Variability of impedivity in normal and pathological breast tissue

Bite wounds have a special position in traumatology because of their high complication rate compared to similar soft tissue wounds caused by other reasons. The authors report in a retrospective study about the results of 525 patients with bite wounds. In 98 (18.7%) cases the wounds were sutured primarily after surgical revision when there was no sign of infection. Antibiotic therapy (a combination of amoxicillin and clavulan acid) was given to 109 patients (20.8%) who had infected wounds or who were at greater risk for infection (e.g., extremely large wounds, large hematoma). Antibiograms were only made when the wounds had already been infected. The total infection rate came to 11.8%, with 5.2% infected facial bites, 11.3% lower extremity, 18.6% upper extremity and 18.8% hand bite wounds. The infection rate after primary suture was 10.2% (3.2% at the face, 18.8% at the upper extremity and 25% each in the hand and the lower extremity). The average time period from the trauma to the first medical treatment amounted to 11 h in the infected wounds and 2 h in the non-infected ones. Cat bites became infected in 37.5%, dog bites in 14.9%.     

Estrogen receptor variants in normal human mammary tissue

BACKGROUND: Several estrogen receptor (ER) variant messenger RNAs (mRNAs) have been identified previously in human breast cancer biopsy samples and cell lines. The relative levels of certain ER variant mRNAs have been observed to increase with breast tumor progression. In vitro assays of the function of polypeptides encoded by some of these variant mRNAs have led to speculation that ER variants may be involved in the progression from hormone dependence to independence in breast cancer. PURPOSE: We set out to establish if ER variant mRNAs are present in normal human breast tissues and, if so, to compare levels of these variants between normal and neoplastic human breast tissues. METHODS: Four human breast tissue samples from reduction mammoplasties and five samples from tissue adjacent to breast tumors were analyzed. The tissue samples were confirmed to be normal (i.e., not malignant) by histopathologic analysis. RNA was extracted immediately from adjacent frozen sections. Human breast tumor specimens originally resected from 19 patients were acquired from a tumor bank and processed in the same way as the normal tissue samples. The RNAs were then reverse transcribed and subsequently amplified with the use of the polymerase chain reaction (PCR). PCR primer sets were designed to detect several different exon-deleted ER variants and a truncated ER variant (i.e., clone 4). A semiquantitative PCR-based method was used to determine the relative expression of exon 5- and exon 7-deleted variants to wild-type ER mRNAs in the nine normal tissues and in 19 ER-positive breast tumor tissues. The Mann-Whitney rank sum test (two-sided) was used to determine P values. RESULTS: ER variant mRNAs corresponding to the clone 4 ER truncated variant and to variants deleted in either exon 2, exon 3, exons 2-3, exon 5, or exon 7 were detected in all normal samples. The results were confirmed by restriction enzyme analyses and sequencing of the PCR products. The expression of exon 5-deleted ER variant relative to the wild-type ER mRNA was significantly lower (P< .001) in normal tissue than in tumor tissue. A similar trend was noted for expression of the exon 7-deleted ER variant mRNA; however, the difference did not achieve statistical significance (P= .476). CONCLUSION: Several ER variant mRNAs are present in normal human breast tissue, but the level of expression of some of these variants may be lower in normal tissue than in tumor tissue. IMPLICATION: These data suggest that the mechanisms generating ER variant mRNAs exist in normal breast tissue and may be deregulated in breast cancer tissues. Further investigation of the role of variant ER expression in development and progression of human breast cancer appears warranted.     

Immunohistochemical identification of the receptor for urokinase plasminogen activator associated with fibrin deposition in normal and ectopic human placenta

The receptor for urokinase plasminogen activator (uPAR) is a key molecule in cell surface-directed plasminogen activation. uPAR binds urokinase plasminogen activator (uPA) and thereby focuses plasminogen activation on the cell surface. Plasmin dissolves fibrin deposits and facilitates cell migration during tissue repair processes by degrading the extracellular matrix. During human implantation and placental development, plasmin is considered important for both trophoblast migration/invasion and for fibrin surveillance. This study examined the expression of uPAR in normal and ectopic human placentae by immunohistochemistry. In first and third trimester normal placentae as well as in tubal ectopic placental tissues, a high uPAR expression was seen in the trophoblast associated with deposits of fibrin-type fibrinoid. Extravillous trophoblast of the basal plate, of the cell islands, and of the cell columns was also positive for uPAR in the first trimester whereas at term the expression of the protein was decreased. Moreover, uPAR immunostaining was observed in decidual cells throughout normal gestation and in endometrial tissues of patients with ectopic pregnancies. These findings suggest that uPAR participates in placental development and in trophoblast invasion particularly in the first trimester of pregnancy and that uPAR is involved in repair mechanisms of the trophoblast and fibrin surveillance.     

A urokinase-sensitive region of the human urokinase receptor is responsible for its chemotactic activity

The role of urokinase-type plasminogen activator (uPA) and its receptor (uPAR/CD87) in cell migration and invasion is well substantiated. Recently, uPA has been shown to be essential in cell migration, since uPA-/- mice are greatly impaired in inflammatory cell recruitment. We have shown previously that the uPA-induced chemotaxis requires interaction with and modification of uPAR/CD87, which is the true chemoattracting molecule acting through an unidentified cell surface component which mediates this cell surface chemokine activity. By expressing and testing several uPAR/CD87 variants, we have located and functionally characterized a potent uPAR/CD87 epitope that mimics the effects of the uPA-uPAR interaction. The chemotactic activity lies in the region linking domains 1 and 2, the only protease-sensitive region of uPAR/CD87, efficiently cleaved by uPA at physiological concentrations. Synthetic peptides carrying this epitope promote chemotaxis and activate p56/p59(hck) tyrosine kinase. Both chemotaxis and kinase activation are pertussis toxin sensitive, involving a Gi/o protein in the pathway.     

Expression and activity of urokinase and its receptor in endothelial and pulmonary epithelial cells exposed to asbestos

Antisera against peptides from the extreme N- and C-terminal regions of the VDR were evaluated. The N-terminal antiserum Ab192 functioned as a general-purpose antibody, being able to supershift the rhVDR in heterodimeric or homodimeric binding complexes in the EMSA, and detect both recombinant and native forms of the receptor on Western blots. The C-terminal antiserum, Ab195, also identified the receptor on Western blots, but in contrast, it displayed differential sensitivity to the conditions employed in the EMSA. In the presence of 1,25(OH)2D3, rhVDR, rhRXR alpha, and nonspecific DNA, Ab195 produced a weak supershift of the heterodimer complex in the EMSA. Significantly, omission of hormone from the binding buffer resulted in a complete shift of the bound complex with the antiserum. A complete supershift was also observed if only the nonspecific DNA was removed. Together these results indicate antiserum sensitivity to the ligand status in the rhVDR C-terminus as part of a DNA-bound heterodimer complex. Inclusion of 9-cis RA resulted in a modest increase in the amount of shifted product relative to 1,25(OH)2D3 alone. Finally, Ab195 completely supershifted the rhVDR homodimer binding complex under all tested conditions, including those analogous to where it was largely ineffective in shifting the heterodimer. Thus, Ab195 is sensitive to the ligand binding status of the VDR, discriminates heterodimer and homodimer binding interactions, and should provide an additional tool for exploring conformational changes induced in the receptor.     

N-linked glycosylation of the ligand-binding domain of the human urokinase receptor contributes to the affinity for its ligand

Variations in glycosylation exist among urokinase plasminogen activator receptors (u-PARs) from different cell types. We have studied the functional role of N-linked carbohydrate within the ligand-binding domain of u-PAR. Treatment with glycosidases demonstrated that all the N-linked carbohydrates on u-PAR are complex-type oligosaccharides. Substitution of a single Asn (Asn52) to Gln by means of site-directed mutagenesis led to an active receptor mutant with a ligand-binding domain devoid of carbohydrate. The cellular distribution, the glycosyl-phosphatidylinositol anchoring, and the conformational stability after solubilization were unaffected by this single substitution. However, ligand binding analysis demonstrated a 4- 5-fold decrease in affinity as compared with the wild type receptor. Two different strategies were used in order to obtain a u-PAR type completely devoid of N-linked carbohydrates. 1) Tunicamycin treatment of wild type u-PAR-expressing cells. 2) Mutation of all glycosylation sites (Hu-PARN5-mut). In neither case, unglycosylated receptors with ligand binding activity were identified. However, immunofluorescence studies demonstrated that the Hu-PARN5-mut was retained inside the cells in the endoplasmic reticulum. The same result was found for Hu-PARN4-mut, where only the glycosylation sites outside the binding domain were mutated. These results demonstrate that some extent of glycosylation of u-PAR is necessary for cellular transport and for molecular maturation events leading to ligand binding activity. Glycosylation of the binding domain per se affects only the affinity of the receptor. The positive modulation of the Asn52 carbohydrate side chain on ligand affinity suggests that the u-PAR glycosylation variants observed in various cell types may have different functional roles.     

Expression of the receptor for urokinase-type plasminogen activator in normal and neoplastic blood cells and hematopoietic tissue

Expression of the receptor for the urokinase type plasminogen activator (uPAR) has been studied by flow cytometry and immunohistology in normal blood and bone marrow cells, in vitro activated lymphoid cells, and tissue samples from reactive lymph nodes (n = 6), thymus (n = 2) and malignant lymphomas (n = 82), or leukemias (n = 32). HL-60 myeloid precursor cells and CD34-positive normal stem cells also were analyzed. In the normal cells, staining was confined to monocytes, macrophages, neutrophils, and myeloid precursors. No labelling was seen of normal or activated lymphoid cells. Purified CD34-positive hematopoietic progenitors were uPAR negative, but expressed uPAR during differentiation in short-term liquid culture stimulated in vitro by recombinant interleukin (IL)-1, IL-3, IL-6, granulocyte-macrophage colony stimulating factor (CSF), granulocyte-CSF, and stem cell factor. Enhanced uPAR expression was also seen in HL-60 cells after induction of differentiation with dimethyl sulfoxide or 1 alpha,25-dihydroxyvitamin D3. In lymphomas and leukemias, the staining pattern was similar to that seen in the normal cells with labelling of monocytic and myeloid that seen in the normal cells with labelling of monocytic and myeloid malignancies, but not of the neoplastic cells in B-cell or T-cell lymphomas or Hodgkin's disease. In conclusion, uPAR is a differentiation marker for myeloid and monocytic cells, and may act to facilitate migration of these cells in normal and pathologic conditions by cell-associated plasminogen activation. Whether expression of uPAR in myeloid and monocytic malignancies relates to their growth and behavior will be an important topic for investigations in the future.     

Protein kinase C mediates up-regulation of urokinase and its receptor in the migrating keratinocytes of wounded cultures, but urokinase is not required for movement across a substratum in vitro

Both in cell culture and in vivo, keratinocytes that are migrating in response to a wound express enhanced levels of both urokinase-type plasminogen activator (uPA) and the uPA cell surface receptor (uPA-R). To explore the mechanism of this up-regulation, keratinocyte cultures were treated proir to wounding with a variety of metabolic and growth factor inhibitors in order to evaluate their effect on uPA and uPA-R expression. Actinomycin D and cycloheximide inhibited the up-regulation of both uPA and uPA-R, as determined by immunohistochemistry, indicating that RNA and protein syntheses are required for their induction in migrating keratinocytes. Neither removal of protein growth factors from the medium nor addition of inhibitory antibodies to a number of growth factors depressed uPA or uPA-R induction; these findings suggest that a variety of exogenous or endogenous growth factors [i.e., basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), amphiregulin, and tumor necrosis factor-alpha (TNF-alpha) do not have a critical role in the induction of uPA or uPA-R. In contrast, when protein kinase C (PKC) was either down-regulated with bryostatin 5 or inhibited with Ro31-8220 or staurosporine, the expression of both uPA and uPA-R was greatly decreased in migrating keratinocytes. Furthermore, pharmacologic activation of PKC enhanced uPA levels in non-wounded cultures. These data suggest that the enhanced expression of uPA and uPA-R in migrating keratinocytes is mediated by selective activation of PKC in these cells, perhaps secondary to alterations in the cytoskeleton induced by wounding. To test the requirement for uPA during keratinocyte migration in vitro, the extent of migration was quantified in the presence and absence of a variety of inhibitors in the wounded culture model. Migration was not altered by actinomycin D, cycloheximide, any of the above growth factor inhibitors, anti-uPA antibodies, a variety of inhibitors of uPA or plasmin enzymatic activity, or exogenous uPA. The independence of keratinocyte migration in vitro from uPA was further suggested by experiments which combined the phagokinetic assay of migration and the zymographic assay for pericellular uPA activity; no relationship was observed between pericellular uPA activity and the motility of individual cells.     

Mitogenic effects of urokinase on melanoma cells are independent of high affinity binding to the urokinase receptor

The structural and functional properties of the urokinase-type plasminogen activator (u-PA) that are involved in the mitogenic effect of this proteolytic enzyme on human melanoma cells M14 and IF6 and the role of the u-PA receptor (u-PAR) in transducing this signal were analyzed. Native u-PA purified from urine induced a mitogenic response in quiescent IF6 and M14 cells that ranged from 25 to 40% of the mitogenic response obtained by fetal calf serum. The half-maximum response in M14 and IF6 cells was reached at u-PA concentrations of approximately 35 and 60 nM, respectively. Blocking the proteolytic activity of u-PA resulted in a 30% decrease of the mitogenic effect, whereas inhibition of plasmin activity did not alter the mitogenic effect. No mitogenic response was elicited by low molecular weight u-PA, lacking the growth factor domain and the kringle domain. The ATF domain of u-PA induced a mitogenic response that was similar to complete u-PA. Defucosylated ATF and recombinant u-PA purified from Escherichia coli lacking all post-translational modifications did not induce a mitogenic response. Blocking the interaction of u-PA with u-PAR, using a specific monoclonal antibody, did not alter the mitogenic effect induced by u-PA. The binding of radiolabeled u-PA to M14 and IF6 cells was characterized by high affinity binding mediated by u-PAR and low affinity binding to an unknown binding site. These results demonstrate that proteolytically inactive u-PA is able to induce a mitogenic response in quiescent melanoma cells in vitro by a mechanism that involves the ATF domain but is independent of high affinity binding to u-PAR. Furthermore, it suggests that u-PA is able to bind with low affinity to a hitherto unidentified membrane associated protein that could be involved in u-PA-induced signal transduction.     

Plastic remodeling of the synaptic apparatus of skeletal muscles under physiological and pathological conditions

The synaptic apparatus of skeletal muscles undergoes continuous remodelling, by adapting to the functioning level of the peripheral neuromuscular apparatus under normal conditions, compensating and restoring its impaired function in pathology. This reorganization has specific features depending on the factors that influence it, such as increased function, decreased or no function, neuronal and nerve damage, synaptic toxin intoxication, autoantibody action, disturbances of trophic influences, etc. Damage to any link of the neuromuscular apparatus due to the above factors is likely to be an essential condition for remodelling.     

Langerhans cells in normal and pathological vocal cord mucosa

The Langerhans cells in samples of histologically normal and pathological vocal cord mucosa were counted after identification using S-100 polyclonal antibody. Langerhans cells were commonly seen in vocal cord polyp epithelium but were infrequent in normal cord mucosa. They were also identified in samples of squamous carcinoma, severe dysplasia and chronic inflammation.     

Characterization of folate receptor from normal and neoplastic murine tissue: influence of dietary folate on folate receptor expression

Membrane-associated folate receptors (FRs) have been detected in many mammalian species, and multiple isoforms have been identified. The pharmacological properties of FRs from murine kidney, liver, and six murine tumors were characterized. Murine kidney expressed primarily folate-binding protein 1, analogous to human FR-alpha, whereas murine liver expressed predominantly folate-binding protein 2, analogous to human FR-beta. Five of six murine tumors expressed high-affinity FRs with pharmacological properties consistent with folate-binding protein 1 isoform expression. Restriction of dietary folate resulted in significant changes in the FR expression in most murine tissues. Kidney and tumor FRs showed a decreased affinity for folic acid, suggesting a change in isoform expression in response to a low folate diet. Density of the FR in the kidney decreased, and, in contrast, density of the FR in all tumors increased. The response of the liver to a low folate diet was unique in that there were no detectable changes in affinity or density of liver FR. Changes in dietary folate that modulate FR isoform expression may have relevance for cancer patients treated with antifolates.     

Endothelial and macrophage upregulation of urokinase receptor expression in human renal cell carcinoma

The binding of urokinase-type plasminogen activator (u-PA) to a specific cell surface receptor (uPA-R) has been shown to enhance plasminogen activation, a process involved in extracellular matrix degradation and cell migration during angiogenesis and tumor growth. We investigated the expression of u-PA and uPA-R in renal cell carcinomas (n = 11). By immunohistochemistry using monoclonal and polyclonal anti-uPA-R antibodies, we found that tumoral capillary endothelial cells (von Willebrand factor and CD31 positive cells) overexpressed uPA-R, whereas vascular endothelial cells of the normal human kidney do not. In addition, tumor-associated macrophages (CD68-positive cells) strongly expressed uPA-R. In contrast, few tumoral cells and stromal fibroblasts expressed uPA-R. By in situ hybridization using a cDNA S35-labeled probe specific for uPA-R, we confirmed the local expression of uPA-R messenger RNA. We also detected the induction of u-PA in tumoral capillary endothelial cells and in tumor-associated macrophages. In two cases, tumoral cells themselves were also stained by anti-u-PA antibodies in focal areas. Finally tissue-type plasminogen activator (t-PA) was also overexpressed by tumoral capillary endothelial cells as compared with endothelial cells of normal human kidney vessels. These findings indicate an active invasive phenotype of endothelial cells in renal cell carcinoma and suggest a role for the plasminogen activation system in tumoral angiogenesis and invasion.     

Time series analysis of glottal airflow in normal and pathological phonation

Time series analysis of glottal airflow was carried out on 26 normal controls and 40 patients with voice disorders, using a modification of Isshiki's original technique which uses a hot-wire flowmeter, taking cycle-by-cycle fluctuations into consideration. The mean flow rate and mean AC/DC were shown to have significant differences among normal and patient groups. The standard deviations of AC/DC and AC/DC perturbation were calculated from the AC/DC value of 50 cycles and shown not to vary significantly among the normal and patient groups. The relationship between AC/DC and perceptual impression of voice was also studied among 20 selected patients with breathy voices. Using Spearman's rank correlation coefficient, this relationship was found to have statistical significance (P < .05).