Preconditioning in the absence or presence of sustained ischemia modulates myocardial Cx43 protein levels and gap junction distribution.

In the heart, brief repeated episodes of ischemia prior to a sustained occlusion (ischemic preconditioning; PC) significantly delay the onset of necrosis and arrhythmogenesis. Ischemia has been reported to influence gap junction organization and connexin43 (Cx43) content, but whether PC affects these structures is not known. We investigated the effect of PC (2 cycles of 5-min ischemia plus 10-min reperfusion) followed by prolonged reperfusion without concomitant regional coronary occlusion on the myocardial Cx43 content and its spatial distribution in rabbit hearts. We also compared the effect of sustained ischemia with or without PC on Cx43 spatial distribution. In experiments with PC only, there was an initial decrease in Cx43 levels within the ischemic zone followed by a progressive increase after 48 h reperfusion. End-to-end immunolabeling of Cx43 was augmented in the ischemic region between 24 and 48 h reperfusion; labeling was not uniquely confined to myocyte abutments, but was also dispersed along the sarcolemma. Cx43 immunolabelling was more intense and diffuse in hearts subjected to PC before sustained coronary occlusion (compared to non-PC). These data indicate that gap junctions are significantly altered during brief episodes of ischemia. Reorganization of the gap junction complex could contribute to PC-mediated reductions in cardiac arrhythmias.

Comparative effect of danazol and a GnRH agonist on monocyte chemotactic protein-1 expression by endometriotic cells.

PROBLEM: Endometriosis is associated with a chronic inflammatory process, and the increased number of activated peritoneal macrophages is one of the major hallmarks of this process. The medical treatment of the disease, which is based on the creation of an hypoestrogenic milieu unfavorable to the growth of endometriotic lesions, is often associated with a reduced peritoneal inflammation. The aim of this study was to investigate the ability of current therapeutic agents to modulate, through a direct mechanism, the expression by endometriotic cells of monocyte chemotactic protein-1 (MCP-1), a chemokine endowed with the potent faculty of recruiting and activating macrophages. METHOD OF STUDY: Cells were stimulated with interleukin-1 beta (IL-1beta) to induce MCP-1 expression. MCP-1 protein secretion and mRNA steady-state levels were evaluated by ELISA and northern blot, respectively. RESULTS: Our results show that danazol concentrations (10(-7) -10(-5) M), taking into account the therapeutic levels found in the plasma of treated patients, inhibited MCP-1 protein and mRNA steady-state levels in endometriotic cells, whereas buserelin acetate (0.1-10 ng/mL), a GnRH agonist, had no significant effect. Dexamethasone, an anti-inflammatory glucocorticoid, used at concentrations varying between 10(-12) and 10(-6) M, also displayed a dose-dependent inhibitory action. CONCLUSIONS: These results put into prominence the capability of danazol to directly inhibit the expression of a potent monocyte chemotactic and activating factor by ectopic endometrial cells shedding more light on the mechanisms underlying the clinical effects of hormonal therapeutic agents used in the treatment of endometriosis.

Decreased expression of the decoy interleukin-1 receptor type II in human endometriosis.

Many of the biological changes occurring in the endometrium during the menstrual cycle bear a striking resemblance to those associated with inflammatory and reparative processes. Hence, it would not be surprising to find that cytokines known for their pro-inflammatory properties, such as interleukin-1 (IL-1), could play a key role in the physiology of this tissue and that their action would be tightly controlled by local mechanisms. In the present study, immunohistochemical and Western blot analyses show that in normal women (n = 39), the endometrial tissue expresses, in a cycle-dependent manner, the IL-1 receptor type II (IL-1RII), a molecule of which the only biological property known to date is that of capturing IL-1, inhibiting thereby its binding to the functional type I IL-1 receptor. IL-RII immunostaining was particularly intense within the lumen of the glands and at the apical side of surface epithelium. Interestingly, the intensity of staining was markedly less pronounced in the endometrium of women with endometriosis (n = 54), a disease believed to arise from the abnormal development of endometrial tissue outside the uterus, especially in the early stages of the disease (stages I and II). This study is the first to show the local expression in endometrial tissue of IL-1RII, a potent and specific down-regulator of IL-1 action and its decreased expression in women suffering from endometriosis.

Estradiol amplifies interleukin-1-induced monocyte chemotactic protein-1 expression by ectopic endometrial cells of women with endometriosis.

Endometriosis, one of the most frequently occurring gynecological disorders, is estrogen dependent and is often associated with immunological changes. These include increased macrophage activation and infiltration into the endometriotic implants themselves as well as the peritoneal cavity where the implants often develop. Despite the critical role estrogens play in the development of endometriosis, the biochemical mechanisms of their action remain unclear. In the present study we report that estradiol (E2) enhances endometriotic cell responsiveness to the proinflammatory cytokine interleukin-1beta by up-regulating interleukin-1-induced monocyte chemotactic protein-1 (MCP-1) expression at the level of both protein secretion and messenger ribonucleic acid (mRNA) synthesis, whereas progesterone had no significant effects. According to mRNA half-life experiments, E2 action does not seem to be due to increased MCP-1 mRNA stability but, rather, to a higher level of transcription, as shown by run-on analysis. Interestingly, immunohistochemical analysis of MCP-1 expression in endometriotic tissue showed intense immunostaining in both epithelial glands and stroma regardless of the menstrual cycle phase, which is consistent with the cell culture data and indicates that MCP-1 expression is not subject to cyclic variation. The findings of the present study for the first time provide evidence that E2 up-regulates, although in an indirect way, the expression of a potent chemotactic and activating factor by ectopic endometrial cells, which may occur locally in the inflammatory site and contribute to peritoneal macrophage recruitment and activation, and reveal a new means of E2 action in the pathophysiology of endometriosis.

Physiological and cytogenetic characterization of immortalized human endometriotic cells containing episomal simian virus 40 DNA.

The study of misplaced endometrial cells, which abnormally implant and grow outside the uterine cavity, is of considerable interest for the understanding of the pathophysiology of endometriosis. However, endometriotic cells, particularly epithelial cells, required for primary cell culture are not easily available. We report here the characterization of an endometriotic cell line immortalized after infection of primary endometriotic cell cultures with simian virus 40. Transformed cells express T-antigen, and blot hybridization analysis showed that the viral genome is present as an episome. Cytogenetic analysis revealed a polyploid karyotype with numerical and structural rearrangements involving mainly the same chromosomes (6, 10, 11, 15, and 17). The cell line has been maintained in culture for over 80 passages and was still proliferating without any noticeable change in the biological properties investigated. Transformed endometriotic cells expressed both progesterone and estradiol receptors and were stimulated by these ovarian hormones to secrete monocyte chemotactic protein-1, a factor that may play an important role in the recruitment and activation of peritoneal macrophages. In addition, this response was enhanced in interleukin-1-treated cells. Taken together, these findings support the view that this cell line may be an interesting tool for the study of the pathophysiology of endometriosis.

Increased expression of monocyte chemotactic protein-1 in the endometrium of women with endometriosis.

The pathogenesis of endometriosis, a disease widely believed to arise from an aberrant growth of endometrial tissue outside the uterus, is still unclear. We have previously observed that cytokine-stimulated endometrial cells of women with endometriosis secrete in vitro increased amounts of monocyte chemotactic protein-1 (MCP-1). This factor may be important in the recruitment and activation of peritoneal macrophages observed in endometriosis patients. The present study reports that, in the presence of the disease, such an up-regulation of MCP-1 expression arises in vivo and can be encountered in situ in the intrauterine endometrium. In women with endometriosis, MCP-1 expression was elevated in endometrial glands, both at the level of the protein (immunohistochemistry) and the mRNA (in situ hybridization). This was observed throughout the menstrual cycle and varied according to the stage of the disease. These findings strongly argue in favor of the presence of pathophysiological changes in the eutopic endometrium of patients with endometriosis and make plausible MCP-1 as a key effector cell mediator involved in the pathogenesis of the disease.

T-cell tolerance toward a transgenic beta-cell antigen and transcription of endogenous pancreatic genes in thymus.

Transgenic mice expressing T antigen (Tag) in pancreatic beta cells establish systemic tolerance toward this self-protein. The self-tolerance in two families of rat insulin promoter (RIP)-Tag mice, expressing different levels of Tag protein, has been characterized. These mice have impaired antibody responses to Tag, show diminished Tag-specific T-cell proliferation, and evidence an inability to generate Tag-specific cytotoxic T cells. The existence of systemic tolerance toward a beta-cell-specific protein motivated examination of transgene expression in the thymus. Indeed, low levels of Tag mRNA were detected intrathymically. Remarkably, this expression is a valid property of the insulin gene regulatory region, since insulin RNA was also expressed in the thymus of nontransgenic mice. RNA for other pancreatic genes was also detected in the thymus, thus raising the possibility that many tissue-specific genes could be expressed intrathymically during immunological development and induction of self-tolerance. These results raise important questions for future research into the role of the thymus in tolerance induction toward so-called tissue-specific antigens.

Isolation of nonobese diabetic mouse T-cells that recognize novel autoantigens involved in the early events of diabetes.

Insulin-dependent diabetes mellitus (IDDM) is thought to result from chronic, cell-mediated, autoimmune islet damage. Our aim was to identify the earliest T-cell autoantigen in IDDM, reasoning that this antigen could be causally involved in the initiation of the disease. Identification of the earliest beta-cell-specific autoantigen is extremely important in allowing advances in prevention and treatment of initial events in the development of inflammatory insulitis that precedes beta-cell destruction and overt diabetes. Therefore, we analyzed the proliferative responses of peripheral T-cells from young, female nonobese diabetic (NOD) mice to extracts of pancreatic beta-cell lines. We were able to demonstrate that T-cells responsive to beta-cell antigens exist in the peripheral lymphoid tissue of these mice in the absence of deliberate priming before the manifestation of histologically detectable insulitis. T-cell lines and clones isolated from the peripheral lymphatic tissues of young, unimmunized, female NOD mice were also shown to react with extracts of beta-cells. Fractionation of the beta-cell extracts showed that these T-cell clones recognized multiple beta-cell-specific autoantigens but none of the previously reported putative autoantigens (glutamic acid decarboxylase [GAD]65, GAD67, Hsp65, insulin, ICA 69, carboxypeptidase-H, and peripherin). Thus, we can conclude that these responses are specific for novel beta-cell autoantigens. Finally, NOD T-cell proliferative responses were also seen to an extract of human islets suggesting potential shared antigenic determinants between human and mouse beta-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Pancreatic beta cells cultured from individual preneoplastic foci in a multistage tumorigenesis pathway: a potentially general technique for isolating physiologically representative cell lines.

Culturing and comparing the discrete stages of tumorigenesis provide a route to defining important components of the cancer phenotype and, in addition, present the opportunity to establish cell cultures more representative of normal cells than the ultimate malignant cancer cells. Herein we report that preneoplastic foci in one multistep tumorigenesis pathway can be cultured in vitro and show that they preserve distinctive characteristics of the normal cells from which they arose, pancreatic beta cells. In the RIP1-Tag2 line of transgenic mice, which express the simian virus 40 T antigen in insulin-producing beta cells, pancreatic islets develop into vascularized tumors in a multistage pathway. We established conditions for reproducible derivation of beta-cell lines from individual hyperplastic islets that have not yet developed into solid tumors. Most of these cell lines, designated beta HC, release insulin at physiological concentrations of glucose. In contrast to tumor-derived lines (beta TC), which are not properly regulated, the ability of the beta HC lines to respond correctly to glucose correlated with maintenance of normally depressed levels of low-Km hexokinases. Glutamic acid decarboxylase (GAD), an early autoantigen in type I diabetes, was detected in most of the beta HC lines. The relative levels of the two forms of this enzyme (GAD65 and GAD67) varied significantly between the different cell lines, suggesting independent regulation. Class I major histocompatibility complex antigens were detected on the beta HC cells, and the levels of surface major histocompatibility complex expression correlated with their capacity to serve as targets in a cytotoxic T-cell killing assay. The beta HC lines will be of value for studies of beta-cell physiology, autoantigenicity, and tumor development. This work suggests the possibility of culturing preneoplastic stages of other cancers, both to address the mechanisms of transformation and to provide a source of cells that maintain important qualities of their normal progenitors.

Determinants of the B-cell response against a transgenic autoantigen.

The failure to induce self-tolerance of simian virus 40 large tumor antigen (T antigen) expressed in the pancreatic beta cells of transgenic mice results in an autoimmune response against this protein and the cells that synthesize it. In every transgenic mouse with delayed onset of T-antigen expression and consequent nontolerance, B cells, T cells, and macrophages are attracted to and infiltrate the pancreatic islets. In contrast, the incidence, onset, and intensity of the B-cell response to produce anti-T-antigen autoantibodies vary considerably with genetic background. Thus the initial attraction of lymphocytes to the cells synthesizing a non-self antigen can be separated from the activation of a B-cell response against it. Haplotypes of the major histocompatibility complex (MHC) differentially influence the character of the autoimmune response, with H-2d and H-2k conferring a high incidence of humoral autoimmunity. Additional non-MHC linked genes are also implicated in control of the B-cell response.

Expression of two forms of prolactin receptor in rat ovary and liver.

The screening of a size-selected cDNA library from the ovary revealed the existence of a second form of PRL receptor in the rat. The polypeptide sequence deduced from cDNAs has a much longer cytoplasmic domain (357 amino acids) than the form previously identified in the liver (57 amino acids). Nucleotide sequence analysis and comparison with rabbit, mouse, and human PRL receptor cDNAs suggests that the two forms of rat PRL receptor result from alternative splicing of a primary transcript. Complementary DNAs encoding the long form of the receptor were also found in a library prepared from estradiol-treated rat liver, although they represent a minor fraction of total PRL receptor cDNAs obtained from this tissue. DNA polymerase chain reaction amplification of cDNA confirmed the presence of the two receptor forms in both the ovary and liver. Northern analysis, using probes that specifically hybridize with either form of mRNA, indicates a major transcript of 1.8 kilobases (kb) in estradiol-treated liver, which encodes the receptor with a short cytoplasmic domain, while the long form of the receptor is encoded by mRNAs of 2.5 and 3 kb. In the ovary, a complex pattern of hybridization to multiple mRNAs (1.8-5.5 kb) is obtained with the probe specific to the long form, and essentially only a 5.5-kb mRNA is obtained with the probe specific to the short form. The predicted size of the mature form of the long PRL receptor (PRL-R2) is 591 amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Mucoadhesion of hydroxypropylmethacrylate nanoparticles to rat intestinal ileal segments in vitro.

The purpose of this study was to evaluate the adhesion of HPMA nanoparticles to mucus using a perfused rat ileum test system. Radiolabeled nanoparticles were prepared and deposited onto rat ileal segments in vitro. The segments were perfused and the perfusate was collected in fractions and assayed for radioactivity. Between 10 and 50% of the radioactivity was eliminated over the first 120-sec perfusion, whereas the remaining activity was firmly attached to the ileum. Among the variables tested, the time interval between nanoparticle deposition and perfusion played the major role, indicating that the mucus-nanoparticle interaction is likely to result from the diffusion of polymers into the mucus and of mucin into the polymeric matrix.

New method for the preparation of cyanoacrylic nanoparticles with improved colloidal properties.

The purpose of this study was to prepare nanoparticles with a size significantly smaller than 0.1 micron. It was shown that when sulphur dioxide was dissolved in the cyanoacrylic monomer at a high concentration, subsequent anionic polymerization in an aqueous phase produced particles as small as 10 nm. Moreover, the obtained particles displayed an important negative charge which improve their stability against aggregation. Finally, nanoparticles were successfully prepared in double-distilled water, thereby avoiding the use of dextran which can induce anaphylactoid reactions.

Identification of a cDNA encoding a long form of prolactin receptor in human hepatoma and breast cancer cells.

Human PRL receptor cDNA clones from hepatoma (Hep G2) and breast cancer (T-47D) libraries were isolated by using a rat PRL receptor cDNA probe. The nucleotide sequence predicts a mature protein of 598 amino acids with a much longer cytoplasmic domain than the rat liver PRL receptor. Although this extended region has additional segments of localized sequence identity with the human GH receptor, there is no identity with any consensus sequences known to be involved in hormonal signal transduction. This cDNA will be a valuable tool to better understand the role of PRL in the development and growth of human breast cancer.

Multiple regulation of prolactin receptor gene expression in rat liver.

Sex steroids are major regulators of PRL receptor expression in rat liver. Using a probe encoding the rat PRL receptor we have studied receptor mRNA levels in female rat liver during ontogeny and in response to estrogen treatment. Steady state mRNA levels were determined by Northern blot and densitometric analysis. Messenger RNA levels have been compared to the number of binding sites, which was assessed by Scatchard analysis of [125I]ovine PRL binding in membrane preparations. Our results show that steady state mRNA and binding levels of PRL receptors are both regulated by development and estrogens, but that binding does not exactly parallel mRNA levels. From the developmental stages of prepuberty to adult, receptor numbers increase 8-fold, whereas mRNA levels increase 3-fold. Estrogen treatment stimulates receptor levels 6-fold, but mRNA levels are only increased 3-fold. These results suggest that PRL receptor gene expression in rat liver is regulated at the transcriptional or posttranscriptional level as well as at the translational level.

[Prolactin receptor: characterization by monoclonal antibodies and cloning of complementary DNA]. / Récepteur de la prolactine: caractérisation par des anticorps monoclonaux et clonage de l'ADN complémentaire.

Rat liver prolactin receptor has been partially characterized and purified to homogeneity using monoclonal antibodies. Pure receptor was digested with trypsin and amino acid sequence of receptor fragments determined. This allowed us to clone the prolactin receptor cDNA. Our approach to clone the receptor cDNA consisted of synthesizing oligonucleotides corresponding to the amino acid sequence of receptor fragments, and to screen a cDNA library. Sequencing reveals that prolactin receptor is a 291 amino acid protein, containing an extracellular domain of 210 residues, a single transmembrane segment of 24 amino acids and a cytoplasmic domain of 57 amino acids. Introduction of the prolactin receptor cDNA into various cell types demonstrates that the single protein is sufficient to bind prolactin with the same affinity and specificity reported for the prolactin receptor.

Identification and sequence analysis of a second form of prolactin receptor by molecular cloning of complementary DNA from rabbit mammary gland.

Two lambda gt11 clones containing fragments of cDNA encoding the prolactin receptor from rabbit mammary gland were isolated using a rat liver prolactin receptor cDNA probe. An 1848-base-pair open reading frame encodes a mature prolactin-binding protein of 592 amino acids that contains three domains: (i) the extracellular, amino-terminal, prolactin-binding region of 210 residues; (ii) the transmembrane region of 24 residues; and (iii) the intracellular, carboxyl-terminal domain of 358 residues. This latter domain is much longer than the cytoplasmic domain (57 residues) previously described for the rat liver prolactin receptor. In addition, the sequence identity of this form of prolactin receptor with the growth hormone receptor is extended in the cytoplasmic domain.

Purification, cloning, and expression of the prolactin receptor.

The rat liver prolactin receptor has been purified to homogeneity, and partial amino acid sequences have been obtained. The structure of the receptor has been deduced from a single complementary DNA clone. The mature protein of 291 amino acids has a relatively long extracellular region, a single transmembrane segment, and a short (57 amino acids) cytoplasmic domain. With the rat cDNA used as a probe, the prolactin receptor in rabbit mammary gland and human hepatoma cells has also been isolated. These tissues contain a second, longer form of the receptor (592 and 598 amino acids, respectively). Both the short and long forms of the prolactin receptor show regions of strong sequence identity with the human and rabbit growth hormone receptors, suggesting that the prolactin and growth hormone receptors originate from a common ancestor.