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High-risk human papilloma virus (HPV) is the leading cause of cervical cancer. HPV oncogenes are responsible for the development of malignancy, and the E6 oncoprotein that HPV expresses induces the degradation of tumour suppressor protein p53 (p53). This degradation leads to the upregulation of p16; however, unidentified proteins may also serve a role in the development and progression of cervical cancer. Therefore, the aim of the present study was to analyse the expression levels of E6, p53, p16, MDM2 proto-oncogene (MDM2) and galectin-3 (gal-3) in cervical cancer specimens. A total of 250 cervical cancer tissue slides were used. The expression of E6, p53, p16, MDM2 and gal-3 was analysed with immunohistochemical methods and a semi-quantitative scoring. SPSS software was used for the statistical evaluation of staining results and survival analysis of patients with cervical cancer. Cervical cancer specimens demonstrated significantly increased E6 staining with advanced T-status and increased International Federation of Gynecology and Obstetrics classification. E6, p53 and p16 demonstrated significantly different expression levels in squamous epithelial tissue compared with adenocarcinomas. MDM2 and gal-3 demonstrated positively correlated expression levels in cervical cancer. In addition, gal-3 expression was correlated with poor prognosis in p16-negative cases. A negative correlation between the expression of E6 and a mutated form of p53 was also identified in cervical cancer. p53 mutation was demonstrated to be common in cervical cancer, and gal-3 and MDM2 appeared to act in a combined manner in this type of tumour. As gal-3 is overexpressed in the cervical cancer tissue of patients with poor prognosis, the use of gal-3 inhibitors should be investigated in future studies.

Background: Adapted from results in the field of cervical cancer, a direct connection between HPV infection and oropharyngeal carcinoma development could be established. Aim of this study was to evaluate p16 and TA-MUC1 in laryngeal cancer and their correlation to diagnostic, since TA-MUC1 is primarily restricted to malignancies. Methods: Paraffin-embedded laryngeal cancer specimens (n=129) and normal tissue (n=5) were analyzed for TA-MUC1 expression using hPankoMab-GEXTM antibody and evaluated according the immunoreactive score. Survival was assessed via log-rank test and Kaplan-Meier-survival analysis. Results: Significant correlation with tumor grading and staging was exhibited by TA-MUC1staining, while being negative in normal tissues. Expression of p16 significantly increased in T4 compared to T1 tumors. Significant differences in overall survival were found in correlation to TNM-classification, grading and relapse. TA-MUC1 showed a positive trend correlating to p16. Conclusion: Because of this positive trend, we suggest a HPV association in head and neck tumors. Most likely due to an insufficient quantity of HPV-positive patients, no statistical significance could be established. However, targeting TA-MUC1 would improve tumor therapy by linking hPankoMab-GEXTM to the overexpressed galectin. Systematic analysis of HPV-association should be performed generally in laryngeal cancer to gain further information about the interaction of HPV and malignancies.

Objective Trophoblast cells synthesize a variety of hormones, in which hCG plays a major role. On the strength of special enzymes they are capable of catalyzing the reaction cortisol cortisone. In vitro experiments showed the influence on ACTH- and cortisol secretion by CRH, ACTH and prednisolon. In this study we describe the influence of cortisol (prednisolon) on hCG production of trophoblast cells in vitro. Material and methods Trophoblast cells were prepared from human term placentae by standard trypsin-DNAse dispersion of villous tissue followed by a percoll gradient centrifugation step. After adjusting the cell suspension to a defined cell concentration of 1 x 10 (6) cells/ml cells were cultivated. The addition of prednisolon followed every eight hours. The samples were collected after 24 hours for a total of 96 hours also from unstimulated cultures. Culture supernatants were assayed for hCG by enzyme-immunometric methods. Results The addition of prednisolon (50 microg/ml) stimulates the concentration of hCG in a time-depending manner. Conclusions The trophoblast cell shows an increase in the concentration of hCG after stimulation with cortisol. For the first time an influence of cortisol (prednisolon) on hCG production could be demonstrated in cultured trophoblast cells.

The glycoprotein, glycodelin A (GdA) is a main product of the maternal decidua in the first trimester of pregnancy and is secreted into the amniotic fluid. The purpose of this study was to investigate the effect of GdA on secretion and surface markers of isolated first trimester trophoblasts in vitro. Cytotrophoblasts were prepared from human first trimester placentae and incubated with varying concentrations of GdA or transfected separately with the expression plasmid of GdA. Supernatants were assayed for human chorionic gonadotropin (hCG) protein concentrations. Expression of human placental lactogen (hPL), mucin 1 (MUC1) and the Thomsen–Friedenreich (TF) epitope was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry. Glycodelin A induced a reduced expression of hPL compared with unstimulated controls. Expression of MUC1 was not affected by GdA. Freshly isolated trophoblast cells showed no TF expression but became positive for this antigen after 96 h of cultivation. GdA-stimulated trophoblast cells inhibited TF expression after 96 h of cultivation. GdA plasmids induced a significantly higher hCG production in transfected cells than in cells transfected with the empty plasmid. The results obtained in this study suggest that GdA is involved in the differentiation of trophoblast cells. The treatment of GdA plasmid transfected trophoblast cells stimulated hCG production in isolated trophoblast cells and inhibited hPL and TF expression, suggesting a functional link between hCG and GdA.

Type X collagen has so far not been reported to occur in human intervertebral discs. The objective of this study was therefore to investigate the occurrence of type X collagen in human lumbar intervertebral discs during ageing and degeneration. Ninety intervertebral discs with adjacent endplates were excised in toto from individuals (0-86 years) without known spinal disease and were processed for routine decalcified histology. Appropriate slices of each disc were processed for immunohistochemistry using a type-specific, monoclonal antibody raised against human type X collagen. Each intervertebral disc was examined for macroscopic and histomorphological features of disc degeneration. Immunohistochemically, a positive specific type X staining was observed in the hypertrophic zone of the growth plate and only in the interstitial matrix of juvenile (2 years) nucleus pulposus. In adult discs, type X collagen could be localized in conjunction with advanced disc degeneration and first occurred in the disc matrix (i.e., pericellular region) of a 47-year-old specimen. Positive type X staining of the disc matrix was more frequently found in senile (70 years) discs with end stages of disc degeneration. This study provides the first evidence for the occurrence of type X collagen in human lumbar intervertebral discs and it appears that type X collagen is re-expressed in late stages of disc degeneration.

The time-dependent appearance of signs of cell death was investigated in human skin wounds using in situ end labeling of DNA fragments (ISEL). In the dermal layer an average of not more than 0.3 positively stained fibroblastic cells/0.01 cm x 0.01 cm was found up to a postinfliction interval of approximately 6 h. Average numbers exceeding 1 positive cell/0.01 cm x 0.01 cm were first detectable in a skin wound after 24 h. Therefore, average numbers greater than 1 labeled cell/ 0.01 cm x 0.01 cm indicate a postinfliction interval of approximately 1 day. An increase in the average number of positively stained cells occurred with increasing wound age. Values exceeding 3 cells/0.01 cm x 0.01 cm were first detectable 19 days after wound infliction. Accordingly, values of more than 3 labeled cells indicate a postinfliction interval of approximately 3 weeks or more. Since low numbers of labeled fibroblastic cells or even negative results were found in wounds of advanced age, only positive results provide information which can be useful for a forensic age estimation of human skin wounds.

Cholesteatoma is a disease with an epithelial state of hyperproliferation based on a disturbed cell-matrix interaction. An analysis of the basement membrane, which can modulate epithelial proliferation, reveals a dysfunction of this layer between cholesteatoma and stroma. Cholesteatoma specimens were obtained from six patients undergoing middle ear surgery. Immunohistochemical staining was performed using antibodies against the major components of basement membranes such as laminin, heparan sulfate proteoglycan (HSPG), collagen IV and fibronectin. In cholesteatoma epithelium, we found a fragmented basement membrane with small amounts of laminin and HSPG showing only occasional positively stained fragments while collagen IV was almost completely absent. In contrast, fibronectin demonstrated an intensive staining especially in the stroma adjacent to the basement membrane. This staining pattern resembles the initial rearrangement of the epithelial basement membrane in human skin wounds and provides evidence for a lost contact inhibition on the provisional fibronectin-layer as a reason for the hyperproliferative nature of cholesteatoma lesions.

Estradiol and progesterone mediate their actions by binding to classical nuclear receptors, estrogen receptor α (ERα) and estrogen receptor β (ERβ) and progesterone receptor A and B (PR-A and PR-B) and the non-classical G protein-coupled estrogen receptor (GPER). Several animal knock-out models have shown the importance of the receptors for growth of the oocyte and ovulation. The aim of our study was to identify GPER in human granulosa cells (GC) for the first time. Moreover, the effect of different doses of gonadotropins on estrogen and progesterone receptors in the human ovary should be investigated as follicle stimulating hormone (FSH) and luteinizing hormone (LH) are also responsible for numerous mechanisms in the ovary like induction of the steroid biosynthesis. Human GC were cultured in vitro and stimulated with different doses of recombinant human FSH or LH. Receptor expression was analyzed by immunocytochemistry and quantitative real-time RT-PCR. GPER could be identified for the first time in human GC. It could be shown that high concentrations of LH increase GPER protein expression. Furthermore FSH and LH increased ERβ, PR-A and PR-B significantly on protein level. These findings were verified for high doses of FSH and LH on mRNA level. ERα was not affected with FSH or LH. We assume that gonadotropins induce GPER, ERβ and PR in luteinized granulosa cells.

The retinoid X receptor α (RXRα) is a nuclear hormone receptor that is able to bind other nuclear receptors in a heterodimeric complex, thereby activating gene transcription. Recently, we identified enhanced expression of RXRα in extravillous trophoblasts (EVT) and villous trophoblasts (VT) of miscarried placentas. In addition, an increased number of apoptotic EVT was present in miscarried placentas. In this study, on the basis of immunocytochemical analysis, western blots, and quantitative real-time reverse transcription PCR, we could demonstrate a reduced expression of RXRα in choriocarcinoma cell lines and in human VTs after stimulation with the retinoids 9-cis-retinoic acid and all-trans-retinoic acid and the prostaglandin 15-deoxy-Δ12,14-prostaglandin J2. Furthermore, a simultaneous expression of RXRα and the apoptotic marker M30 CytoDEATH in EVT of miscarried placentas from the first trimester was shown. In EVT of control placentas from legal termination of pregnancies, no co-expression of RXRα and M30 could be detected. A likely conclusion is that RXRα plays an important role in the induction of apoptosis. Downregulation of RXRα, as observed in the tested choriocarcinoma cells and trophoblasts, might serve as a protection against apoptosis and miscarriage. In conclusion, RXRα represents a potential target in the treatment of recurrent miscarriages.

We review the current knowledge on alterations of the major basement membrane (BM) components and their cellular integrin receptors in benign and malignant tumors of epithelial and mesenchymal origin. While benign tumors usually exhibit a continous BM, recent analyses provide evidence that invasive growth of carcinomas coincides with (a) a loss in a proper BM, (b) changes in the type of integrin receptor expression and (c) the retained ability of certain tumor cells to synthesize matrix components. This latter aspect has been regarded as a potentially beneficial 'host' mechanism against invasive growth. This assumption is strongly supported by the finding of a positive correlation between the extent of BM loss and both a lesser degree of tumor differentiation and a worse prognosis of tumor growth. The resulting concept indicates that in carcinomas an imbalance in the cell-matrix interaction is the leading element in invasive growth. In mesenchymal tumors a somewhat different role of the BM can be observed. Thus, the qualitative and quantitative expression of major BM components in benign mesenchymal tumors closely relates to the BM pattern of normal tissues providing a histogenetically oriented classification of benign mesenchymal tumors. Most well-differentiated sarcomas retain a BM pattern close to that of the histogenetically related tissue, although in poorly differentiated sarcomas no such attribution to a histogenetic orientation of the tumor cells can be found.

The tissue localization was analysed of collagen X during human fetal and juvenile articular cartilage-bone metamorphosis. This unique collagen type was found in the hypertrophic cartilage zone peri- and extracellularly and in cartilage residues within bone trabeculae. In addition, occasionally a slight intracellular staining reaction was found in prehypertrophic proliferating chondrocytes and in chondrocytes surrounding vascular channels. A slight staining was also seen in the zone of periosteal ossification and occasionally at the transition zone of the perichondrium to resting cartilage. Our data provide evidence that the appearance of collagen X is mainly associated with cartilage hypertrophy, analogous to the reported tissue distribution of this collagen type in animals. In addition, we observed an increased and often "spotty" distribution of collagen X with increasing cartilage "degeneration" associated with the closure of the growth plate. In basal hypertrophic cartilage areas, a co-distribution of collagens II and X was found with very little and "spotty" collagen III. In juvenile cartilage areas around single hypertrophic chondrocytes, co-localization of collagens X and I was also detected.

In 62 human skin wounds (surgical wounds, stab wounds and lacerations after surgical treatment) we analyzed the immunohistochemical localization of collagen IV in the epithelial basement membrane. In 27 of these wounds the distribution of collagen VII, which represents a specific component of the basement membrane of stratified epithelia, was also analyzed. We were able to demonstrate a virtually identical co-distribution of both collagen IV and VII in the wound area with no significant time-dependent differences in the appearance of both collagen types. Fragments of the epithelial basement membrane could be detected in the wound area from as early as 4 days after wounding and after 8 days a complete restitution of the epithelial basement membrane was observed. In all cases with a wound age of more than 21 days the basement membrane was completely reformed over the former lesional area. The period between 8 and 21 days after wounding was characterized by a wide variability ranging from complete restitution to deposition of basement membrane fragments or total lack of the epidermal basement membrane.

KEYWORDS CLASSIFICATION: Antibodies;Antigens,Tumor-Associated,Carbohydrate;Cell Line,Tumor;Cell Proliferation;drug effects;Female;Galectin 1;Germany;Glycophorin;Humans;immunology;Immunoglobulin M;metabolism;mechanisms of carcinogenesis;pharmacology;Research;Trophoblastic Neoplasms;Trophoblasts. Galectin-1 (gal-1), a member of the mammalian beta-galactoside-binding proteins, recognizes preferentially Galbeta1-4GlcNAc sequences of several cell surface oligosaccharides. We demonstrate histochemically that the lectin recognizes appropriate glycotopes on the syncytiotrophoblast and extravillous trophoblast layer from second trimester human placenta and on BeWo chorion carcinoma cells. Gal-1 binding to BeWo cells was diminished by the Thomsen-Friedreich (TF)-disaccharide (Galbeta1-3GalNAc-) conjugated to polyacrylamide (TF-PAA). Gal-1 also inhibited BeWo cell proliferation in a concentration-dependent manner. Similar antiproliferative effects were also observed with an anti-TF monoclonal antibody (mAb, A78-G/A7). Therefore, we conclude that ligation of Galbeta1-4GlcNAc and Galbeta1-3GalNAc epitopes on BeWo cells may have regulatory effects on cell proliferation.

Galectin-1 (gal-1), a member of the mammalian beta-galactoside-binding proteins, recognizes preferentially Galbeta1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. As demonstrated histochemically, the lectin recognizes appropriate glycoepitopes on the syncytiotrophoblast and on chorionic carcinoma cells (BeWo). Freshly isolated trophoblast cells and trophoblast tumor cells Jeg3 did not bind gal-1. BeWo cells in contrast to Jeg3 form a syncytium in vitro and synthesize progesterone as well as hCG. BeWo cells were used as an approach to study the effects of gal-1 on hormone production. The lectin decreased cellular hCG and progesterone production as well as hCGbeta gene transcription as measured by real-time RT-PCR. Gal-1 mediated inhibition of cellular progesterone production was reduced in the presence of a Thomsen-Friedenreich (TF)-polyacrylamide conjugate. Inhibition of cellular hCG and progesterone production was also induced by anti-TF monoclonal antibodies. The results demonstrate that ligation of Galbeta1-4GlcNAc and Galbeta1-3GalNAc (TF) epitopes on BeWo cells may have regulatory effects on hCG and progesterone production.

In the present study, we have compared the staining pattern of the Solophenyl-Red 3 BL-method for the visualization of collagen type III with the immunohistochemical staining in serial sections from 7 skin wounds (wound age 3 days up to 4 weeks) to elucidate the specificity of the histochemical staining method. Large amounts of collagen type III were clearly detectable in the investigated wounds using the immunohistochemical technique. In the sections stained with Solophenyl-Red, however, only 3 out of 7 skin lesions showed a significant positive red staining at the wound margin or in the granulation tissue, while the adjacent normal connective tissue revealed a typical intensive staining. Using polarization microscopy no characteristic bright green fibrils, as reported for collagen type III, could be seen in the wound areas without positive Solophenyl-Red staining. Since the localization of collagen type III detected by immunohistochemistry and the presumed distribution of this collagen type by the Solophenyl-Red method was not identical, the histochemical polarization method has to be regarded as non-specific for visualization of this collagen type.

Conflicting data have been reported on the spatial distribution of type X-collagen expression in osteoarthritis, and no concise data exist on a possible correlation between type X-collagen expression and clinical and radiological alterations. Well defined clinical and radiological data were compared with histopathological and immunohistochemical findings to investigate the expression of type-X collagen in osteoarthritis of the hip joint. Femoral heads were obtained in toto from 11 patients undergoing routine hip arthroplasty for femoral neck fractures (n = 3) or osteoarthritis (n = 8) and from 13 patients (age: 12 days to 69 years) without any evidence of hip-joint pathology. Whole coronal sections from the femoral head were decalcified for routine histology and immunohistochemical analysis with use of type-specific monoclonal antibodies to type-X collagen. Our results demonstrate that type-X collagen is consistently found in osteoarthritic cartilage and is absent from normal adult cartilage (including the region of calcified cartilage). Except for the occurrence of type-X collagen in the middle zone of articular cartilage in advanced stages of osteoarthritis, there is no specific change in the staining pattern or intensity for the collagen during osteoarthritis, particularly when the staining is related to clinical and radiological parameters. Hardly more than 20% of the extracellular matrix stained for type-X collagen; therefore, we suggest that, in most cases, this type of collagen may not play a direct biomechanical role in the weakening of osteoarthritic cartilage but rather may contribute indirectly to a disturbance of the disc biomechanics by altering matrix-molecule interaction. However, expression of type-X collagen may indicate a change in chondrocyte phenotype that consistently coincides with the formation of chondrocyte clusters, one of the first alterations in osteoarthritis visible on histologic examination.

We report on the morphological and trace element findings of several internal organs from an Egyptian mummy approximately dating from the year 950 B.C. according to 14C-analysis. By use of a multidisciplinary approach we succeeded in discovering evidence for severe and presumably recurrent pulmonary bleeding during life. This was suggested by the finding of massive haemosiderin deposits in the lung and a selectively and markedly elevated level of iron in trace element analysis of the lung tissue. Furthermore, we observed an enhanced deposition of birefringent particles in the lung tissue, without significant fibrosis. The histological analysis of liver, stomach and intestine confirmed the macroscopic organ diagnoses without evidence of any major pathological processes. In addition, analysis for various drugs revealed a significant deposition of tetrahydrocannabinol (THC), nicotine and cocaine in several organs of the mummy. The concentration profiles additionally provide evidence for a preferential inhalation of THC, while nicotine and cocaine containing drugs seem to have been consumed orally.

The distribution of the interstitial collagens I, II and III was analyzed immunohistochemically in cartilage and bone samples from 32 patients with degenerative osteoarthrosis at various morphological stages. The alterations observed showed a very patchy, focal distribution demonstrating significant heterogeneity in the tissue reaction. In minor osteoarthrotic lesions a focal pericellular deposition of collagens III and I was seen, while the majority of the interterritorial matrix reacted exclusively with collagen II antibodies. These changes were first seen in the superficial cartilage layer. At the more advanced stages of osteoarthrosis, particularly when osteophytic bone spur formation was present, extensive changes in the expression of collagen types in the pericellular matrix was revealed with extensive and overlapping localization of collagens I, II and III in the whole cartilage. These observations support the suggestion that degenerative cartilage shows a collagen type “switch”. In addition, it was demonstrated that the interterritorial cartilage matrix was still mainly composed of collagen II even in advanced lesions. These observations may explain some of the previous discrepancies reported.

Background: Epidermal growth factor (EGF) displays manifold functions in theplacenta including tuningof invasion. Recent reports have demonstrated that activation of signal transducer and activator of transcription (STAT) proteins, including STAT3 and STAT5 actively participate in tumor development and progression. STAT5 is an intracellular proteindownstreamtyrosine-kinase receptors,which stimulates proliferation and cell cycle progression. Several cytokines involved in the regulation of trophoblast behaviourhavebeen shown tomediate their effects though STAT3, but less is known about STAT5 activation in pregnancy. The aim of this study was to assess correlation of STAT5 and STAT3 phosphorylation. Materials and methods: JAR, BeWo and JEG3 choriocarcinoma cells as well as HTR-8/SVneo immortalized trophoblast cells were stimulated with IL-2, IL-11, leukemia inhibitory factor (LIF), epidermal growth factor (EGF) or granulocyte macrophage-colony stimulating factor (GM-CSF) by applying diverse concentrations and times. The effect of the cytokines and growth factors on the expression, phosphorylation and localization of STATs were determined by Western blotting and immunocytochemistry. Results: Only EGF induces STAT5 phosphorylation (Tyr 694) in all analyzed cell lines, while the other cytokines, except IL2, induce different intensity of STAT3 phosphorylation (Tyr 705) depending on cytokine and cell line. Discussion: It can be concluded that cytokines that activate STAT3 and STAT5 in trophoblastic cells are different. The activating cytokines are highly expressed in the placenta. Therefore, it can be assumed that simultaneous activation occurs frequently andmay further influence functions and behaviour of cells.

We monitored the immunohistochemically determined amount of surfactant-producing alveolar type II cells in human fetal lung using polyclonal antibodies against apoprotein B and C of human pulmonary surfactant. Lungs of 30 dead-born fetuses without lung affection aged between 15 and 38 weeks of gestation were evaluated and the surface density of surfactant-producing alveolar type II cells was determined by morphometry. In lungs of fetuses with a gestational age less than 22 weeks no relevant number of positively reacting cells could be found. Between the 22nd and 29th week a progressive increase with considerable inter-individual variability was observed. From the 30th week on the number of the type II pneumocytes appeared rather constant without further significant increase. We provide evidence that the immunohistochemical detection of surfactant-producing alveolar type II cells is useful for the determination of the age of unknown and especially fragmented fetuses: The lack of surfactant-producing alveolar type II cells in fetal lungs before the 22nd week allows a rather safe distinction between fetal lungs of higher age from those of lesser age. Between the 22nd and 29th week an age-dependent increase in the number of these cells occurs with wide inter-individual variability allowing only an approximate age determination. In particular, this may be an important piece of information in fragmented fetal corpses. Furthermore, the number of surfactant-producing alveolar type II cells provides additional information on pulmonary maturation and may thus be helpful in the estimation of a theoretical survival chance.

Human skin wounds (n = 62) with a wound age between 5 h and 6 weeks were investigated. The appearance of cell-associated pericellular basement membrane components collagen type IV, laminin and heparan sulfate proteoglycan (HSPG) in myofibroblasts was evaluated by immunohistochemistry. Laminin and HSPG were first detectable around myofibroblasts approximately 1.5 days after wounding. Collagen type IV did not appear before the 4th day after wound infliction. In wounds more than 7 days old, 94% of the cases showed fibroblastic cells positively staining for laminin, 70% of the wounds contained fibroblastic cells positive for HSPG and in 63% a positive reaction for collagen type IV was obtained around these cells. The numbers of the cases as well as of the cells positively stained for laminin exceeded the corresponding values for HSPG and especially for collagen type IV. The pericellular appearance of laminin or HSPG around myofibroblasts, therefore, indicates a wound age of at least approximately 1.5 days. The pericellular localization of collagen type IV indicates a survival time of approximately 4 days or more. Since these proteins are still detectable in the pericellular region of myofibroblasts in skin wounds with advanced wound age (6 weeks) further information for the time-estimation of older human skin lesions cannot be obtained. A semiquantitative analysis revealed no significant correlation between the number of positively stained cells and the wound age, rendering this parameter unsuitable for a practicable time-estimation of human wounds.

We analyzed the distribution of fibronectin in routinely embedded tissue specimens from 53 skin wounds and 6 postmortem wounds. In postmortem wounds a faint but focal positive staining was exclusively found at the margin of the specimens which dit not extend into the adjacent stroma. Vital wounds were classified into 3 groups. The first comprising lesions with wound ages ranging from a few seconds to 30 min, the second comprising those with wound ages upt to 3 weeks, and the third group with lesions more than 3 weeks old. Ten out of 17 lesions with a wound age up to 30 min showed a clear positive reaction within the wound area. Three specimens in this group were completely negative, while in 4 additional cases the result was not significantly different from postmortem lesions. These 7 cases were characterized by acute death with extremely short survival times (only seconds). In wounds up to 3 weeks old fibronectin formed a distinct network containing an increasing number of inflammatory cells corresponding to the wound age. In 2 cases with a survival time of 17 days and in all wounds older than 3 weeks fibronectin was restricted to the surface of fibroblasts and to parallel arranged fibers in the granulation tissue without any network structures. We present evidence that fibronectin is a useful marker for vital wounds with a survival time of more than a few minutes. Fibronectin appears before neutrophilic granulocytes migrate into the wound area. Since a faint positive fibronectin staining is seen in postmortem lesions and bleedings, we propose that only those wounds which show strong positive fibronectin staining also extending into the adjacent stroma should be regarded as vital.

Background Galectin-1 (gal-1) belongs to the family of β-galactoside-binding proteins which primarily recognizes the Galβ1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. The lectin recognizes correspondent glycoepitopes on human breast cancer cells. Galectin-1 is expressed both in normal and malignant tissues. Lymphatic organs naturally possessing high rates of apoptotic cells, express high levels of Galectin-1. Furthermore galectin-1 can initiate T cell apoptosis. Binding of galectin-1 to trophoblast tumor cells presenting the oncofetal Thomsen-Friedenreich (TF) carbohydrate antigen inhibits tumor cell proliferation. In this study we examined the impact galectin-1 has in vitro on cell proliferation, apoptotic potential and metabolic activity of MCF-7 and T-47D breast cancer cells in dependence to their expression of the Thomsen-Friedenreich (TF) tumor antigen. Methods For proliferation and apoptosis assays cells were grown in presence of 10, 30 and 60 μg gal-1/ml medium. Cell proliferation was determined by a BrdU uptake ELISA. Detection of apoptotic cells was done by M30 cyto death staining, in situ nick translation and by a nucleosome ELISA method. Furthermore we studied the impact galectin-1 has on the metabolic activity of MCF-7 and T-47D cells in a homotypic three-dimensional spheroid cell culture model mimicking a micro tumour environment. Results Gal-1 inhibited proliferation of MCF-7 cells (strong expression of the TF epitope) but did not significantly change proliferation of T-47D cells (weak expression of the TF epitope). The incubation of MCF-7 cells with gal-1 raised number of apoptotic cells significantly. Treating the spheroids with 30 μg/ml galectin-1 in addition to standard chemotherapeutic regimes (FEC, TAC) resulted in further suppression of the metabolic activity in MCF-7 cells whereas T-47D cells were not affected. Conclusions Our results demonstrate that galectin-1 can inhibit proliferation und metabolic cell activity and induce apoptosis in breast tumor cell lines with high expression levels of the Thomsen-Friedenreich (TF) antigen in monolayer and spheroid cell culture models.