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1.
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.  相似文献   

2.
OBJECTIVES: To determine collagenase activity and evaluate matrix metalloproteinase (MMP)-8 and MMP-13 in horses with chronic obstructive pulmonary disease (COPD). ANIMALS: 12 horses with COPD and 12 healthy control horses. PROCEDURE: Collagenase activity was determined by use of an assay for degradation of type-I collagen. Western immunoblot analysis was used to identify interstitial collagenases MMP-8 and MMP-13 in tracheal epithelial lining fluid (TELF). Immunocytochemistry and in situ hybridization were used to determine cellular expression of these 2 collagenases in cells in bronchoalveolar lavage fluid (BALF). RESULTS: Collagenase activity was approximately 7 times higher in samples obtained from horses with COPD, compared with control horses. During stabling, horses with COPD had significantly higher collagenase activity than after being maintained on summer pasture, when activity was similar to that of control horses. Immunoreactivity of MMP-8 and MMP-13 was significantly increased in TELF of horses with COPD, compared with healthy horses. In TELF, a positive correlation was detected between immunoreactivity of MMP-8 and MMP-13 and the amount of degradation of type-I collagen. Macrophages and epithelial cells were the major cellular sources of MMP-8 and MMP-13. CONCLUSIONS AND CLINICAL RELEVANCE: Increased collagenase activity in TELF indicates active ongoing disease and, thus, may reflect lung tissue changes in horses with COPD. Measurements of collagenase activity and MMP immunoreactivity may provide additional diagnostic tools to identify the active phase of chronic lung disease.  相似文献   

3.
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4.
Inflammation causes epithelial cell sloughing and basement membrane (BM) exposure in canine pulmonary eosinophilia (PE), leading to degradation of the epithelial cell attachment component, laminin-5 gamma2-chain, into small molecular weight fragments. The subsidence of inflammation after treatment down-regulates degradation. Laminin-5 gamma2-chain levels and molecular forms in bronchoalveolar lavage fluid (BALF) were analysed semiquantitatively by Western immunoblotting to compare PE affected (n=20) and healthy dogs (n=16) as well as PE dogs (n=6) before and after corticosteroid treatment. PE dogs expressed significantly elevated levels of total (P<0.01), 36 kDa (P<0.05) and 53 kDa (P<0.05) laminin-5 gamma2-fragments. The 36 Da fragment decreased significantly (P<0.05) after treatment. The laminin-5 gamma2-chain degradation products may be linked to epithelial cell sloughing and BM exposure or healing.  相似文献   

5.
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6.
OBJECTIVE: To determine immunoreactivity of matrix metalloproteinase (MMP)-1, -3, and -13 in cartilaginous tumors of dogs, correlate expression of MMP with histologic grade of tumors and clinical outcome of dogs, and compare MMP immunoreactivity between chondrosarcomas and chondromas. SAMPLE POPULATION: Formalin-fixed, paraffin-embedded tissues obtained from samples of naturally occurring chondrosarcomas (n = 31) and chondromas (8) of dogs that were submitted to our veterinary medical diagnostic laboratory. PROCEDURE: Histologic sections from each sample were stained with H&E and monoclonal antibody to MMP-1, -3, and -13 by use of an avidin-peroxidase immunohistochemical technique. For each section, histologic grade (I, II, or III) and immunohistochemical expression (0, 1, 2, or 3) were evaluated. Clinical outcome was obtained from medical records or interviews with referring veterinarians and scored as a good outcome, moderate outcome, or poor outcome. Correlations among variables and differences between chondrosarcomas and chondromas were analyzed. RESULTS: Samples from chondrosarcomas had significantly higher immunoreactivity of MMP-1 and -13, compared with immunoreactivity in samples from chondromas. In chondrosarcomas, a significant positive correlation (r, 0.386) was found between MMP-1 and -13 immunoreactivities, and a significant negative correlation (r, -0.390) was detected between MMP-3 and -13 immunoreactivities. CONCLUSIONS AND CLINICAL RELEVANCE: A significant increase in expression of collagenases (MMP-1 and -13) in chondrosarcomas, compared with expression in chondromas, suggests that collagenases may play an important role in tumor progression, and possibly metastasis, in chondrosarcomas of dogs.  相似文献   

7.
The aim of the present work was to evaluate whether concentrations of the carboxy-terminal cross-linked fragment of type II collagen (CTX-II), the activities of matrix metalloproteinase-2 and -9 (MMP-2/-9) and Myeloperoxidase (MPO) in canine synovial fluids (SF) can reflect structural alterations of articular cartilage in dogs with fragmented medial coronoid process (FMCP). Elbow joints with FMCP underwent radiographic and arthroscopic examination. Commercially available assays were used to analyze SF for CTX-II concentration and MMP-2/-9 activity. MPO activity was measured by o-dianisidine-assay. The MMPs were further evaluated by zymography. CTX-II concentration and MMP-2 activity showed age-dependent trends in controls. Increased enzyme activities of MPO and MMP-2/-9 were found in diseased dogs. MMP-9activity seems suitable to underline the subjective assessment of the degree of cartilage damage. These initial data of the study suggest that MPO and MMP-2/9 may be used as objective biomarkers in the diagnosis of canine osteoarthritis due to FMCP.  相似文献   

8.
Immunologic variables in dogs with eosinophilic bronchopneumopathy (EBP) have not been extensively evaluated. The aim of this study was to determine immunoglobulin (Ig) concentrations and to perform phenotypic subtyping of lymphocytes in the bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) of 12 dogs with EBP at the time of diagnosis (TD) and to compare these data with those obtained in healthy dogs, as well as in EBP dogs after antibiotic therapy (TAB) and during corticosteroid treatment (TM). Matched samples of serum and BALF were used to determine Ig concentrations (IgG, IgM, and IgA) by capture enzyme-linked immunosorbent assay (ELISA), from which a secretory index (SI) was calculated. Lymphocyte subpopulations were studied in the BALF and PB by flow cytometry. Log values of BALF IgM and IgA were significantly higher (0.64+/-0.05 and 1.06+/-0.13, respectively) in EBP dogs at TD than in controls and then tended to decrease at TM (0.55+/-0.03 and 1.02+/-0.17, respectively). A calculated SI for IgA was not significantly increased. In the BALF of dogs with EBg the CD4: CD8 was significantly (P < .05) higher (22.6+/-30.3) than in controls (3.2+/-1.9), due to significantly higher CD4+ T cells and lower CD8+ T cells. At TM, the BALF T-cell percentages returned to normal (2.4+/-0.6). We propose that the influx of eosinophils into the airway of dogs with EBP is at least in part mediated by cytokines derived from CD4+ T cells. Further studies of canine cytokines and chemokines will help determine whether canine EBP involves type I hypersensitivity mechanisms regulated by Th2 lymphocytes.  相似文献   

9.
OBJECTIVE: To determine reference values for cytologic examination results of bronchoalveolar lavage fluid (BALF) and to investigate effects of repeated lavages on pulmonary health and on results of cytologic examination of BALF in dogs. ANIMALS: 16 healthy adult Beagles. PROCEDURE: All dogs underwent pulmonary lavage to obtain BALF. Eleven dogs were repeatedly lavaged 6 times at 5- to 7-week intervals. Analyses for total and differential cell counts and for viability of cells before and after cell processing were performed. Arterial blood gas analysis before and after bronchoalveolar lavage was used to study the safety of the lavage procedure. Histologic and radiologic examinations were used to study effects of repeated lavages on pulmonary health. RESULTS: Mean (+/- SD) cell count was 104 +/- 69 cells/microl, comprising 75 +/- 7% alveolar macrophages, 13 +/- 6% lymphocytes, 5 +/- 4% neutrophils, 4 +/- 5% eosinophils, 2 +/- 2% mast cells, 0.6 +/- 0.7% epithelial cells, and 0.3 +/- 0.4% plasma cells. Centrifugation of samples and washing of cells caused significant cell loss (59 +/- 13%). Repeated lavages did not cause significant variations in cell counts of BALF or results of arterial blood gas analysis, thoracic radiography, or histologic examination of pulmonary specimens. Only a moderate, although significant, decrease in arterial oxygen content was observed after bronchoalveolar lavage. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis indicated that several lavages performed at 5- to 7-week intervals can safely and reliably be used to study the kinetics of pathologic processes in pulmonary tissues or for evaluation of therapeutic efficacy.  相似文献   

10.
A sensitive enzyme-linked immunosorbent assay was used for the simultaneous assessment of the amount of von Willebrand factor (vWF) in canine plasma and its ability to bind to canine collagen in vitro. In 60 normal dogs, there was close correlation between the concentration of vWF and its activity as determined by vWF-collagen binding. In 14 dogs with type I expressions of von Willebrand's disease, the ratio of vWF antigen to collagen binding activity was normal or only slightly increased. In 7 dogs with type II expressions of the disease, this ratio was consistently elevated suggesting a significant functional deficiency of the protein. Plasma from 3 dogs with type III von Willebrand's disease had little collagen binding activity because of the severe quantitative deficiency of the protein. The described assay permits the rapid assessment of both the quantity and quality of vWF in a dog. This information is necessary for the detection and characterization of canine von Willebrand's disease, particularly the type II expressions, which cannot be diagnosed by quantitative vWF assays alone.  相似文献   

11.
Western immunoblotting studies of canine sera using Malassezia pachydermatis extracts have shown that infected dogs commonly have antibodies that recognize multiple antigens. However, reported patterns of immunoreactivity vary between different laboratories. Since culture duration influences the antigenic composition of lipid-dependent Malassezia when probed with human sera, we investigated whether the in vitro growth phase of M. pachydermatis influences immunoreactivity to canine sera. Extracts of M. pachydermatis CBS1879 grown in Sabouraud's liquid medium at 37°C for 2, 4, 6, 8 and 10 days were prepared by mechanical disruption, centrifugation, dialysis and lyophilization. Yeast growth phase was assessed by sequential colony counts and optical density measurements. Patterns of IgG immunoreactivity in high ( n  = 3) and low ( n  = 3) titre sera were compared using extracts prepared at each time point by SDS-PAGE and western immunoblotting. Protein bands of 62 and 49 kDa were recognized by all sera, and five sera recognized 98 and 68 kDa bands. Proteins of 188, 66, 58, 57, 38, 28 and 17 kDa were only recognized by high titre sera. All high titre sera recognized more bands in exponential phase (day 2) extracts when compared with decline phase (days 8–10) extracts, and two of these sera showed most bands in stationary phase (days 4–6) extracts. Bands of 62 and 57 kDa were primarily detected in exponential and early stationary phase extracts. Antigenic variation in extracts of M. pachydermatis prepared during different growth phases might explain discrepancies between previous laboratory studies of immunity to this yeast.
Funding: Government of Malaysia.  相似文献   

12.
We report the effects of mouldy hay/straw exposure, inhaled hay dust suspension (HDS) and inhaled lipopolysaccharide (LPS) on bronchoalveolar lavage fluid (BALF) gelatinolytic matrix metalloproteinase (MMP) levels and degree of activation in healthy (n = 6) and heaves- (previously termed chronic obstructive pulmonary disease) affected (n = 6 or 7) horses. Gelatinolytic MMPs in BALF were quantified by zymography, and gelatinases were shown by Western immunoblotting to be MMP-2 and MMP-9. Hay/straw and HDS challenges increased BALF total gelatinolytic activity only in heaves horses, with the majority of gelatinolytic activity comprising pro- and active MMP-9. The 5 h duration hay/straw challenge increased BALF gelatinolytic MMP activity in heaves horses at 5 and 24 h after the start of this challenge, with activity returning to baseline by Day 4. In contrast to hay/straw and HDS challenges, LPS inhalation increased BALF gelatinolytic MMP activity in both groups. For all challenges, absolute BALF neutrophil counts were highly significantly correlated (P<0.0001) with levels of proMMP-9 and active MMP-9, but not with levels of MMP-2 (P>0.05). As gelatinolytic MMPs are pro-inflammatory agents, they may contribute to lung dysfunction and tissue destruction in heaves horses exposed to airborne organic stable dusts.  相似文献   

13.
OBJECTIVE: To evaluate inhibitory effects of synthetic matrix metalloproteinase (MMP) inhibitors in vitro on gelatinolytic and collagenolytic activities in tracheal epithelial lining fluid (TELF) of horses with recurrent airway obstruction (RAO). ANIMALS: 10 horses with RAO and 5 healthy control horses. PROCEDURES: Substrate-based functional assays, collagen I and gelatin degradation, were used to measure endogenous collagenolytic and gelatinolytic activities in TELF. In vitro inhibition of MMP activity in TELF with 2 chemically modified tetracyclines (CMTs; CMT-3 and CMT-8) and 2 bisphosphonates (BPs; zoledronate and pamidronate) was evaluated. RESULTS: CMT-3, CMT-8, zoledronate, and pamidronate in a dose-dependent manner inhibited TELF type I collagenolytic and gelatinolytic activities, although no complete inhibition of TELF type I collagenolytic and gelatinolytic activities was achieved with the inhibitor concentrations of 25 to 500 microM tested. The CMTs inhibited pathologically induced collagen I degradation more effectively than BPs. Of the tested CMTs, CMT-3 was the most effective inhibitor of gelatinolytic activity, and the efficiency of CMT-3 corresponded with that of the BPs. CONCLUSIONS AND CLINICAL RELEVANCE: An increase in MMP activity in the equine respiratory tract may potentially be inhibited by administration of CMTs or BPs. Distinct synthetic MMP inhibitors may eventually provide an additional means for pharmacologic treatment by decreasing ongoing active tissue destructive inflammation associated with chronic lung disease. The MMP inhibitors such as CMTs and BPs that are targeted to solely inhibit a pathologic increase in MMP activities provide the advantage of minimal adverse effects that are characteristics of other excessively potent MMP inhibitors.  相似文献   

14.
Matrix metalloproteinases (MMPs) are a group of calcium- and zinc-dependent endopeptidases that are involved in maintaining the extracellular matrix. MMP-2 and MMP-9 are thought to be related to the disruption of the blood-brain-barrier (BBB) by their ability to cleave type IV collagen, the main component of the basal membrane. To establish the presence of MMP-2 and MMP-9 in the pathogenesis of canine cerebral leishmaniasis, we examined the levels of these metalloproteinases in the cerebrospinal fluid (CSF) and serum of dogs with visceral leishmaniasis and neurological symptoms (n=16) and in the CSF and serum of uninfected healthy dogs (n=10) using zymography. In the CSF of dogs with cerebral leishmaniasis there was a massive presence of active MMP-2, whereas only the levels of both proMMP-2 and proMMP-9 were elevated in the serum. Although the detected MMP activity in the CSF might merely be related to CNS inflammation, these enzymes may also play a collaborative role in the disease progression. Both MMP-2 and MMP-9 are known to target critical constituents of the BBB, and once activated, they may promote cerebral barrier breakdown, allowing the entrance of inflammatory cells and proteins within the nervous system milieu.  相似文献   

15.
Forty-nine cutaneous plasmacytomas in 46 dogs were studied. Tumors occurred at solitary sites in middle-aged to old dogs (mean age, 9.7 years) and most commonly involved the skin of the digits, lips, and ears. Initial diagnosis was made on the basis of light microscopic morphologic findings. Tumors were graded according to the extent of cellular differentiation and immunoreactivity to a panel of immunohistochemical markers (cytokeratins, canine IgG F[ab]2, neurofilament, neuron-specific enolase, S-100 protein, and vimentin). Immunoreactivity was limited to antibodies directed at canine IgG F(ab)2 and vimentin. Vimentin immunoreactivity was usually greater than that of canine IgG F(ab)2, but there was no correlation between immunoreactivity and histologic grade of the tumors. Thirty-six of 39 dogs (92.3%) followed (mean follow-up, 13 months) were cured by surgical excision. The results of this study indicate that canine cutaneous plasmacytomas are benign neoplasms that should be included in the differential diagnosis of cutaneous round cell tumors in dogs.  相似文献   

16.
The aims of this study were to evaluate the effect of age on bronchoscopic features and bronchoalveolar lavage fluid (BALF) cellularity in dogs. Thirty healthy beagle dogs from three age groups were included: young dogs (10 months to 4.5 years of age; n = 8), middle-aged dogs (5–8 years old; n = 13) and older dogs (>8 years; n = 9). Haematology, thoracic radiography, bronchoscopy and bronchoalveolar lavage were performed; bronchoscopic findings were scored and BALF total and differential cell counts were determined. The total bronchoscopic score was higher in older dogs; these dogs had more irregular bronchial mucosa, more prominent mucosal vessels and bronchiectasis. Younger dogs had a higher percentage of neutrophils in BALF compared with middle-aged and old dogs and a higher percentage of lymphocytes in BALF compared with middle-aged dogs. The results show that age has an effect on bronchoscopic features of airways and the composition of BALF in the dog.  相似文献   

17.
In western immunoblotting studies of canine sera using Malassezia pachydermatis extracts, the reported patterns of immunoreactivity vary between different laboratories. Because the duration of culture influences the antigenic composition of lipid-dependent Malassezia spp. when probed with human sera, we investigated whether the in vitro growth phase of M. pachydermatis influences immunoreactivity using canine sera. Extracts of M. pachydermatis CBS 1879 grown in Sabouraud's liquid medium at 37 degrees C for 2, 4, 6, 8 and 10 days were prepared by mechanical disruption, centrifugation, dialysis and lyophilization. Yeast growth phase was assessed by sequential colony counts and optical density measurements. Patterns of IgG immunoreactivity in high (n = 3) and low (n = 3) titre sera were compared using extracts prepared at each time point by sodium dodecyl sulphate polyacrylamide gel electrophoresis and western immunoblotting. Protein bands of 62 and 49 kDa were recognized by all sera, and 98 and 68 kDa bands were recognized by five sera. Proteins of 188, 66, 58, 57, 38, 28 and 17 kDa were only recognized by high titre sera. All high titre sera used recognized more bands in exponential phase (d2) extracts when compared with decline phase (d8-d10) extracts, and two of these sera showed most bands in stationary phase (d4-d6) extracts. Bands of 62 and 57 kDa were primarily detected in exponential and early stationary phase extracts. There is variation in antigenic expression in different growth phases of M. pachydermatis, which might explain discrepancies between previous laboratory studies of canine immunity to this yeast.  相似文献   

18.
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.  相似文献   

19.
Gelatinolytic activity was analysed to study whether elevated activity previously found at the tracheal level of the respiratory tract of horses with chronic obstructive pulmonary disease (COPD) could also be found at the lower part of the respiratory tract. Furthermore, presence and significance of the gelatinolytic matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in respiratory secretions of healthy and COPD horses were determined. Elevated gelatinolytic matrix metalloproteinases were detected in bronchoalveolar and tracheobronchial secretions from COPD horses. The main pathologically elevated MMP was characterised to be MMP-9. Significantly increased MMP-9 activities as measured by gelatin zymography and Western blotting were found in all the respiratory samples from COPD horses compared to healthy horses. Elevation of active MMP-9 paralleled with increased gelatinase-associated lipocalin levels. Bronchoalveolar lavage fluid (BALF) epithelial cells, macrophages, neutrophils and lymphocytes expressed MMP-9 immunoreactivity demonstrated by immunocytochemistry and MMP-9 mRNA was expressed by bronchial epithelial cells of lung tissue section shown by in situ hybridisation. MMP-2 seems not to play a major role in chronic lung inflammation. No clear differences in MMP-2 or MMP-14 (a potent MMP-2 activator) levels were found when comparing the samples from COPD or healthy horses. These results suggests that MMP-9 could serve as a potential diagnostic marker for the active ongoing tissue remodelling in the acute phase of equine COPD. Increased gelatinolytic activity could be found at both tested respiratory tract levels. Therefore, tracheal epithelial lining fluid (TELF) samples can usefully serve as diagnostic material for detection of increased levels of the main gelatinolytic MMP, MMP-9, representing the entire diseased lung.  相似文献   

20.
Western immunoblotting studies of canine sera using Malassezia pachydermatis extracts have shown that infected dogs commonly have antibodies that recognize multiple antigens. However, reported patterns of immunoreactivity vary between different laboratories. Since culture duration influences the antigenic composition of lipid‐dependent Malassezia when probed with human sera, we investigated whether the in vitro growth phase of M. pachydermatis influences immunoreactivity to canine sera. Extracts of M. pachydermatis CBS1879 grown in Sabouraud's liquid medium at 37°C for 2, 4, 6, 8 and 10 days were prepared by mechanical disruption, centrifugation, dialysis and lyophilization. Yeast growth phase was assessed by sequential colony counts and optical density measurements. Patterns of IgG immunoreactivity in high (n = 3) and low (n = 3) titre sera were compared using extracts prepared at each time point by SDS‐PAGE and western immunoblotting. Protein bands of 62 and 49 kDa were recognized by all sera, and five sera recognized 98 and 68 kDa bands. Proteins of 188, 66, 58, 57, 38, 28 and 17 kDa were only recognized by high titre sera. All high titre sera recognized more bands in exponential phase (day 2) extracts when compared with decline phase (days 8–10) extracts, and two of these sera showed most bands in stationary phase (days 4–6) extracts. Bands of 62 and 57 kDa were primarily detected in exponential and early stationary phase extracts. Antigenic variation in extracts of M. pachydermatis prepared during different growth phases might explain discrepancies between previous laboratory studies of immunity to this yeast. Funding: Government of Malaysia.  相似文献   

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