UVA-induced metabolic changes in non-malignant skin cells and the potential role of pyruvate as antioxidant.

The exposure to UVA (320-400 nm) irradiation is a major threat to human skin concerning photoaging and carcinogenesis. It has been shown that UVA irradiation can induce reactive oxygen species (ROS) and DNA mutations, such as 8-hydroxydeoxyguanosine. Furthermore, UVA induces the expression of photoaging-associated matrix metalloproteases (MMPs), especially of matrix metalloprotease 1 (MMP 1) and matrix metalloprotease 3 (MMP 3). In addition to this, it was recently shown that UVA-induced ROS also increase glucose metabolism of melanoma cells, however, the influence of UVA on the glucose metabolism of non-malignant cells of the human skin has, so far, not been investigated in detail. Here, we investigated the UVA-induced changes in glucose metabolism and the functional relevance of these changes in primary fibroblasts-normal non-malignant cells of the skin. These cells showed an UVA-induced enhanced glucose consumption and lactate production and changes in pyruvate production. As it has been proposed that pyruvate could have antioxidant properties we tested the functional relevance of pyruvate as protective agent against UVA-induced ROS. Our initial experiments support earlier publications, demonstrating that pyruvate treated with H2O2 is non-enzymatically transformed to acetate. Furthermore, we show that this decarboxylation of pyruvate to acetate also occurs upon UVA irradiation. In addition to this, we could show that in fibroblasts pyruvate has antioxidant properties as enhanced levels of pyruvate protect cells from UVA-induced ROS and partially from a DNA mutation by the modified base 8-hydroxydeoxyguanosine. Furthermore, we describe for the first time, that the interaction of UVA with pyruvate is relevant for the regulation of photoaging-associated MMP 1 and MMP 3 expression.

Investigation of the HelioVital filter foil revealed protective effects against UVA1 irradiation-induced DNA damage and against UVA1-induced expression of matrixmetalloproteinases (MMP) MMP1, MMP2, MMP3 and MMP15.

The damaging effects of solar ultraviolet (UV) radiation exposure to human skin are well known and can reach from accelerated skin aging (photoaging) to skin cancer. Much of the damaging effects of solar UVA (320-400 nm) radiation is associated with the induction of reactive oxygen species (ROS), which are capable to cause oxidative damage to DNA like the oxidized guanosine 8-hydroxy-2' -deoxyguanosine (8-OHdG). Therefore, new UV protective strategies, have to be tested for their efficiency to shield against UV induced damage. We investigated the protective effects of HelioVital sun protection filter foil against UVA1 irradiation in skin cells. It could be shown, that HelioVital sun protection filter foil has protective effects against UVA1 irradiation induced changes in matrix metalloproteinase (MMP) expression. Furthermore a UVA1-dependant regulation of MMP15 in human fibroblasts could be shown for the first time in this context. In addition, this study demonstrated the protective effect of the HelioVital filter film against UVA1-induced ROS production and DNA damage. These results could pave the way for clinical studies with HelioVital filter foil shielding against the damaging effects of phototherapy and other forms of irradiation therapy, thereby increasing the safety and treatment opportunities of these forms of therapy.

Association of polymorphous light eruption with NOD-2 and TLR-5 gene polymorphisms.

BACKGROUND: Polymorphous light eruption (PLE) is a common, immunologically mediated, photosensitive skin disease. After ultraviolet-B (UV-B) irradiation, patients with PLE show reduced Langerhans cell (LC) depletion in the epidermis, which results in a non-suppressive microenvironment in the skin. Interestingly, severe acute graft-versus-host disease (aGvHD) occurred in stem cell transplanted patients that showed no or incomplete depletion of LCs after UVB irradiation. Genetic variation in nucleotide-binding oligomerization domain 2 (NOD-2) and toll-like receptor 5 (TLR-5) genes also confers susceptibility to aGvHD. OBJECTIVES: We hypothesized that PLE is associated with genetic variation in the NOD-2 and TLR-5 genes. METHODS: We investigated single-nucleotid polymorphisms (SNPs) of NOD-2 (R702W, G908R, 3020Cins) and TLR-5 (A592S, P616L, N392STOP) in skin biopsies of patients with PLE (n = 143) and in healthy controls (n = 104) using restriction fragment length polymorphism analysis. RESULTS: The frequency of NOD-2 alleles with the SNP R702W was significantly higher in PLE than in controls (31.8% vs. 6.3%; P < 0.0001), and homozygous carriers of this mutation were more common in PLE (27.9% vs. 0%; P < 0.0001). For SNP 3020Cins, the allele frequency (7.3% vs. 0.7%; P = 0.0025) and the number of heterozygotes (14.7% vs. 1.3%; P = 0.0019) were higher in PLE. The frequency of alleles with the N392STOP SNP of the TLR5 gene, which is associated with a truncated, non-functional receptor, was significantly higher in PLE (21% vs. 5%; 7% vs. 1% homozygotes, 28% vs. 8% heterozygotes; P < 0.0001). The other SNPs did not differ significantly. CONCLUSIONS: This study yielded a high frequency of functional SNPs in the NOD-2 and TLR-5 genes in PLE. The same SNPs are associated with aGvHD and there are similarities in the reaction of LCs after UVB irradiation between aGvHD and PLE. This leads to the hypothesis that patients with PLE may be more susceptible to developing GvHD after stem cell transplantation, an assumption that needs to be investigated further.

[Sun protection of human skin: from A to Z]. / Sonnenschutz der menschlichen Haut: Grundlagen.

Solar radiation contains about 6.8% ultraviolet (UV) radiation. UV radiation is still regarded as one of the most important risk factors for both nonmelanoma skin cancer (NMSC; predominantly basal cell carcinoma and squamous cell carcinoma) and malignant melanoma (MM). To avoid induction and persistence of UV-induced mutations, our skin is armed with an arsenal of endogenous protective mechanisms such as induction of cell cycle arrest, repair mechanisms, immunosurveillance and the initiation of various types of cell death. Exogenous sun protection includes a range of behaviors such as avoiding extensive sun exposure, wearing UV-proof clothing and appropriate application of topical sunscreens.

Chondrogenic potential of IL-10 in mechanically injured cartilage and cellularized collagen ACI grafts.

OBJECTIVE: The application of adjunctive mediators in Autologous chondrocyte implantation (ACI) techniques might be useful for improving the dedifferentiated chondrocyte phenotype, to support neocartilage formation and inhibit post-traumatic cartilage destruction. In this study we examined if (a) interleukin 10 treatment can cause chondrogenic phenotype stabilization and matrix preservation in mechanically injured cartilage and if (b) IL-10 can promote chondrogenesis in a clinically applied collagen scaffold for ACI treatment. MATERIALS AND METHODS: For (a) bovine articular cartilage was harvested, subjected to an axial unconfined injury and treated with bovine IL-10 (1-10,000 pg/ng/ml). For (b) a post-operatively remaining ACI graft was treated with human IL-10. Expression levels of type I/II/X collagen, SOX9 and aggrecan were measured by qPCR (a,b). After 3 weeks cell death was analyzed (nuclear blebbing and TUNEL assay) and matrix composition was determined by GAG measurements and immunohistochemistry (aggrecan, type I/II collagen, hyaluronic acid). STATISTICS: One way ANOVA analysis with Bonferroni's correction. RESULTS: (a) IL-10 stabilized the chondrogenic phenotype after injurious compression and preserved matrix integrity. This was indicated by elevated expression of chondrogenic markers COL2A1, ACAN, SOX9, while COL1A1 and COL10A1 were reduced. An increased GAG content paralleled this and histological staining of type 2 collagen, aggrecan and toluidine blue were enhanced after 3 weeks. (b) IL-10 [100 pg/ml] improved the chondrogenic differentiation of human chondrocytes, which was accompanied by cartilaginous matrix formation after 3 weeks of incubation. CONCLUSION: Interleukin-10 is a versatile adjuvant candidate to control the post-injurious environment in cartilage defects and promote chondrogenesis in ACI grafts.

IL-10 ameliorates TNF-α induced meniscus degeneration in mature meniscal tissue in vitro.

BACKGROUND: Joint inflammation causes meniscus degeneration and can exacerbate post-traumatic meniscus injuries by extracellular matrix degradation, cellular de-differentiation and cell death. The aim of this study was to examine whether anti-inflammatory interleukin-10 exerts protective effects in an in vitro model of TNF-α-induced meniscus degeneration. METHODS: Meniscus tissue was harvested from the knees of adult cows. After 24 h of equilibrium explants were simultaneously treated with bovine TNF-α and IL-10. After an incubation time of 72 h cell death was measured histomorphometrically (nuclear blebbing, NB) and release of glycosaminoglycans (GAG, DMMB assay) and nitric oxide (NO, Griess-reagent) were analysed. Transcription levels (mRNA) of matrix degrading enzymes, collagen type X (COL10A1) and nitric oxide synthetase 2 (NOS2) were measured by quantitative real time PCR. TNF-α-dependent formation of the aggrecanase-specific aggrecan neoepitope NITEGE was visualised by immunostaining. Differences between groups were calculated using a one-way ANOVA with a Bonferroni post hoc test. RESULTS: Administration of IL-10 significantly prevented the TNF-α-related cell death (P .001), release of NO (P .003) and NOS2 expression (P .04). Release of GAG fragments (P .001), NITEGE formation and expression of MMP3 (P .007), -13 (P .02) and ADAMTS4 (P .001) were significantly reduced. The TNF-α-dependent increase in COL10A1 expression was also antagonized by IL-10 (P .02). CONCLUSION: IL-10 prevented crucial mechanisms of meniscal degeneration induced by a key cytokine of OA, TNF-α. Administration of IL-10 might improve the biological regeneration and provide a treatment approach in degenerative meniscus injuries and in conditions of post-traumatic sports injuries.

IL-10 reduces apoptosis and extracellular matrix degradation after injurious compression of mature articular cartilage.

OBJECTIVE: The aim of this study was to examine whether anti-inflammatory interleukin-10 (IL-10) exerts chondroprotective effects in an in vitro model of a single mechanical injury of mature articular cartilage. METHOD: Articular cartilage was harvested from the femoro-patellar groove of adult cows (Bos taurus) and cultured w/o bovine IL-10. After 24 h of equilibration explants were subjected to an axial unconfined compression (50% strain, velocity 2 mm/s, held for 10 s). After 96 h cell death was measured histomorphometrically (nuclear blebbing, NB) and the release of glycosaminoglycans (GAG, DMMB assay) and nitric oxide (NO, Griess-reagent) were analyzed. mRNA levels of matrix degrading enzymes and nitric oxide synthetase were measured by quantitative real time PCR. Differences between groups were calculated using a one-way ANOVA with a Bonferroni post hoc test. RESULTS: Injurious compression significantly increased the number of cells with NB, release of GAG and nitric oxide and expression of MMP-3, -13, ADAMTS-4 and NOS2. Administration of IL-10 significantly reduced the injury related cell death and release of GAG and NO, respectively. Expression of MMP-3, -13, ADAMTS-4 and NOS2 were significantly reduced. CONCLUSION: Joint injury is a complex process involving specific mechanical effects on cartilage as well as induction of an inflammatory environment. IL-10 prevented crucial mechanisms of chondrodegeneration induced by an injurious single compression. IL-10 might be a multipurpose drug candidate for the treatment of cartilage-related sports injuries or osteoarthritis (OA).

Loss of spatial organization and destruction of the pericellular matrix in early osteoarthritis in vivo and in a novel in vitro methodology.

OBJECTIVES: Current repair procedures for articular cartilage (AC) cannot restore the tissue's original form and function because neither changes in its architectural blueprint throughout life nor the respective biological understanding is fully available. We asked whether two unique elements of human cartilage architecture, the chondrocyte-surrounding pericellular matrix (PCM) and the superficial chondrocyte spatial organization (SCSO) beneath the articular surface (AS) are congenital, stable or dynamic throughout life. We hypothesized that inducing chondrocyte proliferation in vitro impairs organization and PCM and induces an advanced osteoarthritis (OA)-like structural phenotype of human cartilage. METHODS: We recorded propidium-iodine-stained fetal and adult cartilage explants, arranged stages of organization into a sequence, and created a lifetime-summarizing SCSO model. To replicate the OA-associated dynamics revealed by our model, and to test our hypothesis, we transduced specifically early OA-explants with hFGF-2 for inducing proliferation. The PCM was examined using immuno- and auto-fluorescence, multiphoton second-harmonic-generation (SHG), and scanning electron microscopy (SEM). RESULTS: Spatial organization evolved from fetal homogeneity, peaked with adult string-like arrangements, but was completely lost in OA. Loss of organization included PCM perforation (local micro-fibrillar collagen intensity decrease) and destruction [regional collagen type VI (CollVI) signal weakness or absence]. Importantly, both loss of organization and PCM destruction were successfully recapitulated in FGF-2-transduced explants. CONCLUSION: Induced proliferation of spatially characterized early OA-chondrocytes within standardized explants recapitulated the full range of loss of SCSO and PCM destruction, introducing a novel in vitro methodology. This methodology induces a structural phenotype of human cartilage that is similar to advanced OA and potentially of significance and utility.

Estrogen reduces mechanical injury-related cell death and proteoglycan degradation in mature articular cartilage independent of the presence of the superficial zone tissue.

OBJECTIVE: To study the effect of 17ß-estradiol (E2) and the superficial zone (SFZ) on cell death and proteoglycan degradation in articular cartilage after a single injurious compression in vitro. METHOD: Cartilage explants from the femoropatellar groove of 2 year old cows with or without the SFZ were cultured serum-free with physiological concentrations of E2 and injured by an unconfined single load compression (strain 50%, velocity 2 mm/s). After 96 h cell death was measured histomorphometrically (nuclear blebbing (NB) and TUNEL staining) and release of glycosaminoglycans (GAG) by DMMB assay. RESULTS: Injurious compression increased significantly the number of cells with NB and TUNEL staining and release of GAG. Physiological concentrations of E2 prevented the injury-related cell death and reduced the GAG release significantly in a receptor-mediated manner (shown by co-stimulation with the antiestrogen fulvestrant/faslodex/ICI-182,780). The presence of the SFZ did not alter the NB response to either the mechanical injury or E2, but reduced the overall release of GAG significantly. CONCLUSION: E2 prevents injury-related cell death and GAG release, and might be useful for the development of treatment options for either cartilage-related sports injuries or osteoarthritis (OA). The SFZ does not seem to play an important role in (1) the E2-related tissue response and (2) the mechanically-induced cell death in deeper regions of the explants and GAG release. The latter might be related to the unconfined nature of the injury model.

Response of mature meniscal tissue to a single injurious compression and interleukin-1 in vitro.

OBJECTIVE: To study mechanical overload of mature meniscal tissue under normal and pro-inflammatory conditions in vitro. METHOD: Three days after a single unconfined compression (strain: 25-75%, strain rate 1/s) of meniscal explants from 16 to 24 months-old cattle combined with interleukin-1-treatment (IL-1, 10 ng/ml) release of glycosaminoglycans (GAGs; dimethylmethylene blue (DMMB) assay), lactate dehydrogenase (LDH; cytotoxicity detection kit), and nitric oxide (NO; Griess assay), as well as gene transcription (quantitative reverse transcription polymerase chain reaction (RT-PCR)) and numbers of cells with condensed nuclei (CN; histomorphometry) were determined. RESULTS: Mean peak stresses during compression were about five (25%), 11 (50%), and 30 MPa (75%), respectively. GAG and LDH release and numbers of CN increased whereas NO production and mRNA levels of matrix metalloproteinase (MMP)-2, -3 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 decreased strain-dependently after compression. IL-1 induced an increase in GAG and NO release as well as MMP-2, -3 and ADAMTS-4 levels, but had no impact on the LDH release and slightly increased numbers of CN. However, in combination with compression the tissue responses were reduced and LDH and CN levels were increased compared to IL-1 alone. CONCLUSION: Our data suggest that a single impact compression induces cell damage and release of GAG and reduces the NO production and transcription of certain matrix-degrading enzymes. It also reduces the capacity of meniscal tissue to respond to IL-1, which might be related to the cell damage and suggests that the compression-related GAG release might rather be the result of immediate extracellular matrix-damage than a cell-mediated event. This, however, needs to be confirmed in future studies.

Tumor risk by tissue engineering: cartilaginous differentiation of mesenchymal stem cells reduces tumor growth.

OBJECTIVE: Implantation of autologous chondrocytes (AC) is a promising option for the treatment of cartilage defects, but problems with cell harvesting, dedifferentiation, or the donor age limit the clinical outcome. Mesenchymal stem cells (MSC) gain much interest because of their simple isolation and multipotential differentiation capacity along with their immunosuppressive properties. The latter might introduce tumor manifestation. The influence of undifferentiated and chondrogenically differentiated MSC or AC on tumor growth and metastasis formation was investigated in a murine melanoma model. METHODS: Allogeneic melanoma cells and either syngeneic MSC (C3H10T1/2, transduced with enhanced green fluorescent protein gene) or AC were co-injected at a distance of 3 cm into the contra lateral groins of five mice/group, and evaluated macroscopically and histologically after 4 weeks. RESULTS: Undifferentiated MSC migrated to the tumor site and induced strong tumor growth and metastasis formation. Even avital MSC promoted tumor growth and spreading, but insignificantly without detectable MSC at the tumor site. Chondrogenically differentiated MSC did not migrate and had a significantly lower impact on tumor growth and spreading; AC had no measurable influence on melanoma cells. CONCLUSIONS: Our data suggest that differentiation of MSC reduces MSC-dependent promotion of latent tumors and that native AC do not introduce any increased risk of tumor growth. The question of how far MSC should be differentiated prior to clinical application should be addressed in further studies.

[Meniscal degeneration score and NITEGE expression : immunohistochemical detection of NITEGE in advanced meniscal degeneration]. / Meniskusdegenerationsscore und NITEGE-Expression : Immunhistochemischer NITEGE-Nachweis in der schwergradigen Meniskusdegeneration.

BACKGROUND: Meniscal degeneration (MD) is a structural change of fibrous cartilage that is common in orthopaedic diagnostics and relevant for health insurance matters. So far, there has been neither a standardised scoring system nor an immunohistochemical marker for MD. MATERIAL AND METHOD: In this retrospective trial, the meniscal tissue of 60 patients was assessed immunohistochemically for NITEGE (G1 fragment of the proteoglycan aggrecan) expression. NITEGE expression was correlated with defined grades of MD: little (grade 0/1), medium (grade 2), or severe (grade 3). RESULTS: Detection of extracellular NITEGE deposits in grade 2 or 3 MD had a positive predictive value and specificity of 100%, whereas no deposits were found in grade 0/1 MD. Sensitivity in advanced MD was 55%. Detection of extracellular NITEGE correlated positively with the grade of degeneration, as did patient age and the grade of degeneration. The patient age of those with grade 0/1 MD was significantly lower than for grade 3 (p<0.0001). CONCLUSION: The thoroughly defined degeneration score (grade 1 - grade 3 MD) is suitable to assess the severity of degeneration. Extracellular NITEGE deposits can be regarded as an immunohistochemical marker for advanced (grades 2 and 3) MD.

[Histopathological degeneration score of fibrous cartilage. Low- and high-grade meniscal degeneration]. / Histopathologischer Degenerations-Score des Faserknorpels. Low-grade- und High-grade-Meniskusdegeneration.

Although histopathology of meniscal degeneration plays an important role, no criteria to assess severity of the degeneration are available to date. Our aim was to create a histopathological scoring system for meniscal degeneration with good interobserver variability, taking matrix degradation and cellularity in meniscal tissue into consideration. Degeneration is classified as follows: grade 1 (low), grade 2 (intermediate), grade 3 (high). The pattern of NITEGE deposits (G1 fragment of aggrecan) was assessed immunohistochemically (n=38) and compared with the grades of degeneration. In 48% of the patients with grade 2 or 3 degeneration extracellular NITEGE deposits (specificity 100%) were found, whereas grade 1 patients showed no deposits. Extracellular NITEGE deposits correlated positively with the grade of degeneration. In all, 30 cases (10 per grade) were assessed by three pathologists (A, B, C). Grading conformity was 70% for grade 1, 66% for grade 2 and 100% for grade 3. Cohen's Kappa coefficient was 0.6--0.7 between pairs of observers. Combining grade 1 and 2 to low-grade degeneration, compared to a grade-3 high-grade degeneration achieved Kappa coefficients of between 0.93 and 1.0. This reproducible degeneration score for fibrous cartilage could form the basis for the standardized assessment of meniscal degeneration.

Potent inhibition of cartilage biosynthesis by coincubation with joint capsule through an IL-1-independent pathway.

The reason for the increased risk for development of osteoarthritis (OA) after acute joint trauma is not well understood, but the mechanically injured cartilage may be more susceptible to degradative mediators secreted by other tissues in the joint. To establish a model for such interactions, we coincubated bovine cartilage tissue explants together with normal joint capsule and found a profound ( approximately 70%) reduction in cartilage proteoglycan biosynthesis. This reduction is due to release by the joint capsule of a heat-labile and non-toxic factor. Surprisingly, while cultured synovium is a canonical source of interleukin-1 (IL-1), blockade either by soluble IL-1 type II receptor (sIL-1r) or IL-1 receptor antagonist (IL-1RA) had no effect. Combined blockade of IL-1 and tumor necrosis factor alpha (TNF-alpha) also had no effect. To support the clinical relevance of the findings, we harvested joint capsule from post-mortem human knees. Human joint capsule from a normal adult knee also released a substance that caused an approximately 40% decrease in cartilage proteoglycan biosynthesis. Furthermore, this inhibition was not affected by IL-1 blockade with either sIL-1r or IL-1RA. These results suggest that joint capsule tissue from a normal knee joint can release an uncharacterized cytokine that potently inhibits cartilage biosynthetic activity by an IL-1- and TNF-independent pathway.

Homologous plasmid recombination is elevated in immortally transformed cells.

The levels of intramolecular plasmid recombination, following transfection of a plasmid substrate for homologous recombination into normal and immortally transformed cells, have been examined by two independent assays. In the first assay, recovered plasmid was tested for DNA rearrangements which regenerate a functional neomycin resistance gene from two overlapping fragments. Following transformation of bacteria, frequencies of recombinationlike events were determined from the ratio of neomycin-resistant (recombinant) colonies to ampicillin-resistant colonies (indicating total plasmid recovery). Such events, yielding predominantly deletions between the directly repeated sequences, were substantially more frequent in five immortal cell lines than in any of three normal diploid cell strains tested. Effects of plasmid replication or interaction with T antigen and of bacterially mediated rejoining of linear molecules generated in mammalian cells were excluded by appropriate controls. The second assay used limited coamplification of a control segment of plasmid DNA, and of the predicted recombinant DNA region, primed by two sets of flanking oligonucleotides. Each amplified band was quantitated by reference to a near-linear standard curve generated concurrently, and recombination frequencies were determined from the ratio of recombinant/control DNA regions. The results confirmed that recombinant DNA structures were generated within human cells at direct repeats in the transfected plasmid and were markedly more abundant in an immortal cell line than in the diploid normal cells from which that line was derived.

Accuracy and precision in the detection of articular cartilage lesions using magnetic resonance imaging at 1.5 Tesla in an in vitro study with orthopedic and histopathologic correlation.

BACKGROUND: Magnetic resonance (MR) sequences for cartilage visualization have been the target of numerous studies, and the optimal sequence for cartilage imaging remains a matter of debate in the literature. PURPOSE: To compare MR findings with different MR sequences for the detection of cartilage lesions in fresh deep-frozen human cadaveric patellae in an in vitro setting. MATERIAL AND METHODS: Ten cadaveric patellae were imaged on a 1.5T MR scanner with a 2x2 channel carotid sandwich coil and a conventional knee coil, and compared with orthopedic findings and gold-standard histopathology. MR sequences were: a) fat-saturated (FS) proton density-weighted (PDw) turbo spin-echo (TSE) sequence (TR/TE 4000/39 ms); b) T2-weighted (T2w) double-echo steady-state (DESS) 3D water-excitation (we) sequence (TR/TE 17/4.7 ms); c) 3D-PDw-SPACE (sampling perfection with application-optimized contrasts using different flip-angle evolutions)-we sequence (TR/TE 1800/19 ms). Accuracy, Kendall's tau-b correlation, and weighted kappa coefficients were calculated. RESULTS: Accuracy for cartilage lesion detection with the FS PDw-TSE sequence and the carotid coil was 78.3%, and with the knee coil 73.9%. For the T2wDESS-3D-we sequence, the corresponding values were 69.5% and 65.2%, and for the 3D-PDw-SPACE-we sequence 65.2% and 60.8%, respectively. Kendall's tau-b correlation ranged between 0.508 for the 3D-PDw-SPACE-we sequence (knee coil) and 0.720 for the FS PDw-TSE sequence (carotid and knee coil). Weighted kappa coefficient was lowest for the 3D-PDw-SPACE-we sequence (knee coil) at 0.607, and highest for the carotid coil and FS PDw-TSE sequence at 0.779. CONCLUSION: The evaluated FS PDw-TSE sequences are superior in comparison to the T2wDESS-3D-we and 3D-PDw-SPACE-we sequences in the in vitro setting for the detection of cartilage lesions, and are comparable to results reported in the literature.

Direct identification of major histocompatibility complex class I-bound tumor-associated peptide antigens of a renal carcinoma cell line by a novel mass spectrometric method.

Melanoma and renal cell carcinoma (RCC) are thought to be the most immunogenic human tumors. Presently a series of tumor-specific peptides of melanoma is being tested in clinical trials with different immunotherapy protocols. In contrast, only one decameric peptide (SPSSNRIRNT) derived from one (ORF2) of three possible open reading frames (ORFs) of a gene named RAGE (Renal tumor AntiGEn) was shown to be the target for tumor-specific CTLs on renal carcinoma cells. One reason for the lack of identification of tumor antigens on RCC compared with melanoma may be the difficulty in generating tumor-specific CTLs as screening instruments. Therefore, our approach was directly to isolate and identify peptides bound to HLA class I molecules of the HLA-A2 and -B8 homozygous RCC line A-498. High performance liquid chromatography-fractionated peptides eluted with acid from immunoaffinity-purified HLA class I-peptide complexes were sequenced and identified for the first time by the novel and highly sensitive mass spectrometric method matrix-assisted laser desorption ionization-post source decay (MALDI-PSD) from minute amounts of 100 fmol to 1.5 pmol of the fractionated peptide samples. Fourteen peptide sequences first deduced from interpretations of the mass spectra were also shown to fulfill other reliability criteria such as matching the mass spectra of the respective synthetic peptides. Some peptides were identified to be derived from genes preferentially activated in malignant tissues or resulted from a possibly mutated gene. The most promising candidate for a CTL epitope is a decameric peptide (PASKKTDPQK) derived from another possible ORF (ORF5) of the RAGE gene and probably presented in association with HLA-B8. This peptide was synthesized and used for the in vitro induction of CTLs that lysed the A-498 cells and another HLA-B8-positive RCC line significantly more strongly than either other RAGE-positive but HLA-B8-negative RCC lines or K562 cells. Sensitive sequencing by MALDI-PSD thus may provide a powerful method of identifying potentially tumor-specific and HLA-restricted antigens, even on native malignant cells and tissues.

Altered expression of p53 and mdm-2 proteins at diagnosis is associated with early treatment failure in childhood acute lymphoblastic leukemia.

PURPOSE: To determine whether potential alteration in p53 function through p53 gene mutation or mdm-2 overexpression correlates with early treatment failure in childhood acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Diagnostic marrow samples from 34 children were analyzed for p53 gene alterations by western blot and SSCP/DNA sequence analysis and for mdm-2 overexpression by western blot analysis. These samples were derived from two groups of children with ALL: 17 good outcome patients who are in long-term continuous complete remission and 17 poor outcome patients who did not achieve a complete remission or relapsed within 6 months of achieving remission. RESULTS: Two children within the poor outcome group were found to have p53 gene mutations. Furthermore, five poor outcome patients were shown to have greater than 10-fold overexpression of mdm-2 protein compared with the mean level of mdm-2 protein measured in the good outcome group. Aberrant p53 protein expression was found in only one good outcome patient, whereas no good outcome children were found to have elevated levels (> 10-fold) of mdm-2 protein. CONCLUSION: We show for the first time that potential alteration in p53 function in childhood ALL is more common (P = .036) in cases of early treatment failure than in children who remain in long-term continuous remission.

Catecholamines and lipopolysaccharide synergistically induce the release of interleukin-6 from thymic epithelial cells.

The thymus as the major site of T-cell development is exposed to circulating hormones as well as to neurotransmitters released from peripheral nerves. We investigated the influence of catecholamines on the synthesis of interleukin-1 (IL-1) and IL-6 by cultured rat thymic epithelial cells. Basal or lipopolysaccharide (LPS)-stimulated production of IL-1 was not affected by catecholamines. Release of IL-6 was stimulated only scarcely by catecholamines or tumor necrosis factor-alpha (TNF-alpha) and moderately by LPS alone. However, co-stimulation with adrenaline, noradrenaline, or the beta-adrenoceptor agonist isoproterenol (isoprenaline) had an additive (TNF-alpha) or synergistic (LPS) effect on IL-6 release. The synergistic effect was dose-dependent on catecholamine or LPS concentrations. It was mediated by beta-adrenoceptors that are linked to elevation of intracellular cAMP levels, since it was promoted by beta-adrenoceptor agonists and could be blocked by beta-adrenoceptor antagonists. Co-incubation of LPS with agents directly raising cAMP-levels like forskolin or dibutyryl cAMP yielded even stronger IL-6 induction. After co-stimulation IL-6 mRNA was first detected after 3-4 h and a constant increase of IL-6 bioactivity in the culture supernatant was measured for up to 48 h. Since IL-6 is an important factor for thymocyte differentiation and proliferation, the findings demonstrate an influence of neuronal or hormonal catecholamines on the thymic microenvironment that is created by thymic epithelial cells.