Accelerated aging of intervertebral discs in a mouse model of progeria

Intervertebral disc degeneration (IDD) is a common and debilitating disorder that results in reduced flexibility of the spine, pain, and reduced mobility. Risk factors for IDD include age, genetic predisposition, injury, and other environmental factors such as smoking. Loss of proteoglycans (PGs) contributes to IDD with advancing age. Currently there is a lack of a model for rapid investigation of disc aging and evaluation of therapeutic interventions. Here we examined progression of disc aging in a murine model of a human progeroid syndrome caused by deficiency of the DNA repair endonuclease, ERCC1–XPF (Ercc1^?/Δ mice). The ERCC1‐deficient mice showed loss of disc height and degenerative structural changes in their vertebral bodies similar to those reported for old rodents. Compared to their wild‐type littermates, Ercc1^?/Δ mice also exhibit other age‐related IDD characteristics, including premature loss of disc PG, reduced matrix PG synthesis, and enhanced apoptosis and cell senescence. Finally, the onset of age‐associated disc pathologies was further accelerated in Ercc1^?/Δ mice following chronic treatment with the chemotherapeutic agent mechlorethamine. These results demonstrate that Ercc1^?/Δ mice represent an accurate and rapid model of disc aging and provide novel evidence that DNA damage negatively impacts PG synthesis. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1600–1607, 2010     

Early-stage cervical cancer: Tumor delineation by magnetic resonance imaging and ultrasound — A European multicenter trial

ObjectiveTo compare the diagnostic accuracy of ultrasound (US) and magnetic resonance imaging (MRI) in the preoperative assessment of early-stage cervical cancer using pathologic findings as the reference standard.Patients and methodsProspective multi-center trial enrolling 209 consecutive women with early-stage cervical cancer (FIGO IA2–IIA) scheduled for surgery. The following parameters were assessed on US and MRI and compared to pathology: remaining tumor, size, tumor stromal invasion < 2/3 (superficial) or ≥ 2/3 (deep), and parametrial invasion.ResultsComplete data were available for 182 patients. The agreement between US and pathology was excellent for detecting tumors, correctly classifying bulky tumors (> 4 cm), and detecting deep stromal invasion (kappa values 0.84, 0.82, and 0.81 respectively); and good for classifying small tumors (< 2 cm) and detecting parametrial invasion (kappa values 0.78 and 0.75, respectively). The agreement between MRI and histology was good for classifying tumors as < 2 cm, or > 4 cm, and detecting deep stromal invasion (kappa values 0.71, 0.76, and 0.77, respectively). It was moderately accurate in tumor detection, and in assessing parametrial invasion (kappa values 0.52 and 0.45, respectively).The agreement between histology and US was significantly better in assessing residual tumor (p < 0.001) and parametrial invasion (p < 0.001) than the results obtained by MRI. Imaging methods were not significantly influenced by previous cone biopsy.ConclusionUS and MRI are highly accurate for the preoperative assessment of women with early-stage cervical cancer, although US may be more accurate in detecting residual tumors and assessing parametrial invasion.     

A methodology to ensure and improve accuracy of Ki67 labelling index estimation by automated digital image analysis in breast cancer tissue

Introduction

Immunohistochemical Ki67 labelling index (Ki67 LI) reflects proliferative activity and is a potential prognostic/predictive marker of breast cancer. However, its clinical utility is hindered by the lack of standardized measurement methodologies. Besides tissue heterogeneity aspects, the key element of methodology remains accurate estimation of Ki67-stained/counterstained tumour cell profiles. We aimed to develop a methodology to ensure and improve accuracy of the digital image analysis (DIA) approach.

Methods

Tissue microarrays (one 1-mm spot per patient, n = 164) from invasive ductal breast carcinoma were stained for Ki67 and scanned. Criterion standard (Ki67-Count) was obtained by counting positive and negative tumour cell profiles using a stereology grid overlaid on a spot image. DIA was performed with Aperio Genie/Nuclear algorithms. A bias was estimated by ANOVA, correlation and regression analyses. Calibration steps of the DIA by adjusting the algorithm settings were performed: first, by subjective DIA quality assessment (DIA-1), and second, to compensate the bias established (DIA-2). Visual estimate (Ki67-VE) on the same images was performed by five pathologists independently.

Results

ANOVA revealed significant underestimation bias (P < 0.05) for DIA-0, DIA-1 and two pathologists’ VE, while DIA-2, VE-median and three other VEs were within the same range. Regression analyses revealed best accuracy for the DIA-2 (R-square = 0.90) exceeding that of VE-median, individual VEs and other DIA settings. Bidirectional bias for the DIA-2 with overestimation at low, and underestimation at high ends of the scale was detected. Measurement error correction by inverse regression was applied to improve DIA-2-based prediction of the Ki67-Count, in particular for the clinically relevant interval of Ki67-Count < 40%. Potential clinical impact of the prediction was tested by dichotomising the cases at the cut-off values of 10, 15, and 20%. Misclassification rate of 5-7% was achieved, compared to that of 11-18% for the VE-median-based prediction.

Conclusions

Our experiments provide methodology to achieve accurate Ki67-LI estimation by DIA, based on proper validation, calibration, and measurement error correction procedures, guided by quantified bias from reference values obtained by stereology grid count. This basic validation step is an important prerequisite for high-throughput automated DIA applications to investigate tissue heterogeneity and clinical utility aspects of Ki67 and other immunohistochemistry (IHC) biomarkers.     

Muscle Fatigue Increases Metabolic Costs of Ergometer Cycling without Changing VO2 Slow Component

The aim of the present study was to investigate effects of muscle fatigue on oxygen costs of ergometer cycling and slow component of pulmonary oxygen uptake (VO₂) kinetics. Seven young men performed 100 drop jumps (drop height of 40 cm) with 20 s of rest after each jump. After the subsequent hour of rest, they cycled at 70, 105, 140 and 175 W, which corresponded to 29.6 ± 5.4, 39.4 ± 7.0, 50.8 ± 8.4 and 65.8 ± 11.8 % of VO_2peak, respectively, for 6 min at each intensity with 4-min intervals of rest in between the exercise bouts. The VO₂ response to cycling after the exercise (fatigue condition) was compared to ergometer cycling without prior exercise (control condition). From 3rd to 6th min of cycling at 105, 140 and 175 W, VO₂ was higher (p < 0.05-0.01) when cycling in the fatigue compared to the control condition. Slow component of VO₂ kinetics was observed when cycling at 175 W in the control condition (0.17 ± 0.09, l·min^-1, mean ± SD), but tended to decrease in the fatigue condition (0.13 ± 0.15 l·min^-1). In summary, results of the study are in agreement with the hypothesis that muscle fatigue increases oxygen costs of cycling exercise, but does not affect significantly the slow component of pulmonary oxygen uptake (VO₂) kinetics.

Key Points

• Repetitive fatiguing exercise induce an increase in metabolic costs of ergometer cycling exercise.
• It is argued that muscle pain, muscle temperature, elevated pulmonary ventilation and heart rate, shift towards from carbohydrate to fat metabolism are of minor importance in this phenomenon.
• Increased recruitment of type II fibres and impaired force transmission between muscle fibres due to damage of structural proteins appear to play the major role in reducing efficiency of ergometer cycling.

Key Words: Muscle fatigue, energy cost, oxygen uptake, oxygen consumption slow component     

Ex vivo gene therapy-induced endochondral bone formation: comparison of muscle-derived stem cells and different subpopulations of primary muscle-derived cells

Muscle-based gene therapy and tissue engineering hold great promise for improving bone healing. However, the relative advantage of muscle-derived stem cells (MDSCs) or primary muscle-derived cells (MDCs) remains to be defined. We compared the ability of MDSCs and different subpopulations of MDCs (PP1 and PP3) to induce bone formation via ex vivo gene therapy. We were able to efficiently transduce the MDSCs and all the other evaluated populations of MDCs (efficiency of TRANSDUCTION = approximately 80%) by using a retroviral vector expressing human bone morphogenetic protein 4 (BMP4). All the transduced cell populations secreted high levels of BMP4 (140–300 ng/10⁶ cells/24 h), but the MDSCs differentiated toward the osteogenic lineage more effectively than did the other muscle cell populations, as indicated by the expression of alkaline phosphatase, an early osteogenic marker. von Kossa staining indicated that mineralized bone formed as early as 7 days after implantation of any of the BMP4-expressing cell populations into immunocompetent syngeneic mice; however, MDSCs expressing BMP4 produced significantly more bone than did the other MDC populations, as evidenced by both histomorphometry and biochemical analysis. Further investigation revealed that MDSCs expressing BMP4 persisted for a significantly longer period of time at the bone forming sites than did the other BMP4-expressing MDC populations. Additionally, MDSCs expressing BMP4 triggered a smaller infiltration of CD4 lymphocytes within the bone forming areas than did the other MDC populations expressing BMP4. Finally, we demonstrated that MDSCs expressing BMP4 can heal a critical-sized skull bone defect in immunocompetent mice. In summary, this study shows that MDSCs are better than primary MDCs for use as cellular vehicles in BMP4-based ex vivo gene therapy to improve bone healing. The advantage of MDSCs may be attributable, at least in part, to their lower immunogenicity and higher capacity for in vivo survival.     

Slow progressive FSGS associated with an F392L<Emphasis Type="Italic"> WT1</Emphasis> mutation

Constitutional missense mutations in the WT1 gene are usually associated with the Denys-Drash syndrome, characterized by a rapid progressive nephropathy, male pseudohermaphroditism, and an increased risk for Wilms tumor. We report here a patient with scrotal hypospadias and a slow progressive nephropathy due to focal and segmental glomerulosclerosis. WT1 mutation analysis revealed a constitutional missense mutation in exon 9 resulting in an exchange F392L. This mutation has previously been reported by others in a patient with a similar mild course of nephropathy. In contrast, a mutation in the corresponding codon of exon 8 (F364L) was previously found by us in a patient with a very rapid progression to end-stage renal disease. Whether the position of a mutation may influence the course of the nephropathy must be evaluated in a larger patient cohort. The individual tumor risk for this alteration cannot be given at present because neither of the two patients has shown evidence of a Wilms tumor or a gonadoblastoma to date.Petras Kaltenis and Valérie Schumacher contributed equally to the work     

Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue

PURPOSE: Provide a reproducible method for culturing confluent monolayers of hfRPE cells that exhibit morphology, physiology, polarity, and protein expression patterns similar to native tissue. METHODS: Human fetal eyes were dissected on arrival, and RPE cell sheets were mechanically separated from the choroid and cultured in a specifically designed medium comprised entirely of commercially available components. Physiology experiments were performed with previously described techniques. Standard techniques were used for immunohistochemistry, electron microscopy, and cytokine measurement by ELISA. RESULTS: Confluent monolayers of RPE cell cultures exhibited epithelial morphology and heavy pigmentation, and electron microscopy showed extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. The mean transepithelial potential (TEP) was 2.6 +/- 0.8 mV, apical positive, and the mean transepithelial resistance (R(T)) was 501 +/- 138 Omega . cm(2) (mean +/- SD; n = 35). Addition of 100 microM adenosine triphosphate (ATP) to the apical bath increased net fluid absorption from 13.6 +/- 2.6 to 18.8 +/- 4.6 microL . cm(-2) per hour (mean +/- SD; n = 4). In other experiments, VEGF was mainly secreted into the basal bath (n = 10), whereas PEDF was mainly secreted into the apical bath (n = 10). CONCLUSIONS: A new cell culture procedure has been developed that produces confluent primary hfRPE cultures with morphological and physiological characteristics of the native tissue. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied native human fetal and bovine RPE-choroid explants.     

The effect of relaxin treatment on skeletal muscle injuries

BACKGROUND: Injured skeletal muscle can repair itself via spontaneous regeneration; however, the overproduction of extracellular matrix and excessive collagen deposition lead to fibrosis. Neutralization of the effect of transforming growth factor-beta 1, a key fibrotic cytokine, on myogenic cell differentiation after muscle injury can prevent fibrosis, enhance muscle regeneration, and thereby improve the functional recovery of injured muscle. HYPOTHESIS: The hormone relaxin, a member of the family of insulin-like growth factors, can act as an antifibrosis agent and improve the healing of injured muscle. STUDY DESIGN: Controlled laboratory study. METHODS: In vitro: Myoblasts (C2C12 cells) and myofibroblasts (transforming growth factor-beta 1-transfected myoblasts) were incubated with relaxin, and cell growth and differentiation were examined. Myogenic and fibrotic protein expression was determined by Western blot analysis. In vivo: Relaxin was injected intramuscularly at different time points after laceration injury. Skeletal muscle healing was evaluated via histologic, immunohistochemical, and physiologic tests. RESULTS: Relaxin treatment resulted in a dose-dependent decrease in myofibroblast proliferation, down-regulated expression of the fibrotic protein alpha-smooth muscle actin, and promoted the proliferation and differentiation of myoblasts in vitro. Relaxin therapy enhanced muscle regeneration, reduced fibrosis, and improved injured muscle strength in vivo. CONCLUSION: Administration of relaxin can significantly improve skeletal muscle healing. CLINICAL RELEVANCE: These findings may facilitate the development of techniques to eliminate fibrosis, enhance muscle regeneration, and improve functional recovery after muscle injuries.     

Muscle-based gene therapy and tissue engineering for treatment of growth plate injuries

Growth plate injuries may lead to a progressive angular deformity or longitudinal growth disturbance. The authors investigated the feasibility of gene therapy and tissue engineering based on autologous muscle-and adenoviral-mediated gene transfer of insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2) to treat tibial physeal defects in rabbits. The medial half of the left proximal tibial growth plate was completely excised in 44 6-week-old New Zealand white rabbits. Four experimental groups were created: no treatment (I), autologous muscle interposition (II), autologous muscle interposition injected with adIGF-1 (III), and autologous muscle interposition injected with adBMP-2 (IV). Radiographic and histologic assessments were obtained postoperatively. Significant tibial shortening and a compact osseous bridge were observed in groups I and IV. Growth plates remained open in groups II and III. This experiment demonstrates that IGF-1 had a supportive effect on physeal chondrocytes, while BMP-2 caused increased osteogenic activity in the injured growth plates.