Expression Of Laminin Research Articles

Micropillar substrate influences the cellular attachment and laminin expression

Fibroblasts alter their mode of attachment and focal contact when placed on square arrays of silicon pillars. The pillars had 1-microm diameters with identical surface chemistry. Distance between pillars is 9 microm and height of pillars is 1, 5, or 10 microm on substrates. We found that these micropillars, rather than specific interactions, provided more opportunities for mechanical interlocking of the fibroblasts and acted as physical barriers that restrained cell migration. The cellular morphology and behavior is guidable by the height of pillars. In some cases, the fibroblasts filled in the intervals among several pillars; in others, a pillar protruded visibly through the cell body but did not pierce it, the cells were survived. Therefore, fibroblasts were immobilized upon in situ and the cytoplasma migrated outward to the bottom of the substrate subsequently. Laminin plays a critical role in cell attachment to the basement membrane. The results of laminin expression in fibroblasts suggest that pillar pattern appears to change cellular behavior and affect laminin expression significantly.

Abstract C30: Laminin-5 expression in breast cancer culture cells after chemotherapic exposure

Abstract Introduction: Laminin-5, a large heterotrimeric glycoprotein consisting of an alpha 3, beta 3, and gamma 2 chain, is a component of epithelial cell basement membranes that functions as a ligand of the alpha 3 beta 1 and alpha 6 beta 4 integrins to regulate cell adhesion, migration, and morphogenesis. Laminins also regulate cellular movement, growth and differentiation, induce inflammatory cells and integrate the extracellular matrix to epithelia. It is suspect to be a tumor suppressor gene in breast cancer resulting in inhibition of tumor metastasis. In contrast, recent studies describe that as a modify and metastatic gene. The importance of laminin-5 in the development of a variety of cancers makes it an attractive target for cancer therapeutics. This study was conducted to determine the laminin-5 expression after exposure to routine chemotherapy in breast carcinoma culture cells. The concept is that laminin-5 expression change after doxorubicin exposure. Studying chemotherapy-induced gene expression changes in vitro could provide insights into mechanisms of chemotherapy resistance. Material and Methods: Doxorubicin exposure was performed on tumor tissue obtained from ten newly diagnosed carcinoma. The gene expression was determined before and after doxorubicin aggression. PCR amplifications were performed using a 7500 Fast Real-Time PCR System (Applied Biosystems). The gene expression stability over different samples was analyzed using the geNorm software. Results: The cells were cultured for about 30 days. Then their origin was proof by immunohistochemical procedure using citoqueratin and vimentin. After that, the curve of growth was made after exposure to doxorubicin. There was distinct expression of laminin 5 in breast cancer culture cells. When the molecular study was concern there were statistical correlation between the gene expression and the drug resistance. Discussion and Conclusion: Laminin-5 has abnormal expression and its integrin receptors are hallmark of certain tumor types and are believed to promote invasion of colon, breast and skin cancer cells. In invasive cancers, laminins usually become discontinuous or absent around tumor foci, which is attributed to either increased degradation or reduced synthesis. The importance of laminin-5 in the development of a variety of cancers makes it an attractive target for cancer therapeutics. However, laminin-5 based cancer therapies are not without their problems since treatments of necessity must target tumor laminin-5 but not laminin-5 in normal tissues since the latter is essential for maintaining tissue integrity. Future development of new reagents that inhibit the ability of laminin-5 to drive tumor growth and/or dissemination will be greatly facilitated once we better understand not only the precise regulation of laminin-5 proteolytic processing but also the different signaling cascades regulated by laminin-5 in normal versus tumor cells. Gene expression alterations of breast cancer were specific to doxorubicin treatment, and yielded mechanistic insights into resistance to the drug. Gene expression analysis provides more global perspectives on resistance pathways that could be exploited for therapeutic selection. Consideration of these factors should be incorporated in clinical practice after appropriate validation studies are performed to avoid confounding results, making them true prognostic and predictive factors. Financing agency: FAPESP Citation Information: Cancer Res 2009;69(23 Suppl):C30.

An immunohistochemical study of laminin in basal cell carcinoma

Laminins are components of the extracellular matrix that mediate cell adhesion, growth, migration, proliferation and differentiation. Basement membrane (BM) laminins, in particular, may play a role in enhancing carcinoma cell motility. To evaluate the distribution pattern of laminin in basal cell carcinoma (BCC), as regards the basement membrane, cellular cytoplasm, peritumoral lacunae and surface epithelium and to correlate laminin distribution with different variants of BCC. Skin biopsy specimens were obtained from 21 BCC patients for routine histopathological and immunohistochemical study. Laminin was evaluated qualitatively and semiquantitatively using monoclonal mouse antihuman antibody (Dako-Laminin, 4C7. Code No: MO638, which reacts with the terminal globular domain of the α5 chain) The majority of BCC cases showed patchy cytoplasmic distribution of laminin. The BM expression of laminin, in most cases, was well defined, fine and linear with irregular areas of thickening. Staining intensity was moderate in differentiated and mixed variants, weak in superficial spreading and absent in morpheic types. Cytoplasmic and basement membrane laminin is important in the pathogenesis and invasion of BCC. Most laminin was in basement membrane zone (BMZ), and the more differentiated the tumor, the more cytoplasmic and BM staining it expressed.

The Influence of Enzymes on Adhesive Processes in the Abdominal Cavity

Intraperitoneal adhesions remain a common problem after abdominal surgery. However, the advent of targeted, specific agents as a directed therapy against inflammatory and neoangiogenesis raises the prospect of a new approach for anti-adhesion strategies. 70 adult rats were divided into two groups: an enzyme group (35 rats) and a control group (35 rats). Following laparotomy the visceral peritoneum of the cecum and inner layer the abdominal cavity were abraded. In the enzyme group the preparation Wobenzym (a combination of animal and vegetable proteases plus rutoside) dissolved in 2 ml of sterile 0.9% NaCl was administered to the rats through a gastric probe over a period of 21 days. The control animals received the same quantity of saline without the preparation. On the 1st, 3rd, 7th and 21st postoperative days 8 animals from each group were sacrificed. VEGF, bFGF and laminin were detected in the visceral and parietal peritoneum by immunohistochemistry. The mean adhesion grades of the enzyme group were significantly lower than in the control group. A comparison of the two groups showed that the expression of VEGF, bFGF and laminin in the enzyme group was lower than in the control group. Moreover, in the enzyme group the concentration of bFGF and laminin peaked on the 7th day, while in the control group the maximum concentrations peaked on day 21. Our study found that oral poly-enzyme therapy following laparotomy and abrasion of the visceral and parietal peritoneum reduces the extent of postoperative intestinal adhesions. This favorable effect can be explained by the lower levels of angiogenic agents (VEGF, bFGF) and laminin after the administration of hydrolytic enzymes.

Severe asthma: a consequence of over exuberant repair?

Severe asthma: a consequence of over exuberant repair?

STUDY ON EXPRESSION OF LAMININ IN PATIENTS WITH INTRACTABLE EPILEPSY

In this study, we examined differences in serum laminin expression in patients with intractable epilepsy. Our results suggest that elevated laminin may contribute to the pathogenesis of intractable epilepsy. ELISA and western blots were used to measure laminin in the serum of 30 intractable epilepsy patients, 46 nonintractable epilepsy patients, and 20 normal subjects. By ELISA, serum laminin levels were greater in intractable epilepsy patients (177.396 ± 30.602) and nonintractable epilepsy patients (121.915 ± 35.215) than in normal control subjects (67.474 ± 7.197); laminin was significantly greater in the intractable epilepsy group than in the nonintractable epilepsy group. In western blots, the optical density ratio of laminin to ß-actin was 0.871 ± 0.032 for the intractable epilepsy group, 0.686 ± 0.017 for the nonintractable epilepsy group, and 0.385 ± 0.024 for the normal control group. The optical density ratios of the intractable and nonintractable epilepsy groups were higher than those for the normal control group, and the intractable epilepsy group was even greater than the nonintractable epilepsy group. Thus, laminin is significantly increased in epilepsy patients, and this increase is more profound in intractable epilepsy patients.

Basement membrane changes in lichen planopilaris

Lichen planopilaris (LPP) is an inflammatory disease that affects the scalp and tends to produce cicatricial alopecia. The inflammatory process frequently results in the disruption of the basal cell of the external root sheath of the hair follicle. To investigate the alterations in the basement membrane zone (BMZ) in LPP by immunohistochemistry. Skin biopsies from six patients with LPP plus six normal controls were studied by immunohistochemistry with antibodies to the following BMZ components: cytokeratin 5, cytokeratin 14, BP230 (bullous pemphigoid), BP180, plectin, laminin 5, collagen IV and collagen VII. The localization and staining of the hemidesmosome, laminin and collagen components were strikingly different in the inflamed follicular epithelium when compared to the uninvolved follicles or interfollicular epithelium in active LPP lesions. The hemidesmosome-associated complexes were weakly expressed and discontinuous in involved hair follicles. The expression of laminin-5, type IV collagen and type VII collagen was disrupted and not linear along the BMZ with finger-like projections of the staining protruding into the dermis. The expression of the intermediate filaments was normal. These alterations in the BMZ in LPP may explain the abnormal healing at follicular level which leads to irreversible hair loss and scarring in this condition.

Promotion of Tumor Cell Migration by Extracellular Matrix Proteins in Human Pancreatic Cancer

The pathogenesis of pancreatic carcinoma is driven by the tumor cells ability to migrate causing invasion and metastases. The correlation between the aberrant expression of basement membrane proteins and the process of tumor invasion and metastasis has not been fully determined. In the present study, the influence of laminin, fibronectin, and collagen type IV on migratory activity of 5 different cell lines has been investigated at the level of a single tumor cell using 3-dimensional time-lapse microscopy. All investigated cell lines have shown a high baseline migration that varied between 6.2 +/- 3.6 and 20.6 +/- 6.8 microm/h. The addition of laminin, fibronectin, and collagen type IV to collagen type I matrix has significantly increased tumor cell migration. Tumor cell migration was strongly inhibited after treating the tumor cells with anti-beta1 monoclonal antibodies. An abundant and continuous expression of laminin, fibronectin, and collagen type IV was found on the basement membrane of perineurium, which sharply promoted tumor cell invasion. The continuous presentation of the basement membrane proteins by perineurium contributes to the affinity of pancreatic cancer cells for the perineural tumor invasion. Blockade of integrins could represent a possible approach to control the basement membrane-guided tumor spread.

Comparative expression of laminin and smooth muscle actin in the testis and epididymis of poultry and rabbit

The present investigation was conducted to demonstrate laminin and alpha smooth muscle actin (alphaSMA) in the testis and epididymis of adult chickens, Sudani ducks, pigeons, and rabbits. This study may represent the first indication for the presence of laminin in the male reproductive organs of birds and rabbits and might therefore serve as a milestone for further reports. In the testis of chicken, Sudani duck, pigeon, and rabbit, the laminin was localized in the basal lamina of the seminiferous tubules and of the peritubular myoid cells, in the testicular capsule and to a small extent in the vicinity of Leydig cells. The testicular vasculature also exhibited intense laminin immunostaining. Weak laminin staining was additionally seen in the cytoplasm of the duck Sertoli cells. In the epididymis, the basal lamina of the epididymal epithelium showed a distinctly positive reaction in all birds and rabbit. The basal lamina of the periductal myoid cells also showed a positive reaction. In the interductal tissue, laminin immunostaining was particularly observed in chicken, duck and pigeon. Laminin positive reaction was also seen in the epididymal vasculatures of all birds and rabbit. Interestingly, weak to moderate laminin staining was observed in the apical surface of the ciliated cells of the proximal and distal efferent ductules in chicken, duck and pigeon. alphaSMA positive reaction was seen in the testicular capsule and in the peritubular myoid cells of all birds and rabbit. In the testicular capsule, alphaSMA staining was either observed in the inner portion (chicken) or throughout the tunica albuginea (Sudani duck and pigeon), or in the outer aspect (rabbit). Distinct alphaSMA reaction was additionally observed in the testicular vasculature. In the epididymis of all birds and rabbit, the alphaSMA was particularly seen in the periductal and interductal myoid cells as well as in the epididymal vasculatures. No alphaSMA specific staining was however detected in the epididymal epithelium, fibrous lamina propria, and luminal spermatozoa of all birds and rabbits. In conclusion, the distribution of laminin and alphaSMA in the testis and epididymis might point out to their roles in the male reproduction.

Corneal epithelial cell adhesion and growth on EGF‐modified aminated PDMS

Growth factor tethering has significant potential to mediate cellular responses in biomaterials and tissue engineering. We have previously demonstrated that epidermal growth factor (EGF) can be tethered to polydimethylsiloxane (PDMS) substrates and that these surfaces promoted interactions with human corneal epithelial cells in vitro. The goal of the current work was to better understand the specific effects of the tethered growth factor on the cells. The EGF was reacted with a homobifunctional N-hydroxysuccinimide (NHS) polyethylene glycol (PEG) derivative, and then bound to allyamine plasma-modified PDMS. Human corneal epithelial cells were seeded on the surfaces and cultured in serum-free medium for periods of up to 5 days. Cell growth was monitored and quantified by trypsinization and counting with a Coulter counter. Expression of matrix proteins and alpha(6)-integrins was assessed by immunostaining and confocal microscopy. A centrifugation assay was used to determine cell adhesion under an applied detachment force. Binding of EGF was found to significantly increase cell numbers and coverage across the surfaces at 5 days of culture in vitro. Immunofluorescence experiments indicate increased expression of fibronectin, laminin, and alpha(6)-integrins on the EGF-modified surfaces, and expression is localized at the cell-material interface as observed by confocal microscopy. In accordance with these results, the highest quantity of adherent cells is found on the EGF-modified subtrates at 5 days of culture. The results provide initial evidence that binding of EGF may be used to improve the epithelialization of and the adhesion of the cells on a polymeric artificial cornea device.

Fluoride-induced brain damages in suckling mice

In order to reveal mechanisms of brain damages resulted from fluoride toxicity, we treated adult female mice of Swiss Albinos strain by 500 ppm NaF (226 ppm F −) in their drinking water from the 15th day of pregnancy until the day 14 after delivery. All mice were sacrificed on day 14 after parturition. During treatment, levels of thiobarbituric acid reactive substances (TBARS), the marker of lipid peroxidation extend, increased, while the activities of the antioxidant enzymes such as glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT), and the level of glutathione (tGSH) decreased significantly in both cerebellum and cerebrum compared with those of the control group. These results suggested that fluoride enhanced oxidative stress in the brain, thereby disturbing the antioxidant defense of nursing pups. In addition, acetylcholinesterase (AChE) activity in both cerebellum and cerebrum was inhibited after treatment with fluoride. In our previous studies we have found, a decrease in plasma thyroid hormone levels of nursing pups whose mothers were treated by NaF. In cerebellum of mice, migration of neurons from the external granular layer to the internal granular layer occurred postnatally and was dependent upon the presence of thyroid hormone. In hypothyroidism, several neurons failed to complete their migration and died. Key guidance signals to these migrating neurons were provided by laminin, an extracellular matrix protein fixed to the surface of astrocytes. In the present study we examined the expression and distribution of laminin in cerebellum of 14-day-old mice. Immunoreactive laminin was disappeared by postnatal day 14 in cerebellum parenchyma of control pups and was restricted to vasculature despite the continued presence of granular cells in the external granular layer. In contrast, in cerebellum of NaF treated pups, laminin was deposited in organised punctuate clusters in the molecular layer. These data indicated that the disruption of laminin distribution might play a major role in the profound derangement of neuronal migration observed in cerebellum of NaF treated pups.

The roles of extracellular matrix proteins, apoptosis and c-kit positive cells in the pathogenesis of ureteropelvic junction obstruction

To investigate histopathological changes in ureteropelvic junction obstruction (UPJO) from an etiological perspective. Medical records of patients with UPJO were reviewed and pathological specimens collected. Nephrectomy materials from forensic autopsies were taken as controls. Specimens were assessed with light microscopy. Fibronectin, type 4 collagen, laminin, Bax and Bcl-2 expression for apoptosis, together with interstitial cells of Cajal determination with c-kit were determined immunohistochemically. Staining scores were evaluated semiquantitatively. Results were evaluated using Mann-Whitney U-test. Control group comprised 14 children (median age, 3.5 years; 6 months-17 years). Study group comprised 22 children with UPJO (median age, 9 months; 1 month-10 years). Light microscopy revealed non-specific inflammation, epithelial proliferation and atrophy with fibrosis in the smooth muscle of the UPJ in all patients. Fibronectin, type 4 collagen and laminin were found to be significantly increased in UPJO at the intrafascicular space of smooth muscle and the matrix of stroma. Bcl-2 expression was increased in UPJO. c-Kit was unable to stain interstitial cells of Cajal, but staining for mast cells was significant. High expression of fibronectin, laminin and type 4 collagen may indicate a relation to the pathogenesis of UPJO. Defective kidney morphogenesis, during branching and tubulogenesis of ureteric bud, may be responsible for this congenital pathology.

Age-dependent changes in the gut environment restrict the invasion of the hindgut by enteric neural progenitors

The enteric nervous system (ENS) develops from neural crest cells (NCCs) that enter the foregut and hindgut to become enteric neural-crest-derived cells (ENCCs). When these cells of neural crest origin fail to colonize the terminal hindgut, this aganglionic region becomes non-functional and results in a condition in humans known as Hirschsprung's disease (HSCR). One of the genes associated with HSCR is endothelin receptor type B (Ednrb). To study the development of colonic aganglionosis we have utilized a novel knockout mouse (Ednrb(flex3/flex3)), in which the expression of a null Ednrb allele and YFP is confined to NCCs. We have identified two primary cellular defects related to defective EDNRB signaling. First, ENCC advance in Ednrb(flex3/flex3) embryos is delayed shortly after NCCs enter the gut. Apart from this early delay, Ednrb(flex3/flex3) ENCCs advance normally until reaching the proximal colon. Second, as Ednrb(flex3/flex3) ENCCs reach the colon at E14.5, they display migratory defects, including altered trajectories and reduced speed, that are not dependent on proliferation or differentiation. We constructed grafts to test the ability of donor ENCCs to invade a recipient piece of aganglionic colon. Our results indicate that the age of the recipient, and not the age or genotype of donor ENCCs, determines whether the colon is invaded. We identify changes in laminin expression that are associated with the failure of ENCCs to invade recipient tissue. Together, our data suggest that a defect in pre-enteric Ednrb(flex3/flex3) NCCs results in delayed colonic arrival, which, due to environment changes in the colon, is sufficient to cause aganglionosis.

Cancer progression is associated with increased expression of basement membrane proteins in three-dimensional in vitro models of human oral cancer

Background Although basement membrane was traditionally considered an inert barrier that tumour cells had to cross before invasion into the surrounding stroma, recent studies suggest that basement membrane components are not only degraded during tumour progression, but also newly synthesised at the invasive front. Objective This study aimed at evaluating (1) the expression of basement membrane proteins in human oral carcinogenesis and (2) the role that epithelial–mesenchymal interactions play on it, by using an in vitro oral cancer progression model. Material and methods In vitro three-dimensional (3D) organotypic cultures of normal, early neoplastic and neoplastic human oral mucosa were developed by growing primary normal human oral keratinocytes, dysplastic human oral keratinocytes (DOK cell line), and neoplastic human oral keratinocytes (PE/CA-PJ15 cell line) on type I collagen biomatrices, with or without primary fibroblasts isolated from normal human oral mucosa. The cultured tissues were immunohistochemically assessed for the expression of the major basement membrane proteins laminin-332, type IV collagen, and fibronectin. Results Expression of laminin-332, type IV collagen, and fibronectin was gradually more pronounced in neoplastic models when compared to normal mucosa models, and, with the exception of laminin-332, it was further enhanced by presence of fibroblasts. Deposition of type IV collagen at the epithelium–biomatrix interface occurred only in presence of fibroblasts, as well as the extracellular matrix deposition of fibronectin. Conclusions These findings, obtained in a 3D in vitro model that closely mirrors the in vivo human oral cancer progression, show an enhanced basement membrane protein expression during human oral cancer progression that is dependent on the epithelial–mesenchymal environment, respectively the existence of fibroblasts.

Regulated laminin‐332 expression in human islets of Langerhans

Laminin-332 (LN-332) is a basement membrane component known to exert a beneficial effect on rat pancreatic beta cells in vitro. In this work, we analyzed the expression of LN-332 in human islets, its expression after inflammatory insults by cytokines, and the molecular mechanisms responsible for this effect. By Western blotting and RT-PCR, we showed that LN-332 was expressed in isolated human islets. By immunofluorescence on pancreas sections, we observed that labeling was confined to endocrine cells in islets. Confocal microscopy analysis on isolated islet cells revealed that labeling was granular but did not colocalize with hormone secretory granules. LN-332 was most abundant in cultured islets compared to freshly isolated islets and was found in culture medium, which suggests that it was secreted by islets. When islets were exposed to interleukin (IL)-1beta, expression and secretion of LN-332 increased as compared to control. No effect was observed with tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K) activity, inhibited culture- and IL-1beta-induced LN-332 expression in islets. These results show that LN-332, known to have some beneficial effect on beta cells in vitro, is produced and secreted by endocrine islet cells and is up-regulated by stressing conditions such as culture and IL-1beta-exposure.

Immunolocalization of laminin and integrin in regenerating junctional epithelium of mice after gingivectomy

The expression patterns of adhesive proteins and extracellular matrix proteins in regenerating gingival epithelium after gingivectomy are unknown. The aim of this study was to examine the expression of laminin 1, laminin gamma(2) (a specific component of laminin 5), integrin beta(4) and integrin alpha(3) in the regenerating gingival epithelium in order to understand the mechanism of wound healing during reconstitution of the sulcular environment. The palatal gingivae of the maxillary molars of Institute of Cancer Research mice were excised, and the regenerating tissues were examined 1, 3, 5, 7 and 14 days later. Fresh, non-fixed and non-decalcified frozen sections were prepared and stained using immunofluorescence. At 1 day post-surgery, intense expression of laminin gamma(2), integrin beta(4) and integrin alpha(3) was distinct in the frontal margin of the regenerating oral epithelium. Laminin gamma(2) was diffusely detected on the root surface and in connective tissues beneath the regenerating oral epithelium at 3 and 5 days. At 7 days, laminin gamma(2) was intermittently recognizable in the internal basal lamina (IBL) close to tooth-facing cells, while laminin gamma(2), integrin beta(4) and integrin alpha(3) were observed in the IBL and in the external basal lamina (EBL) of the regenerating junctional epithelium at 14 days. These results suggest that secretion of laminin 5 in the connective tissue may induce epithelial cell migration, and that binding of laminin 5 to integrin alpha(6)beta(4) and integrin alpha(3)beta(1) in the IBL may provoke cell adhesion and migration of cells facing the tooth on the enamel surface of the regenerating junctional epithelium.

Effect of the dental adhesive, 4‐META/MMA‐TBB resin, on adhesion and keratinization of regenerating oral epithelium

The 4-META/MMA-TBB [4-(2-methacryloxyethyl)trimellitic anhydride/methyl methacrylate-tributylborane] resin is widely used as a dental adhesive. It has also been applied in the dressing of gingival wound surfaces following periodontal surgery. However, its effect on the regeneration and/or cell attachment of the oral epithelium remains to be clarified. To evaluate the effect of the resin applied as a wound dressing, we investigated expression of laminin 5, integrin beta(4) and cytokeratin 14 in regenerating oral epithelium treated with this resin following gingivectomy from the viewpoint of cell attachment and differentiation. The resin was applied to the entire wound surface in rats after gingival surgery, and regenerating epithelium was examined immediately and at 1, 3, 5, 7 and 14 days later. The resin was removed 2 weeks after application in some animals and tissue further examined at 1, 3, 5 and 7 days later. Regenerating epithelium under the resin was not keratinized, but became keratinized immediately after removal of the resin. Laminin 5 and integrin beta(4) were immunolocalized in the basal lamina, the internal basal lamina, in marginal cells of the regenerating epithelium and at the resin-regenerating epithelium interface. Cytokeratin 14 localized in the regenerating epithelium underneath the resin, as well as in healthy and regenerated junctional epithelial cells. These results suggest that this resin covers the wound surface and that the regenerating epithelium biologically adheres to the resin during the initial process of its regeneration.

Immunohistochemical expression of basement membrane proteins of verrucous carcinoma of the oral mucosa

Squamous cell carcinoma (SCC) of the oral cavity is an extremely invasive tumour of stratified squamous epithelium that spreads throughout degradation of the basement membrane (BM) and extra-cellular matrix. Oral verrucous carcinoma (VC) is a rare low-grade variant of oral SCC that penetrates into the subepithelial connective tissue. It also has a different clinical behaviour from classical oral SCC. We investigated the immunohistochemical expression of laminin, laminin-5, collagen IV and fibronectin in VC, severe epithelial dysplasia (SED) and SCC in order to analyse if the pattern of these molecules expression contributes to the differences in the biological behaviour of these diseases. The staining pattern of laminin was less intensive in SCC compared with SED and VC, and collagen IV expression was increased in VC compared with SED. Discontinuities of laminin, collagen IV and fibronectin were more evident in SED than in VC. This study indicates that VC has a biological behaviour different from SED or SCC, observable by immunohistochemistry in the BM zone.

The effects of tamoxifen and estradiol on myometrial differentiation and organization during early uterine development in the CD1 mouse

We used a neonatal mouse model to examine the histogenesis of uterine adenomyosis, and to test whether adenomyosis is due to an abnormality in myometrial differentiation, or in extracellular matrix proteins expression. We also studied the effects of tamoxifen and estradiol on uterine development, myometrial differentiation, and organization. Female CD1 pups were treated with oral tamoxifen (1 mg/kg) (n=27) or estradiol (0.1 mg/kg) (n=24) from age 1 to 5 days. Uteri from control (n=27) and treated mice were obtained on days 2, 5, 10, 15, and 42 of age. We examined the sections histologically, using image analysis and immunohistochemistry for alpha-smooth muscle actin (alpha-SMA), desmin, vimentin, laminin, fibronectin, and estrogen receptor-alpha. Following tamoxifen exposure, all uteri showed adenomyosis by 6 weeks of age (seen as early as day 10). The inner myometrium showed thinning, lack of continuity, disorganization, and bundling. alpha-SMA expression was normal. Desmin expression normally showed a wave of maturation that was absent in tamoxifen-treated mice. In the estradiol group, adenomyosis was not observed. All uterine layers were normally developed, but hypertrophied. The inner myometrium retained its circular arrangement. There was no difference in the localization of laminin or fibronectin between groups (laminin expression was reduced in the tamoxifen treated uteri). Vimentin could not be detected in all groups. Our results suggest that the development of the inner myometrium is particularly sensitive to estrogen antagonism, and can be affected by steroid receptors modulation. Disruption of the inner myometrium may play a role in the development of uterine adenomyosis.

Disintegration of human spermatozoal membranes in seminal plasma decreases the binding capacity of integrins.

The in vitro binding capacity of spermatozoal integrins to matrix components after disintegration of sperm membranes was evaluated. The percentage of spermatozoa with functionally-relevant integrins was determined before and after devitalization of spermatozoa, which were resuspended in seminal plasma or in culture medium. The devitalization was performed by cryoshock or by incubation of spermatozoa with triton X-100 in a concentration ranging from 0.01 to 1.0%. The spermatozoal integrins were detected by the binding of anti-integrin antibodies and flow cytometry and the functional activity was monitored by the binding of the spermatozoa to the matrix components in a cell attachment assay. The seminal plasma decreased the binding of anti-integrin antibodies to the spermatozoal surface and the binding of spermatozoa to ligands and matrix components. respectively. In contrast, the expression of fibronectin and laminin on spermatozoa was increased. Not all spermatozoa, which expressed integrins on their surface bound to the ligands in the cell attachment assay. These results suggest that the detectable integrins only partially exert functional relevance. It can be concluded that the spermatozoa with fragile plasma membranes are more prone to functional inactivation of their integrins by the seminal plasma.