Long-term evaluation of mesenchymal stem cell therapy in a feline model of chronic allergic asthma.

BACKGROUND: Mesenchymal stem cells (MSCs) decrease airway eosinophilia, airway hyperresponsiveness (AHR), and remodelling in murine models of acutely induced asthma. We hypothesized that MSCs would diminish these hallmark features in a chronic feline asthma model. OBJECTIVE: To document effects of allogeneic, adipose-derived MSCs on airway inflammation, AHR, and remodelling over time and investigate mechanisms by which MSCs alter local and systemic immunologic responses in chronic experimental feline allergic asthma. METHODS: Cats with chronic, experimentally induced asthma received six intravenous infusions of MSCs (0.36-2.5 × 10E7 MSCs/infusion) or placebo bimonthly at the time of study enrollment. Cats were evaluated at baseline and longitudinally for 1 year. Outcome measures included: bronchoalveolar lavage fluid cytology to assess airway eosinophilia, pulmonary mechanics and clinical scoring to assess AHR, and thoracic computed tomographic (CT) scans to assess structural changes (airway remodelling). CT scans were evaluated using a scoring system for lung attenuation (LA) and bronchial wall thickening (BWT). To assess mechanisms of MSC action, immunologic assays including allergen-specific IgE, cellular IL-10 production, and allergen-specific lymphocyte proliferation were performed. RESULTS: There were no differences between treatment groups or over time with respect to airway eosinophilia or AHR. However, significantly lower LA and BWT scores were noted in CT images of MSC-treated animals compared to placebo-treated cats at month 8 of the study (LA P = 0.0311; BWT P = 0.0489). No differences were noted between groups in the immunologic assays. CONCLUSIONS AND CLINICAL RELEVANCE: When administered after development of chronic allergic feline asthma, MSCs failed to reduce airway inflammation and AHR. However, repeated administration of MSCs at the start of study did reduce computed tomographic measures of airway remodelling by month 8, although the effect was not sustained at month 12. Further study of MSC therapy including repeated MSC administration is warranted to assess impact on remodelling in chronic asthma.

Comparison of direct and indirect bronchoprovocation testing using ventilator-acquired pulmonary mechanics in healthy cats and cats with experimental allergic asthma.

Airway hyperresponsiveness (AHR) is a key feature of asthma and can be measured using bronchoprovocation. Direct (methacholine, MCh) or indirect (adenosine-5-monophosphate, AMP; or mannitol) bronchoprovocants are used in human patients, the latter inducing AHR only with pre-existing airway inflammation. The present study compared the responses to direct (MCh) and indirect (mannitol, AMP) bronchoprovocation in healthy and asthmatic cats (n=6/group). The order of bronchoprovocant was randomized using a published table of random numbers and there was a 1-month washout before crossover to the next treatment. Pulmonary mechanics were measured in anesthetized and mechanically ventilated cats using a critical care ventilator. Saline at baseline and increasing doses of each bronchoprovocant were aerosolized for 30 s, followed by 4 min of data collection between doses. The endpoint for each bronchoprovocant was reached when airway resistance exceeded 200% of baseline values (EC200Raw). There was a significant difference (P<0.001) in the airway response of asthmatic vs. healthy cats over the range of MCh concentrations, despite there being no significant difference in the EC200Raw between the groups. Response to MCh was significantly greater (P<0.05) in asthmatic than in healthy cats at MCh concentrations as low as 0.0625 mg/mL. For AMP, a small subset of asthmatics (n=2/6) responded at low concentrations; four asthmatic cats and all healthy cats failed to respond even to the highest concentrations of AMP. One asthmatic cat but no healthy cats responded to mannitol. In conclusion, MCh discriminated asthmatic from healthy cats but neither AMP nor mannitol was an effective bronchoprovocant in this model.

Characterization of an NBTI-sensitive equilibrative nucleoside transporter in vascular smooth muscle.

Adenosine plays a significant role in various physiological and regulatory processes including coronary vasodilatation. In the current study, a high-affinity adenosine transporter in freshly dissociated porcine coronary smooth muscle (PCSM) cells and cultured human coronary smooth muscle (HCSM) cells was characterized. Kinetic analysis of the transport process revealed a V(max) of 82+/-17 pm/mg protein/min and a K(m) of 4.3+/-2.1 microm for PCSM cells, whereas a K(m) of 4.8 microm and V(max) of 254 pm/mg/min was observed for cultured HCSM. Concentration-dependent inhibition of adenosine uptake by S-(4-nitrobenzyl)-6-thioinosine (NBTI) was observed in both PCSM (IC(50), 0.08 microm) and HCSM (0.1 microm) cells. Both cell types also demonstrate a high-affinity, single binding site for NBTI (PCSM, B(max) 144.8+/-23 fmol/mg protein and K(d) 1.1+/-0.35 nm; HCSM, B(max) 672+/-62 fmol/mg protein and K(d) 0.45+/-0.14 nm). Adenosine uptake in these cells was not affected by extracellular sodium concentration. RT-PCR analysis of mRNA from individually selected PCSM and HCSM cells demonstrated expression of an NBTI-sensitive equilibrative transporter. Smooth muscle cells isolated from porcine brachial and femoral arteries also transported adenosine at levels similar to that of coronaries. These data demonstrate that vascular coronary smooth muscle possess an NBTI-sensitive equilibrative transporter for adenosine which could function in regulation of vasodilation.

Selective transport of adenosine into porcine coronary smooth muscle.

Adenosine (ADO), an endogenous regulator of coronary vascular tone, enhances vasorelaxation in the presence of nucleoside transport inhibitors such as dipyridamole. We tested the hypothesis that coronary smooth muscle (CSM) contains a high-affinity transporter for ADO. ADO-mediated relaxation of isolated large and small porcine coronary artery rings was enhanced 12-fold and 3.4-fold, respectively, by the transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI). Enhanced relaxation was independent of endothelium and was selective for ADO over synthetic analogs. Uptake of [(3)H]ADO into freshly dissociated CSM cells or endothelium-denuded rings was linear and concentration dependent. Kinetic analysis yielded a maximum uptake (V(max)) of 67 +/- 7.0 pmol. mg protein(-1). min(-1) and a Michaelis constant (K(m)) of 10. 5 +/- 5.8 microM in isolated cells and a V(max) of 5.1 +/- 0.5 pmol. min(-1). mg wet wt(-1) and a K(m) of 17.6 +/- 2.6 microM in intact rings. NBTI inhibited transport into small arteries (IC(50) = 42 nM) and cells. Analyses of extracellular space and diffusion kinetics using [(3)H]sucrose indicate the V(max) and K(m) for ADO transport are sufficient to clear a significant amount of extracellular adenosine. These data indicate CSM possess a high-affinity nucleoside transporter and that the activity of this transporter is sufficient to modulate ADO sensitivity of large and small coronary arteries.

Inhaled carbon monoxide concentration during halothane or isoflurane anesthesia in horses.

OBJECTIVE: The purpose of this study was to assess carbon monoxide (CO) exposure during equine anesthesia with either halothane (H) or isoflurane (I) delivered in a circle rebreathing system. STUDY DESIGN: Prospective clinical investigation. ANIMALS: Fifty client-owned horses. METHODS: Horses were randomly assigned for anesthetic maintenance with H (n = 26) or I (n = 24). Two large animal anesthetic machines were used and assigned to a single agent for 2-4 weeks at a time. Machines were disassembled and soda lime changed prior to switching anesthetic agents. Inhalant anesthetic concentration and CO concentration were measured in gas samples obtained from the inspiratory limb of the anesthetic circuit. Values were recorded at 15 minute intervals for 90 minutes. Soda lime status (new or used) and mode of ventilation (spontaneous or mechanical) were also recorded. Data were analyzed using a five-factor ANCOVA with repeated measures. RESULTS: Inspired CO concentration for H and I increased from 1 +/- 3 and 6 +/- 11 ppm at baseline to 54 +/- 33 and 21 +/- 18 ppm at 90 min, respectively (mean +/- sd). H was associated with significantly greater CO concentrations than I at 30 to 90 min, although baseline CO was significantly greater in the I group than the H group. Oxygen flow rates were 9.9 +/- 0.5 L/min at baseline for H and I, and 5.0 +/- 0.4 and 5.0 +/- 0.7 L/min at 90 min for H and I, respectively. There were no significant differences between groups for O2 flow at any time point. Neither mechanical ventilation nor new versus used soda lime affected CO concentration. CONCLUSIONS: Significantly higher concentrations of CO were recorded during the administration of H than I. CLINICAL RELEVANCE: Levels of CO observed during the administration of either H or I for 90 minutes to horses were not clinically significant.

Cardiopulmonary effects of bilateral hemithorax ventilation and diagnostic thoracoscopy in dogs.

OBJECTIVE: To describe alterations in respiratory and cardiovascular variables during diagnostic thoracoscopy, using bilateral hemithorax ventilation with sustained pneumothorax. ANIMALS: 7 adult dogs. PROCEDURE: Each dog was anesthetized and instrumented for 2 episodes of cardiopulmonary monitoring that were performed at an interval of more than 14 days. The first anesthetic episode served as a control procedure for the thoracoscopy treatment performed during the second anesthetic episode. Multiple cardiopulmonary variables were evaluated by comparing changes from baseline values within treatments and between treatments. RESULTS: Arterial oxygen tension decreased significantly from baseline values during thoracoscopy but was unchanged during sham treatment. Arterial carbon dioxide tension, clinical shunt fraction, and systemic mean arterial pressure increased during thoracoscopy. In contrast, these variables were unaffected by the sham treatment. Heart rate and cardiac index increased during sham and thoracoscopy treatments; however, the increase was significantly greater during thoracoscopy. Total peripheral vascular resistance significantly decreased from baseline values for both treatments, but the decrease was greater during thoracoscopy. Significant changes were not observed for oxyhemoglobin saturation or pulmonary vascular resistance during either treatment. Dogs recovered without major clinical complications. CONCLUSIONS: Significant changes were found for several cardiopulmonary variables during bilateral hemithorax ventilation with sustained pneumothorax for diagnostic thoracoscopy of clinically normal dogs. CLINICAL RELEVANCE: Diagnostic thoracoscopy with bilateral hemithorax ventilation and sustained pneumothorax is well tolerated in clinically normal dogs and may provide a diagnostic modality enabling intrathoracic procedures with less morbidity than thoracotomy for dogs with intrathoracic disease.

Evaluation of biopsy specimens obtained during thoracoscopy from lungs of clinically normal dogs.

OBJECTIVE: To determine whether lung biopsy specimens obtained during thoracoscopy, using a commercially available ligature, can provide an adequate amount of tissue for histologic evaluation and to characterize changes in the lungs and thoracic cavity that result from the procedure. ANIMALS: 6 mixed-breed dogs. PROCEDURE: All dogs underwent 2 anesthetic episodes. The first anesthetic episode was a sham procedure. During the second anesthetic episode, each dog underwent a thoracoscopic procedure to obtain a lung biopsy specimen, using a commercially available ligature. Biopsy specimens were assessed subjectively by means of histologic evaluation. Samples for arterial blood gas analysis were obtained, and thoracic radiography was performed after surgery. Dogs were evaluated daily for 14 days after thoracoscopy and then were euthanatized. Tissues were evaluated grossly and histologically. RESULTS: Excellent intraoperative visibility and biopsy specimens adequate for histologic evaluation were obtained from all dogs. Significant differences were not found between arterial blood gas values of sham- and thoracoscopy-treated dogs for samples obtained 0.25, 2, and 24 hours after extubation. Examination of thoracic radiographs obtained 2 and 24 hours after thoracoscopy revealed minimal localized pathologic changes. All dogs were clinically normal 24 hours after thoracoscopy, and major postoperative complications were not detected. Gross and histologic findings of specimens obtained during necropsy revealed changes localized to biopsy and trocar sites. CONCLUSIONS: Thoracoscopic placement of ligatures allowed procurement of lung lobe biopsy specimens from clinically normal dogs without complications. CLINICAL RELEVANCE: This procedure may provide a safe and minimally invasive means of obtaining lung biopsy specimens from clinically affected dogs.

Isolated right-ventricular hypertrophy associated with severe pulmonary vascular apolipoprotein A1-derived amyloidosis in a dog.

A 12-year-old dachshund was referred for respiratory distress, coughing, and weight loss. Cyanosis, dyspnea, tachypnea, and harsh lung sounds were noted on physical examination. Polycythemia with an increased number of nucleated red blood cells; right atrial enlargement; severe interstitial-to-alveolar pattern in all lung fields; and peripheral, echogenic, pulmonary masses were observed. Cytological examination of pulmonary aspirates indicated possible pulmonary carcinoma. The dog was euthanized at the owner's request. Isolated right-ventricular hypertrophy and pulmonary arteriopathy with amyloid deposits of apolipoprotein A1 were identified upon necropsy and histopathology. Pulmonary vascular amyloidosis should be considered in the differential diagnoses of respiratory distress in aged dogs.

Regional anesthesia of the infraorbital and inferior alveolar nerves during noninvasive tooth pulp stimulation in halothane-anesthetized dogs.

OBJECTIVE: To document that regional anesthesia of the infraorbital and inferior alveolar nerves would abolish reflex-evoked muscle action potentials (REMP) in the digastricus muscle during noninvasive stimulation of tooth pulp in halothane-anesthetized dogs. DESIGN: Prospective study. ANIMALS: 9 healthy female dogs between 2 and 6 years old. PROCEDURE: Dogs were anesthetized using halothane. An alligator clip anodal electrode was attached to the tooth to be stimulated, and a platinum needle cathodal electrode was inserted in adjacent gingival mucosa. The cathodal and anodal electrodes were moved to the left upper and lower canine, fourth premolar, and first molar teeth for sequential stimulation. Baseline recording of REMP was made for each tooth. Catheters were inserted percutaneously in the infraorbital and mandibular canals. Saline (0.9% NaCl) solution was injected at each catheterized site in 3 control dogs, and chloroprocaine hydrochloride was injected at each catheterized site in 6 test dogs. Each tooth was stimulated every 10 minutes for 90 minutes (test dogs) or every 10 minutes for 30 minutes and at 90 minutes (control dogs), and REMP was recorded. RESULTS: REMP was abolished within 10 minutes in all test dogs, except during stimulation of the lower first molar in 1 dog. In 4 dogs, duration of blockade was less than 90 minutes. The REMP was not restored within 90 minutes for the upper teeth in 1 dog and within 2 hours for all teeth in another dog. At 24 hours, REMP was restored for all teeth except the lower left canine in 1 dog. The REMP was restored for the lower left canine in that dog at 96 hours. The REMP was not abolished at any time in control dogs. CLINICAL IMPLICATIONS: Regional anesthesia of the infraorbital and inferior alveolar nerves may effectively provide analgesia for dental procedures in dogs.

Effect of pertussis toxin and B-oligomer on platelet-activating factor-induced generation of inositol phosphates in porcine alveolar macrophages.

OBJECTIVE: To test the hypothesis that platelet-activating factor (PAF) induces inositol phosphate turnover through a receptor-linked, pertussis toxin-sensitive guanine nucleotide-binding (G) protein-dependent pathway in porcine alveolar macrophages. DESIGN: Randomized complete block design was used with 2 or 3 replicates/block. ANIMALS: Porcine alveolar macrophages were obtained by lavage of excised lungs from Yorkshire-type pigs (mean +/- SEM, 21 +/- 2 kg). PROCEDURE: Phospholipase C activation was assessed, using anion exchange chromatography to measure accumulation of inositol phosphates in [3H]myo-inositol-labeled alveolar macrophages. Macrophages were incubated with saline solution, pertussis toxin (4.75 nM), or B-oligomer (4.75 nM) for 2 hours. Cells then were washed and incubated for 5 minutes with PAF (0, 0.1, 1.0, or 10 microM; n = 15). Results were expressed as total inositol phosphates (inositol monophosphate, bisphosphate, trisphosphate, and tetrakisphosphate). RESULTS: Concentrations of total inositol phosphates were significantly (P < 0.05) increased to 162 +/- 7, 172 +/- 4, and 194 +/- 9% of control in response to 0.1, 1.0, and 10 microM PAF, respectively. Pertussis toxin attenuated the PAF-induced increase in total inositol phosphates by approximately 50% (P < 0.05). The B-oligomer of pertussis toxin failed to modify PAF-induced increases in total inositol phosphates. The specific PAF receptor antagonist WEB 2086 markedly attenuated PAF-induced. (10 microM) increase in inositol phosphates. CONCLUSIONS: We conclude that PAF stimulates accumulation of inositol phosphates through a specific receptor and that a pertussis toxin-sensitive G protein is involved in the signal transduction process leading to activation of phospholipase C in porcine alveolar macrophages.

Differential effects of tumor necrosis factor-alpha and platelet-activating factor on bovine pulmonary artery endothelial cells in vitro.

The effects of tumor necrosis factor alpha (TNF alpha) and platelet-activating factor (PAF) on monolayer permeability, cytotoxicity, and release of prostacyclin (measured as the stable metabolite 6-ketoprostaglandin [PG]F1 alpha) and thromboxane (TX)B2 were investigated in bovine pulmonary artery endothelial cells (BPAEC). After 4 h of incubation, TNF alpha (2000 U/mL) induced an increase in steady-state 125I-albumin permeability across the BPAEC monolayer (2.9 +/- 0.3%/h vs. 1.8 +/- 0.3%/h in control monolayers; n = 7, p < .05), and induced release of 6-keto-PGF1 alpha (2581 +/- 226 pg/mL vs. 863 +/- 164 pg/mL in controls; n = 16, p < .05) and TXB2 (204 +/- 14 pg/mL vs. 105 +/- 23 pg/mL in controls; n = 10, p < .05). PAF-incubation was also associated with increased 6-keto-PGF1 alpha and TXB2 release (4157 +/- 471 pg/mL and 276 +/- 32 pg/mL, respectively), but did not markedly alter morphology or increase 125I-albumin permeability. Specific tritiated deoxyglucose release and specific LDH release were unaffected by both treatments. These results indicate that TNF alpha contributed directly to increased BPAEC permeability without cytotoxicity or requirement for other serum or cellular components. However, PAF did not directly alter endothelial barrier function despite increased release of 6-keto-PGF1 alpha and TXB2.

Effects of clenbuterol hydrochloride on pulmonary gas exchange and hemodynamics in anesthetized horses.

We evaluated the effects of clenbuterol HCl (0.8 micrograms/kg, of body weight, IV), a beta 2 agonist, on ventilation-perfusion matching and hemodynamic variables in anesthetized (by IV route), laterally recumbent horses. The multiple inert gas elimination technique was used to assess pulmonary gas exchange. Clenbuterol HCl induced a decrease in arterial oxygen tension (from 57.0 +/- 1.8 to 49.3 +/- 1.2 mm of Hg; mean +/- SEM) as a result of increased shunt fraction (from 6.6 +/- 2.1 to 14.4 +/- 3.1%) and ventilation to regions with high ventilation-perfusion ratios. In contrast, no changes in these variables were found in horses given sterile water. In horses given clenbuterol HCl, O2 consumption increased from 2.23 +/- 0.18 to 2.70 +/- 0.14 ml.min-1.kg-1, and respiratory exchange ratio decreased from 0.80 +/- 0.02 to 0.72 +/- 0.01. Respiratory exchange ratio and O2 consumption were not significantly modified in sterile water-treated (control) horses. Clenbuterol HCl administration was associated with increased cardiac index (from 57.4 +/- 4.0 to 84.2 +/- 6.3 ml.min-1.kg-1), decreased total peripheral vascular resistance (from 108.3 +/- 9.3 to 47.6 +/- 2.8 mm of Hg.s.kg.ml-1), and decreased pulmonary vascular resistance (from 31.3 +/- 3.8 to 13.6 +/- 0.7 mm of Hg.s.kg.ml-1). Our findings indicated that clenbuterol HCl may potentiate hypoxemia as a result of increased shunt fraction in horses anesthetized by the IV route, and caused changes in hemodynamic variables that were consistent with its ability to stimulate beta 2-adrenergic receptors.

Differential effects of WEB 2086 and SRI 63-441 on TNF-alpha-induced alterations in cardiopulmonary function.

We hypothesized that platelet-activating factor (PAF) and cyclooxygenase products might be important mediators of the cardiopulmonary effects induced by tumor necrosis factor (TNF-alpha) in anesthetized pigs. A 6-h infusion of human recombinant TNF-alpha caused hypoxemia, leukopenia, thrombocytopenia, decreased cardiac index (CI), increased pulmonary vascular resistance (PVR) and increased mean pulmonary arterial (Ppa) and intratracheal (Pt) pressures. Administration of the PAF receptor antagonist SRI 63-441 or indomethacin blocked the early (0.25-0.5 h) and attenuated the later increases in PVR and Ppa; indomethacin also attenuated the increase in Pt and hypoxemia associated with TNF-alpha infusion. WEB 2086 did not attenuate the TNF-alpha-induced alterations in CI, PVR, Pt, or PaO2. The in vivo specificity of SRI 63-441 and WEB 2086 was tested by infusing exogenous PAF, prostaglandin (PG) F2 alpha, U-46619 [thromboxane (Tx)A2 receptor mimetic], or arachidonic acid (AA) before and during administration of SRI 63-441 or WEB 2086. Both antagonists blocked the cardiopulmonary effects induced by exogenous PAF. SRI 63-441, but not WEB 2086, significantly attenuated the increased PVR caused by PGF2 alpha, U-46619, and AA. We conclude that SRI 63-441 is a less specific PAF receptor antagonist in vivo compared with WEB 2086 and that cyclooxygenase products, but not PAF, contribute significantly to the cardiopulmonary responses induced by exogenously infused TNF-alpha in pigs.

Duration of etomidate-induced adrenocortical suppression during surgery in dogs.

Plasma cortisol concentrations were compared in canine surgical patients given etomidate (2 mg/kg of body weight, IV) or thiopental sodium (12 mg/kg, IV) for anesthetic induction. Blood samples to determine plasma concentrations of etomidate were obtained at 0, 5, 10, 15, and 30 minutes and 1, 2, 3, 4, 5, 6, 8, 12, and 24 hours after induction. Adrenocortical function was evaluated before surgery by use of adrenocorticotropic hormone stimulation tests. Dogs in both induction groups had high plasma cortisol concentrations after induction. Dogs given thiopental had a significant increase (P less than 0.05) in plasma cortisol concentration from baseline at 2, 3, 4, 5, 6, 8, and 12 hours after induction. Dogs given etomidate had a significant increase (P less than 0.05) in plasma cortisol concentration from baseline at 5, 6, and 8 hours after induction. A comparison of plasma cortisol concentrations determined at 2, 3, 4, 5, and 6 hours after induction with thiopental or etomidate revealed a higher (P less than 0.05) concentration in dogs given thiopental. The disposition of etomidate was best described by a 2-compartment model, with a redistribution half-life of 0.12 +/- 0.04 minute and a terminal half-life of 1.70 +/- 0.27 minute. Plasma cortisol concentrations did not correlate with plasma etomidate concentrations. We conclude that, compared with thiopental, a single bolus injection of etomidate reduces the adrenocortical response to anesthesia and surgery from 2 to 6 hours after induction. Because cortisol concentrations were significantly higher than baseline, and because cardiopulmonary function is maintained after a single bolus injection of etomidate, it can be considered a safe induction agent in dogs.