Minna M. Rajamäki

Metalloproteinase inhibition reduces lung injury and improves survival after cecal ligation and puncture in rats

BACKGROUND Neutrophil activation with concomitant matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) release has been implicated in the development of sepsis-induced acute lung injury. We hypothesized that COL-3, a chemically modified tetracycline known to inhibit MMP-2 and MMP-9, would reduce lung injury and improve survival in rats following cecal ligation and puncture (CLP). METHODS Sprague-Dawley rats were separated into five groups: 1) sham CLP+ carboxymethylcellulose (CMC; vehicle for COL-3, n = 6); 2) sham CLP + COL-3 (n = 6); 3) CLP + CMC (n = 10); 4) CLP + single-dose (SD) COL-3 administered concomitant with CLP (n = 9); and 5) CLP + multiple-dose (MD) COL-3 administered concomitant with CLP and at 24 h after CLP (n = 15). Rats were sacrificed at 168 h (7 days) or immediately after death, with survival defined as hours after CLP. Histological lung assessment was made based on neutrophil infiltration, alveolar wall thickening, and intraalveolar edema fluid. Lung MMP-2 and MMP-9 levels were assessed by immunohistochemistry. MMP-2 and MMP-9 levels were correlated with survival by simple regression analysis. RESULTS The mortality of rats in the cecal ligation and puncture without treatment group (CLP + CMC) was 70% at 168 h. A single dose of COL-3 in the CLP + COL-3 (SD) group significantly reduced mortality to 54%. Furthermore, with a repeat dose of COL-3 at 24 h after CLP, mortality was significantly reduced to 33%. Pathologic lung changes seen histologically in the CLP + CMC group were significantly reduced by COL-3. A significant reduction in lung tissue levels of MMP-2 and MMP-9 was noted in both groups treated with COL-3. Reduction of MMP-2 and MMP-9 levels correlated with improved survival. CONCLUSION Inhibition of MMP-2 and MMP-9 by COL-3 in a clinically relevant model of sepsis-induced acute lung injury reduces pulmonary injury and improves survival in a dose-dependent fashion. Our results suggest that prophylactic treatment with COL-3 in high-risk patients may reduce the morbidity and mortality associated with sepsis-induced acute respiratory distress syndrome.

Clinical, bronchoscopic, histopathologic, diagnostic imaging, and arterial oxygenation findings in West Highland White Terriers with idiopathic pulmonary fibrosis.

BACKGROUND Idiopathic pulmonary fibrosis (IPF) is a chronic, interstitial lung disease primarily affecting West Highland White Terriers (WHWTs). OBJECTIVE To describe the clinicopathological and diagnostic imaging features in WHWTs with IPF. ANIMALS Twelve WHWTs with IPF and 14 healthy control WHWTs. METHOD Prospective study. Clinical signs and findings of physical examination, blood and arterial blood gas analyses, radiography, high-resolution computed tomography (HRCT), bronchoscopy and bronchoalveolar lavage (BAL) of IPF dogs were obtained and compared with controls. Histopathologic changes in IPF dogs were evaluated. RESULTS Mean partial pressure of oxygen was significantly lower in IPF (mean ± SD, 65.5 ± 15.4 mmHg) than in controls (99.1 ± 7.8 mmHg, P<.001). The alveolar-arterial oxygen gradient was significantly higher in IPF (50.1 ± 17.3 mmHg) than in controls (17.5 ± 4.9 mmHg, P<.001). In HRCT, ground glass opacity (GGO) was detected in all IPF dogs, traction bronchiectasis in 4, and honeycombing in 1. Bronchoscopic airway changes were noted in all IPF dogs. On BAL fluid (BALF) cytology, the total cell count (TCC) was higher in IPF dogs, and the numbers but not the percentages of macrophages, neutrophils, and mast cells were increased. On histopathology, multifocal or diffuse interstitial fibrosis, type II pneumocyte hyperplasia, prominent intraalveolar macrophages, distortion of alveolar architecture, and emphysematous change were detected. CONCLUSION AND CLINICAL IMPORTANCE IPF causes substantial hypoxemia. In HRCT, GGO is a consistent finding. IPF dogs have concurrent airway changes and an increase in BALF TCC.

Detecting early kidney damage in horses with colic by measuring matrix metalloproteinase -9 and -2, other enzymes, urinary glucose and total proteins

BackgroundThe aim of the study was to investigate urine matrix metalloproteinase (MMP-2 and -9) activity, alkaline phosphatase/creatinine (U-AP/Cr) and gamma-glutamyl-transpeptidase/creatinine (U-GGT/Cr) ratios, glucose concentration, and urine protein/creatinine (U-Prot/Cr) ratio and to compare data with plasma MMP-2 and -9 activity, cystatin-C and creatinine concentrations in colic horses and healthy controls. Horses with surgical colic (n = 5) were compared to healthy stallions (n = 7) that came for castration. Blood and urine samples were collected. MMP gelatinolytic activity was measured by zymography.ResultsWe found out that horses with colic had significantly higher urinary MMP-9 complex and proMMP-9 activities than horses in the control group. Colic horses also had higher plasma MMP-2 activity than the control horses. Serum creatinine, although within reference range, was significantly higher in the colic horses than in the control group. There was no significant increase in urinary alkaline phosphatase, gamma-glutamyltranspeptidase or total proteins in the colic horses compared to the control group. A human cystatin-C test (Dako Cytomation latex immunoassay® based on turbidimetry) did not cross react with equine cystatin-C.ConclusionThe results indicate that plasma MMP-2 may play a role in the pathogenesis of equine colic and urinary MMP-9 in equine kidney damage.

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.

Evaluation of plasma activity of matrix metalloproteinase-2 and -9 in dogs with myxomatous mitral valve disease

OBJECTIVE To investigate whether plasma activity of matrix metalloproteinase (MMP)-2 and -9 was associated with severity of myxomatous mitral valve disease (MMVD) in dogs and to assess potential associations between MMP activity and dog characteristics, echocardiographic variables, systolic arterial blood pressure (SAP), heart rate, cardiac troponin I (cTnI) concentration, and C-reactive protein concentration. ANIMALS 75 client-owned dogs. PROCEDURES Severity of MMVD was assessed by use of echocardiography. Plasma activity of latent (pro-MMP) and active MMP-2 and -9 was analyzed via zymography. Plasma concentration of cTnI was analyzed with a high-sensitivity cTnI assay, and C-reactive protein concentration was analyzed with a canine-specific ELISA. RESULTS Pro-MMP-9, active MMP-9, and pro-MMP-2 were detected, but active MMP-2 was not. No significant differences were found in MMP concentrations among the 4 MMVD severity groups. Activity of pro-MMP-9 decreased with decreases in SAP and was higher in male dogs than in female dogs. Activity of MMP-9 decreased with increases in left ventricular end-systolic dimension and with decreases in SAP and cTnI concentration. Left ventricular end-systolic dimension was the variable most strongly associated with MMP-9 activity. No associations were found between the activity of pro-MMP-2 and investigated variables. CONCLUSIONS AND CLINICAL RELEVANCE Plasma MMP-9 activity decreased with increases in the end-systolic left ventricular internal dimension and decreases in SAP. Hence, evaluation of MMP-9 activity has the potential to provide unique information about the myocardial remodeling process in dogs with MMVD.

Serum C‐Reactive Protein as a Diagnostic Biomarker in Dogs with Bacterial Respiratory Diseases

Background C‐reactive protein (CRP) is a major acute‐phase protein in dogs. Serum concentrations are low in healthy animals, but increase rapidly after inflammatory stimuli. Objective The aim of the study was to investigate CRP concentrations in various respiratory diseases of dogs and to determine if CRP can be used as a biomarker in the diagnosis of bacterial respiratory diseases. Animals A total of 106 privately owned dogs with respiratory diseases (17 with bacterial tracheobronchitis [BTB], 20 with chronic bronchitis [CB], 20 with eosinophilic bronchopneumopathy [EBP], 12 with canine idiopathic pulmonary fibrosis [CIPF], 15 with cardiogenic pulmonary edema [CPE], and 22 with bacterial pneumonia [BP]) and 72 healthy controls. Methods The study was conducted as a prospective cross‐sectional observational study. CRP was measured in serum samples. Diagnosis was confirmed by clinical and laboratory findings, diagnostic imaging, and selected diagnostic methods such as cytological and microbiological analysis of respiratory samples, echocardiography, and histopathology. Results Dogs with BP had significantly higher CRP concentrations (median, 121 mg/L; interquartile range, 68–178 mg/L) than dogs with BTB (23, 15–38, P = .0003), CB (13, 8–14, P < .0001), EBP (5, 5–15, P < .0001), CIPF (17, 10–20, P < .0001), or CPE (19, 13–32, P < .0001) and healthy controls (14, 8–20, P < .0001). Dogs with BTB had significantly higher CRP concentrations than dogs with CB (P = .001) or EBP (P < .0001) and healthy controls (P = .029). Conclusion and Clinical Importance These results indicate that CRP has potential for use as an additional biomarker, especially in the diagnostics of BP.

Comparison of results for weight-adjusted and fixed-amount bronchoalveolar lavage techniques in healthy Beagles

OBJECTIVE To compare recovery of epithelial lining fluid (ELF) in bronchoalveolar lavage fluid (BALF) by use of weight-adjusted or fixed-amount volumes of lavage fluid in dogs. ANIMALS 13 healthy Beagles. PROCEDURES Dogs were allocated to 2 groups. In 1 group, the right caudal lung lobe was lavaged on the basis of each dogs weight (2 mL/kg, divided into 2 aliquots) and the left caudal lung lobe was lavaged with a fixed amount of fluid (50 mL/dog, divided into 2 aliquots). In the second group, the right and left caudal lung lobes were lavaged by use of the fixed-amount and weight-adjusted techniques, respectively. The BALF was collected by use of bronchoscopy. A recovery percentage ≥ 40% was required. The proportion of ELF was calculated by use of the following equation: (concentration of urea in BALF/concentration of urea in serum) × 100. RESULTS Mean ± SD proportion of ELF in BALF was 2.28 ± 0.39% for the weight-adjusted technique and 2.89 ± 0.89% for the fixed-amount technique. The SDs between these 2 techniques differed significantly (calculated by comparing 2 covariance structures [unstructured and compound symmetry] in a repeated-measures mixed ANOVA). CONCLUSIONS AND CLINICAL RELEVANCE The findings strongly suggested that use of a weight-adjusted bronchoalveolar lavage technique provided a more uniform ELF recovery, compared with that for a fixed-amount bronchoalveolar lavage technique, when urea was used as a marker of dilution. A constant ELF fraction can facilitate more accurate comparisons of cellular and noncellular constituents in BALF among patients of various sizes.

Long‐Term Outcome and Use of 6‐Minute Walk Test in West Highland White Terriers with Idiopathic Pulmonary Fibrosis

Background Idiopathic pulmonary fibrosis (IPF) is an incurable interstitial lung disease occurring mainly in West Highland White Terriers (WHWTs). The effects of IPF on survival and on exercise tolerance in WHWTs are unknown. Objectives To evaluate survival, prognostic factors, and exercise tolerance in WHWTs with IPF. Animals Privately owned WHWTs; 15 with IPF and 11 healthy controls. Methods Prospective case‐control study conducted in 2007–2012. For survival, descriptive statistics and Kaplan–Meier (KM) survival curves with Cox proportional hazard ratios were performed. For the prognostic factor study, KM curves, Cox regression analysis, and logistic regression models were used. The 6‐minute walk test (6MWT) was used for measurement of exercise tolerance. Results The median IPF‐specific survival of deceased WHWTs (7/15) with IPF was 32 (range 2–51) months from onset of clinical signs. The risk of death from birth in WHWTs with IPF in age‐adjusted Cox model was significantly higher (hazard ratio 4.6; 95% confidence interval 1.05–19.74, P = .04) than in control WHWTs. No significant prognostic factors were identified. In 6MWT, WHWTs with IPF walked a shorter distance, median 398 m (range 273–519 m), than healthy controls, median 492 m (420–568 m), P = .05, and the partial pressure of oxygen in arterial blood in diseased dogs had a moderate positive correlation with walking distance (Kendall′s tau‐b = 0.69, P = .06). Conclusion and Clinical Importance IPF had a negative impact on life expectancy, but individual survival varied considerably. 6MWT proved to be a well‐tolerated, noninvasive test to evaluate exercise tolerance.

Idiopathic Pulmonary Fibrosis in West Highland White Terriers

Canine idiopathic pulmonary fibrosis (CIPF) is a chronic, progressive, interstitial lung disease affecting mainly middle-aged and old West Highland white terriers. Other dogs, especially terriers, have been diagnosed with the disease. The cause is largely unknown, but it is likely to arise from interplay between genetic and environmental factors. CIPF shares several features with human idiopathic pulmonary fibrosis. This article summarizes the current literature; describes the findings in physical examination, arterial blood gas analysis, bronchoscopy, bronchoalveolar lavage, diagnostic imaging, and histopathology; compares the canine and human diseases; gives an overview of potential treatments; and discusses biomarker research.

Co-infections with Respiratory Viruses in Dogs with Bacterial Pneumonia

Background Bacterial pneumonia (BP) is an inflammation of the lower airways and lung parenchyma secondary to bacterial infection. The pathogenesis of BP in dogs is complex and the role of canine respiratory viruses has not been fully evaluated. Objectives The aim of this study was to investigate the occurrence of viral co‐infections in dogs with BP and to assess demographic or clinical variables as well as disease severity associated with viral co‐infections. Animals Twenty household dogs with BP caused by opportunistic bacteria and 13 dogs with chronic (>30 days) tracheobronchitis caused by Bordetella bronchiseptica (BBTB). Methods Prospective cross‐sectional observational study. Diagnosis was confirmed by clinical and laboratory findings, diagnostic imaging, and cytologic and microbiologic analysis of bronchoalveolar lavage or transtracheal wash fluid. Canine parainfluenza virus (CPIV), canine adenovirus, canine herpes virus, canine influenzavirus, canine distemper virus, canine respiratory coronavirus (CRCoV) and canine pneumovirus, as well as B. bronchiseptica and Mycoplasma spp. were analyzed in respiratory samples using PCR assays. Results CPIV was detected in 7/20 and CRCoV in 1/20 dogs with BP. Respiratory viruses were not detected in dogs with BBTB. There were no significant differences in clinical variables between BP dogs with and without a viral co‐infection. Conclusion and Clinical Importance Respiratory viruses were found frequently in dogs with BP and may therefore play an important role in the etiology and pathogenesis of BP. Clinical variables and disease severity did not differ between BP dogs with and without viral co‐infection.