Friction properties of a new silk fibroin scaffold for meniscal replacement.

The menisci protect the articular cartilage by reducing contact pressure in the knee. To restore their function after injury, a new silk fibroin replacement scaffold was developed. To elucidate its tribological properties, friction of the implant was tested against cartilage and glass, where the latter is typically used in tribological cartilage studies. The silk scaffold exhibited a friction coefficient against cartilage of 0.056, which is higher than meniscus against cartilage but in range of the requirements for meniscal replacements. Further, meniscus friction against glass was lower than cartilage against glass, which correlated with the surface lubricin content. Concluding, the tribological properties of the new material suggest a possible long-term chondroprotective function. In contrast, glass always produced high, non-physiological friction coefficients.

Antioxidative therapy in an ex vivo human cartilage trauma-model: attenuation of trauma-induced cell loss and ECM-destructive enzymes by N-acetyl cysteine.

OBJECTIVE: Mechanical trauma of articular cartilage results in cell loss and cytokine-driven inflammatory response. Subsequent accumulation of reactive oxygen (ROS) and nitrogen (RNS) species enhances the enzymatic degradation of the extracellular matrix (ECM). This study aims on the therapeutic potential of N-acetyl cysteine (NAC) in a human ex vivo cartilage trauma-model, focusing on cell- and chondroprotective features. DESIGN: Human full-thickness cartilage explants were subjected to a defined impact trauma (0.59 J) and treated with NAC. Efficiency of NAC administration was evaluated by following outcome parameters: cell viability, apoptosis rate, anabolic/catabolic gene expression, secretion and activity of matrix metalloproteinases (MMPs) and proteoglycan (PG) release. RESULTS: Continuous NAC administration increased cell viability and reduced the apoptosis rate after trauma. It also suppressed trauma-induced gene expression of ECM-destructive enzymes, such as ADAMTS-4, MMP-1, -2, -3 and -13 in a dosage- and time-depending manner. Subsequent suppression of MMP-2 and MMP-13 secretion reflected these findings on protein level. Moreover, NAC inhibited proteolytic activity of MMPs and reduced PG release. CONCLUSION: In the context of this ex vivo study, we showed not only remarkable cell- and chondroprotective features, but also revealed new encouraging findings concerning the therapeutically effective concentration and treatment-time regimen of NAC. Its defense against chondrocyte apoptosis and catabolic enzyme secretion recommends NAC as a multifunctional add-on reagent for pharmaceutical intervention after cartilage injury. Taken together, our data increase the knowledge on the therapeutic potential of NAC after cartilage trauma and presents a basis for future in vivo studies.

A chitin-binding lectin from stinging nettle rhizomes with antifungal properties.

Rhizomes of stinging nettle contain a small-sized lectin that exhibits binding specificity toward chitin. This lectin inhibits growth of several phytopathogenic and saprophytic chitin-containing fungi in vitro. The antifungal action of the nettle lectin differs from the action of chitinases, which are a ubiquitous class of antifungal plant proteins. Moreover, the nettle lectin acts synergistically with chitinase in inhibiting fungal growth. The nettle lectin may be a promising candidate for possible applications in the genetic engineering of disease-resistant crops.

Characterization of a migrative subpopulation of adult human nasoseptal chondrocytes with progenitor cell features and their potential for in vivo cartilage regeneration strategies.

BACKGROUND: Progenitor cells display interesting features for tissue repair and reconstruction. In the last years, such cells have been identified in different cartilage types. In this study, we isolated a migrative subpopulation of adult human nasoseptal chondrocytes with progenitor cell features by outgrowth from human nasal septum cartilage. These putative progenitor cells were comparatively characterized with mesenchymal stem cells (MSC) and human nasal septum chondrocytes with respect to their cellular characteristics as well as surface marker profile using flow cytometric analyses. Differentiation capacity was evaluated on protein and gene expression levels. RESULTS: The migrative subpopulation differentiated into osteogenic and chondrogenic lineages with distinct differences to chondrocytes and MSC. Cells of the migrative subpopulation showed an intermediate surface marker profile positioned between MSC and chondrocytes. Significant differences were found for CD9, CD29, CD44, CD90, CD105 and CD106. The cells possessed a high migratory ability in a Boyden chamber assay and responded to chemotactic stimulation. To evaluate their potential use in tissue engineering applications, a decellularized septal cartilage matrix was either seeded with cells from the migrative subpopulation or chondrocytes. Matrix production was demonstrated immunohistochemically and verified on gene expression level. Along with secretion of matrix metalloproteinases, cells of the migrative subpopulation migrated faster into the collagen matrix than chondrocytes, while synthesis of cartilage specific matrix was comparable. CONCLUSIONS: Cells of the migrative subpopulation, due to their migratory characteristics, are a potential cell source for in vivo regeneration of nasal cartilage. The in vivo mobilization of nasal cartilage progenitor cells is envisioned to be the basis for in situ tissue engineering procedures, aiming at the use of unseeded biomaterials which are able to recruit local progenitor cells for cartilage regeneration.

FMIP, a novel Fms-interacting protein, affects granulocyte/macrophage differentiation.

Hematopoietic cell growth, differentiation, and commitment to a restricted lineage are guided by a set of cytokines acting exclusively on cells expressing the corresponding cytokine receptor. The macrophage colony stimulating factor (M-CSF, also termed CSF-1) and its cognate receptor, the tyrosine kinase c-Fms, are essential for monocyte and macrophage development. The underlying molecular mechanism, however, is poorly understood. Here we identified a novel Fms-interacting protein (FMIP, MW 78 kDa) which binds transiently via its N-terminal 144 residues to the cytoplasmic domain of activated Fms-molecules. Binding of FMIP was paralleled by rapid tyrosine phosphorylation within the binding domain which drastically reduced its ability to associate with Fms. Binding was specific as evidenced by co-immunoprecipitation and association with recombinant GST-Fms fusion proteins. No binding was observed with the tyrosine phosphorylated cytoplasmic domains of c-Kit, TrkA, c-Met, and the insulin receptor. The role of FMIP in hematopoietic differentiation was studied in the bipotential myeloid progenitor cell line, FDC-P1Mac11. Overexpression of FMIP prevented M-CSF induced macrophage differentiation. Instead, cells differentiated into granulocytes. Our data suggest that the level of FMIP expression could form a threshold that decides about differentiation either into macrophages or into granulocytes.

Apoptosis during castration-induced regression of the prostate is Fos dependent.

Apoptotic cell death was shown to be accompanied or preceded by an elevated expression of the c-fos protooncogene and DNA binding activity of transcription factor AP-1. We used Fos-deficient mice to study the role of c-Fos during programmed cell death in the prostate. In normal mice apoptosis is induced in the prostate within 2-4 days after castration. Histological features of reduced secretory activity and morphological signs of programmed cell death become obvious. No apparent decrease in secretory activity and no epithelial cell death were observed in Fos-deficient animals after castration. Fragmentation of nuclear DNA was measured by in situ terminal transferase reaction. DNA fragmentation was observed in the prostate epithelium of control mice after castration whereas no similar fragmentation was found in Fos-deficient animals. After castration an AP-1 complex accumulated in the prostate of Fos deficient mice which mainly consists of FosB, Fra-2 and JunD whereas in control animals the AP-1 complex in addition contained c-Fos. Our data strongly suggest that c-Fos is required for programmed cell death of prostate epithelial cells.

Identification of a second Grb2 binding site in the v-Fms tyrosine kinase.

Tyrosine autophosphorylation of the v-Fms oncogene product results in the formation of high-affinity binding sites for cellular proteins containing Src homology 2 (SH2) domains. These proteins transduce various mitogenic and morphogenic signals. As reported previously, Y696KNI in the kinase insert domain of v-Fms binds to the growth factor receptor bound protein 2 (Grb2), a stimulator of the Ras/Raf1 pathway. Here, we mapped Y921TNL within the C-terminal domain of Fms as a novel autophosphorylation site. We demonstrate that this site constitutes a second Grb2 binding site: a recombinant fusion protein (residues 904-944) containing phosphorylated Y921 bound Grb2 from FDCP-1Mac11 cell extracts significantly more efficiently than a corresponding protein (residues 617-759) containing Y696. A yeast two-hybrid system which allowed the formation of a functional Fms tyrosine kinase was employed to quantify binding of Grb2. Fms-protein containing either one of the two phosphorylation sites bound Grb2 equally well, binding was increased for proteins carrying both sites. In contrast, the simultaneous substitution of Y696 and Y921 by phenylalanines abolished Grb2 binding. Mouse NIH3T3 cells expressing the Y921F mutant Fms-protein showed a substantially higher content of fibronectin network than wild-type transformed cells and had largely lost their serum independent growth phenotype.

Cloning and transfer of genes for antifungal compounds from Erwinia herbicola to Escherichia coli.

Erwinia herbicola CHS1065 produces antifungal compounds highly related to herbicolin A. In 11 Tn5 mutants that have lost the antifungal activity, the transposon insertion is located on a 170-kb plasmid present in CHS1065. This plasmid was designated pHER1065. When analyzing these antifungal mutants, it was found that the genes for the biosynthesis of the antifungal compounds were organized in at least two clusters on pHER1065. Upon insertion of the aphII gene of Tn5 and genes for plasmid mobilization in pHER1065, the plasmid could be stably introduced into Escherichia coli. All the E. coli exconjugants expressed an antifungal activity that was quantitatively and qualitatively comparable to the activity produced by E. herbicola CHS1065. Amino acid analysis and molecular weight determinations of the antifungal compound produced by CHS1065 were identical to those of herbicolin A.

Characterization and heterologous expression of the tetL gene and identification of iso-ISS1 elements from Enterococcus faecalis plasmid pJH1.

The tetracycline-resistance (TcR) determinant of the Enterococcus faecalis plasmid pJH1 has been identified and located on a 2.2-kb RsaI-EcoRI fragment. The fragment was cloned in Escherichia coli, and specified TcR in this host. The nucleotide (nt) sequence of the cloned fragment showed the presence of an open reading frame (ORF) of 1374 bp, designated tetL. The nt sequence of tetL from pJH1 was identical to that of the tetL present on pLS1 from Streptococcus agalactiae. Upstream of the pJH1 tetL, part of another ORF was found that, except for two single-nt substitutions, was identical to an iso-ISS1 element from Lactococcus lactis. Hybridization studies indicated the presence of several ISS1-like elements in plasmid pJH1, but not on the En. faecalis chromosome. To study its usefulness as a marker in Gram+ organisms, the pJH1 tetL was cloned on the broad-host-range plasmid pNZ124, resulting in pNZ280, that was found to give resistance to 40 micrograms Tc/ml in Lc. lactis and Bacillus subtilis.

Radiotherapy of testicular intraepithelial neoplasia (TIN): a novel treatment regimen for a rare disease.

PURPOSE: Testicular intraepithelial neoplasia (TIN) is a consistent precursor of most invasive germ cell tumors, currently treated by radiotherapy with 20 Gy, which destroys TIN but preserves Leydig cells. Nevertheless, analysis has shown dose-dependent dysfunction even with low therapeutic doses of 20 Gy in some cases. Therefore, we tested a dose reduction regimen by delivering smaller fractional doses to enhance the tolerance of Leydig cells. METHODS AND MATERIALS: Between 1993 and 1999, 9 patients were treated for TIN in a prospective multicenter trial. A total dose of 13 Gy was administered in 10 fractions of 1.3 Gy. Hormonal levels of follicle-stimulating hormone, luteinizing hormone, and testosterone were assayed serially. RESULTS: During a median follow-up time of 36 months, no patient showed evidence of local disease. A first postradiation biopsy was obtained 3-12 months after radiotherapy; 5 patients underwent a second biopsy 2-3 years after treatment. All biopsies showed a Sertoli cell-only pattern. Follicle-stimulating hormone levels continued to increase 1 year after radiotherapy, signaling eradicated spermiogenesis. Luteinizing hormone and testosterone remained within the normal range 2 years after radiotherapy. CONCLUSIONS: In the treatment of TIN, there seems to be a dose reduction potential to 13 Gy by lowering single fractional doses, which enhances the therapeutic ratio in favor of the Leydig cells.

Endocrine profiles after radiotherapy in stage I seminoma: impact of two different radiation treatment modalities.

BACKGROUND AND PURPOSE: In patients with stage I seminoma treated with elective lymph node irradiation, testicular scatter doses are often thought to be responsible for later disturbances in fertility. We studied the influence of radiation field extensions and testicular doses on hormonal function. MATERIALS AND METHODS: FSH (follicle stimulating hormone) and LH (luteinizing hormone) were evaluated before radiotherapy (RT) and by serial analyses after treatment for 4 years. Twenty-three patients were irradiated by hockey stick fields with a mean dose of 31.9 Gy (+/-4.7 SD) and a mean scatter dose of 54 8 cGy (+/-16.6 SD). Twenty-one patients received limited RT to the paraaortic nodes with 28.1 Gy (+/-2.4 SD). The mean testicular dose was only 25 cGy (+/-7.8 SD). All patients had normal pre-treatment hormonal values. RESULTS: Six months after the end of RT, mean FSH values were significantly elevated in the hockey stick group (P = 0.032), returning to normal after 3 years. The increase in LH was also significant, but stayed within normal ranges. Limited RT resulted in a minimal, dose-dependent increase of FSH; no changes in LH were noted. CONCLUSIONS: In patients with a normal hormonal status after semicastration, FSH is a reliable monitor for transient radiation-induced effects. To avoid treatment-related disturbances in spermatogenesis, scatter doses should be reduced to less than 20 cGy.

[MRT of bladder carcinoma: tumor staging and gadolinium contrast behavior]. / MRT des Harnblasenkarzinoms: Tumorstaging und Gadoliniumkontrastverhalten.

33 patients with tumours of the urinary bladder were studied via MR, both with and without the paramagnetic contrast medium Gd-DTPA. Results were compared with the final pathological classification after TUER and bimanual palpation. The signal intensity ratio of tumour tissue/fat and tumour/muscle were calculated on T1 weighted images and after GD-DTPA and examined for their statistical significance. The increase in signal intensity of the tumours was statistically significant (Wilcoxon test p less than 0.01). There was no advantage for the T2 weighted images compared with Gd-contrasted enhanced T1 weighted images. MRI staging was correct in 28 out of 33 cases (accuracy 84.8%). Because of the relatively short acquisition time of T1 weighted images and the specific tumour enhancement, administration of Gd-DTPA proved to be of help in the staging of the carcinoma of the bladder. Two small tumours and regressive changes in central areas of tumours were recognised after Gd-DTPA.

Double-blind study with Serocytol "Muqueuse urinaire" and "S.R.E." in patients with urological disorders.

A double-blind study with Serocytol "Muqueuse urinaire" (immune serum against mucosa of the urinary tract) and Serocytol "S.R.E." (immune serum against reticulo-endothelial system) and placebo was designed for treatment of different bladder diseases (interstitial cystitis sui generis, after radiotherapy and in connection with multiple transurethral resections of bladder tumour, cystitis granularis). Twenty-four patients (17 women and 5 men, aged 41-75, with interstitial cystitis, and 2 women, aged 42 and 75, with cystitis granularis) were included in this study. According to the procedure and after breaking the code 13 patients were treated with Serocytol and 11 patients with the placebo. Nine out of 13 patients who were treated with Serocytol showed an improvement of their condition. In 1 patient the condition was unchanged. Three patients treated with the active compound dropped out of the study during the treatment phase. In one instance the patient did not appear for further check-up; in another the therapy was discontinued after 3 weeks, and in one case a general exanthema occurred. Of the 11 patients treated with placebo, there was an improvement in 2 patients, whereas in 9 cases their status was unchanged. Serocytol and placebo were well tolerated by most of the patients. There were no side effects resulting from the almost daily suppository application over a period of 2 months. No changes in the blood counts, electrolytes, liver and renal function could be found in any of these patients.

Sonographic staging after nephrectomy for tumours.

Sonography for postoperative staging after malignant renal tumours was evaluated in this study based on multiple examinations in 63 patients. Sonography permits a critical examination of the liver, the abdomen in general, the retroperitoneal space, including the renal fossa and solitary kidney. Local recurrences after hypernephroma operation were found in 5.3% and after nephrectomy for renal pelvic carcinoma in 68%. Retroperitoneal lymph node metastases appeared in 17% of the patients operated on renal pelvic carcinoma and in 7.6% after hypernephroma operation. These data indicate that special attention should be drawn to the renal fossa after tumour nephrectomy. The higher incidence of recurrences in the renal fossa, and metastatic lymph node involvement in patients following nephrectomy for renal pelvic carcinoma compared with the situation after hypernephroma operation might be explained by the difference of the lymph drainage between the renal pelvis and renal parenchyma which, in addition, includes a separate ontogenetic development. The majority of secondary lesions in hypernephroma patients occur as lung and bone metastases which have been discovered by conventional X-ray examination. These data are not statistically evaluated in the study.

Effect of intratesticular administration of BCG on testicular function (preliminary report).

Four patients suffering from carcinoma of the prostate (2 in stage C and 2 in stage D) in whom orchidectomy was indicated clinically, were enrolled in the study. Two patients were given a single injection of 25 units of BCG per testis and the other two received 50 units intratesticularly. Testicular biopsy at 0, 4 and 16 weeks showed the effects of the procedure in the tubular and interstitial compartment. The tubules were partially or completely atrophied, Sertoli cells were vacuolated. In the interstitium, mononuclear infiltration was evident. The reaction was more intense in patients receiving 50 units. The plasma testosterone after a slight drop in the first month returned to normal and pretreatment levels, respectively, and remained so during the eight months of observation. There was no significant change either in plasma oestradiol and prolactin or in T3, T1 and cortisol levels. FSH and LH, however, increased beyond the basal levels after an initial drop. The general condition of the patients remained good. All four patients gained weight. No side effects other than scrotal swelling during the first few weeks were either seen or reported by the patients. The swelling subsided after four weeks. This preliminary study indicates that there is a partial or total destruction of geminal elements. As the plasma testosterone levels remained undiminished in these four cases over a period of 8 months, it is apparent that testosterone production is not affected by intratesticularly administered BCG.

Differential effects of p38MAP kinase inhibitors on the expression of inflammation-associated genes in primary, interleukin-1beta-stimulated human chondrocytes.

BACKGROUND AND PURPOSE: A main challenge in the therapy of osteoarthritis (OA) is the development of drugs that will modify the disease. Reliable test systems are necessary to enable an efficient screening of therapeutic substances. We therefore established a chondrocyte-based in vitro cell culture model in order to characterize different p38MAPK inhibitors. EXPERIMENTAL APPROACH: Interleukin-1beta (IL-1beta)-stimulated human OA chondrocytes were treated with the p38MAPK inhibitors Birb 796, pamapimod, SB203580 and the new substance CBS-3868. Birb 796- and SB203580-treated cells were analysed in a genome-wide microarray analysis. The efficacy of all inhibitors was characterized by quantitative gene expression analysis and the quantification of PGE(2) and NO release. KEY RESULTS: Microarray analysis revealed inhibitor-specific differences in gene expression. Whereas SB203580 had a broad effect on chondrocytes, Birb 796 counteracted the IL-1beta effect more specifically. All p38MAPK inhibitors significantly inhibited the IL-1beta-induced gene expression of COX-2, mPGES1, iNOS, matrix metalloproteinase 13 (MMP13) and TNFRSF11B, as well as PGE(2) release. Birb 796 and CBS-3868 showed a higher efficacy than SB203580 and pamapimod at inhibiting the expression of COX-2 and MMP13 genes, as well as PGE(2) release. In the case of mPGES1 and TNFRSF11B gene expression, CBS-3868 exceeded the efficacy of Birb 796. CONCLUSIONS AND IMPLICATIONS: Our test system could differentially characterize inhibitors of the same primary pharmaceutical target. It reflects processes relevant in OA and is based on chondrocytes that are mainly responsible for cartilage degradation. It therefore represents a valuable tool for drug screening in between functional in vitro testing and in vivo models.