Bladder cancer biomarker array to detect aberrant levels of proteins in urine.

Bladder cancer (BCa) is a serious malignancy of the urinary tract worldwide and also prominent for its high rate of recurrence incorporating 50% of all treated patients. To reduce relapse of BCa, lifelong surveillance of patients is essential leading to high treatment costs. The gold standard for the diagnosis of bladder cancer is cystoscopy. It is very sensitive, but due to high costs and its invasive nature this method for routine diagnosis of bladder cancer remains questionable. Because of this and the required surveillance of patients suffering from bladder cancer, urine based markers represent a new potential field of investigation. Literature at the National Center of Biological Information (NCBI) was retrieved for a potential marker panel offering specific protein signatures and used to develop a sensitive and accurate chip assay to monitor BCa. Discovery of possible bladder cancer protein markers is compiled by extensive literature search including 1077 recently (15.01.2008-20.03.2014) published research articles. Validation of this literature is done by selection based on prior defined inclusion and exclusion criteria. A set of six putative biomarkers (VEGF, IL-8, MMP-9, MMP-7, survivin and Cyfra 21.1) was identified and a non-invasive microarray developed to be used for further clinical validation. Investigation regarding optimized urine preparation and assay development, to enhance assay sensitivity for the marker panel, was carried out. This protein based BCa chip enables the fast (within 5 h), simultaneous, easy to operate, cheap, early and non-invasive determination of BCa and is ready for clinical evaluation.

Cementless acetabular reconstruction and structural bone-grafting in dysplastic hips. Surgical technique.

BACKGROUND: Studies of acetabular reconstruction with use of cement and bulk bone graft have demonstrated increasing rates of cup failure in patients with dysplastic hips seven years after total hip arthroplasty. Comparable data on the long-term results of bulk bone-grafting done in conjunction with cementless implants are limited. The aim of this study was to review the clinical and radiographic results of autologous bulk bone-grafting in conjunction with a cementless cup. METHODS: From 1987 to 1992, forty-seven patients (forty women and seven men, with an average age of 50.4 years) who had developmental dysplasia of the hip underwent fifty-six total hip arthroplasties and received a structural graft in combination with a cementless Harris-Galante type-I cup. All patients were followed prospectively. In fifty-five hips, implant migration was measured with single-image radiographic analysis. RESULTS: After an average duration (and standard deviation) of 10.2 +/- 2.9 years, three patients (four hips) had died. In the surviving patients, four implants had been revised and two had radiographic evidence of loosening. With use of revision and loosening as end points, the eleven-year survival rates were 91.6% and 88.9%, respectively. Of the fifty implants that had no loosening, fourteen had measurable cup migration, thirty-five had no migration, and one implant could not be measured. All migrations but one were progressive. With loosening used as the end point, the survival rate at eleven years was 100% for the implants with no migration; however, the survival rate for the cups that had migrated was 69.3% (p = 0.0012). CONCLUSIONS: The eleven-year survival rate for the spherical press-fit cups in combination with bulk bone-grafting is satisfactory, given the complexity of these reconstructions. However, the difference between the survival of the implants that had migrated and those that had not was significant. We expect that the thirteen implants with progressive acetabular migration at the time of the latest follow-up are at risk for loosening, which will increase the revision rate for this series in the coming years.

Cementless acetabular reconstruction and structural bone-grafting in dysplastic hips.

BACKGROUND: Studies of acetabular reconstruction with use of cement and bulk bone graft have demonstrated increasing rates of cup failure in patients with dysplastic hips seven years after total hip arthroplasty. Comparable data on the long-term results of bulk bone-grafting done in conjunction with cementless implants are limited. The aim of this study was to review the clinical and radiographic results of autologous bulk bone-grafting in conjunction with a cementless cup. METHODS: From 1987 to 1992, forty-seven patients (forty women and seven men, with an average age of 50.4 years) who had developmental dysplasia of the hip underwent fifty-six total hip arthroplasties and received a structural graft in combination with a cementless Harris-Galante type-I cup. All patients were followed prospectively. In fifty-five hips, implant migration was measured with single-image radiographic analysis. RESULTS: After an average duration (and standard deviation) of 10.2 +/- 2.9 years, three patients (four hips) had died. In the surviving patients, four implants had been revised and two had radiographic evidence of loosening. With use of revision and loosening as end points, the eleven-year survival rates were 91.6% and 88.9%, respectively. Of the fifty implants that had no loosening, fourteen had measurable cup migration, thirty-five had no migration, and one implant could not be measured. All migrations but one were progressive. With loosening used as the end point, the survival rate at eleven years was 100% for the implants with no migration; however, the survival rate for the cups that had migrated was 69.3% (p = 0.0012). CONCLUSIONS: The eleven-year survival rate for the spherical press-fit cups in combination with bulk bone-grafting is satisfactory, given the complexity of these reconstructions. However, the difference between the survival of the implants that had migrated and those that had not was significant. We expect that the thirteen implants with progressive acetabular migration at the time of the latest follow-up are at risk for loosening, which will increase the revision rate for this series in the coming years.

A phase I clinical and pharmacokinetic study of the camptothecin glycoconjugate, BAY 38-3441, as a daily infusion in patients with advanced solid tumors.

BACKGROUND: The aim of this study was to define the maximum tolerated dose (MTD), dose-limiting toxicity (DLT) and pharmacokinetics of the camptothecin glycoconjugate BAY 38-3441, administered as an infusion for 30 min on two separate schedules every 3 weeks. PATIENTS AND METHODS: A total of 81 patients with advanced solid tumors were treated with BAY 38-3441 either at doses of 20, 40, 67, 100, 140, 210, 315, 470 and 600 mg/m2/day for 1 day every 3 weeks (single-dose schedule), or at doses of 126, 189, 246, 320 and 416 mg/m2/day once daily for three consecutive days every 3 weeks (3-day schedule). Plasma sampling was performed to characterize the pharmacokinetics of BAY 38-3441 and camptothecin with these schedules. RESULTS: DLTs included renal toxicity, granulocytopenia and thrombocytopenia on the single-day schedule at doses > or = 470 mg/m2/day, and diarrhea and thrombocytopenia on the 3-day schedule at doses > or = 320 mg/m2/day. Other non-DLTs were gastrointestinal, dermatological and hematological. Pharmacokinetics of BAY 38-3441 and camptothecin appear to be dose-dependent, but not linear. CONCLUSIONS: Renal toxicity was dose-limiting for BAY 38-3441 using 30-min infusions on the single-dose schedule. Dose escalation to 470 mg/m2/day is feasible using a 2-h infusion. However, because of the superior safety profile, we recommend the 3-day schedule for BAY 38-3441 at a dose of 320 mg/m2/day as 30-min infusions for further phase II studies.

Overexpression of the ATP-dependent helicase RecG improves resistance to weak organic acids in Escherichia coli.

Increased resistance to several weak organic acids was conferred on Escherichia coli by overexpression of the ATP-dependent helicase RecG and, to a lesser extent, by overexpressing the helicase RuvAB. This property of helicases was identified by reproducible selection of recG-bearing clones from genomic libraries of the acetate-resistant species Acetobacter aceti and Staphylococcus capitis. We show that overexpression of RecG from both species, but also from E. coli, increased the maximum biomass concentration attained by E. coli cultures that were grown in the presence of various weak organic acids and uncouplers. Furthermore, overexpression of RecG from A. aceti significantly improved the maximum growth rates of E. coli under weak organic acid challenge. Based on the known role of RecG in DNA replication/repair, our data provide a first indication that weak organic acids negatively affect DNA replication and/or repair, and that these negative effects may be counteracted by helicase activity.

Myocardial perfusion and coronary flow reserve assessed by positron emission tomography in patients after Fontan-like operations.

Ventricular dysfunction in patients after Fontan-like operations (FLOs) is a serious complication that might contribute to poor long-term results. Ischemic heart disease will have debilitating consequences on a Fontan heart. Ten patients (15.8 +/- 5.01 years) after FLO had transesophageal echocardiography and cardiac catheterization 9.3 +/- 4.2 years after surgery. Myocardial perfusion was assessed by NH3-positron emission tomography (rest/adenosine) and compared with that of 10 healthy adults (26.1 +/- 6.3 years). Ventricular function was normal in 4 and reduced in 6 patients; end systolic and end diastolic meridional wall stress was significantly elevated in the FLO group. Coronary angiography revealed no stenosis of the coronaries. Compared to normals, myocardial blood flow (MBF) at rest was higher in the FLO group (0.99 +/- 0.25 vs 0.77 +/- 0.17 ml/g/min, p <0.05), whereas MBF after vasodilatation (2.12 +/- 0.78 vs 3.10 +/- 0.85 ml/g/min, p <0.05) and coronary flow reserve (CFR) was reduced (2.5 +/- 0.88 vs 4.1 +/- 1.01, p <0.05), especially in those with impaired ventricular function. Coronary vascular resistance after vasodilatation was elevated in the FLO group (38.2 +/- 17.4 vs 24.5 +/- 8.3 mmHg/ml/g/min, p <0.05). Altered MBF, increased meridional wall stress, and impaired CFR are common findings in FLO. Attenuated CFR and reduced ventricular function are significantly correlated and may be risk factors for the long-term outcome.

Metabolic flux response to phosphoglucose isomerase knock-out in Escherichia coli and impact of overexpression of the soluble transhydrogenase UdhA.

Blocking glycolytic breakdown of glucose by inactivation of phosphoglucose isomerase (Pgi) in Escherichia coli led to a greatly reduced maximum specific growth rate. Examination of the operational catabolic pathways and their flux ratios using [U-(13)C(6)]glucose-labeling experiments and metabolic flux ratio analysis provide evidence for the pentose phosphate (PP) pathway as the primary route of glucose catabolism in the knock-out mutant. The resulting extensive flux through the PP pathway disturbs apparently the reducing power balance, since overexpression of the recently identified soluble transhydrogenase UdhA improves significantly the growth rate of the Pgi mutant. The presented results provide first evidence that UdhA restores the cellular redox balance by catalyzing electron transfer from NADPH to NADH.

Immunohistochemical analysis of major TGF-beta isoforms and their receptors in laryngeal carcinomas.

We analyzed immunohistochemically the tissue distribution of the three major transforming growth factors-beta isoforms (TGF-beta1, -2, -3) and their receptors (TBR-I, -II and -III) in tissue samples from 38 patients with laryngeal squamous cell carcinomas. Besides a qualitative evaluation, the number of the respectively labeled cells was determined by morphometric analysis. In all tumor samples a significant staining of most tumor cells was seen both for the TGF-beta isoforms and the TBRs. Similarly, the majority of stromal cells were labeled. On semiserial sections, there were only minor differences in the distribution pattern and in the number of labeled cells between the three TGF-beta isoforms and the TBRs, suggesting that most tumor cells are actively involved in the neosynthesis of TGF-betas and TBRs; accordingly, at least most tumor cells seem to be capable of producing more than one TGF-beta form and in parallel several TBRs. With decreasing tumor cell differentiation the number of TGF-beta- and TBR-positive tumor cells decreased slightly (but not to a statistically significant degree). Interestingly, the stromal cells were labeled for TGF-betas and TBRs to a lower extent than the epithelial cells, and there was no significant difference between non-tumor-associated control stroma and the immediate peritumoral stroma. Our observations suggest an even, enhanced level of TGF-beta production in laryngeal squamous cell carcinomas, which may explain some well-known side-effects of tumor growth, such as stromal desmoplasia. In addition, the presence of immunoreactive TBR-proteins in the vast majority of tumor cells excludes the mere absence of TBRs in those carcinomas as the cause for inappropriate TGF-beta function in the tumor cells. This in turn suggests that molecular alterations either of the TBR-proteins non-affecting the synthesis and turnover or downstream alterations of the TGF-beta signaling pathway may be main reasons for the loss of response of the tumor cells to the enhanced amounts of TGF-betas.

Bacillus subtilis metabolism and energetics in carbon-limited and excess-carbon chemostat culture.

The energetic efficiency of microbial growth is significantly reduced in cultures growing under glucose excess compared to cultures growing under glucose limitation, but the magnitude to which different energy-dissipating processes contribute to the reduced efficiency is currently not well understood. We introduce here a new concept for balancing the total cellular energy flux that is based on the conversion of energy and carbon fluxes into energy equivalents, and we apply this concept to glucose-, ammonia-, and phosphate-limited chemostat cultures of riboflavin-producing Bacillus subtilis. Based on [U-(13)C(6)]glucose-labeling experiments and metabolic flux analysis, the total energy flux in slow-growing, glucose-limited B. subtilis is almost exclusively partitioned in maintenance metabolism and biomass formation. In excess-glucose cultures, in contrast, uncoupling of anabolism and catabolism is primarily achieved by overflow metabolism, while two quantified futile enzyme cycles and metabolic shifts to energetically less efficient pathways are negligible. In most cultures, about 20% of the total energy flux could not be assigned to a particular energy-consuming process and thus are probably dissipated by processes such as ion leakage that are not being considered at present. In contrast to glucose- or ammonia-limited cultures, metabolic flux analysis revealed low tricarboxylic acid (TCA) cycle fluxes in phosphate-limited B. subtilis, which is consistent with CcpA-dependent catabolite repression of the cycle and/or transcriptional activation of genes involved in overflow metabolism in the presence of excess glucose. ATP-dependent control of in vivo enzyme activity appears to be irrelevant for the observed differences in TCA cycle fluxes.

Stoichiometric growth model for riboflavin-producing Bacillus subtilis.

Rate equations for measured extracellular rates and macromolecular composition data were combined with a stoichiometric model to describe riboflavin production with an industrial Bacillus subtilis strain using errors in variables regression analysis. On the basis of this combined stoichiometric growth model, we explored the topological features of the B. subtilis metabolic reaction network that was assembled from a large amount of literature. More specifically, we simulated maximum theoretical yields of biomass and riboflavin, including the associated flux regimes. Based on the developed model, the importance of experimental data on building block requirements for maximum yield and flux calculations were investigated. These analyses clearly show that verification of macromolecular composition data is important for optimum flux calculations.

Myocardial blood flow and flow reserve after coronary reimplantation in patients after arterial switch and ross operation.

BACKGROUND: Coronary reimplantation is used in therapy for congenital heart disease, such as in the arterial switch (ASO) and Ross operations. The adequacy of myocardial perfusion may remain a matter of concern. The aim of the present study was to stratify the effect of coronary reimplantation on myocardial perfusion and to highlight the clinical relevance of any attenuation in myocardial perfusion. METHODS AND RESULTS: A total of 21 children with transposition of the great arteries at a mean interval of 11.2+/-2.9 years after ASO and 9 adolescents at a mean interval of 4.2+/-2.1 years after the Ross procedure were investigated. All patients were asymptomatic and had a normal exercise capacity. On stress echocardiography, 2 of the ASO patients had dyskinetic areas within the left ventricular myocardium, and 5 had adenosine-induced perfusion defects on positron emission tomography. No coronary obstruction was detected on coronary angiography in any patient, but a common finding was right coronary dominance and a small caliber of the distal part of the left anterior descending artery. Coronary flow reserve (CFR) was significantly reduced in all patients after ASO when compared with 10 normal healthy volunteers (age, 25.6+/-5.3 years). CFR was normal in the 9 patients who had the Ross operation (age, 19.2+/-7.6 years); exercise-induced perfusion defects were not detected in the Ross patients. CONCLUSIONS: Children after ASO are asymptomatic, without clinical signs of coronary dysfunction. In contrast to patients who had the Ross operation, stress-induced perfusion defects and an attenuated CFR were documented. The prognostic implications of these findings and the clinical consequences are unclear; nevertheless, close clinical follow-up of ASO patients is mandatory.

Divergence in distribution and prognostic significance of major basement components in laryngeal carcinomas.

In previous studies, we demonstrated a loss of major basement membrane (BM) components in laryngeal squamous cell carcinomas and provided initial evidence that this was of potential prognostic significance. In our current study, we extended the panel of BM antibodies and enlarged our study group in order to perform a multivariate statistical analysis. We analyzed 26 laryngeal squamous cell carcinomas immunohistochemically for the distribution of the BM-components collagen IV, collagen VII, laminin-1, laminin-5, perlecan and fibronectin. The resulting data were correlated with clinical prognostic factors and statistical correlation coefficients were determined for independent uni- and multivariate analysis. All carcinomas analyzed revealed defects of the peritumoral BM with more extensive loss of collagen VII than collagen IV, laminin-1, perlecan and fibronectin. Laminin-5 in contrast was present even in poorly differentiated tumors showing an enhanced intracytoplasmatic staining in the tumor cells. Furthermore, our statistical analysis did not show independent prognostic significance of any of the BM-components. Our observations indicate a divergence between the loss of several major BM-components (collagens IV, VII, laminin-1, perlecan) and an enhanced deposition of laminin-5. This suggests a severely altered cell-matrix interaction, since laminin-5 links the collagen VII-containing anchoring fibrils to cell receptors of the integrin type.

Chloride binding by the AML1/Runx1 transcription factor studied by NMR.

It is known that the DNA binding Runt domain of the AML1/Runx1 transcription factor coordinates Cl(-) ions. In this paper we have determined Cl(-) binding affinities of AML1 by (35)Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl(-) with a dissociation constant (K(d,Cl)) of 34 mM. If CBFbeta is added to form a 1:1 complex, the K(d,Cl) value increases to 56 mM. Homology modeling suggests that a high occupancy Cl(-) binding site overlaps with the DNA binding surface. NMR data show that DNA displaces this Cl(-) ion. Possible biological roles of Cl(-) binding are discussed based on these findings.

Crystallization and preliminary studies of the DNA-binding runt domain of AML1.

The acute myeloid leukaemia 1 (AML1) protein belongs to the Runx family of transcription factors and is crucial for haematopoietic development. The genes encoding Runx1 and its associated factor CBF beta are the most frequent targets for chromosomal rearrangements in acute human leukaemias. In addition, point mutations of Runx1 in acute leukaemias and in the familial platelet disorder FPD/AML cluster within the evolutionary conserved runt domain that binds both DNA and CBF beta. Here, the crystallization of the Runx1 runt domain is reported. Crystals belong to space groups C2 and R32 and diffract to 1.7 and 2.0 A resolution, respectively.

The quality of a liver graft.

Because transplantation success is influenced by the quality of the graft, the objective of this study was to find parameters to evaluate transplant livers in the recipient centre. In 64 liver grafts, the venous effluates of a portal back-table flush were investigated for various parameters. Amongst them, glutathione S-transferase (GST), glutamate dehydrogenase (GLDH) and the leucocyte count were found superior in predicting graft survival. Using the combination of these parameters, 100-day graft survival of between 95% (all parameters positive) and 0% (all parameters negative) was predicted. We concluded that good liver grafts are characterized by a low width of injury (cytosolic component: GST), a low depth of injury (mitochondrial component: GLDH), as well as by a potential to induce tolerance (passenger leucocytes). Perfusate analysis seems to be a valuable tool to recognize problematic grafts in advance and to quantify the "graft factor" in considerations concerning quality control.

Relationship between viral load in blood, cerebrospinal fluid, brain tissue and isolated microglia with neurological disease in macaques infected with different strains of SIV.

The role of the viral burden in the brain for the pathogenesis of human immunodeficiency virus-associated neurological disorders is still unclear. To address this issue, we have quantified the viral load in plasma, cerebrospinal fluid (CSF) and brain tissue of macaques infected with simian immunodeficiency virus (SIV). We discovered that the viral strain used for infection determines the replicative capacity in microglial cells as well as the extent of neuropathological lesions and the occurrence of neurological symptoms. Moreover, the viral load in the brain parenchyma correlated with the development of overt neurological disease whereas the one in plasma did not. By comparing the viral load in three different compartments, we demonstrated that the viral burden in the CSF is influenced both by the viral replication in the periphery as well as in the brain parenchyma. According to these results, it is not the absolute amount of viral load in the CSF but rather the viral antigen contributed by the viral production within the brain which correlates with the development of neurological disease. In longitudinal studies, we observed that this autochthonous virus production, as evidenced by a ratio of the viral load in CSF to the one in plasma, takes place for a prolonged period of time before overt neurological signs are manifested. This finding suggests that this ratio could be used as a prognostic marker for immunodeficiency virus-induced neurological disease.

Estimation of P-to-O ratio in Bacillus subtilis and its influence on maximum riboflavin yield.

Simultaneous growth and riboflavin overproduction were investigated using a previously developed stoichiometric model of Bacillus subtilis metabolism. A fit of model predictions to experimental data was used to obtain estimates of fundamental energetic parameters of B. subtilis. Although multiple solutions describe the experimental data, evidence for a P-to-O ratio of about 1(1/3) mole of ATP produced per atom of oxygen consumed in oxidative phosphorylation was provided by genomic analysis of electron transport components, because no homologue of the proton-translocating NADH dehydrogenase I was found in the B. subtilis genome database. These results allow us to devise a rational metabolic engineering strategy to improve riboflavin production. The potential influence of increased energy coupling in oxidative phosphorylation on riboflavin yield is discussed. Higher coupling is most significant under carbon-limiting conditions in slow-growing cells, that is, in fed-batch processes of industrial interest.

Tissue distribution of major matrix metalloproteinases and their transcripts in human breast carcinomas.

The key event of invasive growth of malignant epithelial tumors is the dissolution of the peritumoral basement, membrane (BM). Accordingly, numerous immunohistochemical studies have shown that particularly in breast carcinomas there is an almost complete loss of the BM, even in well-differentiated carcinomas. In order to find out the significance of tumor-associated BM-degradation we localized major matrix metalloproteinases (MMPs) as the most important proteolytic enzymes for connective tissue dissolution. As a prerequisite, we had to identify antibodies reacting specifically on paraffin-embedded tissue material. Extensive pretesting of MMP-2, -3 and -9 antibodies of various sources provided evidence that only a small proportion of the antibodies analyzed showed a specific, positive staining result, as most of the commercial antibodies did not react on the paraffin material or revealed non-specific staining results. Using the specifically reacting antibodies, we analyzed material from 65 cases of invasive ductal breast cancer by immunohistochemistry for the localization of MMP-2, -3 and -9 and by the non-radioactive in-situ hybridization technique for the localization of the MMP-3-mRNA. The specificity of the in-situ hybridization was analyzed using the sense control. We observed a distinct positive immunoreaction for MMP-2, -3 and -9 over both invasive, as well as non-invasive tumor cells, without apparent differences in the staining intensity. Remarkably, there was a significant staining of tumor cell complexes undergoing lymphangiotic dissemination. In addition to this tumor cell staining pattern, a positive immunoreaction, although to reduced proportion, was observed over peritumoral fibroblastic and endothelial stroma cells. Normal breast tissue also revealed a positive immunostaining of epithelial and stromal cells. Using in-situ hybridization, we observed mRNA expression for MMP-3 both in tumor and stroma cells, comparable to the protein data. Normal breast epithelia reacted weakly positive for MMP-3-mRNA. Our data indicate that there is a major active expression of important MMPs in invasive breast carcinomas as the possible cause for the matrix dissolution. These MMPs are synthesized both by tumor and peritumoral stroma cells which may interact with each other. However, the de-novo synthesis and the amount of immunoreactive enzyme protein does not seem to be significantly enhanced in invasive versus noninvasive tumor areas or normal breast epithelia, indicating that other mechanisms, such as enzyme activation and/or differences in the levels of proteinase inhibitors may be biologically essential factors.

Expression of TGF-beta 1 protein and mRNA and the effect on the tissue remodeling in laryngeal carcinomas.

TGF-beta is one of the most effective suppressors of cell proliferation and acts as a potent inducer of matrix formation by promoting the neosynthesis of the extracellular matrix and inhibiting the synthesis of matrix degrading enzymes. In this study we analysed the expression of TGF-beta 1 protein and mRNA in normal laryngeal mucosa and in invasive laryngeal carcinomas, to find out any differences in the amount of expressed TGF-beta 1 and to identify the cells which are actively involved in the synthesis of TGF-beta 1. In addition, we analysed the biological effect of TGF-beta 1 on the rate of tumor cell proliferation and the amount of neosynthesis of the extracellular matrix. The study comprised a series of 24 laryngeal squamous cell carcinomas (SCC) of different degree of tumor cell differentiation and 10 cases of normal laryngeal mucosa, which was immunhistochemically analyzed using antibodies against TGF-beta 1, the cell proliferation antigen Ki-67 (Mib-1 antibody) and major interstitial collagen types (III, V, VI) and tenascin. The TGF-beta 1-mRNA was identified by non-radioactive in-situ hybridization using a 360bp cDNA-clone for TGF-beta 1. All immunostainings and the in-situ hybridization were quantitatively evaluated by morphometric analysis and subjected to a statistical evaluation. In the normal laryngeal mucosa we found TGF-beta 1 protein mainly in the suprabasal epithelial cell layer and in some stroma cells below the epithelium. The mRNA of TGF-beta 1 was located in the basal cell layer of the normal mucosa as well as in the stroma cells next to the squamous cell epithelial. All cases of invasive carcinoma analysed showed a positive cytoplasmatic staining for TGF-beta 1 in the tumor cells as well as in stroma cells with a significantly more intense staining in the tumor cells than in the stroma cells. The in-situ hybridisation for TGF-beta 1-mRNA provided also positively stained tumor as well as stroma cells. Again, the amount of positively labeled tumor cells outnumbered the amount of stained stroma cells. In addition, we found a dramatic increase of the proliferation index in the invasive laryngeal carcinomas which was correlated with the amount of TGF-beta 1 expression. The collagens III, V and VI as well as tenascin were found strongly in the invasive carcinomas when compared to the normal mucosa, with statistically positive correlation. In conclusion, we found that invasive tumor growth of the larynx is associated with an increase of the TGF-beta 1 protein and mRNA expression. The synthesis of TGF-beta 1 is mainly performed by the tumor cells, but also to a lesser extent by the stroma cells. We provide evidence, that the TGF-beta 1 is biologically active on the neofomation of extracellular matrix components. In contrast the lack of a suppressive effect on the proliferative capacity of the tumor cells, indicates a "dissociation" of the TGF-beta effect in SCCs. A possible reason for this dissociation may lie in a partial dysfunction of the TGF-beta receptors of the tumor cells or in defects of the intracellular signaling pathways.

Morphological aspects of altered basement membrane metabolism in invasive carcinomas of the breast and the larynx.

In the present study we compared the localization of major basement membrane (BM) components and their mRNAs between invasive carcinomas of the breast (adenocarcinomas) and larynx carcinomas (squamous cell carcinomas, SCC), in order to determine the extent of BM production and deposition in malignant tumors of biologically different behaviour. Thus, breast carcinomas usually show a rapid locoregional/systemic spread, while the laryngeal SCCs normally show a more locally restricted growth pattern. While normal mammary glands and laryngeal mucosa revealed an intact epithelial BM as evidenced by a continuous linear staining for collagen IV, laminin-1, heparan sulfate proteoglycan (perlecan) and fibronectin-as well as collagen VII in the larynx mucosa-, this continuous staining was lost in the invasive carcinomas, however, affecting the two tumor types differently. In the breast carcinomas, a complete loss was seen even in well differentiated tumors affecting the various BM components similarly, while in the SCCs well differentiated carcinomas had retained significantly more BM material than poorly differentiated ones. In the SCCs, an "early" loss of collagen VII contrasted with a "later" loss of collagen IV, laminin, perlecan and fibronectin the extent of which was, however, associated with a decreasing degree of differentiation. In contrast to the protein findings, by use of the in-situ hybridization we observed a significant expression of mRNA for collagen IV, perlecan and fibronectin. The resulting pattern was comparable between both tumor types and not significantly related to the tumor cell differentiation. Both tumor cells and stroma cells were positively labelled with a more extensive labelling of the stroma cells. Our observations indicate a similar upregulation of the mRNAs for BM-components in breast and larynx carcinomas, but significant differences in the BM-protein deposition so that either major differences in presumed BM-proteolysis or further translational defects are suggested. Furthermore, it can be speculated that the far lesser amount of BM-material in the breast carcinomas may be linked to the more aggressive metastatic spread of those tumors, particularly when compared to the SCCs.