High Levels Of Alpha Research Articles

Dendritic cell immunization route determines integrin expression and lymphoid and nonlymphoid tissue distribution of CD8 T cells.

Exogenous dendritic cells (bone marrow-derived dendritic cell (BMDC)) display restricted trafficking in vivo after injection into mice, but the route(s) by which they generate gut-homing effector cells is unclear. Mesenteric lymph nodes (LN) and spleen were differentially targeted by i.p. and i.v. administration of BMDC, respectively, whereas mediastinal LN were targeted by both routes. BMDC injected by either route activated CD8(+) T cells to up-regulate both alpha(4)beta(1) and alpha(4)beta(7) integrins. However, the lymphoid compartment in which activation occurred determined their expression kinetics, magnitude, and population distribution. Only T cells activated in mesenteric LN after i.p. immunization expressed high levels of alpha(4)beta(7), which also correlated with localization to small intestine. These alpha(4)beta(7)(high) cells also redistributed to mediastinal LN in a manner sensitive to treatment with alpha(4)beta(7) blocking Abs, but not to mucosal addressin cell adhesion molecule-1 blocking Abs. Our results demonstrate the importance of lymphoid compartment, as dictated by immunization route, in determining integrin expression on activated T cells and their distribution in lymphoid and nonlymphoid tissues.

Comparative anatomy of α2 and β adrenoceptors in the adult and developing brain of the marine teleost the red porgy (Pagrus pagrus, Sparidae): [3H]clonidine and [3H]dihydroalprenolol quantitative autoradiography and receptor subtypes immunohistochemistry

The present study aimed to determine the anatomic distribution and developmental profile of alpha(2) and beta adrenoceptors (AR) in marine teleost brain. Alpha 2 and beta adrenoceptors were studied at different developmental stages by using [(3)H]clonidine and [(3)H]dihydroalprenolol, respectively, by means of in vitro quantitative autoradiography. Furthermore, immunohistochemical localization of the receptor subtypes was performed to determine their cellular distribution. Saturation studies determined a high-affinity component of [(3)H]clonidine and [(3)H]dihydroalprenolol binding sites. High levels of both receptors were found in preglomerular complex, ventral hypothalamus, and lateral torus. Dorsal hypothalamus and isthmus included high levels of alpha(2) AR, whereas pretectum and molecular and proliferative zone of cerebellum were specifically characterized by high densities of beta AR. From the first year of life, adult levels of both AR were found in most medial telencephalic, hypothalamic, and posterior tegmental areas. Decreases in both receptors densities with age were prominent in ventral and posterior telencephalic, pretectal, ventral thalamic, hypothalamic, and tegmental brain regions. Immunohistochemical data were well correlated with autoradiography and demonstrated the presence of alpha(2A), alpha(2C), beta(1), and beta(2) AR subtype-like immunoreactivity. Both the neuronal (perikaryal or dendritic) and the glial localization of receptors was revealed. The localization and age-dependent alterations in alpha(2) and beta AR were parallel to plasticity mechanisms, such as cell proliferation in periventricular thalamus, hypothalamus, and cerebellum. In addition, the biochemical characteristics, distribution pattern, and neuronal or glial specificity of the receptors in teleost brain support a similar profile of noradrenergic transmission in vertebrate brain evolution.

Isolation of human epidermal stem cells by adherence and the reconstruction of skin equivalents

The isolation of human epidermal stem cells is critical for their clinical applications. In the present study, we isolated three populations of epidermal keratinocytes according to their ability to adhere to collagen type IV: i.e., rapidly adhering (RA), slowly adhering (SA), and non-adhering (NA) cells. The aim of this study was to characterize RA cells and to investigate the possibility of using these cells for epidermis reconstruction. To identify RA cells, flow cytometric analysis was performed using anti-alpha(6) integrin and anti-CD71 antibodies. RA cells express high levels of alpha(6) integrin and low levels of CD71, which are considered as markers of an epidermal stem cell nature. Furthermore, electron microscopy showed that RA cells are small and have a high nuclear to cytoplasmic ratio, whereas SA and NA cells have well-developed cellular organelles and abundant tonofilaments. Western blot analysis showed that RA cells are slow cycling and express p63, a putative epidermal stem cell marker, whereas SA and NA cells express c-Myc, which is known to regulate stem cell fate. To compare epidermal regenerative abilities, skin equivalents (SEs) were made using RA, SA, and NA cells. The epidermis constructed from RA cells was well formed compared to those formed from SA or NA cells. In addition, only SEs with RA cells expressed alpha(6) integrin and beta(1) integrin at the basal layer. These results indicate that RA cells represent epidermal stem cells and are predominately comprised of stem cells. Therefore, the isolation of RA cells using a simple technique offers a potential route to their clinical application, because they are easily isolated and provide a high yield of epidermal stem cells.

Significance of hormone receptor status and tumor vessels in normal, hyperplastic and neoplastic endometrium.

The aims of this study were to identity the roles of tumor vessels and hormone receptor status in normal, hyperplastic, and neoplastic endometrium, and to explore their relationships with other prognostic factors of endometrial adenocarcinoma. Endometrial curettage specimens of proliferative phase and secretory phase endometrium, simple hyperplasia with or without atypia, complex hyperplasia with or without atypia, and grade 1 adenocarcinoma were examined for estrogen receptor alpha (ER alpha), progesterone receptor (PgR), Ki-67 labeling index (LI), cyclin D1, microvessel density (MVD), and area of venules (AV) using an immunoperoxidase method. The results showed high levels of ER alpha in complex hyperplasia, and high levels of PgR in simple hyperplasia without atypia. Expression of ER alpha in the endometrium decreased in a stepwise manner from complex hyperplasia without atypia to grade 1 adenocarcinoma. Expression of PgR in the endometrium decreased in a stepwise manner from simple hyperplasia without atypia to grade 1 adenocarcinoma. In contrast, the expressions of Ki-67 LI, cyclin D1, MVD and AV in the endometrium increased in a stepwise manner from normal, simple or complex hyperplasia with or without atypia to grade 1 adenocarcinoma. These changes may become irreversible on progression from simple or complex hyperplasia to neoplasia.

Synthesis, Biochemical Properties and Molecular Modelling Studies of Organometallic Specific Estrogen Receptor Modulators (SERMs), the Ferrocifens and Hydroxyferrocifens: Evidence for an Antiproliferative Effect of Hydroxyferrocifens on both Hormone‐Dependent and Hormone‐Independent Breast Cancer Cell Lines

A series of ferrocene derivatives based upon the structure of the antiestrogenic drug tamoxifen or of its active metabolite hydroxytamoxifen has been prepared and named by analogy ferrocifens and hydroxyferrocifens. This series includes 1-[4-(O(CH(2))(n)NMe(2))phenyl]-1-phenyl-2-ferrocenyl-but-1-ene and 1-[4-(-O(CH(2))(n)NMe(2))phenyl]-1-(4-hydroxyphenyl)-2-ferrocenyl-but-1-ene, with n=2, 3, 5 and 8, and 1-[4-(-O(CH(2))(2)NMe(2))phenyl]-1-(4-hydroxyphenyl)-2-ferrocenylethene. Most of these molecules have been synthesised by McMurry cross-coupling of the appropriate ketones, except for the ethene complexes, which were prepared by a four-step reaction sequence starting from the ferrocenylacetic acid. All these compounds were obtained as mixtures of Z and E isomers. The isomers were separated in the cases of the ferrocenyl derivatives of tamoxifen and hydroxytamoxifen (n=2). No isomerisation of the Z and E isomers occurred in DMSO after one day, while a 50:50 mixture of the isomers was obtained within one hour in chloroform. The X-ray structure of (E)-1-[4-(-O(CH(2))(2)NMe(2))phenyl]-1-(4-hydroxyphenyl)-2-ferrocenyl-but-1-ene has been determined. The relative binding affinity (RBA) values of the hydroxyferrocifens for the estrogen receptor alpha (ERalpha) was good to moderate, with values decreasing progressively with the length of the basic chain. The RBA values found for the estrogen receptor beta (ERbeta) are equal to or slightly less than those found for the alpha form. The lipophilicity of the hydroxyferrocifens are superior to the values found for estradiol and increase with lengthening of the chain. The antiproliferative effects of the four hydroxyferrocifens with n=2, 3, 5 and 8 were studied on four breast cancer cell lines (MCF7, MDA-MB231, RTx6 and TD5) possessing different levels of ERalpha. On MCF7 cells containing high levels of ERalpha, hydroxyferrocifens behave as antiestrogens. At a molarity of 1 microM the effect is close to that of hydroxytamoxifen (used for reference) when n=2 or 5, more marked when n=3, and weaker when n=8. Ferrocene alone has no effect. For the MDA-MB231 cells, classed as a hormone-independent breast cancer cell line, on the other hand, the hydroxyferrocifens show remarkable antiproliferative behaviour while the hydroxytamoxifen is completely inactive. Hydroxyferrocifens therefore show the unique property of being active both on hormone-dependent and on hormone-independent breast cancer cell lines. The molecular modelling study provides some clues for understanding of the antagonist effect of these molecules, while an additional cytotoxic effect due to the vectorised ferrocenyl unit is revealed in some occasions.

Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle.

Estrogenic effects of 7alpha-methyl-17alpha-ethynylestradiol: a newly discovered tibolone metabolite.

Tibolone is a synthetic steroid that is prescribed to postmenopausal women for relief of climacteric symptoms and prevention of osteoporosis. It has been reported to be metabolized in a tissue-selective manner to three steroids that collectively have weak estrogenic, progestogenic, and androgenic activities. Recently, a new tibolone metabolite, 7alpha-methyl-17alpha-ethynyl-17beta-estradiol (7alpha-Me-EE2), was identified in women. In this report, we describe the pre-clinical estrogenic activities of this metabolite and compare these effects to those obtained with 17alpha-ethynyl-17beta-estradiol (EE2) and 17beta-estradiol (E2). In an in vitro ligand-binding assay, 7alpha-Me-EE2 bound to both human estrogen receptor (ER)-alpha and -beta with IC(50)'s of 1.2 and 3.0 nM, respectively. Using MCF-7 human breast cancer cells that express high levels of ER-alpha, 7alpha-Me-EE2 transactivated an estrogen response element (ERE)-tk-luciferase reporter gene construct with an EC(50) of 0.021 nM. Likewise, 7alpha-Me-EE2 stimulated MCF-7 breast cancer cell proliferation with an EC(50) of 0.002 nM. In immature female rats, subcutaneous (s.c.) administration of 7alpha-Me-EE2 stimulated uterine wet weight gain with an ED(50) of 0.2 microg/kg. Moreover, 7alpha-Me-EE2 induced uterine complement component C3 gene expression, an estrogenic marker of epithelial cell stimulation, with an ED(50) of 0.5 microg/kg. When compared to EE2 and E2, 7alpha-Me-EE2 exhibited equivalent or greater potencies and efficacies in these assays. In summary, these results indicate that 7alpha-Me-EE2 is a very potent estrogen. This steroid appears to be the most potent estrogenic metabolite of tibolone identified to date, and additional studies are, therefore, warranted regarding the role of this metabolite in the biological actions of the drug.

Identification of a factor that links apoptotic cells to phagocytes

Apoptotic cells are rapidly engulfed by phagocytes to prevent the release of potentially noxious or immunogenic intracellular materials from the dying cells, thereby preserving the integrity and function of the surrounding tissue. Phagocytes engulf apoptotic but not healthy cells, indicating that the apoptotic cells present a signal to the phagocytes, and the phagocytes recognize the signal using a specific receptor. Here, we report a factor that links apoptotic cells to phagocytes. We found that milk fat globule-EGF-factor 8 (MFG-E8), a secreted glycoprotein, was produced by thioglycollate-elicited macrophages. MFG-E8 specifically bound to apoptotic cells by recognizing aminophospholipids such as phosphatidylserine. MFG-E8, when engaged by phospholipids, bound to cells via its RGD (arginine-glycine-aspartate) motif--it bound particularly strongly to cells expressing alpha(v)beta(3) integrin. The NIH3T3 cell transformants that expressed a high level of alpha(v)beta(3) integrin were found to engulf apoptotic cells when MFG-E8 was added. MFG-E8 carrying a point mutation in the RGD motif behaved as a dominant-negative form, and inhibited the phagocytosis of apoptotic cells by peritoneal macrophages in vitro and in vivo. These results indicate that MFG-E8 secreted from activated macrophages binds to apoptotic cells, and brings them to phagocytes for engulfment.

Molecular determinants of glioma cell migration and invasion.

Migration and invasion are important prerequisites for the infiltrative and destructive growth patterns of malignant gliomas. Infiltrative growth prevents complete tumor resection and causes significant neurological morbidity and mortality. The authors assessed the expression of matrix metalloproteinases (MMPs) at messenger RNA and protein levels, MMP-2 and MMP-9 activities, and expression levels of a panel of anti- and proapoptotic proteins of the BCL-2 family. They then correlated their findings with alpha(v)beta3 integrin expression and the migratory and invasive potentials in 12 human malignant glioma cell lines. Multiple MMPs were expressed by most cell lines. The levels of MMP-2 and MMP-3 and the activities of MMP-2 and MMP-9 correlated with tumor cell invasion. Migration and invasion were also correlated. Although the expression levels of alpha(v)beta3 integrin did not predict migration or invasion, a neutralizing alpha(v)beta3 integrin antibody inhibited migration and invasion selectively in cell lines that contained a high level of alpha(v)beta3 integrin expression, thus indicating the important role of alpha(v)beta3 integrin for migration and invasion in this subset of cell lines. An expression pattern of BCL-2 family proteins that favor resistance to apoptosis was associated with enhanced migration, invasion, and MMP activity. Wild-type p53 cell lines migrated farther than mutant p53 cell lines. Activities of MMP-2 and MMP-9 are the best predictors of glioma cell invasion. The alpha(v)beta3 integrin mediates migration and invasion in a subset of glioma cell lines, but these processes do not depend on alpha(v)beta3 integrin expression. Antiapoptotic BCL-2 family protein expression is a predictor of efficient migration and invasion.

αvβ6 Integrin Upregulates Matrix Metalloproteinase 9 and Promotes Migration of Normal Oral Keratinocytes

The integrin alpha v beta 6 is a fibronectin receptor that is undetectable on normal keratinocytes in situ, but is increased significantly in wound healing and in culture-established keratinocytes, suggesting that it may promote changes associated with cell motility. Using normal human oral keratinocytes we have shown that cultured cells express relatively high levels of alpha v beta 6 and this integrin has a functional role in both cell adhesion and migration towards fibronectin. We provide experimental evidence that the increased expression of alpha v beta 6 by normal human oral keratinocytes results in coordinate changes, which promote a more migratory phenotype. Thus increased expression of alpha v beta 6 results in a fibronectin-dependent increase in pro-matrix metalloproteinase 9, matrix metalloproteinase 9 activity increases normal human oral keratinocyte migration, and this may be further dependent on plasmin activation. The results suggest a key role for alpha v beta 6 in these processes and indicate a coordinated link between alpha v beta 6 expression and upregulation of matrix metalloproteinase 9. It appears that alpha v beta 6 may function in normal human oral keratinocyte migration through matrix-metalloproteinase-9-dependent and -independent mechanisms.

Erythrocyte metallothionein in relation to other biochemical zinc indices in pregnant and nonpregnant women.

Erythrocyte metallothionein (E-MT) is considered a promising index of zinc status in humans, since it may be more sensitive than other biochemical indices to changes in dietary zinc. However, conditions of high zinc demand with substantial redistribution of tissue zinc and specific changes in hormone profile, such as pregnancy, may have an influence on E-MT levels in addition to dietary zinc. In this study, we compared E-MT concentrations in relation to other biochemical zinc indices in healthy pregnant women at delivery (n = 40) and non-pregnant women (n = 22) with similar habitual dietary zinc intakes (average 13.3 mg/d). Pregnant women had lower serum zinc and albumin-bound serum zinc, but higher levels of alpha 2-macroglobulin-bound serum zinc than the nonpregnant women. Erythrocyte zinc (E-Zn) was similar in both groups, but E-MT (mean +/- SE) was slightly but significantly (p < 0.05) higher in the pregnant women (2.9 +/- 0.09 nmol/g protein) compared to nonpregnant women (2.6 +/- 0.06 nmol/g protein). A significant correlation was observed between E-MT and E-Zn in the nonpregnant women (r = 0.70; p < 0.001), consistent with the role of intracellular zinc in the regulation of metallothionein synthesis. However, such correlation was not observed in the pregnant women, suggesting that E-MT levels in pregnancy may be influenced by factors related to the pregnant state.

Endodermal sinus tumor of the ovary in pregnancy

The ultrasonographic evidence of an irregular mixed mass, cystic and solid, in retrouterine area and the high levels of alpha(1)-FP values in a young pregnant woman, indicated the possibility of an ovarian germ cell neoplasia. Respecting the patient's will, pregnancy was protracted until the 36th week. A cesarian section allowed the releasing of a vital fetus and the removal of a neoplastic mass which proved to be an endodermal sinus tumor (EST) at stage Ia. Then the patient, the 12th case of EST in pregnancy reported in the literature, underwent 5 PVB chemotherapy cycles and, 3 years after the intervention, appears disease-free.

Alpha 1-antitrypsin blocks the release of transforming growth factor-alpha from MCF-7 human breast cancer cells.

Human breast cancer cells synthesize and release a variety of growth-modulating substances in response to estrogen stimulation, and it is generally accepted that the growth-promoting effects of estrogens are due at least in part to this autocrine/paracrine mechanism. Several of these growth-modulating substances, including transforming growth factor-alpha (TGF alpha) and its analogs, have been shown to require pericellular proteolysis for activation or release. Recently, we reported that MCF-7 human breast cancer cells are able to synthesize alpha 1-antitrypsin (alpha 1-AT), the major elastase inhibitor in human serum, and that there is a negative correlation between anchorage-independent growth of MCF-7 cells in soft agar and synthesis of alpha 1-AT. The studies we present here were undertaken to gain an understanding of the mechanisms responsible for this observation. We show that release of TGF alpha from its membrane-bound precursor on MCF-7 cells is blocked by alpha 1-AT whether the cells were maintained in the presence or absence of estradiol and that there is a clear dose-response relationship between the alpha 1-AT concentration and both the release of TGF alpha and growth in soft agar. Consistent with this, TGF alpha release was increased in the presence of antibody to alpha 1-AT. In contrast, TGF alpha release and growth in soft agar were not blocked by peptide inhibitors specific for trypsin- or chymotrypsin-like enzymes. The alpha 1-AT concentration required for a half-maximal effect is lower for inhibition of TGF alpha release than it is for inhibition of colony formation (0.4 vs. 1.5 mumol/L). However, both values are in the range of concentrations one might expect at the cell surface in vivo. A new MCF-7 cell subline producing 10-fold higher levels of alpha 1-AT than its parent cell line was constructed by stable transfection of MCF-7 ML cells (a subline producing low levels of alpha 1-AT) with an alpha 1-AT complementary DNA. Growth in soft agar and release of TGF alpha were significantly decreased in cells transfected with the alpha 1-AT complementary DNA compared to those in cells transfected with vector alone, although, TGF alpha expression was the same. The above observations support a model for growth regulation in human breast ductal epithelial cells in which growth factor activation and release are dependent on the coordinate action of proteases and protease inhibitors. This model would predict that alpha 1-AT can act as a tumor suppressor in inhibiting the growth of breast cancer cells.

Integrin function in osteoclasts.

Integrins are a large family of heteromeric cell surface receptors composed of non-covalently bound alpha and beta subunits which interact with extracellular matrix molecules, serum constituents and the adhesion molecules of the immunoglobulin family. The extracellular domains of many integrins recognize the RGD (Arg-Gly-Asp) tripeptide found in several extracellular macromolecules such as fibronectin, vitronectin, fibrinogen and osteopontin. The vitronectin receptor, alpha v beta 3 integrin, is highly expressed in osteoclasts, the bone resorbing cells, and binds many of these RGD containing proteins including osteopontin, which is abundant in bone. Antibodies to alpha v beta 3, RGD peptides and RGD containing proteins such as echistatin, and kistrin were shown to inhibit bone resorption in vitro and in vivo. The identity of the alpha v beta 3 natural ligand and its mode of action in bone are so far not known. In addition to the very high levels of alpha v beta 3, mammalian osteoclasts also express alpha 2 beta 1, a collagen/laminin receptor and alpha v beta 1, another vitronectin receptor. Signaling events that follow substrate recognition by osteoclasts are not well understood. RGD containing peptides and proteins modulate [Ca2+] transients in osteoclasts and phosphatidylinositol 3-kinase and pp60c-src are associated with alpha v beta 3 in these cells. alpha v and beta 3 genes were shown to be regulated by the calciotropic hormone 1,25(OH)2D3 and by a number of cytokines known to be modulators of bone metabolism. In summary, elucidation of the interactions of osteoclast integrins with components of bone matrix, may lead to further understanding of the mechanism of bone resorption.

Uptake of alpha-(L)-iduronidase produced by retrovirally transduced fibroblasts into neuronal and glial cells in vitro.

The uptake of recombinant alpha-(L)-iduronidase into glial and neuronal cells, produced by retrovirally transduced NIH3T3 fibroblasts, was studied. We demonstrate that: (1) neuronal and glial cells take up alpha-(L)-iduronidase released into the medium by retrovirally transduced fibroblasts expressing high levels of alpha-(L)-iduronidase; (2) both glial and neuronal cells express the cation independent mannose-6-phosphate receptor responsible for lysosomal enzyme uptake; and (3) uptake of the lysosomal enzyme can be blocked by excess free mannose-6-phosphate, but not glucose-6-phosphate. Thus, various brain cells take up alpha-(L)-iduronidase, possibly through a cation independent mannose-6-phosphate receptor mediated pathway, and this uptake is higher in actively dividing or immature brain cells. Consequently, (1) neuronal metabolism ought to be capable of cross correction by enzyme provided by genetically engineered and transplanted cells provided by bone marrow transplantation (BMT); (2) that BMT could have a more beneficial effect on neurological function if performed as early as possible; and (3) given that the uptake mechanism of glial cells has a higher capacity, it might be easier to target diseases like the leukodystrophies in which lysosomal enzymes are needed in glial cells, compared to diseases where lysosomal enzymes ought to be delivered into neurons.

Adenovirus Vector Infection of Chronic Lymphocytic Leukemia B Cells

Adenovirus vectors have several features that make them attractive for potential use in gene therapy, including a broad tissue tropism and an ability to infect quiescent or postmitotic cells. In light of this, we examined whether recombinant adenovirus vectors could transfer genes into neoplastic cells of patients with chronic lymphocytic leukemia (CLL), a leukemia of "resting" B cells. Using high-titer recombinant adenovirus vectors, we found we could transfer genes encoding beta-galactosidase or murine CD80 (B7-1) into the CLL B cells of all patients tested (n = 10). The efficiency of gene transduction into CLL B cells was approximately 100 to 1,000-fold lower than into HeLa cells at any given multiplicity of infection (MOI). At a MOI of 500, 10% to 70% of the CLL B cells from different patients were made to express the transgene, as assessed by multiparameter flow cytometric analysis. Sustained levels of expression with little loss in the percentage of infected cells were maintained for up to 9 days, at which point the analysis was stopped. We found that CLL B cells have markedly lower expression levels of integrins that facilitate internalization of adenovirus particles into target cells, perhaps accounting, in part, for the reduced efficiency of adenovirus-mediated gene transfer compared with that in HeLa cells. Although HeLa cells express high levels of alpha(v)beta5, and detectable amounts of alpha(v)beta3, we find CLL cells from all patients tested express only low amounts of alpha(v)beta3, and no detectable alpha(v)beta5. Activation of CLL cells via CD40 cross-linking enhances expression of alpha(v)beta3, and induces expression of alpha(v)beta5. This phenotypic change is associated with a fivefold increase in the efficiency of adenovirus-mediated gene transfer into such activated CLL B cells. This study demonstrates that adenovirus vectors can transduce genes into CLL B cells and that the efficiency of gene transduction is enhanced by activation via CD40 cross-linking. This is the first demonstration that high proportions of CLL B cells can be made to express a selected transgene, suggesting that such gene transfer methods may become useful for the study of the pathogenesis and/or treatment of this disease.

Attachment, spreading and migration of melanoma cells on vitronectin. The rôle of alpha V beta 3 and alpha V beta 5 integrins.

Recent in situ studies suggest the alpha V beta 3 integrin is a tumour progression marker in melanoma. We analyzed 5 human melanoma cell lines for their expression of the vitronectin binding alpha V beta 3 and alpha V beta 5 integrins using flow cytometry. The role of these receptors in cell attachment, spreading and migration was investigated using attachment assays, video time lapse spreading and migration assays and with function blocking monoclonal antibodies. Cell lines derived from later stages of tumor progression exhibited high levels of alpha V beta 3 expression, whereas no similar correlation with alpha V beta 5 expression was identified. Cell attachment, spreading and migration response on vitronectin correlated well with the expression level of the alpha V beta 3 but not the alpha V beta 5 vitronectin receptor. Blocking of the alpha V beta 3 integrin resulted in a significant decrease in cell attachment, spreading and motility whereas the function blocking antibody against the alpha V beta 5 integrin only inhibited cell attachment in cell lines with the highest level of expression of this integrin. Taken together, our study indicates that the level of expression of the alpha V beta 3 and alpha V beta 5 integrins is heterogeneous in melanoma cell lines and that the alpha V beta 5 integrin, if present, may function only during the initial cell attachment whereas the alpha V beta 3 plays an important role in cell spreading and cell migration as well.

Differential expressions of collagen types IV, III, and I during the development of invasive trophoblasts in rats.

We examined the differential expressions of collagen types IV, III and I in the developing feto-maternal placental tissue of pregnant rats by a combination of in situ hybridization and immunohistochemistry. At day 9.5 of gestation, polygonal invasive cytotrophoblasts from the ectoplacental cone, which was modifying the maternal central artery, revealed intensely expressed alpha 1 (IV) and alpha 1 (III) collagen mRNAs. The localization patterns of these translated products, collagen type IV and procollagen type III, were slightly different in the invasive cytotrophoblasts. Collagen type IV densely deposited intracellularly and intercellularly in the maternal central artery and in the thickened basement membranes of the cytotrophoblasts. However, expression of alpha 1 (I) collagen mRnA and procollagen type I was hardly detectable in the cytotrophoblasts. At day 13 of gestation, a high level of alpha 1 (IV) collagen mRNA was expressed in the cytotrophoblastic cell layer (trophospongium) and in the invasive large cytotrophoblasts. A moderate level of alpha 1 (III) collagen mRNA was also expressed mainly in the cytotrophoblasts, while alpha 1 (I) collagen mRNA was expressed at very low levels. Interestingly, procollagen type III failed to show linear immunoreactivity in the subepithelial extracellular matrix beneath the maternal artery with the invasive cytotrophoblasts. Additional quantitative analyses of these type IV, III, and I collagen mRNA levels in in situ hybridization experiments between several cell types also revealed significant differences individually. Electron-microscopic study detected no cross-striated collagen fibers in the thickened basement membrane-like structures adjacent to the invasive cytotrophoblasts. Fibrillar and basement membrane collagen gene expressions, their protein syntheses, and the processing of these procollagens seems to be developmentally regulated in the invasive cytotrophoblasts during the organization of feto-maternal placental tissue. The remodeling of the maternal central artery by the invasive cytotrophoblasts is important for ensuring the adequate blood supply to the developing placenta and fetus.

Neuronal Nicotinic Receptor a6 Subunit mRNA is Selectively Concentrated in Catecholaminergic Nuclei of the Rat Brain

Although the neuronal nicotinic receptor alpha 6 subunit was cloned several years ago, its functional significance remains to be investigated. Here we describe an in situ hybridization study of the mRNA for this subunit in the adult rat central nervous system using oligonucleotide probes. Specific alpha 6 mRNA labelling was restricted to a few nuclei throughout the brain; it was particularly high in several catecholaminergic nuclei [the locus coeruleus (A6), the ventral tegmental area (A10) and the substantia nigra (A9)] at levels significantly higher than those found for any other known nicotinic receptor subunit mRNA. Labelling for alpha 6 mRNA was also detected at lower levels in the reticular thalamic nucleus, the supramammillary nucleus and the mesencephalic V nucleus. Some cells of the medial habenula (medioventral part) and of the interpeduncular nucleus (central and lateral parts) were also labelled. The distribution of alpha 6 mRNA was compared with the distribution of the other known nicotinic acetylcholine receptor subunit mRNAs. In several nuclei, the expression of alpha 6 was complementary to those of other alpha subunits. Moreover, some of the cell groups (such as the substantia nigra, the ventral tegmental area and the locus coeruleus) previously thought to contain mainly alpha 3 mRNA in fact were found to contain high levels of alpha 6 mRNA. Finally, we found extensive colocalization of alpha 6 and beta 3, indicating the possible existence of nicotinic receptor hetero-oligomers containing both subunits. The present results show that alpha 6 is the major nicotinic acetylcholine receptor alpha subunit expressed in dopaminergic cell groups of the mesencephalon and noradrenergic cells of the locus coeruleus. This suggests the involvement of the alpha 6 subunit in some of the major functions of central nicotinic circuits, including the modulation of locomotor behaviour and reward.

Heterogeneity of collagen synthesis in normal and systemic sclerosis skin fibroblasts. Increased proportion of high collagen-producing cells in systemic sclerosis fibroblasts.

The goal of this study was to quantitatively analyze the distribution of collagen synthesis in normal and systemic sclerosis (SSc) fibroblast populations in order to determine the extent of activation in SSc populations. We used quantitative in situ hybridization to assess the population distribution of type I collagen synthesis. Fibroblast cultures were derived from both clinically involved and uninvolved skin regions of patients with SSc, and from healthy adults, and assessed for levels of alpha 1(I) procollagen messenger RNA (mRNA). Dermal fibroblasts from both patients with SSc and normal adults were heterogeneous for distribution of alpha 1(I) procollagen mRNA when assessed by in situ hybridization, with a wide range of grains per cell. In contrast, clones of neonatal fibroblasts showed a relatively homogeneous distribution of grain counts. Involved SSc skin fibroblasts had a larger proportion of cells in the high collagen-producing mRNA subpopulation (mean +/- SEM 28.4 +/- 6.85%), compared with normal fibroblasts (1.75 +/- 1.44%) and uninvolved fibroblasts (9.6 +/- 6.73%). Conversely, within the low collagen-producing mRNA subpopulation, involved fibroblasts had a smaller proportion of cells (mean +/- SEM 14.0 +/- 5.63%) than did uninvolved fibroblasts (37.8 +/- 13.69%), while normal fibroblasts had a majority of the cells in this subpopulation (53.5 +/- 8.68%). These results suggest that only a specific subset of fibroblasts are activated in SSc, as evidence by an increased proportion of cells with high levels of alpha 1(I) procollagen mRNA. Differences between the SSc and normal fibroblast populations appeared to be quantitative rather than qualitative. This may be a result of either clonal selection or selective activation in the pathogenesis of SSc.