IL-6 and IL-8 production from cultured human endothelial cells stimulated by infection with Rickettsia conorii via a cell-associated IL-1 alpha-dependent pathway.

Mediterranean spotted fever due to infection by Rickettsia conorii, is characterized by a general vasculitis. This vasculitis is thought to be due to a direct injury to endothelial cells induced by R. conorii. However, production and activity of cytokines on endothelial cells is an important pathway in inflammation, and part of the underlying mechanism of vasculitis. In the present studies, human umbilical vein endothelial cells (HUVEC) infected with R. conorii actively secrete high levels of IL-8 and IL-6 (P < 0.002, and P < 0.03, respectively, compared with uninfected cells). IL-1alpha, IL-1beta, or TNFalpha were not detected in the culture supernates. Nevertheless, IL-6 and IL-8 production was due, in a large part, to a cell-associated form of IL-1 alpha expressed on R. conorii-infected HUVEC, since production of these cytokines was suppressed by 80% (P = 0.0001) and 85% (P < 0.04) by the addition of IL-1 receptor antagonist, or anti-IL-1alpha antibodies (60% inhibition, P < 0.01 and 65% inhibition, P < 0.05, respectively) and IL-1alpha was measured after lysis of R. conorii-infected HUVEC but not in uninfected cells (P < 0.01). Rickettsial lipopolysaccharide does not seem to be involved, since polymyxin B did not reduce cytokine secretion. On the contrary, infection by intracellular R. conorii appears to be necessary to induce IL-1alpha and subsequently IL-8, since formalin-fixed R. conorii did not induce cytokine production. These observations demonstrate that R. conorii-infected HUVEC secrete IL-6 and IL-8 via the induction of cell-associated IL-1alpha, providing a possible mechanism for the vasculitis observed in Mediterranean spotted fever.

A monoclonal antibody inhibits adhesion to fibronectin and vitronectin of a colon carcinoma cell line and recognizes the integrins alpha v beta 3, alpha v beta 5, and alpha v beta 6.

Using whole viable human colon carcinoma HT29 cells as immunogen, we produced a monoclonal antibody (mAb) termed 69-6-5. The antibody was functionally selected on its anti-cell-spreading activity. By immunoprecipitation of surface radiolabeled cell lysates from HT29-D4 cells (an HT29 cell clone), mAb 69-6-5 recognized a molecular complex resembling integrin heterodimers. Sequential immunodepletions with mAb to the integrin alpha v subunit demonstrated that this complex was composed of alpha v-containing integrins. Accordingly, mAb 69-6-5 reacted with integrin alpha v beta 3 immunopurified from melanoma cells and integrins alpha v beta 5 and alpha v beta 6 immunopurified from pancreatic carcinoma cells. In cell adhesion assays, the 69-6-5 mAb was able to inhibit strongly in a dose-dependent manner arginine-glycine-aspartic acid-mediated adhesion of HT29-D4 cells to vitronectin, fibronectin, or ProNectin F but not to laminin or collagen. Immunoprecipitations with beta chain-specific antisera indicated that these cells express integrins alpha v beta 5 (receptor for vitronectin) and alpha v beta 6 (receptor for fibronectin) but neither alpha v beta 1 nor alpha v beta 3. In summary, these results indicated that mAb 69-6-5 reacts with several alpha v integrins and that it can effectively interfere with the adhesive functions of at least alpha v beta 5 and alpha v beta 6, which represent the major receptors on HT29-D4 cells responsible for their adhesion on vitronectin and fibronectin.

Leukosialin (CD43) behavior during adhesion of human monocytic THP-1 cells to red blood cells.

To understand the modulation and the behavior of glycocalyx elements during adhesion, we explored one of its components, the CD43 molecule, on human monocytic THP-1 cells exposed to cytokine stimulation and its redistribution during heterotypic adhesion to opsonized erythrocytes. First we demonstrated by immunofluorescence and immunoprecipitation that CD43 is dys-sialylated in monocytic THP-1 cells stimulated by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) and stimulation increased correlated to heterotypic adhesion. CD43 anti-adhesive effect seemed to be related to sialic acid moeties because an increase in adhesion was also induced by sialidase treatment and by monoclonal antibodies recognizing sialic acid-dependent epitopes on CD43. Second, a redistribution of CD43 molecules was observed after adhesion, resulting in the exclusion of CD43 molecules from contact areas as demonstrated by immunofluorescence and by ultrastructural immunogold localization. We therefore demonstrated in monocytic THP-1 cells that some glycocalyx molecules can be modulated by cytokines and redistributed during adhesion. These results support the concept that CD43 can regulate cell interactions.

Attachment of spores of the human pathogenic fungus Rhizopus oryzae to extracellular matrix components.

Fungi of the order Mucorales determine various infections involving principally the respiratory tract. In spite of their medical importance, little is known about their mechanisms of adherence to the host tissues. Thus we have attempted to define the morphological stages involved in the adherence process of Rhizopus oryzae which is the main causative agent of mucormycoses. The study of the kinetics of germination and adherence to plastic revealed that attachment occurred prior to germination and decreased dramatically with germ tube formation. This correlates with important modifications of the cell wall of the fungus with respect to both carbohydrate composition and distribution of anionic sites. Moreover, the attachment of spores to extracellular matrix components immobilized onto wells of polystyrene microtiter plates has been investigated. Spores adhered readily to immobilized laminin or type IV collagen, but not to fibronectin or the glycosaminoglycans. Attachment to laminin and collagen was dose-dependent and specific. Adhesion was not inhibited by the different carbohydrates tested, suggesting that a lectin was not involved in these interactions. Finally, immunofluorescence revealed that laminin and type IV collagen interacted exclusively with spores and mother cells of germ tubes. Thus, the recognition of laminin or collagen by spores may participate in their adherence to epithelial basement membranes exposed after epithelial tissue damage which frequently accompanies the predisposing factors for mucormycoses.

Localization of alpha-melanocyte-stimulating hormone in rat brain and pituitary.

The distribution of alpha-melanocyte-stimulating hormone (alpha-MSH) was studied in the rat brain with an immunoperoxidase technique. alpha-MSH-containing cells were found in the arcuate nucleus of the hypothalamus. Cells staining for alpha-MSH were also localized in the intermediate lobe of the pituitary. alpha-MSH-containing nerve fibers extended throughout regions of the hypothalamus, thalamus, and midbrain. Two weeks after hypophysectomy, alpha-MSH-positive cells anf fibers were still present in the brain. These results indicate that alpha-MSH of non-pituitary origin is synthesized and stored by neural structures in the rat brain. The detection of alpha-MSH by radioimmunoassay in the rat brain and pituitary supports these observations.

Influence of thyrotropin-releasing hormone on the secretion of thyrotropin in neonatal rats.

Two hours after the ip administration of TRH antiserum, no change in serum TSH concentration was observed in the rat from birth through day 5 of life. Under the same conditions, a significant reduction in serum TSH was observed in 7- to 14-day-old rats. Similarly, in neonatal hypothyroidism, TSH levels did not change in 1-day-old rats, whereas a significant decrease was observed on day 7. The administration of synthetic TRH during the neonatal period induced a significant increase of serum TSH in the newborn; however, TSH release by the pituitary gland increased progressively from days 3-10. Immunoreactive TRH was undetectable in the serum of newborn rats. Adult levels were reached when the rats were 10 days old. It is concluded that neonatal pituitary-thyroid function in the rat is not physiologically dependent upon TRH secretion, although synthetic TRH is able to stimulate the secretion of TSH at birth.

Influence of endogenous somatostatin on growth hormone and thyrotropin secretion in neonatal rats.

When gestating rats were injected iv with an antiserum to somatostatin (SRIF-AS) during the last week of gestation, serum GH levels in fetuses and 6-h-old newborn rats were not significantly different from controls. Similarly, 2 h after the ip administration of SRIF-AS, no change in serum GH concentration was observed in 2-h-old rats. However, under the same conditions, a significant increase in serum GH was observed in 24-h-old rats and in 2- to 60-day-old rats. The injection of SRIF-AS neither changed basal serum TSH levels during the neonatal development nor in the adult stage. A significant increase in TRH-induced TSH release was observed after the third postnatal day. It is concluded that endogenous SRIF plays a physiological role in GH release by 24 h of age in the rat and that the fall in GH secretion that normally occurs during the first week of life is due to the development of inhibitory mechanisms mediated by hypothalamus SRIF. Additionally the results suggest that the influence of SRIF upon TSH secretion is present before that of TRH.

The primate thyroid gland contains receptors for androgens.

The gonadal steroids have long been known to modulate thyroid function. Most studies suggest that the gonadal steroids act indirectly through the hypothalamic-pituitary axis to modulate thyroid function. The following studies were conducted to determine whether there are receptors for androgens in the thyroid itself. Cytosols from male and female euthyroid patients were analyzed for the presence of androgen with the synthetic analog methyltrienolone [( 3H]R1881). No evidence of androgen receptors was found in any of the cytosols prepared from female patients. In all males studied, androgen receptors were found in concentrations ranging from 100-150 fmol/10 mg DNA for the cytosols and from 690-2800 fmol/10 mg DNA for the nuclear extracts. The receptors had a dissociation constant (Kd) of approximately 5-10 X 10(-10) M for the cytosol and approximately 10-15 X 10(-10) M for the nuclear extracts. In addition to the human studies, studies in baboons were conducted to determine the possible cell type which might contain receptors for androgens. Male and female baboons were injected with [3H] dihydrotestosterone and killed between 1 and 1 1/2 h later. The thyroids were removed and processed for autoradiography. In autoradiograms from animals injected with [3H]dihydrotestosterone, nuclear localization of radioactivity was found in virtually all of the follicular cells. Also, label was found overlying the colloid, with heaviest labeling near the cells. These data suggest that there may be direct actions of androgens on follicular cells.

Laminin-mediated adhesion in metastatic rat rhabdomyosarcoma cell lines involves prominent interactions with the laminin E8 fragment.

In vitro attachment assays were carried out to assess adhesion between two basement membrane proteins, type IV collagen and laminin, and rat rhabdomyosarcoma (RMS) cell lines with different metastatic potentials. Whereas cells did not adhere to type IV collagen, adhesion to laminin appeared to be very sensitive as maximal adhesion was achieved in dose-response assays with only nanograms of laminin. Adhesion was mediated by interactions between coated laminin and cell surface components, probably receptors, but not endogenous laminin. Laminin-mediated adhesion of RMS cell lines was compared with that of the MCF-7 (human mammary carcinoma) and the L6 (rat myoblast) cell lines. In dose-response assays, RMS cell lines required 10 times less laminin to reach half-maximal attachment rates than MCF-7 and L6 cell lines. Two laminin fragments, P1 and E8, which are structurally and immunologically distinct as shown by alpha-helix content, SDS-PAGE and monoclonal antibody mapping, supported adhesion by RMS cells and L6 myoblasts, but MCF-7 adhered only to P1. This fragment was 10 times less active than laminin in RMS cell lines. Attachment in dose-response assays and adhesion inhibition studies by antibodies revealed that E8 accounted for the activity of laminin in RMS cell adhesion. Adhesion in the RMS cell lines was dominated by interaction with E8 regardless of metastatic potential.

Parallel release of ACTH, beta-endorphin, alpha-MSH and beta-MSH-like immunoreactivities in rat anterior pituitary cells in culture.

Specific radioimmunoassays (RIAs) for ACTH, beta-endorphin, alpha-MSH and beta-MSH were used to identify the immunoreactive components released during incubation of rat anterior pituitary cells in primary culture. Such measurements were performed after separation of the incubation media by chromatography on Sephadex G-50 or G-75. The ACTH-RIA measured approximately equal amounts of 13 and 4.5K ACTH while equal proportions of components migrating at the position of beta-LPH and beta-endorphin were measured in the beta-endorphin RIA system. Immunoreactive components migrating at the position of gamma-LPH and alpha-MSH were measured in the beta-MSH and alpha-MSH RIA systems, resp. 3 purified corticotropin-releasing fractions (CRF) prepared from porcine hypothalami and increasing concentrations of N6,O2'-dibutyryl cyclic AMP and theophylline led to parallel release of ACTH, beta-endorphin, beta-MSH and alpha-MSH immunoreactivities while preincubation with dexamethasone led to a 30-60% inhibition of the release of all peptides. The present data show that the release of ACTH, beta-LPH, beta-endorphin, gamma-LPH and alpha-MSH-like immunoeactivities occurs in parallel in anterior pituitary cells in culture both under basal and acute stimulatory or inhibitory conditions of release.

Parallel stimulation of ACTH, beta-LPH + beta-endorphin and alpha-MSH release by alpha-adrenergic agents in rat anterior pituitary cells in culture.

Characteristics of the alpha-adrenergic stimulation of ACTH, beta-endorphin + beta-LPH and alpha-MSH release were studied in rat anterior pituitary cells in primary culture. Parallel changes of ACTH, beta-endorphin + beta-LPH and alpha-MSh release were found under all stimulatory and inhibitory conditions by natural and synthetic catecholamine agonists and antagonists. (-)Epinephrine and (-)norepinephrine lead to a 8--10-fold stimulation of peptide release at ED50 values of 20 and 90 nM, respectively. The stereoselectivity of the alpha-adrenergic stimulatory action on peptide release is indicated by a 100-fold higher activity of (-)- than (+)norepinephrine while (-)epinephrine is 10 times more potent than the corresponding (+) stereoisomer. The involvement of a typical alpha-adrenergic mechanism in the control of release of ACTH, beta-endorphin and related peptides in rat anterior pituitary gland is indicated by the following order of potency of a series of catecholaminergic agents (ED50 values): (-)epinephrine (20 nM) greater than (-)norepinephrine (90 nm) greater than phenylephrine (400 nM) greater than isoproterenol (6000 nM). The stimulatory effect of (-)epinephrine or phenylephrine is completely reversed by low concentrations of the alpha-adrenergic antagonist phentolamine while the beta-adrenergic antagonist propranolol has no effect up to 10 muM. Beside providing an easily accessible pure population of post-synaptic alpha-adrenergic receptors having potential applications as a model for other less accessible alpha-adrenergic brain systems, the present data suggest the possibility of the direct involvement of a catecholamine in the physiological control of ACTH secretion in the rat anterior pituitary gland.

Immunohistochemical detection of laminin in 98 human breast carcinomas: a light and electron microscopic study.

The distribution of laminin was studied in 98 breast carcinomas with antilaminin and the avidin-biotin-peroxidase complex method. Laminin was observed within vascular and epithelial basement membranes. Laminin displayed a continuous linear pattern in intraductal carcinomas, and it was heterogeneously distributed, with a discontinuous linear pattern, in invasive carcinomas. No intracellular laminin staining was detected. Electron microscopic study showed laminin immunostaining in the lamina densa of basement membranes in nonneoplastic breast tissue. In tumors, laminin immunostaining frequently revealed multilayered basement membranes and abnormal multilayered basement membranes in blood vessels in the tumor stroma. These data suggest that laminin immunostaining, as a new approach to the heterogeneous basement membrane changes occurring in carcinomas, should permit better understanding of cell diffusion processes and of stroma-tumor cell interactions. The consistent extracellular distribution of laminin in contact with the stroma indicates that the latter plays an important role in the assembly of basement membrane components.

Regulation of opioid gene expression.

Opioid peptides are synthesized in the form of large precursors, which contain the information for more than one biologically active peptide. Using recombinant DNA technology, three opioid precursors have been sequenced: pro-opiomelanocortin (POMC), proenkephalin and prodynorphin. Analysis of the structures of these three precursors and their corresponding genes show striking similarities suggesting a common evolutionary mechanism. Regulation of POMC gene expression has been analyzed in different rat tissues. Detection of POMC mRNA in brain tissues supports the hypothesis that ACTH and endorphin peptides are synthesized in these tissues. Quantitation of POMC mRNA levels in pituitaries of rats subjected to adrenalectomy and glucocorticoid treatment shows that the feedback effect of the glucocorticoids occurs at the level of the rate of transcription of POMC mRNA.

Influence of haloperidol on ACTH and beta-endorphin secretion in the rat.

The injection of haloperidol, a dopamine receptor blocker, was followed by a large increase of plasma ACTH and beta-endorphin-like immunoreactivity (beta-EI) in the rat. This effect was prevented when the rats were previously treated with corticosteroids. These results suggest that catecholamines inhibit ACTH and beta-endorphin secretion in the rat.

beta-Endorphin is present in high concentration in the hypophysial portal vessels of rats.

beta-Endorphin-like immunoreactivity (beta-ELI) was measured in hypophysial portal and arterial blood of intact, adrenalectomized and hypophysectomized rats. beta-ELI levels were 61 times higher in the long portal vessels than in the general circulation. Circulating, and especially portal, levels of beta-ELI were significantly increased after adrenalectomy. After removal of the pituitary gland, the mean level of beta-ELI in portal blood was significantly lower than in intact rats. beta-ELI in portal blood displayed the same chromatographic properties as synthetic beta-endorphin.

Modulable Southern and Northern blot hybridization apparatus for routine screening of gene amplification and expression.

An apparatus for Northern and Southern blot hybridization is described. It allows from one to twenty-four blots to be processed at the same time, with different probes. All the pre-, post- and hybridization steps are performed without handling the filters and the experimenter is totally protected from beta radiations. The development of such modulable materials has become necessary since Southern and Northern techniques are becoming routine assays in hospitals, particularly in the field of oncology, in prognosis and for hereditary diseases, as an antenatal diagnosis procedure.

Corticolipotropin immunoreactivity in silent chromophobe adenomas: a light and electron microscopic study.

Twenty silent human pituitary adenomas were morphologically studied. Immunoperoxidase methods showed numerous adrenocorticotropic hormone-immunoreactive tumor cells in 14 cases by light microscopy and in one additional case by electron microscopy. Three of these cases were positive for beta-endorphin and one for beta-lipotropin by electron microscopy. These immunoreactions were found in undifferentiated tumors as well as in oncocytic adenomas, and could not be related to the presence of basophils by light microscopy. The peptides so detected in silent adenomas may have no biological activity and may correspond to common precursor molecule subunits.

[Invasion, metastases of solid tumors. Interaction of tumor cells with tissue and vascular basement membranes]. / Invasion, métastases des tumeurs solides. Interactions cellules tumorales/membranes basales tissulaires et vasculaires.

Since early antiquity malignant tumors have been recognized and characterized by: 1) their particular local-regional invasive properties. Developing irregularly from the primary tumor mass, the invasion of adjacent tissues often displays classical crab-like images which distinguish, macroscopically and microscopically, malignant tumors from benign tumors. 2) their metastatic capacities. Even though they have been taught since the time of Hippocrates, the specific clinical characteristics of malignant tumors have only recently been apprehended and studied on cellular and biochemical levels. The development of the metastatic phenomenon is a complex chain of events limited to only a few tumor subpopulations. Like every biological study knowledge has progressed over the past years only by concentration of efforts on some of these stages: Invasion associated with disorganization and destruction of basement membranes; Migration of metastatic tumor cells in the stroma and bridging of vascular basal membranes; Colonization of target tissues by recognition and penetration of specific vascular membranes, establishment and concentric development within these tissues. These three stages result from successive interactions between multiple cellular and biochemical phenomena. This review is a schema of our present knowledge of tumor invasion, and more precisely of that concerning the relations between tumor cells and extracellular matrices, particularly basal membranes, leading to a definition of the cellular phenotype with a high metastatic potential.

Laminin immunodetection in tumorous and nontumorous disorders of human thyroid.

Laminin distribution in tissues from surgically removed thyroids consisting in normal gland (5), Basedow's disease (5), thyroiditis (5), follicular adenomas (8), papillary carcinomas (8), follicular carcinomas (6) was studied using rabbit laminin antibody raised against murine laminin. Immunoperoxidase technique (Avidin-Biotin-Peroxidase Complex) was performed on (1) paraffin sections of fixed tissue (2) frozen sections for light microscopy examination. Vibratome thick sections (100 micron) and pre-embedding technique were used for electron microscopy study. Positive staining, was obtained only on frozen and vibratome sections and was found within basement membranes but never in epithelial cell cytoplasm. Laminin had a similar distribution in follicular adenomas, Basedow's disease and normal tissue. Nests of damaged cells in Hashimoto's thyroiditis lacked positive laminin immunostaining. In papillary carcinomas positive staining was found beneath the epithelial cells along the cores. In well differentiated follicular carcinomas the perifollicular laminin staining was preserved, whereas in poorly differentiated follicular carcinomas laminin staining was barely visible or absent.

Estrogen receptor immunocytochemical assay (ERICA) and laminin detection in 130 breast carcinomas and computerized (Samba 200) multiparametric quantitative analysis on tissue sections.

An estrogen receptor immunocytochemical assay (ER-ICA) was applied to 130 malignant breast carcinomas and the results were compared to those of steroid binding assays performed on cytosol extracts of the same tumors. Also laminin (lam) distribution was studied in the same tumors. A semi quantitative analysis and a computerized image analysis system (SAMBA 200 TITN) were used to evaluate the positive ER and lam immunostaining. Positive ER immunostaining was always located in the nucleus of tumor cells and of normal cells in adjacent breast tissue. When immunohistochemical staining was correlated to biochemical assay there was a 88% correlation staining intensity and percentage of positive cells significantly increased (p less than 0.01) with cytosolic ER levels. Lam was observed within vascular and epithelial basement membranes (BMs). Lam staining displayed a continuous linear pattern in intraductal carcinomas or was heterogeneously distributed with a discontinuous linear pattern in invasive carcinomas. No intracellular lam staining was detected. In tumors, laminin immunostaining revealed often multilayered BMs and abnormal multilayered BMs in blood vessels in the tumor stroma. These results indicated that (ER-ICA) is to date the most reliable histochemical method for ER detection and correlated in 88% of the cases with ER biochemical assay ER-ICA provides additional information for heterogeneous ER distribution within tumors ER-ICA as a qualitative method is unable to replace the quantitative ER determination obtained with biochemical assay ER-ICA based on ER antigenic site detection is complementary to biochemical assay based on ER functional site determination laminin immunostaing constitutes a new approach to the heterogeneous BM changes occurring in carcinomas, and permits a better understanding of cell diffusion processes and of stroma-tumor cells interactions: the consistent extracellular lam distribution in contact with the stroma, indicates that the latter plays an important role in the assembly of BM components the SAMBA 200 permits a reliable accurate evaluation of the percentage of the immunostained cells and surfaces.