Multimodal imaging of pancreatic beta cells in vivo by targeting transmembrane protein 27 (TMEM27).

AIMS/HYPOTHESIS: Non-invasive diagnostic tools specific for pancreatic beta cells will have a profound impact on our understanding of the pathophysiology of metabolic diseases such as diabetes. The objective of this study was to use molecular imaging probes specifically targeting beta cells on human samples and animal models using state-of-the-art imaging modalities (fluorescence and PET) with preclinical and clinical perspective. METHODS: We generated a monoclonal antibody, 8/9-mAb, targeting transmembrane protein 27 (TMEM27; a surface N-glycoprotein that is highly expressed on beta cells), compared its expression in human and mouse pancreas, and demonstrated beta cell-specific binding in both. In vivo imaging was performed in mice with subcutaneous insulinomas overexpressing the human TMEM27 gene, or transgenic mice with beta cell-specific hTMEM27 expression under the control of rat insulin promoter (RIP-hTMEM27-tg), using fluorescence and radioactively labelled antibody, followed by tissue ex vivo analysis and fluorescence microscopy. RESULTS: Fluorescently labelled 8/9-mAb showed beta cell-specific staining on human and mouse pancreatic sections. Real-time PCR on islet cDNA indicated about tenfold higher expression of hTMEM27 in RIP-hTMEM27-tg mice than in humans. In vivo fluorescence and PET imaging in nude mice with insulinoma xenografts expressing hTMEM27 showed high 8/9-mAb uptake in tumours after 72 h. Antibody homing was also observed in beta cells of RIP-hTMEM27-tg mice by in vivo fluorescence imaging. Ex vivo analysis of intact pancreas and fluorescence microscopy in beta cells confirmed these findings. CONCLUSIONS/INTERPRETATION: hTMEM27 constitutes an attractive target for in vivo visualisation of pancreatic beta cells. Studies in mouse insulinoma models and mice expressing hTMEM27 demonstrate the feasibility of beta cell-targeted in vivo imaging, which is attractive for preclinical investigations and holds potential in clinical diagnostics.

Molecular and neuronal substrate for the selective attenuation of anxiety.

Benzodiazepine tranquilizers are used in the treatment of anxiety disorders. To identify the molecular and neuronal target mediating the anxiolytic action of benzodiazepines, we generated and analyzed two mouse lines in which the alpha2 or alpha3 GABAA (gamma-aminobutyric acid type A) receptors, respectively, were rendered insensitive to diazepam by a knock-in point mutation. The anxiolytic action of diazepam was absent in mice with the alpha2(H101R) point mutation but present in mice with the alpha3(H126R) point mutation. These findings indicate that the anxiolytic effect of benzodiazepine drugs is mediated by alpha2 GABAA receptors, which are largely expressed in the limbic system, but not by alpha3 GABAA receptors, which predominate in the reticular activating system.

Hippocampal alpha 5 subunit-containing GABA A receptors are involved in the development of the latent inhibition effect.

Hippocampal GABA(A) receptors containing the alpha 5 subunit have been implicated in the modulation of hippocampal-dependent learning, presumably via their tonic inhibitory influence on hippocampal glutamatergic activity. Here, we examined the expression of latent inhibition (LI)--a form of selective learning that is sensitive to a number of manipulations targeted at the hippocampal formation, in alpha 5(H105R) mutant mice with reduced levels of hippocampal alpha 5-containing GABA(A) receptors. A single pre-exposure to the taste conditioned stimulus (CS) prior to the pairing of the same CS with LiCl-induced nausea was effective in reducing the conditioned aversion against the taste CS in wild-type mice--thus constituting the LI effect. LI was however distinctly absent in male alpha 5(H105R) mutant mice. Hence, a partial loss of hippocampal alpha 5 GABA(A) receptors is sufficient to alter one major form of selective learning, albeit this was not seen in the female. This observed phenotype suggests that specific activation of these extrasynaptic GABA(A) receptors may confer therapeutic potential against the failure to show selectivity in learning by human psychotic patients.

L-arginine-dependent reactive nitrogen intermediates as mediators of tumor cell killing by activated macrophages.

The capacities of lymphokines and of various microbes to induce in a pure population of bone marrow-derived mononuclear phagocytes tumoricidal activity and/or the production of L-arginine-dependent reactive nitrogen intermediates, measured by the release of nitrite, were comparatively assessed. These parameters were found to be closely correlated in a variety of experimental situations, i.e., enhanced by a surplus of L-arginine and abrogated by N-monomethyl-L-arginine, a selective inhibitor of L-arginine-dependent effector mechanisms. In other macrophage/tumor cell combinations, such correlation was less obvious or not at all detectable, suggesting that, in these models, L-arginine-dependent reactive nitrogen intermediates are not or not alone responsible for the mediation of tumoricidal activity by activated macrophages. Collectively, the present findings suggest that the mechanism of tumor cell killing by activated macrophages may differ, depending on the tumor cell type and the pathway of macrophage activation. Among the various effector mechanisms considered to be involved in tumor cell killing by activated macrophages, L-arginine-dependent reactive nitrogen intermediates appear to hold a major role.

Pathways of tumour cell killing by activated macrophages: both tumour cell membrane and nucleus can be the primary target.

Plasma membrane and nucleus can be primary targets of tumour cell killing by activated macrophages (AMø). Necrotic-type cytotoxicity with loss of membrane integrity and cytoplasmic swelling was expressed by AMø from normal and from perforin-deficient mice, indicating that perforin was not involved. Incubation with AMø consistently triggered the release of thymidine from prelabelled targets, whereas chromatin condensation and small DNA fragments were only occasionally detected. It is shown by means of Pulsed-Field Gel Electrophoresis that DNA degradation in target cells is a slowly progressing process that may stop at any time, indicating that nuclear-type killing doesnot necessarily lead to the formation of low molecular weight fragments. Neither Fas nor the p55 tumour necrosis factor receptor appear to be involved in signalling nuclear-type killing. Accordingly, AMø do mediate membrane- and nuclear-type killing but the mechanisms differ from those identified in T cell cytotoxicity.

Pharmacology of recombinant gamma-aminobutyric acidA receptors rendered diazepam-insensitive by point-mutated alpha-subunits.

Amino acids in the alpha- and gamma-subunits contribute to the benzodiazepine binding site of GABA(A)-receptors. We show that the mutation of a conserved histidine residue in the N-terminal extracellular segment (alpha1H101R, alpha2H101R, alpha3H126R, and alpha5H105R) results not only in diazepam-insensitivity of the respective alphaxbeta2,3gamma2-receptors but also in an increased potentiation of the GABA-induced currents by the partial agonist bretazenil. Furthermore, Ro 15-4513, an inverse agonist at wild-type receptors, acts as an agonist at all mutant receptors. This conserved molecular switch can be exploited to identify the pharmacological significance of specific GABA(A)-receptor subtypes in vivo.

A chemiluminescent assay for mycoplasmas in cell cultures.

A chemiluminescent assay for the detection of mycoplasma contamination of cell cultures is described. Cells (and supernatant) derived from mycoplasma-contaminated cultures stimulate a burst of luminol-dependent chemiluminescence in cell suspensions containing phagocytic effector cell types. The assay conditions for spleen cells, human and bovine polymorphonuclear leucocytes as the responder or indicator cells have been optimized. The chemiluminescent assay can be utilized for both monolayer and suspension cell cultures and is more sensitive than colony formation on agar plates and electron microscopy. Results are obtained within 3-5 h including the time required for the preparation of the indicator cells. CL can be measured in the tritium window of standard liquid scintillation spectrometers after switching off the coincidence circuit.

Antitumor activity of bacteria and bacterial products: enhancement of the tumor-protective effect of bacteria by lipoteichoic acid.

The ability of some microbial agents and/or their products to affect local tumor growth was assessed in the D-12 DA rat ascites tumor model. Various bacteria and bacterial products markedly enhanced tumor resistance when injected i.p. several days before tumor cell challenge. The tumor-protective effect of these compounds was amplified further by lipoteichoic acid (LTA) inoculated i.p. a few days after tumor cell challenge. Under these conditions, the majority of animals did not exhibit progressive tumor growth.

Hippocampal alpha5 subunit-containing GABAA receptors modulate the expression of prepulse inhibition.

Prepulse inhibition (PPI) refers to the phenomenon in which a low-intensity prepulse stimulus attenuates the reflexive response to a succeeding startle-eliciting pulse stimulus. The hippocampus, among other structures, is believed to play an important role in the modulation of PPI expression. In alpha5(H105R) mutant mice, the expression of the alpha5 subunit-containing GABA(A) receptors in the hippocampus is reduced. Here, we report that PPI was attenuated, and spontaneous locomotor activity was increased in alpha5(H105R) mutant mice. These effects were apparent in both genders. Thus, alpha5 subunit-containing GABA(A) receptors, which are located extrasynaptically and are thought to mediate tonic inhibition, are important regulators of the expression of PPI and locomotor exploration. Post-mortem analyses of schizophrenia brains have consistently revealed structural abnormalities of a developmental origin in the hippocampus. There may be a possibility that such abnormalities include disturbance of alpha5 GABA(A) receptor function or distribution, given that schizophrenia patients are known to exhibit a PPI deficit. Our data further highlight that the potential use of alpha5-selective inverse agonists to treat hippocampal-related mnemonic dysfunction needs to be considered against the possibility that such compounds may be adversely associated with deficient sensorimotor gating.

A schizophrenia-related sensorimotor deficit links alpha 3-containing GABAA receptors to a dopamine hyperfunction.

Overactivity of the dopaminergic system in the brain is considered to be a contributing factor to the development and symptomatology of schizophrenia. Therefore, the GABAergic control of dopamine functions was assessed by disrupting the gene encoding the alpha3 subunit of the GABA(A) receptor. alpha3 knockout (alpha3KO) mice exhibited neither an obvious developmental defect nor apparent morphological brain abnormalities, and there was no evidence for compensatory up-regulation of other major GABA(A)-receptor subunits. Anxiety-related behavior in the elevated-plus-maze test was undisturbed, and the anxiolytic-like effect of diazepam, which is mediated by alpha2-containing GABA(A) receptors, was preserved. As a result of the loss of alpha3 GABA(A) receptors, the GABA-induced whole-cell current recorded from midbrain dopamine neurons was significantly reduced. Spontaneous locomotor activity was slightly elevated in alpha3KO mice. Most notably, prepulse inhibition of the acoustic startle reflex was markedly attenuated in the alpha3KO mice, pointing to a deficit in sensorimotor information processing. This deficit was completely normalized by treatment with the antipsychotic D2-receptor antagonist haloperidol. The amphetamine-induced hyperlocomotion was not altered in alpha3KO mice compared with WT mice. These results suggest that the absence of alpha3-subunit-containing GABA(A) receptors induces a hyperdopaminergic phenotype, including a severe deficit in sensorimotor gating, a common feature among psychiatric conditions, including schizophrenia. Hence, agonists acting at alpha3-containing GABA(A) receptors may constitute an avenue for an effective treatment of sensorimotor-gating deficits in various psychiatric conditions.

Abilities of activated macrophages to manifest tumoricidal activity and to generate reactive nitrogen intermediates: a comparative study in vitro and ex vivo.

The abilities of lymphokines and heat-killed bacteria to induce and to maintain tumoricidal activity and/or the secretion of reactive nitrogen intermediates (RNI) were comparatively assessed in bone marrow-derived mononuclear phagocytes (BMM phi) in vitro and in adherent peritoneal cells (APC) ex vivo. In showing that the kinetics of tumoricidal activity and of secretion of RNI induced by macrophage-activating agents in BMM phi and/or in peritoneal cells do largely parallel each other, the present findings provide evidence for a role of RNI in tumor cell killing by activated macrophages both in vitro and in vivo.

Induction, maintenance, and reinduction of tumoricidal activity in bone-marrow-derived mononuclear phagocytes by macrophage-activating lymphokines.

Rat bone-marrow-derived mononuclear phagocytes, induced to differentiate in vitro from precursors and virtually homogeneous with respect to the cell lineage, were the source of effector cells. These effector cells do not manifest spontaneous cytolytic activity in the resting state, but readily acquire marked long-term tumoricidal activity upon incubation with macrophage-activating lymphokines (MAF). MAF-induced tumoricidal activity of bone marrow-derived effector cells decays rapidly. However, in sharp contrast to tissue macrophages, bone marrow-derived mononuclear phagocytes retain in vitro responsiveness to a primary exposition to MAF over a period of several weeks, postcytolytic mononuclear phagocytes recover reactivity to MAF after a variable time interval.

Induction of interleukin 1 secretion and of tumoricidal activity in macrophages are not closely related phenomena.

Rat bone marrow-derived mononuclear phagocytes (BMM phi), induced to differentiate in vitro and homogeneous with respect to the cell lineage, were interacted with various macrophage-activating agents. The effect of these agents on the secretion of interleukin 1 (IL 1) activity and on the development of tumoricidal capacity was comparatively assessed. The findings show that IL 1 secretion is considerably enhanced by macrophage-activating factor and by recombinant interferon-gamma but remained unaffected or was even suppressed by heat-killed C. parvum or Listeria monocytogenes. In contrast, lymphokines as well as C. parvum and Listeria were similarly potent in inducing in BMM phi a tumoricidal state. The findings indicate that induction of IL 1 secretion and of tumoricidal activity are not necessarily closely linked phenomena.

Tumor-promoting phorbol esters induce macrophage differentiation from bone marrow precursors.

The tumor-promoting phorbol diester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), induces in liquid cultures of rat and mouse bone marrow cells a sequence of events strikingly similar to those initiated by colony-stimulating activity known to regulate growth. Changes include stimulation of DNA synthesis and induction of cell proliferation, enhancement of adherence to the substratum, increase in lysozyme secretion, the expression of plasma membrane receptors for the Fc portion of IgG and the capability of manifesting lymphokine-induced, immunologically nonspecific long-term cytotoxicity. These findings indicate that TPA selectively stimulates precursors of the mononuclear phagocyte lineage to proliferate and to differentiate into macrophages, thus mimicking the effects of macrophage colony-stimulating activity.

Macrophage response to viruses, protozoa, and fungi: secretory and cellular activities induced in resting unprimed bone marrow-derived mononuclear phagocytes.

The secretory (tumor necrosis factor, TNF-alpha; nitrite) and cellular response (mitochondrial respiration, TNF-alpha-independent tumoricidal activity) of a pure, lymphocyte-free population of resting, unprimed rat bone marrow-derived mononuclear phagocytes (BMM phi) to direct interaction with viruses, protozoa, and fungi was assessed and compared with that triggered by bacterial agents and interferon-gamma (IFN-gamma). Viruses (herpes simplex, vaccinia, poliomyelitis, vesicular stomatitis, lymphocytic choriomeningitis, Sendai), protozoa (Trypanosoma brucei, Giardia lamblia), and fungi (Penicillium, Trichosporon, Fusarium, Rhizopus, Aspergillus, Geotrichum species) affected primarily the secretion of TNF-alpha and mitochondrial respiration of BMM phi; their effects on the secretion of nitrite and on tumoricidal activity were at best marginal. Collectively, the macrophage response to viruses, protozoa, and fungi was less varied and less marked than that to bacterial agents (intact organisms, peptidoglycan, lipoteichoic acid, lipopolysaccharide) and IFN-gamma.

Tumor-host interactions in the rabbit V2 carcinoma: stimulation of cathepsin B in host fibroblasts by a tumor-derived cytokine.

Organ cultures of explanted V2 carcinoma specimens as well as cultured V2 carcinoma cells produced a cytokine which stimulated rabbit skin fibroblasts to synthesize increased amounts of cathepsin B. The cytokine was released by the tumor cells as a heterogeneous family of polypeptides: two inactive forms (Mr = 55,000 and 68,000) which could be activated by limited proteolysis with trypsin and three active forms with Mr values of 12,000, 16,000 and 18,000. The treatment of inactive cytokine-containing tumor-conditioned media with trypsin, followed by chromatographic separation of the products, suggested that the high-Mr inactive components may represent precursors of the active forms. Cathepsin B was immunolocalized in the tumor-host interzone in co-cultures of tumor and host tissues. Some other possible activities of the tumor cytokine which emerged from previous studies, such as the induction of host cells to produce increased levels of collagenase and extracellular matrix, as well as the stimulation of host cell proliferation, are discussed in the light of the new findings and are proposed as an important mechanism in tumor invasion.

Firm persistent binding between activated macrophages and tumor cells is not a prerequisite for the mediation of cytolysis.

The present study seeks to reassess the roles of macrophage activation and persistent firm binding to tumor cells as a prerequisite for tumoricidal activity. To this end, macrophage effector populations from various tissues, expressing diverse functional activities, were made to interact in vitro with different suspension tumor cell lines. The resultant binding and cytolytic effector cell capacities were determined. Macrophage activation appears to be an absolute prerequisite for binding and for killing of tumor cells. Activated macrophages firmly bind and form clusters with D-12 rat fibrosarcoma cells, which are relatively resistant to macrophage-mediated tumoricidal action. In contrast, P-815 murine mastocytoma cells, highly susceptible to the lytic activity of macrophages, bind rather poorly with activated macrophages and do not form clusters. Since susceptible target cells are readily killed by activated rat macrophages in the absence of persistent firm contact, it appears that firm binding and killing are not causally related events.

The interaction of macrophages and bacteria: Escherichia coli species, bacterial lipopolysaccharide, and lipid A differ in their ability to induce tumoricidal activity and the secretion of reactive nitrogen intermediates in macrophages.

The ability of nine Escherichia coli strains, and of bacterial lipopolysaccharide (LPS)3 and lipid A preparations, to elicit in a pure population of bone marrow-derived mononuclear phagocytes (BMM phi) tumoricidal activity and/or the generation of reactive nitrogen intermediates (RNI) was compared. Generally, low concentrations of E. coli organisms were able to trigger the generation of RNI: however, for induction of tumoricidal activity, higher concentrations were required. Nonisogenic E. coli species exhibited different ability; isogenic E. coli organisms that differed only in the expression of K antigen exhibited similar ability to elicit the macrophage activities. LPS proved to be highly efficient in triggering the secretion of reactive nitrogen intermediates; lipid A was clearly less potent, but evidence is presented to suggest that this was due to the diminished solubility of these reagents. On the other hand, all LPS and lipid A samples were very poor inducers of tumoricidal activity. Although RNI secretion and expression of tumoricidal activity are both strongly dependent on L-arginine, various evidence suggests that the two functions are not closely correlated and are induced by different bacterial structures.

Macrophage response to microbial pathogens: modulation of the expression of adhesion, CD14, and MHC class II molecules by viruses, bacteria, protozoa and fungi.

The ability of inactivated viruses, bacteria, protozoa and fungi to modulate the expression of CD14, CD49d, CD49f, CD11a (LFA-1), and CD54 (ICAM-1) molecules in unprimed bone marrow-derived mononuclear phagocytes (BMM phi) was investigated by means of flow cytometry. Incubation with bacterial agents resulted in the large majority of experimental situations in enhanced expression of these macrophage surface molecules. In contrast, viruses and fungi down-regulated the expression of several adhesion receptors, especially integrins. Amplification of MHC class II expression triggered in macrophages by interferon gamma was clearly inhibited by viruses, bacteria, protozoa and fungi. The findings explain earlier results showing that, under the same experimental conditions, bacterial agents are, for the most part, potent stimulators of secretory and cell-mediated macrophage activities while viruses, protozoa and fungi are poor in this respect.

Lymphokines and bacteria, that induce tumoricidal activity, trigger a different secretory response in macrophages.

The abilities of various macrophage-activating agents to trigger tumoricidal activity and/or the secretion of prostaglandin E2 (PGE2), interleukin 6 (IL 6) and transforming growth factor beta (TGF beta) in bone marrow-derived mononuclear phagocytes (BMM phi) in vitro were comparatively assessed. Induction of tumoricidal activity by lymphokines, that is only short-lived, was not associated with enhanced secretion of these activities by BMM phi; in contrast, incubation with heat-killed facultative intracellular bacteria resulted in persisting tumoricidal activity and in marked enhancement of the secretion of IL 6 and PGE2, but not of TGF beta activity. These findings support the concept that the pattern of the secretory response induced in macrophages by lymphokines differs from that triggered by bacteria and that the rapid decay of lymphokine-induced tumoricidal activity is not due to autocrine macrophage deactivation mediated by one of these agents alone.