Spinal and cervical nodal metastases in a patient with glioblastoma.

This article presents the rare case of a 54-year-old gentleman with primary glioblastoma developing multiple extracranial metastases 7 months after diagnosis. Initially, the patient complained of progressive headaches, confusion, and weakness of the left arm. Magnetic resonance imaging of the brain showed a right temporoparietal tumor with substantial surrounding subcortical edema and midline shift to the left. Two consecutive craniotomies resulted in complete microsurgical resection of the lesion. Histology was consistent with a World Health Organization grade IV, IDH-wildtype glioblastoma. Further treatment was standard chemoradiation including intensity-modulated radiotherapy with oral temozolomide chemotherapy. Seven months after diagnosis, the cranial lesion progressed, and the patient developed painful metastases in multiple bones and suspicious right-sided cervical lymph nodes. Immunohistochemistry and molecular signature supported the case of a metastatic glioblastoma. Further treatment was palliative radiotherapy of the spinal lesions along with symptomatic pain management. Extracranial metastasis of glioblastoma is a rare complication of which only a few cases have been reported in the literature. Little is known about the precise mechanisms of tumor dissemination and the appropriate treatment.

Radiochemotherapy and interstitial brachytherapy for cervical cancer: clinical results and patient-reported outcome measures.

OBJECTIVE: To evaluate clinical results and long-term patient-reported outcome measures (PROMs) on quality of life in cervical cancer patients following radiochemotherapy (RCT) and brachytherapy (BT) as definitive treatment. MATERIALS AND METHODS: Between 2003 and 2023, a total of 132 patients with advanced cervical cancer were evaluated for possible treatment. Patients treated by postoperative RCT, palliative radiotherapy, and those treated for recurrent disease were excluded. Thus, 46 patients receiving standard RCT and BT as their curative treatment were included in this study. PROMs were assessed prospectively by patients' self-completion of the EORTC-QLQ-C30 and EORTC-QLQ-CX24 questionnaires. RESULTS: Five-year overall survival (OS), distant metastases-free survival (DMFS), and pelvic tumor-free survival rates (PTFS) were 53%, 54%, and 83%, respectively. A significant impact on OS was seen for FIGO (International Federation of Gynecologic Oncology) stage (IIB-IIIA: 79% vs. IIIB-IVA: 33%, p = 0.015), for overall treatment time (OTT; 50-65 d: 64% vs. > 65 d: 38%, p = 0.004), and for rectal D2cc (≤ 73 Gy: 50% vs. > 73 Gy: 38%, p = 0.046). The identical parameters were significantly associated with DMFS (FIGO stage: p = 0.012, OTT: p = 0.008, D2cc: p = 0.024). No parameters with a significant influence on PTFS were seen. In multivariate analysis, an impact of FIGO stage on OS (p = 0.05) and DMFS (p = 0.014) was detected, and of rectal D2cc on DMFS (p = 0.031). The overall QoL score was 63/100. Cognitive function was the least impaired (84/100), while role functioning was the worst (67/100). On the symptom scale, insomnia (46/100), fatigue (41/100), dyspnea (32/100), pain (26/100), and financial difficulties (25/100) were scored the worst. According to EORTC-QLQ-CX24, peripheral neuropathy (36/100) and lymphedema (32/100) occurred most frequently. Impaired sexual/vaginal functioning (32/100) and body image (22/100) were also frequently recorded. CONCLUSION: In patients with advanced cervical cancer, a combination of RCT and BT remains an excellent treatment option. In terms of patient-reported long-term quality of life, specific support is needed to alleviate symptoms including lymphedema, peripheral neuropathy, and impaired sexual activity.

Adenocarcinoma of the oesophagus: neoadjuvant chemoradiation and radical surgery : Long-term results.

PURPOSE: To retrospectively evaluate long-term treatment results following neoadjuvant chemoradiation (CRT) and radical surgery in patients with advanced adenocarcinoma (AC) of the oesophagus. PATIENTS AND METHODS: Between 2005 and 2015, a total of 102 consecutive patients with a median age of 64 years (range, 44-86 years) and AC of the oesophagus were evaluated of whom 84 received a full CRT. A group of 51 patients was treated with neoadjuvant intent followed by radical surgery. A total dose of 50.4 Gy with mostly weekly paclitaxel/fluorouracil chemotherapy was administered. Six to eight weeks following CRT, a transthoracic subtotal oesophageal and proximal gastric resection was performed. Survival curves for overall survival and no evidence of disease (NED) survival (primary endpoints) were calculated according to Kaplan-Meier, and possible prognostic factors were evaluated by the log-rank test as well as by a Cox regression analysis. RESULTS: Median follow-up time of the surviving patients was 48 months (range, 14-134 months). Overall and NED survival rates for patients of the study group (n = 51) were 40 and 32%, respectively, at 5 years. Age (p = 0.04), ypT category (p = 0.1) and the development of distant metastases (p = 0.05) were identified as (marginally) independent prognostic variables with impact on survival. Median survival time for patients of the study group (n = 51) was 45 ± 18 months (95%CI 9-81 months). Clear resection margins were achieved in 46/51 patients (92%). Regression rates with complete regression rare residual cancer and increased number of residual cells, but predominantly fibrosis were 33, 41, and 10%, respectively. Patterns of failure revealed local with distant recurrence in 2/51 (4%), regional recurrence alone in 2/51 (4%), and distant metastases in 27/51 (53%) patients. CONCLUSION: Neoadjuvant CRT in patients with AC of the oesophagus followed by thoracoabdominal surgery is a locally very effective concept. A significant tumour regression in almost 75% of the patients may stimulate prospective trials on the omission of radical surgery for some elderly patients. Due to a high rate of distant metastases further investigations in terms of effective systemic therapy may be warranted.

NEK1 mutations cause short-rib polydactyly syndrome type majewski.

Defects of ciliogenesis have been implicated in a wide range of human phenotypes and play a crucial role in signal transduction and cell-cycle coordination. We used homozygosity mapping in two families with autosomal-recessive short-rib polydactyly syndrome Majewski type to identify mutations in NEK1 as an underlying cause of this lethal osteochondrodysplasia. NEK1 encodes a serine/threonine kinase with proposed function in DNA double-strand repair, neuronal development, and coordination of cell-cycle-associated ciliogenesis. We found that absence of functional full-length NEK1 severely reduces cilia number and alters ciliar morphology in vivo. We further substantiate a proposed digenic diallelic inheritance of ciliopathies by the identification of heterozygous mutations in NEK1 and DYNC2H1 in an additional family. Notably, these findings not only increase the broad spectrum of ciliar disorders, but suggest a correlation between the degree of defective microtubule or centriole elongation and organization and the severity of the resulting phenotype.

Proteomic analysis of synovial fluid from the osteoarthritic knee: comparison with transcriptome analyses of joint tissues.

OBJECTIVE: The pathophysiology of the most common joint disease, osteoarthritis (OA), remains poorly understood. Since synovial fluid (SF) bathes joint cartilage and synovium, we reasoned that a comparative analysis of its protein constituents in health and OA could identify pathways involved in joint damage. We undertook this study to perform a proteomic analysis of knee SF from OA patients and control subjects and to compare the results to microarray expression data from cartilage and synovium. METHODS: Age-matched knee SF samples from 10 control subjects, 10 patients with early-stage OA, and 10 patients with late-stage OA were compared using 2-dimensional difference-in-gel electrophoresis and mass spectrometry (MS). MS with a multiplexed peptide selected reaction monitoring assay was used to confirm differential expression of a subset of proteins in an independent OA patient cohort. Proteomic results were analyzed by Ingenuity Pathways Analysis and compared to published synovial tissue and cartilage messenger RNA profiles. RESULTS: Sixty-six proteins were differentially present in healthy and OA SF. Three major pathways were identified among these proteins: the acute-phase response signaling pathway, the complement pathway, and the coagulation pathway. Differential expression of 5 proteins was confirmed by selected reaction monitoring assay. A focused analysis of transcripts corresponding to the differentially present proteins indicated that both synovial and cartilage tissues may contribute to the OA SF proteome. CONCLUSION: Proteins involved in the acute-phase response signaling pathway, the complement pathway, and the coagulation pathway are differentially regulated in SF from OA patients, suggesting that they contribute to joint damage. Validation of these pathways and their utility as biomarkers or therapeutic targets in OA is warranted.

Preoperative Radiochemotherapy in Rectal Cancer: Is There an Impact of Oxaliplatin on Pathologic Complete Response and Survival Rates under "Real World" Conditions?

This study aimed to evaluate the benefit of additional administration of oxaliplatin during fluorouracil-based neoadjuvant radiochemotherapy (nRCT) in terms of pathologic complete remission (pCR), disease-free survival (DFS), and overall survival (OS) in patients with advanced rectal cancer. Between 2006 and 2021, 669 patients (pts) were diagnosed with locally advanced rectal cancer, of whom a total of 414 pts with nRCT were identified and included in the study. A total of 283 pts were treated by nRCT using concurrent chemotherapy with fluorouracil or capecitabine; 131 pts were treated using a combination of fluorouracil or capecitabine and oxaliplatin. Propensity score matching analyses (PSM) with 114 pts in each group were used to balance the patients' characteristics. OS, DFS, pCR-rate, and potential prognostic factors were compared between the two groups. The median follow-up time was 59.5 weeks in the fluorouracil-group and 43 weeks in the fluorouracil/oxaliplatin group (p = 0.003). After PSM, the pCR-rate (including sustained clinical complete remission) was 27% (31/114 pts) in the fluorouracil/oxaliplatin group and 16% (18/114 pts) in the fluorouracil-group (p = 0.033). There was no difference between these two groups for both 10-year OS and DFS neither before nor after PSM, respectively (OS: 72.6% vs. 55.4%, p = 0.066, and 67.8% vs. 55.1%, p = 0.703, and DFS: 44.8% vs. 46.8%, p = 0.134, and 44.7% vs. 42.3%, p = 0.184). Multivariate analysis identified regression grading according to Dworak grade 4 (HR: 0.659; CI: 0.471-0.921; p = 0.015) and age over 60 years (HR: 2.231; CI: 1.245-4.001; p = 0.007) as independent predictors for OS. In conclusion, the addition of oxaliplatin to fluorouracil during nRCT significantly improved pCR-rate without having an impact on survival.

PD-1 and PD-L1 expression predict regression and prognosis following neoadjuvant radiochemotherapy of oesophageal adenocarcinoma.

Background and purpose: PD-1 and PD-L1 are involved in anticancer immunosurveillance, and their expression may be predictive for therapeutic effectiveness of specific antibodies. Their influence on response to neoadjuvant radiochemotherapy (RCT) and prognosis in patients with oesophageal adenocarcinoma (OAC) remains to be defined. Materials and methods: Between 10/2004 and 06/2018, complete pre-RCT biopsy-specimens were available from 76 patients with locally advanced, non-metastatic OAC scheduled for trimodality therapy. We evaluated intra- and peritumoural expression of CD8, PD-1 and PD-L1 in pre-treatment specimens to determine their influence on tumour regression grade and survival. PD-1 and PD-L1 expression were considered positive (+) if ≥1% of all cells were stained positive, otherwise negative (-); densities of CD8+ cells were categorized as being high (Hi) or low (Lo) according to the median. Results: A negative PD-L1 expression in peritumoural cells predicted a poor tumour regression (RD 0.24 [95% CI 0.03-0.44], p = 0.023). A positive PD-1 expression in intra- as well as peritumoural cells was identified as an unfavourable prognostic factor (HR 0.52 [95% CI 0.29-0.93], p = 0.028; HR 0.50 [0.25-0.99], p = 0.047, respectively). With respect to CD8+ infiltration, positive PD-1 and PD-L1 expressions attenuated its favourable prognostic effect in intratumoural area (LoCD8/PD1 + vs. HiCD8/PD1-: HR 0.25 [0.09-0.69], p = 0.007; LoCD8/PDL1+ vs. HiCD8/PDL1-: HR 0.32 [0.12-0.89], p = 0.028) and were associated with negative outcome when seen in peritumoural area (HiCD8/PD1+ vs. LoCD8/PD1-: HR 0.29 [0.11-0.74], p = 0.010); HiCD8/PDL1+ vs. LoCD8/PDL1-: HR 0.33 [0.12-0.90], p = 0.031). Conclusions: PD-1 and PD-L1 expression were identified to be of predictive and prognostic value in patients with OAC, particularly when considering CD8+ infiltration. Further validation by a large size dataset is required.

ADAM15 exerts an antiapoptotic effect on osteoarthritic chondrocytes via up-regulation of the X-linked inhibitor of apoptosis.

OBJECTIVE: To investigate the capacity of ADAM15, a disintegrin metalloproteinase that is up-regulated in osteoarthritic (OA) cartilage, to protect chondrocytes against apoptosis induced by growth factor deprivation and genotoxic stress. METHODS: Caspase 3/7 activity was determined in primary OA and ADAM15-transfected T/C28a4 chondrocytes upon exposure to the DNA-damaging agent camptothecin or serum withdrawal. Camptothecin-induced cytotoxicity was determined by measuring cellular ATP content. (Anti-)apoptotic proteins were analyzed by immunoblotting, and levels of messenger RNA (mRNA) for X-linked inhibitor of apoptosis (XIAP) were determined using real-time polymerase chain reaction. RNA interference was applied for down-regulation of ADAM15 and XIAP expression. Immunohistochemistry analysis of normal and OA cartilage samples was performed using XIAP- and ADAM15-specific antibodies. RESULTS: ADAM15-transfected chondrocytes cultured on a collagen matrix displayed significantly reduced caspase 3/7 activity upon serum or intermittent matrix withdrawal, compared with vector-transfected control cells. Apoptosis induction by camptothecin exposure also led to significantly elevated caspase 3/7 activity and reduced cell viability of the vector-transfected compared with ADAM15-transfected chondrocytes. Increased levels of activated caspase 3 and cleaved poly(ADP-ribose) polymerase were detected in the vector controls. XIAP, an inhibitor of activated caspase 3, was significantly up-regulated ( approximately 3-fold) at the protein and mRNA levels in ADAM15-transfected chondrocytes upon camptothecin treatment. Specific down-regulation of either ADAM15 or XIAP in OA chondrocytes led to significant sensitization to camptothecin-induced caspase 3/7 activity. Immunohistochemical analysis revealed low to moderate XIAP expression in normal specimens and markedly increased XIAP staining, colocalizing with ADAM15, in OA cartilage. CONCLUSION: ADAM15 conveys antiapoptotic properties to OA chondrocytes that might sustain their potential to better resist the influence of death-inducing stimuli under pathophysiologic conditions.

Molecular characterization of commonly used cell lines for bone tumor research: a trans-European EuroBoNet effort.

Usage of cancer cell lines has repeatedly generated conflicting results provoked by differences among subclones or contamination with mycoplasm or other immortal mammalian cells. To overcome these limitations, we decided within the EuroBoNeT consortium to characterize a common set of cell lines including osteosarcomas (OS), Ewing sarcomas (ES), and chondrosarcomas (CS). DNA fingerprinting was used to guarantee the identity of all of the cell lines and to distinguish subclones of osteosarcoma cell line HOS. Screening for homozygous loss of 38 tumor suppressor genes by MLPA revealed deletion of CDKN2A as the most common event (15/36), strictly associated with absence of the CDKN2A (p16) protein. Ten cell lines showed missense mutations of the TP53 gene while another set of nine cell lines showed mutations resulting in truncation of the TP53 protein. Cells harboring missense mutations expressed high levels of nuclear TP53, while cell lines with nonsense mutations showed weak/absent staining for TP53. TP53(wt) cell lines usually expressed the protein in 2-10% of the cells. However, seven TP53(wt) osteosarcomas were negative for both mRNA and protein expression. Our analyses shed light on the correlation between immunohistochemical and genetic data for CDKN2A and TP53, and confirm the importance of these signaling pathways. The characterization of a substantial number of cell lines represents an important step to supply research groups with proven models for further advanced studies on tumor biology and may help to make results from different laboratories more comparable.

Population doublings and percentage of S100-positive cells as predictors of in vitro chondrogenicity of expanded human articular chondrocytes.

The aim of this study was to investigate the interconnection between the processes of proliferation, dedifferentiation, and intrinsic redifferentiation (chondrogenic) capacities of human articular chondrocyte (HAC), and to identify markers linking HAC dedifferentiation status with their chondrogenic potential. Cumulative population doublings (PD) of HAC expanded in monolayer culture were determined, and a threshold range of 3.57-4.19 PD was identified as indicative of HAC loss of intrinsic chondrogenic capacity in pellets incubated without added chondrogenic factors. While several specific gene and surface markers defined early HAC dedifferentiation process, no clear correlation with the loss of intrinsic chondrogenic potential could be established. CD90 expression during HAC monolayer culture revealed two subpopulations, with sorted CD90-negative cells showing lower proliferative capacity and higher chondrogenic potential compared to CD90-positive cells. Although these data further validated PD as critical for in vitro chondrogenesis, due to the early shift in expression, CD90 could not be considered for predicting chondrogenic potential of HAC expanded for several weeks. In contrast, an excellent mathematically modeled correlation was established between PD and the decline of HAC expressing the intracellular marker S100, providing a direct link between the number of cell divisions and dedifferentiation/loss of intrinsic chondrogenic capacity. Based on the dynamics of S100-positive HAC during expansion, we propose asymmetric cell division as a potential mechanism of HAC dedifferentiation, and S100 as a marker to assess chondrogenicity of HAC during expansion, of potential value for cell-based cartilage repair treatments.

Matrix biochemistry and cell biology of dedifferentiated chondrosarcomas.

Dedifferentiated chondrosarcoma is an uncommon mesenchymal neoplasm comprised of two different components, low-grade conventional chondrosarcoma and high-grade non-cartilaginous sarcoma. In order to gain better insight into the biology of this tumor, we investigated a large series of dedifferentiated chondrosarcomas by looking at the composition of the extracellular tumor matrix within each of the distinct histological components. Our results showed that the well-differentiated portion of the tumors showed matrix components largely similar to conventional chondrosarcomas or enchondromas. In contrast, the high-grade portions showed a variety of staining patterns related to the matrix being formed. Cartilage-specific proteoglycans and collagens were consistently absent, except in areas showing a chondroblastic osteosarcoma histomorphology. Instead, the most dominant immunostaining was received for type I collagen. Type III and VI collagens were concentrated in the areas showing a fibroblastic phenotype. Our results lend further support to the notion that dedifferentiated chondrosarcoma represents transdifferentiation of a cell towards various blastic mesenchymal cell lineages, most commonly osteoblastic and fibroblastic, but occasionally chondroblastic as well. There was no difference in the clinical outcome of patients with differing high-grade tumor types, emphasizing that grade is a more important predictor of biological behavior than the direction of tumor differentiation.

Residual lymph node metastasis in stage 4 neuroblastoma--advantage of radio-guided surgery?

Log-term prognosis of children suffering from high-risk neuroblastomas is characterized by a shortened event-free survival, especially if metastases remain after chemotherapy. We report the case of a 3-year-old boy afflicted with a stage 4 neuroblastoma and persistent residual lymph node metastases despite the administration of a various number of treatment modalities. The insertion of a MIBG (metaiodobenzylguanidine) single-photon emission computed tomography (SPECT)-CT and radio-guided surgery implementing a hand held gamma probe finally allowed the exact localization and resection of the suspected lymphatic tissue. As a consequence, the child has been under event-free remission for 20 months. Because study-based knowledge is missing due to the small number of affected patients, individual case reports are helpful to improve future treatment strategies.

Soft tissue metastasis of thyroid carcinoma in the knee region mimicking a paraarticular inflammatory lesion.

We report a case of a 57-year-old male patient presenting with a painful mass in the popliteal fossa of the left knee, while the X-ray being unremarkable, MRI suggested a paraarticular lesion such as an inflamed paraarticular ganglion. A biopsy showed a poorly differentiated metastasis of a papillary thyroid carcinoma, the patient had been operated on 8 years ago. This case emphasizes that in patients with malignancies such as papillary thyroid carcinomas long-term courses (over years) with several phases of tumor spread occur finally leading to filiae in any location. Thus, in such patients, a metastatic lesion even in unusual places such as the periarticular soft tissue should be included in the differential diagnosis.

Cytotoxic and immunosuppressive inflammatory cells predict regression and prognosis following neoadjuvant radiochemotherapy of oesophageal adenocarcinoma.

BACKGROUND AND PURPOSE: Tumour infiltrating lymphocytes (TIL) and tumour associated macrophages (TAM) play a key role in anticancer immunosurveillance. We studied their influence on response to neoadjuvant radiochemotherapy (RCT) and prognosis in patients with oesophageal adenocarcinoma (OAC). MATERIALS AND METHODS: Between 10/2004 and 06/2018, pre-RCT biopsy-specimens were available from 76 patients with locally advanced, non-metastatic OAC scheduled for trimodality therapy. We evaluated intra- and peritumoural expression of FoxP3+-, CD8+-TIL and CD68+-, CD163+-TAM, contemplating cell density, cell ratios and cell-to-cell distances to determine a possible influence on tumour regression grade (TRG) and survival. Median follow-up time for all patients was 18 months (IQR 9-43), and 54 months (25-97) for surviving patients. Data were analysed using risk analysis, logrank test and Cox regression. RESULTS: Poor tumour regression was detected for cN+ (RR 0.77 [95% CI 0.66-0.90], p = 0.001), low intratumoural FoxP3+/CD8+ ratio (RR 0.75 [0.60-0.96], p = 0.020), high peritumoural CD163+/CD68+ ratio (RR 0.77 [0.60-0.99], p = 0.045) and high intratumoural TAM density (RD -0.44 [-0.82 to -0.06], p = 0.023). Apart from poor resection quality and TRG, pretherapeutic high peritumoural CD8+ infiltration (HR 2.36 [1.21-4.61], p = 0.012) and short intratumoural FoxP3+ to CD8+ cell-to-cell distances in middle ranged CD8+ density (HR 2.55 [1.00-6.52], p = 0.050) were significant unfavourable prognostic factors in multivariate analysis. CONCLUSIONS: Immunologic parameters, such as CD8+-, FoxP3+-TIL and CD68+-, CD163+-TAM, were identified to be of independent predictive and prognostic value in patients with OAC. Further and independent validation of these biomarkers by a large size dataset may urgently be contemplated.

ROR2 blockade as a therapy for osteoarthritis.

Osteoarthritis is characterized by the loss of the articular cartilage, bone remodeling, pain, and disability. No pharmacological intervention can currently halt progression of osteoarthritis. Here, we show that blocking receptor tyrosine kinase-like orphan receptor 2 (ROR2) improves cartilage integrity and pain in osteoarthritis models by inhibiting yes-associated protein (YAP) signaling. ROR2 was up-regulated in the cartilage in response to inflammatory cytokines and mechanical stress. The main ligand for ROR2, WNT5A, and the targets YAP and connective tissue growth factor were up-regulated in osteoarthritis in humans. In vitro, ROR2 overexpression inhibited chondrocytic differentiation. Conversely, ROR2 blockade triggered chondrogenic differentiation of C3H10T1/2 cells and suppressed the expression of the cartilage-degrading enzymes a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5. The chondrogenic effect of ROR2 blockade in the cartilage was independent of WNT signaling and was mediated by down-regulation of YAP signaling. ROR2 signaling induced G protein and Rho-dependent nuclear accumulation of YAP, and YAP inhibition was required but not sufficient for ROR2 blockade-induced chondrogenesis. ROR2 silencing protected mice from instability-induced osteoarthritis with improved structural outcomes, sustained pain relief, and without apparent side effects or organ toxicity. Last, ROR2 silencing in human articular chondrocytes transplanted in nude mice led to the formation of cartilage organoids with more and better differentiated extracellular matrix, suggesting that the anabolic effect of ROR2 blockade is conserved in humans. Thus, ROR2 blockade is efficacious and well tolerated in preclinical animal models of osteoarthritis.

Regulation of Gdf5 expression in joint remodelling, repair and osteoarthritis.

Growth and Differentiation Factor 5 (GDF5) is a key risk locus for osteoarthritis (OA). However, little is known regarding regulation of Gdf5 expression following joint tissue damage. Here, we employed Gdf5-LacZ reporter mouse lines to assess the spatiotemporal activity of Gdf5 regulatory sequences in experimental OA following destabilisation of the medial meniscus (DMM) and after acute cartilage injury and repair. Gdf5 expression was upregulated in articular cartilage post-DMM, and was increased in human OA cartilage as determined by immunohistochemistry and microarray analysis. Gdf5 expression was also upregulated during cartilage repair in mice and was switched on in injured synovium in prospective areas of cartilage formation, where it inversely correlated with expression of the transcriptional co-factor Yes-associated protein (Yap). Indeed, overexpression of Yap suppressed Gdf5 expression in chondroprogenitors in vitro. Gdf5 expression in both mouse injury models required regulatory sequence downstream of Gdf5 coding exons. Our findings suggest that Gdf5 upregulation in articular cartilage and synovium is a generic response to knee injury that is dependent on downstream regulatory sequence and in progenitors is associated with chondrogenic specification. We propose a role for Gdf5 in tissue remodelling and repair after injury, which may partly underpin its association with OA risk.

FOXO3a: a novel player in thyroid carcinogenesis?

The forkhead box transcription factor FOXO3a has recently been identified as central mediator of the cellular response to oxidative stress inducing cell cycle arrest or apoptosis. The aim of our study was to investigate the regulation of FOXO3a in the thyroid and to determine whether alterations in FOXO3a activity occur in thyroid carcinogenesis. In vitro, we demonstrate that FOXO3a activity is negatively regulated by the PI3K/Akt cascade promoting increased phosphorylation and cytoplasmatic accumulation of FOXO3a with decreased transcription of the target genes p27kip (CDKN1B) and Bim (BCL2L11), but increased expression of GADD45A. By contrast, we show that H(2)O(2) exposure activates FOXO3a in thyrocytes with JNK (MAPK8)-mediated nuclear accumulation of FOXO3a and increased expression of the cell cycle arrest genes p27kip and GADD45A. In vivo, we observed a marked cytoplasmatic accumulation of FOXO3a in differentiated thyroid cancers versus an exclusive nuclear accumulation in follicular adenoma and normal thyroid tissue. Moreover, this cytosolic accumulation of FOXO3a correlated with an increased phospho-Akt expression in thyroid malignancies and was accompanied by decreased expression of the FOXO targets p27kip and Bim and an increase in GADD45A mRNA expression in the thyroid cancers. Our data suggest FOXO3a as a novel player of cellular stress response in the thyroid, mediating the thyrocyte's fate either to survive or to undergo apoptosis. Furthermore, PI3K-dependent FOXO3a inactivation may be a novel pathomechanism for the escape from apoptosis in thyroid cancer cells, in particular in follicular thyroid carcinoma.

Response of HEK293 and CHO cells overexpressing fusiogenic syncytin-1 to mitochondrion-mediated apoptosis induced by antimycin A.

Apoptosis is essential for the regulation of cellular homeostasis in the placenta and is also involved in the pathophysiology of pregnancy-related diseases such as pre-eclampsia and intrauterine growth restriction (IUGR). Syncytin-1, a fusiogenic glycoprotein of endogenous-retroviral origin expressed in human trophoblasts, facilitates placental syncytium formation and is found reduced in pre-eclamptic placentas. We focus here on the mitochondrial apoptotic pathway and investigate whether the overexpression of syncytin-1 in HEK293-52 (human embryonic kidney cells) and CHO-52 cells influences the apoptotic response to the mitochondrial inhibitor antimycin A (AA). After the induction of apoptosis by 5 microM AA and incubation for up to 36 h in the absence of serum, the mean apoptotic rate was reduced by 15-30% in syncytin-1 transfected cells compared with mock-transfectants. After 12 h of challenge with AA we found lower cytochrome c levels in the cytoplasmic protein fraction and higher amounts in the mitochondrial fraction in syncytin-1 transfectants compared with mock-transfectants. We observed a decreased Mitotracker Red staining of mitochondria following AA challenge for 24 h in mock-treated CHO cells, in particular, compared with syncytin-1 transfectants. Moreover, we found a reduced activation of caspase 9 in syncytin-1 transfected HEK293-52 cells after 48 h of apoptotic challenge compared to mock-transfectants. However, a high expression of anti-apoptotic Bcl-x(L) was found in both cell types. Using syncytin-1 transfected HEK293-52 cells and CHO-52 cells, we provide initial evidence that syncytin-1 may exert its anti-apoptotic function at the mitochondrial level. A reduced release of cytochrome c followed by a diminished activation of caspase 9 is a possible mechanism.

Metalloproteinases in juvenile angiofibroma--a collagen rich tumor.

Matrix metalloproteinases (MMPs) act in diverse physiological and pathological conditions such as tumor growth and angiogenesis by cleaving extracellular matrix and nonmatrix substrates. MMPs with gelatinase/collagenase activity have not yet been studied in juvenile angiofibroma, a unique fibrovascular tumor with prominent collagen expression. Quantitative real-time polymerase chain reaction studies, Western blot analysis, immunofluorescence studies, gel zymography, and in situ zymography were used to analyze MMP-1, MMP-2, MMP-9, MMP-13, MMP-14, TIMP-1, and TIMP-2 in 9 juvenile angiofibromas and 2 inferior nasal turbinate specimens. Quantitative real-time polymerase chain reaction found significantly elevated expression of MMP-2, MMP-9, and MMP-14 (P < .05) in tumor tissue compared with the inferior nasal turbinate specimens. Western blot analysis detected more prominent MMP-1, MMP-2, and MMP-9 protein levels in juvenile angiofibromas compared with inferior nasal turbinates, but not MMP-13, MMP-14, TIMP-1, and TIMP-2. Immunofluorescent staining proved a mainly stromal localization of the analyzed MMPs. Only MMP-9 and MMP-14 were also detected in vessel walls. MMP-1, MMP-2, and MMP-13 also stained mast cells. Gel zymography indicated increased MMP-2 and MMP-9 gelatinase activity in juvenile angiofibromas compared with inferior nasal turbinates. Finally, in situ zymography detected very high stromal gelatinase/collagenase activity. This study indicates significant expression of MMPs with gelatinase/collagenase activity in juvenile angiofibromas with evidence of a disturbed balance of MMPs to TIMPs toward enhanced MMP activity. These MMPs are assumed to be involved in tumor pathology with an influence on tumor growth and angiogenesis.

SOX gene expression in human osteoarthritic cartilage.

OBJECTIVE: While the developmental role of the SOX transcription factors in fetal chondrocyte differentiation is well documented, much less is known about the expression of SOX family members in normal and osteoarthritic adult cartilage. Therefore, the aim of the present study was to present a thorough analysis of SOX gene expression in normal and osteoarthritic human adult cartilage. METHODS: RNA from normal and osteoarthritic knee cartilage from human adults was analyzed by gene expression profiling using GeneChip technology (Affymetrix) and quantitative real time PCR. RESULTS: Most members of the SOX transcription factor family showed no or very low expression levels in normal and osteoarthritic cartilage from adults. In contrast, SOX9 expression was fairly high in normal cartilage, amounting to approximately 20% of GAPDH levels. SOX9 transcript levels were substantially reduced in osteoarthritis. SOX6 levels were reduced, albeit starting from a low basis expression in normal tissue. CONCLUSION: The presented data indicate that the role of the SOX transcription factor family in adult human cartilage is most probably restricted to a few members, with SOX9 being the most prominent. Furthermore, the reduction of SOX9 and SOX6 transcript levels in osteoarthritic chondrocytes might be responsible for the loss of phenotypic stability of osteoarthritic chondrocytes.