Myocardial collagen deposition and inflammatory cell infiltration in cats with pre-clinical hypertrophic cardiomyopathy.

The histological features of feline hypertrophic cardiomyopathy (HCM) have been well documented, but there are no reports describing the histological features in mild pre-clinical disease, since cats are rarely screened for the disease in the early stages before clinical signs are apparent. Histological changes at the early stage of the disease in pre-clinical cats could contribute to an improved understanding of disease aetiology or progression. The aim of this study was to evaluate the histological features of HCM in the left ventricular (LV) myocardium of cats diagnosed with pre-clinical HCM. Clinically healthy cats with normal (n = 11) and pre-clinical HCM (n = 6) were identified on the basis of echocardiography; LV free wall dimensions (LVFWd) and/or interventricular septal wall (IVSd) dimensions during diastole of 6-7 mm were defined as HCM, while equivalent dimensions <5.5 mm were defined as normal. LV myocardial sections were assessed and collagen content and inflammatory cell infiltrates were quantified objectively. Multifocal areas of inflammatory cell infiltration, predominantly lymphocytes, were observed frequently in the left myocardium of cats with pre-clinical HCM. Tissue from cats with pre-clinical HCM also had a higher number of neutrophils and a greater collagen content than the myocardium of normal cats. The myocardium variably demonstrated other features characteristic of HCM, including arteriolar mural hypertrophy and interstitial fibrosis and, to a lesser extent, myocardial fibre disarray and cardiomyocyte hypertrophy. These results suggest that an inflammatory process could contribute to increased collagen content and the myocardial fibrosis known to be associated with HCM.

Plasma pharmacokinetics of alfaxalone after a single intraperitoneal or intravenous injection of Alfaxan(®) in rats.

Alfaxalone (3α-hydroxy-5α-pregnane-11, 20-dione) is a neuroactive steroid with anaesthetic properties and a wide margin of safety. The pharmacokinetic properties of alfaxalone administered intravenously and intraperitoneally in rats (n = 28) were investigated. Mean t(1/2elim) for 2 and 5 mg/kg i.v. was 16.2 and 17.6 min, respectively, but could not be estimated for IP dosing, due to sustained plasma levels for up to 60 min after injection. Clp for i.v. injection was calculated at 57.8 ± 23.6 and 54.3 ± 6.8 mL/min/kg, which were 24.5% and 23% of cardiac output, respectively. The observed C(max) was 3.0 mg/L for IP administration, and 2.2 ± 0.9 and 5.2 ± 1.3 mg/L for 2 and 5 mg/kg i.v. administration, respectively. AUC(0-60) was 96.2 min.mg/L for IP dosing. The relative bioavailability for IP dosing was 26% and 28% compared to i.v. dosing. Differences in t(1/2elim) and Cl(p) from previous pharmacokinetic studies in rats are likely due to variations in alfaxalone formulation rather than sex differences. Alfaxan® given IP caused sustained levels of alfaxalone, no apnoea and longer sleep times than i.v. dosing, although immobilization was not induced in 30% of rats given Alfaxan® IP. A pharmacodynamic study of the effects of combining IP injection of Alfaxan® with other premedication agents is worthwhile, to determine whether improved anaesthesia induction could ultimately provide an alternative anaesthetic regimen for rats.

Complement C5a: impact on the field of veterinary medicine.

Complement C5a is a pro-inflammatory polypeptide produced during activation of the complement cascade in response to foreign antigens or tissue damage secondary to physical or chemical injury. C5a, via activation of the C5a receptor (C5aR or CD88), is a major inflammatory mediator involved in a number of diseases, including some of veterinary relevance. Greater understanding of the role of C5a has been possible with the availability of gene knockout mice, specific antibodies and peptide agonists/antagonists. This review outlines the functions of C5a and its role in the development of disease, including neoplastic conditions and sepsis, in animals of veterinary importance. The application of C5aR agonist and antagonist analogues to combat those conditions is also discussed.

Inhibition of immune-complex mediated dermal inflammation in rats following either oral or topical administration of a small molecule C5a receptor antagonist.

1. Initiation of a peritoneal Arthus reaction by deposition of immune-complexes results in vascular leakage, polymorphonuclear leukocyte (PMN) infiltration, and tumour necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6) production. We now demonstrate in rats that oral administration of the C5a receptor antagonist AcPhe[Orn-Pro-D-Cyclohexylalanine-Trp-Arg] (AcF-[OPdChaWR]; 1 - 10 mg kg(-1) 30 min prior to immune-complex deposition) inhibits these inflammatory markers in the peritoneal Arthus reaction. 2. Initiation of a dermal Arthus reaction resulted in a significant increase in vascular leakage, PMN infiltration, systemic production of TNFalpha and pathological changes in the dermis. 3. Pretreatment of rats with AcF-[OPdChaWR] either intravenously (1 mg kg(-1) 10 min prior to immune-complex deposition) or orally (1 - 10 mg kg(-1) 30 min prior to immune-complex deposition) significantly inhibited immune-complex mediated dermal vascular leakage and systemic cytokine production. Topical pretreatment with AcF-[OPdChaWR] (400 microg site(-1) in 10% dimethyl sulphoxide 10 min prior to immune-complex deposition) also inhibited vascular leakage, as well as histopathological changes associated with a dermal Arthus reaction. 4. Oral administration of 3 mg kg(-1) AcF-[OPdChaWR] resulted in the appearance of the drug in plasma within 5 min, with peak blood levels approximately 0.3 microM reached within 20 min. The plasma elimination half-life was approximately 70 min. The oral activity and bioavailability of AcF-[OPdChaWR], its activity when applied topically to the skin, suggest that small molecule C5a receptor antagonists may have therapeutic utility in dermal inflammatory disorders involving complement activation. 5. This is the first demonstration for either an orally or topically active C5a receptor antagonist, and suggests that small molecule C5a antagonists may have therapeutic utility when given by multiple routes of application.

Transforming growth factor-beta1 induces alpha-smooth muscle actin expression and fibronectin synthesis in cultured human retinal pigment epithelial cells.

BACKGROUND: Proliferative vitreoretinopathy is a serious complication of retinal detachment, yet its pathogenesis is not fully understood. Retinal pigment epithelial cells and glial cells are found in the fibrous membranes in proliferative vitreoretinopathy. Many cytokines are involved in the pathology. Transforming growth factor (TGF)-beta, a cytokine found in serum, has been shown to be an important factor regulating the synthesis of fibrous extracellular matrix in proliferative vitreoretinopathy. METHODS: Cultured human retinal pigment epithelial cells were used in the experiments. The effects of TGF-beta1 on phenotype and function in retinal pigment epithelial cells were recorded as changes in the expression of alpha-smooth muscle actin and fibronectin synthesis using immunohistochemistry and enzyme-linked immunosorbent assay, respectively. RESULTS: TGF-beta1 induced the expression of alpha-smooth muscle actin (P < 0.0001, n = 3), and significantly increased the synthesis of fibronectin by cultured human retinal pigment epithelial cells (P < 0.01, n = 4). CONCLUSIONS: Elevated levels of TGF-beta1 in proliferative vitreoretinopathy may contribute to phenotype changes in retinal pigment epithelial cells leading to matrix deposition and contraction. Since the elevated levels of TGF-beta1 may emanate from a number of diverse sources in proliferative vitreoretinopathy, developing an antagonist to TGF-beta1 may offer an approach to the treatment of proliferative vitreoretinopathy.

Lyprinol: anti-inflammatory and uterine-relaxant activities in rats, with special reference to a model for dysmenorrhoea.

Lyprinol exhibits anti-inflammatory activity distinct from that of most NSAIDs, controlling chronic but not acute inflammation. Unlike Cox-1 inhibitors (aspirin, meclofenamic acid) it is not gastro-toxic. Predosing rats with Lyprinol can modify both (i) the spontaneous and (ii) the oxytocin-induced contractions of the uterus. In humans there is anecdotal evidence that Lyprinol can relieve dysmenorrhea. This report explores the concept that the uterotrophic actions of Lyprinol are conditioned by: the intrinsic profile of estrogenic hormones and progestagens and, certain extrinsic stimuli. Evidence from in vitro studies indicates that Lyprinol is not a smooth muscle relaxant and that its uterotrophic mechanism is not that of a cyclo-oxygenase inhibitor, but may mimic that of a leukotriene receptor antagonist.

Cell phenotype as a target of drug therapy in chronic inflammatory diseases.

Many diseases share common pathological changes which could in principle be targets for new drugs. Vascular leakage of plasma and migration of cells into perivascular tissues are common to chronic inflammatory diseases such as asthma, atherosclerosis, arthritis, and proliferative nephropathy as well as some non-inflammatory proliferative disorders such as diabetes mellitus. Individual components of plasma have been shown to stimulate cellular proliferation, matrix deposition and phenotypic change, leading to tissue-damaging structural changes. Whereas most anti-inflammatory drugs either downregulate expression of inflammatory mediators or inhibit their actions on cells, there are alternate potential therapeutic strategies described here based on moderating vascular leakage or its consequences in chronic diseases. The hypothesis is that drugs that can modify a cell's phenotype could be used to limit structural changes which accompany inflammation and thus reduce permanent debility resulting from these diseases. Such drugs include the differentiating agents being developed for cancer therapy.

Homologous serum-stimulated fibronectin synthesis in human retinal pigmented epithelial cells.

PURPOSE: Fibronectin expression has been recorded in subretinal membranes from patients with proliferative vitreoretinopathy (PVR). Retinal pigmented epithelial (RPE) cells are a major cellular component of PVR membranes and synthesize fibronectin. We investigated the effects of human serum (as a model of vascular leakage in vivo) on the expression of fibronectin by cultured human RPE cells and compared the responses to those stimulated by fetal bovine serum (FBS). METHODS: Human RPE cells were incubated in M199 with 1, 10 and 40% human adult serum or FBS for 72 h. RESULTS: Retinal pigmented epithelial cells expressed maximum extracellular matrix fibronectin when exposed to 40% human serum using immunohistochemistry. Using ELISA to quantify fibronectin, 10 and 40% human serum significantly increased the total fibronectin (P < 0.01; n = 4), whereas FBS did not affect fibronectin expression. CONCLUSIONS: The results show that human serum contains substances stimulating fibronectin synthesis in human RPE cells.

The effects of salbutamol, beclomethasone, and dexamethasone on fibronectin expression by cultured airway smooth muscle cells.

Possible mechanisms of adverse drug effects in asthma include worsening of cellular hyperplasia and stimulation of extracellular matrix deposition. In this study, salbutamol, dexamethasone and beclomethasone were investigated to ascertain their ability to induce mitogenesis and stimulate fibronectin expression in cultured canine airway smooth muscle cells. In cells maintained in serum-free media for 72 h, salbutamol (1 nM-10 microM) caused mitogenesis. The control cells had 2.57 +/- 0.34 x 10(5) cells per ml (mean +/- SEM, N = 13), while salbutamol (1 microM) caused a maximal increase in cell number to 3.57 +/- 0.23 x 10(5) cells/ml (P < 0.01). In cells stimulated to replicate by addition of either fetal bovine serum or canine serum, no additional mitogenic effect of salbutamol was seen. Salbutamol did not have a detectable quantitative effect on fibronectin matrix expression. The glucocorticoids, beclomethasone and dexamethasone, significantly altered fibronectin expression by cultured airway smooth muscle cells. Beclomethasone increased fibronectin expression, while dexamethasone decreased expression.

Effects of a new C5a receptor antagonist on C5a- and endotoxin-induced neutropenia in the rat.

A new C5a receptor antagonist, the cyclic peptide Phe-[Orn-Pro-D-cyclohexylalanine-Trp-Arg], (F-[OPdChaWR]), was tested for its ability to antagonize the neutropenic effects of both C5a and endotoxin in rats. Human recombinant C5a (2 microg kg(-1) i.v.) caused rapid neutropenia, characterized by an 83% decrease in circulating polymorphonuclear leukocytes (PMNs) at 5 min. Administration of F-[OPdChaWR] (0.3-3 mg kg(-1) i.v.), did not affect the levels of circulating PMNs but, when given 10 min prior to C5a, it inhibited the C5a-induced neutropenia by up to 70%. Administration of E. Coli lipopolysaccharide (LPS, 1 mg kg(-1) i.v.) also caused neutropenia with an 88% decrease in circulating PMNs after 30 min. When rats were pretreated with F-[OPdChaWR] (0.3 - 10 mg kg(-1) i.v.) 10 min prior to LPS, there was a dose-dependent antagonism of the neutropenia caused by LPS, with up to 69% reversal of neutropenia observed 30 min after LPS administration. These findings suggest that C5a receptor antagonists may have therapeutic potential in the many diseases known to involve either endotoxin or C5a.

Arterially perfused eye model of uveitis.

OBJECTIVE: To develop an in vitro model of uveitis based on an ex situ perfused eye to evaluate the anti-inflammatory activity of new pharmacological products. PROCEDURE: Eyes were removed from more than 60 dogs and 9 horses immediately after euthanasia and perfused with nutrient medium through the lateral long ciliary artery. Perfused eyes produced aqueous humour, and perfusion pressure was adjusted to obtain an intraocular pressure in the physiological range. When the eyes were treated with histamine, a complement C5a analogue peptide and hydrogen peroxide, typical signs of uveitis were produced. These included miosis, vascular leakage, reduced intraocular pressure, reduced flow of perfusate and, in some eyes, conjunctival oedema. RESULTS: Canine eyes showed a decrease in intraocular pressure and a decrease in perfusate flow rate when challenged with 100 mumol/L hydrogen peroxide. Flunixin meglumine (5 mumol/L), ketoprofen (5 mumol/L), indomethacin (5 mumol/L) as well as a new drug pirfenidone (10 mumol/L) prevented changes in intraocular pressure induced by hydrogen peroxide, but did not significantly moderate the mediator-induced changes in perfusate flow. CONCLUSIONS: This model is suitable for evaluating potential anti-inflammatory activity of drugs without having to induce uveitis in an experimental animal. The technique is suitable for species that range in size from cats to horses.

Pirfenidone reduces fibronectin synthesis by cultured human retinal pigment epithelial cells.

PURPOSE: Pirfenidone is a novel anti-fibrotic drug that has been shown to inhibit fibroblast growth and collagen synthesis induced by transforming growth factor (TGF)-beta1. In the present study we investigated the ability of pirfenidone to moderate fibronectin synthesis by cultured human retinal pigment epithelial (RPE) cells maintained in media containing 1% foetal bovine serum when stimulated with TGF-beta1. METHODS: Primary human RPE cultures were used. Treatments included TGF-beta1, pirfenidone and pirfenidone with TGF-beta1. After 72 h treatments, cell growth was determined by cell counting and fibronectin was measured by ELISA. RESULTS: Transforming growth factor-beta1 (1-10 ng/mL) increased the production of soluble fibronectin, while pirfenidone (300 micromol/L) significantly reduced the TGF-beta1-induced synthesis of fibronectin. Pirfenidone alone had no effect on fibronectin synthesis by cultured RPE cells. CONCLUSION: We conclude that the anti-fibrotic effect of pirfenidone may be partly mediated through inhibition of TGF-beta1-induced fibronectin synthesis.

Vascular leakage stimulates phenotype alteration in ocular cells, contributing to the pathology of proliferative vitreoretinopathy.

Plasma leaking from damaged retinal blood vessels can have a significant impact on the pathologies of the posterior segment of the eye. Inflammation in the eye and metabolic change resulting from diabetes mellitus causes vascular leakage with alteration of the phenotype of retinal pigment epithelial (RPE) cells and fibrocytes, resulting in changes in cell function. Phenotypically altered cells then significantly contribute to the pathogenesis of retinopathies by being incorporated into tractional membranes in the vitreous, where they secrete matrix molecules, such as fibronectin, and express altered cell surface antigens. We hypothesize that there is a direct relationship between the leaking of plasma and the proliferation and phenotypic change of RPE cells and fibroblasts, thus exacerbating the pathology of retinal disease. If the hypothesis is correct, control of vascular leakage becomes an important target of therapy in proliferative vitreoretinopathy.

Homologous serum increases fibronectin expression and cell adhesion in airway smooth muscle cells.

This study describes altered patterns of growth and upregulation of fibronectin expression of cultured canine airway smooth muscle cells grown in homologous serum, which provides a model of the vascular leakage occurring in asthma, compared to fetal bovine serum (FBS). Cells were incubated in increasing concentrations of serum (2.5-40%) for 72 hours. Both homologous serum and FBS caused cellular proliferation which reached a maximum increase at 2.5-5% serum concentration. Differences in the cellular responses to the two types of sera were noted at higher concentrations of sera. At a concentration of 40% FBS, airway smooth muscle cells increased in number by 307 +/- 16% (n = 5) compared to serum-free control cells, whereas in canine serum the increase in growth was significantly smaller, 239 +/- 25% (n = 7) (P < 0.05). Airway fibrocytes similarity treated increased in number by 256 +/- 43% (n = 3) in 40% FBS, but exhibited a reduction in cell number to 80 +/- 10% (n = 3) of controls in 40% homologous serum (P < 0.05). Smooth muscle cells demonstrated a dose-dependent increase in fibronectin expression when grown in homologous serum but not in FBS, suggesting phenotypic change occurred in these cells when exposed to homologous serum. These data suggest that the leakage of plasma in the asthmatic airway may trigger phenotypic change in both airway smooth muscle cells and airway fibrocytes leading to cellular proliferation and expression of extracellular matrix molecules. These in vitro changes are consistent with the histological findings in clinical asthma.

Airway smooth muscle proliferation in asthma: the potential of vascular leakage to contribute to pathogenesis.

Proliferation of the non-vascular smooth muscle in the walls of the bronchi and bronchioles is a prominent histopathological feature of asthma and is thought to contribute to airway hyperreactivity and narrowing. Increased vascular permeability with plasma leakage is also a feature of asthma pathology and causes submucosal oedema. We hypothesize that, in asthmatics, the accumulation of enriched plasma in the environment surrounding airway smooth muscle promotes respiratory smooth muscle mitogenesis and hyperplasia. This situation represents the in vivo correlate of the increase in airway smooth muscle cell growth seen in vitro with increasing concentrations of serum in the culture medium. Thus, we hypothesize that vascular leakage in the airways in asthma is a primary pathogenic event leading to airway smooth muscle hyperplasia and hypertrophy, and consequently airway narrowing, promoting the characteristic bronchial hyperreactivity associated with narrowing of the airway lumen.

Investigations of breakdowns in protection provided by living Babesia bovis vaccine.

Field investigations of protection afforded by live Babesia bovis vaccine in Australia revealed that a ninefold increase in vaccine failures occurred in the period from 1985 to 1990. Laboratory trials using 189 experimental cattle were conducted to evaluate the protection afforded by the Babesia bovis strain used in the commercial vaccine during this time. Four isolates from clinical cases of babesiosis in vaccinated cattle were assessed. The results showed that the strain used in the vaccine during the 5 year period was poorly protective against three isolates while a recently isolated and prepared vaccine strain was strongly protective. Circumstantial evidence is provided that indicates the vaccine failures were due to change in the field populations of Babesia bovis, rather than change in the strain used in the vaccine. Implications of the results for the future of Babesia bovis vaccines are discussed.

Elimination of Theileria buffeli infections from cattle by concurrent treatment with primaquine phosphate and halofuginone lactate.

Fifty splenectomised calves naturally infected with Theileria buffeli were treated with primaquine phosphate (ICI, UK) and halofuginone lactate (Hoechst, Australia) either separately or in combination. Infections in treated calves were monitored for up to 26 weeks by examining Giemsa stained peripheral blood films for piroplasms and by an immunofluorescent antibody test. When used alone neither of the drugs eliminated infection. The most successful results were obtained when two treatments of halofuginone lactate, at a rate of 1 mg kg-1 body weight and six treatments of primaquine phosphate, at a rate of 2 mg kg-1 body weight, were administered concurrently. No theilerial relapses were observed in 14 of 16 calves so treated, and no antibody to T. buffeli was detected in these calves beyond the twelfth week after treatment. The results have application in the preparation of Theileria-free calves for use in the production of living vaccines against babesiosis and anaplasmosis.

Transmission of Theileria buffeli to cattle by Haemaphysalis bancrofti fed on artificially infected mice.

Larval and nymphal Haemaphysalis bancrofti became infected with Theileria buffeli following the intraperitoneal inoculation of infected bovine blood into CBA mice on which they were feeding. Subsequent instars of fed ticks were released on calves and transmitted theileriosis at each of 10 attempts. Suspensions made from ticks moulted in vitro and then incubated for 4 days at 37 degrees C were also infective for calves inoculated subcutaneously. The findings provide a convenient method for infecting ticks with field isolates of Theileria.

Heterologous antibody responses of calves to Anaplasma centrale and A. marginale.

Antigens of Anaplasma centrale, Onderstepoort isolate, and A. marginale, Wacol isolate, were analysed by a Western blotting technique. Sera from A. centrale-infected calves reacted to 41- and 38-kDa antigens in A. centrale and a 41-kDa antigen in A. marginale. Serum collected during the primary reaction from an A. marginale-infected calf reacted only to the 41-kDa antigen of A. marginale; heterologous antibody response to the 41-kDa antigen of A. centrale did occur later during the infection, but remained markedly weaker than the homologous response. The serologic cross-reactivity to this 41-kDa Anaplasma antigen confirms that it is common to the genus and also that it is a heterogeneous complex.