Health-related quality of life in cutaneous T-cell lymphoma: A post hoc analysis of a phase 3 trial in mycosis fungoides and Sézary syndrome.

BACKGROUND: Mycosis fungoides (MF) and Sézary syndrome (SS) are common subtypes of cutaneous T-cell lymphoma that primarily affect the skin but may spread to the lymph nodes, viscera and blood. The symptom burden may compromise health-related quality of life (HRQL). The phase 3 MAVORIC study (ClinicalTrials.gov identifier NCT01728805) in patients with relapsed/refractory MF/SS reported improved HRQL with mogamulizumab compared with vorinostat. OBJECTIVES: Use baseline (pre-treatment) data from the MAVORIC study to describe the symptom burden of MF/SS and identify characteristics associated with worse HRQL. METHODS: Data were from 372 adults with stage IB-IVB histologically confirmed relapsed/ refractory MF or SS. Associations between demographic and medical history variables and worse HRQL (Skindex-29, ItchyQol and Functional Assessment of Cancer Therapy - General [FACT-G]) were determined by regression models. RESULTS: In the cohort of 372 adults, 70% were white; 42% were female; mean age was 63 (SD 13.0) years. Fifty-five per cent had MF and 45% had SS; 77% had advanced (stage IIB-IV) disease, involving the skin in all patients and the blood and/or nodes in 66%. HRQL scores showed impairment versus normative means (where available), with the greatest impact on Symptoms and Emotions in the Skindex-29, Functioning in the ItchyQol, and Functional Wellbeing in the FACT-G. In regression analysis, worse HRQL across all domains and total score was associated with being female and younger, worse mSWAT score and worse itch for the Skindex-29 (n = 352), and being female, younger, Black/African American, worse performance status and worse itch for the ItchyQol (n = 369). Associations across domains and total score were not found for the FACT-G. Associations between domains and demographic/medical history were seen for all instruments. CONCLUSIONS: The symptoms of advanced MF/SS compromise all HRQL domains. Treatment goals and therapeutic choice should be informed by individual patients' disease burden.

The effect of a novel, digital physical activity and emotional well-being intervention on health-related quality of life in people with chronic kidney disease: trial design and baseline data from a multicentre prospective, wait-list randomised controlled trial (kidney BEAM).

BACKGROUND: Physical activity and emotional self-management has the potential to enhance health-related quality of life (HRQoL), but few people with chronic kidney disease (CKD) have access to resources and support. The Kidney BEAM trial aims to evaluate whether an evidence-based physical activity and emotional wellbeing self-management programme (Kidney BEAM) leads to improvements in HRQoL in people with CKD. METHODS: This was a prospective, multicentre, randomised waitlist-controlled trial, with health economic analysis and nested qualitative studies. In total, three hundred and four adults with established CKD were recruited from 11 UK kidney units. Participants were randomly assigned to the intervention (Kidney BEAM) or a wait list control group (1:1). The primary outcome was the between-group difference in Kidney Disease Quality of Life (KDQoL) mental component summary score (MCS) at 12 weeks. Secondary outcomes included the KDQoL physical component summary score, kidney-specific scores, fatigue, life participation, depression and anxiety, physical function, clinical chemistry, healthcare utilisation and harms. All outcomes were measured at baseline and 12 weeks, with long-term HRQoL and adherence also collected at six months follow-up. A nested qualitative study explored experience and impact of using Kidney BEAM. RESULTS: 340 participants were randomised to Kidney BEAM (n = 173) and waiting list (n = 167) groups. There were 96 (55%) and 89 (53%) males in the intervention and waiting list groups respectively, and the mean (SD) age was 53 (14) years in both groups. Ethnicity, body mass, CKD stage, and history of diabetes and hypertension were comparable across groups. The mean (SD) of the MCS was similar in both groups, 44.7 (10.8) and 45.9 (10.6) in the intervention and waiting list groups respectively. CONCLUSION: Results from this trial will establish whether the Kidney BEAM self management programme is a cost-effective method of enhancing mental and physical wellbeing of people with CKD. TRIAL REGISTRATION: NCT04872933. Registered 5th May 2021.

Contact sensitisation in oral lichen planus: An Australian perspective.

BACKGROUND: The role of contact sensitisation in the pathogenesis of oral lichen planus (OLP) has not been clearly defined. OBJECTIVE: We aimed to evaluate relevant contact sensitisers in OLP. METHODS: A retrospective study was conducted on OLP patients who underwent patch testing from 1 January 2006 to 31 December 2020 at an Australian tertiary dermatology institution, compared to cheilitis patients patch tested over the same time period. RESULTS: Ninety-six OLP patients and 152 cheilitis patients were patch tested during the 15-year period. Seventy-one (73.9%) OLP patients and 100 (65.8%) cheilitis patients recorded one or more relevant reactions. Forty-three (44.8%), 22 (22.9%), 21 (21.9%) and 17 (17.7%) OLP patients had relevant reactions to mercury-related chemicals, amalgam, spearmint and carvone, respectively, compared to 6 (3.9%), 3 (2.0%), 4 (2.6%) and 0 (0%) cheilitis patients, respectively (p-value <0.001 each). Four (4.2%) OLP patients had relevant positive reactions to sodium metabisulfite, compared to none in the cheilitis group (p-value 0.021). CONCLUSION: While dental amalgam is used less frequently these days, we report that mercury (found in amalgam) and additionally spearmint and carvone are relevant sensitisers in OLP in Australia. Sodium metabisulfite may also be a relevant sensitiser in OLP, which has not previously been reported.

Symptom-burden in people living with frailty and chronic kidney disease.

BACKGROUND: Frailty is independently associated with worse health-related quality of life (HRQOL) in chronic kidney disease (CKD). However, the relationship between frailty and symptom experience is not well described in people living with CKD. This study's aim was to evaluate the relationship between frailty and symptom-burden in CKD. METHODS: This study is a secondary analysis of a cross-sectional observational study, the QCKD study (ISRCTN87066351), in which participants completed physical activity, cardiopulmonary fitness, symptom-burden and HRQOL questionnaires. A modified version of the Frailty Phenotype, comprising 3 self-report components, was created to assess frailty status. Multiple linear regression was performed to assess the association between symptom-burden/HRQOL and frailty. Logistic regression was performed to assess the association between experiencing symptoms frequently and frailty. Principal Component Analysis was used to assess the experienced symptom clusters. RESULTS: A total of 353 patients with CKD were recruited with 225 (64%) participants categorised as frail. Frail participants reported more symptoms, had higher symptom scores and worse HRQOL scores. Frailty was independently associated with higher total symptom score and lower HRQOL scores. Frailty was also independently associated with higher odds of frequently experiencing 9 out of 12 reported symptoms. Finally, frail participants experienced an additional symptom cluster that included loss of appetite, tiredness, feeling cold and poor concentration. CONCLUSIONS: Frailty is independently associated with high symptom-burden and poor HRQOL in CKD. Moreover, people living with frailty and CKD have a distinctive symptom experience. Proactive interventions are needed that can effectively identify and address problematic symptoms to mitigate their impact on HRQOL.

Pooled analysis of two randomized, double-blind trials comparing proposed biosimilar LA-EP2006 with reference pegfilgrastim in breast cancer.

BACKGROUND: Following the functional and physicochemical characterization of a proposed biosimilar, comparative clinical studies help to confirm biosimilarity by demonstrating similar safety and efficacy to the reference product in a sensitive patient population. PATIENTS AND METHODS: LA-EP2006 is a proposed biosimilar that has been developed for pegfilgrastim, a long-acting form of granulocyte colony-stimulating factor for the prevention of neutropenia. The current analysis reports data pooled from two independent, multinational, prospective, randomized, controlled, double-blind phase III studies of similar design comparing the safety and efficacy of reference pegfilgrastim with LA-EP2006 in patients with breast cancer receiving myelotoxic (neo)adjuvant TAC (docetaxel, doxorubicin, and cyclophosphamide) chemotherapy and requiring granulocyte colony-stimulating factor. RESULTS: A total of 624 patients were randomized in the PROTECT-1 and PROTECT-2 studies (NCT01735175; NCT01516736) (LA-EP2006: n = 314; reference: n = 310). Baseline characteristics of patients were well balanced across treatment groups. The primary end point, mean duration of severe neutropenia in the first chemotherapy cycle was similar in both the LA-EP2006 and reference groups (1.05 ± 1.055 days versus 1.01 ± 0.958 days), with a treatment difference of - 0.04 days [95% confidence interval (CI): -0.19 to 0.11] that met the equivalence criteria (the 95% CI were within the defined margin of ±1 day). Secondary end points, such as the nadir of absolute neutrophil count and the incidence of febrile neutropenia, were also similar between LA-EP2006 and reference pegfilgrastim. The safety and tolerability profile of LA-EP2006 was similar to that observed with reference pegfilgrastim, and there were no reports of neutralizing antibodies. CONCLUSIONS: This pooled analysis confirms, as a part of totality of evidence approach, that the proposed biosimilar pegfilgrastim LA-EP2006 has a comparable efficacy and safety profile to reference pegfilgrastim in patients with breast cancer receiving TAC chemotherapy. CLINICAL TRIAL NUMBERS: NCT01735175 and NCT01516736.

Lubricin/proteoglycan 4 increases in both experimental and naturally occurring equine osteoarthritis.

OBJECTIVE: The goals of this study were (1) to quantify proteoglycan 4 (PRG4) gene expression; (2) to assess lubricin immunostaining; and (3) to measure synovial fluid lubricin concentrations in clinical and experimental models of equine carpal osteoarthritis (OA). DESIGN: Lubricin synovial fluid concentrations and cartilage and synovial membrane PRG4 expression were analyzed in research horses undergoing experimental OA induction (n = 8) and in equine clinical patients with carpal OA (n = 58). Lubricin concentrations were measured using a custom sandwich enzyme-linked immunosorbent assay, and PRG4 expression was quantified using qRT-PCR. Lubricin immunostaining was assessed in synovial membrane and osteochondral sections in the experimental model. RESULTS: Lubricin concentrations increased in synovial fluid following induction of OA, peaking at 21 days post-operatively in OA joints vs sham-operated controls (331 ± 69 µg/mL vs 110 ± 19 µg/mL, P = 0.001). Lubricin concentrations also increased in horses with naturally occurring OA as compared to control joints (152 ± 32 µg/mL vs 68 ± 4 µg/mL, P = 0.003). Synovial membrane PRG4 expression increased nearly 2-fold in naturally occurring OA (P = 0.003), whereas cartilage PRG4 expression decreased 2.5-fold (P = 0.025). Lubricin immunostaining was more pronounced in synovial membrane from OA joints as compared to controls, with intense lubricin localization to sites of cartilage damage. CONCLUSIONS: Although PRG4 gene expression decreases in OA cartilage, synovial membrane PRG4 expression, synovial fluid lubricin concentrations and lubricin immunostaining all increase in an equine OA model. Lubricin may be elevated to protect joints from post-traumatic OA.

A radiologic and anatomic assessment of injectate spread following transmuscular quadratus lumborum block in cadavers.

We performed bilateral transmuscular quadratus lumborum blocks in six cadavers using iodinated contrast and methylene blue. Computed tomography imaging was performed in four cadavers and anatomical dissection was completed in five. This demonstrated spread to the lumbar paravertebral space in 63% of specimens, laterally to the transversus abdominis muscle in 50% and caudally to the anterior superior iliac spine in 63% of specimens. There was no radiographic evidence of spread to the thoracic paravertebral space. Anatomical dissection revealed dye staining of the upper branches of the lumbar plexus and the psoas major muscle in 70% of specimens. Further clinical studies are required to confirm if the quadratus lumborum block might be a suitable alternative to lumbar plexus block.

Efficacy of the nanoparticle-drug conjugate CRLX101 in combination with bevacizumab in metastatic renal cell carcinoma: results of an investigator-initiated phase I-IIa clinical trial.

BACKGROUND: Anti-angiogenic therapies are effective in metastatic renal cell carcinoma (mRCC), but resistance is inevitable. A dual-inhibition strategy focused on hypoxia-inducible factor (HIF) is hypothesized to be active in this refractory setting. CRLX101 is an investigational camptothecin-containing nanoparticle-drug conjugate (NDC), which durably inhibits HIF1α and HIF2α in preclinical models and in gastric cancer patients. Synergy was observed in the preclinical setting when combining this NDC and anti-angiogenic agents, including bevacizumab. PATIENTS AND METHODS: Patients with refractory mRCC were treated every 2 weeks with bevacizumab (10 mg/kg) and escalating doses of CRLX101 (12, 15 mg/m(2)) in a 3 + 3 phase I design. An expansion cohort of 10 patients was treated at the recommended phase II dose (RP2D). Patients were treated until progressive disease or prohibitive toxicity. Adverse events (AEs) were assessed using CTCAE v4.0 and clinical outcome using RECIST v1.1. RESULTS: Twenty-two patients were response-evaluable in an investigator-initiated trial at two academic medical centers. RCC histologies included clear cell (n = 12), papillary (n = 5), chromophobe (n = 2), and unclassified (n = 3). Patients received a median of two prior therapies, with at least one prior vascular endothelial tyrosine kinase inhibitor therapy (VEGF-TKI). No dose-limiting toxicities were observed. Grade ≥3 AEs related to CRLX101 included non-infectious cystitis (5 events), fatigue (3 events), anemia (2 events), diarrhea (2 events), dizziness (2 events), and 7 other individual events. Five of 22 patients (23%) achieved partial responses, including 3 of 12 patients with clear cell histology and 2 of 10 patients (20%) with non-clear cell histology. Twelve of 22 patients (55%) achieved progression-free survival (PFS) of >4 months. CONCLUSIONS: CRLX101 combined with bevacizumab is safe in mRCC. This combination fulfilled the protocol's predefined threshold for further examination with responses and prolonged PFS in a heavily pretreated population. A randomized phase II clinical trial in mRCC of this combination is ongoing.

A chondrocyte infiltrated collagen type I/III membrane (MACI® implant) improves cartilage healing in the equine patellofemoral joint model.

UNLABELLED: Autologous chondrocyte implantation (ACI) has improved outcome in long-term studies of joint repair in man. However, ACI requires sutured periosteal flaps to secure the cells, which precludes minimally-invasive implantation, and introduces complications with arthrofibrosis and graft hypertrophy. This study evaluated ACI on a collagen type I/III scaffold (matrix-induced autologous chondrocyte implantation; MACI(®)) in critical sized defects in the equine model. METHODS: Chondrocytes were isolated from horses, expanded and seeded onto a collagen I/III membrane (ACI-Maix™) and implanted into one of two 15-mm defects in the femoral trochlear ridge of six horses. Control defects remained empty as ungrafted debrided defects. The animals were examined daily, scored by second look arthroscopy at 12 weeks, and necropsy examination 6 months after implantation. Reaction to the implant was determined by lameness, and synovial fluid constituents and synovial membrane histology. Cartilage healing was assessed by arthroscopic scores, gross assessment, repair tissue histology and immunohistochemistry, cartilage glycosaminoglycan (GAG) and DNA assay, and mechanical testing. RESULTS: MACI(®) implanted defects had improved arthroscopic second-look, gross healing, and composite histologic scores, compared to spontaneously healing empty defects. Cartilage GAG and DNA content in the defects repaired by MACI implant were significantly improved compared to controls. Mechanical properties were improved but remained inferior to normal cartilage. There was minimal evidence of reaction to the implant in the synovial fluid, synovial membrane, subchondral bone, or cartilage. CONCLUSIONS: The MACI(®) implant appeared to improve cartilage healing in a critical sized defect in the equine model evaluated over 6 months.

Development of the Osteoporosis Assessment Questionnaire--physical Function (OPAQ-PF): an osteoporosis-targeted, patient-reported outcomes (PRO) measure of physical function.

UNLABELLED: We have developed a short, patient-reported outcome questionnaire--the Osteoporosis Assessment Questionnaire--Physical Function (OPAQ-PF)--that assesses the impact of osteoporosis on physical function. OPAQ-PF contains 15 items in three domains (mobility, physical positions, and transfers) and has content validity in osteoporosis patients with and without a history of fracture. INTRODUCTION: This paper describes the development of the Osteoporosis Assessment Questionnaire--Physical Function (OPAQ-PF), a patient-reported outcome (PRO) questionnaire based on OPAQ v.2.0 (60 items, 14 domains) that assesses the impact of osteoporosis on physical function. METHODS: OPAQ v.2.0 was administered to patients with osteoporosis. Item response theory methodology and clinical judgment were used to retain/eliminate items. The resulting instrument was modified during two sets of concept elicitation and cognitive debriefing interviews with osteoporosis patients. RESULTS: Item response theory-based analysis of OPAQ v.2.0 (n = 1,478) coupled with clinician input resulted in the generation of a 21-item, six-domain instrument with a frequency response format. Interview data from 32 participants were used to modify this version and led to generation of the final instrument, OPAQ-PF. This final version has a severity response format and contains 15 items in three domains (mobility, physical positions, and transfers) that group together to provide an overall assessment of physical function in patients with osteoporosis. Twenty-two of the 32 interview participants (69 %) had previously sustained a fracture. Symptoms occurred primarily in these patients. CONCLUSIONS: OPAQ-PF represents a brief, focused, PRO instrument that assesses physical function in patients with osteoporosis, specifically related to mobility, physical positions, and transfers. This questionnaire has content validity in osteoporosis patients who have, and have not, sustained a prior fracture.

Osteoporosis Assessment Questionnaire-Physical Function (OPAQ-PF): a psychometrically validated osteoporosis-targeted patient reported outcome measure of daily activities of physical function.

UNLABELLED: The purpose of this study was to evaluate the measurement properties of the Osteoporosis Assessment Questionnaire-Physical Functioning (OPAQ-PF). Based on this study, the OPAQ-PF has confirmed unidimensionality and acceptable reliability, construct validity, and sensitivity to change in a recent fracture/no recent fracture osteoporosis sample. METHODS: Dimensionality was established through exploratory and confirmatory factor analysis. Patients completed three patient reported outcome (PRO) measures and four performance-based measures (PBMs) at baseline to enable an evaluation of construct validity. Patients without a recent fracture completed the OPAQ-PF 2 weeks after baseline to enable an evaluation of test-retest reliability. Ability to detect change and interpretation of change were investigated following completion of the OPAQ-PF 12 and 24 weeks postbaseline by patients with a recent fracture. RESULTS: A prospective psychometric validation study in 144 postmenopausal women, with moderate to severe osteoporosis, 37 of whom had experienced a recent fragility fracture (<6 weeks). Unidimensionality was established for the OPAQ-PF by factor analysis. The OPAQ-PF had good internal consistency (α = 0.974) and test-retest reliability (mean intraclass correlation coefficient (ICC) 0.993. The OPAQ-PF differentiated between patients with/without recent fracture, and by severity of osteoarthritis; it correlated strongly with hypothesized-related scales and PBMs (r > 0.3, p < 0.001). Ability to detect change was established with high correlations between changes in OPAQ-PF score and changes in global concept scores in recent fracture patients (r ≥ 0.6, 24-week change). Effect size of change on OPAQ-PF score increased by level of global change (p < 0.001). Anchor-based methods identified an OPAQ-PF change of 10 at an individual patient level and 20 at a group level as meaningful to patients. CONCLUSIONS: The OPAQ-PF has confirmed unidimensionality and acceptable reliability, construct validity, and sensitivity to change in a recent fracture/no recent fracture osteoporosis sample.

Patient-reported fatigue and its impact on patients with systemic lupus erythematosus.

Fatigue is a hallmark symptom of systemic lupus erythematosus (SLE), often associated with flares, side effects of treatment, and extensive organ damage and may have a significant impact on health-related quality of life (HrQoL). To date, the experience of fatigue in patients with SLE is underexplored. This study explored the experience of fatigue in patients with SLE and its impact on their lives through qualitative interviews. This cross-sectional qualitative study was conducted with 22 adult patients with SLE, recruited from two clinical sites in the United States. In-person semi-structured interviews were conducted and thematic analysis was performed focusing on the experience of fatigue in SLE. Results indicated that 21 out of 22 patients experienced fatigue due to SLE. Patients reported that fatigue was variable in nature in terms of both severity and frequency. Fatigue was described as having an impact on multiple aspects of a patient's life: emotions, cognition, work, activities of daily living, leisure activities, social activities, and family activities. Understanding how patients with SLE describe the symptom of fatigue and how it impacts their lives is the key to better understanding how to measure fatigue in clinical studies evaluating new treatments for SLE.

The Limitations of Frailty Assessment Tools in ANCA-Associated Vasculitis.

Anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) can be associated with a high burden of morbidity and mortality in an ageing population. It is increasingly recognised that individualised management is needed. Few studies have looked specifically at frailty related outcomes in AAV and a gap remains in understanding the application of frailty assessment tools in these patients. We carried out a single centre, cohort study between 2017 to 2022. Forty-one patients who had newly diagnosed or relapsing AAV and aged ≥65 years were included. The Clinical Frailty Scale (CFS) score at presentation was assessed by health care practitioners and interval CFS scores were carried out a minimum of 6 weeks from diagnosis. The aim was to determine if patients living with frailty had worse outcomes or if their perceived frailty improved with immunosuppressive treatment. The median CFS at diagnosis was 4 (vulnerable) and this remained at follow up. There was no significant interval change in CFS (P=0.16) suggesting that the patients did not become frailer and instead there was a tendency towards improved frailty scores at re-assessment. There was no significant difference in end stage kidney disease between those with higher (>5) or lower (≤5) CFS (P=1.0), although crude mortality was higher among those with an initial CFS >5 (P=0.03). Overall, we demonstrated that CFS has limitations in determining patients that may be frail as a result of disease burden with the potential to improve with treatment and clinicians should be mindful of this when making decisions relating to management.

Development of a conceptual model of health-related quality of life for systemic lupus erythematosus from the patient's perspective.

AIMS: The aim of this study was to evaluate the impact of systemic lupus erythematosus (SLE) on the lives of patients in order to inform the development of a conceptual model. METHODS: Twenty-two participants with SLE (defined as meeting four of the 11 ACR criteria) were recruited for this study. Semi-structured, in-person interviews were conducted with each participant, exploring the symptoms experienced and the impact on the patient's life. Thematic analysis of interview transcripts was conducted in ATLAS.ti software to identify areas of impact and explore the interrelationships between concepts to inform the development of a conceptual model. RESULTS: Almost all participants were female (95%); the sample was diverse in terms of age (mean age of 45.5 years; age range of 20-60 years), ethnicity (59% black/African American) and disease duration. Commonly reported symptoms were pain, fatigue/tiredness and skin problems. Qualitative analysis revealed seven themes relating to the impact of SLE symptoms on patient's Health Related Quality of Life (HRQL): emotions, social, family and leisure activities, daily activities, cognition, appearance, employment activities and independence. The interrelationships between symptoms, impacts and symptom triggers are illustrated in a conceptual model. CONCLUSIONS: The conceptual model illustrates the wide-reaching impact of SLE symptoms on a patient's HRQL, and the potential broad impact of a treatment that improves SLE symptoms.

Olive oil polyphenol oleuropein inhibits smooth muscle cell proliferation.

OBJECTIVES: The Mediterranean diet, with a high content of olive oil, is associated with a reduced risk of coronary artery disease. The aim of this study was to determine the effect of oleuropein, one of the polyphenols in olive oil, on vascular smooth muscle cell (SMC) proliferation in vitro. DESIGN: This was an experimental study. MATERIALS AND METHODS: Bovine vascular SMCs were cultured in the presence of 100 µM of oleuropein. On days 1, 3 and 5, cell number was counted. Cell cycle analysis was performed by flow cytometry. Cell cycle regulators were assessed by immunoblotting. RESULTS: Cell proliferation in the presence of oleuropein was significantly inhibited by 92%. Cell cycle analysis revealed that oleuropein treatment blocked cells in the G1-S phase compared with the non-treated group. Among G1 phase regulators, retinoblastoma protein, cyclinD, p21 and p27 were not affected by oleuropein, but extracellular signal-regulated kinase 1/2 (ERK1/2) activation was inhibited. Growth of SMC treated with 100 µM of the mitogen-activated protein (MAP) kinase/ERK kinase 1 (MEK1) inhibitor PD98059 was also significantly inhibited by 70%. CONCLUSIONS: Oleuropein inhibited SMC proliferation through a cell cycle block between the G1 and the S phases, which may be regulated by ERK1/2. These results suggest a mechanism by which olive oil consumption may have beneficial effects on cardiovascular mortality by inhibiting SMC proliferation.

Cytokine and catabolic enzyme expression in synovium, synovial fluid and articular cartilage of naturally osteoarthritic equine carpi.

REASONS FOR PERFORMING STUDY: Understanding the expression of catabolic and anabolic genes during osteoarthritis progression should help to identify the major mediators of the disease. OBJECTIVE: To compare the cytokine and anabolic marker concentrations in synovium, synovial fluid and cartilage between normal and osteoarthritic joints. METHODS: Carpi from horses age 2-11 years were used. Tissues were harvested at the time of surgery or euthanasia, and RNA was isolated for RT-PCR analysis. Tumour necrosis factor alpha (TNFα), interleukin-1beta (IL-1ß), aggrecanase 1 (ADAMTS-4), aggrecanase 2 (ADAMTS-5), matrix metalloproteinase-13 (MMP-13), interleukin 17 (IL-17) and collagen type I alpha 1(Col-1) expression were determined in synovium. TNFα, IL-1ß, ADAMTS-4, ADAMTS-5, MMP-13, IL-17, collagen type IIB (Col-2B), and aggrecan expression were determined in cartilage. TNFα concentration in the synovial fluid was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Expression of TNFα, ADAMTS-5 and MMP-13 was significantly increased in synovial tissue from OA joints. Synovial membrane IL-1ß abundance showed only moderate elevations in OA, without reaching significant levels. Cytokine expression was increased significantly in OA cartilage samples, particularly TNFα, IL-1ß, ADAMTS-4 and MMP-13; and collagen type I expression was significantly increased in synovial tissues from OA groups. Collagen type II message was diminished in mild and moderate stages of OA, but rebounded to significant elevations in severely degenerate joints. Conversely, aggrecan levels significantly declined in cartilage from all OA groups. Synovial fluid TNFα peptide concentration was significantly increased in severe OA cases. CONCLUSION: TNFα was increased in all degrees of equine OA, and was abundantly expressed in synovial membrane and cartilage. IL-1ß was overexpressed in OA cartilage, but not to a significant extent in synovium. POTENTIAL RELEVANCE: Control of TNFα should be considered further as a target in the treatment of OA. ADAMTS-4 may be the primary aggrecanase causing cartilage breakdown in OA.

Development of a novel equine whole transcript oligonucleotide GeneChip microarray and its use in gene expression profiling of normal articular-epiphyseal cartilage.

REASONS FOR PERFORMING STUDY: No large scale equine microarray is available commercially to allow genomic and transcriptional profiling of the majority of genes that would define the genetic basis of equine disease. OBJECTIVES: To generate a whole transcript target labelled GeneChip to interrogate the equine transcriptome and validate chip performance using RNA samples derived from organs, articular cells and normal cartilage. METHODS: Equine mRNA and selected equine gene sequences derived from perfect cross-hybridisation of equine RNA on human microarray GeneChips, were used to design a custom equine gene microarray. Sequence data were used as a template for generation of a glass-slide based 5'-3' multi-exon-encompassing gene chip. The microarray was characterised using RNA derived from organs including spleen, liver, brain and kidney, and RNA from cultured chondrocytes, cartilage, synovial tissue and stem cells, employing a whole transcript target labelling assay to sample mRNA across the 5'-3' spectrum. RESULTS: The custom microarray simultaneously interrogated over 12,300 equine specific genes. Probing the chip with mixtures of total RNA derived from parenchymatous organs and articular tissues resulted in 61.7 and 62.8% present calls, respectively. This gene chip provided expression information on up to 90% of the key molecules in important signalling, metabolic and development pathways. Cartilage specific matrix genes were abundantly expressed in normal articular cartilage, but surprisingly high levels of collagen types I, III, V and XI, reflected expression from the epiphyseal layers of maturing articular epiphyseal cartilage. CONCLUSION: An oligonucleotide microarray with over 12,300 probe sets was generated by uniquely combining a labelling strategy incorporating expressed sequence tags from the entire transcriptome and supplementing selected human sequences that cross-hybridised with the horse. Validation showed robust performance of the microarray. POTENTIAL RELEVANCE: This array may be a useful tool to elucidate the pathogenesis of equine diseases.

The low VEGF production allele of the +936C/T polymorphism is strongly associated with increased risk for oral cancer.

PURPOSE: Based on the well-established role of vascular endothelial growth factor (VEGF) in tumor-associated angiogenesis in several cancer types and its undefined role in oral oncogenesis, we investigated the possible association of an expression-regulating polymorphism (+936C/T) with risk for oral squamous cell carcinoma (OSCC). METHODS: We studied the allele frequencies of the +936C/T polymorphism in DNA samples of 144 patients with OSCC and 153 healthy controls matched by age, gender and ethnicity, using restriction fragment length polymorphism typing analysis. RESULTS: The low-expression T allele was significantly increased in the total patient group compared to controls (P = 0.008), due to a significant over-representation of C/T heterozygotes compared to C/C homozygotes (P = 0.007). The same pattern was observed in most patient subgroups and more noticeably in patients with a positive family history of cancer (P = 0.001). Interestingly, the increase in T allele frequency was only significant in patients at cancer stages I and II (P = 0.006). CONCLUSIONS: This study clearly indicates that the low-VEGF-production T allele is strongly associated with increased risk for OSCC. In addition, the impressive T allele frequency increment in patients with a positive family cancer history suggests that this allele may also be involved in other malignancies. The fact that this significant increase was observed only in patients with early cancer stages may imply that low VEGF levels might hinder subsequent tumorigenesis. Our findings might be the result of either unidentified properties of the +936 C/T polymorphism or of a strong linkage disequilibrium between this polymorphism and another genetic locus.

Using an AraC-based three-hybrid system to detect biocatalysts in vivo.

Recent methods to create large libraries of proteins have greatly advanced the discovery of proteins with novel functions. However, one limitation in the discovery of new biocatalysts is the screening or selection methods employed to find enzymes from these libraries. We have developed a potentially general method termed QUEST (QUerying for EnzymeS using the Three-hybrid system), which allows the construction of an easily screened or selected phenotype for, in theory, any type of enzymatic reaction. The method couples the in vivo concentration of an enzyme's substrate to changes in the transcriptional level of a reporter operon. Using the arabinose operon activator AraC, we constructed a system capable of detecting the fungal enzyme scytalone dehydratase (SD) in bacteria, and demonstrated its sensitivity and usefulness in library screening.

Genetic modification of chondrocytes with insulin-like growth factor-1 enhances cartilage healing in an equine model.

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 x 10(7) AdIGF-1 modified chondrocytes and the contralateral joint received 2 x 10(7) naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months. Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model. The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation.