Tyrosine hydroxylase variants influence protein expression, cellular localization, stability, enzymatic activity and the physical interaction between tyrosine hydroxylase and GTP cyclohydrolase 1.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis catalyzing the tetrahydrobiopterin (BH4 )-dependent hydroxylation of tyrosine to L-DOPA. Here, we analyzed 25 TH variants associated with various degrees of dopa-responsive dystonia and evaluate the effect of each variant on protein stability, activity and cellular localization. Furthermore, we investigated the physical interaction between TH and human wildtype (wt) GTP cyclohydrolase 1 (GTPCH) and the effect of variants on this interaction. Our in vitro results classify variants according to their resistance to proteinase K digestion into three groups (stable, intermediate, unstable). Based on their cellular localization, two groups of variants can be identified, variant group one with cytoplasmic distribution and variant group two forming aggregates. These aggregates do not correlate with loss of enzymatic activity but nevertheless might be a good target for molecular chaperones. Unfortunately, no obvious correlation between the half-life of a variant and its enzymatic activity or between solubility, stability and enzymatic activity of a given variant could be found. Excitingly, some variants disrupt the physical interaction between TH and human wildtype GTPCH, thereby interfering with enzymatic activity and offering new druggable targets for therapy. Taken together, our results highlight the importance of an in-depth molecular analysis of each variant in order to be able to classify groups of disease variants and to find specific therapies for each subgroup. Stand-alone in silico analyses predict less precise the effect of specific variants and should be combined with other in vitro analyses in cellular model systems.

Immunoadsorption in a dog with severe immune mediated hemolytic anemia.

Immune mediated hemolytic anemia (IMHA) is a life-threatening disease with severe, acute hemolysis as a result of an autoimmune response directed against erythrocyte surface antigens. In veterinary medicine, IMHA is usually treated with immunosuppressants and often multiple blood transfusions. In human medicine, immunoadsorption (IA) is an established therapy for antibody removal in immune-mediated diseases. A female, spayed, five-year-old, 28 kg Entlebucher Mountain dog was presented with regenerative anemia and positive autoagglutination diagnosed as immune-mediated hemolytic anemia to the veterinary emergency service. Conventional treatment consisting immunosuppression with prednisolone and mycophenolate failed to improve hemolysis. As hematocrit dropped daily, multiple blood transfusions of blood group DEA 1 negative were required. IA was initiated at day 3 with COM.TEC and ADAsorb platforms and a LIGASORBstaphylococcus antitoxin A column. IA with citrate anticoagulation was performed over the treatment time of 77 minutes with a blood flow of 50 mL/min. Total plasma volume of 1.6 L was processed. Complications consisted of vomitus and lid swelling, shivering, excessive clotting in the tubing after a calcium bolus and hypotension. After IA, hemolysis stopped immediately, plasma concentrations of immunoglobulin G, immunoglobulin M and bilirubin decreased, and hematocrit remained stable. The dog was discharged without further hemolysis 4 days after immunoadsorption with immunosuppressive therapy. IA is a promising adjunctive therapy in severe cases of canine IMHA, but it cannot be concluded to which degree IA or concurrent immunosuppression contributed to cessation of hemolysis in the present case.

Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model.

The CD95 (Fas/APO-1) death-inducing signaling complex (DISC) is essential for the initiation of CD95-mediated apoptotic and nonapoptotic responses. The CD95 DISC comprises CD95, FADD, procaspase-8, procaspase-10, and c-FLIP proteins. Procaspase-8 and procaspase-10 are activated at the DISC, leading to the formation of active caspases and apoptosis initiation. In this study we analyzed the stoichiometry of the CD95 DISC. Using quantitative western blots, mass spectrometry, and mathematical modeling, we reveal that the amount of DED proteins procaspase-8/procaspase-10 and c-FLIP at the DISC exceeds that of FADD by several-fold. Furthermore, our findings imply that procaspase-8, procaspase-10, and c-FLIP could form DED chains at the DISC, enabling the formation of dimers and efficient activation of caspase-8. Taken together, our findings provide an enhanced understanding of caspase-8 activation and initiation of apoptosis at the DISC.

Improvement of in vitro potency assays by a resting step for clinical-grade chimeric antigen receptor engineered T cells.

BACKGROUND: Chimeric antigen receptor engineered T (CAR-T) cell therapy is a promising approach currently revolutionizing the field of cancer immunotherapy. However, data concerning clinical-grade CAR-T cell stability and functionality after months of cryopreservation have not been released by companies so far. To investigate the effect of cryopreservation on CAR-T cells and to further optimize the potency assays, we performed this study. METHODS: A third generation of CD19 CAR-T cells was manufactured according to Good Manufacturing Practice (GMP) requirements, which is applied to patients in an ongoing clinical phase 1 study. Quality control tests for sterility, endotoxin and mycoplasma were performed for each batch. Stability in terms of viability, recovery, transduction efficiency and functional capacity was determined using microscopy, multiparametric flow cytometry as well as chromium-51 release tests. RESULTS: Up to 90days of cryopreservation had no influence on viability, recovery and transduction efficiency of CAR-T cells. However, higher cell concentration for cryopreservation could alter the cell viability and recovery but not the transduction efficiency. Moreover, directly after thawing, both the quantity and quality of the functionality of CAR-T cells were transiently hampered by the negative effects of cryopreservation. Notably, the impaired functionality could be fully restored and even strengthened after an overnight resting process. DISCUSSION: Cryopreservation is a challenge for the functional activity of CAR-T cells. However, CAR-T cells regain their potency by overnight incubation at 37°C, which mimics the clinical application setting. Therefore, an overnight resting step should be included in in vitro potency assays.

Myofibroblasts have an impact on expression, dimerization and signaling of different ErbB receptors in OSCC cells.

INTRODUCTION: Receptors of the ErbB family belong to the key players in cancer development and are targets of several therapeutic approaches. Their functional dependency on the tumor microenvironment, especially on CAFs is albeit still poorly understood. Our objective was to investigate the impact of CAF secretome on ErbB receptor expression and signaling behavior in OSCC. METHODS: Stimulation of PE/CA-PJ15 OSCC cells with conditioned media of TGF-ß1-activated fibroblasts was used as model system for CAF to cancer cell communication. Thereby costimulation with inhibitors against matrix metalloproteinases (MMPs), epidermal growth factor receptor (EGFR), MAPK/ERK kinase (MEK), phosphoinositide-3 kinase (PI3-K), signal transducer and activator of transcription 3 (Stat3) or knockdown of Her3 by siRNA was utilized for detailed investigation of the expression, dimerization and signaling pattern of ErbB in western blot and coimmunoprecipitation. RESULTS: Our results show that soluble factors in activated fibroblast secretome stimulate metalloproteinase activity in the membrane of cancer cells. Thereby ligands are released that activate EGFR and subsequently upregulates EGFR expression via the STAT3 pathway. Simultaneously, the expression of PKCɛ was enhanced via a PI3-kinase/Akt-mediated pathway and a negative feedback regulation loop on EGFR downstream signaling generated. Furthermore, the activated fibroblasts secretome stimulated the highly oncogenic hetero-dimerization between HER3 and p95HER2. That protein association is inversely dependent on the expression level of HER3. CONCLUSIONS: Our results demonstrate that the activated fibroblasts secretome can induce a counterbalanced regulation of protein expression, downstream signaling and the dimerization patterns of different ErbB receptor subtypes in the cancer cell. Thus, the combinatorial targeting of CAFs and selective ErbB receptor subtype inhibitors may provide a useful approach in cancer therapy.

Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells.

Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients' outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFß1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCMTGF, FCMPDGF) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCMB). FCMTGF stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCMTGF≫FCMPDGF induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCMTGF>FCMPDGF) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies.

A large and diverse brain organoid dataset of 1,400 cross-laboratory images of 64 trackable brain organoids.

Brain organoids represent a useful tool for modeling of neurodevelopmental disorders and can recapitulate brain volume alterations such as microcephaly. To monitor organoid growth, brightfield microscopy images are frequently used and evaluated manually which is time-consuming and prone to observer-bias. Recent software applications for organoid evaluation address this issue using classical or AI-based methods. These pipelines have distinct strengths and weaknesses that are not evident to external observers. We provide a dataset of more than 1,400 images of 64 trackable brain organoids from four clones differentiated from healthy and diseased patients. This dataset is especially powerful to test and compare organoid analysis pipelines because of (1) trackable organoids (2) frequent imaging during development (3) clone diversity (4) distinct clone development (5) cross sample imaging by two different labs (6) common imaging distractors, and (6) pixel-level ground truth organoid annotations. Therefore, this dataset allows to perform differentiated analyses to delineate strengths, weaknesses, and generalizability of automated organoid analysis pipelines as well as analysis of clone diversity and similarity.

Intradermal testing and serum allergen-specific IgE-testing in cats with naturally occurring feline bronchial disease.

OBJECTIVES: While feline asthma (FA) is considered to be of allergic origin, the etiology of feline chronic bronchitis (CB) to date is unknown. Aim of the study was to compare the results of intradermal testing (IDT) and serum testing for allergen-specific immunoglobulin E (SAT) in cats diagnosed with FA and CB. MATERIAL AND METHODS: Twenty-seven client-owned cats with clinical signs, suggestive of feline inflammatory bronchial disease (FBD) were prospectively enrolled in the study. Patients were assigned to 3 groups based on results of bronchoalveolar-lavage-fluid (BALF)-cytology: FA (n=8), CB (n=10), or cats with a physiological BALF cytology (PB; n=9). A standardized IDT for 27 allergens was performed in all cats. In addition, allergen-specific IgE was measured in serum samples using an FcεRIα-ELISA. The number of positive reactions in both tests was compared between groups, and agreement between test results of both tests was evaluated. RESULTS: Regarding the number of positive reactions, no statistically significant difference was detected between groups in IDT (p=0.65) and SAT (p=0.51). When comparing the 2 test systems, a weak correlation was found for the allergens Tyrophagus putrescentiae (k=0.256), Dermatophagoides farinae (k=0.276), and rye (k=0.273). The most commonly observed reactions were to house dust mites, storage mites, rye and nettle in IDT and to sheep sorrel, storage mites, and house dust mites in SAT. CONCLUSION AND RELEVANCE: IDT and SAT in cats with feline inflammatory bronchial disease (FBD) cannot be used interchangeably for allergen detection. Sensitization to environmental allergens can occur in cats with and without airway inflammation. Therefore, a positive test result should always be assessed in context with clinical signs and allergen exposure.

An AI-based segmentation and analysis pipeline for high-field MR monitoring of cerebral organoids.

Cerebral organoids recapitulate the structure and function of the developing human brain in vitro, offering a large potential for personalized therapeutic strategies. The enormous growth of this research area over the past decade with its capability for clinical translation makes a non-invasive, automated analysis pipeline of organoids highly desirable. This work presents a novel non-invasive approach to monitor and analyze cerebral organoids over time using high-field magnetic resonance imaging and state-of-the-art tools for automated image analysis. Three specific objectives are addressed, (I) organoid segmentation to investigate organoid development over time, (II) global cysticity classification and (III) local cyst segmentation for organoid quality assessment. We show that organoid growth can be monitored reliably over time and cystic and non-cystic organoids can be separated with high accuracy, with on par or better performance compared to state-of-the-art tools applied to brightfield imaging. Local cyst segmentation is feasible but could be further improved in the future. Overall, these results highlight the potential of the pipeline for clinical application to larger-scale comparative organoid analysis.

Expression of the E-cadherin repressors Snail, Slug and Zeb1 in urothelial carcinoma of the urinary bladder: relation to stromal fibroblast activation and invasive behaviour of carcinoma cells.

Epithelial-mesenchymal transition (EMT) is regulated by interaction of carcinoma and stromal cells and crucial for progression of urinary bladder carcinoma (UBC). Therefore, the influence of activated fibroblasts on the expression of E-cadherin repressors as well as EMT and invasion in UBC was investigated. A correlative analysis of the immunohistochemical expression of fibroblast (ASMA, S100A4, FAP, SDF1, PDGFRß) and EMT (Snail, Slug, Zeb1, E-cadherin) markers was performed on 49 UBC cases of different stages. The impact of distinguishable growth factor stimulated fibroblasts on invasion, EMT, and E-cadherin repressor expression was investigated in an invasion model. In situ, invasiveness was significantly correlated to the loss of membranous E-cadherin (E-cad_m) and increased Snail, Slug, Zeb1 in tumour cells, as well as to increased ASMA, S100A4, and PDGFRß in stromal cells. A significant correlation to nodal metastasis could be evidenced for the loss of E-Cad_m, and for an increase in S100A4 and PDGFRß. Comparison of stromal and EMT markers revealed significant correlations of ASMA to Snail and Slug; of S100A4 to the loss of E-cad_m and Zeb1; and of PDGFRß to the loss of E-Cad_m, Slug and Zeb1. In vitro, TGFß1 induced myofibroblasts were the strongest attractants, while aFGF or TGFß1/aFGF stimulated fibroblasts were the most potent EMT inductors. As shown here for the first time, distinct sub-populations of fibroblasts are to various extents associated with EMT and tumour progression in UBC. These relevant findings might be the basis for the identification of new diagnostic markers and therapeutic targets selectively affecting tumour supporting CAF effects.

Differential vascular expression and regulation of oncofetal tenascin-C and fibronectin variants in renal cell carcinoma (RCC): implications for an individualized angiogenesis-related targeted drug delivery.

The study was aimed at determining the vascular expression of oncofetal fibronectin (oncfFn) and tenascin-C (oncfTn-C) isoforms in renal cell carcinoma (RCC) and its metastases which are well-known targets for antibody-based pharmacodelivery. Furthermore, the influence of tumour cells on endothelial mRNA expression of these molecules was investigated. Evaluation of vascular ED-A(+) and ED-B(+) Fn as well as A1(+) and C(+) Tn-C was performed after immunofluorescence double and triple staining using human recombinant antibodies on clear cell, papillary and chromophobe primary RCC and metastases. The influence of hypoxic RCC-conditioned medium on oncfFn and oncfTn-C mRNA expression was examined in human umbilical vein endothelial cells (HUVEC) by real time RT-PCR. There are RCC subtype specific expression profiles of vascular oncfFn and oncfTn-C and corresponding patterns when comparing primary tumours and metastases. Within one tumour, there are different vessel populations with regard to the incorporation of oncfTn-C and oncfFn into the vessel wall. In vitro tumour-derived soluble mediators induce an up regulation of oncfTn-C and oncfFn mRNA in HUVEC which can be blocked by Avastin(®). Vascular expression of oncFn and oncTn-C variants depends on RCC subtype and may reflect an individual tumour stroma interaction or different stages of vessel development. Therefore, oncFn or oncTn-C variants can be suggested as molecular targets for individualized antibody based therapy strategies in RCC. Tumour-derived VEGF could be shown to regulate target expression.

Comparative analysis of oncofetal fibronectin and tenascin-C expression in right atrial auricular and left ventricular human cardiac tissue from patients with coronary artery disease and aortic valve stenosis.

Aortic valve stenosis (AVS) and coronary artery disease (CAD) are accompanied by changes in the cardiac extra cellular matrix (cECM) including the re-expression of oncofetal fibronectin (Fn) and tenascin-C (Tn-C) variants. Human antibodies against these variants are usable for targeted therapy. Aim of the study was the comparative analysis of cECM remodelling in tissue samples from right atrial auricle (RAA) and left ventricular septum (LVS). RAA and LVS specimens from 30 patients (17 × AVS; 13 × AVS+CAD) were analysed with respect to histological changes and ECM remodelling using PCR based ECM gene expression profiling. Re-expression of ED-A(+) Fn and A1(+) Tn-C was investigated on the mRNA and on the protein level. For immunofluorescence, human recombinant small immunoprotein (SIP) format antibodies were used. There was a positive correlation of the grade of histological changes in RAA and corresponding LVS samples (r = 0.695). ECM gene expression levels were higher in LVS compared to RAA. For 24 genes, a corresponding relevant (>2.5-fold) up- or down-regulation in RAA and LVS occurred. Using SIP antibodies, a positive correlation of protein deposition levels in RAA and corresponding LVS (r = 0.818) could be shown for ED-A(+) Fn. Cardiac tissue remodelling is likely a process involving the entire heart reflected by intra-individually comparable histology and cECM changes in RAA and LVS samples. ED-A(+) Fn might be an excellent target for an antibody-mediated delivery of diagnostic or therapeutic agents. The RAA is a valuable and representative tool to evaluate cardiac remodelling and to plan individualized therapy.

EGF/TGFß1 co-stimulation of oral squamous cell carcinoma cells causes an epithelial-mesenchymal transition cell phenotype expressing laminin 332.

Epithelial-mesenchymal transition (EMT) is suggested to be crucial for the development of an invasive and metastatic carcinoma cell phenotype. Therefore, the definition of this phenotype is of great clinical interest. We recently evidenced vimentin positive cells in oral squamous cell carcinoma (OSCC) invasive front expressing laminin γ2 chain mRNA implicating an EMT origin of these cells. To further elucidate the nature of these cells, we have investigated the relation between EMT criteria and laminin-332 expression in a cell culture model of transforming growth factor beta-1 (TGFß1)/epithelial growth factor (EGF) long time co-stimulation. We demonstrate that in contrast to TGFß1 or EGF alone, co-stimulation induces phenotype transition in OSCC cells which fulfils the criteria of EMT in terms of vimentin up-regulation and E-cadherin down-regulation on protein level as well as cell scattering. Furthermore, cells displayed a strongly enhanced invasiveness and adhesion to type I-IV collagens. Phenotype transition is accompanied by an enhanced expression of laminin-332, especially of its γ2 chain. We further analyse the expression of extracellular matrix related genes by RT-PCR profiling. With respect to strongly enhanced proteins, data confirm the EMT phenotype of co-stimulated OSCC cells and expression of laminin-332. Furthermore, alpha catenin, collagen type 16, the integrin α7 and ß1 chains, and MMP11 are suggested as candidates with potential role in EMT in OSCC. In summary we are able to show that EMT in OSCC is mediated by multiple growth factors and is accompanied by laminin γ2 chain up-regulation evidencing the existence of an intermediate Vim(+) /Ln332(+) EMT phenotype as seen in situ.

A comparative analysis of oncofetal fibronectin and tenascin-C incorporation in tumour vessels using human recombinant SIP format antibodies.

Tumour angioneogenesis is associated with the reexpression of oncofetal fibronectin (oncFn) and tenascin-C (oncTn-C) splice variants, which may serve as targets for antibody-based pharmacodelivery. Knowledge of the vascular distribution and organization in different tumours is of importance for the understanding of tumour vessel formation and might be crucial for therapy. Therefore, human SIP format antibodies against Fn ED-A, Fn ED-B and Tn-C A and C splice domains were used for immunofluorescence labelling in renal, lung, oral, colon, breast and urinary bladder carcinoma specimens and in a renal carcinoma xenograft. The spatial relation to stroma, vessels and vascular basement membrane (vBM) was analysed including CD31 and laminin alpha4 chain antibodies. Renal cell carcinomas and atypical carcinoid of the lung revealed vessel-restricted oncFn and/or oncTn-C depositions; all other entities showed a variable stroma positivity including vessels. The individual pattern of oncFn/oncTn-C incorporation in the vBM depended on tumour type, vessel size and intratumoural heterogeneity. There was a stratification of the vessel wall showing luminal oncFn and extraluminal oncTn-C depositions. As shown in the xenograft, perivascular oncTn-C is provided by carcinoma cells. In conclusion, tumours differ in the pattern of Fn or Tn-C isoform positivity in the vessel wall, potentially representing a tumour type specific endothelial cell-tumour cell-stromal cell interaction. Carcinoma cells themselves are involved in vascular Tn-C matrix organization. Up to antigen distribution, Fn and Tn-C domain antibodies may serve as vehicles for antiangiogenetic and antifibrotic agents; oncFn/oncTn-C based targeting should be adapted individually.

Stromal laminin chain distribution in normal, hyperplastic and malignant oral mucosa: relation to myofibroblast occurrence and vessel formation.

BACKGROUND: The contribution of stromal laminin chain expression to malignant potential, tumour stroma reorganization and vessel formation in oral squamous cell carcinoma (OSCC) is not fully understood. Therefore, the expression of the laminin chains alpha2, alpha3, alpha4, alpha5 and gamma2 in the stromal compartment/vascular structures in OSCC was analysed. METHODS: Frozen tissue of OSCC (9x G1, 24x G2, 8x G3) and normal (2x)/hyperplastic (11x) oral mucosa was subjected to laminin chain and alpha-smooth muscle actin (ASMA) immunohistochemistry. Results were correlated to tumour grade. The relation of laminin chain positive vessels to total vessel number was assessed by immunofluorescence double labelling with CD31. RESULTS: Stromal laminin alpha2 chain significantly decreases and alpha3, alpha4, alpha5 and gamma2 chains and also ASMA significantly increase with rising grade. The amount of stromal alpha3, alpha4 and gamma2 chains significantly increased with rising ASMA positivity. There is a significant decrease in alpha3 chain positive vessels with neoplastic transformation. CONCLUSIONS: Mediated by myofibroblasts, OSCC development is associated with a stromal up-regulation of laminin isoforms possibly contributing to a migration promoting microenvironment. A vascular basement membrane reorganization concerning alpha3 and gamma2 chain laminins during tumour angioneogenesis is suggested.

Expression of Snail is associated with myofibroblast phenotype development in oral squamous cell carcinoma.

Snail is a regulator of epithelial-mesenchymal transition (EMT) and considered crucial to carcinoma metastasis, myofibroblast transdifferentiation, and fibroblast activation. To investigate the role of Snail in oral squamous cell carcinoma (OSCC), its immunohistochemical expression was analysed in 129 OSCC samples and correlated to nodal metastasis, histological grade, E-cadherin, and alpha smooth-muscle-actin (alpha SMA). The results were compared to findings in 23 basal cell carcinomas (BCC). Additionally, the influence of TGF beta 1 and EGF on Snail, E-cadherin, vimentin, and alpha SMA expression was analysed in two OSCC cell lines. As a result, Snail-positive cells were mainly found in the stroma of the OSCC invasive front without statistically significant correlation to histological grade or nodal metastasis. Snail was co-localised to alpha SMA but not to E-cadherin or cytokeratin and showed a significant correlation to the loss of membranous E-cadherin. All BCCs were Snail negative. In OSCC culture, the growth-factor-mediated EMT-like phenomenon was accompanied by alpha SMA down-regulation. In summary, Snail expression in OSCC is a stromal phenomenon associated with the myofibroblast phenotype and not related to growth-factor-mediated transdifferentiation of the carcinoma cells themselves. Consequently, Snail immunohistochemistry cannot contribute to the prediction of the metastatic potential. Furthermore, stromal Snail expression is suggested to be the result of mutual paracrine interaction of fibro-/myofibroblasts and dedifferentiated carcinoma cells leading to the generation of a special type of carcinoma-associated fibroblasts.

IIICS de novo glycosylated fibronectin as a marker for invasiveness in urothelial carcinoma of the urinary bladder (UBC).

PURPOSE: The urothelial carcinoma is the most frequent malignancy of the urinary bladder (UBC). The transition into invasive growth is accompanied by several histological changes including an oncofoetal reorganization of the extracellular matrix. Recently, the occurrence of oncofoetal fibronectin with an O-linked glycosylation in the IIICS region (oncf Fn) was shown to be present in urine from UBC patients and was recommended as a tumour marker. Until now there are no data available regarding the source and distribution of oncf Fn in UBC and its value for the assessment of invasiveness. METHODS: oncf Fn was analysed in noninvasive and invasive UBC using immunohistochemistry and western blot. Additionally, the mRNA expression of the IIICS splicing region was evaluated by quantitative real time RT-PCR. RESULTS: Immunohistochemical results reveal a highly significant correlation of oncf Fn to invasiveness. Papillary tumours regularly show no positivity. In western blot, invasive UBC show a strongly increased amount of the 250 kDa oncf Fn. Additionally, several smaller bands could be shown suggesting a proteolytic processing of Fn. The mRNA of the IIICS region shows a 21.5-fold increase in invasive UBC compared with noninvasive carcinomas. CONCLUSIONS: In summary, immunohistochemistry of oncf Fn is a valuable histological marker for invasiveness of urothelial carcinoma of the urinary bladder. The restricted and invasion-associated tissue distribution of immunoreactivity enables to monitor the recurrence of invasive UBC by a quantitative evaluation of IIICS O-linked glycosylated Fn in urine.

Mesenchymal cells contribute to the synthesis and deposition of the laminin-5 gamma2 chain in the invasive front of oral squamous cell carcinoma.

Tumour progression in oral squamous cell carcinoma (OSCC) is associated with a reorganisation of extracellular matrix. Laminin-5 (Ln-5) plays an important role for tumour migration and shows an increased expression in areas of direct tumour/stroma interactions. We have previously shown stromal spot like Ln-5/gamma2 chain deposits distant from the basement membrane region. In this study we have analysed which cell type is responsible for Ln-5/gamma2 chain synthesis in situ. Furthermore, we studied its spatial relation to TGF-beta1 as well as the Ln-5 modulating enzymes matrix metalloproteinase (MMP) 2, membrane type-1 (MT1-) MMP and bone morphogenetic protein (BMP-) 1 by different techniques including triple immunofluorescence labelling and in situ hybridisation in OSCC. We found that the stromal spot-like Ln-5 deposits occurred in the invasive front in the vicinity of mesenchymal cells and vessel structures. In particular, not only carcinoma cells but also mesenchymal cells were shown to express the Ln-5/gamma2 chain mRNA. Moreover, stromal Ln-5 deposits showed a spatial association with TGF-beta1 as well as with MT1-MMP and BMP-1. Based on these findings we suggest that mesenchymal cells contribute to the promotion of tumour cell migration as well as vessel formation in OSCC by providing and organising promigratory Ln-5 fragments.

Cardiopulmonary response during whole-body vibration training in patients with severe COPD.

Several studies in patients with chronic obstructive pulmonary disease (COPD) have shown that whole-body vibration training (WBVT) has beneficial effects on exercise capacity. However, the acute cardiopulmonary demand during WBVT remains unknown and was therefore investigated in this study. Ten patients with severe COPD (forced expiratory volume in 1 s: 38±8% predicted) were examined on two consecutive days. On day one, symptom-limited cardiopulmonary exercise testing was performed on a cycle ergometer. The next day, six bouts of repeated squat exercises were performed in random order for one, two or three minutes either with or without WBVT while metabolic demands were simultaneously measured. Squat exercises with or without WBVT induced comparable ventilatory efficiency (minute ventilation (VE)/carbon dioxide production (V'CO2 ): 38.0±4.4 with WBVT versus 37.4±4.1 without, p=0.236). Oxygen uptake after 3 min of squat exercises increased from 339±40 mL·min-1 to 1060±160 mL·min-1 with WBVT and 988±124 mL min-1 without WBV (p=0.093). However, there were no significant differences between squat exercises with and without WBVT in oxygen saturation (90±4% versus 90±4%, p=0.068), heart rate (109±13 bpm versus 110±15 bpm, p=0.513) or dyspnoea (Borg scale 5±2 versus 5±2, p=0.279). Combining squat exercises with WBVT induced a similar cardiopulmonary response in patients with severe COPD compared to squat exercises without WBVT. Bearing in mind the small sample size, WBVT might be a feasible and safe exercise modality even in patients with severe COPD.

Differential expression of tenascin-C splicing domains in urothelial carcinomas of the urinary bladder.

PURPOSE: Through alternative splicing of the extracellular matrix protein tenascin-C (Tn-C) primary transcript nine type III homology repeats can be independently included or omitted. Large, low spliced Tn-C variants (Tn-C(L)) are preferentially expressed during tissue remodelling processes like tumour invasion to modulate cell migration. The study was aimed to evaluate the differential expression of Tn-C splicing domains in urinary bladder carcinoma with respect to the invasive behaviour. METHODS: The deposition and synthesis of the Tn-C splicing domains A1-D was analysed in 34 urinary bladder carcinomas by semiquantitative immunohistochemistry using domain specific antibodies and by RT-PCR. Results were correlated to tumour stage and grade. RESULTS: There is a significant increase of Tn-C(L) with higher tumour stage and grade. Immunohistochemistry revealed a more restricted distribution pattern of A1, B, and/or D domain containing Tn-C variants to invasive tumours, tumour vessels, and to destructed muscle. The mRNA expression patterns of the domains A1-A3 are similar among the different carcinomas. Stronger differences exist in the region from the B to D domain. In general, the domains AD1/C are rarely expressed. AD1 domain expression seems to be connected with compact invasion pattern. CONCLUSION: In urinary bladder carcinoma a differential expression of Tn-C splicing variants exists in dependence of tumour type, vascularization, and invasive behaviour. Therefore, the detection of different Tn-C splicing domains could be useful for assessment of muscle invasion, tumour surveillance, as well as target structures for antibody based tumour detection and therapy.