Cerebrospinal fluid and blood lactate concentrations as prognostic biomarkers in dogs with meningoencephalitis of unknown origin.

Meningoencephalitis of unknown origin (MUO) is a common inflammatory disease of the central nervous system. Several studies investigated finding prognostic factors, but results are contradictory. The aim of this study was to determine the concentrations of blood lactate (Blood-L) and cerebrospinal fluid lactate (CSF-L) in dogs with MUO for prognostic purposes. A total of 45 dogs with MUO (MUO group) and 11 with idiopathic epilepsy (IE group) were included. In the MUO group, 22 dogs were treated with prednisolone + cytosine arabinoside, 17 with prednisolone ± cyclosporine, and six received no treatment. In the MUO group, there was a strong-moderate positive correlation between Blood-L and CSF-L (ρ = 0.63557; P < 0.0001), a strong-moderate negative correlation between survival and CSF-L (ρ= -0.50210; P < 0.0004), and a weak negative correlation between survival and Blood-L (ρ= -0.35685; P < 0.0220). Dogs with a favourable response to treatment at 1 month had lower initial concentrations of Blood-L and CSF-L (P < 0.0010; P < 0.0037), and those with a worse response had higher values (P < 0.0497; P < 0.0004). Dogs that remained stable with treatment showed lower CSF-L concentrations (P < 0.0013). Dogs with Blood-L>4 mmol/L (P < 0.03) and/or CSF-L> 4 mmol/L (P < 0.009) had lower survival rates with the latter also showing more severe signs, probably indicating severe neuronal damage. These findings suggest that concentrations of CSF-L and Blood-L in dogs with MUO could be used as prognostic indicators.

Canine stem cell factor augments expression of matrix metalloproteinase-9 by CD34 cells.

BACKGROUND: Canine models have proved to be predictive of clinical findings in human bone marrow (BM) transplantation; consequently, the utilization of dogs is an excellent tool for supporting therapeutic purposes. Considering the role of growth factors in homing and mobilization of hematopoietic progenitors, the aim of this work was to evaluate whether canine stem cell factor (cSCF) contributes to matrix metalloproteinase (MMP)-9 secretion by CD34 cells. METHODS: The study was carried out in a cell population selected by immunomagnetic techniques using the anti-canine CD34 monoclonal antibody (MAb) 3B4 produced by us. Secretion of MMP-9 was evaluated by zymography. RESULTS: Analyzes of canine CD34(+) cells guaranteed that the MAb 3B4 was optimum for selecting a subset population with defined characteristics of primitive hematopoietic cells. The isolated cells were able to proliferate onto irradiated pre-established stroma, giving rise to mature neutrophils. There was also a 20-fold enrichment in the long-term culture-initiating cell content when the isolated population was added to irradiated cultures, with respect to the starting mononuclear cell population. DISCUSSION: We have provided the first evidence that canine BM CD34(+) cells constitutively express MMP-9 and the role of cSCF in up-regulating the secretion of this enzyme. The fact that cSCF augments expression of MMP-9 together with the ability of the isolated CD34(+)cells to proliferate onto irradiated pre-established stroma enables further investigations to determine whether the secretion of MMP-9 mediated by cSCF is one of the factors that enhance migration, homing and repopulation of primitive hemopoietic cells.

Phospholipase A2 activity and exocytosis of the ram sperm acrosome: regulation by bivalent cations.

Previous work has shown that the sequence leading to exocytosis of the sperm acrosome involves at least three Ca(2+)-requiring processes, the first one probably represented by breakdown of the polyphosphoinositides and the final one by membrane fusion. We have investigated whether phospholipase A2 (PLA2) represents the intermediate Ca(2+)-requiring event by stimulating ram spermatozoa with the ionophore A23187 and various bivalent cations. Spermatozoa prelabelled with [14C]arachidonic acid and treated with ionophore and millimolar Ca2+ showed a considerable release of arachidonic acid; parallel sperm samples similarly treated underwent acrosomal exocytosis. Mn2+ was capable of completely substituting for Ca2+, even if residual Ca2+ in the system was chelated with EGTA: both arachidonic acid release and acrosomal exocytosis took place after treatment with A23187, EGTA and Mn2+. Neither Mg2+ nor Ba2+ promoted arachidonic acid release or exocytosis. The effects of Sr2+ were more complex and allowed us to probe the sequence of events leading to membrane fusion. Both arachidonic acid release and exocytosis occurred after treatment with A23187 and Sr2+ but none of these responses were seen if EGTA was also included. These results suggest that residual micromolar Ca2+ is either needed for Sr2+ to fully promote PLA2 activity, or that micromolar Ca2+ is needed for one or more upstream events that may in turn serve to activate PLA2. Evidence for or against the first possibility was sought by examining PLA2 activity in sperm sonicates. Enzyme activity was maximal in the presence of any bivalent cation and it was not reduced (in the case of Sr2+) or only reduced slightly (Mg2+, Mn2+, Ba2+) if residual Ca2+ was chelated with EGTA; this indicates that Sr2+ can promote PLA2 activity in the total absence of Ca2+. The second possibility was explored by treating spermatozoa with A23187 for 5 min (to allow for complete phosphoinositide breakdown; Roldan and Harrison (1989) Biochem. J. 259, 397-406), and then adding EGTA and Sr2+. This resulted in neither arachidonic acid release nor exocytosis, thus indicating that another as yet unidentified Ca(2+)-dependent event may occur before PLA2 activation.

Detection of canine cytokine gene expression by reverse transcription-polymerase chain reaction.

Further characterization of the canine immune system will greatly benefit from the availability of tools to detect canine cytokines. Our interest concerns the study on the role of cytokines in canine visceral leishmaniasis. For this purpose, we have designed specific primers using previously published sequences for the detection of canine IL-2, IFN-gamma and IL10 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). For IL-4, we have cloned and sequenced this cytokine gene, and developed canine-specific primers. To control for sample-to-sample variation in the quantity of mRNA and variation in the RT and PCR reactions, the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a housekeeping gene, were determined in parallel. Primers to amplify G3PDH were designed from consensus sequences obtained from the Genbank database. The mRNA levels of the cytokines mentioned here were detected from ConA-stimulated peripheral mononuclear cells derived from Leishmania-infected dogs. A different pattern of cytokine production among infected animals was found.

Optimal conditions for simultaneous measurement of platelet aggregation and ATP secretion in canine whole blood.

This paper describes the optimal conditions for simultaneous evaluation of platelet aggregation and secretion capacity in canine whole blood using a Whole Blood Lumi-Aggregometer (Chrono-Log Corporation, Havertown, Pensylvania). For this purpose, the potential influence of several parameters was investigated using collagen, adenosinediphosphate (ADP), arachidonic acid (AA) and thrombin as platelet agonists. Results indicate that optimal experimental conditions to obtain reliable results include: allowing blood samples to stand at room temperature 60 minutes after blood collection, analysing samples within 3 hours from time of collection, adjusting platelet numbers to a final concentration of 150 000 microl(-1)and mixing the sample with isotonic saline (1:1) before adding the platelet agonist. The use of different platelet agonists offers variable results: collagen (0.5, 1 and 5 microg ml(-1)) is suitable for simultaneous platelet aggregation and adenosintriphosphate (ATP) secretion measurements; 1 UI ml(-1)of thrombin induced maximum ATP secretion;AA (0.5 and 1 mM) and ADP (5, 10 and 25 microM) did not give consistent results. The method described in this study has important clinical applications since it allows easy and quick platelet function evaluation in pathologic states.

Phospholipase A2 activation and subsequent exocytosis in the Ca2+/ionophore-induced acrosome reaction of ram spermatozoa.

In ram spermatozoa treatment with Ca2+ and A23187 or ionomycin stimulated the release of arachidonic acid (20:4) and exocytosis of the acrosome in a time- and concentration-dependent manner. Diacylglycerol did not appear to be the source of 20:4. On the other hand, generation of 20:4 was significantly correlated with breakdown of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine under a variety of conditions, thus indicating that 20:4 release was due to phospholipase A2 activity. Generation of 20:4 preceded acrosomal exocytosis. Moreover, it was significantly correlated with exocytosis when spermatozoa were stimulated with Ca2+ and A23187 or ionomycin for different periods of time or with different ionophore concentrations. Treatment with Ro 31-4493, a compound that has been found to inhibit sperm phospholipase A2 activity in vitro, considerably reduced both the release of 20:4 and exocytosis; addition of exogenous 20:4 or lysophosphatidylcholine overcame the inhibitory effect of Ro 31-4493. Spermatozoa preincubated with several unsaturated fatty acids, including 20:4, underwent exocytosis much more rapidly when treated with Ca2+/A23187. Exogenous lysophosphatidylcholine also enhanced acrosomal exocytosis and this effect was mimicked by an alkyl-containing analogue (1-O-alkyl-glycerophosphorylcholine = lyso-platelet activating factor). These results indicate that phospholipase A2 plays a fundamental role in the exocytosis of the acrosome elicited by Ca2+ and ionophore stimulation. Therefore, it is possible that activation of this enzyme constitutes an essential Ca2+-dependent event underlying exocytosis in response to physiological stimuli.

Diradylglycerols stimulate phospholipase A2 and subsequent exocytosis in ram spermatozoa. Evidence that the effect is not mediated via protein kinase C.

We tested the hypothesis that the role of diacylglycerol (DAG) in sperm acrosomal exocytosis is related to the activation of phospholipase A2, and that this effect is not mediated via protein kinase C. Treatment of [14C]arachidonic acid-labelled ram spermatozoa with Ca2+ and the ionophore A23187 stimulated both liberation of arachidonic acid and acrosomal exocytosis. No changes in [14C]DAG or [14C]monoacylglycerol were found after stimulation of spermatozoa, thus suggesting that arachidonic acid may be released exclusively via phospholipase A2. An increase in the endogenous levels of diradylglycerols (DRGs), resulting from exposure either to the DAG kinase inhibitor R 59022 or to exogenous 1-oleoyl-2-acetyl-sn-glycerol or 1,2-dioctanoyl-sn-glycerol, led to an increase in both phospholipase A2 activity and exocytosis when cells were stimulated with A23187 and Ca2+. Addition of DRGs that do not stimulate protein kinase C(1,3-dioctanoylglycerol, 1-O-hexadecyl-2-acetyl-rac-glycerol) also resulted in an increase in phospholipase A2 activity and exocytosis. On the other hand, phorbol esters (phorbol 12,13-dibutyrate; phorbol 12-myristate 13-acetate) did not enhance enzyme activity or exocytosis. Finally, exposure to 1-O-hexadecyl-2-O-methyl-rac-glycerol, a compound known to inhibit protein kinase C, did not affect phospholipase A2 activity or acrosomal exocytosis. We therefore conclude that in spermatozoa the messenger role of DAG is related to the activation of phospholipase A2, which in turn would generate an array of metabolites directly or indirectly involved in bringing about exocytosis of the acrosome.

A clinical case of canine mycotic pneumonia.

A bronchopneumonic process diagnosed as mycotic in origin is described. The dog fully recovered after 120 days of treatment with ketoconazole. Determination of the serological level of anti-IgG against Aspergillus was very useful in the follow-up, because the clinical improvement of the animal was evident long before the antibody level dropped significantly.

Canine long-term bone marrow culture neutrophil production and functionality.

This in vitro study has been conducted to determine the optimal experimental conditions under which to produce canine neutrophils in long-term bone marrow cultures (LTBMC), establish functional parameters of neutrophils obtained from LTBMC and peripheral blood and to ascertain whether these cells display physiological similarities. Our aim is to provide an experimental model, enabling a correlation between hemopoietic injury and neutrophil functionality. The authors demonstrate for the first time that canine neutrophils grown in cultures are able to produce oxyradicals capable of killing bacterial products. Moreover, culture-grown neutrophils contain gelatinase granules, a marker of terminal neutrophil differentiation, and express a specific surface antigen. The results described in this article illustrate the development of a dynamic system that mimics physiological hemopoiesis.