Role of ß3 subunit of the GABA type A receptor in triple negative breast cancer proliferation, migration, and cell cycle progression.

Triple negative breast cancer (TNBC) is known for its heterogeneous nature and aggressive onset. The unresponsiveness to hormone therapies and immunotherapy and the toxicity of chemotherapeutics account for the limited treatment options for TNBC. Ion channels have emerged as possible therapeutic candidates for cancer therapy, but little is known about how ligand gated ion channels, specifically, GABA type A ligand-gated ion channel receptors (GABAAR), affect cancer pathogenesis. Our results show that the GABAA ß3 subunit is expressed at higher levels in TNBC cell lines than non-tumorigenic cells, therefore contributing to the idea that limiting the GABAAR via knockdown of the GABAA ß3 subunit is a potential strategy for decreasing the proliferation and migration of TNBC cells. We employed pharmacological and genetic approaches to investigate the role of the GABAA ß3 subunit in TNBC proliferation, migration, and cell cycle progression. The results suggest that pharmacological antagonism or genetic knockdown of GABAA ß3 subunit decreases TNBC proliferation and migration. In addition, GABAA ß3 subunit knockdown causes cell cycle arrest in TNBC cell lines via decreased cyclin D1 and increased p21 expression. Our findings suggest that membrane bound GABAA receptors containing the ß3 subunit can be further developed as a potential novel target for the treatment of TNBC.

Predicting pathogen-specific CD8 T cell immune responses from a modeling approach.

The primary CD8 T cell immune response constitutes a major mechanism to fight an infection by intra-cellular pathogens. We aim at assessing whether pathogen-specific dynamical parameters of the CD8 T cell response can be identified, based on measurements of CD8 T cell counts, using a modeling approach. We generated experimental data consisting in CD8 T cell counts kinetics during the response to three different live intra-cellular pathogens: two viruses (influenza, vaccinia) injected intranasally, and one bacteria (Listeria monocytogenes) injected intravenously. All pathogens harbor the same antigen (NP68), but differ in their interaction with the host. In parallel, we developed a mathematical model describing the evolution of CD8 T cell counts and pathogen amount during an immune response. This model is characterized by 9 parameters and includes relevant feedback controls. The model outputs were compared with the three data series and an exhaustive estimation of the parameter values was performed. By focusing on the ability of the model to fit experimental data and to produce a CD8 T cell population mainly composed of memory cells at the end of the response, critical parameters were identified. We show that a small number of parameters (2-4) define the main features of the CD8 T cell immune response and are characteristic of a given pathogen. Among these parameters, two are related to the effector CD8 T cell mediated control of cell and pathogen death. The parameter associated with memory cell death is shown to play no relevant role during the main phases of the CD8 T cell response, yet it becomes essential when looking at the predictions of the model several months after the infection.

Inter-laboratory reproducibility of the LC-ELSD method for the control of composition of sesame oil recently introduced in the Ph. Eur.

Evaporative light-scattering detection (ELSD) is an increasingly important technology for the control of substances for pharmaceutical use. A LC-ELSD method has recently been introduced in the Ph. Eur. monograph for sesame oil, refined to control the composition of triglycerides. A collaborative study was run to assess the reproducibility of the new method and to confirm that it was suitable for pharmacopoeial use.

Dendritic cells are essential for priming but inefficient for boosting antitumour immune response in an orthotopic murine glioma model.

The prognosis of malignant gliomas remains dismal and alternative therapeutic strategies are required. Immunotherapy with dendritic cells (DCs) pulsed with tumour antigens emerges as a promising approach. Many parameters influence the efficacy of DC-based vaccines and need to be optimised in preclinical models. The present study compares different vaccine schedules using DCs loaded with tumour cell lysate (DC-Lysate) for increasing long-term survival in the GL26 orthotopic murine glioma model, focusing on the number of injections and an optimal way to recall antitumour immune response. Double vaccination with DC-Lysate strongly prolonged median survival compared to unvaccinated animals (mean survival 87.5 days vs. 25 days; p < 0.0001). In vitro data showed specific cytotoxic activity against GL26. However, late tumour relapses frequently occurred after 3 months and only 20% of mice were finally cured at 7 months. While one, two or three DC injections gave identical survival, a boost using only tumour lysate after initial DC-Lysate priming dramatically improved long-term survival in vaccinated mice, compared to the double DC-Lysate group, with 67.5% of animals cured at 7 months (p < 0.0001). In vitro data showed better specific CTL response and also the induction of specific anti-GL26 antibodies in the DC-Lysate/Lysate group, which mediated Complement Dependent Cytotoxicity. These experimental data may be of importance for the design of clinical trials that currently use multiple DC injections.

Comparison of the ecotoxicological impact of the triazines Irgarol 1051 and atrazine on microalgal cultures and natural microalgal communities in Lake Geneva.

The antifouling herbicide Irgarol 1051 has been detected in recent years in numerous estuaries, marinas, harbors and coastal areas, and in some harbors on Lake Geneva, but so far only a few studies have investigated the ecotoxicological effects of this compound on microalgae. The purpose of this study was to assess the ecotoxicological impact of Irgarol 1051 on the algal communities of Lake Geneva, and to compare its phytotoxicity to that of the common triazine herbicide, atrazine. We investigated the response of phytoplanktonic and periphytonic algal communities and single-species isolates collected from the lake, to the PS II inhibitor Irgarol 1051 (growth, proxy of photosynthetic activity and community structure). A short-term bioassay was developed based on in vivo fluorescence, together with nanocosm experiments with natural algal communities, and single-species tests on algal strains isolated from the lake. The toxicity of Irgarol 1051 towards periphyton and phytoplankton was shown to be higher than that of atrazine. Indications of the tolerance induced by this triazine in the algal communities of Lake Geneva, suggests that even at the levels of contamination reported in some parts of the lake, Irgarol 1051 is already exerting selection pressure. Information about sensitivities, selection and tolerance from laboratory experiments are used to explain the observations in natural microalgal communities from the lake.

Nitric oxide attenuates the expression of transforming growth factor-beta(3) mRNA in rat cardiac fibroblasts via destabilization.

Transforming growth factor-beta (TGF-beta) has been implicated in the development of interstitial fibrosis in cardiac hypertrophy. NO has been regarded as a potent inhibitor of cardiac fibroblast growth, albeit the modulation of cellular events associated with interstitial fibrosis remains undefined. In this regard, the regulation of TGF-beta mRNA expression by the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) was examined in neonatal rat cardiac fibroblasts. SNAP treatment for 4 hours decreased TGF-beta(3) mRNA levels, an effect mimicked by 8-bromo-cGMP. TGF-beta(3) mRNA, however, had returned to levels observed in the untreated cells after a 24-hour exposure to SNAP, whereas a decreased expression persisted with 8-bromo-cGMP. In contrast to TGF-beta(3), TGF-beta(1) mRNA levels were modestly increased in response to cGMP-generating molecules. The treatment with actinomycin D for at least 8 hours did not appreciably alter TGF-beta(3) mRNA levels. By contrast, SNAP treatment caused a rapid decrease of TGF-beta(3) mRNA with a half-life of 3.3+/-0.2 hours, thereby supporting a mechanism of destabilization. The pretreatment with SNAP inhibited angiotensin II-stimulated protein synthesis and the concomitant expression of TGF-beta(3) mRNA. These data reveal a disparate pattern of TGF-beta(1) and TGF-beta(3) mRNA regulation by NO and highlight a novel mechanism of destabilization contributing to the decreased expression of TGF-beta(3) mRNA. The modulation of both basal and angiotensin II-stimulated TGF-beta(3) mRNA expression provides a mechanism by which NO may influence the progression of interstitial fibrosis.

beta-Adrenergic stimulation of rat cardiac fibroblasts promotes protein synthesis via the activation of phosphatidylinositol 3-kinase.

beta -adrenergic agonists stimulate neonatal rat cardiac fibroblast growth, albeit the identity of the signaling event(s) remains equivocal. Isoproterenol (ISO) treatment increased intracellular cyclic AMP levels; however, cyclic AMP-elevating agents had no effect on protein synthesis. The tyrosine kinase inhibitor tyrphostin A25, and the inhibition of ras processing by the farnesyltransferase inhibitor BMS-191563 attenuated ISO-stimulated protein synthesis. Concomitant with increased protein synthesis, ISO stimulated extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3-K) activity. The MEK1/2 inhibitor PD098059 abrogated ISO-stimulated ERK activity, albeit the increase in protein synthesis was unaffected. By contrast, LY294002 inhibited both ISO-stimulated PI3-K activity, and protein synthesis. ISO treatment did not increase the expression of transforming growth factor-beta(1)(TGF-beta(1)) mRNA, whereas a significant decrease in the steady-state mRNA level of TGF- beta(3)was observed. This latter effect was mimicked by cyclic AMP-elevating agents. Angiotensin II (AII) activation of the AT(1)receptor increased protein synthesis, but in contrast to ISO, the growth response was not inhibited by either tyrphostin A25 or BMS-191563, and was associated with the concomitant expression of both TGF-beta(1)and TGF-beta(3)mRNAs. Analogous to ISO, AII treatment increased ERK and PI3-K activity, and PI3-K was required for protein synthesis. These findings are the first to highlight the activation of PI3-K by a Gs(alpha)-coupled receptor, and its essential role in beta -adrenergic as well as AT(1)receptor-mediated protein synthesis in neonatal rat cardiac fibroblasts. However, despite the conserved role of PI3-K, additional disparate signaling pathways are recruited by ISO and AII, which may differentially influence fibroblast phenotype.

Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector.

Limb-girdle muscular dystrophies 2C-F represent a family of autosomal recessive diseases caused by defects in sarcoglycan genes. The cardiomyopathic hamster is a naturally occurring model for limb-girdle muscular dystrophy caused by a primary deficiency in delta-sarcoglycan. We show here that acute sarcolemmal disruption occurs in this animal model during forceful muscle contraction. A recombinant adeno-associated virus vector encoding human delta-sarcoglycan conferred efficient and stable genetic reconstitution in the adult cardiomyopathic hamster when injected directly into muscle. A quantitative assay demonstrated that vector-transduced muscle fibers are stably protected from sarcolemmal disruption; there was no associated inflammation or immunologic response to the vector-encoded protein. Efficient gene transduction with rescue of the sarcoglycan complex in muscle fibers of the distal hindlimb was also obtained after infusion of recombinant adeno-associated virus into the femoral artery in conjunction with histamine-induced endothelial permeabilization. This study provides a strong rationale for the development of gene therapy for limb-girdle muscular dystrophy.

Different classes of coactivators recognize distinct but overlapping binding sites on the estrogen receptor ligand binding domain.

We have analyzed interaction of coactivators with the wild-type estrogen receptor alpha (ER), HEG0, and a mutant, L536P-HEG0, which is constitutively active in several transiently transfected cells and a HeLa line that stably propagates an estrogen-sensitive reporter gene. Different classes of coactivators do not recognize the ER ligand binding domain (LBD) in the same manner. Steroid receptor coactivator-1 (SRC-1), amplified in breast cancer-1 (AIB-1), transcriptional intermediary factor-1 (TIF-1), transcriptional intermediary factor-2 (TIF-2), and receptor interacting protein 140 (RIP140) interacted with HEG0 and L536P-HEG0 in the presence of estradiol, but generally not in the presence of anti-estrogens. However, ICI164,384 stimulated some interaction of RIP140 with LBDs. SRC-1, AIB-1, and RIP140 interacted constitutively with the L536P ER, whereas TIF-1 and TIF-2 interacted only weakly in the absence of hormone. Reciprocal competition for binding to the ER LBD was observed between different classes of coactivators. Moreover, coexpression of RIP140 blocked enhanced transactivation by HEG0 observed in the presence of TIF-2, suggesting that RIP140 may play a negative role in ER signaling. We conclude that constitutive activity of L536P-HEG0 is manifested to similar degrees in different cell types and likely arises from constitutive coactivator binding; different classes of coactivators recognize distinct but overlapping binding sites on the ER LBD. Finally, the observation that L536P-HEG0 interacted constitutively with AIB-1, a coactivator that has been implicated in ER signaling in breast and ovarian cancer, suggests that similar mutations in the ER may contribute to hormone-independent proliferation of breast and ovarian cells.

Interactions of human skin fibroblasts with monomeric or fibrillar collagens induce different organization of the cytoskeleton.

Fibrillar collagens represent the most abundant extracellular matrix components surrounding fibroblasts. Although there is a large heterogeneity in the collagen composition and in the physiological functions of different tissues, interactions between cells and native collagens monomers are mediated by only two integrins, the alpha1beta1 and alpha2beta1 integrins. In tissue, fibroblasts are exposed to collagen polymers, supramolecular assemblies which might play a role on the availability of the cell-binding sites at the surface of the fibrils. We have addressed this issue by investigating the patterns of adhesion structures in normal human skin fibroblasts exposed to collagen monomers or polymers. Our results showed that cell morphology, cell adhesion pattern, actin organization, and distribution of integrin subunits, talin, vinculin, and phosphotyrosine-containing proteins are dependent on the supramolecular organization of the collagens. In particular, compared to monomers, collagen polymers induced a looser organization of the actin network and a linear clustering of integrins, talin, vinculin, and phosphotyrosine-containing proteins. These results emphasize the role of the physical state of collagen on cellular interactions and underline the role of the extracellular matrix in the phenotypic modulation of fibroblasts. Furthermore, our studies suggest the existence of a local heterogeneity in the biological activity of collagen fibrils.

Adhesion complexes formed by OVCAR-4 cells on laminin 1 differ from those observed on fibronectin.

Cell adhesion to laminin 1 or to fibronectin is mediated by distinct sets of integrins and is differentially regulated by protein kinase C (PKC). It suggests that upon integrin ligation to laminin 1 or to fibronectin different intracellular signaling pathways could be activated. we have therefore investigated the formation of signaling complexes induced during cell adhesion to laminin 1 or to fibronectin. Following cell adhesion to laminin 1 the re-arrangement of the cytoskeleton was slower than that observed on fibronectin and it was activated by treating the cells with H-7, an inhibitor of PKC. Conversely, treatment of laminin-adhering cells with a PKC activator resulted in a rapid disorganization of the actin cyto skeleton while a similar treatment had no effect on fibronectin-adhering cells. These results suggested that the structural organization of the adhesion complexes might be substrate-specific and might correspond to a different arrangement of cytoskeletal and/or cytoplasmic proteins. Reflection interference contrast microscopy (RICM) images revealed that cell-substratum contacts formed on laminin 1 were not well differentiated in contrast to those developed on fibronectin. However, immunofluorescence staining revealed a similar organisation of actin microfilaments, talin and phosphotyrosyl-containing proteins on both substrates. In contrast, differences were observed for vinculin distribution within cells spread on fibronectin or on laminin 1. Following cell adhesion to fibronectin most of the vinculin appeared as thick patches at the tips of the actin stress fibers while in laminin-adhering cells vinculin was recruited into thin streaks localized at the end of only some actin stress fibers.

Detection of panel-reactive anti-HLA class I antibodies by enzyme-linked immunosorbent assay or lymphocytotoxicity. Results of a blinded, controlled multicenter study.

A soluble HLA ELISA for the detection of anti-HLA class I IgG antibodies was developed and compared to complement-dependent microlymphocytotoxicity. ELISA plates were coated with a panel of sHLA class I antigens isolated from the culture supernatants of 46 different EBV-transformed phenotyped B-cell lines. After the incubation of the coated plates with test serum, bound antibodies were detected using a peroxidase-conjugated anti-human IgG antibody. Absorbance was read using an ELISA plate reader and assay results were analyzed by computer. Antibody specificities were determined by Fisher's exact test tail analysis. The reproducibility of ELISA assay results was evaluated in a blinded, controlled multicenter study. A total of 102 serum specimens from patients on waiting lists to receive kidney transplants were tested five times by ELISA in five different laboratories. The correlation coefficients (r) of %PRA values determined by ELISA ranged from 0.89 to 0.96, and the average agreement on qualitative assay results (antibody positive vs antibody negative) was 98%. Endpoint titration of several serum specimens demonstrated equivalent sensitivity of ELISA and microlymphocytotoxicity (using the anti-globulin antibody protocol). Most of the antibody specificities determined by ELISA were in agreement with specificities determined by microlymphocytotoxicity. To evaluate the correlation of ELISA and microlymphocytotoxicity (CDC) assay results the same 102 specimens were tested six times by CDC in five different laboratories. The interlaboratory correlation coefficient (r) of %PRA values determined by microlymphocytotoxicity ranged from 0.57 to 0.94, and the average agreement on qualitative assay results was 85%. A comparison of ELISA with microlymphocytotoxicity was performed using consensus microlymphocytotoxicity results. This showed a high correlation (r = 0.81) of %PRA values determined by ELISA and microlymphocytotoxicity. This demonstrates that the detection of anti-HLA class I antibodies by soluble HLA ELISA is a reliable alternative to microlymphocytotoxicity testing.

Ovine aortic smooth muscle cells allow the replication of visna-maedi virus in vitro.

Visna-maedi virus induces in sheep an interstitial lung disease characterised by an accumulation of smooth muscle cells (SMC) or myomatosis. Infection by HIV-1 has been recently associated with disorders of the vessel-derived cells: primary pulmonary hypertension, coronary artery disease and smooth muscle tumors in humans. We hypothesized that, besides their regular targets (i.e. macrophages and lymphocytes), lentiviruses could infect smooth muscle cells. Smooth muscle cell cultures derived from ovine aorta were infected with visna-maedi virus strain K1514. The cultured cells were smooth muscle cells as demonstrated by their antigenic expression of alpha-actin and vimentin. The lentiviral infection of the smooth muscle cells was demonstrated by a typical cytopathic effect (syncytia), the expression of virus specific antigens, and the presence of genomic RNA detected by Northern blot analysis and RT PCR. The detection of a reverse transcriptase activity, the presence of viral RNA in supernatants of infected smooth muscle cells detected by RT PCR and their ability to infect ovine permissive fibroblasts demonstrated a productive infection. The ability of smooth muscle cells to be infected by lentiviruses may participate in the pathogenesis of the tissue damage associated with the lentiviruses such as myomatosis in sheep and vascular disease in humans.

Typing of a panel of soluble HLA class I antigens by enzyme-linked immunosorbent assay.

Two ELISA assays were developed to test the reactivity of soluble sHLAs with anti-HLA class I mAbs of IgG or IgM isotype. A panel of 40 different alleles of sHLA antigens was produced using 57 lymphoblastoid B-cell lines, which had been generated from class-I-phenotyped PBLs. Using 14 mAbs, the expression of 13 different sHLA antigens (sHLA-A2, -A3, -A11, -A24, -A29, -B7, -B8, -B13, -B14, -B27, -B44, -B57, and -B58) by 43 different cell lines was confirmed. In addition, the expected absence of these alleles from the culture supernatant of 11 cell lines was confirmed. Cross-reactivities of mAbs observed by microlymphocytotoxicity assays were also detected by ELISA. The results of this extensive analysis confirmed previous results demonstrating that sHLA class I typing by ELISA correlated with HLA class I cell typing by microlymphocytotoxicity [1-3]. Furthermore, additional information about the fine specificities of two mAbs was obtained. An anti-B27/44 IgM mAb appeared to react only with sHLA-B44 but not with sHLA-B27; mAb CR11-351, previously reported to react with HLA-A2, 28, bound also to sHLA-A1, -3, -11, and -A24.

Components of postprandial thermogenesis in relation to meal frequency in humans.

Experiments on dogs have shown that the size of the meal has no effect on the early cephalic postprandial thermogenesis, and that four small meals are more thermogenic than a larger meal with the same total caloric content as the four meals. A study was repeated on human subjects who were fed during alternating weeks either one large meal (653 kcal (1 kcal = 4.1855 kJ)) or four small meals (163 kcal) at 40-min intervals. Oxygen consumption and respiratory exchange ratio determinations indicated (i) larger overall increase in postprandial thermogenesis with the four meals than with one meal and (ii) an enhancement of glucose utilization with the large meal compared with greater lipid utilization with the four meals. On the basis of indirect evidence from previous investigations it is suggested that the enhanced thermogenesis observed in the four-meal experiment is due to lipid mobilization caused by repeated stimulation of the sympathetic nervous system with palatable food. Blood analysis indicated a reduced elevation of plasma glucose in the four-meal experiment. The variations of insulin and C-peptide exactly paralleled those observed for glucose. It is concluded that the increased frequency of feeding significantly reduces insulin secretion in subjects fed a relatively high carbohydrate meal. In addition to this beneficial effect, increasing the number of meals increased thermogenesis and fat utilization.

Characteristics and specificity of the inhibition of liver glucose-6-phosphatase by arachidonic acid. Lesser inhibitability of the enzyme of diabetic rats.

The effect of arachidonic acid (delta 4Ach) on liver glucose-6-phosphatase (Glc6Pase) has been studied in vitro using untreated and detergent-treated microsomes prepared from fed and 48-h-fasted normal rats and from streptozotocin-induced diabetic rats. Glc6Pase of both untreated and detergent-treated microsomes (60 micrograms protein/ml) is inhibited by delta 4Ach in a dose-dependent manner between 10-100 microM. The inhibition is very rapid and does not depend on preincubation of microsomes in the presence of delta 4Ach. It does depend on the concentration of microsomal membranes and on the concentration of glucose 6-phosphate: it is more pronounced at low Glc6P concentrations than at high. As a consequence, the enzyme displays sigmoidal kinetics in the presence of delta 4Ach. Hill coefficients (equal to 1 in the control experiments) of about 1.4 were determined in the presence of 50 microM delta 4Ach, indicating a clear positive cooperative dependency of the Glc6Pase upon its substrate in the presence of delta 4Ach. The delta 4Ach inhibition is fully reversible in the presence of bovine serum albumin. The inhibition does not depend on the metabolism of delta 4Ach through the prostaglandin synthase (cyclooxygenase) or arachidonate 12-lipoxygenase pathways since it is not affected by indomethacin and nordihydroguaiaretic acid. Several other unsaturated fatty acids are able to inhibit the enzyme within the same concentration range. In contrast, saturated fatty acids, the arachidonic acid methyl ester and numerous other lipid compounds containing esterified unsaturated fatty acids do not inhibit Glc6Pase within the same concentration range. The enzyme of fed rats was inhibited in the same manner as the enzyme of 48-h-fasted rats. However, Glc6Pase of untreated microsomes from diabetic rats was less inhibitable by delta 4Ach than the Glc6Pase of normal rats. This difference does not persist after solubilization of the membrane lipids by detergent treatment.

Cold wind stimulation reflex.

The bradycardia induced by cold wind blown on the face and the early cephalic release of insulin induced by feeding have been shown to be caused by a vagal reflex stimulation. An experiment was designed to determine whether cold wind blown on the face would induce both pancreatic and cardiac stimulation. A 4 degrees C wind blown on the face for 4 min produced a rapid and persistent bradycardia, which interestingly persisted for up to 35 min after the test. The effect on respiration rate is more gradual and vanishes immediately after cold wind stimulation. Cold wind produced a slight reduction of insulin secretion, as evidenced by the fall of both plasma insulin and C-peptide, and caused a significant increase in plasma norepinephrine. These results suggest that the cold wind action of the vagus nerve is exerted on the heart and that of the sympathetic on the pancreas, whereas during the cephalic phase of feeding a vagal influence is observed on the pancreas and a sympathetic action on the heart. The mechanisms of the quantitative and qualitative control of these autonomic responses are not known and deserve further investigation.