European League Against Rheumatism recommendations for the inclusion of patient representatives in scientific projects.

OBJECTIVE: To develop recommendations to enable successful inclusion of the patient perspective in European League Against Rheumatism (EULAR)-funded scientific research projects. METHODS: The EULAR standardised operational procedures for guideline development were followed. A systematic literature review was presented during a first task force meeting, including 3 rheumatologists, 1 rheumatologist/epidemiologist, 2 allied health professionals, 2 representatives of arthritis research organisations and 7 patient representatives, resulting in 38 statements. A Delphi method was carried out to reduce and refine the statements and agree on a set of eight. Next, a survey among a wider group of experts, professionals and patient representatives (n=42), was completed. Feedback from this wider group was discussed at the second meeting and integrated in the final wording of the recommendations. Subsequently, the level of agreement of the group of experts (n=81) was re-evaluated. RESULTS: The project resulted in a definition of patient research partner and agreement on a set of eight recommendations for their involvement in research projects. These recommendations provide practical guidance for organising patient participation, capturing (1) the role of patient research partners, (2) phase of involvement, (3) the recommended number, (4) recruitment, (5) selection, (6) support, (7) training and (8) acknowledgement. CONCLUSION: Collaboration between patients and professionals in research is relatively new. Trials or effectiveness studies are not yet available. Nevertheless, it is possible to define recommendations for the inclusion of patients in research following a solid expert opinion based consensus process.

Extracellular ATP and UTP stimulate cartilage proteoglycan and collagen accumulation in bovine articular chondrocyte pellet cultures.

Bovine articular chondrocytes were maintained in high density pellet cultures with and without serum and nucleotide triphosphates for different periods of time. Despite half-lives in culture of about 3 h, adenosine triphosphate and uridine triphosphate in the presence of serum increased sulphated glycosaminoglycan and collagen deposition above control levels. In the presence of serum a single dose of uridine triphosphate on the first day of culture was sufficient to induce significant increases in subsequent proteoglycan and collagen deposition. We conclude that both adenine triphosphate and uridine triphosphate are anabolic for articular chondrocytes, and that this effect on the chondrocyte is long-term.

Evidence of in situ stability of the type IV collagen triple helix in human inflammatory bowel disease using a denaturation specific epitope antibody.

A peptide specific antibody (AH1OW1) was raised against an epitope, AH10 (aa 449-463), of the alpha1(IV) chain adjacent to a cleavage site for matrix metalloproteinases (MMP)-2 and -9 within the triple helix of type IV collagen. The antibody only reacted with denatured and reduced preparations of type IV collagen, or with pepsin isolated type IV collagen digested with MMP-2 and MMP-9. The specificity of this antibody for the denatured triple helix was demonstrated by the lack of staining with pre-immune antibody and by pre-incubation of AH1OW1 antibody with excess AH10 peptide epitope. The AH1OWI antibody was used to detect whether proteolysis of type IV collagen occurs in ulcerative colitis, an inflammatory bowel condition often characterised by a large influx of granulocytes and macrophages and an associated tissue destruction. However, no evidence of in situ proteolysis of the basement membrane type IV collagen was observed. Only in the most actively inflamed mucosa was staining with AH1OW1 antibody observed in the mucosal connective tissue. Digestion of frozen sections of bowel with MMP-1, MMP-2, MMP-3 and MMP-9 did not result in the exposure of the AH10 epitope. These data demonstrate the stability of intact type IV collagen and indicate that susceptibility of alpha1(IV) chain to digestion with MMP-2 and MMP-9 may require other proteolytic/denaturing events in the molecule.

Effect of an exercise programme for elderly patients with heart failure.

AIMS: Benefit from exercise training programmes for patients with chronic heart failure has been demonstrated in relatively young (mean age 60 years) and predominantly male subjects. This study was undertaken to assess the effect of an exercise programme for older subjects more representative of the general heart failure population. METHODS AND RESULTS: Twenty-two patients with chronic heart failure of mean age 81+/-4 years were recruited into the study. Twenty-five percent were female and 31% were in chronic atrial fibrillation. A crossover study design was employed. The programme consisted of once weekly exercise sessions tailored to the abilities of elderly subjects and lasted for 12 weeks. Fifteen subjects completed the exercise component and nine the control component. The programme resulted in a 20% relative increase in 6-min walk test distance (ANCOVA: P<0.012). There was no improvement in quality of life as measured by the Living With Heart Failure Questionnaire, but the majority of subjects reported subjective improvement in wellbeing. CONCLUSION: Elderly patients with chronic heart failure can benefit from an appropriately designed exercise programme and should not be excluded from future large scale trials.

Differential detection of type II collagen N-terminal and C-terminal denaturation epitopes in degrading cartilage.

AIMS: To investigate the relative stability of collagen metabolites in degrading cartilage. METHODS: New antipeptide antibodies to denaturation epitopes located in the N-terminal and C-terminal regions of the type II collagen helix have been made and characterized. Type II collagen fragments in the conditioned medium from cultures of degrading bovine nasal cartilage were detected by immunoblotting with the new antisera as well as by N-terminal sequencing. The antibodies were also used in immunohistochemical studies of normal and osteoarthritic human cartilage. RESULTS: Type II collagen fragments with an apparent molecular mass of approximately 30 kDa were detected in cartilage conditioned media using antibody AH12L3, which recognizes N-terminal epitope AH12. The N-terminal sequence of one of these fragments matched exactly a sequence in the N-terminal region of type II collagen. Antibody AH9L2, which recognizes C-terminal epitope AH9, did not bind to any protein bands in the immunoblotted culture medium. In immunohistochemical studies, antibody AH12L3 detected extensive regions of degraded collagen in osteoarthritic cartilage and a more restricted pattern of staining in nonarthritic cartilage. Far less immunostaining was apparent in all cartilage specimens with antibody AH9L2. CONCLUSIONS: These results indicate that the N-terminal region of type II collagen is more resistant to proteolysis than the C-terminal region, an observation that has important implications for the choice of epitopes that are likely to be good markers of damage to cartilage collagen in patients with arthritis.

Why patients choose to purchase a hearing aid privately.

A questionnaire survey was undertaken of patients who had recently purchased a private hearing aid. They were asked their reasons for choosing a private aid in preference to an NHS aid. Cosmetic considerations and the failings of Health Service provisions were the most commonly cited reasons.

Distribution of two alternatively spliced variants of the type II collagen N-propeptide compared with the C-propeptide in bovine chondrocyte pellet cultures.

We have analyzed the distribution of type II collagen N- and C-propeptides in the cell layers and culture medium of bovine articular chondrocyte pellet cultures. Two splice variants of the type II collagen N-propeptide were detected by immunoblotting and immunoassay, using a new anti-peptide antibody, while the C-propeptide was detected using a monoclonal antibody. Type II collagen molecules containing the N-propeptide were detected weakly in cell layers, but not in tissue culture medium of chondrocyte pellet cultures, and both splice variants were observed. Free N-propeptide could not be detected in cell layers or medium. Type II procollagen molecules containing the C-propeptide were detected strongly in cell layers, but not in tissue culture medium, while the free C-propeptide was detected in both cell layers and medium. Since the N- and C-propeptides must be synthesized in a 1:1 molar ratio, we conclude that the N-propeptide is metabolized more quickly than the C-propeptide in this system. Our model can be used to study regulation of procollagen synthesis and propeptidase activity.

Human cathepsin K cleaves native type I and II collagens at the N-terminal end of the triple helix.

Cathepsin K (EC 3.4.22.38) is a recently described enzyme that has been shown to cleave type I collagen in its triple helix. The aim of this study was to determine if it also cleaves type II collagen in the triple helix and to identify the helical cleavage site(s) in types I and II collagens. Soluble human and bovine type II collagen, and rat type I collagen, were incubated with cathepsin K before the reaction was stopped with trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64). Analysis by SDS/PAGE of the collagen digests showed that optimal activity of cathepsin K against native type II collagen was between pH 5.0 and 5.5 and against denatured collagen between pH 4.0 and 7.0. The enzyme cleaved telopeptides as well as the alpha1(II) chains, generating multiple fragments in the range 90-120 kDa. The collagenolytic activity was not due to a contaminating metalloenzyme or serine proteinase as it was not inhibited by 1,10-phenanthroline, EDTA or 3,4-dichloroisocoumarin. Western blotting with anti-peptide antibodies to different regions of the alpha1(II) chain suggested that cathepsin K cleaved native alpha1(II) chains in the N-terminal region of the helical domain rather than at the well-defined collagenase cleavage site. This was confirmed by N-terminal sequencing of one of the fragments, revealing cleavage at a Gly-Lys bond, 58 residues from the N-terminus of the helical domain. By using a similar approach, cathepsin K was found to cleave native type I collagen close to the N-terminus of its triple helix. These results indicate that cathepsin K could have a role in the turnover of type II collagen, as well as type I collagen.