Micro- and Nanopatterned Silk Substrates for Antifouling Applications.

A major problem of current biomedical implants is the bacterial colonization and subsequent biofilm formation, which seriously affects their functioning and can lead to serious post-surgical complications. Intensive efforts have been directed toward the development of novel technologies that can prevent bacterial colonization while requiring minimal antibiotics doses. To this end, biocompatible materials with intrinsic antifouling capabilities are in high demand. Silk fibroin, widely employed in biotechnology, represents an interesting candidate. Here, we employ a soft-lithography approach to realize micro- and nanostructured silk fibroin substrates, with different geometries. We show that patterned silk film substrates support mammal cells (HEK-293) adhesion and proliferation, and at the same time, they intrinsically display remarkable antifouling properties. We employ Escherichia coli as representative Gram-negative bacteria, and we observe an up to 66% decrease in the number of bacteria that adhere to patterned silk surfaces as compared to control, flat silk samples. The mechanism leading to the inhibition of biofilm formation critically depends on the microstructure geometry, involving both a steric and a hydrophobic effect. We also couple silk fibroin patterned films to a biocompatible, optically responsive organic semiconductor, and we verify that the antifouling properties are very well preserved. The technology described here is of interest for the next generation of biomedical implants, involving the use of materials with enhanced antibacterial capability, easy processability, high biocompatibility, and prompt availability for coupling with photoimaging and photodetection techniques.

Microglia induce myelin basic protein-specific T cell anergy or T cell activation, according to their state of activation.

Microglial cells are non-professional antigen-presenting cells (APC) the function of which is still controversial. Here, we studied the function of microglia derived from H-2(u) mice. We show that these microglia express a low level of B7.2 and CD40 and, interestingly, lack surface expression of B7.1. Resting and IFN-gamma-activated microglia were unable to activate naive and primed myelin basic protein (MBP)-specific CD4(+) T cells in the presence of MBP and encephalomyelitic MBP Ac1-11 peptide. Furthermore, in the presence of Ac1-11 peptide, CD4(+) TCR-transgenic T cells became anergized. Microglia became professional APC only after a multistep activation process involving both stimulation through cytokines [granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-gamma] and cognate signaling (B7-CD28 and CD40-CD40 ligand interactions). As such they were able to present MBP to both unprimed and primed T cells. Co-culture of microglia with GM-CSF up-regulated co-stimulatory molecules, in particular B7.1. Additional activation with IFN-gamma induced MHC class II and CD40 up-regulation. CD40-CD40 ligand interaction significantly enhanced microglial ability to prime TCR-transgenic T cells and was essential for presentation of MBP to in vivo primed non-transgenic T cells. We propose that microglia may serve different functions under different inflammatory conditions, depending on the cytokine milieu and the type of cognate interaction they are involved in.

Localization of calcitonin gene-related peptide mRNA in developing olfactory axons.

During development of the olfactory pathway, calcitonin gene-related peptide (CGRP) expression is regulated both temporally and spatially. We had previous evidence that between E13 and E19 CGRP mRNA was present at the level of olfactory axons but the resolution of light-microscope in situ hybridization did not permit the axons to be distinguished from the closely apposed ensheathing cells. In this study, the localization of CGRP mRNA was studied at early developmental stages (E13-15) through in situ hybridization at the transmission electron-microscope (TEM) level. CGRP transcripts were observed exclusively in axons and not in ensheathing cells. The distribution of transcripts in the axons suggests that they are associated with intermediate filaments rather than microtubules. In addition, a careful ultrastructural analysis provided evidence that polysomes and membrane-bound ribosomes are present in such axons, suggesting that the peptide could be synthesized locally.

Subcellular localization of the oncoprotein MTG8 (CDR/ETO) in neural cells.

The t(8;21) translocation associated with acute myeloid leukemia (AML) disrupts two genes, the AML1 gene also known as the core binding factor A2 (CBFA2) on chromosome 21, and a gene on chromosome 8, hereafter referred to as MTG8, but also known as CDR and ETO. Extensive information is available on AML1, a member of the CBF family of transcription factors, containing a highly conserved domain, the runt box, of the Drosophila segmentation gene runt. This gene is essential for the hematopoietic development and is found disrupted in several leukemias. In contrast, the function of the MTG8 gene is poorly understood. The predicted protein sequence shows two unusual, putative zinc-fingers, three proline-rich regions, a PEST domain and several phosphorylation sites. In addition, we found a region encompassing aa 443-514 predicted to have a significant propensity to form coiled coil structures. MTG8 displays a high degree of similarity with nervy, a homeotic target gene of Drosophila, expressed in the nervous system. Human and mouse wild-type MTG8 are also highly expressed in brain relative to other tissues. For these reasons, we set out to investigate the expression and subcellular localization of the MTG8 protein in neural cells. Immunohistochemical experiments in a 12.5-day-old mouse embryo clearly showed that the protein was expressed in the neural cells of the developing brain and the spinal cord. In primary cultures of hippocampal neurons of 2-3 day-old mice, MTG8 was found in the nucleus, in the cytoplasm and as fine granules in the neurites. Cytoplasmic localization of the protein was observed in Purkinje cells of both human and mouse cerebellum. The molecular mass of MTG8 in total human and mouse brain was analysed by immunoblotting and determined to be between 70 and 90 kDa. Isoforms with the same molecular mass were demonstrated in synaptosomes isolated from mouse forebrain. The evidence of MTG8 in the nucleus and cytoplasm of neural cells suggests a specific mechanism regulating the subcellular localization of the protein.

Induction of apoptosis in Kaposi's sarcoma spindle cell cultures by the subunits of human chorionic gonadotropin.

OBJECTIVES: Elucidation of the mechanisms of the previously shown growth-inhibitory action of human chorionic gonadotropin (hCG) on Kaposi's sarcoma (KS) cells and the role of the luteinizing hormone/hCG receptor (hCGR). DESIGN AND METHODS: Analysis of KS tissues and cultured spindle-type KS cells for the presence of the hCGR using 125I-hCG binding and reverse transcriptase-polymerase chain reaction; analysis of several hCG preparations (urinary, recombinant, isolated alpha and beta subunits); analysis of apoptosis mechanisms by several assays including using z-Val-Ala-Asp-fluoromethylketone (zVAD-FMK), a known apoptosis-inhibitory drug. RESULTS: First, we found that some urinary preparations of hCG (e.g., CG-10, Steris Profasi) were indeed KS-killing but others (such as Pregnyl, Choragon, Serono Profasi) were not. Secondly, recombinant subunits (alpha as well as beta) of hCG were KS cell-killing but recombinant intact hCG was not. Thirdly, the hCGR message and protein were undetectable in KS. Fourthly, CG10-induced cell death occurred by apoptosis and KS cells could be rescued by preincubation with zVAD-FMK. Finally, we also found that normal peripheral blood lymphocytes (PBL) were killed by CG-10. CONCLUSION: It is proposed that the action of subunits or subunit fragments of hCG, mediated by a putative orphan receptor (as opposed to the hCGR) and executed by interleukin-1-converting enzyme (ICE)-like protease(s), constitutes a novel apoptosis mechanism effective towards KS cells, but PBLs and possibly other cells as well. These results provide a basis for testing in vitro the therapeutic efficacy of hCG preparations which, in turn, should improve current clinical trials with 'hCG' in patients who have KS.

The Ras Guanine nucleotide Exchange Factor CDC25Mm is present at the synaptic junction.

CDC25Mm, a mouse Ras-Guanine nucleotide Exchange Factor, is specifically expressed as a product of 140 kDa (p140) in the postnatal and adult brain. Immunohistochemical analysis indicates that it is present throughout the brain particularly concentrated in discrete punctate structures. Subcellular fractionation of the mouse brain shows that p140 is present in synaptosomes but not in highly purified synaptic vesicles. Moreover, isolated postsynaptic densities (PSDs) are largely enriched in CDC25Mm. This protein can be phosphorylated by calcium/calmodulin kinase II, the most abundant protein in PSDs. Altogether these results suggest that CDC25Mm is present at synaptic junctions and that it may be involved in synaptic signal transduction leading to Ras activation.

Differential regulation of indoleamine 2,3-dioxygenase expression by nitric oxide and inflammatory mediators in IFN-gamma-activated murine macrophages and microglial cells.

Induction of indoleamine 2,3-dioxygenase (IDO) and nitric oxide synthase (NOS) is involved in the immunomodulatory roles of IFN-gamma and evidence suggests that these pathways are functionally cross-regulated. We report here that nitric oxide (NO) negatively modulates the expression of IDO activity in IFN-gamma-primed macrophages, but not in microglial cells from mouse. In MT2 macrophages, the induction of IDO activity by IFN-gamma was further increased by the presence of NOS inhibitors, whereas culturing of IFN-gamma-activated MT2 cells with NO generators produced a marked reduction of IDO activity expression. Conversely, neither NOS inhibitors nor exogenous NO affected the induction of the enzyme activity in N11 microglial cells after IFN-gamma activation. LPS and picolinic acid, two costimulatory agents that up-regulate inducible NOS in activated cells, regulated IDO induction differently in the two cell lines. LPS and picolinic acid caused a significant decrease of IDO activity in IFN-gamma-activated MT2 cells. This effect, however, did not appear to be mediated by the ability of LPS and picolinic acid to stimulate NO production. In N11 cells, LPS further stimulated the enzyme activity and picolinic acid had no effect. Northern blot analysis revealed that, in MT2 macrophages, NOS inhibitors increased the levels of IDO mRNA, while a reduction was observed with picolinic acid. No changes in IDO mRNA levels were detected in N11 cells. Consistent with the functional heterogeneity of phagocytes, the reported results indicate the existence of marked differences in the regulation of IDO expression between murine macrophages and microglial cells.

Characterization of the major histocompatibility complex class II binding site on LAG-3 protein.

The lymphocyte activation gene-3 (LAG-3), selectively transcribed in human activated T and NK cells, encodes a ligand for major histocompatibility complex (MHC) class II molecules. Like CD4, LAG-3 ectodomain is composed of four Ig-like domains (D1-D4). Nothing is known about the LAG-3 regions or residues required to form a stable MHC class II binding site. In contrast to CD4, soluble LAG-3 molecules stably interact with MHC class II molecules expressed on the cell surface. In addition, the first two N-terminal domains of soluble LAG-3 (D1 and D2) molecules, alone, are capable of binding MHC class II. From a LAG-3 model structure, we designed mutants and tested their ability to bind MHC class II molecules in an intercellular adhesion assay. We found residues on the membrane-distal, CDR1-2-containing top face of D1 that are essential for either binding or repulsing MHC class II proteins. Most of these residues are clustered at the base of a large extra-loop structure that is a hallmark of the LAG-3 D1 Ig-like domain. In addition, as for CD4, oligomerization of LAG-3 on the cell surface may be required to form a stable MHC binding site because mutation of three residues in the ABED beta-strands containing side of D1 results in a dominant negative effect (i.e., binding inhibition of coexpressed wild-type LAG-3).

The beta-core fragment of human chorionic gonadotrophin inhibits growth of Kaposi's sarcoma-derived cells and a new immortalized Kaposi's sarcoma cell line.

OBJECTIVE: Kaposi's sarcoma (KS), a condition often associated with HIV infection, is more common in men than in women; pregnancy and sex hormones could be involved. Urinary human chorionic gonadotrophin (hCG) has been reported to inhibit the growth of KS cell lines, with great variability among preparations. Urinary hCG often contains free forms of the hCG subunits and a fragment of the free beta-subunit, the beta-core, which may have biological activity. We compared the effect of the beta-core fragment, the beta-subunit, recombinant and urinary hCG on KS immortal and spindle cells. DESIGN AND METHODS: A new immortal KS cell line was phenotypically and karyotypically characterized. The effects on growth of this cell line and of primary KS spindle cells by hCG and its purified derivatives were tested. Induction of apoptosis was demonstrated using acridine orange/ethidium bromide staining. RESULTS: The beta-core fragment harboured the most potent growth inhibitory activity on a molar basis. After 72 h of treatment with the beta-core, 60-70% of KS cells show apoptotic nuclei. No effects were observed on endothelial cells. CONCLUSIONS: The beta-core fragment of hCG proved to be the most effective part of the hCG molecule, inducing growth inhibition and apoptosis of KS cells. Thus, the beta-core could be the most appropriate hCG derivative for the therapy of KS.

A highly precise reporter gene bioassay for type I interferon.

We describe the setting up and validation of a reporter gene assay for type I IFN based on monkey Vero cells transfected with pMx-Luc, a plasmid carrying the luciferase gene under the control of the type I IFN inducible mouse Mx1 promoter. Vero cells were stably transfected with pMx-Luc and clone 3-143/5 was selected on the basis of luciferase inducibility by IFN-beta. A linear dose-response relationship was found between 1 and 16 IU/ml IFN-beta. The assay was shown to be specific for IFN-alpha and -beta as no effect by a number of other cytokines including IFN-gamma could be detected. In order to render the IFN-beta reporter gene assay protocol more suitable for routine assays, a 3 x 3 balanced parallel line assay design was applied using a 96-well luminometer for luminescence measurement. The assay was shown to be precise with a coefficient of variation of less than 9%. This assay is characterized by high precision coupled to high efficiency, as reflected by a very short assay duration (1 day), when compared to the classical cytopathic effect assays for IFNs and the previously published IFN reporter gene assay based on growth hormone measurement (Lleonart, R., Näf, D., Browning, H. and Weissmann, C. (1990) A novel, quantitative bioassay for type 1 interferon using a recombinant indicator cell line. Biotechnology 8, 1263-1267).

Cloning of murine LAG-3 by magnetic bead bound homologous probes and PCR (gene-capture PCR).

In recent years PCR-based gene cloning strategies have found wide application in molecular biology, due to the power, speed, and relative simplicity of the PCR methodology. We have set up a novel PCR cloning strategy to isolate homologous genes, which is based on the capture of the cDNA sequence(s) of interest with a biotinylated probe and streptavidin-coupled magnetic beads followed by PCR amplification of the selected molecules. This method does not require sequence information on 5' and 3' regions of the cDNA of interest and permits gene isolation to be sensitive, fast, simple, and specific even when the conventional screening procedures give rise to high backgrounds. By using this technique, which we propose to call gene-capture PCR (GC-PCR) cloning, we were able to isolate the full-length murine lymphocyte activation gene 3 (LAG-3) cDNA from total RNA of activated thymocytes. The GC-PCR technique represents a powerful tool for easy isolation not only of homologous genes from related species, but also of genes sharing conserved regions of suitable length, gene variants, and gene encoding proteins where only limited knowledge of the amino acid sequence exists.

Oncogene expression is modulated by recombinant human interferon-beta in human breast-cancer cells.

The effect of recombinant human interferon-beta on growth and oncoprotein expression was investigated in several human breast-cancer cell lines with different characteristics. All cell lines tested were sensitive to the antiproliferative action of the drug, regardless of their estrogen sensitivity. The maximal inhibition of cell proliferation was seen after 6 days of treatment. In estrogen-sensitive CG-5 and ZR-75-1 cells, but not in MDA-MB-453 estrogen-insensitive cells, a reduction in c-myc and c-erbB2 oncoproteins occurred after 48-72 hr and became more pronounced after 120-168 hr of treatment, suggesting that this down-regulation is not direct but is mediated by undefined molecular mechanisms. The time-course of the IFN-mediated decrease in oncoproteins seems to indicate that this event is not strictly related to the IFN-regulation of cell proliferation. The expression of c-erbB2 and c-myc was also analyzed, after recombinant human interferon-beta treatment, at the mRNA level in CG-5 cells. Surprisingly, no statistically significant variation of c-erbB2 or of c-myc mRNA was found either before or after 120-168 hr. Thus, we surmise that the observed reduction of oncoproteins may be due to post-transcriptional mechanisms.

Synaptic activation of NF-kappa B by glutamate in cerebellar granule neurons in vitro.

Neuronal proliferation, migration, and differentiation are regulated by the sequential expression of particular genes at specific stages of development. Such processes rely on differential gene expression modulated through second-messenger systems. Early postnatal mouse cerebellar granule cells migrate into the internal granular layer and acquire differentiated properties. The neurotransmitter glutamate has been shown to play an important role in this developmental process. We show here by immunohistochemistry that the RelA subunit of the transcription factor NF-kappa B is present in several areas of the mouse brain. Moreover, immunofluorescence microscopy and electrophoretic mobility-shift assay demonstrate that in cerebellar granule cell cultures derived from 3- to 7-day-old mice, glutamate specifically activates the transcription factor NF-kappa B, as shown by binding of nuclear extract proteins to a synthetic oligonucleotide reproducing the kappa B site of human immunodeficiency virus. The use of different antagonists of the glutamate recpetors indicates that the effect of glutamate occurs mainly via N-methyl-D-aspartate (NMDA)-receptor activation, possibly as a result of an increase in intracellular Ca2+. The synaptic specificity of the effect is strongly suggested by the observation that glutamate failed to activate NF-kappa B in astrocytes, while cytokines, such as interleukin 1 alpha and tumor necrosis factor alpha, did so. The effect of glutamate appears to be developmentally regulated. Indeed, NF-kappa B is found in an inducible form in the cytoplasm of neurons of 3- to 7-day-old mice but is constitutively activated in the nuclei of neurons derived from older pups (8-10 days postnatal). Overall, these observations suggest the existence of a new pathway of trans-synaptic regulation of gene expression.

Determination of haptoglobin expression in IL-6 treated HepG2 cells by ELISA and by RNA hybridization--evaluation of a quantitative method to measure IL-6.

Interleukin-6 (IL-6) is known to be an important modulator of acute phase (AP) protein expression in hepatocytes both in vivo and in vitro. In the present study the inducing activity of IL-6 on the expression of the AP protein haptoglobin (HP) by the human hepatoma cell line HepG2, has been evaluated. HP mRNA inducibility was analysed by Northern and slot-blot hybridization, while HP protein was detected by means of an ELISA procedure. A dose-response relationship from 0.3 to 4.8 ng/ml of a human recombinant IL-6 preparation derived from a Chinese hamster ovary (CHO) cell line was observed after 48 h of treatment. Comparable results were obtained by analysing both HP mRNA expression and HP protein secretion. Detectable induction of HP protein secretion was observed with as little as 25 pg/ml of IL-6. The effect of IL-6 was potentiated by dexamethasone, while an inhibition on HP mRNA inducibility could be prevented by lowering the foetal calf serum (FCS) concentration to 1%. Preliminary data indicate that neither IL-1 beta nor TNF-alpha were able to induce significantly HP mRNA expression and protein secretion. The activity ratio between two IL-6 preparations (from CHO and E. coli cells) obtained with a conventional IL-6 bioassay (i.e., T1165 cell growth assay) was comparable to that obtained in the induction of HP expression. The nominal specific activity of the CHO-derived IL-6 was two to three times higher with both responses.

Cloned microglial cells but not macrophages synthesize beta-endorphin in response to CRH activation.

The properties of microglial cell clones, obtained from embryonic mouse brain primary cultures immortalized with recombinant retroviruses, have been investigated and compared with the properties of macrophage clones similarly obtained. Macrophage clones differed from microglial clones in some functions but shared most of the immunological properties. Interestingly, microglial cells were able to produce beta-endorphin, and this production was regulated differently in microglial cell clones when compared with macrophages clones. Although lipopolysaccharide (LPS) treatment induces an increase in beta-endorphin concentration in both cell types, only microglial clones and primary microglial cell cultures respond to the neuroendocrine stimulus corticotropin releasing hormone (CRH). In addition, in these cells, beta-endorphin release is regulated by a classical neurotransmitter, such as noradrenaline, adding some evidence of communication between neurons and microglial cells.

Developmentally regulated expression of CGRP in the mouse olfactory pathway.

The pattern of expression of the neuropeptide CGRP and its encoding mRNA has been determined by immunohistochemistry and in situ hybridization in the mouse olfactory pathway during development. Specific CGRP transcripts are first detected at E13 followed by the appearance of the peptide at E15. Both peptide and transcript are present until birth; their expression then appears to be down-regulated since postnatally the peptide is only observed in some olfactory receptor neurons. A monoclonal antibody that specifically recognizes the neurofilament subunit NF-M has been used in order to identify olfactory and trigeminal axons. Our results demonstrate that CGRP is expressed in olfactory neurons and their axons during development, thus supporting further its role as a differentiation factor during olfactory bulb ontogenesis.

Inducible nitric oxide synthase activity of cloned murine microglial cells.

Nitric oxide (NO) is a short-lived diffusable molecule now believed to participate in multiple physiologic functions in the CNS including neurotransmission and the maintenance of vascular tone. Previously, we reported that cell lines obtained by retroviral immortalization of tissue macrophages (M phi) could be induced to synthesize nitrite (NO2-), a stable end product of the NO synthetic pathway. We have further characterized the induction and activity of this pathway in a panel of seven microglial clones derived from primary embryonic mouse brain cultures. Like M phi, these clones were found to release high levels of NO2- in response to recombinant interferon-gamma (rIFN-gamma) as a priming signal together with either bacterial lipopolysaccharide (LPS) or exogenous recombinant tumor necrosis factor-alpha (rTNF-alpha). As previously demonstrated for M phi, phagocytosis of zymosan particles during induction of enzyme activity enhanced subsequent NO2- production, which is of interest in light of the postulated phagocytic role of microglia within the CNS. Biochemical characterization of enzyme activity in intact microglial clones and in isolated cytosolic fractions indicates that the microglial NO synthase present in these murine cell clones represents the M phi-like isotype. These findings suggest that microglial cells could represent a major source of NO within the CNS.

Social support, adversities and emotional distress in an Italian community sample.

The association of social support with emotional distress in relation to adversities such as social problems, physical health and undesirable life events was assessed in an Italian community sample of 222 men and 224 women. Univariate comparisons and logistic regression analyses showed that neither the quality of a confiding core relationship nor social support from kin confidants was related to adversities. In women only, social support from friend confidants exerted a statistically significant independent main effect together with social problems and undesirable life events in producing a greater probability of emotional distress. The implications of these findings are discussed considering the socio-cultural characteristics of Italian families and individual coping strategies.

Effect of natural beta-interferon on estrogen receptor mRNA of breast cancer cells.

We have demonstrated that natural beta-interferon (beta-IFN) enhances estrogen receptor (ER) mRNA of a human breast cancer cell line, CG-5. Cells were sensitive to the effect of beta-IFN at concentrations ranging from 10 to 100 IU/ml. The increase of ER mRNA was seen after 48 hr of treatment in at least three separate experiments. Our results are in agreement with the previously observed enhancement of receptor protein. In addition, they suggest that the IFN-induced promotion of the antiproliferative activity of drugs which act via ER may be due, in part, to increased receptor synthesis.