The Matrilin-3 T298M mutation predisposes for post-traumatic osteoarthritis in a knock-in mouse model.

OBJECTIVE: The human matrilin-3 T303M (in mouse T298M) mutation has been proposed to predispose for osteoarthritis, but due to the lack of an appropriate animal model this hypothesis could not be tested. This study was carried out to identify pathogenic mechanisms in a transgenic mouse line by which the mutation might contribute to disease development. METHODS: A mouse line carrying the T298M point mutation in the Matn3 locus was generated and features of skeletal development in ageing animals were characterized by immunohistology, micro computed tomography, transmission electron microscopy and atomic force microscopy. The effect of transgenic matrilin-3 was also studied after surgically induced osteoarthritis. RESULTS: The matrilin-3 T298M mutation influences endochondral ossification and leads to larger cartilage collagen fibril diameters. This in turn leads to an increased compressive stiffness of the articular cartilage, which, upon challenge, aggravates osteoarthritis development. CONCLUSIONS: The mouse matrilin-3 T298M mutation causes a predisposition for post-traumatic osteoarthritis and the corresponding knock-in mouse line therefore represents a valid model for investigating the pathogenic mechanisms involved in osteoarthritis development.

Crtap and p3h1 knock out zebrafish support defective collagen chaperoning as the cause of their osteogenesis imperfecta phenotype.

Prolyl 3-hydroxylation is a rare collagen type I post translational modification in fibrillar collagens. The primary 3Hyp substrate sites in type I collagen are targeted by an endoplasmic reticulum (ER) complex composed by cartilage associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1) and prolyl cis/trans isomerase B, whose mutations cause recessive forms of osteogenesis imperfecta with impaired levels of α1(I)3Hyp986. The absence of collagen type I 3Hyp in wild type zebrafish provides the unique opportunity to clarify the role of the complex in vertebrate. Zebrafish knock outs for crtap and p3h1 were generated by CRISPR/Cas9. Mutant fish have the typical OI patients' reduced size, body disproportion and altered mineralization. Vertebral body fusions, deformities and fractures are accompanied to reduced size, thickness and bone volume. Intracellularly, collagen type I is overmodified, and partially retained causing enlarged ER cisternae. In the extracellular matrix the abnormal collagen type I assembles in disorganized fibers characterized by altered diameter. The data support the defective chaperone role of the 3-hydroxylation complex as the primary cause of the skeletal phenotype.

Antimicrobial peptides derived from the cartilage.-specific C-type Lectin Domain Family 3 Member A (CLEC3A) - potential in the prevention and treatment of septic arthritis.

OBJECTIVE: To investigate the antimicrobial activity of peptides derived from C-type Lectin Domain Family 3 Member A (CLEC3A), shed light on the mechanism of antimicrobial activity and assess their potential application in prevention and treatment of septic arthritis. DESIGN: We performed immunoblot to detect CLEC3A peptides in human cartilage extracts. To investigate their antimicrobial activity, we designed peptides and recombinantly expressed CLEC3A domains and used them to perform viable count assays using E.coli, P.aeruginosa and S.aureus. We investigated the mechanism of their antimicrobial activity by fluorescence and scanning electron microscopy, performed ELISA-style immunoassays and transmission electron microscopy to test for lipopolysaccharide binding and surface plasmon resonance to test for lipoteichoic acid (LTA) binding. We coated CLEC3A peptides on titanium, a commonly used prosthetic material, and performed fluorescence microscopy to quantify bacterial adhesion. Moreover, we assessed the peptides' cytotoxicity against primary human chondrocytes using MTT cell viability assays. RESULTS: CLEC3A fragments were detected in human cartilage extracts. Moreover, bacterial supernatants lead to fragmentation of recombinant and cartilage-derived CLEC3A. CLEC3A-derived peptides killed E.coli, P.aeruginosa and S.aureus, permeabilized bacterial membranes and bound lipopolysaccharide and LTA. Coating CLEC3A antimicrobial peptides (AMPs) on titanium lead to significantly reduced bacterial adhesion to the material. In addition, microbicidal concentrations of CLEC3A peptides in vitro displayed no direct cytotoxicity against primary human chondrocytes. CONCLUSIONS: We identify cartilage-specific AMPs originating from CLEC3A, resolve the mechanism of their antimicrobial activity and point to a novel approach in the prevention and treatment of septic arthritis using potent, non-toxic, AMPs.

TGF-ß1 differentially modulates the collagen VI α5 and α6 chains in human tendon cultures.

Collagen VI is a microfibrillar collagen with a potential regulatory role in tendon repair mechanism. We studied the expression of collagen VI α5 and α6 chains in normal human tendon fibroblast cultures, both under basal condition and in response to TGF-ß1, a potent regulator of tendon healing. Under basal condition, we found that the α5 chain was expressed, although to a lesser extent with respect to the α3 chain; in contrast, the α6 chain was absent. The treatment with TGFß1 induced an opposite effect on the expression of the α5 and α6 chains; in fact, while the α5 chain was dramatically reduced, the α6 chain was induced and released in the culture medium. These data indicate that collagen VI α5 and α6 chains are differentially involved in tendon matrix homeostasis. The α6 chain may represent a new potential biomarker for monitoring TGFß1-related events in tendon, as healing and fibrotic scar formation.

Parvalbumin- and vasoactive intestinal polypeptide-expressing neocortical interneurons impose differential inhibition on Martinotti cells.

Disinhibition of cortical excitatory cell gate information flow through and between cortical columns. The major contribution of Martinotti cells (MC) is providing dendritic inhibition to excitatory neurons and therefore they are a main component of disinhibitory connections. Here we show by means of optogenetics that MC in layers II/III of the mouse primary somatosensory cortex are inhibited by both parvalbumin (PV)- and vasoactive intestinal polypeptide (VIP)-expressing cells. Paired recordings revealed stronger synaptic input onto MC from PV cells than from VIP cells. Moreover, PV cell input showed frequency-independent depression, whereas VIP cell input facilitated at high frequencies. These differences in the properties of the two unitary connections enable disinhibition with distinct temporal features.

A photoinduced mixed-valence state in an organic bis-triarylamine mixed-valence compound with an iridium-metal-bridge.

Upon irradiation a mixed-valence (MV) state is formed in a donor-iridium(III)-acceptor triad by a photoinduced electron transfer process. The resulting radical and intervalence charge transfer (IV-CT) absorptions cover a wide spectral range (3200-400 nm). These results were supported by spectroelectrochemistry, fs-time resolved pump-probe spectroscopy and assisted by TD-DFT calculations.

Defective collagen VI α6 chain expression in the skeletal muscle of patients with collagen VI-related myopathies.

Collagen VI is a non-fibrillar collagen present in the extracellular matrix (ECM) as a complex polymer; the mainly expressed form is composed of α1, α2 and α3 chains; mutations in genes encoding these chains cause myopathies known as Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and myosclerosis myopathy (MM). The collagen VI α6 chain is a recently identified component of the ECM of the human skeletal muscle. Here we report that the α6 chain was dramatically reduced in skeletal muscle and muscle cell cultures of genetically characterized UCMD, BM and MM patients, independently of the clinical phenotype, the gene involved and the effect of the mutation on the expression of the "classical" α1α2α3 heterotrimer. By contrast, the collagen VI α6 chain was normally expressed or increased in the muscle of patients affected by other forms of muscular dystrophy, the overexpression matching with areas of increased fibrosis. In vitro treatment with TGF-ß1, a potent collagen inducer, promoted the collagen VI α6 chain deposition in the ECM of normal muscle cells, whereas, in cultures derived from collagen VI-related myopathy patients, the collagen VI α6 chain failed to develop a network outside the cells and accumulated in the endoplasmic reticulum. The defect of the α6 chain points to a contribution to the pathogenesis of collagen VI-related disorders.

SPAG7 is a candidate gene for the periodic fever, aphthous stomatitis, pharyngitis and adenopathy (PFAPA) syndrome.

Periodic fever, aphthous stomatitis, pharyngitis and adenopathy (PFAPA) syndrome is an auto-inflammatory disease for which a genetic basis has been postulated. Nevertheless, in contrast to the other periodic fever syndromes, no candidate genes have yet been identified. By cloning, following long insert size paired-end sequencing, of a de novo chromosomal translocation t(10;17)(q11.2;p13) in a patient with typical PFAPA syndrome lacking mutations in genes associated with other periodic fever syndromes we identified SPAG7 as a candidate gene for PFAPA. SPAG7 protein is expressed in tissues affected by PFAPA and has been functionally linked to antiviral and inflammatory responses. Haploinsufficiency of SPAG7 due to a microdeletion at the translocation breakpoint leading to loss of exons 2-7 from one allele was associated with PFAPA in the index. Sequence analyses of SPAG7 in additional patients with PFAPA point to genetic heterogeneity or alternative mechanisms of SPAG7 deregulation, such as somatic or epigenetic changes.

The matrilin-3 VWA1 domain modulates interleukin-6 release from primary human chondrocytes.

OBJECTIVE: We previously demonstrated the ability of matrilin-3 to modulate the gene expression profile of primary human chondrocytes (PHCs) toward a state favoring cartilage catabolism. The structure within matrilin-3 responsible for the induction of these catabolic genes is unknown. Here, we investigated the potential of matrilin-3 (MATN3) and truncated matrilin-3 proteins, in both monomeric and oligomeric form, to stimulate interleukin (IL)-6 release in PHCs. METHODS: We expressed full-length matrilin-3 oligomers, matrilin-3 von Willebrand factor A (VWA) domain oligomers, matrilin-3 four epidermal growth factor (EGF) domain oligomers, matrilin-3 monomers without oligomerization domains, matrilin-3 VWA domain monomers, and matrilin-3 4EGF monomers. We then incubated PHCs in the absence or presence of full-length matrilin-3 or one of the truncated matrilin-3 proteins and finally determined the release of IL-6 in cell-culture supernatants. RESULTS: The addition of full-length matrilin-3 oligomers, matrilin-3 VWA domain oligomers, and, less pronounced, matrilin-3 monomers without oligomerization domains, and matrilin-3 4EGF-oligomers to the cell-culture medium led to a significant induction of IL-6 in PHCs. DISCUSSION: Based on recombinant expression of different matrilin-3 domains in both monomeric and oligomeric form, this work demonstrated that the VWA1 domain of matrilin-3 is primarily responsible for the induction of IL-6 release and that the oligomerization of the VWA1 domain markedly promotes its activity.

Matrilin-3 mutations that cause chondrodysplasias interfere with protein trafficking while a mutation associated with hand osteoarthritis does not.

Several mutations in the extracellular matrix protein matrilin-3 cause a heterogeneous disease spectrum affecting skeletal tissues. We introduced three disease causing point mutations leading to single amino acid exchanges (R116W, T298M, C299S) in matrilin-3 and expressed the corresponding proteins in primary articular chondrocytes to elucidate pathogenic mechanisms at the cellular level. Expression levels, processing, and the secretion pattern of a mutation linked to hand osteoarthritis (T298M) were similar to the wildtype protein, whereas the two other mutants were poorly expressed and hardly detectable in supernatants of transiently transfected cells. Using immunofluorescence staining, we demonstrated that mutants R116W and C299S are retained and accumulate within the endoplasmatic reticulum (ER). Their further trafficking to the Golgi compartment seems to be disturbed, whereas T298M is secreted normally. In cells transfected with the wildtype and T298M constructs, a matrilin-3 containing filamentous network was formed surrounding the cells, whereas in the case of R116W and C299S such structures were completely absent. These observations are similar to those for mutations in the cartilage oligomeric matrix protein (COMP) leading to multiple epiphyseal dysplasia and pseudoachondroplasia suggesting that retention and accumulation of cartilage proteins in the ER might be a general mechanism involved in the pathogenesis of chondrodysplasias.

Matrilin-3 in human articular cartilage: increased expression in osteoarthritis.

OBJECTIVE: Matrilin-3 is a member of the recently described matrilin family of extracellular matrix proteins containing von Willebrand factor A-like domains. The matrilin-3 subunit can form homo-tetramers as well as hetero-oligomers together with subunits of matrilin-1 (cartilage matrix protein). It has a restricted tissue distribution and is strongly expressed in growing skeletal tissues. Detailed information on expression and distribution of extracellular matrix proteins is important to understand cartilage function in health and in disease like osteoarthritis (OA). METHODS: Normal and osteoarthritic cartilage were systematically analysed for matrilin-3 expression, using immunohistochemistry, Western blot analysis, in situ hybridization, and quantitative PCR. RESULTS: Our results indicate that matrilin-3 is a mandatory component of mature articular cartilage with its expression being restricted to chondrocytes from the tangential zone and the upper middle cartilage zone. Osteoarthritic cartilage samples with only moderate morphological osteoarthritic degenerations have elevated levels of matrilin-3 mRNA. In parallel, we found an increased deposition of matrilin-3 protein in the cartilage matrix. Matrilin-3 staining was diffusely distributed in the cartilage matrix, with no cellular staining being detectable. In cartilage samples with minor osteoarthritic lesions, matrilin-3 deposition was restricted to the middle zone and to the upper deep zone. A strong correlation was found between enhanced matrilin-3 gene and protein expression and the extent of tissue damage. Sections with severe osteoarthritic degeneration showed the highest amount of matrilin-3 mRNA, strong signals in in situ hybridization, and prominent protein deposition in the middle and deep cartilage zone. CONCLUSION: We conclude that matrilin-3 is an integral component of human articular cartilage matrix and that the enhanced expression of matrilin-3 in OA may be a cellular response to the modified microenvironment in the disease.

Characterization of the mouse matrilin-4 gene: a 5' antiparallel overlap with the gene encoding the transcription factor RBP-l.

We have isolated and characterized the gene encoding mouse matrilin-4 (Matn4), an extracellular matrix protein present in a broad spectrum of tissues. The gene spanned 16 kb, consisted of 12 exons, and localized to chromosome 2. As in all known matrilin genes, the last intron, separating the exons coding for the coiled-coil domain, did not follow the GT-AG rule and belonged to the subgroup of introns having AT-AC at the ends. Matn4 contained two exons in the 5' UTR that could be alternatively spliced. We localized a major and a minor transcription start site to two different untranslated exons: exon 0a and exon 0b. Matn4 divergently overlapped 5' with the gene encoding RBP-L (for recombining binding protein suppressor of hairless-like; Rbpsuhl), a transcription factor with homology to RBP-JK. Exon 1 of Rbpsuhl was located in the second intron of Matn4, whereas exon 0a, the first exon of Matn4, was located in the second intron of Rbpsuhl. The second exons of the respective genes overlapped in an antisense orientation. We mapped the major transcription start of Rbpsuhl to a position approximately 150 nt upstream of the splice acceptor site of the first intron, leading to the synthesis of a truncated variant of RBP-L probably missing the amino-terminal 121 amino acid residues. We analyzed the expression of the different Matn4 and Rbpsuhl transcripts by quantitative RT-PCR; this showed the highest expression for both genes in lung and brain. In situ hybridization of brain sections showed a partially overlapping expression pattern for the two genes.

Molecular structure, processing, and tissue distribution of matrilin-4.

Matrilin-4 is the most recently identified member of the matrilin family of von Willebrand factor A-like domain containing extracellular matrix adapter proteins. Full-length matrilin-4 was expressed in 293-EBNA cells, purified using affinity tags, and subjected to biochemical characterization. The largest oligomeric form of recombinantly expressed full-length matrilin-4 is a trimer as shown by electron microscopy, SDS-polyacrylamide gel electrophoresis, and mass spectrometry. Proteolytically processed matrilin-4 species were also detected. The cleavage occurs in the short linker region between the second von Willebrand factor A-like domain and the coiled-coil domain leading to the release of large fragments and the formation of dimers and monomers of intact subunits still containing a trimeric coiled-coil. In immunoblots of calvaria extracts similar degradation products could be detected, indicating that a related proteolytic processing occurs in vivo. Matrilin-4 was first observed at day 7.5 post-coitum in mouse embryos. Affinity-purified antibodies detect a broad expression in dense and loose connective tissue, bone, cartilage, central and peripheral nervous systems and in association with basement membranes. In the matrix formed by cultured primary embryonic fibroblasts, matrilin-4 is found in a filamentous network connecting individual cells.

Expression of matrilin-1, -2 and -3 in developing mouse limbs and heart.

The expression of matrilin-1, -2 and -3 was studied in the heart and limb during mouse development. Matrilin-1 is transiently expressed in the heart between days 9.5 and 14.5 p.c. Matrilin-2 expression was detected in the heart from day 10.5 p.c. onwards. In the developing limb bud, both matrilin-1 and -3 were observed first at day 12.5 p.c. Throughout development matrilin-3 expression was strictly limited to cartilage, while matrilin-1 was also found in some other forms of connective tissue. Matrilin-2, albeit present around hypertrophic chondrocytes in the growth plate, was mainly expressed in non-skeletal structures. The complementary, but in part overlapping, expression of matrilins indicates the possibility for both redundant and unique functions among the members of this novel family of extracellular matrix proteins.

Mediation training enhances conflict management by healthcare personnel.

OBJECTIVES: Mediation training can prepare healthcare professionals to manage conflict effectively in today's changing healthcare system. The primary purpose of the study was to measure the perceived comfort level of healthcare professionals with conflict before and after mediation training and to determine the extent to which mediation principles were applied within and outside the work setting. Secondary objectives were to observe firsthand transfer of skills and to identify subjects' perceptions of the impact of mediation training. STUDY DESIGN: A cross-sectional, descriptive experimental design was used. PATIENTS AND METHODS: Over a 3-year period, 173 healthcare personnel, chosen from a community not-for-profit hospital, a health maintenance organization, a managed care insurance company, and a skilled nursing rehabilitation setting, received 25 hours of mediation training; of the personnel who underwent training, 130 participated in the pre- and posttraining survey. A Likert scale with a Cronbach alpha of .82 was used to measure perceived differences in comfort level with conflict before and after the training intervention. RESULTS: The comfort level of healthcare personnel with conflict increased significantly (P < .01 or P < .001) for all groups of participants after training. The mean pretraining score was 5.92, compared with a mean score of 7.57 after training. Active listening, summarizing, and reframing were the mediation skills most often used by participants after training. Skills were transferred to interactions with patients and peers, and participants noted that they were able to intervene successfully early in problem cases. CONCLUSIONS: Mediation training significantly increased healthcare workers' comfort level with conflict, and the skills were transferable to the healthcare workplace. Mediation training in healthcare settings can help resolve conflicts with clients at an early stage and prevent progression to costly litigation.

Health technology assessment in Luxembourg.

Luxembourg's public health insurance is a compulsory insurance for all employees, self-employed professionals, farmers, and pensioners. It is financed through contributions of the insured people, as well as by state taxes. Providers of health care are mainly private nonprofit institutions and self-employed professionals. All healthcare procedures are defined in fee schedules determined by a common decision of the Ministers of Social Security and Health according to proposals of a board of experts. The relative value of a service is also determined by the corresponding fee schedule. Hospitals are financed by individual budgets negotiated between each hospital and the health insurance. These hospital budgets do not cover services provided in hospitals by medical specialists, who are reimbursed on a fee-for-service basis. A low on hospital planning and organization allows the government to restrict the installation in hospitals of very expensive equipment or of equipment for which there is only a limited need in Luxembourg hospitals. Until recently there has been limited interest in or use of health technology assessment (HTA). However, large hospital investments have provoked some interest in the last few years. The Ministry of Health has asked for some HTA studies when a concrete decision had to be taken. Luxembourg decision makers have become more aware that HTA may help them to become more informed about the short- and long-term consequences of the application of health technology.

Molecular structure and tissue distribution of matrilin-3, a filament-forming extracellular matrix protein expressed during skeletal development.

Matrilin-3 is a recently identified member of the superfamily of proteins containing von Willebrand factor A-like domains and is able to form hetero-oligomers with matrilin-1 (cartilage matrix protein) via a C-terminal coiled-coil domain. Full-length matrilin-3 and a fragment lacking the assembly domain were expressed in 293-EBNA cells, purified, and subjected to biochemical characterization. Recombinantly expressed full-length matrilin-3 occurs as monomers, dimers, trimers, and tetramers, as detected by electron microscopy and SDS-polyacrylamide gel electrophoresis, whereas matrilin-3, purified from fetal calf cartilage, forms homotetramers as well as hetero-oligomers of variable stoichiometry with matrilin-1. In the matrix formed by cultured chondrosarcoma cells, matrilin-3 is found in a filamentous, collagen-dependent network connecting cells and in a collagen-independent pericellular network. Affinity-purified antibodies detect matrilin-3 expression in a variety of mouse cartilaginous tissues, such as sternum, articular, and epiphyseal cartilage, and in the cartilage anlage of developing bones. It is found both inside the lacunae and in the interterritorial matrix of the resting, proliferating, hypertrophic, and calcified cartilage zones, whereas the expression is lower in the superficial articular cartilage. In trachea and in costal cartilage of adult mice, an expression was seen in the perichondrium. Furthermore, matrilin-3 is found in bone, and its expression is, therefore, not restricted to chondroblasts and chondrocytes.