Cleft lip and/or palate with monogenic autosomal recessive transmission in Pyrenees shepherd dogs.

OBJECTIVE: To document the genetic background of Pyrenees shepherd dogs as it relates to the incidence of cleft lip and/or cleft palate, to describe the phenotype, and to determine possible candidate genes. DESIGN: Pedigree analysis was performed and blood samples were taken from five affected pups, their siblings, and parents. Seven candidate genes were selected and linkage analysis was performed. Further methods used included sequencing and histology. RESULTS: In 37 litters consisting of 163 pups, we found 47 affected pups in a total population of 2104. The male:female ratio was 1:0.96. Affected pups showed isolated cleft lip and/or cleft palate; no attendant disorders have been reported. Despite a high degree of relationship, two affected pups displayed a cleft palate (- H S H -) and a cleft lip with or without cleft palate (L A -) cleft formation. Histology of affected pups showed that the medial edge epithelium remained intact and did not undergo an epithelial-mesenchymal transformation. There was no evidence for linkage between the trait and TGFb3 or Msx1. Subsequent sequencing excluded the coding sequence of Fst as well. CONCLUSION: Pedigree analysis showed that cleft palate is not genetically distinct from cleft lip with or without cleft palate but is inherited in this breed as a monogenic autosomal recessive trait. Linkage analysis and sequencing excluded TGFb3, Msx1, and Fst as candidate genes. Histology of affected pups showed that the medial edge epithelium is still intact.

Growth characterization of neo porcine cartilage pellets and their use in an interactive culture model.

OBJECTIVE: The aim of this study was to evaluate the growth characteristics of freshly isolated porcine chondrocytes in high-density pellet cultures and to preliminary investigate their use in an interactive in vitro model with synovial fibroblast cell lines to study rheumatoid arthritis (RA). DESIGN: 1.8x10(6) chondrocytes/cm2 were seeded in 48-multiwell plates. Thickness, cell number and cell distribution in pellet cross sections were documented over a 22-day-long period. Alcian blue staining, type I and type II collagen staining, real-time reverse transcriptase polymerase polymerase chain reaction (RT-PCR) and high performance liquid chromatography (HPLC) were used to characterize cartilage extracellular matrix (ECM) formation, and cell proliferation was demonstrated by Ki67 staining. Furthermore, 2-week-old chondrocyte pellets were co-cultured for additional 2 weeks with two human synovial fibroblast cell lines derived from a normal donor (non-invasive cell line) and a RA patient (invasive-aggressive (IA) cell line), respectively. RESULTS: Chondrocyte pellets from 11 individual preparations showed a significant increase in pellet thickness from 44+/-19 microm (day 3) to 282+/-19 microm (day 22). Calculation of chondrocyte distribution, cell number and pellet thickness indicated that pellet growth was due to ECM formation and not cell proliferation. This was also confirmed by low numbers of Ki67 positive chondrocytes and absence of cell clusters. HPLC, messenger RNA-analysis, histochemistry and antibody staining verified the expression of ECM components such as type II collagen, whereas type I collagen expression was very low. In contrast to the non-aggressive synovial fibroblast cell line, the IA synovial fibroblast cell line clearly showed cartilage invasion. CONCLUSION: Pellet formation of freshly isolated chondrocytes followed a reproducible developmental kinetics and showed typical immature hyaline cartilage properties. Such uniform cartilage pellets are very useful as a substrate for interactive cell culture models that simulate diseases like RA.

Identification of novel human aggrecan T cell epitopes in HLA-B27 transgenic mice associated with spondyloarthropathy.

The pathology of ankylosing spondylitis, reactive arthritis, and other spondyloarthropathies (SpA) is closely associated with the human leukocyte class I Ag HLA-B27. A characteristic finding in SpA is inflammation of cartilage structures of the joint, in particular at the site of ligament/tendon and bone junction (enthesitis). In this study, we investigated the role of CD8+ T cells in response to the cartilage proteoglycan aggrecan as a potential candidate autoantigen in BALB/c-B27 transgenic mice. We identified four new HLA-B27-restricted nonamer peptides, one of them (no. 67) with a particularly strong T cell immunogenicity. Peptide no. 67 immunization was capable of stimulating HLA-B27-restricted, CD8+ T cells in BALB/c-B27 transgenic animals, but not in wild-type BALB/c mice. The peptide was specifically recognized on P815-B27 transfectants by HLA-B27-restricted CTLs, which were also detectable by HLA tetramer staining ex vivo as well as in situ. Most importantly, analysis of the joints from peptide no. 67-immunized mice induced typical histological signs of SpA. Our data indicate that HLA-B27-restricted epitopes derived from human aggrecan are involved in the induction of inflammation (tenosynovitis), underlining the importance of HLA-B27 in the pathogenesis of SpA.