Biological variation of 16 biochemical analytes estimated from a large clinicopathologic database of dogs and cats.

BACKGROUND: Biochemical measurements are commonly evaluated using population-based reference intervals; however, there is a growing trend toward reassessing results with within-subject variation (CVI). OBJECTIVES: We aimed to estimate the CVI of 16 biochemical analytes using a large database of dogs and cats, which refers to the results of routine health checkups. METHODS: Pairs of sequential results for 16 analytes were extracted from a database of adult patients. The second result was divided by the first result to produce the ratio of sequential results (rr), and the frequency distribution of rr was plotted. From the plots, the coefficient of variation (CVrr) was calculated. Analytical variation (CVA) was calculated using quality control data, and CVI was estimated as follows: CV I = CV rr / 2 1 / 2 2 - CV A 2 1 / 2 . Estimated CVI was compared with previously reported CVI using the Bland-Altman plot analysis. RESULTS: From the database, 9078 data points from 3610 dogs and 3743 data points from 1473 cats were extracted, with 5468 data pairs for dogs and 2270 for cats. Sampling intervals ranged from 10 to 1970 days (median 366) for dogs and 23 to 1862 days (median 365) for cats. Bland-Altman analysis showed most CVI plots fell within the limits of agreement; however, positive fixed biases were observed in both dogs and cats. CONCLUSIONS: Our study introduces a novel approach of estimating CVI using routine health checkup data in dogs and cats. Despite biases, our method holds promise for clinical application in assessing the significance of measurement result differences.

Large granular lymphocyte lymphoma in the skin and urinary bladder of a dog.

A 10-year-old female Cavalier King Charles Spaniel presented with hematuria, pollakiuria and skin rash. Based on the histopathological and cytological examination of the skin and bladder mucosa, the dog was diagnosed with large granular lymphocytic (LGL) lymphoma of the bladder and skin. The dog responded well to the initial chemotherapy with nimustine for 3 months. Since recurrence of skin erosion and bladder wall thickening were observed, the dog was subsequently administered chemotherapy with other anticancer drugs, including chlorambucil, vincristine, doxorubicin, L-asparaginase, cytosine arabinoside, and cyclophosphamide. The dog survived for 11 months and died due to tumor-related disseminated intravascular coagulation. This is the first report of a canine case of LGL lymphoma in the skin and bladder.

Distribution of regulatory T cells in inflammatory colorectal polyps of miniature dachshunds.

Inflammatory colorectal polyp (ICRP) is an emerging disease in Miniature Dachshunds (MDs). Animals with this disease exhibit multiple polyps with severe neutrophil infiltration that respond to immunosuppressive therapy. Macrophages in polypoid lesions have been described to play an important role in neutrophil infiltration in the lesion by producing IL-8. In contrast, IL-10, an anti-inflammatory cytokine, was also reported to be upregulated in polypoid lesions, but its significance in the pathogenesis of ICRP has not been clarified. Regulatory T cells (Tregs) are the main source of IL-10 production and contribute to the maintenance of intestinal homeostasis. Therefore, the objective of this research was to compare the distribution of Tregs in polypoid lesions of ICRPs and the association between the distribution and expression of pro- or anti-inflammatory cytokines. Tissue biopsy specimens of polypoid lesions were collected from 28 MDs with ICRP. Those of macroscopically non-polypoid colonic mucosa from 24 MDs with ICRPs and 21 control dogs were further included as controls. Real-time quantitative polymerase chain reaction was used to quantify gene expression of IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-17, IL-22, IFN-γ, TNF-α, TGF-ß, and forkhead box protein P3 (Foxp3) in each tissue sample. The numbers of Foxp3-positive cells (Tregs) and ionized calcium binding adapter molecule 1 (Iba-1)-positive cells (macrophages) were determined by immunohistochemistry. The gene expression of IL-1ß, IL-6, IL-8, TNF-α, IFN-γ, IL-17, IL-10, TGF-ß, and Foxp3 was significantly upregulated in polypoid lesions relative to control levels. The numbers of Foxp3-positive Tregs and Iba-1-positive macrophages were significantly increased in polypoid lesions compared to those in the non-polypoid colonic mucosa of MDs with ICRPs and control dogs. The upregulation of IL-10 was moderately correlated with the distribution of Tregs in polypoid lesions from MDs with ICRPs. In addition, the relative upregulation of IL-1ß, IL-6, and IL-8 in polypoid lesions, compared to expression in non-polypoid colonic mucosa of MDs with ICRPs, was significantly greater than that of IL-10. These results indicate that increases in Treg numbers and anti-inflammatory cytokines in polypoid lesions comprise reactive changes in response to the inflammation, which warrants further investigation.

Detection of anti-neutrophil cytoplasmic antibodies in dogs with immune-mediated inflammatory diseases.

Anti-neutrophil cytoplasmic antibody (ANCA) is a type of autoantibody against neutrophil cytoplasm. In veterinary medicine, few studies have reported the detection of ANCA in dogs, and most of these studies were performed in dogs with inflammatory bowel disease (IBD). The aim of this study was to evaluate whether ANCA is detected in dogs with immune-mediated inflammatory diseases (IMIDs) other than IBD. Serum samples were collected before treatment initiation from 40 client-owned dogs with various diseases and 22 healthy beagle dogs; the dogs were classified into two groups: IMID group (n = 16) and control group (n = 46). ANCA was detected using the indirect fluorescent antibody test. Of the 16 dogs in the IMID group, 13 (81.3%) tested positive for ANCA. In contrast, of the 46 dogs in the control group, 13 (28.03%) tested positive for ANCA. Moreover, a significant association between ANCA positivity and IMIDs was identified in the IMID group than in the control group (P = 0.0003). In the control group, however, dogs with bacterial infection showed a relatively high rate of ANCA positivity. Therefore, ANCA positivity was observed in dogs with both IMIDs and bacterial infection. This suggested that ANCA positivity is associated with diseases accompanied by neutrophil activation and infiltration.

Variation of amino acid sequences of serum amyloid a (SAA) and immunohistochemical analysis of amyloid a (AA) in Japanese domestic cats.

Amyloid A (AA) amyloidosis, a fatal systemic amyloid disease, occurs secondary to chronic inflammatory conditions in humans. Although persistently elevated serum amyloid A (SAA) levels are required for its pathogenesis, not all individuals with chronic inflammation necessarily develop AA amyloidosis. Furthermore, many diseases in cats are associated with the elevated production of SAA, whereas only a small number actually develop AA amyloidosis. We hypothesized that a genetic mutation in the SAA gene may strongly contribute to the pathogenesis of feline AA amyloidosis. In the present study, genomic DNA from four Japanese domestic cats (JDCs) with AA amyloidosis and from five without amyloidosis was analyzed using polymerase chain reaction (PCR) amplification and direct sequencing. We identified the novel variation combination of 45R-51A in the deduced amino acid sequences of four JDCs with amyloidosis and five without. However, there was no relationship between amino acid variations and the distribution of AA amyloid deposits, indicating that differences in SAA sequences do not contribute to the pathogenesis of AA amyloidosis. Immunohistochemical analysis using antisera against the three different parts of the feline SAA protein-i.e., the N-terminal, central, and C-terminal regions-revealed that feline AA contained the C-terminus, unlike human AA. These results indicate that the cleavage and degradation of the C-terminus are not essential for amyloid fibril formation in JDCs.

Effects of serum amyloid A on matrix metalloproteinase-9 production in feline lymphoma-derived cell lines.

Serum amyloid A (SAA) concentration and plasma matrix metalloproteinase-9 (MMP-9) levels are increased in cats with lymphoma. In the present study, the association between SAA and MMP-9 production was evaluated using recombinant feline SAA (rfSAA) and three feline lymphoma-derived cell lines: 3201, MS4, and MCC. MMP-9 mRNA expression was significantly increased by rfSAA stimulation only in MCC cells. Secreted MMP-9 protein in culture media was confirmed by gelatin zymography, with clear bands of MMP-9 detected in MCC cells following rfSAA stimulation. A significant increase in semi-quantified MMP-9 levels was observed with 5 and 25µg/ml of rfSAA stimulation. The infiltrative activities of feline lymphoma cells, assessed by the matrigel transwell assay, showed that rfSAA stimulated cell infiltration in MCC cells, in addition to MMP-9 expression. Although the response to rfSAA stimulation varied between cell lines, the results showed that rfSAA can stimulate MMP-9 production and infiltration of feline lymphoma-derived cells. The findings of this study have identified a novel role for SAA in the progression of some forms of feline lymphoma.

Assessment of severity and changes in C-reactive protein concentration and various biomarkers in dogs with pancreatitis.

Canine pancreatitis is a relatively common disorder, and its mortality rate remains high. However, prognostic factors for pancreatitis based on evidence are limited. Moreover, the relationship between changes in C-reactive protein (CRP) concentration-an important prognostic factor for human patients with acute pancreatitis-and the prognosis of dogs with pancreatitis has not been widely studied. Therefore, we examined prognostic factors for canine pancreatitis during the first medical examination and evaluated the usefulness of serial CRP measurements during hospitalization. Sixty-five dogs met the inclusion criteria, including 22 that were hospitalized and treated. In Study 1, a multivariate analysis revealed that three factors- decreased platelet count and a marked (greater than 1,000 µg/l) elevation of specific canine pancreatic lipase (Spec cPL) concentration at the first medical examination, as well as elevated blood urea nitrogen (BUN) and/or creatinine (CRE) level-were significantly different between the survivors and nonsurvivors. Moreover, CRP concentrations on the third and fourth days were significantly different between the two groups in Study 2. An evaluation of the decreased platelet count, remarkable elevation of Spec cPL concentration at the first medical examination, elevation of BUN and/or CRE as well as serial CRP concentration measurements may be useful for predicting the prognosis of canine pancreatitis.

Measurement of feline lipase activity using a dry-chemistry assay with a triolein substrate and comparison with pancreas-specific lipase (Spec fPL(TM)).

Pancreatic lipase immunoreactivity (Spec fPL) is currently considered to be the most accurate blood test for the diagnosis of feline pancreatitis. In this study, we measured lipase activity in cats using a newer catalytic lipase assay of dry-chemistry system (FDC-v-LIP) to determine the reference range and compared the results with those for Spec fPL. Based on the results of healthy cats, the reference range of FDC-v-LIP was determined to be less than 30 U/l. FDC-v-lip did not show a strong correlation with Spec fPL in cats with various diseases, which resulted in the low sensitivity and positive predictive value. However, the relatively high (>90%) specificity and negative predictive value indicated that FDC-v-LIP could be a useful patient-side screening test for the exclusion of feline pancreatitis.

Serum amyloid A promotes invasion of feline mammary carcinoma cells.

The serum amyloid A (SAA) concentration is higher in mammary tumors with metastases in both humans and animals. In the present study, the direct effects of recombinant feline SAA (rfSAA) protein on invasiveness of feline mammary carcinoma cells were evaluated. As an indicator of invasiveness, matrix metalloproteinase-9 (MMP-9) expression was investigated in 4 feline mammary carcinoma cell lines of different origins. In 3 of 4 cell lines, MMP-9 expression was significantly increased by rfSAA stimulation. The invasive capacities of feline mammary carcinoma cells were also stimulated by rfSAA. The findings of this study have identified a novel role for SAA in mammary tumorigenesis and suggest that therapeutic strategies targeting SAA may provide new alternatives in treating tumor invasion and metastasis.

Differential expression of CD45 isoforms in canine leukocytes.

CD45 is one of the most abundant molecules expressed on the white blood cell surface in various mammals. In this study, we investigated the differential expression of CD45 isoforms in normal canine white blood cells. It has been shown that all canine nucleated blood cells express CD45. We characterized two major isoforms of canine CD45 derived from alternative splicing: a higher molecular weight isoform, CD45RA, and a lower molecular weight isoform, CD45RO. The nucleotide sequences of the two isoforms were identical, except for the region corresponding to a part in the extracellular domain. Flow cytometry analysis using an antibody that recognizes CD45RA, but not CD45RO, revealed that granulocytes did not express CD45RA, and monocytes express low levels of CD45RA. We further analyzed the expression levels of CD45RA in each lymphocyte subpopulation and found that the expression of CD45RA on CD21+ B cells was uniform. On the other hand, expression of CD45RA on CD3+ T cells was variable. Upon stimulation of lymphocytes with Con A, the CD45RA+ fraction increased, indicating that not only the phenotypes but also the activation status influences the isoform expression pattern of CD45. Our finding provides a basic knowledge of the expression of canine CD45, which could be a tool to study lymphocytes with various phenotypes, developmental stages, and activation status.

Feline serum amyloid A protein as an endogenous Toll-like receptor 4 agonist.

Serum amyloid A (SAA) is one of the major acute phase proteins and a biomarker of infection or inflammation in humans and cats. In humans, cytokine-like functions of SAA protein have been determined, and SAA is considered to be an important factor in immune responses. However, there are no reports about the functions of SAA protein in cats. In the present study, the functions of feline SAA protein on peripheral monocytes were investigated by using TNF-α production as an indicator. In feline peripheral blood monocytes, SAA protein stimulated the transcription of TNF-α within 2h and induced TNF-α secretion in time- and dose-dependent manners. The production of TNF-α by SAA stimulation in feline monocytes was found to be mediated by the activation of nuclear factor-kappa B (NF-κB). Moreover, SAA-stimulated TNF-α production was prevented by a Toll-like receptor 4 (TLR4) antagonist. On the basis of these results, feline SAA was demonstrated to be an endogenous agonist of TLR4 for the stimulation of TNF-α production and secretion by peripheral monocytes. These results suggest that feline SAA can play an important role in the regulation of inflammation and immune responses as it does in humans.

Serum amyloid A as a prognostic marker in cats with various diseases.

Serum amyloid A (SAA) is reported not only as a marker for the presence of inflammation but also as a prognostic indicator in human beings. In cats, however, there is no report on the association between SAA concentration and prognosis. The objective of the current study was to evaluate SAA concentration as a prognostic marker in diseased cats. A total of 175 cats with neoplastic diseases, inflammatory diseases, and other diseases were retrospectively recruited, and the medical records of these cats, including follow-up data on mortality, were reviewed. Cats were divided into 2 groups according to SAA concentration, and differences in survival between each group were assessed. Median survival time of cats in the elevated SAA (>0.82 mg/l) group was significantly shorter than that in the nonelevated SAA (≤0.82 mg/l) group (P < 0.001). Furthermore, by multivariate analysis, SAA concentration was shown as a significant and independent prognostic marker in cats with various diseases (P = 0.015). Serum amyloid A concentration in diseased cats is a useful predictive indicator of prognosis regardless of diagnosis.

Serum amyloid A uptake by feline peripheral macrophages.

Serum amyloid A (SAA) is one of the major acute phase proteins in cats and humans. SAA concentrations increase in response to the inflammatory status and secondary amyloid A amyloidosis has been documented in cats. In order to control the SAA concentration, it is important to clarify how the SAA protein is metabolized. Although the details of SAA metabolism in the body remain unknown, human and murine research indicates that macrophages play a key role in SAA uptake. The objectives of this study were to demonstrate SAA uptake by feline macrophages and to evaluate the effects of lipopolysaccharide (LPS) and dexamethasone (Dex) on SAA uptake. The concentration of recombinant feline SAA added to a feline macrophage culture was decreased in a time-dependent manner and was significantly reduced after a 24-h incubation, as demonstrated by enzyme linked immunosorbent assay (ELISA). SAA uptake into feline peripheral macrophages was demonstrated by immunofluorescence microscopy. Pretreatment to macrophages with LPS did not affect this decrease in the SAA concentration, but this was significantly blocked by Dex pretreatment. In conclusion, SAA was incorporated by feline macrophages and pretreatment with Dex inhibited SAA uptake by macrophages in this study. Further investigation is needed to determine the molecules that influence SAA uptake by macrophages and the effect of clinical glucocorticoid usage on the SAA concentration in cats.

Time-course monitoring of serum amyloid A in a cat with pancreatitis.

Time-course changes in the concentration of serum amyloid A (SAA), a major acute phase protein, were measured in a cat with pancreatitis over an 831-day period and compared with changes in WBC count and feline trypsin-like immunoreactivity (fTLI). SAA concentration was increased at the onset of the disease and gradually decreased over 5 days of treatment with an improvement in the clinical condition. In contrast, fTLI concentration and WBC count were not increased at the onset of the disease but increased gradually during the 5 days of treatment. Long-term monitoring from days 68 to 831 revealed a good correlation between SAA concentration and the reoccurrence of clinical signs in the cat; however, WBC count did not increase even with the exacerbation of disease. These findings suggest that the SAA concentration may be a useful marker for evaluating response to treatment and disease exacerbation in feline pancreatitis.

Verification of measurement of the feline serum amyloid A (SAA) concentration by human SAA turbidimetric immunoassay and its clinical application.

Serum amyloid A (SAA) is one of the major acute phase proteins in cats that has potential to be used as an inflammatory marker. A previous study showed that the human SAA turbidimetric immunoassay (hSAA-TIA) could be used to measure the SAA concentration in cats. The objectives of the present study were to assess use of hSAA-TIA for determining the feline SAA concentration and to evaluate its clinical application. Recombinant feline SAA protein (rfSAA) was expressed in Escherichia coli and purified for SDS-PAGE and immunoblot analysis with anti-human SAA antibodies. The concentration of rfSAA was determined by ELISA and hSAA-TIA. Plasma SAA concentrations were measured in healthy and diseased cats by hSAA-TIA. The time-courses changes in the SAA and alpha1-acid glycoprotein (AGP) concentrations in the cats after ovariohysterectomy were investigated. In SDS-PAGE, rfSAA was detected as a clear band that reacted with anti-human SAA antibodies. There was significant correlation between the SAA concentration measured by ELISA and hSAA-TIA. The SAA concentration of the diseased cats (n=263) was significantly increased (P<0.01; 0.0-88.9 mg/l, mean: 7.52 mg/l) compared with that in the healthy cats (n=26; 0.0-0.9 mg/l, mean: 0.14 mg/l). No correlation was observed between SAA and WBC in the diseased cats. The SAA concentration changed more rapidly and remarkably than the AGP concentration after ovariohysterectomy. The present study revealed that hSAA-TIA is useful for determination of the feline SAA concentration. Measurement of the SAA concentration, in addition to the WBC count, would be clinically valuable as a routine test to detect inflammation.