Failed cartilage repair for early osteoarthritis defects: a biochemical, histological and immunohistochemical analysis of the repair tissue after treatment with marrow-stimulation techniques.

PURPOSE: To examine the entire repair tissue resulting from marrow-stimulation techniques in patients with early osteoarthritis. METHODS: The repair tissue and adjacent articular cartilage after failed marrow-stimulation techniques (microfracture and Pridie drilling) of 5 patients (47-65 years old) with cartilage defects and radiographic early osteoarthritis (Kellgren-Lawrence grading 1 and 2) was removed during total joint arthroplasty (mean time until analysis: 8.8 months), analysed by histology, polarized light microscopy, immunohistochemistry, biochemistry and by histological score systems. RESULTS: Macroscopic cartilage repair assessment revealed ICRS grades of II (nearly normal) and III (abnormal). Cartilage defects were mostly completely filled with a fibrocartilaginous tissue that had small and large fissures. Cartilage-specific stains of the repair tissue were more intense than the surrounding native cartilage but reduced compared with normal articular cartilage. The subchondral bone was incompletely restored. A new tidemark was absent. The repair tissue always showed positive immunoreactivity for types II and X collagen, and was sometimes positive for type I collagen. Proteoglycan contents of the repair tissue were generally higher than of the surrounding cartilage. The repair tissue was always more cellular than the adjacent articular cartilage. Histological scoring of the repair tissue revealed a mean Sellers score of 17.6 ± 3.0 and an ICRS grading of 7-9. CONCLUSION: Failed marrow stimulation of articular cartilage defects in patients with early osteoarthritis is characterized by fibrocartilaginous repair. The balance of cell number to extracellular matrix is shifted towards an increased cell number in this tissue. Articular cartilage repair did not reach the quality of normal hyaline articular cartilage. LEVEL OF EVIDENCE: IV.

Genomic alterations and allelic imbalances are strong prognostic predictors in osteosarcoma.

PURPOSE: Osteosarcoma, the most common primary malignant tumor of the bone, is characterized by complex karyotypes with numerous structural and numerical alterations. Despite attempts to establish molecular prognostic markers at the time of diagnosis, the most accepted predictive factor remains the histologic evaluation of necrosis after neoadjuvant chemotherapy. The present approach was carried out to search for genome-wide recurrent loss of heterozygosity and copy number variations that could have prognostic and therapeutic impact for osteosarcoma patients. EXPERIMENTAL DESIGN: Pretherapeutic biopsy samples of 45 osteosarcoma patients were analyzed using Affymetrix 10K2 high-density single nucleotide polymorphism arrays. Numerical aberrations and allelic imbalances were correlated with the histologically assessed response to therapy and clinical follow-up. RESULTS: The most frequent genomic alterations included amplifications of chromosome 6p21 (15.6%), 8q24 (15.6%, harboring MYC), and 12q14 (11.1%, harboring CDK4), as well as loss of heterozygosity of 10q21.1 (44.4%). All these aberrations and the total degree of heterozygosity of each tumor were significantly associated with an adverse outcome of patients and were used to define a chromosomal alteration staging system with a superior predictive potential compared with the histologic regression grading. CONCLUSIONS: Structural chromosomal alterations detected by single nucleotide polymorphism analysis provide a simple but robust parameter to anticipate response to chemotherapy. The proposed chromosomal alteration staging system might therefore help to better predict the clinical course of osteosarcoma patients at the time of initial diagnosis and to adapt neoadjuvant treatment in patients resistant to the current protocols.

Cytokine plasma levels: reliable predictors for radiation pneumonitis?

BACKGROUND: Radiotherapy (RT) is the primary treatment modality for inoperable, locally advanced non-small-cell lung cancer (NSCLC), but even with highly conformal treatment planning, radiation pneumonitis (RP) remains the most serious, dose-limiting complication. Previous clinical reports proposed that cytokine plasma levels measured during RT allow to estimate the individual risk of patients to develop RP. The identification of such cytokine risk profiles would facilitate tailoring radiotherapy to maximize treatment efficacy and to minimize radiation toxicity. However, cytokines are produced not only in normal lung tissue after irradiation, but are also over-expressed in tumour cells of NSCLC specimens. This tumour-derived cytokine production may influence circulating plasma levels in NSCLC patients. The aim of the present study was to investigate the prognostic value of TNF-alpha, IL-1beta, IL-6 and TGF-beta1 plasma levels to predict radiation pneumonitis and to evaluate the impact of tumour-derived cytokine production on circulating plasma levels in patients irradiated for NSCLC. METHODOLOGY/PRINCIPAL FINDINGS: In 52 NSCLC patients (stage I-III) cytokine plasma levels were investigated by ELISA before and weekly during RT, during follow-up (1/3/6/9 months after RT), and at the onset of RP. Tumour biopsies were immunohistochemically stained for IL-6 and TGF-beta1, and immunoreactivity was quantified (grade 1-4). RP was evaluated according to LENT-SOMA scale. Tumour response was assessed according to RECIST criteria by chest-CT during follow-up. In our clinical study 21 out of 52 patients developed RP (grade I/II/III/IV: 11/3/6/1 patients). Unexpectedly, cytokine plasma levels measured before and during RT did not correlate with RP incidence. In most patients IL-6 and TGF-beta1 plasma levels were already elevated before RT and correlated significantly with the IL-6 and TGF-beta1 production in corresponding tumour biopsies. Moreover, IL-6 and TGF-beta1 plasma levels measured during follow-up were significantly associated with the individual tumour responses of these patients. CONCLUSIONS/SIGNIFICANCE: The results of this study did not confirm that cytokine plasma levels, neither their absolute nor any relative values, may identify patients at risk for RP. In contrast, the clear correlations of IL-6 and TGF-beta1 plasma levels with the cytokine production in corresponding tumour biopsies and with the individual tumour responses suggest that the tumour is the major source of circulating cytokines in patients receiving RT for advanced NSCLC.

Naturally occurring T-cell response against mutated p21 ras oncoprotein in pancreatic cancer.

Mutated p21 ras proteins (muRas) are present in approximately 90% of pancreatic adenocarcinomas and express mutants which can function as cancer-specific antigens. To evaluate the frequency and magnitude of the natural T-cell response against muRas in 19 HLA-A2-positive patients with muRas-positive pancreatic carcinomas, antigen-experienced T lymphocytes in fresh peripheral blood mononuclear cells were shown by IFN-gamma enzyme-linked immunospot using muRas peptides (5-21) that encompass both HLA class I (HLA-A2)- and class II-restricted (HLA-DRB1) epitopes. Six of 19 patients (32%) were found to have a specific T-cell response against individual mutation-specific ras(5-21) but not against other ras mutations or wild-type ras. In contrast, none of 19 healthy subjects had T cells specifically secreting IFN-gamma (P = 0.004). The T-cell response consisted of both CD8(+) and CD4(+) T cells but was dominated by CD8 T cells in three of four patients. MuRas(5-14) and muRas(6-14) were shown to specifically induce CD8(+) T-cell mediated cytotoxicity against HLA-A2-positive, muRas-bearing pancreatic carcinoma cells. The T-cell response was not correlated with prognostic or clinical variables such as tumor-node-metastasis status, stage, or survival. In conclusion, a natural T-cell response against muRas proteins that could be exploited for immunostimulatory therapeutic approaches has been shown in a significant proportion of patients with pancreatic cancer.

The bronchiolar epithelium as a prominent source of pro-inflammatory cytokines after lung irradiation.

PURPOSE: To study in detail the temporal and spatial release of the pro-inflammatory cytokines tumor necrosis factor alpha, interleukin (IL)-1alpha, and IL-6 in the lung tissue of C57BL/6 mice after thoracic irradiation with 12 Gy. METHODS AND MATERIALS: C57BL/6J mice were exposed to either sham irradiation or a single fraction of 12 Gy delivered to the thorax. Treated and sham-irradiated control mice were killed at 0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 1 week, 2 weeks, 4 weeks, 8 weeks, 16 weeks, and 24 weeks post-irradiation (p.i.). Real-time multiplex reverse transcriptase polymerase chain reaction was established to evaluate the relative messenger RNA (mRNA) expression of TNF-alpha, IL-1alpha, and IL-6 in the lung tissue of the mice (compared with nonirradiated lung tissue). Immunohistochemical detection methods (alkaline phosphatase anti-alkaline phosphatase, avidin-biotin-complex [ABC]) and automated image analysis were used to quantify the protein expression of TNF-alpha, IL-1alpha, and IL-6 in the lung tissue (percentage of the positively stained area). RESULTS: Radiation-induced release of the pro-inflammatory cytokines TNF-alpha, IL-1alpha, and IL-6 in the lung tissue was detectable within the first hours after thoracic irradiation. We observed statistically significant up-regulations for TNF-alpha at 1 h p.i. on mRNA (4.99 +/- 1.60) and at 6 h p.i. on protein level (7.23% +/- 1.67%), for IL-1alpha at 6 h p.i. on mRNA (11.03 +/- 0.77) and at 12 h p.i. on protein level (27.58% +/- 11.06%), for IL-6 at 6 h p.i. on mRNA (6.0 +/- 3.76) and at 12 h p.i. on protein level (7.12% +/- 1.93%). With immunohistochemistry, we could clearly demonstrate that the bronchiolar epithelium is the most prominent source of these inflammatory cytokines in the first hours after lung irradiation. During the stage of acute pneumonitis, the bronchiolar epithelium, as well as inflammatory cells in the lung interstitium, produced high amounts of TNF-alpha (with the maximal value at 4 weeks p.i.: 9.47% +/- 1.78%), IL-1alpha (with the peak value at 8 weeks p.i.: 14.76% +/- 7.77%), and IL-6 (with the peak value at 8 weeks p.i.: 4.28% +/- 1.33%). CONCLUSIONS: In the present study we have clearly demonstrated the immediate expression of the pro-inflammatory cytokines TNF-alpha, IL-1alpha, and IL-6 in the bronchiolar epithelium in the first hours after lung irradiation. A second, long-lasting release of these cytokines by the bronchiolar and alveolar epithelium, as well as by inflammatory cells, was observed at the onset of acute pneumonitis. Therefore, we postulate that lung irradiation causes immediate epithelial reaction, with the bronchiolar epithelium becoming a significant source of pro-inflammatory cytokines capable of promoting inflammation through recruitment and activation of inflammatory cells.

Increased expression of pro-inflammatory cytokines as a cause of lung toxicity after combined treatment with gemcitabine and thoracic irradiation.

BACKGROUND AND PURPOSE: Preclinical evidence suggesting gemcitabine potentiates the anti-tumor effects of irradiation has resulted in clinical trials to evaluate the treatment efficacy of gemcitabine and concurrent thoracic irradiation in non-small-cell lung cancer (NSCLC). Although these studies demonstrated favorable tumor response, this combined treatment modality was accompanied by severe treatment-related toxicities predominantly of the lung. In an attempt to elucidate the determinants of lung toxicity for gemcitabine, we analyzed the expression of the pro-inflammatory cytokines TNF-alpha, IL-1alpha and IL-6 in the lung tissue of mice treated with gemcitabine and concurrent thoracic irradiation. MATERIALS AND METHODS: Four study groups were defined: C57BL/6J mice that received neither irradiation nor gemcitabine (NT-group), those that received gemcitabine (120 mg/kg intraperitoneal, i.p.) but no irradiation (GEM-group), those that underwent thoracic irradiation (12 Gy) without gemcitabine (XRT-group), and those that received both gemcitabine (120 mg/kg i.p., 2 h before irradiation) and thoracic irradiation (GEM/XRT-group). The mice were sacrificed at 1 h, 1 and 3 days, 1, 2 and 4 weeks post-treatment (p.t.). The mRNA expression of TNF-alpha, IL-1alpha and IL-6 in the lung tissue was quantified by competitive RT-PCR. The cellular origin of the cytokine expression was identified by immunohistochemistry. The cytokine expression was correlated with histopathological alterations. RESULTS: The TNF-alpha, IL-1alpha and IL-6 expression in the lung tissue of the GEM/XRT mice was clearly higher at all assessment time points compared to the NT mice (statistically significant at 1 h, 1 and 3 days, 1, 2 and 4 weeks p.t.), XRT mice (statistically significant at 1 week p.t.) or GEM mice (statistically significant at 1 h, 1 and 2 weeks p.t.). Maximal treatment-induced cytokine expression in the lung tissue of the GEM/XRT mice occurred already at 1 week p.t. (TNF-alpha: 30.9 +/- 5.3/IL-1alpha: 28.3 +/- 5.0/IL-6: 4.9 +/- 0.1 times basal level), and coincides with pathohistologically discernable interstitial pneumonitis. The elevated levels of TNF-alpha and IL-1alpha have been found to correlate with immunohistochemical staining of the bronchiolar epithelium and predominantly of inflammatory cells. CONCLUSIONS: Our data provide evidence that the increased expression of pro-inflammatory cytokines and the induction of a cytokine-triggered inflammatory response may be a determinant of the observed elevated lung toxicity after concurrent treatment with gemcitabine and thoracic irradiation.

The 'subsequent artificial neural network' (SANN) approach might bring more classificatory power to ANN-based DNA microarray analyses.

MOTIVATION: Human decisions often proceed in two steps. Initially those most preferred are chosen followed by a subsequent choice of these preferences. Applying one artificial neural network (ANN), a classification is limited to the preselection process. The final categorization is only possible by a subsequent ANN that distinguishes the pre-chosen classes. Existing strategies using coupled ANNs are discussed and a new approach particularly suited for multiclass classification problems is introduced ('Subsequent ANN', SANN). RESULTS: Evaluating a simulated data base comprising 3 classes, classification results of SANN were obviously superior to those achieved by ANN. To evaluate a real-world data base the microarray benchmark GCM (14 classes) was chosen. The ANN results reached 72%, comparable to previous results. Using SANN, up to 81% of the tumors were correctly classified. AVAILABILITY: Programs used in this work and numerical results are available upon request.

Latent Epstein-Barr virus (EBV) infection and cytomegalovirus (CMV) infection in synovial tissue of autoimmune chronic arthritis determined by RNA- and DNA-in situ hybridization.

In rheumatoid arthritis (RA) viral triggers, especially Epstein-Barr virus (EBV) and cytomegalovirus (CMV), have been suggested. By PCR analysis DNA of several viruses among which EBV, CMV, and parvovirus B19 (B19) has been detected in RA synovial fluid and synovial tissue. In 63 synovial tissues of 29 rheumatoid arthritis (RA), 6 psoriatic arthritis (PsA), 26 reactive arthritis/synovitis (rA/S), and two normal synovial cases, we recently could demonstrate a high percentage of replicative B19 infection within the synovial tissue, being significantly more frequent in autoimmune arthritis. To further investigate the influence of synovial virus infections in rheumatoid arthritis, we now analyzed the same sample of synovial tissues for CMV and EBV infections by DNA-in situ hybridization (CMV), EBER1/2-RNA-in situ hybridization (EBV), and immunohistochemistry. A significant latent EBV infection of synovial lining cells, synovial fibroblasts, and/or infiltrating lymphocytes was identified in 5/29 (17.2 %) RA, 1/6 (16.7%) PsA, and to a much lower degree in 1/26 (3.8%) rA/S specimens. CMV-DNA was detected in 31% of RA, 3/6 (50%) of PsA, and 11.5% of rA/S. Immunohistochemical analysis of CMV early antigen revealed replicative CMV activity in 20.7% of RA and 2/6 (33.3%) of PsA specimens but not in reactive arthritis synovia. Comparative analysis of the EBV-, CMV-, and published B19-data demonstrated that relevant synovial virus infections in general and furthermore double or multiple infections are far more common in autoimmune arthritis than in rA/S. A triple virus infection was found solely in RA in 10.3% of cases. The evidence of increased synovial persistence of EBV, CMV, or B19 either alone or even more as coinciding infections may further reinforce the notion of a primary role of these viruses in autoimmune arthritis.

Lymph node positive prostate cancer: long-term survival data after radical prostatectomy.

PURPOSE: We retrospectively reviewed the outcome in our patients with prostate cancer and regional positive lymph nodes who underwent prostatectomy. MATERIALS AND METHODS: Between January 1984 and December 2002, 147 men were found to have local lymph node metastases after surgery, of whom 135 underwent further androgen ablation, including 88% within 6 weeks after prostatectomy. We especially determined overall, cancer specific and progression-free survival rates. RESULTS: Median patient age was 63.2 years (range 46 to 75 years). Postoperative followup was up to 214 months (median 41.9). There was 1 death secondary to surgery. To date 49 patients (33.3%) had disease progression, including 6 with a prostate specific antigen increase later than 100 months after surgery, and 36 (24.5%) died, including 22 of prostate cancer and 14 of other causes. Overall and cause specific survival probabilities at 5, 10 and 15 years were 76.6% and 86.5%, 60.1% and 73.7%, and 47.2% and 57.9%, respectively. Median overall survival was 144 months and median cancer specific survival was greater than 145 months. Overall progression-free probabilities at 5, 10 and 15 years were 72.7%, 49.8% and 31.6%, respectively. Biochemical progression-free survival rates were 77.4% after 5, 53.0% after 10 and 33.7% after 15 years. CONCLUSIONS: Since three-quarters of our patients were likely not to die of prostate cancer within the 10 years after surgery despite histological evidence of lymph node metastases, radical prostatectomy with or without hormonal therapy is a viable option for patients with local lymph node involvement, particularly in view of long-term survival.

Prenatal diagnosis of congenital mesoblastic nephroma associated with renal hypertension in a premature child.

In the present article, we report, for the first time, a prenatal diagnosis of a congenital mesoblastic nephroma in combination with a post-partum hyperreninemia with hypertension. A newborn was delivered at 35 weeks gestation who had an intrauterine diagnosis of a renal mass as early as 32 weeks gestational age by ultrasound examination. Tumor nephrectomy was performed on day 11 after delivery when an increase in hypertension was observed in the newborn.

Detection of parvovirus B19 capsid proteins in lymphocytic cells in synovial tissue of autoimmune chronic arthritis.

The pathogenic influence of viral agents in chronic inflammatory joint diseases like rheumatoid arthritis has been discussed for many years. More recently, DNA of several viruses, among them parvovirus B19 (B19), was traceable by PCR analysis in synovial fluid and synovial tissue. To investigate the potential role of parvovirus B19 in rheumatoid arthritis, we analyzed the expression of B19 VP1/VP2 proteins by immunohistochemistry in paraffin sections of 63 synovial specimens in rheumatoid arthritis (RA; n = 29), psoriatic arthritis (PSA; n = 6), nonspecific arthritis or synovitis (n = 26), and normal synovia (n = 2). Thereby we could demonstrate replicative virus infection in a variable number of cells in about 90% of rheumatoid specimens and in four of six (66%) cases of psoriatic arthritis, but only in 38% of cases with chronic reactive inflammation and one case of normal synovia. In virus-positive rheumatoid specimens, moreover, the average number of affected cells was significantly higher than in virus-expressing synovia of nonspecific reactive inflammation. These findings support the importance of B19-viral infection in the pathogenesis of chronic arthritis. B19-positive cells in the synovia could be ascribed to CD20- or CD3-positive B- or T-lymphocytes by double immunostaining. Based on these results, B19 infection of lymphocytic cells also seems possible.

Sustained transgene expression in cartilage defects in vivo after transplantation of articular chondrocytes modified by lipid-mediated gene transfer in a gel suspension delivery system.

BACKGROUND: Genetically modified chondrocytes may be able to modulate articular cartilage repair. To date, transplantation of modified chondrocytes into cartilage defects has been restricted to viral vectors. We tested the hypothesis that a recombinant gene can be delivered to sites of cartilage damage in vivo using chondrocytes transfected by a lipid-mediated gene transfer method. METHODS: Isolated lapine articular chondrocytes were transfected with an expression plasmid vector carrying the P. pyralis luciferase gene using the reagent FuGENE 6. Transfected chondrocytes were encapsulated in alginate spheres and implanted into osteochondral defects in the knee joints of rabbits. RESULTS: In vitro, luciferase activity in pCMVLuc-transfected spheres showed an early peak at day 2 post-transfection and remained elevated at day 32, the longest time point evaluated. The number of viable chondrocytes in non-transfected and transfected spheres increased over the period of cultivation. In vivo, luciferase activity was maximal at day 5 post-transfection, declined by day 16, but was still present at day 32. On histological analysis, the alginate-chondrocyte spheres filled the cartilage defects and were surrounded by a fibrous repair tissue composed of spindle-shaped cells. CONCLUSIONS: These data demonstrate the successful introduction of articular chondrocytes modified by lipid-mediated gene transfer in a gel suspension delivery system into osteochondral defects and the sustained expression of the transgene in vivo. This method may be used to define the effects of genes involved in cartilage repair and may provide alternative treatments for articular cartilage defects.

Differential expression of the estrogen receptor beta (ERbeta) in human prostate tissue, premalignant changes, and in primary, metastatic, and recurrent prostatic adenocarcinoma.

BACKGROUND: Estrogen signaling mediated by the estrogen receptor beta (ERbeta) has potential implications in normal and abnormal prostate growth. Few studies have addressed this issue in human prostate tissue leaving conflicting results on the immunolocalization of the ERbeta in benign and neoplastic lesions. METHODS: Using a new monoclonal antibody, the current study reports on the differential expression of the ERbeta in tissue sections from 132 patients with prostate cancer. RESULTS: The prostatic epithelium expressed the ERbeta extensively in secretory luminal cell types and at lower levels in basal cells. Atrophic changes of the peripheral zone (PZ) were more immunoreactive than hyperplastic lesions of the transition zone (TZ). When compared with glandular tissue of the PZ, high-grade prostatic intraepithelial neoplasia (HGPIN) revealed decreased levels of the ERbeta in 30 of 47 cases and was unreactive in six lesions. In informative cases with suitable internal controls, all primary tumors (n = 60), lymph node (n = 7), and bone metastases (n = 5) expressed the ERbeta at variable degree. No correlation was found between the ERbeta status, the primary Gleason grade (P = 0.254), and the pathological stage (P = 0.157). Recurrent adenocarcinoma revealed markedly decreased levels in 15 of 40 cases and was ERbeta negative in five recurrent lesions. CONCLUSIONS: The secretory epithelium is a major target of ERbeta-mediated estrogen signaling in the human prostate. Its downregulation in HGPIN is consistent with chemopreventive effects that the ERbeta may exert on the prostatic epithelium. The continuous expression of the receptor protein at significant levels in untreated primary and metastatic adenocarcinoma indicates that these tumors can use estrogens through an ERbeta-mediated pathway. The partial loss of the ERbeta in recurrent tumors after androgen-deprivation may reflect the androgen-dependence of ERbeta gene expression in human prostate cancer.

Apoptosis resistance of neuroendocrine phenotypes in prostatic adenocarcinoma.

BACKGROUND: Neuroendocrine (NE) differentiation has been implicated in prostate cancer progression and hormone therapy failure. It has been shown that prostate cancer cells with NE features lack proliferation activity in vitro and in vivo. The current study reports on the apoptotic status of NE phenotypes in human prostate cancer. METHODS: Double-label techniques were used for simultaneous demonstration of the endocrine marker chromogranin A (ChrA) and DNA fragmentation assessed by the terminal transferase-mediated biotinylated 16-desoxy-uridine-tri-phosphate (bio-16-dUTP) nick-end-labeling (TUNEL) assay. The material included primary prostatic adenocarcinoma (n = 18), lymph node metastases (n = 5), bone metastases (n = 2), and recurrent lesions (n = 10) showing NE differentiation at the immunohistochemical level. RESULTS: Irrespective of grades, stages, and the degree of NE differentiation, DNA fragmentation was restricted to exocrine (ChrA-negative) tumor cells and was undetectable in most of NE tumor cells expressing ChrA. At least 0.16% of ChrA-positive tumor cells revealed DNA fragmentation assessed by the TUNEL assay. CONCLUSION: The present data suggest that the vast majority of prostate cancer cells with NE features escapes programmed cell death. This escape may contribute significantly to their drug resistance and their malignant potential.