In vivo effects of aspirin and cyclosporine on regulatory T cells and T-cell cytokine production in healthy dogs.

Cyclosporine and aspirin are routinely used in combination to treat immune-mediated hemolytic anemia (IMHA) in dogs. Cyclosporine is a potent immunosuppressive agent that targets T cell production of the cytokines IL-2 and IFN-γ. Low-dose aspirin is often used to inhibit platelet function in dogs with IMHA, since these animals are prone to life-threatening thromboembolic disease. In rodents and humans, aspirin and cyclosporine have both been shown to variably affect T cell cytokine production, and also numbers of circulating regulatory T cells (Tregs). In dogs, it has not yet been determined if concurrent aspirin alters the effects of cyclosporine on T-cell cytokine expression, or if either drug influences Treg numbers. In a crossover study, seven healthy young adult dogs were given either oral high-dose cyclosporine (10 mg/kg Q12 h), oral low-dose aspirin (1 mg/kg Q24 h), oral high-dose aspirin (10 mg/kg Q12 h), or combined low-dose aspirin with cyclosporine, each for 8 days, with a washout of at least 2 weeks after each treatment. Activated T cell cytokine expression (IL-2 & IFN-γ) and percent CD4 + CD25 + FOXP3+ Tregs were evaluated using flow cytometry, both prior to and on the last day of treatment. The difference between pre- and post-treatment values for each group, as well as the difference between treatment groups, was evaluated. Cyclosporine significantly decreased IL-2 and IFN-γ expression when used alone or in combination with low-dose aspirin. High-dose aspirin, but not low-dose aspirin, also significantly decreased IL-2 expression, although the decrease was not as marked as that seen with cyclosporine alone or in combination with aspirin. Neither low-dose nor high-dose aspirin significantly affected IFN-γ expression. No drug or drug combination affected Treg numbers. Low-dose aspirin given with cyclosporine creates the same degree of T-cell cytokine suppression as does cyclosporine alone, suggesting that the two drugs can be used concurrently without significantly altering the immunosuppressive mechanism of action of cyclosporine.

[One-year results on the safety and efficacy of the InnFocus MicroShunt™ depending on placement and concentration of mitomycin C]. / Résultats à un an de l'efficacité et de l'innocuité du MicroShunt InnFocus selon l'emplacement et la concentration de MMC.

PURPOSE: To compare intraocular pressure (IOP) at one year with the InnFocus MicroShunt(®) with or without cataract surgery with according to placement and concentration of mitomycin C (MMC) DESIGN: A retrospective two-center, two-surgeon study (France and Dominican Republic). PATIENTS AND METHODS: Adults with POAG requiring filtering surgery. One MicroShunt(®) was placed in one eye of each patient. The effect of concentration and site of application of MMC was assessed by IOP and medication reduction at one year. RESULTS: Eighty-seven eyes were studied with one-year follow-up. Twenty-three eyes treated with 0.4 mg/mL MMC close to the limbus demonstrated a 55% reduction in IOP from 23.8 ± 5.3 at baseline to 10.7 ± 2.8 mmHg at one year. Topical glaucoma medication/patient was reduced 85% from 2.4 ± 0.9 to 0.3 ± 0.8. Thirty-one eyes treated with 0.2mg/mL MMC close to the limbus demonstrated a 52% reduction in IOP from 27.9 ± 6.7 at baseline to 13.3 ± 3.3 mmHg at one year. Topical glaucoma medication/patient was reduced 88% from 2.5 ± 1.4 to 0.5 ± 1.0. Thirty-three eyes treated with 0.4 mg/mL MMC deep in the pocket demonstrated a 38% reduction in IOP from 25.4 ± 7.9 at baseline to 15.7 ± 4.6 mmHg at one year. Topical glaucoma medication/patient was reduced 72% from 2.9 ± 1.0 to 0.8 ± 1.3. There were no sight-threatening long-term adverse events. CONCLUSION: The InnFocus MicroShunt(®) is a filtering surgery whose efficacy is related to the location of application and concentration of MMC used.

Cyclooxygenase expression and platelet function in healthy dogs receiving low-dose aspirin.

BACKGROUND: Low-dose aspirin is used to prevent thromboembolic complications in dogs, but some animals are nonresponsive to the antiplatelet effects of aspirin ("aspirin resistance"). HYPOTHESIS/OBJECTIVES: That low-dose aspirin would inhibit platelet function, decrease thromboxane synthesis, and alter platelet cyclooxygenase (COX) expression. ANIMALS: Twenty-four healthy dogs. METHODS: A repeated measures study. Platelet function (PFA-100 closure time, collagen/epinephrine), platelet COX-1 and COX-2 expression, and urine 11-dehydro-thromboxane B(2) (11-dTXB(2)) were evaluated before and during aspirin administration (1 mg/kg Q24 hours PO, 10 days). Based on prolongation of closure times after aspirin administration, dogs were divided into categories according to aspirin responsiveness: responders, nonresponders, and inconsistent responders. RESULTS: Low-dose aspirin increased closure times significantly (62% by Day 10, P < .001), with an equal distribution among aspirin responsiveness categories, 8 dogs per group. Platelet COX-1 mean fluorescent intensity (MFI) increased significantly during treatment, 13% on Day 3 (range, -29.7-136.1%) (P = .047) and 72% on Day 10 (range, -0.37-210%) (P < .001). Platelet COX-2 MFI increased significantly by 34% (range, -29.2-270%) on Day 3 (P = .003) and 74% (range, -19.7-226%) on Day 10 (P < .001). Urinary 11-dTXB(2) concentrations significantly (P = .005, P < .001) decreased at both time points. There was no difference between aspirin responsiveness and either platelet COX expression or thromboxane production. CONCLUSIONS AND CLINICAL IMPORTANCE: Low-dose aspirin consistently inhibits platelet function in approximately one-third of healthy dogs, despite decreased thromboxane synthesis and increased platelet COX expression in most dogs. COX isoform expression before treatment did not predict aspirin resistance.

The effects of cyclosporine on platelet function and cyclooxygenase expression in normal dogs.

BACKGROUND: Cyclosporine has been shown to alter platelet plasma membranes and have a hypercoagulable effect in humans, leading to thromboembolic complications. HYPOTHESIS/OBJECTIVES: Our hypothesis was that by modulating platelet reactivity, cyclosporine increases the risk of thromboembolic complications. The objective was to determine the effects of cyclosporine on primary hemostasis in normal dogs. ANIMALS: Eight healthy, intact female dogs. METHODS: A repeated-measures design utilized flow cytometry to evaluate platelet expression of platelet reactivity markers (P-selectin and phosphatidylserine) and COX-1 and COX-2 during the administration of 2 cyclosporine dosages (19 mg/kg q12h [immunosuppressive dosage] and 5 mg/kg q24h [atopy dosage]). Urine 11-dehydro-thromboxane-B(2) (11-dTXB(2) ) concentration was normalized to urine creatinine concentration, and platelet function was analyzed by PFA-100. RESULTS: After a week of the immunosuppressive dosage, all platelet reactivity markers showed a significant decrease in mean fluorescent intensity (MFI). After the atopy dosage, only P-selectin and COX-2 MFI demonstrated a change from baseline, decreasing by 29% (P = .013) and 31% (P = .003), respectively. Urinary 11-dTXB(2) -to-creatinine ratio significantly increased at all time points during the immunosuppressive dosage, but no significant change occurred during administration of the atopy dosage. PFA-100 closure times using collagen/ADP cartridges increased by 62% (P = .008) with the immunosuppressive dosage and decreased by 45% with the atopy dosage (P = .035). No significant changes in closure times occurred with collagen/epinephrine cartridges. CONCLUSIONS AND CLINICAL IMPORTANCE: Our study suggests that, similar to what is observed in humans, cyclosporine alters the platelet plasma membrane and increases thromboxane production in dogs, especially at immunosuppressive dosages.

Pharmacodynamic monitoring of canine T-cell cytokine responses to oral cyclosporine.

BACKGROUND: Pharmacodynamic assays measure the immunosuppressive effects of cyclosporine on T-cells and offer an alternative assessment of efficacy in individual patients. OBJECTIVE: To assess the immunosuppressive effects of high and low dosage cyclosporine on canine T-cells and to develop a novel testing system for individualized dose adjustment. ANIMALS: Seven healthy female Walker hounds. METHODS: Experimental study using a paired comparison design. Flow cytometry was used to measure T-cell expression of IL-2, IL-4, and IFN-γ. Cytokine expression 8 days after oral administration of high and low dosages of cyclosporine was compared to baseline and washout values, respectively. The high dosage was initially 10 mg/kg q12h and was then adjusted to attain established immunosuppressive trough blood drug concentrations (>600 ng/mL). The low dosage was 5 mg/kg q24h. RESULTS: High dosage cyclosporine resulted in significant decreases in IL-2 and IFN-γ expression (P = .0156, P = .0156), but not IL-4 expression (P = .2188). Low dosage cyclosporine was associated with a significant decrease in IFN-γ expression (P = .0156), while IL-2 expression was not affected (P = .1094). CONCLUSIONS AND CLINICAL IMPORTANCE: T-cell function is suppressed at trough blood drug concentrations exceeding 600 ng/mL, and is at least partially suppressed in some dogs at low dosages. Direct evaluation of T-cell function could be an effective, more sensitive alternative to measuring blood drug concentrations for monitoring immunosuppressive therapy.

Platelet cyclooxygenase expression in normal dogs.

BACKGROUND: Human platelets express both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Variation in COX-2 expression could be a mechanism for variable response to aspirin. HYPOTHESIS/OBJECTIVES: The hypotheses were that circulating canine platelets express COX-1 and COX-2, and that aspirin alters COX expression. The objective was to identify changes in platelet COX expression and in platelet function caused by aspirin administration to dogs. ANIMALS: Eight female, intact hounds. METHODS: A single population, repeated measures design was used to evaluate platelet COX-1 and COX-2 expression by flow cytometry before and after aspirin (10 mg/kg Q12h for 10 days). Platelet function was analyzed via PFA-100(®) (collagen/epinephrine), and urine 11-dehydro-thromboxane B(2) (11-dTXB(2)) was measured and normalized to urinary creatinine. Differences in COX expression, PFA-100(®) closure times, and urine 11-dTXB(2 ): creatinine ratio were analyzed before and after aspirin administration. RESULTS: Both COX-1 and COX-2 were expressed in canine platelets. COX-1 mean fluorescent intensity (MFI) increased in all dogs, by 250% (range 63-476%), while COX-2 expression did not change significantly (P = 0.124) after aspirin exposure, with large interindividual variation. PFA-100(®) closure times were prolonged and urine 11-dTXB(2) concentration decreased in all dogs after aspirin administration. CONCLUSIONS AND CLINICAL IMPORTANCE: Canine platelets express both COX isoforms. After aspirin exposure, COX-1 expression increased despite impairment of platelet function, while COX-2 expression varied markedly among dogs. Variability in platelet COX-2 expression should be explored as a potential mechanism for, or marker of, variable aspirin responsiveness.

Tribological aspects of joint intraaricular therapy.

The condition of synovial joints affected by synovitis or degeneration dystrophy has been usually normalized by drugs of antibacterial, anti-inflammatory, immunomodulating and/or lubricating-protective action injected into the joint cavity. These preparations influence, but in different way, the friction in joints and wear of cartilages, even if they belong to the same pharmacological group and follow the same medication mechanism. With the development of a large group of lubricating-and-protective substitutes for the synovial fluid the tribological efficiency of injection drugs has begun to attract attention, whereas no information is available in the literature on pharmacopoeia or orthopedics about the lubricity of anti-inflammatory and antibacterial drugs commonly used as injections. The mechanisms by which structure and properties of lubricating films containing drugs undergo transformation under the influence of biological field in joints remain in fact unknown. In vitro experiments have been conducted to simulate a biofield of a joint; the lubricity of some injection drugs used in orthopedics has been evaluated.

[Impact of a magnetic field on blood separation kinetics in patients with joint diseases].

The paper gives the results of experiments on phase separation of blood in the constant magnetic field that allows the structure of blood to be regulated, without changing its cellular and chemical composition. Blood deposition kinetic relationships were obtained for patients with joint diseases of various etiology (osteoarthritis, osteoarthrosis deformans, endoprosthesis instability, contusions, and joint wounds). They correlate with the severity of an inflammatory process in the joint and its adjacent tissues, with a patient's resistance to the development of pathology, and with red blood cell mobility in the biophysical field of a living organism. Analysis of relationships gives information on concentrations in plasma and hence synovial fluid (the basis of which is blood dialysate) in the liquid-crystalline phospholipid and cholesterol phase that determines the lubricity of synovial fluid and a low friction in the joints. The method may be used for the primary evaluation of efficacy of drugs for joint treatment, which is made in vitro on the blood taken from the patients rather than on the latter.

Cyclosporine A affects the in vitro expression of T cell activation-related molecules and cytokines in dogs.

Cyclosporine is a powerful immunosuppressive drug that is being used with increasing frequency to treat a wide range of immune-mediated diseases in the dog. To date, ideal dosing protocols that will achieve immunosuppression with cyclosporine in dogs remain unclear, and standard methods that can measure effectiveness of immunosuppression have not been established. The aim of our study was to evaluate the effects of in vitro cyclosporine exposure on a panel of molecules expressed by activated T cells to ascertain their potential as biomarkers of immunosuppression in dogs. Blood was drawn from six healthy dogs, and peripheral blood mononuclear cells (PBMC) were isolated and activated. Half of the cells were incubated with 200 ng/mL cyclosporine prior to activation, and the other half were not exposed to cyclosporine. Samples were analyzed using flow cytometry, and the expression of intracellular cytokines IL-2, IL-4, and IFN-γ was evaluated after 6, 12, and 24h of drug exposure. Each cytokine exhibited a time-dependent suppression profile, and all but two samples activated in the presence of cyclosporine showed lower cytokine expression than untreated controls. We also evaluated the expression of the surface T cell activation molecules CD25 and CD95 by flow cytometry after 36 h of drug exposure. Expression of these surface molecules decreased significantly when activated in the presence of cyclosporine. Our results suggest that suppressed expression of the markers related to T cell activation could potentially be utilized as an indicator of the efficacy of cyclosporine therapy in dogs.

[Infrared spectroscopy of synovial fluid and serum from patients with joint diseases].

The infrared spectra of synovial fluid and serum from the same patient were comparatively analyzed. The coordination bond variations in the complex protein-polysaccharide compounds of synovial fluid were shown to correspond to the range of frequencies of 3000-3500 cm(-1) and those of valence bonds in its individual components were in the range of 800-1700 cm(-1). It was established that a reduction in the peak area of 3300 cm(-1) may be an additional indicator in the diagnosis of osteoarthritis. The above results confirm it expedient to treat joints, by injecting serum-based agents.

[Molecular mass distribution of proteins in serum and synovial fluid].

The Laemmli polyacrylamide gel protein electrophoresis method was used to assess the protein molecular mass distribution in the serum and synovial fluid from apparently healthy individuals and patients with knee osteoarthritis of various etiologies. The development of inflammatory complications in joint diseases was shown to be identified on the protein separation spectra as a rise in the synovial fluid concentration of protein fractions with the extremely low molecular mass (to 20 kD). The protein molecular mass distribution in the sera in all the groups and that in the synovial fluid from the apparently healthy joints were virtually equal.

[Immobilization of Saccharomyces yeast cells on electret polyethylene films].

The method of electret-thermal analysis developed in dielectric physics has been applied to monitor bioelectric phenomena that accompany the immobilization of microorganisms on electret substrates. A spectrum of thermo stimulated currents for biofilms consisting of immobilized Saccharomyces cells has been obtained. The spectrum represents a halo in the region of 30-90 degrees C. The immobilization of Saccharomyces cells on electret polyethylene films results in their depolarization that is recorded on the spectra of thermostimulated currents as a reduction of the current peak corresponding to the polyethylene melting point. A hypothesis has been put forward that explains the phenomenon by the absorption of the electric energy of polarized substrates by the cells for the occurrence of metabolic reactions.

Bovine viral diarrhea viruses modulate toll-like receptors, cytokines and co-stimulatory molecules genes expression in bovine peripheral blood monocytes.

We have used noncytopathic (ncp) and cytopathic (cp) Bovine Viral Diarrhea Viruses (BVDV) to determine the expression levels of TLR genes, type I IFN, pro-inflammatory and Th1/Th2 cytokine gene expression in bovine monocytes. In general, both BVDV strains had similar effects. However, we found some significant differences that could be due to biological differences between cp and ncp BVDV strains. TLR3 was significantly up-regulated in 1h ncp, but not in cp BVDV- infected monocytes, whereas TLR7 expression dominated in 24h infection with both BVDV strains. Type I IFN and IL-12 gene expression was also significantly up-regulated in 1h ncp, but not cp BVDV infection that correlated with the enhanced TLR3 gene expression. Both BVDV biotypes suppressed pro-inflammatory cytokines TNF-alpha, IL-1beta, and IL-6, co-stimulatory molecules CD80 and CD86, but did not change Th1 type cytokine IL-12 and INF-gamma, gene expression after 24h infection. We hypothesize that BVDV may escape immune responses by altering the expression of TLR 3 and 7 and their signaling pathways.

Tribological monitoring of drugs used for the treatment of arthropathies.

Lubricating capacity of drugs injected into articular cavity were tested using a pendulum tribometer. The findings indicate that friction coefficient for a pair simulating metallopolymeric articular endoprosthesis is changed under the effect of electromagnetic field after lubrication. Regularities of these changes depend on the nature and mechanism of action of drugs. A method for rapid evaluation of the lubricating characteristics of drugs is proposed for optimizing their therapeutic effect on joints.

Differential detergent fractionation for non-electrophoretic bovine peripheral blood monocyte proteomics reveals proteins involved in professional antigen presentation.

Professional antigen presenting cells (APC), dendritic cells (DC) and their myeloid progenitors, monocytes/macrophages are critical controllers of innate and adaptive immunity. Here we show that differential detergent fractionation (DDF) analysis of bovine monocytes reveals proteins related to antigen pattern recognition, uptake and presentation to immunocompetent lymphocytes. We identify 53 bovine proteins involved in immune function of professional APC. In particular, 13 adhesion molecules, three toll-like receptors (TLR1, 6 and 8), three antigen uptake-related proteins (including mannose receptor [MR] precursor), and eight actin-like proteins involved in active endocytosis were identified. In addition, MHC class I and II-related proteins, cytokines, active substances and growth factors have been identified. We conclude that the DDF approach can provide interpretable and meaningful functional information concerning protein expression profiles associated with monocyte activation, transformation into macrophages and/or immature DC, and maturation of monocyte-derived DC in the presence of multiple bovine pathogens.

Bovine monocytes induce immunoglobulin production in peripheral blood B lymphocytes.

Although a role for monocytes and monocyte-derived dendritic cells (DC) in the activation of T cells is well established, it is less clear to what extent DC and their precursors, monocytes, regulate B cell immune responses. Here we show that regulatory mechanisms similar to those in humans are in place in the bovine immune system. In vitro culture of bovine monocytes with bovine B cells activated by the anti-CD3 triggered CD4+ T cells or through immunoglobulin (Ig) receptor crosslinking induces B cell Ig secretion. Unlike bovine monocyte-derived DC, monocytes do not promote Ig class switching to IgG and IgA in activated peripheral blood B cells. These results suggest that bovine monocytes are capable of directly inducing Ig secretion in activated bovine peripheral blood B cells, but do not provide the signals for B cell Ig class switching.

Bovine dendritic cells generated from monocytes and bone marrow progenitors regulate immunoglobulin production in peripheral blood B cells.

We examined whether bovine monocyte-derived and bone marrow (BM) dendritic cells (DCs) regulate antibody production in activated peripheral blood B cells. DCs were generated from monocytes and BM progenitors in the presence of bovine recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). Monocyte-derived DCs promoted B cells activated by the anti-CD3 triggered CD4(+) T cells or through immunoglobulin M (IgM) receptor to increase the level of IgG secretion. Furthermore, the addition of DCs triggered B cells activated through IgM receptors to produce IgG2 and IgA, thus inducing an isotype switch. BM-derived DCs increased the production of IgG in B cells activated by the anti-CD3 triggered CD4(+) T cells, but unlike monocyte-derived DCs did not have any effect on B cells activated through surface IgM. These data suggest that the regulation of humoral immune responses in cattle depends on the origin of DCs and the mode of B cell activation.

Electret-thermal analysis of blood.

The thermally stimulated discharge (TSD) method, intended for the analysis of charged dielectrics, was used for human blood research. Above-room-temperature TSD spectra of blood consist of three peaks. There are indications that the low-temperature peak (40-50 degrees C) corresponds to the thermally stimulated destruction of hydrate shells surrounding blood components while the mid-temperature peak (70-90 degrees C) is related to thermal denaturation of blood proteins. The intensive high-temperature peak (105-120 degrees C) is observed when a phase transition of blood, accompanied by the formation of a firm dry film of blood, occurs. The position of the high-temperature peak depends on the blood group. Data is discussed which suggests that the spontaneous "quasi-electret effect" of blood relates to the character of the biochemical processes taking place in human organisms. The TSD method might be used as simple and informative means of diagnostics in cooperation with medical and physical investigations.

Seven regions of the genome show evidence of linkage to type 1 diabetes in a consensus analysis of 767 multiplex families.

Type 1 diabetes (T1D) is a genetically complex disorder of glucose homeostasis that results from the autoimmune destruction of the insulin-secreting cells of the pancreas. Two previous whole-genome scans for linkage to T1D in 187 and 356 families containing affected sib pairs (ASPs) yielded apparently conflicting results, despite partial overlap in the families analyzed. However, each of these studies individually lacked power to detect loci with locus-specific disease prevalence/sib-risk ratios (lambda(s)) <1.4. In the present study, a third genome scan was performed using a new collection of 225 multiplex families with T1D, and the data from all three of these genome scans were merged and analyzed jointly. The combined sample of 831 ASPs, all with both parents genotyped, provided 90% power to detect linkage for loci with lambda(s) = 1.3 at P=7.4x10(-4). Three chromosome regions were identified that showed significant evidence of linkage (P<2.2x10(-5); LOD scores >4), 6p21 (IDDM1), 11p15 (IDDM2), 16q22-q24, and four more that showed suggestive evidence (P<7.4x10(-4), LOD scores > or =2.2), 10p11 (IDDM10), 2q31 (IDDM7, IDDM12, and IDDM13), 6q21 (IDDM15), and 1q42. Exploratory analyses, taking into account the presence of specific high-risk HLA genotypes or affected sibs' ages at disease onset, provided evidence of linkage at several additional sites, including the putative IDDM8 locus on chromosome 6q27. Our results indicate that much of the difficulty in mapping T1D susceptibility genes results from inadequate sample sizes, and the results point to the value of future international collaborations to assemble and analyze much larger data sets for linkage in complex diseases.

Colestasis inducida por meloxican / Meloxicam-induced cholestasis

The cholestasis by meloxicam has not been often described. However, we present here the clinic, laboratory, histologic and follow up of a patient with cholestatic hepatitis produced by this drug.