Telomerase activation cooperates with inactivation of p16 in early head and neck tumorigenesis

Alteration of the p16/pRb pathway may cooperate with telomerase activation during cellular immortalization and tumour progression. We studied p16 expression status by immunohistochemistry and telomerase activity using the TRAP assay in 21 premalignant lesions of the head and neck epithelium as well as 27 squamous-cell carcinomas. We also examined expression of other components of the pathway (cyclin D1 and pRb) as well as presence of human papillomavirus genomes which can target these molecules. 4 of 9 mild dysplastic lesions (44%), 8 of 12 moderate/severe dysplastic lesions (67%), and 25 of 27 squamous-cell carcinomas (92%) demonstrated high telomerase activity (P = 0.009). There was a parallel increase with severity of lesions for the trend in proportions of cases demonstrating p16 inactivation or cyclin D1 overexpression (P = 0.02 and P = 0.01, respectively). For Ki67, a marker of cell proliferation, this trend was not significant (P = 0.08). Human papillomavirus infection was only found in 4 cases among the 48 samples tested (8.3%). In conclusion, progression of disease is accompanied by a parallel and continuous increase in telomerase activity and alterations in cell cycle regulators (p16, cyclin D1), as proposed by in vitro models. © 2001 Cancer Research Campaign http://www.bjcancer.co

An Interdisciplinary Diagnostic Approach to Guide Therapy in C3 Glomerulopathy

Since the re-classification of membranoproliferative glomerulonephritis the new disease entity C3 glomerulopathy is diagnosed if C3 deposition is clearly dominant over immunoglobulins in immunohistochemistry or immunofluorescence. Although this new definition is more orientated at the pathophysiology as mediated by activity of the alternative complement pathway C3 glomerulopathy remains a heterogenous group of disorders. Genetic or autoimmune causes are associated in several but not in all patients with this disease. However, prognosis is poorly predictable, and clinicians cannot directly identify patients that might benefit from therapy. Moreover, therapy may range from supportive care alone, unspecific immune suppression, plasma treatment, or plasma exchange to complement inhibition. The current biopsy based diagnostic approaches sometimes combined with complement profiling are not sufficient to guide clinicians neither (i) whether to treat an individual patient, nor (ii) to choose the best therapy. With this perspective, we propose an interdisciplinary diagnostic approach, including detailed analysis of the kidney biopsy for morphological alterations and immunohistochemical staining, for genetic analyses of complement genes, complement activation patterning in plasma, and furthermore for applying novel approaches for convertase typing and complement profiling directly in renal tissue. Such a combined diagnostic approach was used here for a 42-year-old female patient with a novel mutation in the Factor H gene, C3 glomerulopathy and signs of chronic endothelial damage. We present here an approach that might in future help to guide therapy of renal diseases with relevant complement activation, especially since diverse new anti-complement agents are under clinical investigation

'SOSORT consensus paper on brace action: TLSO biomechanics of correction (investigating the rationale for force vector selection)'

BACKGROUND: The effectiveness of orthotic treatment continues to be controversial in international medical literature due to differences in the reported results and conclusions of various studies. Heterogeneity of the samples has been suggested as a reason for conflicting results. Besides the obvious theoretical differences between the brace concepts, the variability in the technical factors can also explain the contradictory results between same brace types. This paper will investigate the degree of variability among responses of scoliosis specialists from the Brace Study Ground of the International Society on Scoliosis Orthopedic and Rehabilitation Treatment SOSORT. Ultimately, this information could be a foundation for establishing a consensus and framework for future prospective controlled studies. METHODS: A preliminary questionnaire on the topic of 'brace action' relative to the theory of three-dimensional scoliosis correction and brace treatment was developed and circulated to specialists interested in the conservative treatment of adolescent idiopathic scoliosis. A particular case was presented (main thoracic curve with minor lumbar). Several key points emerged and were used to develop a second questionnaire which was discussed and full filed after the SOSORT consensus meeting (Milano, Italy, January 2005). RESULTS: Twenty-one questionnaires were completed. The Chêneau brace was the most frequently recommended. The importance of the three point system mechanism was stressed. Options about proper pad placement on the thoracic convexity were divided 50% for the pad reaching or involving the apical vertebra and 50% for the pad acting caudal to the apical vertebra. There was agreement about the direction of the vector force, 85% selecting a 'dorso lateral to ventro medial' direction but about the shape of the pad to produce such a force. Principles related to three-dimensional correction achieved high consensus (80%–85%), but suggested methods of correction were quite diverse. CONCLUSION: This study reveals that among participating SOSORT specialists there continues to be a strongly held and conflicting if not a contentious opinion regarding brace design and treatment. If the goal of a 'treatment consensus' is realistic and achievable, significantly more effort will be required to reconcile these differences

Increased plasma level of terminal complement complex in AMD patients: potential functional consequences for RPE cells

Purpose: Polymorphisms in complement genes are risk-associated for age-related macular degeneration (AMD). Functional analysis revealed a common deficiency to control the alternative complement pathway by risk-associated gene polymorphisms. Thus, we investigated the levels of terminal complement complex (TCC) in the plasma of wet AMD patients with defined genotypes and the impact of the complement activation of their plasma on second-messenger signaling, gene expression, and cytokine/chemokine secretion in retinal pigment epithelium (RPE) cells. Design: Collection of plasma from patients with wet AMD (n = 87: 62% female and 38% male; median age 77 years) and controls (n = 86: 39% female and 61% male; median age 58 years), grouped for risk factor smoking and genetic risk alleles CFH 402HH and ARMS2 rs3750846, determination of TCC levels in the plasma, in vitro analysis on RPE function during exposure to patients' or control plasma as a complement source. Methods: Genotyping, measurement of TCC concentrations, ARPE-19 cell culture, Ca2+ imaging, gene expression by qPCR, secretion by multiplex bead analysis of cell culture supernatants. Main outcome measures: TCC concentration in plasma, intracellular free Ca2+, relative mRNA levels, cytokine secretion. Results: TCC levels in the plasma of AMD patients were five times higher than in non-AMD controls but did not differ in plasma from carriers of the two risk alleles. Complement-evoked Ca2+ elevations in RPE cells differed between patients and controls with a significant correlation between TCC levels and peak amplitudes. Comparing the Ca2+ signals, only between the plasma of smokers and non-smokers, as well as heterozygous (CFH 402YH) and CFH 402HH patients, revealed differences in the late phase. Pre-stimulation with complement patients' plasma led to sensitization for complement reactions by RPE cells. Gene expression for surface molecules protective against TCC and pro-inflammatory cytokines increased after exposure to patients' plasma. Patients' plasma stimulated the secretion of pro-inflammatory cytokines in the RPE. Conclusion: TCC levels were higher in AMD patients but did not depend on genetic risk factors. The Ca2+ responses to patients' plasma as second-messenger represent a shift of RPE cells to a pro-inflammatory phenotype and protection against TCC. We conclude a substantial role of high TCC plasma levels in AMD pathology

Increased plasma level of terminal complement complex in AMD patients: potential functional consequences for RPE cells

PurposePolymorphisms in complement genes are risk-associated for age-related macular degeneration (AMD). Functional analysis revealed a common deficiency to control the alternative complement pathway by risk-associated gene polymorphisms. Thus, we investigated the levels of terminal complement complex (TCC) in the plasma of wet AMD patients with defined genotypes and the impact of the complement activation of their plasma on second-messenger signaling, gene expression, and cytokine/chemokine secretion in retinal pigment epithelium (RPE) cells.DesignCollection of plasma from patients with wet AMD (n = 87: 62% female and 38% male; median age 77 years) and controls (n = 86: 39% female and 61% male; median age 58 years), grouped for risk factor smoking and genetic risk alleles CFH 402HH and ARMS2 rs3750846, determination of TCC levels in the plasma, in vitro analysis on RPE function during exposure to patients’ or control plasma as a complement source.MethodsGenotyping, measurement of TCC concentrations, ARPE-19 cell culture, Ca2+ imaging, gene expression by qPCR, secretion by multiplex bead analysis of cell culture supernatants.Main outcome measuresTCC concentration in plasma, intracellular free Ca2+, relative mRNA levels, cytokine secretion.ResultsTCC levels in the plasma of AMD patients were five times higher than in non-AMD controls but did not differ in plasma from carriers of the two risk alleles. Complement-evoked Ca2+ elevations in RPE cells differed between patients and controls with a significant correlation between TCC levels and peak amplitudes. Comparing the Ca2+ signals, only between the plasma of smokers and non-smokers, as well as heterozygous (CFH 402YH) and CFH 402HH patients, revealed differences in the late phase. Pre-stimulation with complement patients’ plasma led to sensitization for complement reactions by RPE cells. Gene expression for surface molecules protective against TCC and pro-inflammatory cytokines increased after exposure to patients’ plasma. Patients’ plasma stimulated the secretion of pro-inflammatory cytokines in the RPE.ConclusionTCC levels were higher in AMD patients but did not depend on genetic risk factors. The Ca2+ responses to patients’ plasma as second-messenger represent a shift of RPE cells to a pro-inflammatory phenotype and protection against TCC. We conclude a substantial role of high TCC plasma levels in AMD pathology

Nuclear poly(A)-binding protein aggregates misplace a pre-mRNA outside of SC35 speckle causing its abnormal splicing

A short abnormal polyalanine expansion in the polyadenylate-binding protein nuclear-1 (PABPN1) protein causes oculopharyngeal muscular dystrophy (OPMD). Mutated PABPN1 proteins accumulate as insoluble intranuclear aggregates in muscles of OPMD patients. While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice have been established, the molecular mechanisms which trigger pathological defects in OPMD and the role of aggregates remain to be determined. Using exon array, for the first time we have identified several splicing defects in OPMD. In particular, we have demonstrated a defect in the splicing regulation of the muscle-specific Troponin T₃ (TNNT₃) mutually exclusive exons 16 and 17 in OPMD samples compared to controls. This splicing defect is directly linked to the SC₃₅ (SRSF2) splicing factor and to the presence of nuclear aggregates. As reported here, PABPN1 aggregates are able to trap TNNT₃ pre-mRNA, driving it outside nuclear speckles, leading to an altered SC₃₅ -mediated splicing. This results in a decreased calcium sensitivity of muscle fibers, which could in turn plays a role in muscle pathology. We thus report a novel mechanism of alternative splicing deregulation that may play a role in various other diseases with nuclear inclusions or foci containing an RNA binding protein

Pulsed electromagnetic fields after arthroscopic treatment for osteochondral defects of the talus: double-blind randomized controlled multicenter trial

Background. Osteochondral talar defects usually affect athletic patients. The primary surgical treatment consists of arthroscopic debridement and microfracturing. Although this is mostly successful, early sport resumption is difficult to achieve, and it can take up to one year to obtain clinical improvement. Pulsed electromagnetic fields (PEMFs) may be effective for talar defects after arthroscopic treatment by promoting tissue healing, suppressing inflammation, and relieving pain. We hypothesize that PEMF-treatment compared to sham-treatment after arthroscopy will lead to earlier resumption of sports, and aim at 25% increase in patients that resume sports. Methods/Design. A prospective, double-blind, randomized, placebo-controlled trial (RCT) will be conducted in five centers throughout the Netherlands and Belgium. 68 patients will be randomized to either active PEMF-treatment or sham-treatment for 60 days, four hours daily. They will be followed-up for one year. The combined primary outcome measures are (a) the percentage of patients that resume and maintain sports, and (b) the time to resumption of sports, defined by the Ankle Activity Score. Secondary outcome measures include resumption of work, subjective and objective scoring systems (American Orthopaedic Foot and Ankle Society Ankle-Hindfoot Scale, Foot Ankle Outcome Score, Numeric Rating Scales of pain and satisfaction, EuroQol-5D), and computed tomography. Time to resumption of sports will be analyzed using Kaplan-Meier curves and log-rank tests. Discussion. This trial will provide level-1 evidence on the effectiveness of PEMFs in the management of osteochondral ankle lesions after arthroscopy. Trial registration. Netherlands Trial Register (NTR1636)

PABPN1 gene therapy for oculopharyngeal muscular dystrophy

International audienceOculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset muscle disorder characterized by ptosis, swallowing difficulties, proximal limb weakness and nuclear aggregates in skeletal muscles. OPMD is caused by a trinucleotide repeat expansion in the PABPN1 gene that results in an N-terminal expanded polyalanine tract in polyA-binding protein nuclear 1 (PABPN1). Here we show that the treatment of a mouse model of OPMD with an adeno-associated virus-based gene therapy combining complete knockdown of endogenous PABPN1 and its replacement by a wild-type PABPN1 substantially reduces the amount of insoluble aggregates, decreases muscle fibrosis, reverts muscle strength to the level of healthy muscles and normalizes the muscle transcriptome. The efficacy of the combined treatment is further confirmed in cells derived from OPMD patients. These results pave the way towards a gene replacement approach for OPMD treatment

Repair, regenerative and supportive therapies of the annulus fibrosus: achievements and challenges

Lumbar discectomy is a very effective therapy for neurological decompression in patients suffering from sciatica due to hernia nuclei pulposus. However, high recurrence rates and persisting post-operative low back pain in these patients require serious attention. In the past decade, tissue engineering strategies have been developed mainly targeted to the regeneration of the nucleus pulposus (NP) of the intervertebral disc. Accompanying techniques that deal with the damaged annulus fibrous are now increasingly recognised as mandatory in order to prevent re-herniation to increase the potential of NP repair and to confine NP replacement therapies. In the current review, the requirements, achievements and challenges in this quickly emerging field of research are discussed