Evaluation of transformation growth factor beta1, interleukin-10, and interferon-gamma in male symptomatic and asymptomatic dogs naturally infected by Leishmania (Leishmania) chagasi

The aims of this study were to evaluate the immunomodulatory role of TGF-β₁, IL-10, and INF-γ in spleen and liver extracts and supernatant cultures of white spleen cells from male symptomatic and asymptomatic dogs, naturally infected by Leishmania (Leishmania) chagasi. Thirty dogs from Araçatuba, São Paulo, Brazil, an endemic leishmaniosis area, were selected by positive ELISA serological reaction for Leishmania sp. and divided into two groups: asymptomatic (n = 15) and symptomatic (n = 15) consisting of animals with at least three characteristic signs (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotion problems, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy). After euthanasia, spleen and liver fragments were collected for ex vivo quantification of TGF-β₁, IL-10, and INF-γ. Naturally active in vitro produced TGF-β₁ was also evaluated in spleen cell culture supernatant. Spleen and liver extract of asymptomatic dogs had higher mean TGF-β₁ levels than symptomatic dogs. High concentrations of IL-10 were found in spleen, and mainly in liver extract of both groups. Higher INF-γ concentrations were found in spleen extracts of symptomatic dogs, and in liver extracts of asymptomatic dogs. Extract of this cytokine was lower in spleen extract. Although INF-γ is being produced in canine infection, mean levels of TGF-β₁ and IL-10 from spleen and liver extracts were quantitatively much higher; suggesting that immune response in both asymptomatic and symptomatic dogs was predominantly type Th2.     

Exposure to Sarcocystis spp. in horses from Spain determined by Western blot analysis using Sarcocystis neurona merozoites as heterologous antigen

Horses serve as an intermediate host for several species of Sarcocystis, all of which utilize canids as the definitive host. Sarcocystis spp. infection and formation of latent sarcocysts in horses often appears to be subclinical, but morbidity can occur, especially when the parasite burden is large. A serological survey was conducted to determine the presence of antibodies against Sarcocystis spp. in seemingly healthy horses from the Galicia region of Spain. Western blot analyses using Sarcocystis neurona merozoites as heterologous antigen suggested greater than 80% seroprevalance of Sarcocystis spp. in a sample set of 138 horses. The serum samples were further tested with enzyme-linked immunosorbent assays (ELISAs) based on recombinant S. neurona-specific surface antigens (rSnSAGs). As expected for horses from the Eastern Hemisphere, less than 4% of the serum samples were positive when analyzed with either the rSnSAG2 or the rSnSAG4/3 ELISAs. An additional 246 horses were tested using the rSnSAG2 ELISA, which revealed that less than 3% of the 384 samples were seropositive. Collectively, the results of this serologic study suggested that a large proportion of horses from this region of Spain are exposed to Sarcocystis spp. Furthermore, the anti-Sarcocystis seroreactivity in these European horses could be clearly distinguished from anti-S. neurona antibodies using the rSnSAG2 and rSnSAG4/3 ELISAs.     

Neutrophil apoptosis during the resolution of bovine mammary gland injury

The role of neutrophil apoptosis in the resolution of bovine mammary gland injury induced by intramammary administration of physiological buffered saline (PBS) or lipopolysaccharide (LPS) was investigated. Twenty mammary glands of five non-pregnant heifers were used in the two studies and each animal received both stimuli. Samples of cell populations were collected by mammary gland lavages before and 24, 48, 72 and 96 hours after treatment and examined by light microscopy and staining for myeloperoxidase (MPO). A marked influx of neutrophils into the mammary gland was observed 24 hours after stimulation. At the same time, apoptotic neutrophils and MPO-positive macrophages (MAC) were identified in the samples. The numbers increased to reach maximum values at 48 hours after stimulation with PBS and at 72 hours after stimulation with LPS. The observed differences in the length of the resolution period indicate that neutrophil viability can be modulated by delaying the apoptotic process. Apoptosis of neutrophils and their subsequent phagocytosis by MAC can be regarded as a significant mechanism in the removal of neutrophils from the acutely injured mammary glands and, hence, in the resolution of bovine mastitis.     

Adhesion of Escherichia coli K88ab fimbriae to porcine enterocytes: effect on enzymatic activities of the brush border, and cyclic AMP and GMP levels

We have studied the cellular alterations, after in vitro adherence, of purified K88ab fimbriae to membranes of porcine enterocytes. Effects on enzymatic activities as disaccharydases and alkaline phosphatase show low changes. While cAMP levels were decreased (44%), guanylyl cyclase was increased (up to 200%), and levels of cGMP were in consequence significantly affected. This study support the role of cyclic GMP as intracellular mediator for adherence, and suggest their implication in disease, affecting a membrane-mediated mechanisms for guanylate cyclase activation, that is unique in the intestine.     

Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

Background

The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells.

Methods

A total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses.

Results

Both the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles.

Conclusion

We were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive labelling in this structure. It is believed that the formation of the bundles may have been stimulated by cellular signalling mechanisms that have not yet been elucidated.     

Autonomic Innervation of the Equine Urinary Bladder

The distribution and density of intrinsic autonomic nerve fibers and cells were studied in the equine urinary bladder by means of the peroxidase-antiperoxidase immunohistochemical method to localize tyrosine-hydroxylase (TH), and by means of a histochemical technique to detect acetylcholinesterase (AChE) activity. The results suggest that the equine urinary bladder, like that of other mammalian species, possesses a rich autonomic innervation which includes catecholaminergic and acetylcholinesterase positive nerves. At least a part of these nerve fibers have an intrinsic origin from ganglion cell bodies within the bladder wall.     

Intravaginal prostaglandin F2 alpha for the treatment of metritis and pyometra in the bitch

The purpose of this study was to determine whether intravaginal prostaglandin F2 alpha (PGF2 alpha) would be effective for the treatment of metritis or pyometra in the bitch. Seventeen bitches with metritis or pyometra were treated with PGF2 alpha. Prostaglandin F2 alpha (150 micrograms/kg body weight) was administered once or twice daily by infusing 0.3 ml per 10 kg body wt into the vaginal lumen. Bitches were also treated with amoxicillin (15 mg/kg body wt/48 h) and/or gentamicin (4 mg/kg body wt/day) administered as intramuscular (i.m.) injections. Fifteen bitches were treated successfully with intravaginally administered PGF2 alpha for 3 to 12 days and with intramuscularly administered antibiotics for 4 to 12 days. Success of treatment was judged by cessation of vaginal discharge, the absence of fluid in the uterus as determined by ultrasonography, and the overall health status of the animal. As two bitches with pyometra showed clinical deterioration in spite of medical treatment, ovariohysterectomy was performed after the first and the second treatment, respectively. No side effects (salivation, vomiting, diarrhoea, hyperpnoea, ataxia, urination, anxiety, pupillary dilatation followed by contraction) were observed after PGF2 alpha treatment. The disease did not recur during the subsequent oestrous cycles within 12 months after the initial treatment. The results demonstrate that intravaginal administration of PGF2 alpha was effective in 13 dogs (86.6%) with metritis or pyometra, and caused no side effects. Although the study was based on a relatively small number of cases, it is concluded that prostaglandin F2 alpha can be a useful means of treating bitches with metritis or pyometra. However, in severe cases of pyometra ovariohysterectomy is needed.     

Different action of IBMX,isoproterenol and rutin on orthovanadate-induced nitric oxide release in mouse macrophage cells

The effects of cAMP-elevating compounds IBMX (3-isobutyl-1-methylxanthine) and isoproterenol, and that of rutin (an effective superoxide scavenger) were studied on orthovanadate--(a putative protein-phosphotyrosine phosphatase inhibitor) induced nitric oxide (NO) production in J774A.1 mouse macrophage cells. As we previously reported (Koncz and Horváth, 2000), rutin and sodium orthovanadate act synergistically to induce production of high amount of NO in J774A.1 cells. IBMX, an agent that can elevate cAMP level in the cells, can reduce the production of both the LPS- and rutin + orthovanadate-induced NO in macrophages. In contrast, isoproterenol, a non-selective beta-adrenergic receptor agonist, that reduced the LPS-induced NO production in macrophage cells, was unable to reduce the rutin + orthovanadate-induced NO production without negatively affecting cell viability. Moreover, isoproterenol dramatically enhanced the orthovanadate-induced NO synthesis in J774A.1 cells. Our previous study clarified that rutin and orthovanadate, in a specific concentration ratio of both, were able to produce hydrogen peroxide (H2O2). Using 2',7'-dichlorofluorescein-diacetate as a marker for H2O2, isoproterenol alone induced its oxidation but the rutin plus orthovanadate-induced H2O2 production was reduced by isoproterenol. These observations have revealed that, in some cases, H2O2 and superoxide (O2-) scavengers can act in a reverse mode on macrophage cells depending on the presence or absence of orthovanadate.