Autoimmunity to the alpha 3 chain of type IV collagen in glomerulonephritis is triggered by 'autoantigen complementarity'.

'Autoantigen complementarity' is a theory proposing that the initiator of an autoimmune response is not necessarily the autoantigen or its molecular mimic, but may instead be a peptide that is 'antisense/complementary' to the autoantigen. We investigated whether such complementary proteins play a role in the immunopathogenesis of autoimmune glomerulonephritis. Experimental autoimmune glomerulonephritis, a model of anti-glomerular basement membrane (GBM) disease, can be induced in Wistar Kyoto (WKY) rats by immunization with the α3 chain of type IV collagen. In this study, WKY rats were immunized with a complementary α3 peptide (c-α3-Gly) comprised of amino acids that 'complement' the well characterized epitope on α3(IV)NC1, pCol(24-38). Within 8 weeks post-immunization, these animals developed cresentic glomerulonephritis, similar to pCol(24-38)-immunized rats, while animals immunized with scrambled peptide were normal. Anti-idiotypic antibodies to epitopes from c-α3-Gly-immunized animals were shown to be specific for α3 protein, binding in a region containing sense pCol(24-38) sequence. Interestingly, anti-complementary α3 antibodies were identified in sera from patients with anti-GBM disease, suggesting a role for 'autoantigen complementarity' in immunopathogenesis of the human disease. This work supports the idea that autoimmune glomerulonephritis can be initiated through an immune response against a peptide that is anti-sense or complementary to the autoantigen. The implications of this discovery may be far reaching, and other autoimmune diseases could be due to responses to these once unsuspected 'complementary' antigens.

A portable fiberoptic ratiometric fluorescence analyzer provides rapid point-of-care determination of glomerular filtration rate in large animals.

Measurement of the glomerular filtration rate (GFR) is the gold standard for precise assessment of kidney function. A rapid, point-of-care determination of the GFR may provide advantages in the clinical setting over currently available assays. Here we demonstrate a proof of principle for such an approach in a pig and dogs, two species that approximate the vascular access and GFR results expected in humans. In both animal models, a sub-millimeter optical fiber that delivered excitation light and collected fluorescent emissions was inserted into a peripheral vein (dog) or central venous access (pig) by means of commercial intravenous catheters. A mixture of fluorescent chimeras of a small freely filterable reporter and large non-filterable plasma volume marker were infused as a bolus, excited by light-emitting diodes, and the in vivo signals detected and quantified by photomultiplier tubes in both species in less than 60 min. Concurrent standardized 6-h iohexol plasma kidney clearances validated the accuracy of our results for both physiologic and a chronic kidney disease setting. Thus, our ratiometric technique allows for both measurement of plasma vascular volume and highly accurate real-time GFR determinations, enabling clinical decision making in real time.

Pilot evaluation of a vacuum-assisted biopsy instrument for percutaneous renal biopsy in dogs.

Kidney biopsies in dogs are commonly obtained using automated spring-loaded biopsy instruments. Interpretation of biopsies from dogs with glomerular disease requires examination of at least 5-10 glomeruli, with at least two biopsies usually required for full evaluation. The purpose of this study was to compare quality and interpretability of renal biopsies obtained from healthy dogs with a large-gauge, vacuum-assisted biopsy instrument versus two biopsies obtained with a spring-loaded biopsy needle. Twenty dogs were randomized into two groups, and percutaneous, ultrasound-guided renal biopsies were evaluated using standard criteria. There were no significant differences in the number of biopsies that contained renal tissue, cortex, or medulla. Biopsies obtained with either instrument contained an adequate number of glomeruli and an equivalent number of arterioles and severity of tissue compression. Differences included easier penetration of the renal capsule and collection of sufficient tissue for interpretation with only one instrument pass when using the vacuum-assisted device (vs two passes required with the spring-loaded instrument). Before use in client-owned dogs, future studies should evaluate whether these differences are clinically relevant advantages in the diagnostic evaluation of dogs with kidney disease, and determine the prevalence and severity of complications when using this larger gauge device.

Effect of heparin administration on urine protein excretion during the developmental stage of experimentally induced laminitis in horses.

OBJECTIVE: To investigate the effects of heparin administration on urine protein excretion during the developmental stages of experimentally induced laminitis in horses. ANIMALS: 13 horses. Procedures-Horses received unfractionated heparin (80 U/kg, SC, q 8 h; n=7) or no treatment (control group; 6) beginning 3 days prior to induction of laminitis. All horses were given 3 oligofructose loading doses (1 g/kg each) at 24-hour intervals and a laminitis induction dose (10 g of oligofructose/kg) 24 hours following the final loading dose (designated as 0 hours) via nasogastric tube. Serum glucose and insulin concentrations were measured before administration of the first loading dose (baseline) and at 0 and 24 hours; urine protein-to-creatinine (UP:C) ratio was determined at 0 hours and every 4 hours thereafter. Lameness was evaluated every 6 hours, and horses were euthanized when Obel grade 2 lameness was observed. RESULTS: Mean±SD time until euthanasia did not differ significantly between the heparin-treated (28.9±6.5 hours) and control (29.0±6.9 hours) horses. The UP:C ratio was significantly increased from baseline at 20 to 28 hours after induction of laminitis (ie, 4±4 hours before lameness was evident) in control horses but did not change significantly from baseline in heparin-treated horses. Serum glucose or insulin concentration did not change significantly from baseline in either group. CONCLUSIONS AND CLINICAL RELEVANCE: Urine protein excretion increased during the developmental stages of carbohydrate-induced laminitis in horses; administration of heparin prevented that increase, but did not delay onset or decrease severity of lameness.

Diffuse cylindrical bronchiectasis due to eosinophilic bronchopneumopathy in a dog.

A miniature pinscher-cross was evaluated for chronic coughing. Computed tomography and bronchoscopy revealed severe, diffuse, cylindrical bronchiectasis secondary to eosinophilic bronchopneumopathy. Computed tomography is the gold standard for diagnosis of bronchiectasis in humans, and should be further investigated in dogs as a means of characterizing severity and pattern of disease.

Partial characterization of feline myeloperoxidase and investigation of its potential role as an autoantigen in hyperthyroid cats.

OBJECTIVE: To partially characterize the cDNA, amino acid sequence, and tertiary structure of feline myeloperoxidase, describe its cellular location in mature granulocytes, and determine whether hyperthyroid cats have anti-myeloperoxidase antibody. SAMPLE POPULATION: Bone marrow RNA and whole blood from cats of various sources and feline serum samples submitted for measurement of total thyroxine concentration from September 2006 to July 2007. PROCEDURES: Feline myeloperoxidase cDNA was amplified from bone marrow RNA; presumptive splice sites were determined by comparison with human sequences. Intracellular localization of myeloperoxidase in granulocytes was determined by use of immunofluorescence and electron microscopy, and molecular weight and partial tertiary structure were determined by use of immunoblotting of granulocyte lysates. Anti-human myeloperoxidase (hMPO) antibody was detected via ELISA. RESULTS: A 2,493-bp sequence encompassing the 2,160-bp cDNA with presumably the same number and size of exons as hMPO was generated. Translation predicted 85% homology with hMPO. Feline myeloperoxidase was localized to neutrophil primary granules, and immunoblotting revealed heavy and light bands with molecular weights similar to those of hMPO. The prevalence of anti-hMPO antibody did not differ between nonhyperthyroid and hyperthyroid cats or among hyperthyroid cats subclassified by treatment modality. CONCLUSIONS AND CLINICAL RELEVANCE: Moderate homology existed between feline myeloperoxidase and hMPO cDNA and protein. Although findings suggested a similar tertiary structure and function for the 2 proteins, they also suggested that inability to detect a high prevalence of anti-hMPO antibody in hyperthyroid cats may be attributable to antigenic differences between the human and feline proteins rather than a lack of autoantibody.

Determination of and correlation between urine protein excretion and urine protein-to-creatinine ratio values during a 24-hour period in healthy horses and ponies.

OBJECTIVE-To determine whether urine protein-to-creatinine (UP:C) ratio assessment provides an estimate of urine protein excretion (UPE) over a 24-hour period in horses and ponies, establish a preliminary UP:C ratio reference range, and determine UP:C ratio variation over time in healthy equids. ANIMALS-11 female horses and 6 female ponies. PROCEDURES-Urine was collected from all equids at 4-hour intervals for 24 hours. Total 24-hour UPE (mg of protein/kg of body weight) and UP:C ratio were determined; these variables were also assessed in aliquots of urine collected at 4-hour intervals. On 2 additional days, urine samples were also obtained from 6 horses (1 sample/horse/d) to determine day-to-day variation in UP:C ratio. Correlation between 4-hour or 24-hour UPE and UP:C ratio values was assessed. Reference ranges for 24-hour UPE, 24-hour UP:C ratio, and 4-hour UP:C ratios were calculated as central 95th percentiles of observed values. RESULTS-Mean 24-hour UPE (4.28 +/- 2.99 mg/kg) and 24-hour UP:C ratio (0.0 to 0.37) had excellent correlation (R = 0.826; P < 0.001) in both horses and ponies; analysis of 4-hour data also revealed good correlation (R = 0.782; P < 0.001) with these variables. Calculated UPE and UP:C ratio reference ranges were similar to established ranges in other species. Day-to-day variability in UP:C ratio was minimal, and all results were within the reference range calculated by use of the 24-hour urine samples. CONCLUSIONS AND CLINICAL RELEVANCE-Assessment of the UP:C ratio appears to be a reliable method for estimating 24-hour UPE in horses and ponies.

Mucinous gastric carcinoma with abdominal carcinomatosis and hypergastrinemia in a dog.

A 12-year-old, spayed female Australian cattle dog was evaluated for a 5-month history of progressive vomiting. Abdominal radiographs and ultrasound revealed significant gastric wall thickening and a peripancreatic mass, and serum gastrin concentration was increased (127 pg/mL, reference range 10 to 40 pg/mL). Surgical exploration of the abdomen revealed a thickened, firm, and irregular gastric fundus, pylorus, and antrum; nodules were present throughout the spleen and mesentery adjacent to the left limb of the pancreas. Mucinous gastric carcinoma with carcinomatosis was diagnosed by histopathological examination of surgically excised tissues. Unfortunately, severe postoperative complications resulted in euthanasia 10 days after surgery, and a necropsy was not performed. This case is significant, because it is the first report of a mucinous gastric carcinoma associated with hypergastrinemia in a dog.

ANCA patients have T cells responsive to complementary PR-3 antigen.

Some patients with proteinase 3 specific anti-neutrophil cytoplasmic autoantibodies (PR3-ANCA) also have antibodies that react to complementary-PR3 (cPR3), a protein encoded by the antisense RNA of the PR3 gene. To study whether patients with anti-cPR3 antibodies have cPR3-responsive memory T cells we selected conditions that allowed cultivation of memory cells but not naïve cells. About half of the patients were found to have CD4+TH1 memory cells responsive to the cPR3(138-169)-peptide; while only a third of the patients had HI-PR3 protein responsive T cells. A significant number of T cells from patients responded to cPR3(138-169) peptide and to HI-PR3 protein by proliferation and/or secretion of IFN-gamma, compared to healthy controls while there was no response to scrambled peptide. Cells responsive to cPR3(138-169)-peptide were not detected in MPO-ANCA patients suggesting that this response is specific. The HLADRB1(*) 15 allele was significantly overrepresented in our patient group and is predicted to bind cPR3(138-169) peptide with high affinity. Regression analysis showed a significant likelihood that anti-cPR3 antibodies and cPR3-specific T cells coexist in individuals, consistent with an immunological history of encounter with a PR3-complementary protein. We suggest that the presence of cells reacting to potential complementary protein pairs might provide an alternative mechanism for auto-immune diseases.

Autoantigen complementarity: a new theory implicating complementary proteins as initiators of autoimmune disease.

Autoimmune diseases affect approximately 1 in 21 persons in the United States. Treatment often requires long-term cytotoxic therapy. How and why these deleterious diseases occur is unclear. A serendipitous finding in our laboratory using serum from patients with autoimmune vasculitis led us to develop the theory of autoantigen complementarity, a novel concept that may elucidate the etiological and pathogenetic mechanisms underlying autoimmune disease in general. The theory proposes that the inciting immunogen that elicits a cascade of immunological events is not the self-antigen (the autoantigen) or its mimic but rather a protein that is complementary in surface structure to the autoantigen; that is, a protein homologous or identical to the amino acid sequence of translated antisense RNA from the noncoding strand of the autoantigen gene. The cascade begins when this complementary protein initiates the production of antibodies that in turn elicit an anti-antibody or anti-idiotypic response. These anti-idiotypic antibodies can now react with the autoantigen. Strikingly, homology search of complementary proteins yields microbial and fungal proteins, thus indicating that invading micro-organisms can deliver the inciting immunogen. Curiously, approximately 50% of our patients transcribe the complementary protein's antisense RNA. If it transpires that these aberrant RNAs are translated, the complementary protein would be produced by the individual. Here we review published research investigating complementary proteins, anti-idiotypic immune responses, and antisense transcripts, all of which support complementary proteins as initiators of autoimmune disease. In addition, we provide possible microbial and/or fungal organisms that may incite some of the most studied autoimmune diseases. Lastly, we propose mechanisms by which cell-mediated autoimmunity can be triggered by autoantigen complementarity. Based on our data and the contributions of the researchers described in this review, identification of proteins complementary to autoantigens is likely to be informative in most autoimmune diseases. This vein of study is in the early phases; however, we expect "autoantigen complementarity" is an underlying mechanism in many autoimmune diseases.

Prevalence of feline infectious peritonitis in specific cat breeds.

Although known that purebreed cats are more likely to develop feline infectious peritonitis (FIP), previous studies have not examined the prevalence of disease in individual breeds. All cats diagnosed with FIP at a veterinary teaching hospital over a 16-year period were identified. Breed, sex and reproductive status of affected cats were compared to the general cat population and to mixed breed cats evaluated during the same period. As with previous studies sexually intact cats and purebreed cats were significantly more likely to be diagnosed with FIP; males and young cats also had a higher prevalence of disease. Abyssinians, Bengals, Birmans, Himalayans, Ragdolls and Rexes had a significantly higher risk, whereas Burmese, Exotic Shorthairs, Manxes, Persians, Russian Blues and Siamese cats were not at increased risk for development of FIP. Although additional factors doubtlessly influence the relative prevalence of FIP, this study provides additional guidance when prioritizing differentials in ill purebreed cats.

Urinary tract manifestations of protothecosis in dogs.

Records of 13 dogs with systemic infection with Prototheca sp. from 3 veterinary teaching hospitals were reviewed. Acute renal failure secondary to disseminated infection with Prototheca zopfii was diagnosed in 2 dogs. In 1 dog, acute renal failure developed during administration of immunosuppressive drugs for treatment of anterior uveitis. During diagnostic evaluation of this dog, Prototheca sp. organisms were noted in urine sediment and renal biopsy specimens. In the 2nd dog, acute renal failure was diagnosed after treatment for bacterial cystitis. After diagnosis of protothecosis, organisms were successfully isolated by aerobic urine culture. Both dogs with acute renal failure did not respond to conventional medical therapy. In total, Prototheca sp. was noted in urine sediment in 4 of 8 dogs and successfully cultured from urine in 5 of 7 dogs. Four of 5 dogs had organisms noted in the kidneys on histopathologic examination. In all dogs, the species identified was P zopfii. Sensitivity testing of 3 isolates revealed wide differences in in vitro drug resistance. Examination and culture of urine is recommended as a practical method for diagnosis of systemic infection with Prototheca sp.

Clinical Approach to Advanced Renal Function Testing in Dogs and Cats.

Serum creatinine concentration is insensitive for detecting kidney injury and does not assist in differentiation between glomerular versus tubular damage. Advanced renal function tests, including glomerular filtration rate testing, determining fractional excretion of electrolytes, and assay of urine biomarkers, may allow earlier detection of reduced renal function mass, differentiation of renal from non-renal causes of azotemia, and assist with localization of damage. This article reviews the principles, indications, and limitations of these tests and describes their use in sample clinical scenarios.

Isolation and identification of Mycobacterium kansasii from pleural fluid of a dog with persistent pleural effusion.

A 3-year-old spayed female Whippet was examined for cough and respiratory distress. Lung lobe torsion with pleural effusion was diagnosed, and lung lobectomy was performed. Pleural effusion recurred during the following 27 months; conventional bacteriologic cultures of pleural effusion did not result in bacterial growth. A second lung lobectomy, pleuroperitoneal shunt placement. and pericardectomy were subsequently performed. Mycobacterium kansasii was eventually isolated from pleural fluid and identified by polymerase chain reaction amplification and DNA sequencing. The dog was euthanatized before therapeutic response could be evaluated. To our knowledge, this is the first report of M. kansasii infection in a dog. Additionally, this is the first report of mycobacterial isolation from pleural fluid, and one of few reports of antemortem mycobacterial isolation from a body fluid, as opposed to identification in specimens during histologic examination. Routine bacteriologic culture methods are insufficient to isolate mycobacterial agents, and special methods are indicated in dogs with persistent pleural effusion.

Hypercalcemia and high parathyroid hormone-related protein concentration associated with malignant melanoma in a dog.

A 12-year-old Cocker Spaniel with an oral malignant melanoma was evaluated for progressive lethargy and anorexia. No metastases were identified during antemortem evaluation, but severe hypercalcemia was evident. Antemortem diagnostic testing failed to identify a cause for the hypercalcemia. No neoplasms other than the melanoma were identified on postmortem examination. Serum parathyroid hormone-related protein concentration was markedly high, and the melanoma had moderate to marked immunostaining for this protein. Paraneoplastic syndromes are rare in dogs with malignant melanoma.

Detection of canine microalbuminuria using semiquantitative test strips designed for use with human urine.

BACKGROUND: Commercial testing for microalbuminuria in human urine is often performed with point-of-care semiquantitative test strips followed by quantitative testing when indicated. An ELISA that quantifies canine urine albumin concentration has been developed, but semiquantitative test strips for use in the dog are not available. OBJECTIVE: The purpose of this study was to prospectively determine the concordance of canine urine albumin concentrations measured by a commercial human test strip and by ELISA. METHODS: Urine samples were obtained from 67 dogs evaluated for a variety of clinical conditions. Dipstick urinalyses were performed on all samples; clinician discretion determined method of urine collection and performance of urine sediment examination and/or urine culture. Urine albumin concentration was determined using test strips (Clinitek Microalbumin, Bayer Corporation, Elkhart, Ind, USA), and results were compared with those obtained by ELISA. RESULTS: The Clinitek strips correctly determined albumin concentration in 42 of 67 (63%) urine samples tested. Concordance was lowest (48%) for dogs with microalbuminuria (10-300 microg/mL by ELISA). Clinitek strip sensitivity and specificity for correct identification of microalbuminuria were 48% and 75%, respectively. Concordance was lower in dogs with urinary tract infection or hematuria and in samples collected by catheterization. Sensitivity and specificity for correct identification of microalbuminuria after exclusion of dogs with urinary tract infection or hematuria were 59% and 83%, respectively. CONCLUSIONS: These results suggest that the Clinitek strips lack sufficient concordance with results obtained by ELISA to be a reliable screening for test microalbuminuria in the dog. A reliable semiquantitative point-of-care test for canine urine albumin concentrations below those detected by standard urine dipsticks is still needed.

Effects of urinary tract inflammation and sample blood contamination on urine albumin and total protein concentrations in canine urine samples.

BACKGROUND: Urinary tract inflammation and hemorrhage are believed to be common causes of proteinuria in dogs based on results of studies that measured total urine protein concentration. A method to quantify urine albumin (UAlb) concentration in dogs recently has become available; however, the effect of inflammation on albuminuria is unknown. OBJECTIVES: The goals of this study were to determine the effects of urinary tract inflammation, as indicated by pyuria and sample blood contamination, on UAlb concentration and on urine protein:creatinine (UPC) ratio in dogs. METHODS: Urine samples were obtained from dogs with pyuria that were presented to a veterinary teaching hospital or were part of a laboratory colony. To mimic the effects of hematuria, canine whole blood was added to a microscopically normal canine urine sample that had baseline albumin and total protein concentrations below the limits of detection. UAlb concentration was measured using a canine albumin-specific competitive ELISA. UPC ratio was determined using routine methods. RESULTS: Of 70 samples with pyuria, 67% had negligible UAlb concentrations and 81% had normal UPC ratios. UAlb concentration but not UPC ratio was significantly higher (P < 0.05) in samples with concurrent hematuria or bacteriuria. When whole blood was added to normal urine, UAlb concentration did not exceed 1 mg/dL until the sample became visibly pink; the UPC did not exceed 0.4 at any dilution. CONCLUSIONS: Many dogs with pyuria do not have albuminuria or proteinuria; however, albuminuria may be more likely in dogs with pyuria and concurrent hematuria or bacteriuria. Hematuria may not cause an increase in UAlb concentration until it becomes macroscopic and even then may not increase the UPC ratio.

Endoscopic evaluation of the esophagus and stomach in three loggerhead sea turtles (Caretta caretta) and a Malaysian giant turtle (Orlitia borneensis).

Three loggerhead sea turtles (Caretta caretta) and a Malaysian giant turtle (Orlitia borneensis) were presented with suspected or confirmed esophageal foreign bodies. Esophagoscopy was performed on all turtles, and gastroscopy was performed on three turtles. In all cases, endoscopy was easy to perform, and allowed visualization of most upper gastrointestinal features. The papillated esophagus was easy to navigate, but mucosal papillae in the loggerhead sea turtles prevented examination of the underlying mucosa. The stomach was easily entered and examined in both species, but the working endoscope length (100 cm) prevented inspection of the pyloric antrum and the duodenum in all turtles. The turtles in this report may serve as references for future endoscopic examinations of these species.

Clinical approach to advanced renal function testing in dogs and cats.

Serum creatinine concentration is insensitive for detecting kidney injury and does not assist in differentiation between glomerular versus tubular damage. Advanced renal function tests, including glomerular filtration rate testing, determining fractional excretion of electrolytes, and assay of urine biomarkers, may allow earlier detection of reduced renal function mass, differentiation of renal from non-renal causes of azotemia, and assist with localization of damage. This article reviews the principles, indications, and limitations of these tests and describes their use in sample clinical scenarios.