Congenital Tumours and Tumour-Like Lesions in Calves: a Review.

Congenital tumours and tumour-like lesions represent a group of rare disorders in both veterinary and human medicine that arise from tissue remnants and are detected during pregnancy or within the first 2-3 months of life. Different forms of congenital tumours and congenital tumour-like lesions have been reported in calves and their development is poorly understood. They often pose a diagnostic challenge and the referring nomenclature occasionally may be equivocal. Previous reports regarding tumour-like lesions, soft tissue tumours, vascular tumours, round cell tumours and neoplasms of the nervous, peritoneum and urogenital systems are summarized in this review, and the role of genetic factors in the development of these conditions is discussed.

Molecular determinants of alphavirus neuropathogenesis in mice.

Alphaviruses are enveloped viruses with a positive-stranded RNA genome, of the family Togaviridae. In mammals and birds they are mosquito-transmitted and are of veterinary and medical importance. They cause primarily two types of disease: encephalitis and polyarthritis. Here we review attempts to understand the molecular basis of encephalitis and virulence for the central nervous system (CNS) in mouse models. Sindbis virus (SINV) was the first virus to be studied in this way. Other viruses analysed are Semliki Forest virus (SFV), Venezuelan equine encephalitis virus, Eastern equine encephalitis virus and Western equine encephalitis virus. Neurovirulence was found to be associated with damage to neurons in the CNS. It mapped mainly to the E2 region of the genome, and to the nsP3 gene. Also, avirulent natural isolates of both SINV and SFV have been found to have more rapid cleavage of nonstructural proteins due to mutations in the nsP1-nsP2 cleavage site. Immune-mediated demyelination for avirulent SFV has been shown to be associated with infection of oligodendrocytes. For Chikungunya virus, an emerging alphavirus that uncommonly causes encephalitis, analysis of the molecular basis of CNS pathogenicity is beginning. Experiments on SINV and SFV have indicated that virulence may be related to the resistance of virulent virus to interferon action. Although the E2 protein may be involved in tropism for neurons and passage across the blood-brain barrier, the role of the nsP3 protein during infection of neurons is unknown. More information in these areas may help to further explain the neurovirulence of alphaviruses.

Virulence variation among isolates of western equine encephalitis virus in an outbred mouse model.

Little is known about viral determinants of virulence associated with western equine encephalitis virus (WEEV). Here, we have analysed six North American WEEV isolates in an outbred CD1 mouse model. Full genome sequence analyses showed < or =2.7 % divergence among the six WEEV isolates. However, the percentage mortality and mean time to death (MTD) varied significantly when mice received subcutaneous injections of 10(3) p.f.u. of each virus. Two WEEV strains, McMillan (McM) and Imperial 181 (IMP), were the most divergent of the six in genome sequence; McM caused 100 % mortality by 5 days post-infection, whereas IMP caused no mortality. McM had significantly higher titres in the brain than IMP. Similar differences in virulence were observed when McM and IMP were administered by aerosol, intranasal or intravenous routes. McM was 100 % lethal with an MTD of 1.9 days when 10(3) p.f.u. of each virus was administered by intracerebral inoculation; in contrast, IMP caused no mortality. The presence of IMP in the brains after infection by different routes and the lack of observed mortality confirmed that IMP is neuroinvasive but not neurovirulent. Based on morbidity, mortality, MTD, severity of brain lesions, virus distribution patterns, routes of infection and differences in infection of cultured cells, McM and IMP were identified as high- and low-virulence isolates, respectively.

Electroporation of RNA stimulates immunity to an encoded reporter gene in mice.

Electroporation is the application of high-voltage short-duration pulses to transiently permeabilize cells, permitting the cellular uptake of macromolecules, including nucleic acid. Although much attention has been focused on DNA vaccines, antigen-encoding RNA molecules may also stimulate immunity. Several methods are being examined in an effort to enhance the efficacy of nucleic acid delivery. One such method is the application of electroporation. The present study was designed to develop electroporation for use as a method of RNA delivery in conjunction with the Semliki Forest virus (SFV) RNA vector system for stimulation of immunity. Expression of SFV-based ß-galactosidase and luciferase vectors was observed in the muscle after electroporation. Although some tissue damage was induced following intramuscular injection and electroporation with SFV vector RNA encoding LacZ at optimum pulse conditions, immunity to LacZ was efficiently induced. Following two immunizations, there was a higher IgG2a antibody response with the viral vector delivery and a higher IgG1 response in electroporated rSFV-LacZ RNA immunized mice.

Therapeutic and prophylactic applications of alphavirus vectors.

Alphavirus vectors are high-level, transient expression vectors for therapeutic and prophylactic use. These positive-stranded RNA vectors, derived from Semliki Forest virus, Sindbis virus and Venezuelan equine encephalitis virus, multiply and are expressed in the cytoplasm of most vertebrate cells, including human cells. Part of the genome encoding the structural protein genes, which is amplified during a normal infection, is replaced by a transgene. Three types of vector have been developed: virus-like particles, layered DNA-RNA vectors and replication-competent vectors. Virus-like particles contain replicon RNA that is defective since it contains a cloned gene in place of the structural protein genes, and thus are able to undergo only one cycle of expression. They are produced by transfection of vector RNA, and helper RNAs encoding the structural proteins. Layered DNA-RNA vectors express the Semliki Forest virus replicon from a cDNA copy via a cytomegalovirus promoter. Replication-competent vectors contain a transgene in addition to the structural protein genes. Alphavirus vectors are used for three main applications: vaccine construction, therapy of central nervous system disease, and cancer therapy.

The 5' untranslated region as a pathogenicity determinant of Semliki Forest virus in mice.

An investigation of the role of the 5' untranslated region (UTR) of Semliki Forest virus (SFV) in determining pathogenicity in infected mice was carried out by constructing 5' UTR chimeras. Analysis of 5' UTR sequences showed nucleotide differences between virulent and avirulent strains at positions 21, 35 and 42. Reciprocal chimeras incorporating these changes were constructed from avirulent CA7 and rA7[74], and virulent SFV-4 virus, derived from infectious clones, and avirulent A7 and A7[74] plaque-purified stock virus. Survival rates and neuropathology in intranasally (i.n.) infected mice were analysed. While no statistically significant difference between rates of RNA synthesis was detected between strains in cell culture, an increase in survival of infected mice and a reduction in the severity of brain lesions was observed on substitution of the 5' UTR from a stock avirulent virus into an infectious clone where the remainder of the genome was derived from avirulent virus. However, substitution of a 5' UTR from an avirulent stock virus into an infectious clone where the remainder of the genome was from virulent virus did not affect virulence. These results and other studies suggest that control of virulence is polygenic, and that the SFV 5' UTR acts as a pathogenicity determinant in synergy with other determinants in the genome.

Semliki Forest virus vectors expressing the H and HN genes of measles and mumps viruses reduce immunity induced by the envelope protein genes of rubella virus.

A Semliki Forest virus (SFV) recombinant particle vaccine vector was constructed expressing the viral E1 and E2 envelope proteins of the RA27/3 vaccine strain of rubella virus. This vector induced high titres of antibody after intramuscular administration to Balb/C mice, both following initial vaccination and a boost 4 weeks later. This occurred for antibody as measured by ELISA and as measured by a latex agglutination test. However, co-administration of similar particles expressing the measles virus H protein and the mumps virus HN protein with the rubella protein expressing vector resulted in reduction of the anti-rubella immune response.

Deletions in the hypervariable domain of the nsP3 gene attenuate Semliki Forest virus virulence.

Major virulence determinants of Semliki Forest virus (SFV) lie within the non-structural genes that form the replicase complex proteins. Gene exchange between virulent and avirulent viruses has shown that the nsP3 gene, which has essential 5' conserved domains and a non-essential hypervariable 3' domain, is one of the virulence determinants. This protein plays a role in subgenomic 26S and negative-strand RNA synthesis and is thought to function with nsP1 to anchor replication complexes to cell membrane structures. Studies to date have focused on analysing the effect of mutational changes spread over the whole gene on virulence of the virus. The virulent SFV4 virus, derived from an infectious clone, was utilized to analyse the effect on virulence of large deletions in the hypervariable domain of nsP3. Two viruses with different in-frame deletions that spanned this domain showed reduced rates of RNA synthesis and multiplication in cell culture. In adult BALB/c mice, these viruses were avirulent after intramuscular and intraperitoneal inoculation, and brains sampled from infected mice showed minimal or no evidence of pathology. These deleted viruses had greatly reduced virulence when administered by the intranasal route and brains from infected mice showed lesions that were much less severe than those seen in SFV4 infection. Mice surviving infection with the deleted viruses resisted challenge with the virulent L10 strain, indicating induction of protective immunity. This work establishes that deletions in the nsP3 hypervariable domain attenuate virulence after peripheral inoculation and also reduce virulence after intranasal inoculation.

The pyrrolo-1,5-benzoxazepine, PBOX-6, inhibits the growth of breast cancer cells in vitro independent of estrogen receptor status and inhibits breast tumour growth in vivo.

Members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds have been shown to induce apoptosis in a number of human leukemia cell lines of different haematological lineage, suggesting their potential as anti-cancer agents. In this study, we sought to determine if PBOX-6, a well characterised member of the PBOX series of compounds, is also an effective inhibitor of breast cancer growth. Two estrogen receptor (ER)-positive (MCF-7 and T-47-D) and two ER-negative (MDA-MB-231 and SK-BR-3) cell lines were examined. The 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine reduction in cell viability. PBOX-6 reduced the cell viability of all four cell lines tested, regardless of ER status, with IC(50) values ranging from 1.0 to 2.3 microM. PBOX-6 was most effective in the SK-BR-3 cells, which express high endogenous levels of the HER-2 oncogene. Overexpression of the HER-2 oncogene has been associated with aggressive disease and resistance to chemotherapy. The mechanism of PBOX-6-induced cell death was due to apoptosis, as indicated by the increased proportion of cells in the pre-G1 peak and poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, intratumoural administration of PBOX-6 (7.5 mg/kg) significantly inhibited tumour growth in vivo in a mouse mammary carcinoma model (p=0.04, n=5, Student's t-test). Thus, PBOX-6 could be a promising anti-cancer agent for both hormone-dependent and -independent breast cancers.

Alphaviruses and their derived vectors as anti-tumor agents.

The alphaviruses Semliki Forest virus (SFV) and Sindbis virus have recently been developed as prototype anti-cancer agents. These are RNA-containing enveloped viruses that code for only 9 proteins of unique sequence. The standard recombinant SFV vector system consists of suicide particles containing recombinant RNA. In addition, alphavirus vectors capable of limited multiplication in the host are also being developed. Several strategies are being adopted to construct prototype SFV vectors for cancer treatment. These include: 1) construction of both prophylactic and therapeutic vaccines to stimulate immunity to tumor-associated antigens, 2) use of apoptosis induction to destroy tumor cells, which includes both the use of the inherent apoptosis-inducing ability of the vector and the action of pro-apoptotic genes cloned into the vector, and 3) expression of cytokines and other immunoregulatory proteins by the vector that enhance anti-tumor immune responses and/or inhibit tumor cell growth. This includes the use of cytokines such as IL-12 that target angiogenesis. Sindbis virus appears to have a natural tropism for tumor cells that may allow targeting both of the wild-type virus and the vector. This approach may also be useful for targeting metastases. For SFV, neurovirulence and/or neurotropism, as well as other tissue damage, may preclude the use of unmodified replication competent wild-type virus in tumor treatment. However, it may be possible to use such a virus in animals that have been vaccinated, using a vector-derived vaccine.

Calcospherite-like bodies and caseous necrosis in tracheal mucus from a dog with tuberculosis.

A 3-year-old Wirehaired Fox Terrier was presented to the University Veterinary Hospital, University College Dublin, for evaluation of chronic cough of 8-months duration. Bronchoscopy showed a severely dilated collapsed left principal bronchus filled with highly viscous white mucus. Cytologically, globular lipid-like material and round concentrically laminated crystalline structures were evident within the proteinaceous mucus. These findings resembled the calcospherites and granular caseous debris often observed in human tuberculous patients. A Ziehl-Neelsen-stained cytocentrifuged preparation of material obtained by bronchoalveolar lavage revealed a few acid-fast rods within macrophages, suggestive of tuberculosis. At necropsy, granulomas with caseous necrosis were present in the lung parenchyma, bronchial and mediastinal lymph nodes, liver, pancreas, and mesentery. Granulomas were adherent to both kidney capsules and to the diaphragm. Histologically, there was evidence of mild calcification within caseous granulomas, which was confirmed by von Kossa's stain. Using Ziehl-Neelsen stain, acid-fast rods were identified within granulomas; bacterial culture was positive for Mycobacterium bovis. The cytologic findings in this case have not been reported previously in dogs and demonstrate a possible correlation between tuberculosis and calcospherite-like bodies with caseous, globular material in bronchial mucus, similar to that described in human patients.

Effect of intranasal administration of Semliki Forest virus recombinant particles expressing reporter and cytokine genes on the progression of experimental autoimmune encephalomyelitis.

We have initiated studies to determine the feasibility of employing the Semliki Forest virus (SFV) expression system as a central nervous system (CNS) vector. We investigated the effects of infecting Balb/c mice intranasally (i.n.) with recombinant SFV particles expressing the enhanced green fluorescent protein (EGFP) reporter gene. EGFP expression was detected by fluorescence microscopy in the olfactory bulb as early as 1 day postinfection. No pathological changes were associated with infection. Viral RNA could be detected in the olfactory mucosa only, whereas fluorescence was detected in axons in the olfactory bulb, indicating that only the expressed protein was present. A vector expressing interleukin 10 (IL-10) was constructed and shown to induce good cytokine expression in cultured cells. IL-10 expression in the nasal passage and olfactory bulb of infected mice was enhanced following i.n. administration of such particles. Mice induced for experimental autoimmune encephalomyelitis (EAE) were treated i.n. with vectors expressing EGFP and IL-10 and with empty vector. The EGFP-expressing and empty vectors were found to exacerbate EAE, whereas that expressing IL-10 ameliorated EAE. It is concluded that the mice showed a significant biological response when treated i.n. with recombinant SFV particles and that such particles administered by the i.n. route have potential as a noninvasive vector for protein delivery to the CNS.

Inhibition of matrix metalloproteinases ameliorates blood-brain barrier disruption and neuropathological lesions caused by avirulent Semliki Forest virus infection.

Semliki Forest virus (SFV) infection of mice is a useful model of viral neuropathogenesis in animals and avirulent strains such as SFV-A7 induce immune-mediated demyelination and death of neurones by necrosis and apoptosis. Matrix metalloproteinases (MMPs) have been implicated in various diseases including arthritis and cancer in many species. In this report, we show that MMP-2 and MMP-9 expression is induced in the brains of mice infected i.n. with SFV-A7. Treatment of mice with the pan MMP inhibitor GM6001 ameliorated the development of SFV-induced neuropathological lesions via an effect on the integrity of the blood-brain barrier. Low levels of neuronal necrosis and demyelination in GM6001-treated mice correlated with localisation of fibrinogen staining to thin-walled blood vessels and less intense staining of the perivascular neuropil.