Imaging Fibrogenesis in a Diet-Induced Model of Nonalcoholic Steatohepatitis (NASH).

Purpose: Liver fibrosis is the hallmark of chronic nonalcoholic steatohepatitis (NASH) and is characterised by the excessive deposition of extracellular matrix proteins. Early detection and accurate staging of liver fibrosis is critically important for patient management. One of the earliest pathological markers in NASH is the activation of hepatic stellate cells (HSCs) which may be exploited as a marker of fibrogenesis. Activated HSCs secreting factors such as integrin α v ß 3 propagate fibrosis. The purpose of the current study was to assess the utility of the integrin α v ß 3 imaging agent [18F]FtRGD for the early detection of fibrosis in a diet-induced model of NASH longitudinally using PET imaging. Procedures: Mice were fed with either standard chow diet (SD), high-fat diet (HFD), or a choline-deficient, L-amino acid-defined high-fat fibrogenic diet (CDAHFD) to mimic the clinical pathology of liver disease and followed longitudinally for 10 weeks to assess the development of liver fibrosis using [18F]FtRGD positron emission tomography (PET) imaging. Standard blood biochemistry, histological measures, and qPCR were used to quantify integrin α v ß 3, smooth muscle actin, and collagen types 1 and 6 to assess the extent of NASH pathology and accurately stage liver fibrosis. Results: The CDAHFD fibrogenic diet predictably developed hepatic inflammation and steatosis over the 10 weeks studied with little NASH pathology detected in high fat diet-treated animals. Stage 1 fibrosis was detected early by histology at day 21 and progressed to stage 2 by day 35 and stage 3 by day 56 in mice fed with CDAHFD diet only. Noninvasive imaging with [18F]FtRGD correlated well with integrin α v ß 3 and was able to distinguish early mild stage 2 fibrosis in CDAHFD animals compared with standard chow diet-fed animals at day 35. When compared with high fat diet-fed animals, [18F]FtRGD was only able to distinguish later moderate stage 2 fibrosis in CDAHFD animals at day 49. Conclusions: The diet-induced progression of liver fibrosis was confirmed using histology and correlated well with the mRNA of integrin α v ß 3 and extracellular matrix protein expression. [18F]FtRGD showed very good correlation between liver uptake and integrin α v ß 3 expression and similar detection sensitivity to the current clinical gold standard modalities for staging of liver fibrosis.

Postauricular cutaneous mastoid fistula.

Postauricular cutaneous mastoid fistula is a rare condition. The cutaneous mastoid fistula is a very rare complication of chronic suppurative otitis media. The fistula tracts are typically difficult to manage because of the surrounding necrotic skin edges. We describe an unusual case of a postauricular cutaneous mastoid fistula and outline the surgical technique used for closure.

Vaccination of maternally immune pigs with a live Aujeszky's disease vaccine by coarse spray and other routes.

33 ten weeks old passively immune weaners were inoculated with live, attenuated Aujeszky's disease (AD) vaccine, according to four different vaccination protocols: (groups A/A2) 3 x coarse spray vaccination at 10, 11 and 13 weeks of age, (groups B/B2) 1 x coarse spray at 10 weeks of age followed by 1 x intramuscularly at 13 weeks, (C) 1 x intranasal instillation at 10 weeks of age, and (groups D/D2) 2 x intramuscularly at 10 and 13 weeks of age. A further 10 weaners were included as unvaccinated controls (E/E2). Spray vaccination was technically simple to perform but on average, 20% of subjects were reluctant to expose themselves to the spray. Clinical reactions were absent apart from mild fever in one pig from group B. Weight gains between 10 and 17 weeks of age were slightly lower in group A and group B weaners, compared to control unvaccinated pigs and pigs vaccinated by other routes. Virus neutralising (VN) antibody response was extremely uneven between individuals in groups A and B. Group D pigs vaccinated 2 x intramuscularly showed a 3 week lag in developing high levels of antibody but the intramuscular route, as well vaccination by intransal instillation, proved to be the most dependable technique for inducing uniformly high levels of VN antibody. Challenge with virulent ADV at 17 weeks of age resulted in death from Aujeszky's disease of all five control pigs. One pig in group A which had no VN antibody, also died. All other pigs were protected against death.(ABSTRACT TRUNCATED AT 250 WORDS)

Immune responses of specific pathogen free foals to EHV-1 infection.

Four foals were raised under specific pathogen free (SPF) conditions. At 3 to 4 months of age, SPF foals and 1 other non-SPF foal were intranasally inoculated with equine herpes virus type 1 (EHV-1). Clinical signs included depression, fever, inappetence and intermittent coughing. Clinical recovery was complete by seven days but high titres of virus were detected in nasal mucus for at least 10 days after inoculation. Clinical illness was less severe in the non-SPF foal. Interferon was detected in the nasal mucus of all foals from 2 days post infection (dpi), persisting until 8 or 10 dpi. ELISA antibody was detected in serum from 6 dpi. Titres continued to rise throughout the period of observation, and were slightly stimulated by re-inoculation. EHV antibody, identified as belonging to the IgM class by the double sandwich ELISA, was detected from 6 dpi. Peak IgM titres were observed between day 10 and 18, declining to base levels by day 42. Virus neutralizing antibody was detectable in serum from day 14 and rises in titre were parallel to that of total ELISA antibody. Cellular immunity in EHV-1 infected SPF horses was examined by the antibody dependent cytotoxicity (ADCC) test and the specific lymphocyte transformation test. The ability of foal neutrophils to effect ADCC decreased significantly between 3 to 10 days after inoculation. Peripheral blood mononuclear cells (PBMC) displayed reactivity towards EHV-1 antigens from about day 14, with maximum stimulation indices being obtained between 28 and 42 dpi.

Natural killer cells in normal horses and specific-pathogen-free foals infected with equine herpesvirus.

Peripheral blood mononuclear cells (PBMC) from an adult horse and from foals demonstrated natural killer (NK)-type cytotoxicity against a range of xenogeneic and allogeneic cell targets. The human tumour cell line, Chang liver was consistently the most susceptible. Chang liver, rabbit kidney (RK-13), equine sarcoid (ES) and embryonic equine kidney (EEK) cells were more susceptible when presented to horse PBMC than monolayer cultures. Embryonic equine lung (EEL) and murine YAC-1 cells conversely, were more susceptible in a trypsinized state. Horse PBMC demonstrated higher levels of NK-type activity against EEK, EEL and RK-13 cells infected with equine herpesvirus 1 (EHV-1) compared with uninfected cells. Similarly, EEK and EEL cells infected with Semliki forest virus (SFV) were more susceptible. Cytotoxicity against EHV-1-infected EEK cells developed faster, between 4 and 8 h of incubation and reaching a maximum at 24 h. By contrast, cytotoxicity against uninfected fibroblasts was not significant until approximately 16 h of incubation with maximum cytotoxicity observed between 32 h and 48 h. Specific pathogen-free (SPF) foals were inoculated with live EHV-1. PBMC isolated from these foals at different days after inoculation did not display appreciably reduced or elevated NK cytotoxicities against Chang liver cells and EHV-1-infected EEK targets, when compared with that of a PBMC reference from a healthy adult horse.

Prostaglandin E2 receptor activity and susceptibility to natural killer cells.

We have described a high-affinity receptor for prostaglandin E2 (PGE2) present on metastatic murine mammary tumor cells. Pharmacologic antagonism of this receptor increases metastatic potential. In the present study, we have asked whether the binding activity of PGE on tumor target cells plays a role in natural killer (NK)-target cell interactions. We have used three unrelated PGE-receptor antagonists, SC19220, LEO101, and AH6809, to show inhibition of [3H]PGE2 binding to YAC-1 cells and inhibition of PGE2-mediated elevation of intracellular cyclic AMP (cAMP). Addition of any of these three receptor antagonists to standard 4-h 51Cr-release assays inhibits YAC-1 lysis by NK-enriched populations from murine spleen. This is the first report that antagonism of PGE binding affects NK activity. Our studies demonstrate that these effects are mediated through inhibition of target-effector cell conjugate formation. Studies in which effector and target cells were pretreated separately indicate that the PGE-mediated effects are expressed at the target cell level.

The role of macrophage-derived TNFa in the induction of sublethal tumor cell DNA damage.

In previous studies we showed that tumor-associated macrophages isolated from murine mammary tumors are mutagenic to bacteria and mammalian cells and thus may contribute to tumor progression. We reported previously, and confirm here, that inflammatory macrophages induce DNA strand breaks in cultured mammary tumor cells co-incubated at a 1:1 ratio for 1 h. This activity is prevented by inhibitors of arachidonate metabolism or the removal of H2O2 with catalase. In the present study, we show that two antibodies to recombinant murine tumor necrosis factor alpha (rMuTNFa)--a hamster monoclonal antibody (TN3-19.12) and a rabbit polyclonal antibody (Genzyme)--partially protect tumor cells from DNA strand breaks induced by elicited but not resident peritoneal macrophages. Antibody protection was reversed upon the addition of excess exogenous rMuTNFa. Purified rMuTNFa alone was unable to induce DNA strand breaks in the absence of macrophages, indicating that TNFa is necessary but not sufficient to mediate damage. Tumor target cells were completely resistant to the cytotoxic effects of rMuTNFa in the absence of actinomycin D and relatively resistant (in comparison to WEHI 164 clone 13 cells) in its presence. The incomplete protection seen with either catalase or anti-TNF suggests that macrophage-released TNFa, in the presence of other factors, induces non-cytotoxic DNA effects in tumor cells.

Role of the prostaglandin E2 receptor in mammary tumor metastasis.

Both clinical and experimental breast tumors often synthesize high levels of prostaglandins, most notably prostaglandin E2 (PGE2). We have reported previously that metastatic murine mammary tumor cells also express a high-affinity PGE2 receptor. We have now shown that the receptor plays a functional role in the metastasis of two mammary tumor cell subpopulations, lines 66 and 4526. We showed that three agents, LEO101 (LEO Pharmaceuticals), SC19220 (Searle Co.), and AH6809 (Glaxo Co.), antagonize [3H]PGE2 binding to these cells and block PGE2-mediated elevation of intracellular cyclic AMP. Pretreatment of line 66 cells with nontoxic concentrations of any of the three receptor antagonists prior to i.v. injection results in more experimental lung colonies. As shown previously, and confirmed here, pretreatment of these cells with indomethacin (which inhibits endogenous PGE synthesis and therefore increases detectable PGE receptor) inhibits metastasis. Thus, the tumor cell PGE2 receptor contributes to the ability of murine mammary tumor cells to metastasize.

The raising of equine colostrum-deprived foals; maintenance and assessment of specific pathogen (EHV-1/4) free status.

Over a period of two years, a total of 22 full term foals from Welsh Mountain pony mares were raised in conditions that were free from infection by Equid herpesvirus (EHV-1/4). Parturition dates were predicted by monitoring colostrum electrolytes, and the mares allowed to foal naturally under supervision or following induction with intravenous oxytocin. Immediately following birth, foals were separated from their dams and transferred to a specially built, positive pressure isolation unit. They were given antibiotic prophylaxis and fed bovine colostrum during the first 24 h, and then mare's milk replacer until weaned. Out of 22 specific pathogen free (SPF) foals one that had not been given antibiotic prophylaxis died of an E. coli septicaemia aged eight days. Two foals developed a streptococcal upper respiratory tract infection, which responded to antibiotic therapy and did not spread to the rest of the herd. A self limiting upper respiratory tract infection was seen in a fourth foal and mild diarrhoea was observed in six foals. Physical development in all SPF foals appeared normal and behavioural patterns resembled those of conventional handreared foals. Newborn foals were held in a separate quarantine area, within the isolation unit, and checked extensively for evidence of EHV-1/4 infection, before being transferred to the main holding unit. Periodic checks were then made for EHV-1/4 over a period ranging from 2 to 4 months; none of the SPF foals showed evidence of infection with EHV-1/4 in terms of clinical disease, virus isolation, sero-conversion or specific lymphocyte transformation.

The pathogenesis of equine herpesvirus type 1 in the mouse: a new model for studying host responses to the infection.

An infection was established in adult BALB/c mice by means of intranasal inoculation of the AB4 strain of equine herpesvirus type 1 (EHV-1). The acute infection was confined to the respiratory tract and blood. Virus was shown to replicate in the nasal mucosa, trachea and lung for several days producing clinical signs of disease. Viraemia was also detected and a small proportion of peripheral blood cells contained virus at the peak of the infection. Histological and electron microscopic evidence were obtained which proved that productive virus replication occurred in the ciliated epithelial cells lining the bronchi and in pneumocytes in the lung, resulting in the destruction of these cells. Both humoral and cell-mediated responses to the infection were detected and monitored. By means of immunoprophylaxis or chemotherapy it was possible to modify the course of the infection. This infection model has many striking features in common with that observed in the natural host and the observations suggest that the mouse is a convenient and relevant model in which to study both host responses to EHV-1 infection and modification of the pathogenesis by means of immunoprophylaxis or therapy.

Macrophage-mediated induction of DNA strand breaks in target tumor cells.

We have shown previously that macrophages are mutagenic to bacteria (A. M. Fulton et al., Cancer Res., 44: 4308-4311, 1984) and can induce the appearance of drug-resistant variants of murine mammary tumor cells (K. Yamashina et al., Cancer Res., 46: 2396-2401, 1986). The present study asks whether inflammatory macrophages can induce lesions in the DNA of cocultured tumor cells and seeks to determine the mediators of this damage. We quantitated the induction of DNA strand breaks using the technique of fluorometric analysis of DNA unwinding. We report that inflammatory macrophages coincubated with a mammary tumor cell line for 60 min at a 1:1 ratio result in significant numbers of strand breaks in the tumor cell DNA. The degree of damage is equivalent to 300 to 1200 rads of gamma-irradiation. Resident (unstimulated) peritoneal macrophages also induce tumor cell DNA strand breaks. However, inhibitor studies reveal quantitative and qualitative differences in strand breaks induced by inflammatory (elicited) versus resident peritoneal macrophages. Resident macrophages require a longer induction period (60 min) before significant breaks are detected, but induce more breaks than do elicited macrophages, which require only a 5-min coincubation period to induce significant damage. The enzyme catalase, which removes H2O2, protects tumor cells from both macrophage effector populations as does the prostaglandin synthase inhibitor, indomethacin. The superoxide anion scavenger, superoxide dismutase, and the lipoxygenase inhibitor, nordihydroguaiaretic acid, are protective against resident macrophage effects only. The metal chelator, o-phenanthroline, provides limited protection for elicited macrophages but induces total DNA breakage in the presence of resident macrophages. Taken together, our data indicate that the degree of strand breakage is greater for the macrophage population with high arachidonate metabolism and low oxidative metabolism (resident macrophages) and less for the macrophage population with high oxidative and low arachidonate metabolism (MVE-2 elicited macrophages). Inhibitor studies implicate both metabolites of reactive oxygen and arachidonate as mediators of this tumor cell DNA damage, with the relevant mediator dependent upon the particular macrophage population under study.

Relative rates of Aujeszky's disease virus attenuation, as assessed in mice.

Field strains of Aujeszky's disease virus (ADV) were attenuated by heat treatment and serial passage at sub-optimal growth temperatures in chicken embryo fibroblasts (CEF). At chosen passage levels, virus was titrated in cell culture and in mice. For each strain, the pathogenicity was expressed as a mouse lethal index (MLI), defined as the inverse of the log10 (CCID50:LD50). MLIs determined for field strains displayed a wide range of comparatively high values. The attenuation of field strains was accompanied by a rapid fall in MLI values, particularly in the initial stages. Heat-treated ADV attenuated faster than untreated ADV, when passaged at 30 degrees C. Passage at 27 degrees C resulted in considerably accelerated attenuation compared to passage at 30 degrees C, in the case of both untreated and heat-treated ADV. MLIs were determined for attenuated ADV strains that had been tested in 6-day-old piglets. Low MLI values were found to correlate with low virulence in piglets and high MLI values with virulence.