Antimicrobial resistance patterns of bacteria isolated from canine urinary samples submitted to a New Zealand veterinary diagnostic laboratory between 2005-2012.

AIMS: To identify and describe culture and antimicrobial resistance (AMR) patterns in bacteria isolated from canine urinary samples submitted to a New Zealand veterinary diagnostic laboratory. METHODS: Records from a veterinary diagnostic laboratory were examined for bacterial isolates cultured from canine urine samples between January 2005 and December 2012. Culture and susceptibility results were compiled with information on the age, sex and breed of dog. Repeat submissions were removed. Susceptibility was assessed using results of the Kirby-Bauer disk diffusion method, for a standard panel including amoxicillin-clavulanic acid (AMC), cefovecin (from 2010-2012), cephalothin, clindamycin, enrofloxacin and trimethoprim-sulphonamide (TMS). RESULTS: A total of 5,786 urine samples were submitted for analysis, and 3,135 bacterial isolates were cultured from 2,184 samples. Of these 3,135 isolates, 1,104 (35.2%) were Escherichia coli, 442 (14.1%) were Staphylococcus spp., 357 (11.4%) Proteus mirabilis and 276 (8.8%) were Enterococcus spp. The frequency of culture-positive samples increased with increasing age in both female and male dogs (p<0.001). The percentage of E. coli isolates resistant to AMC and cephalothin increased between 2005 and 2012 (p<0.001), as did resistance to enrofloxacin (p=0.022), but there was no change in resistance to TMS (p=0.696). Enrofloxacin was the antimicrobial with the least resistance shown by the four most common bacteria isolated during the course of the study. CONCLUSIONS AND CLINICAL RELEVANCE: The results of this study provide important regional information regarding the prevalence of bacterial uropathogens and their susceptibility patterns. There was an increase in resistance to some commonly used antimicrobials in the treatment of urinary tract infections. Having access to regional antimicrobial susceptibility results is crucial when forming guidelines for the use of antimicrobials for the treatment of urinary tract infections. Given changes in practising habits and antimicrobial usage over time, ongoing monitoring and surveillance of resistance in pathogens is needed.

Policies for the vaccination of cats and dogs in New Zealand veterinary practices.

AIMS: To determine current practices and attitudes towards vaccination of dogs and cats of veterinarians in New Zealand; the methods used for informing clients on which vaccines to use, and the preferred site for vaccination of cats. METHODS: A postal questionnaire was sent to all 483 listed veterinary practices in New Zealand during February 2012. Some questions were specific to pet dogs, cats, or working farm dogs. Responses were categorised according to practice type and geographical region of the respondent. Factors associated with respondent recommendation of annual vaccination with modified live viral (MLV) vaccines were examined using logistic regression analysis. Vaccines that were considered to be essential for every animal were defined as core; those that may be recommended for animals whose location or lifestyle placed them at risk, were defined as non-core. RESULTS: There were 204 useable returns, equivalent to a response rate of 42.2%, distributed across the country. Annual vaccination with MLV vaccines of dogs was recommended by 54/198 (27.3%) respondents, and of cats by 107/181 (59.1%) respondents. Factors associated with the recommendation of annual administration of MLV vaccines to dogs included being a companion animal practice, a desire for policies on vaccination to be left to individual clinics, and having one veterinarian in the practice. Administration of the final vaccination for puppies was recommended at ≥14 weeks old by 55/185 (29.7%) respondents, and for kittens at ≥13 weeks old by 42/183 (23%) respondents. Of respondents that administered MLV vaccines annually, 62/103 (60.2%) believed reducing the frequency of vaccination would reduce income, and 52/103 (50.5%) considered it would have a negative effect on animal health. Advice to enable clients to decide which non-core vaccines were administered was given by 181/199 (91%) respondents. Factors considered when recommending a vaccine included consideration of risk to individual patients (190/203; 93.6%), requirements of boarding kennels/catteries (165/203; 81.3%) and clinic vaccination policy (142/203; 70%). The preferred site for administering MLV vaccines to cats was the dorsal neck or inter-scapular region (137/198; 69.2%). Amongst respondents, 18 wanted disease surveillance information to allow for truly informed decisions to be made about vaccination. CONCLUSIONS AND CLINICAL RELEVANCE: Veterinarians can now compare their own vaccination practices and attitudes with those of veterinarians nationally, and internationally. There is a need for national surveillance information and for continued education of the public and commercial kennel and cattery owners for optimal vaccination strategies to be developed.

Attitudes and perceptions of veterinary paraprofessionals in New Zealand to postoperative pain in dogs and cats.

AIM: To survey the attitudes and perceptions of veterinary paraprofessionals in New Zealand to postoperative pain in dogs and cats. METHODS: In December 2011, veterinary paraprofessionals (VP) from throughout New Zealand were invited to participate in an online survey. Eleven questions, which were divided into five sections, were used to determine demographic information, the respondents' assessment of pain after commonly performed surgeries in dogs and cats, their opinions on provision of analgesia, who had responsibility for pain monitoring and the use of any formal pain scoring system in the practice. RESULTS: Data from 165 respondents were able to be used, and 162 (98%) respondents to the survey were female. According to the respondents' estimates, fracture repair in dogs and repair of diaphragmatic hernias in cats had the highest pain score following surgery. Neutering procedures involving dogs were scored higher than for cats (p<0.01). All respondents agreed that animals benefit from perioperative analgesia. The veterinary nurse was reported to be predominantly responsible for monitoring pain in animals postoperatively by 116/165 (70.3%) respondents. Of 165 respondents, 154 (93%) considered that their knowledge of pain and assessment of pain could be enhanced. CONCLUSIONS: This survey reflects the attitudes and perceptions of a sample of VP in New Zealand to postoperative pain in dogs and cats. The results indicate that all respondents believe that surgery results in sufficient pain to warrant analgesic therapy. Routine neutering surgeries were considered to be more painful in dogs than in cats. The current survey also provides information to educators on potential areas of focus, given that 93% of respondents felt that their knowledge of pain and assessment of pain could be enhanced.

The prevalence of ocular lesions associated with hypertension in a population of geriatric cats in Auckland, New Zealand.

AIMS: To provide an estimate of the prevalence of ocular lesions associated with hypertension in geriatric cats in Auckland, New Zealand and to evaluate the importance of examination of the ocular fundi of cats over eight years of age. METHODS: A total of 105 cats ≥8 years of age were examined and clinical signs recorded. Blood was collected for the laboratory measurement of the concentrations of blood urea nitrogen (BUN), glucose and creatinine in serum, urine was collected for determination of urine specific gravity (USG), and blood pressure (BP) was measured using high definition oscillometry equipment. A cat was determined to have systemic hypertension with a systolic BP ≥160 mm Hg and a diastolic BP ≥100 mm Hg. Each animal had an ocular fundic examination using a retinal camera to diagnose ocular lesions associated with hypertension, including retinopathies, choroidopathies and optic neuropathies. RESULTS: Blood pressure was successfully recorded in 73 cats. Of these, 37 (51%) had no hypertensive ocular lesions and no underlying disease diagnosed, 24 (33%) had no hypertensive ocular lesions detected, but underlying disease such as chronic kidney disease, hyperthyroidism or diabetes mellitus was diagnosed, and 12 (16%) cats had evidence of hypertensive ocular lesions. Ten of the cats with hypertensive ocular lesions were hypertensive at the time of the first visit and two were normotensive. One additional cat had hypertensive ocular lesions, but it was not possible to obtain consistent BP readings in this animal. Chronic kidney disease was the most commonly diagnosed concurrent disease in cats with hypertensive ocular lesions (n=6). Mean systolic BP for cats with hypertensive ocular lesions (168.0 (SE 6.29) mm Hg) was higher than for those with no ocular lesions (144.7 (SE 3.11) mm Hg) or those with no lesions but with underlying disease (146.0 (SE 4.97) mm Hg) (p=0.001). CONCLUSIONS: Ocular fundic examination of cats over eight years of age allows identification of cats with hypertensive ocular lesions, often before the owner or veterinarian is aware the cat has a problem with its vision. This may result in diagnosis of systemic hypertension allowing early treatment and resolution of lesions. CLINICAL RELEVANCE: The current study demonstrates that ocular lesions resulting from hypertension occur frequently enough in cats in Auckland to support the recommendation for fundic examination in cats over eight years of age as part of the routine physical examination.

Systemic effects of periodontal disease in cats.

BACKGROUND: Periodontal disease in cats is a local disease that may have systemic consequences that are affected by treatment. OBJECTIVE: To test the hypotheses that systemic health indices would be correlated with the severity of periodontitis, and would improve with treatment. ANIMALS AND METHODS: Apparently otherwise healthy cats from an in-bred colony were randomly assigned to a treatment group (n = 30), or a control group (n = 18), which was left untreated for 3 months. Periodontal disease was scored at baseline in the treatment group according to calculus, gingivitis, and alveolar bone loss measured from dental radiographs. Blood, urine and saliva were collected from both groups before, and 16, 45, and 90 days after dental treatment. Assays included haematology, urinalysis, serum biochemistry, serum IgG, salivary IgA, lymphocyte subsets and proliferation, and plasma malonyldialdehyde (MDA). Correlations between the severity of periodontitis and assays at baseline were assessed, and the effect of treatment determined using linear mixed model methodology. RESULTS: The severity of periodontitis was associated with age, bodyweight, total globulins (Globs), Alanine aminotransferase, and IgG, and negatively associated with albumin, haemoglobin, haematocrit, and Aspartate aminotransferase (AST). Treatment significantly reduced IgG, total Globs, AST, and eosinophils, and increased cholesterol. Other leucocyte assays and plasma MDA concentrations were not affected by the treatment. Cats ate dry food faster 1 week after, than they did 1 week before treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Although the clinical significance of these findings are unknown, we conclude that periodontitis is not simply a localized disease, but also impacts on systemic health and wellbeing.

Bone volume and regional density of the central tarsal bone detected using computed tomography in a cross-sectional study of adult racing greyhounds.

AIMS: To determine whether left-to-right asymmetry of the central tarsal bone (CTB) of racing greyhounds was detectable using computed tomography (CT) in live dogs; to quantify the asymmetry in terms of average bone volume, volumetric bone mineral density (vBMD), and dorsal cortical shape, and to determine if age, gender, bodyweight, number of starts or history of tarsal injury were significant covariates. METHODS: One trainer supplied 11 male and seven female, unrelated, skeletally mature, actively racing greyhounds, including dogs with a history of tarsal injury diagnosed by the trainer and/or track veterinarian (n=8), and dogs without a history of tarsal injury (n=10). Using CT, standardised parameters of the CTB were measured including volume and average vBMD of the left and right CTB, vBMD of regions within the CTB, and bone shape. RESULTS: There was no difference in the volumes of the left and right CTB and no association with number of racing starts. Volume of CTB in dogs with a history of tarsal injury was greater than in dogs with no history of injury (p<0.001). Mean vBMD of the right was greater than the left CTB (p=0.004), and was independent of history of injury, gender, bodyweight, and number of starts. Males with a history of injury weighed more than those with no history of injury (p=0.004). The region of greatest difference in vBMD between right and left limbs was the centrodorsomedial aspect of the CTB. Middle plantar ligament enthesiopathies and fractures of tarsal bones other than the central tarsal bone were identified in dogs with and without a history of tarsal injury. CONCLUSIONS: Significant left-to-right asymmetry of the CTB in racing greyhounds was detected using CT. Contrary to previous suggestions, the asymmetry was not associated with the number of racing starts. We propose that the majority of the adaptive modelling of the CTB occurred rapidly following the onset of counter-clockwise training, with little further modelling throughout the racing career of the dog, however further investigation is warranted. CLINICAL RELEVANCE: This study described a technique using CT for imaging the CTB in live dogs, which opens the way for a longitudinal study of bone modelling of the CTB in response to training and racing in a counter-clockwise direction.

Seasonal variation in the hip score of dogs as assessed by the New Zealand Veterinary Association hip dysplasia scheme.

AIM: To determine whether there is a seasonal variation in the phenotypic hip score of dogs born in New Zealand as assessed by the New Zealand Veterinary Association (NZVA) canine hip dysplasia (CHD) scheme. METHODS: Data from dogs born in New Zealand between 1988 and 2009 that have been scored for CHD were retrospectively evaluated for the effect of month of birth on radiographic phenotype. Data included both the total score and the subtotal score, comprising Norberg's angle, the subluxation score and changes to the cranial acetabular edge, for each dog. Datasets were created for all breeds combined and for the four most populous breeds using the scheme (German Shepherd dog, Labrador Retriever, Golden Retriever and Rottweiler) and stratified according to month of birth and season. Due to the skewed nature of the data, a Kruskal-Wallis Rank Sum test was used to test for statistical significance. Additionally, χ² analysis was performed using the median of each dataset (proportion above/below the median). The null hypothesis was that there would be no effect of month of birth, and hence seasonality, on hip phenotype for dogs born and scored in New Zealand by the NZVA. RESULTS: For all breeds combined, month of birth had an effect on total and subtotal NZVA CHD scores (p<0.001) with a lower total hip score in the autumn months of March and April than other months. When individual large breed data were analysed, there was an effect of month of birth on total and subtotal scores for the Labrador Retriever and the Rottweiler (p ≤ 0.05), but not the German Shepherd dog or Golden Retriever breeds. CONCLUSIONS: Being born in the autumn was associated with a protective effect on hip phenotype in some breeds. These results suggest that weather and/or another seasonal factor may have a significant environmental effect on the phenotype of the coxofemoral joint. CLINICAL RELEVANCE: The protective effect of being born in autumn suggests that a decreased level of exercise during subsequent development over winter may positively impact on final coxofemoral joint conformation. Whilst statistically significant, the magnitude of the sparing effect is not likely to be clinically relevant. However, this study, in concert with other studies, may suggest that the effects of exercise can be manipulated to improve hip phenotype.

Long-term outcomes after partial tarsal arthrodesis in working farm dogs in New Zealand.

AIM: To report the long-term outcome (return to work and owner satisfaction) for working farm dogs in New Zealand after partial tarsal arthrodesis for proximal intertarsal and/or tarsometatarsal tarsal joint injury. METHODS: Working farm dogs that underwent partial tarsal arthrodesis were identified by diagnosis and breed code via a search of the medical records of Massey University Veterinary Teaching Hospital (MUVTH) and Veterinary Specialist Orthopaedic Services (VetSOS). Fourteen dogs fulfilled the selection criteria and were all actively in work on farms at the time of injury. Data from case files were analysed, and the ability of the dog to work after surgery and owners' satisfaction were assessed using a questionnaire sent to owners at a median follow-up interval of 57 months. RESULTS: Following partial tarsal arthrodesis, 7/14 (50%) dogs could perform normal work duties required while 4/14 (29%) dogs could perform most of the duties they had undertaken before injury, though some allowance had to be made for reduced performance. Thirteen owners were satisfied or very satisfied with the outcome of surgery and 12/14 thought the financial investment required for the surgery was worthwhile. Of the three dogs that did not return to work, infection requiring implant removal (one dog) and less than optimal post-operative limb alignment (two dogs) were assessed to contribute to the poor outcome. Two other dogs that required implant removal due to infection did successfully return to work. CONCLUSIONS: In this limited cases series, partial tarsal arthrodesis in working dogs had a good prognosis for return to work. Any conclusion regarding the benefit of a particular surgical technique requires a prospective evaluation with a larger number of animals. It is important to minimise the risk of infection and optimally align the tarsus to avoid a poor outcome. CLINICAL RELEVANCE: New Zealand veterinarians can now provide more accurate prognostic information to owners whose working dogs suffer tarsal trauma requiring partial arthrodesis.

Trends in the phenotypic hip status of selected breeds of dog as measured by the New Zealand Veterinary Association Hip Dysplasia scheme (1990-2008).

AIM: To determine whether there has been improvement in the phenotypic hip dysplasia status in four susceptible dog breeds as measured by the New Zealand Veterinary Association (NZVA) Canine Hip Dysplasia (CHD) scheme. METHODS: A retrospective analysis of the NZVA CHD database was performed using records of all German Shepherd dogs, Labrador Retrievers, Golden Retrievers and Rottweilers that had undergone evaluation for hip dysplasia between 1990 and 2008. The effect of date of birth on the total hip score was analysed using linear regression, including the covariates of age and gender. When a significant effect of date of birth on total score was noted, ordinal logistic regression was performed to determine the probability of different grades of the Norberg angle and subluxation scores by year of birth; these categories being most indicative of laxity of the coxofemoral joint. Given the known heritability of hip phenotype, determined using radiological measurements, the hypothesis was that if sufficient selection pressure has been applied there would have been a trend towards a lower total score over time. RESULTS: For Labrador Retrievers (n=1,451), Golden Retrievers (n=896) and Rottweilers (n=313), there was no effect of date of birth on total score over the period of the study (p>0.1). For German Shepherd dogs (n=1,087), there was a significant trend to a lower total score over time (p=0.0003). However the actual size of the effect was small. Ordinal logistic regression on the Norberg angle and subluxation scores for German Shepherd dogs demonstrated a significant lowering of grade in both of these measures of hip laxity. CONCLUSIONS: This study failed to show significant improvement in the phenotypic hip status of three out of the four most populous large-dog breeds in the NZVA CHD database. Even in the German Shepherd dog, the trend towards a lower total score did not represent a substantial change. Lack of evidence of phenotypic improvement may be due to insufficient selection pressure over the course of the study, selective usage of the scheme (and thus a biased sample), or deficiencies within the NZVA CHD scoring method itself. CLINICAL RELEVANCE: Greater improvement might be possible if use of the scheme (or an equivalent) is made a compulsory requirement for registration of pedigree breeding stock, if greater selection pressure is applied and/or if pedigree data are included to enable estimations of breeding value.

Reduction in the incidence of elbow dysplasia in four breeds of dog as measured by the New Zealand Veterinary Association scoring scheme.

AIM: To determine if there has been any reduction in the incidence of elbow dysplasia in four popular large-dog breeds as measured by the New Zealand Veterinary Association (NZVA) scoring scheme. METHODS: A retrospective analysis of the NZVA elbow dysplasia database was performed using records of all German Shepherd dogs, Labrador Retrievers, Golden Retrievers and Rottweilers that had undergone evaluation since the scheme's inception in 1992. The data for each dog included date of birth, date of radiography, gender, grade of left and right elbow (0, 1, 2 or 3), and accredited or dysplastic status. Ordinal logistic regression was used to model the grade of the worst-affected elbow over time. The model included age at scoring and gender as additional variables. Given the known heritability of elbow dysplasia, the hypothesis was that if the NZVA scheme effectively identifies elbow dysplasia, and that dog breeders have been using the data responsibly, there should have been a trend towards a lower incidence of dogs graded dysplastic over time. RESULTS: In all four breeds, there was a significant trend towards lower grades of the worst-affected elbow over time. For German Shepherd dogs the incidence of elbow dysplasia (worst elbow grade not zero) fell from 75% to 47% between dogs born in 1991 vs those born in 2008. The corresponding figures were a drop from 86% to 68% for the Labrador Retriever, from 89% to 77% for Golden Retrievers, but only 98% to 95% for Rottweilers. In the Rottweiler and Golden Retriever, gender had a significant effect on the worst elbow grade. In the Golden Retriever, age at scoring also had a significant effect. CONCLUSIONS: There has been a significant reduction in the incidence of elbow dysplasia in four popular large-dog breeds as scored by the NZVA elbow dysplasia scoring scheme. The limitations of the study are the non-compulsory nature of the elbow dysplasia scheme, and the potential bias caused by dog breeders or veterinarians pre-screening potential submissions. The results therefore may not represent those of the overall population. CLINICAL RELEVANCE: The incidence of elbow dysplasia, as measured by the NZVA elbow dysplasia scheme, has reduced in the four breeds investigated since the scheme's inception. The New Zealand Kennel Club (NZKC) and the veterinary profession can confidently support the NZVA scoring scheme, and should promote its use by dog breeders.

A survey of diseases of working farm dogs in New Zealand.

AIMS: To record the incidence of specific diseases affecting working farm dogs; identify any apparent breed predispositions; determine the most important causes of loss from death, euthanasia, or retirement of the dog; and identify potential interventions that could reduce the incidence of disease. METHODS: A national questionnaire-based cross-sectional study was conducted to identify the incidence of specific diseases, and circumstances of trauma affecting working farm dogs that presented to rural veterinary practices over a 12-month period, between April 2008 and April 2009. The practices were convenience-sampled on the basis of geography and size. RESULTS: Sixty-six practices were approached, and veterinarians from 30 practices agreed to participate, with representation from all major rural regions of New Zealand. Data were provided on 2,214 presentations, excluding revisits, of which 51% were for Huntaways and 39% Heading dogs. Trauma was identified as a cause of injury in 848 (38%) visits. Huntaways were apparently over-represented in cases of constipation, gastric dilatation-volvulus (GDV), theriogenological problems, laryngitis, hip dysplasia, and degenerative lumbosacral disease. In contrast, Heading dogs were over-represented among cases of multiple ligamentous injury of the stifle, disruption of the gastrocnemius or Achilles tendon, tarsal injuries, and hip luxation. Traumatic injury involved injury by stock (20%), automotive incidents (19%), transit across fence lines (16%), and dog bites (12%). Loss occurred following 10% of visits, of which trauma was known to be involved in 32%. The most important non-traumatic causes of loss were GDV, degenerative joint disease, mammary neoplasia and diseases involving the female reproductive tract, cardiac disease, and poisoning. CLINICAL RELEVANCE: Several important diseases appear amenable to intervention through nutrition, neutering, or behavioural modification, including constipation, GDV, theriogenological problems, dog-bite injuries, and laryngitis. Altering modes of transit across fence lines has the potential to reduce large numbers of serious orthopaedic injuries to working farm dogs.

Effects of corticosterone treatment on growth, development, and the corticosterone response to handling in young Japanese quail (Coturnix coturnix japonica).

Corticosterone, a glucocorticoid secreted during stress responses, has a range of actions that help birds respond to stressors. Although effects of corticosterone treatment have been described in several avian species, the impacts of defined increases in plasma corticosterone on early development and on corticosterone stress responses are little known. These issues were addressed by providing quail with different doses of corticosterone in drinking water from days 8 to 38 post-hatch. The corticosterone dose consumed by each bird during treatment days 15-30 was calculated by measuring water intake. The corticosterone dose was inversely, but weakly, correlated with weights of the bursa, thymus, spleen, liver, testes, oviduct, muscle, and body, and positively correlated with peritoneal fat deposition. When birds were divided into groups based on their corticosterone intake, weights of the spleen, thymus, bursa, muscle, testes, and oviduct were significantly reduced in birds receiving the highest doses; with the exception of muscle, similar reductions were also observed in birds receiving medium doses, and thymic growth was inhibited in birds receiving low doses. The acute corticosterone stress response was measured by handling birds for 15 min. Plasma corticosterone was transiently increased at 15 min in control birds in response to the handling stressor. Some birds consuming low doses of corticosterone had corticosterone responses similar to control birds. Initial corticosterone concentrations were elevated in birds consuming higher doses of corticosterone. Plasma corticosterone in these birds decreased from 0 to 15 min, then increased from 15 to 30 min. The initial decrease could be due to corticosterone clearance, whilst the increase could indicate that the birds had a greater response than control birds to isolation as a stressor. Corticosterone treatment may have reduced the strength of corticosterone negative feedback within the hypothalamo-pituitary-adrenal axis. The results indicate that individuals and organs differ in their sensitivity to corticosterone. Moreover, elevated plasma corticosterone may disrupt the acute corticosterone stress response, and may thus reduce the ability of birds to cope with stressors.

Surfactant lipid peroxidation damages surfactant protein A and inhibits interactions with phospholipid vesicles.

The goal of these studies was to examine the effect of lipid peroxidation (LPO) on the function of surfactant protein A (SP-A). First, the optimal dialysis conditions for quantitative removal of EDTA and redoxactive metals from reagents were established. Surfactant phospholipids were incubated with free radical generators in the absence or presence of the SP-A or with BSA as a control. We found that SP-A inhibited copper-initiated LPO to an extent similar to BSA (P < 0.05). Exposure of SP-A to LPO was associated with an increase in the level of SP-A-associated carbonyl moieties and a marked reduction in SP-A-mediated aggregation of liposomes. LPO initiated by an azo-compound also resulted in enhanced protein oxidation and markedly inhibited SP-A-mediated liposome aggregation. The kinetics of aggregation of auto-oxidized and nonoxidized liposomes by nonoxidized SP-A was similar, suggesting that SP-A has similar affinities for oxidized and nonoxidized lipids. Oxidative inactivation of SP-A did not occur upon direct incubation of the protein with malondialdehyde alone. We conclude that exposure of SP-A to LPO results in oxidative modification and functional inactivation of SP-A by phospholipid radicals.

Secretion of surfactant protein C, an integral membrane protein, requires the N-terminal propeptide.

Proteolytic processing of surfactant protein C (SP-C) proprotein in multivesicular bodies of alveolar type II cells results in a 35-residue mature peptide, consisting of a transmembrane domain and a 10-residue extramembrane domain. SP-C mature peptide is stored in lamellar bodies (a lysosomal-like organelle) and secreted with surfactant phospholipids into the alveolar space. This study was designed to identify the peptide domain of SP-C required for sorting and secretion of this integral membrane peptide. Deletion analyses in transiently transfected PC12 cells and isolated mouse type II cells suggested the extramembrane domain of mature SP-C was cytosolic and sufficient for sorting to the regulated secretory pathway. Intratracheal injection of adenovirus encoding SP-C mature peptide resulted in secretion into the alveolar space of wild type mice but not SP-C (-/-) mice. SP-C secretion in null mice was restored by the addition of the N-terminal propeptide. The cytosolic domain, consisting of the N- terminal propeptide and extramembrane domain of mature SP-C peptide, supported secretion of the transmembrane domain of platelet-derived growth factor receptor. Collectively, these studies indicate that the N-terminal propeptide of SP-C is required for intracellular sorting and secretion of SP-C.

Pulmonary surfactant proteins A and D are potent endogenous inhibitors of lipid peroxidation and oxidative cellular injury.

The lung is composed of a series of branching conducting airways that terminate in grape-like clusters of delicate gas-exchanging airspaces called pulmonary alveoli. Maintenance of alveolar patency at end expiration requires pulmonary surfactant, a mixture of phospholipids and proteins that coats the epithelial surface and reduces surface tension. The surfactant lining is exposed to the highest ambient oxygen tension of any internal interface and encounters a variety of oxidizing toxicants including ozone and trace metals contained within the 10 kl of air that is respired daily. The pathophysiological consequences of surfactant oxidation in humans and experimental animals include airspace collapse, reduced lung compliance, and impaired gas exchange. We now report that the hydrophilic surfactant proteins A (SP-A) and D (SP-D) directly protect surfactant phospholipids and macrophages from oxidative damage. Both proteins block accumulation of thiobarbituric acid-reactive substances and conjugated dienes during copper-induced oxidation of surfactant lipids or low density lipoprotein particles by a mechanism that does not involve metal chelation or oxidative modification of the proteins. Low density lipoprotein oxidation is instantaneously arrested upon SP-A or SP-D addition, suggesting direct interference with free radical formation or propagation. The antioxidant activity of SP-A maps to the carboxyl-terminal domain of the protein, which, like SP-D, contains a C-type lectin carbohydrate recognition domain. These results indicate that SP-A and SP-D, which are ubiquitous among air breathing organisms, could contribute to the protection of the lung from oxidative stresses due to atmospheric or supplemental oxygen, air pollutants, and lung inflammation.

Segmental regulation of pulmonary vascular permeability by store-operated Ca2+ entry.

An intact endothelial cell barrier maintains normal gas exchange in the lung, and inflammatory conditions result in barrier disruption that produces life-threatening hypoxemia. Activation of store-operated Ca2+ (SOC) entry increases the capillary filtration coefficient (Kf,c) in the isolated rat lung; however, activation of SOC entry does not promote permeability in cultured rat pulmonary microvascular endothelial cells. Therefore, current studies tested whether activation of SOC entry increases macro- and/or microvascular permeability in the intact rat lung circulation. Activation of SOC entry by the administration of thapsigargin induced perivascular edema in pre- and postcapillary vessels, with apparent sparing of the microcirculation as evaluated by light microscopy. Scanning and transmission electron microscopy revealed that the leak was due to gaps in vessels >/= 100 micrometer, consistent with the idea that activation of SOC entry influences macrovascular but not microvascular endothelial cell shape. In contrast, ischemia and reperfusion induced microvascular endothelial cell disruption independent of Ca2+ entry, which similarly increased Kf,c. These data suggest that 1) activation of SOC entry is sufficient to promote macrovascular barrier disruption and 2) unique mechanisms regulate pulmonary micro- and macrovascular endothelial barrier functions.

Ca(2+)-inhibitable adenylyl cyclase and pulmonary microvascular permeability.

Intracellular mechanisms responsible for endothelial cell disruption are unknown, although either elevated cytosolic Ca2+ ([Ca2+]i) or decreased adenosine 3',5'-cyclic monophosphate (cAMP) promotes permeability. Recent identification that Ca(2+)-inhibitable adenylyl cyclase establishes an inverse relationship between [Ca2+]i and cAMP in macrovascular endothelial cells provided a possible mechanism of development of permeability. However, these data utilized an in vitro model; lacking was evidence supporting 1) expression of Ca(2+)-inhibitable adenylyl cyclase in pulmonary microvascular endothelium and 2) Ca2+ inhibition of adenylyl cyclase and cAMP content as a paradigm for inflammatory mediator-induced permeability in the intact circulation. We therefore addressed these issues in microvascular endothelial cells derived from rat lung and in an isolated perfused rat lung preparation. Results demonstrate expression of a Ca(2+)-inhibitable adenylyl cyclase in microvascular endothelial cells. Furthermore, data suggest that Ca2+ inhibition of adenylyl cyclase is necessary for development of microvascular permeability in the intact circulation. We conclude Ca2+ inhibition of cAMP represents a critical step in genesis of microvascular permeability in the intact pulmonary circulation.

Inhaled nitric oxide pretreatment but not posttreatment attenuates ischemia-reperfusion-induced pulmonary microvascular leak.

BACKGROUND: Ischemia-reperfusion (I/R) pulmonary edema probably reflects a leukocyte-dependent, oxidant-mediated mechanism. Nitric oxide (NO) attenuates leukocyte-endothelial cell interactions and I/R-induced microvascular leak. Cyclic adenosine monophosphate (cAMP) agonists reverse and prevent I/R-induced microvascular leak, but reversal by inhaled NO (INO) has not been tested. In addition, the role of soluble guanylyl cyclase (sGC) activation in the NO protection effect is unknown. METHODS: Rat lungs perfused with salt solution were grouped as either I/R, I/R with INO (10 or 50 ppm) on reperfusion, or time control. Capillary filtration coefficients (Kfc) were estimated 25 min before ischemia (baseline) and after 30 and 75 min of reperfusion. Perfusate cell counts and lung homogenate myeloperoxidase activity were determined in selected groups. Additional groups were treated with either INO (50 ppm) or isoproterenol (ISO-10 microM) after 30 min of reperfusion. Guanylyl cyclase was inhibited with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ-15 microM), and Kfc was estimated at baseline and after 30 min of reperfusion. RESULTS: (1) Inhaled NO attenuated I/R-induced increases in Kfc. (2) Cell counts were similar at baseline. After 75 min of reperfusion, lung neutrophil retention (myeloperoxidase activity) and decreased perfusate neutrophil counts were similar in all groups. (3) In contrast to ISO, INO did not reverse microvascular leak. (4) 8-bromoguanosine 3',5'-cyclic monophosphate (8-br-cGMP) prevented I/R-induced microvascular leak in ODQ-treated lungs, but INO was no longer effective. CONCLUSIONS: Inhaled NO attenuates I/R-induced pulmonary microvascular leak, which requires sGC activation and may involve a mechanism independent of inhibition of leukocyte-endothelial cell interactions. In addition, INO is ineffective in reversing I/R-induced microvascular leak.