L5-L6 vertebral junction that is likely predisposes horses to catastrophic fracture. Knowledge of these findings should encourage assessment of the lumbar vertebrae, therefore increasing detection of mild vertebral injuries and preventing catastrophic racehorse and associated jockey injuries.

Herpes simplex virus with highly reduced gD levels can efficiently enter and spread between human keratinocytes.

The rapid spread of herpes simplex virus type 1 (HSV-1) in mucosal epithelia and neuronal tissue depends primarily on the ability of the virus to navigate within polarized cells and the tissues they constitute. To understand HSV entry and the spread of virus across cell junctions, we have previously characterized a human keratinocyte cell line, HaCaT. These cells appear to reflect cells infected in vivo more accurately than many of the cultured cells used to propagate HSV. HSV mutants lacking gE/gI are highly compromised in spread within epithelial and neuronal tissues and also show defects in cell-to-cell spread in HaCaT cells, but not in other, nonpolarized cells. HSV gD is normally considered absolutely essential for entry and cell-to-cell spread, both in cultured cells and in vivo. Here, an HSV-1 gD mutant virus, F-US6kan, was found to efficiently enter HaCaT cells and normal human keratinocytes and could spread from cell to cell without gD provided by complementing cells. By contrast, entry and spread into other cells, especially highly transformed cells commonly used to propagate HSV, were extremely inefficient. Further analyses of F-US6kan indicated that this mutant expressed extraordinarily low (1/500 wild-type) levels of gD. Neutralizing anti-gD monoclonal antibodies inhibited entry of F-US6kan, suggesting F-US6kan utilized this small amount of gD to enter cells. HaCaT cells expressed high levels of an HSV gD receptor, HveC, and entry of F-US6kan into HaCaT cells could also be inhibited with antibodies specific for HveC. Interestingly, anti-HveC antibodies were not fully able to inhibit entry of wild-type HSV-1 into HaCaT cells. These results help to uncover important properties of HSV and human keratinocytes. HSV, with exceedingly low levels of a crucial receptor-binding glycoprotein, can enter cells expressing high levels of receptor. In this case, surplus gD may be useful to avoid neutralization by anti-gD antibodies.

Herpes simplex virus gE/gI sorts nascent virions to epithelial cell junctions, promoting virus spread.

Alphaherpesviruses spread rapidly through dermal tissues and within synaptically connected neuronal circuitry. Spread of virus particles in epithelial tissues involves movement across cell junctions. Herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseudorabies virus (PRV) all utilize a complex of two glycoproteins, gE and gI, to move from cell to cell. HSV gE/gI appears to function primarily, if not exclusively, in polarized cells such as epithelial cells and neurons and not in nonpolarized cells or cells that form less extensive cell junctions. Here, we show that HSV particles are specifically sorted to cell junctions and few virions reach the apical surfaces of polarized epithelial cells. gE/gI participates in this sorting. Mutant HSV virions lacking gE or just the cytoplasmic domain of gE were rarely found at cell junctions; instead, they were found on apical surfaces and in cell culture fluids and accumulated in the cytoplasm. A component of the AP-1 clathrin adapter complexes, mu1B, that is involved in sorting of proteins to basolateral surfaces was involved in targeting of PRV particles to lateral surfaces. These results are related to recent observations that (i) HSV gE/gI localizes specifically to the trans-Golgi network (TGN) during early phases of infection but moves out to cell junctions at intermediate to late times (T. McMillan and D. C. Johnson, J. Virol., in press) and (ii) PRV gE/gI participates in envelopment of nucleocapsids into cytoplasmic membrane vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004-4016, 2000). Therefore, interactions between the cytoplasmic domains of gE/gI and the AP-1 cellular sorting machinery cause glycoprotein accumulation and envelopment into specific TGN compartments that are sorted to lateral cell surfaces. Delivery of virus particles to cell junctions would be expected to enhance virus spread and enable viruses to avoid host immune defenses.

The extracellular domain of herpes simplex virus gE is sufficient for accumulation at cell junctions but not for cell-to-cell spread.

Herpes simplex virus (HSV) expresses a number of membrane glycoproteins, including gB, gD, and gH/gL, that function in both entry of virus particles and movement of virus from an infected cell to an uninfected cell (cell-to-cell spread). However, a complex of HSV glycoproteins gE and gI (gE/gI) is required for efficient cell-to-cell spread, especially between cells that form extensive cell junctions, yet it is not necessary for entry of extracellular virions. We previously showed that gE/gI has the capacity to localize specifically to cell junctions; the glycoprotein complex was found at lateral surfaces of cells in contact with other cells but not at those lateral surfaces not forming junctions or at apical surfaces. By virtue of these properties, gE/gI is an important molecular handle on the poorly understood process of cell-to-cell spread. Here, we show that the cytoplasmic domain of gE is important for the proper delivery of gE/gI to lateral surfaces of cells. Without this domain, gE/gI is found on the apical surface of epithelial cells, and more uniformly in the cytoplasm, although incorporation into the virion envelope is unaffected. However, even without proper trafficking signals, a substantial fraction of gE/gI retained the capacity to accumulate at cell junctions. Therefore, the extracellular domain of gE can mediate accumulation of gE/gI at cell junctions, if the glycoprotein can be delivered there, probably through interactions with ligands on the opposing cell. The role of phosphorylation of the cytoplasmic domain of gE was also studied. A second mutant HSV type 1 was constructed in which three serine residues that form a casein kinase II phosphorylation site were changed to alanine residues, reducing phosphorylation by 70 to 80%. This mutation did not affect accumulation at cell junctions or cell-to-cell spread.

Induction of B cell lymphomas by overexpression of a Myb oncogene truncated at either terminus.

The c-myb oncogene encodes a nuclear transcriptional transactivator that is often terminally truncated in hematopoietic tumors. To directly assess the tumorigenic activity of full length and terminally-truncated variants of c-myb, we have overexpressed several structurally-altered forms of myb within an avian retroviral vector and have shown that overexpression of truncated (but not full length) myb transforms both myeloid cells in vitro and mesenchymal cells in vivo. In vivo infection with these truncated myb viruses is now shown to induce metastatic B cell lymphomas in a significant minority of animals. Evaluation of the lymphomas revealed two distinct mechanisms of myb-induced tumorigenesis. In most of the lymphomas, proviral DNA inserted into the endogenous chicken c-myb gene and promoted the expression of a 5'-truncated myb transcript encoding an amino terminal truncated protein. In comparison, some animals infected with a virus encoding a carboxyl (C) terminal truncated myb (T-myb) developed non-insertional B cell lymphomas that directly expressed the provirally-encoded T-myb gene. The lymphomagenic T-myb protein lacks 214 C terminal amino acids including all of the myb transcription inhibition domain. This novel lymphomagenic activity for a C terminal truncated myb suggests that a loss of regulatory sequences at either end of c-myb is sufficient to create a B cell-specific transforming gene.

Tumor cells induced by the v-src oncogene are heterogeneous for expression of markers of mesenchyme differentiation.

The observation that v-src-induced tumors contain tumor cells of differing morphology, notably fibroblastoid or polygonal, raised the question as to whether the tumor cells are also heterogeneous with respect to expression of markers of cellular differentiation. Of the markers tested here, consistent reactivity for tumor tissue was noted only for antibody probes reactive to muscle actin (HHF35, alpha sm-1) or to procollagen type I (SP1. D8); for any given tumor, whether induced by v-src DNA or by Rous sarcoma virus, each of these markers was found only in a subpopulation of tumor cells. The observation of marker heterogeneity in the one v-src DNA-induced tumor examined here that typed as monoclonal suggests that v-src-induced transformation is consonant with a degree of plasticity in the phenotypes of the clonal progeny of a single transformant.

Immune-based resistance to the formation of v-src-induced distal tumors.

Although v-src, the oncogene of Rous sarcoma virus, has been shown to be capable of inducing lethal tumors at visceral sites distal to the primary tumor mass, the mechanisms opposing visceral tumor formation remain to be elucidated. In the present study, we show that visceral tumors, many of which represent a metastasis spawned by the primary mass, are found only in hosts exhibiting reduced levels of tumor immunity. We conclude that it is the weakness of the tumor immune response, rather than a lack of expression of tumor antigen on visceral tumor cells, that is a major underpinning of the formation of v-src-induced visceral tumors.

Tumor immunity generated in the course of regression of v-src-induced sarcomas.

Previous studies have indicated that the regression versus progression of v-src-DNA-induced sarcomas is dependent on chicken line. As a first step in analyzing the role of tumor immunity as a determinant of this line dependence, experiments were undertaken to ascertain whether an antisarcoma immune response is generated in the course of sarcoma growth in TK chickens, a regressor line. To assay for this response, test TK chickens in which v-src-induced wing web sarcomas had regressed, as well as control TK chickens that had not been exposed to v-src, were challenged in protocols known to yield v-src-dependent sarcoma formation and monitored for challenge sarcoma growth. Compared with the control chickens, the test chickens showed a significant resistance to the sarcomagenic challenge. These results raise the possibility that the antisarcoma response that is inducible in regressor lines, as demonstrated here in terms of a protective effect against a subsequent sarcomagenic challenge, may also underlie the regression of v-src-induced primary sarcomas.