Histologic evidence for a humoral immune response in synovitis associated with cranial cruciate ligament disease in dogs.

OBJECTIVE: To investigate histopathological features of synovium from dogs with cranial cruciate ligament disease (CCLD) to seek mechanisms of osteoarthritis (OA) associated with CCLD. STUDY DESIGN: Retrospective, single-institution case series. ANIMALS: Thirty client-owned dogs. METHODS: Synovial biopsies (n = 30) obtained from stifles with CCLD were assessed by using two synovitis histopathology grading systems (Krenn and Hospital for Special Surgery [HSS]). The Krenn synovitis score was interpreted as "no synovitis," "low-grade," or "high-grade," while inflammatory subtype (low, mixed, or high) was determined by a computational algorithm within the HSS system. Comparison of synovitis scores was based on degree of CCL rupture and presence of meniscal tears. RESULTS: Histopathological changes and synovitis scores were similar regardless of degree of rupture (partial n = 5, complete n = 25) or presence of meniscal injury (n = 12) and were characterized by hyperplastic and lymphoplasmacytic synovitis with increased vascularity (30/30) and the presence of hemosiderin deposits (28/30), binucleated plasma cells (28/30), mucoid change (25/30), and Mott cells (16/30). Thirteen (43%) specimens were consistent with high-grade synovitis according to the Krenn system, while 11 (37%) specimens fit into the high-inflammatory subtype with the HSS system. CONCLUSION: Synovitis associated with canine CCLD in this study population was lymphoplasmacytic and was often highly inflammatory, with the presence of cells pertaining to humoral immunity. Humoral immune responses may play key roles in the synovitis associated with CCLD. CLINICAL SIGNIFICANCE: Modulation of biological factors that provoke humoral immune responses may mitigate symptoms of OA that persist and progress even after surgical treatment of CCLD in dogs.

Differential gene expression in skin RNA of horses affected with degenerative suspensory ligament desmitis.

BACKGROUND: Equine degenerative suspensory ligament desmitis (DSLD) is a systemic connective tissue disorder first identified in Peruvian Paso horses but afflicting other horse breeds as well. Inappropriate accumulation of proteoglycans in connective tissues, most prominently in tendons and ligaments, leads to progressive and debilitating lameness and pain. It is largely unknown what drives the overproduction of proteoglycans, but our previous studies suggest involvement of bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-ß (TGFß) family, impacting synthesis of proteoglycans. To identify potential players in pathogenesis of DSLD a new approach utilizing next generation sequencing was undertaken. METHODS: Next generation sequencing was performed using RNA extracted from skin biopsies of six control Peruvian Pasos and six horses with DSLD (4 Peruvian Pasos and 2 warmbloods). The CuffDiff result sets were validated with algorithms used to run them. This was based on the determined false discovery rates derived from the P values adjusted for multiple testing for any given result. RESULTS: Bioinformatics analysis of transcriptomes revealed differential expression of over 1500 genes, including increased expression of genes for several growth factors (most prominently BMP2, FGF5, CTGF, many members of the EGF family), and mediators of signaling (Fos, Myc, MAPK system), and keratins. Two genes encoding for enzymes involved in synthesis of hyaluronan were also overexpressed. Gene expression was decreased for protein cores of many proteoglycans, several growth factors, most collagens, and many peptides with immune function. CONCLUSIONS: The overexpression of BMP2 correlates well with our previous data. However, the decrease in expression of numerous proteoglycans was unexpected. A mutation in a gene of a less characterized proteoglycan and/or glycosyltransferase with subsequent increased production of hyaluronan and/or a proteoglycan(s) undetected in our study could account for the systemic proteoglycan deposition. Decreased collagen gene expression indicates abnormal connective tissue metabolism. The increased expression of keratin genes and FGF5 supports reports of skin abnormalities in DSLD. Underexpression of immune function genes corresponds with lack of inflammation in DSLD tissues. Finally, though the proteoglycan and/or glycosaminoglycan abundant in DSLD has not been identified, we validated our previous data, including overexpression of BMP2, and systemic nature of DSLD due to disturbed metabolism of the extracellular matrix.

Histopathological and electron microscopic study in dogs with patellar luxation and skin hyperextensibility.

Patellar luxation is abnormal displacement of the patella from the femoral trochlear groove. It is seen primarily in small breed dogs and causes pain and limited mobility of the stifle joint. This study aimed to investigate the relationship among patellar luxation, skin extension, and skin collagen fibril diameter. Nine dogs with patellar luxation and five clinically normal dogs were enrolled in the study. We measured the skin extension and investigated the ultrastructure of the skin and patellofemoral ligament by histopathology and transmission electron microscopy. The mean skin extension in dogs with patellar luxation was 18.5 ± 5.5% which is greater than the reference value (14.5%). Mean skin extension in controls was 8.8 ± 1.7% and was within the normal range. In dogs with patellar luxation, histopathology of the skin and patellofemoral ligament showed sparse and unevenly distributed collagen fibers. Transmission electron microscopy identified poorly organized, irregularly shaped, thin collagen fibrils. Collagen fibril thickness in dogs with patellar luxation was significantly less than fibril thickness in controls (P<0.001). There was a significant negative correlation (ρ= -0.863; P<0.001) between skin collagen fibril diameter and skin extension. Skin extension was correlated with patellar luxation and disease severity. Dogs with patellar luxation, joint dysplasia, and hyperextensible skin appear to be pathologically related. This might represent a phenotype of the Ehlers-Danlos syndrome, a hereditary connective tissue disorder in humans.

Inflammatory pattern of the infrapatellar fat pad in dogs with canine cruciate ligament disease.

BACKGROUND: Despite the importance of inflammation during the pathogenesis of cranial cruciate ligament disease (CCLD) in dogs and despite the latest knowledge suggesting a significant role of adipose tissue in osteoarthritis, the infrapatellar fat pad (IFP) was up to now mostly disregarded in veterinary investigations. In the present study, the inflammatory activity of the IFP, the main adipose structure within the stifle joint, was thoroughly investigated to evaluate its potential impact in the pathogenesis of this common disease of our canine companions. Samples of IFP, subcutaneous adipose tissue (ScAT) of the thigh and synovial fluid in both diseased (n = 36) and healthy control (n = 23) dogs were tested for their immune cell composition but also for interleukins (IL-1ß, IL-6, IL-8, IL-10), degradative enzymes (MMP-1, MMP-3, MMP-13, TIMP-2, iNOS) and adipokines (leptin and adiponectin). Characterization of the immune cell composition was ascertained by fluorescence activated cell sorting. Gene expression and protein release of the inflammatory markers was determined by real RT-qPCR and ELISA. RESULTS: IFPs of dogs with CCLD had a significantly increased immune cell count with T cells (CD3) as the most abundant immune cells. T cells and macrophages (CD14) were significantly increased compared to healthy controls or corresponding ScAT. In addition, IFPs of dogs with CCLD demonstrated a significant increase on gene as well as protein level of multiple inflammatory indicators (IL-1ß, IL-6, MMP-1, MMP-13) compared to the other tissues. TNFα was only increased on gene expression. Adipokine analysis showed higher secretion of adiponectin and lower leptin secretion in IFP from dogs with CCLD than from controls. In the synovial fluid from dogs with CCLD concentrations of IL-1ß, MMP-1, MMP-13 as well as leptin were significantly increased compared to the synovial fluid from healthy control dogs. CONCLUSIONS: The present study indicates that the IFP is a potential contributory factor in the pathogenesis of CCLD, due to its inflammatory phenotype and the proximity within the stifle joint. To determine the extent of this possible inter-relationship, further studies need to be undertaken.

COL9A2 and COL9A3 mutations in canine autosomal recessive oculoskeletal dysplasia.

Oculoskeletal dysplasia segregates as an autosomal recessive trait in the Labrador retriever and Samoyed canine breeds, in which the causative loci have been termed drd1 and drd2, respectively. Affected dogs exhibit short-limbed dwarfism and severe ocular defects. The disease phenotype resembles human hereditary arthro-ophthalmopathies such as Stickler and Marshall syndromes, although these disorders are usually dominant. Linkage studies mapped drd1 to canine chromosome 24 and drd2 to canine chromosome 15. Positional candidate gene analysis then led to the identification of a 1-base insertional mutation in exon 1 of COL9A3 that cosegregates with drd1 and a 1,267-bp deletion mutation in the 5' end of COL9A2 that cosegregates with drd2. Both mutations affect the COL3 domain of the respective gene. Northern analysis showed that RNA expression of the respective genes was reduced in affected retinas. These models offer potential for studies such as protein-protein interactions between different members of the collagen gene family, regulation and expression of these genes in retina and cartilage, and even opportunities for gene therapy.

Antinuclear antibodies: presence and specificity in autoimmune connective tissue disease in the dog.

Autoimmune connective tissue disease in both animals and humans present as multi-systemic disorders. Clinical signs referable to joints, musculoskeletal system and Dermatopathies are present. Autoantibody production is a regular feature of systemic autoimmune disease and these antibodies have diagnostic value in both human and veterinary medicine. In this study, we demonstrate that many of the commonly used diagnostic techniques developed for use with human patients may also be adapted for use in dogs. However, in some instances, 'canine specific' autoantibodies, distinct from analogous human antibodies may be detected in sera from dogs with a diagnosis of autoimmune disease. This finding indicates that dogs may develop 'dog-specific' autoimmune connective tissue disease.

Collagen dysplasia in a litter of Garafiano shepherd dogs.

A connective tissue disease resembling the human Ehlers-Danlos syndrome is reported in three Garafiano shepherd dogs from the same litter. The clinical signs included skin hyperextensibility and fragility, joint laxity, and ocular lesions. Microscopical studies showed abnormalities in the packing of collagen in fibrils and fibres in the skin. Systemic connective tissue defects were demonstrated in one necropsied puppy.

Canine lymphomatoid granulomatosis: an immunophenotypic analysis of three cases.

Three dogs had a history of multiple progressive lesions affecting the skin, subcutis or skeletal muscles. The lesions developed over a period of several months, and each case demonstrated late cardiopulmonary complications. Post-mortem examination revealed multicentric, angio-destructive, lymphohistiocytic, proliferative lesions typical of the rare disorder lymphomatoid granulomatosis. Immunohistochemical examination demonstrated variable CD3 antigen expression by the atypical cell population in two of the three cases. This provides the first evidence that canine lymphomatoid granulomatosis may be a form of atypical T-cell lymphoma similar to the comparable disorder that occurs in man.

Heterogeneity in dermatosparaxis is shown by contraction of collagen gels.

Dermal fibroblasts from sheep exhibiting a mild form of dermatosparaxis were able to contract reconstituted, fibrillar collagen gels at the same rate as control dermal fibroblasts, indicating a normal interaction between the cells and a collagenous matrix. An extract from dermatosparactic skin was shown, after partial purification, to have N-proteinase activity, although the level of activity was much lower than found in normal skin. These data show that dermatosparaxis is a heterogeneous disease, since in the severe forms of the disease the defect has been characterized as an absence of N-proteinase and an inability of the cells to interact with and contract collagen gels.

[Soft tissue lesions of the equine carpus: roentgenological and echographic studies]. / Weke delen letsels van de carpus van het paard: Röntgenologisch en echografisch onderzoek.

Common soft tissue disorders of the equine carpus are fluctuating or firm soft tissue swellings, wounds and draining tracts. Survey radiography may show the size, position and origin of the swellings and reveals soft tissue calcification, accumulation of air and radiopaque foreign material. Contrast radiography enables accurate visualization of the size, shape, position and origin of fluctuating soft tissue swellings, demonstrates abnormal intersynovial communication and allows precise demonstration of the extent of puncture wounds and draining tracts. Ultrasonography allows differentiation between a firm solid or thick-walled cystic lesion, clearly reveals injuries to tendons, muscle and ligaments, reveals minor irregularities of the wall and the lumen of distended tendon sheaths and may demonstrate radiolucent foreign material more clearly than contrast radiography.

An inherited connective tissue disease in the horse.

The hyperextensible, fragile skin of two related horses was compared with the skin of eight normal horses. Skin sections were examined by light microscopy and transmission electron microscopy. The deep dermal layer of the dorsal abdomen was much thinner in the affected horses, and contained bundles of collagen fibers which were more loosely packed. Within individual fibers, the fibrils were frequently curved and nonparallel rather than straight and parallel. Both of the affected animals had a greater range of fibril diameters than a normal horse. They had some unusually thick fibrils with very irregular outlines in cross-sections, not observed in the normal animal. Other skin samples were subjected to acetic acid extraction, pepsin digestion, amino acid analysis and polyacrylamide gel electrophoresis. In the skin of the two affected horses, the proportion of total extracted collagen which was acid-soluble was twice as high as in two normal horses. Collagen types I and III were present in similar proportions in normal and affected horses, and the collagen chains were of normal molecular weights. The disorder resembles the group described by Minor (Minor RR: Am J Pathol 98: 226, 1980) as 'dominant collagen packing defect I' which has been reported in dogs, mink, and cats, and which shares features with Ehlers Danlos Syndrome I, II, and III in man. The pedigree data available for these horses suggest an autosomal recessive mutation, but are also consistent with autosomal dominant inheritance.

A defective cell surface collagen-binding protein in dermatosparactic sheep fibroblasts.

Fibroblasts from dermatosparactic sheep fail to contract collagen gels and show a reduced attachment to collagenous substrates. By comparing collagen-binding membrane proteins of normal (+/+), homozygote (-/-), and heterozygote (+/-) fibroblasts, we present evidence that the interaction of normal fibroblasts with native type I collagen involves a protein of apparent Mr = 34,000 which is absent from dermatosparactic fibroblasts and seems to be related to anchorin CII. This conclusion was reached from the following experiments: (a) On a blot of membrane proteins from normal fibroblasts radioactively labeled type I collagen bound predominantly to a protein band of 34 kD; dermatosparactic membranes revealed only a small amount of binding to a component with a molecular mass of 47 kD. (b) After separation of normal fibroblast membrane proteins on type I collagen-Sepharose, a collagen-binding component of 34 kD was found which was absent from the corresponding fraction of dermatosparactic membranes. (c) Antibodies to anchorin CII stained the surface of normal (+/+), but not of dermatosparactic (-/-) fibroblasts and labeled a 34-kD component after immunoblotting of normal fibroblast membrane proteins. (d) After metabolic labeling of fibroblasts with [35S]methionine and immunoprecipitation with anti-anchorin CII, 40- and 34-kD components were precipitated from extracts of normal fibroblasts, while the latter component was absent from affected cells. Similar differences were found after immunoblotting of membranes from whole normal or affected skin. These data indicate that dermatosparaxis of sheep involves a molecular defect of a collagen-binding protein. Therefore this disease represents a model to study the complex interaction of cells with the extracellular matrix on a molecular level.

Rhesus monkeys (Macaca mulatta) as a model for calcium pyrophosphate dihydrate crystal deposition disease.

Calcium pyrophosphate dihydrate crystal deposition disease (CPDD) was recognized in 4 of 30 free-ranging rhesus macaques. By means of tissue radiography, focal radiodensities were noted in lumbar intervertebral discs, menisci, and articular cartilage. Crystal deposits were identified as calcium pyrophosphate dihydrate (Ca2P2O7 X 2H2O) by means of X-ray diffraction. The pathogenesis of calcium pyrophosphate dihydrate arthropathy in man remains elusive. However, with the recognition of this arthritis in a well defined population of aged nonhuman primates, a model now exists to facilitate the study of this disease.

A mild form of ovine dermatosparaxis.

Dermatosparaxis is an inherited disease which results from a deficiency in procollagen aminopropeptidase activity. A form of this disease in sheep has been examined which is much milder than the previously reported instance in sheep. Partially processed pN alpha 1 and pN alpha 2 chains are present in the skin of affected animals and have been characterized by gel electrophoresis, amino acid analysis, susceptibility to collagenase digestions and by segment-long-spacing aggregate formations. Fully processed collagen is also present in the skin of affected animals indicating that some aminopropeptidase activity is present. Extracts from the skin of affected animals were shown to contain an aminopropeptidase activity which was about 25% of the activity found in similar extracts from normal animals.

Peripheral neuropathy in the Twitcher mutant. A new experimental model of endoneurial edema.

The Twitcher mouse (Twi/Twi) is a recently identified mutant experimental model for human globoid leukodystrophy. Affected mice develop neurologic abnormalities with demyelination of white matter and peripheral nerve due to an inherited enzyme deficiency. The neuropathy has unusual pathologic features:severe interstitial edema and infiltration by eosinophils. To investigate its pathogenesis and to identify the mechanism of demyelination, we studied vascular permeability and measured endoneurial fluid pressure. Significantly increased endoneurial fluid pressure was detected in clinically affected animals (average, 6.4 cm H2O) versus controls (1.7 cm H2O), and these data are the first measurements of EFP to be reported in mice. Increased vascular permeability to horseradish peroxidase was visualized by electron microscopy with leakage of horseradish peroxidase between endothelial cells and flooding of the endoneurial interstitium. Numerous eosinophils were present in the interstitium, as well as some polymorphonuclear cells, occasional erythrocytes, and degranulating mast cells. Abnormalities of nerve fibers included swelling of Schwann cells with intracytoplasmic inclusions, demyelination, and remyelination. As well as being a model for globoid leukodystrophy, the Twitcher is the first spontaneously occurring experimental model for endoneurial edema and increased endoneurial fluid pressure.