Pulmonary function,airway cytology and bronchoalveolar lavage fluid drug concentration after aerosol administration of cefquinome to horses

The administration of antibiotics by aerosol to horses suffering from respiratory infections may partially circumvent the limitations of antimicrobial therapy, e.g. large injection volumes, low bioavailability and risk of diarrhoea. Only injectable formulations are available currently and usually contain other substances that could irritate the mucosa and induce coughing and bronchospasm. In addition, the quality of the aerosol, particularly in terms of the delivery of antibiotics to the deep parts of the lung, is unknown. Although used under field conditions, cefquinome delivered by aerosol has never been studied in horses. This study examined the safety of cefquinome injectable solution, administered by aerosol at a dose of 225 mg/inhalation to 7 healthy horses, by assessing (1) pulmonary function before and 15 min after a single inhalation, at the first day (Day 1) and the fifth day (Day 5) of a 5 day period treatment; and (2) the inflammatory status of the lung, i.e. percentage neutrophils and myeloperoxidase concentration, based on bronchoalveolar lavage (BAL) at D1 and D5. In addition, cefquinome concentrations were measured in bronchoalveolar lavage fluid after aerosol, intravenous (i.v.) and intramuscular (i.m.) administrations. A single aerosol of cefquinome injectable solution did not induce any immediate nor delayed pulmonary side effects in healthy horses and produced cefquinome concentrations in bronchoalveolar lavage (BAL) within 30 min that were higher than the minimal inhibitory concentration of the main equine respiratory pathogens. These results should stimulate further studies, especially in horses suffering from bronchial hyper‐reactivity. Aerosol delivery of antibiotics may well have a role in equine therapeutics.     

Clinical findings,bronchoalveolar lavage fluid cytology and matrix metalloproteinase-2 and -9 in canine pulmonary eosinophilia

We characterized clinical and clinicopathological features, and the involvement of gelatinolytic matrix metalloproteinases (MMP-2 and -9) in canine pulmonary eosinophilia (PE). Study material consisted of 20 PE dogs and 16 healthy beagles. All dogs underwent a similar clinical examination and bronchoalveolar lavage (BAL). Analysis for cell count and differential cell count of BAL fluid (BALF), arterial blood gas analysis before and after BAL, and thoracic radiographs before BAL and after treatment were obtained. Twelve dogs were re-evaluated and six relavaged. MMP-2 and MMP-9 in BALF were analysed by zymography, Western immunoblotting and immunocytochemistry.In the PE dogs, BALF, cell count, number and percentage of eosinophils, and numbers of macrophages, lymphocytes, neutrophils, mast cells and epithelial cells were all significantly elevated. Blood eosinophilia was detected in half of the PE dogs. Three PE dogs had mild hypoxaemia. The BAL procedure had an equal effect on PE and healthy dogs' arterial blood gas values. Bronchointerstitial densities were seen in PE dogs' radiographs. Treatment of PE decreased BALF cell count, eosinophil count and percentage and diminished radiographic changes. Gelatinolytic activity was higher in PE dogs' BALF. BALF macrophages and epithelial cells were the principal sources of the MMP-9.     

IL-4, IL-5 and IFN-gamma mRNA expression in pulmonary lymphocytes in equine heaves

Heaves is a common condition of horses of cold climate that is characterized by small airway inflammation and obstruction following exposure of susceptible horses to moldy hay and straw. It has been shown that helper T lymphocytes (Th) orchestrate the inflammatory response in asthma and in various animal models of allergic lung diseases by the release of Th2-type cytokines. Results of previous studies indicate that a predominant expression of Th2-type response by airway cells may also be present in heaves. To evaluate the temporal mRNA expression of Th1 (IFN-gamma) and Th2 (IL-4, IL-5) type cytokines in heaves and their relationship to clinical disease, we studied the pulmonary mechanics and cytokine mRNA expression (IL-4, IL-5 and IFN-gamma) in bronchoalveolar lavage lymphocytes of horses with heaves (n=6) and control (n=6) before and after 24h and 9 days of continuous natural inhalation challenge. Starting 24h after challenge horses with heaves, but not control horses, had a significant increase in pulmonary elastance and the number of lymphocytes expressing mRNA for IL-4 and IL-5. These changes were further increased at 9 days, at which time the number of cells positive for IFN-gamma mRNA was decreased. In this study we have shown that BAL lymphocytes of horses with heaves during clinical exacerbation have a predominant Th2-type cytokine response and that this response coincides in time with the presence of airway obstruction.     

Serum and bronchoalveolar lavage fluid endothelin-1 concentrations as diagnostic biomarkers of canine idiopathic pulmonary fibrosis

Background: Diagnosis of canine idiopathic pulmonary fibrosis (IPF) is challenging. Endothelin‐1 (ET1) is a biomarker of IPF in humans, but whether ET1 can detect and differentiate IPF from other canine respiratory diseases is unknown. Objective: To evaluate whether measurement of the concentration of ET1 in serum and bronchoalveolar lavage fluid (BALF) can be used to distinguish canine IPF from chronic bronchitis (CB) and eosinophilic bronchopneumopathy (EBP). Animals: Twelve dogs with IPF, 10 dogs with CB, 6 dogs with EBP, 13 privately owned healthy West Highland White Terriers (WHWT), and 9 healthy Beagle dogs. Methods: Prospective, case control study. ET1 concentration was determined by ELISA in serum and in BALF. Results: No significant difference in serum ET1 concentration was detected between healthy Beagle dogs and WHWT. Serum ET1 concentration was higher in dogs with IPF (median interquartile range; 2.32 pg/mL, 2.05–3.38) than healthy Beagle dogs (1.28, 1.07–1.53; P < .001), healthy WHWT (1.56, 1.25–1.85; P < .001), dogs with EBP (0.94 0.68–1.01; P = .001), and dogs with CB (1.54 0.74–1.82; P = .005). BALF ET1 concentration was below the detection limit in healthy WHWT and in dogs with CB, whereas it was measurable in all dogs with IPF. A cut‐off serum concentration of 1.8 pg/mL had a sensitivity of 100% and a specificity of 81.2% for detection of IPF, with an area under the receiver operating characteristic curve of 0.818. Conclusions and Clinical Importance: Serum ET1 can differentiate dogs with IPF from dogs with EBP or CB. ET1 can be detected in BALF of dogs with IPF.     

Cyclosporine A inhibits the mRNA expressions of IL-2, IL-4 and IFN-gamma, but not TNF-alpha, in canine mononuclear cells

The effects of the calcineurin inhibitors cyclosporine A (CsA) and FK506 on the mRNA expressions of various cytokines were evaluated in dogs to determine whether the effects of CsA and FK506 in dogs were similar to those in humans. The mRNA expression levels of the cytokines IL-2, IL-4, IFN-gamma and TNF-alpha were measured in PHA-stimulated canine PBMC using real-time RT-PCR after incubation with CsA or FK506 for 5 hr. Both reagents inhibited IL-2, IL-4 and IFN-gamma mRNA expressions in a dose-dependent manner. However, CsA hardly inhibited the mRNA expression of TNF-alpha. These findings are important for assessing the indications of CsA treatment in dogs.     

Cytologic, microbiologic, and biochemical analysis of bronchoalveolar lavage fluid obtained from 24 healthy cats

Twenty-four healthy cats underwent bronchoscopy and bronchoalveolar lavage to determine the normal cytologic environment of the lower respiratory tract of cats. Initial screening to ensure the health of the study population included complete histories, physical examinations, thoracic radiography, CBC, serologic tests for feline leukemia virus, feline immunodeficiency virus, and occult heartworm, and sugar and Baermann fecal flotation. In 18 cats, protected catheter brush samples of airway secretions from the lavaged lung segment were taken for culture of aerobic and anaerobic bacteria and mycoplasma. Bronchial lavage fluid (5 sequential 10-ml aliquots of normal saline solution) was pooled and filtered with cotton gauze. The unspun sample was used for determination of a total nucleated cell count. Lavage fluid was cytocentrifuged and 500 cells/slide were scored for determination of the cellular differential. Activity of lactate dehydrogenase and concentrations of total protein and IgG within the supernatant were measured, and assays were performed to detect the presence of IgA and IgM. Complete histologic evaluation of the lavaged lung of each of 6 random-source cats was performed after differential cell counting revealed 18% eosinophils within bronchoalveolar lavage fluid recovered from this group. Alveolar macrophages were the predominant cells encountered; however, a quarter of all cells recovered were eosinophils. A significant relationship was not found between the abundance of eosinophils in the lavage fluid, and either isolation of aerobic bacteria, high total nucleated cell counts, total protein concentrations, or activity of lactate dehydrogenase. Histologic evaluation of the lungs of 5 of 6 random-source cats revealed normal lungs in 2 cats, and minimal abnormal change in 3 others. Evaluation of the lungs from 1 random source cat revealed acute, mild eosinophilic bronchiolitis. We conclude that large numbers of eosinophils may be retrieved from the bronchoalveolar lavage fluid of healthy cats.     

猪白细胞介素-4基因的克隆与表达

利用RT-PCR技术,从被刺激诱导的PBMC中克隆IL-4基因,序列分析表明:克隆的猪IL-4基因序列与GenBank上登录的IL-4基因的核苷酸和氨基酸序列同源性分别为99%和97.8%.然后用表达型引物从T载体上扩增IL-4基因,双酶切PCR产物后与表达载体pGEX连接,构建重组表达质粒pGEX-IL-4,用IPTG诱导表达,表达产物经SDS-PAGE分析表明,表达出38 ku融合蛋白,占菌体总蛋白的30%以上,并且以包涵体的形式存在,这为猪重组IL-4规模化生产和疫苗佐剂的研制奠定了基础.     

Blood concentrations of the cytokines IL-1beta,IL-6, IL-10, TNF-alpha and IFN-gamma during experimentally induced swine dysentery

Background

Knowledge of the cytokine response at infection with Brachyspira hyodysenteriae can help understanding disease mechanisme involved during swine dysentery. Since this knowledge is still limited the aim of the present study was to induce dysentery experimentally in pigs and to monitor the development of important immunoregulatory cytokines in blood collected at various stages of the disease.

Methods

Ten conventional pigs (~23 kg) were orally inoculated with Brachyspira hyodysenteriae B204^T. Eight animals developed muco-haemorrhagic diarrhoea with impaired general body condition. Blood was sampled before inoculation and repeatedly during acute dysentery and recovery periods and cytokine levels of IL-1β, IL-6, Il-10, TNF-α and IFN-γ were measured by ELISA.

Results

IL-1β was increased at the beginning of the dysentery period and coincided with the appearance of Serum amyloid A and clinical signs of disease. TNF-α increased in all animals after inoculation, with a peak during dysentery, and IL-6 was found in 3 animals during dysentery and in the 2 animals that did not develop clinical signs of disease. IL-10 was found in all sick animals during the recovery period. IFN-γ was not detected on any occasion.

Conclusion

B. hyodysenteriae inoculation induced production of systemic levels of IL-1β during the dysentery period and increased levels of IL-10 coincided with recovery from dysentery.     

Correlation and discriminant analysis between clinical, endoscopic, thoracic X-ray and bronchoalveolar lavage fluid cytology scores, for staging horses with recurrent airway obstruction (RAO)

As recurrent airway obstruction (RAO) is progressive and as medical history is frequently unknown by owners, it's important to suggest a score model to characterize RAO stages for a more accurate diagnosis and treatment. The authors correlated clinical (CS), endoscopic (ES), thoracic X-ray (XRS) and bronchoalveolar lavage fluid (BALFS) scores in horses with RAO, in an attempt to establish relevance of each factor's contribution for the characterization of RAO stages and to suggest a staging method. Thirty horses with RAO and ten healthy controls were studied. Pearson correlation coefficients were determined between CS, ES, XRS and BALFS. Only significant correlation coefficients (>0.60) were considered. One way variance analyses were used to compare the two groups. A discriminant analysis model was adjusted on the RAO staging method suggested. There was a significant correlation coefficient between the CS cough, nostril flare and abdominal lift, all the mucus ES (0.61-0.84), the XRS interstitial pattern, bronchial radiopacity and thickening and tracheal thickening (0.67-0.78) and the BALFS neutrophil percentages (0.63-0.84). These variables (e.g., cough) which presented a significant correlation coefficient were considered relevant and chosen for a score model to characterize RAO stages. The ten healthy controls were attributed stage 0 and the 30 RAO horses were attributed stages 1 (4 horses), 2 (7 horses), 3 (10 horses) and 4 (9 horses). There was also a significant correlation coefficient between all the relevant variables and the RAO stage (0.61-0.89). Furthermore, discriminant analysis of the RAO staging method showed 92.5% of original grouped cases and 85.0% of cross-validated grouped cases correctly classified, having confirmed major contribution of the same variables that had significant correlation coefficients. Even though further confirmation by lung functional testing is desirable, the significant correlation between relevant variables and RAO stage and the results of RAO staging discriminant analysis support the suggestion of our score model for the characterization of RAO stages.     

Use of ELISPOT and ELISA to evaluate IFN-gamma, IL-10 and IL-4 responses in conventional pigs

ELISPOT and ELISA were standardised for pig interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and interleukin-4 (IL-4) with the aim to study the evolution of the immune response in conventional pigs from birth to 6 months of age and also to compare results of both techniques. Five pigs were bled at 1, 6, 9, 12 and 22 weeks of age and peripheral blood mononuclear cells (PBMC) were stimulated with phytohemagglutinin. The frequencies of cytokine secreting cells (CSC) and the levels of secreted cytokines were compared. For IFN-gamma the mean of CSC increased with age (p<0.05) from an average of 486/10(6) PBMC at first week of age to 1256/10(6) PBMC at 22 weeks of age. No correlation was found between the number of IFN-gamma CSC and the cytokine levels obtained by ELISA. For IL-10, frequencies of CSC did not increase with age of pigs, having a low of 315/10(6) PBMC at first week of age and a high of 1485/10(6) PBMC at six weeks. Comparison of ELISA and ELISPOT results for IL-10 showed a certain degree of correlation (r=0.74; p<0.05). Spontaneous secretion was observed in unstimulated cultures. For IL-4, frequencies of CSC were low (50-70/10(6) PBMC). In this case, comparison of ELISA and ELISPOT could not be done because cytokine levels in culture supernatants were often below the detection limit of the IL-4 ELISA. All these values can serve as a reference for future studies and also, our observations suggest that ELISPOT and ELISA should be carefully interpreted and do not necessarily correlate.     

Interleukin-8 concentration and neutrophil chemotactic activity in bronchoalveolar lavage fluid of horses with chronic obstructive pulmonary disease following exposure to hay

OBJECTIVE: To analyze effects of hay dust exposure on interleukin-8 (IL-8) concentration, percentage of neutrophils, and neutrophil chemotactic activity in bronchoalveolar lavage fluid (BALF) of horses with chronic obstructive pulmonary disease (COPD). ANIMALS: 16 healthy horses and 29 horses with COPD. PROCEDURE: IL-8 concentration, percentage of neutrophils, and neutrophil chemotactic activity in BALF were measured. Values were analyzed with respect to hay dust exposure.These variables were also measured in 5 asymptomatic horses with COPD after the induction of clinical signs by changing feed from silage to hay. RESULTS: L-8 concentrations and chemotactic activity in BALF were greater in horses with COPD, compared with healthy horses, and greater in horses with COPD exposed to hay dust, compared with nonexposed affected horses. An increase in IL-8 concentration accompanied by an increase in percentage of neutrophils in BALF and development of clinical signs of COPD were induced in asymptomatic horses with COPD by changing feed from silage to hay. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure of horses with COPD to hay dust components resulted in an increase in IL-8 secretion at the bronchoalveolar surface. This chemokine may play a role in the pathogenesis of COPD, because it causes neutrophil accumulation in the bronchoalveolar space. Our results underscore the importance of eliminating dust sources for the treatment and prevention of COPD in horses.     

Clinical signs, evaluation of bronchoalveolar lavage fluid, and assessment of pulmonary function in horses with inflammatory respiratory disease

OBJECTIVE: To evaluate the association among clinical signs, results of cytologic evaluation of bronchoalveolar lavage (BAL) fluid, and measures of pulmonary function in horses with inflammatory respiratory disease. ANIMALS: 9 healthy horses, 5 horses with inflammatory airway disease (IAD), and 9 horses with chronic obstructive pulmonary disease (COPD). PROCEDURES: Clinical examination, lung function tests, and BAL were performed on each horse. RESULTS: Standard lung mechanics of horses with exacerbated COPD differed significantly from those of healthy horses; however, there were few differences among horses with IAD, horses with COPD during remission, and healthy horses. Most variables for forced expiration (FE) in horses with COPD or IAD differed significantly from those for healthy horses. Results of clinical examination had low to moderate sensitivity and predictive values for a diagnosis of COPD (range, 67 to 80%). Results of FE tests had high sensitivity, specificity, and predictive values for a diagnosis of COPD (79 to 100%), and results of standard lung mechanics tests had low sensitivity and predictive values (22 to 69%). Percentage of neutrophils in BAL fluid was highly sensitive (100%) but moderately specific (64%) for a diagnosis of COPD. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical examination is moderately accurate for establishing a diagnosis of COPD. Forced expiration tests can specifically detect early signs of airway obstruction in horses with COPD and IAD that may otherwise be inapparent. Cytologic evaluation of BAL fluid allows early detection of inflammatory respiratory disease, but it is not specific for COPD.     

Synergistic effects of IL-2, IL-12 and IL-18 on cytolytic activity, perforin expression and IFN-gamma production of porcine natural killer cells

Natural killer (NK) cells are one of the main cellular components of the innate immune system. They play an important role in the immune response against infections as well as tumour cells and therefore have two major properties: production of immune regulatory cytokines and chemokines as well as cytolytic destruction of particular target cells. The existence of NK cells in swine is well known as well as the phenotype of resting NK cells, but their response following activation by cytokines is still poorly understood. Therefore, we tested the influence of the immune regulatory cytokines IL-2, IL-12 and IL-18 on cytolytic activity, phenotype, IFN-gamma production and the accumulation of perforin in cytoplasm of peripheral blood mononuclear cells (PBMC) as well as purified NK cells. NK cells were enriched from PBMC using a magnetic cell separation (MACS) strategy with monoclonal antibodies against CD3, CD21 and SWC3, thereby removing T-, B- and myeloid cells. Respective fractions were used in flow cytometry (FCM) based cytolytic assays with the human tumour cell line K562 as target. After stimulation with the cytokines described above, the NK cell enriched CD3(-)CD21(-)SWC3(-) fraction showed an evident increase in the cytolytic activity compared to PBMC. This enhanced cytolytic activity was accompanied by a strong enrichment of IFN-gamma producing cells when a combination of all three cytokines (IL-2/IL-12/IL-18) was used; as determined in ELISPOT assays and intracellular staining of IFN-gamma in FCM. Also, the combination of these three cytokines led to an accumulation of perforin in the cytoplasm and an up-regulation of CD25 compared to control cultures incubated in medium without cytokines. The experiments performed clearly indicate a stimulatory role and strong synergistic effects of the investigated cytokines in the activation of porcine NK cells in vitro, inducing IFN-gamma, perforin production and cytotoxicity against target cells.     

Cytological analysis of equine bronchoalveolar lavage fluid. Part 3: The effect of time,temperature and fixatives

Bronchoalveolar lavage fluid (BALF) samples are often subject to time delays, possibly with temperature fluctuations, between collection and processing. The aim of this study was to evaluate the effects of time, temperature and 2 different fixatives on equine BALF cytology, in order to develop guidelines for optimal equine BALF storage conditions. Total nucleated cell count (TCC), differential cell counts (DCC), absolute cell counts (ACC), cell viability, cell morphology and bacterial growth of BALF samples stored at 4, 18 (+/- addition of formalin- or alcohol-based fixatives) and 38 degrees C were monitored serially over a 72 h period. The time taken for a significant reduction in TCC and cell viability of unfixed BALF samples decreased as the storage temperature increased. There was no diagnostically significant difference in DCC or ACC over this time-course at any temperature. Unfixed BALF samples showed significant bacterial growth by 24 h at 4 degrees C, and 8 h at 18 and 38 degrees C; and poor morphology by 48 h at 4 degrees C, 24 h at 18 degrees C and 8 h at 38 degrees C. Fixed BALF samples showed poor morphology with Leishman's stain compared to unfixed samples.