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Biomedical subjects

F Granucci

Publications and source records attributed to F Granucci.

32 records · Page 2Linked to original sources
PubMed1998

Rabbit monoclonal Fab derived from a phage display library.

Rabbit monoclonal antibodies (RmAb) are not routinely obtained by eukaryotic cell fusion techniques. Therefore, we have applied phage display technology to produce a recombinant rabbit Fab molecule directed against the KLH model antigen. The Fab fragments selected from the rabbit phage display library were subcloned in an expression vector to permit the production of a fusion protein comprising a dimer of bacterial alkaline phosphatase (phoA). This fusion protein was directly produced into the periplasmic space of Escherichia coli. We show that a crude extract containing these conjugates can be used in a direct enzyme immunoassay, as exemplified in the case of the KLH antigen.

Alkaline Phosphatase↗
PubMed1998

Molecular mimicry by herpes simplex virus-type 1: autoimmune disease after viral infection.

Viral infection is sometimes associated with the initiation or exacerbation of autoimmune disease, although the underlying mechanisms remain unclear. One proposed mechanism is that viral determinants that mimic host antigens trigger self-reactive T cell clones to destroy host tissue. An epitope expressed by a coat protein of herpes simplex virus-type 1 (HSV-1) KOS strain has now been shown to be recognized by autoreactive T cells that target corneal antigens in a murine model of autoimmune herpes stromal keratitis. Mutant HSV-1 viruses that lacked this epitope did not induce autoimmune disease. Thus, expression of molecular mimics can influence the development of autoimmune disease after viral infection.

Adoptive Transfer↗
PubMed1997

Self gamma2a(b) protein is presented in vivo by gamma2a(b) B cells but not by dendritic cells.

We have previously shown that IgG2a(b) Ig does not induce tolerance in MHC class II restricted CD4 T cells in a TCR transgenic model and that anti-IgG2a(b) transgenic T cells specific for peptide 435-451 are indeed present in the periphery where they interact with gamma2a(b)-positive B cells. We also observed that because T cell tolerance depends on the presentation of self peptides, it was probable that IgG2a(b) was not easily processed and presented in vivo. In this study, we have investigated the presentation of naturally processed gamma2a(b) (435-451) determinants to specific T cells. Dendritic cells, macrophages, and B cells were purified from the spleens of Igh-1b mice. These cells were then functionally tested for the presence of specific peptide-MHC complexes. The results showed that, in vivo, gamma2a(b)-producing B cells, but not dendritic cells, are the only APCs able to present this self peptide. This indicates that recognition of the IgG2a(b)-self peptide is exclusively mediated by T-B cell interaction.

Animals↗
PubMed1997

Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures.

The signals controlling the checkpoints of dendritic cells (DC) maturation and the correlation between phenotypical and functional maturational stages were investigated in a defined model system of growth factor-dependent immature mouse DC. Three sequential stages of DC maturation (immature, mature, and apoptotic) were defined and characterized. Immature DC (stage 1) had low expression of costimulatory molecules, highly organized cytoskeleton, focal adhesion plaques, and slow motility; accordingly, they were very efficient in antigen uptake and processing of soluble proteins. Further, at this stage most of major histocompatibility complex class II molecules were within cytoplasmic compartments consistent with a poor allostimulatory capacity. Bacteria or cytokines were very efficient in inducing progression from stage 1 towards stage 2 (mature). Morphological changes were observed by confocal analysis including depolymerization of F-actin and loss of vinculin containing adhesive structures which correlates with acquisition of high motility. Antigen uptake and presentation of native protein antigen was reduced. In contrast, presentation of immunogenic peptides and allostimulatory activity became very efficient and secretion of IL-12 p75 was detectable after antigen presentation. This functional DC maturation ended by apoptotic cell death, and no reversion to the immature phenotype was observed.

Animals↗
PubMed1996

Ig-specific T cell receptor-transgenic T cells are not deleted in the thymus and are functional in vivo.

The mechanisms that induce T cell tolerance to circulating self-proteins are still controversial, and both the deletion and selection of autoreactive T cells have been observed in the thymus of transgenic mouse models. To address the question of the induction of tolerance to circulating self-constituents, a T cell receptor-transgenic mouse specific for the serum protein immunoglobulin (Ig) gamma and (IgG2ab) was generated. The choice of an allotype-specific T cell also allowed the generation of transgenic control mice not expressing the self-antigen. It was found that the transgenic T cells were not deleted in the thymus, did not become tolerant in the periphery, and regulated the function of gamma 2ab-positive B cells as shown by the lack of IgG2ab protein in the serum of the transgenic mice. In spite of this activity in vivo, the transgenic T cells did not proliferate in vitro in response to the allotype-specific peptide. Interestingly, antigen-specific T cell proliferation could be restored if the transgenic mice were previously challenged to induce IgG2ab responses. After this challenge, IgG2ab protein in the serum of the transgenic mice could be partially restored, although still remaining much lower than in control mice. In addition, there was a dramatic increase in serum IgE levels, suggesting that newly generated gamma 2ab-secreting B cells can be induced to switch to IgE in the presence of allotype-specific T cells. These results indicate that Ig-specific T cells may represent a late-acting form of T cell help for the regulation of the IgG2a-to-IgE class switch.

Animals↗
PubMed1995

The beta-endorphin inhibition of mitogen-induced splenocytes proliferation is mediated by central and peripheral paracrine/autocrine effects of the opioid.

In this study we show that the opioid peptide beta-endorphin exerts a tonic inhibitory effect on the proliferative response of splenocytes to the polyclonal mitogen phytohemoagglutinin throughout two separate sites of action: one central and one peripheral. The intracerebroventricular administration of beta-endorphin, in fact, induces a significant inhibition of splenocyte proliferation. In contrast, both the intracerebroventricular and the peripheral administration of anti-beta-endorphin gamma globulins induce a significant increase in proliferation. Moreover, an increase of splenocyte proliferation was observed also after the intravenous administration of gamma globulins and intraperitoneal naloxone, and this effect was still present in hypophysectomized rats. The data reported suggest that beta-endorphin exerts a tonic inhibitory effect on proliferation, acting centrally, and peripherally throughout a paracrine/autocrine mechanism. FACS experiments show that the effect observed is not the consequence of an alteration of lymphocyte trafficking induced by the opioid.

Analysis of Variance↗
PubMed1994

Immortalization of multipotent growth-factor dependent hemopoietic progenitors from mice transgenic for GATA-1 driven SV40 tsA58 gene.

The transcription factor GATA-1 is required for the normal development of erythroid cells. GATA-1 is also expressed in other hemopoietic cells, suggesting that it might be initially activated in a multipotent progenitor. To immortalize GATA-1-expressing progenitors, we generated mice transgenic for a thermosensitive SV40 T gene, driven by the GATA-1 promoter-enhancer. Immortalized marrow cells grow in culture at 32 degrees C but not at 38 degrees C, and are dependent on erythropoietin (Epo) or interleukin 3 (IL-3). Epo dependent cells express hemoglobin, high levels of GATA-1, GATA-2 and NF-E2 p45 mRNAs, and are positive for stem cell antigen 2 (Sca-2) and the early myeloid marker ER-MP12. IL-3 dependent cells can be derived from Epo dependent lines, and are hemoglobin-, Sca-2- and ER-MP12-negative, have low GATA-1 and NF-E2 p45 mRNA levels, and express myeloid markers Mac-1, F4/80 and Gr-1. Brief treatment of Epo dependent cells with myeloid growth factors (plus Epo) leads to the induction of Mac-1, F4/80 and Gr-1, concomitant with the disappearance from most cells of Sca-2, ER-MP12 and GATA-1 driven T antigen nuclear expression. Thus, the immortalized Epo dependent cells have the property of a progenitor capable of differentiation towards either the erythroid or myeloid lineages. These cells initiate transcription of a proportion of GATA-1 RNA molecules at an upstream promoter, previously known to be expressed only in testis cells.

Animals↗
PubMed1994

Retroviral immortalization of phagocytic and dendritic cell clones as a tool to investigate functional heterogeneity.

We have developed a method to generate immortalized phagocytic and dendritic cell clones from various mouse tissues such as spleen, thymus, brain and bone marrow. The clones were phenotypically characterized and shown to retain the ability to respond to immune or inflammatory signals, e.g., IFN-gamma. Functional cytokine activity and nitric oxide production were maintained in activated macrophages, microglial and dendritic cell clones. Immune functions, such as antigen presentation was exhibited by all clones whereas tissue-specific properties such as the ability to respond to corticotropin-releasing hormone and produce beta-endorphin was shown in microglial cell clones but not in macrophage cell clones, indicating that heterogeneity of cells of the mononuclear-phagocytic lineage can be maintained in vitro after the immortalization procedure. Moreover, the continuous proliferation of the clones could be inhibited by various stimuli and further differentiation of the cells could be achieved in vitro.

Animals↗
PubMed1994

Modulation of cytokine expression in mouse dendritic cell clones.

Dendritic cells (DC) play an essential role in the induction of primary immune responses; however, very little information is available on cytokine production by DC. Here we determined the cytokine gene expression profile of two immortalized DC clones, CB1 and D2SC/1, both generated from mouse spleen but differing in their activation requirements. Among the cytokines tested, only transforming growth factor-beta 1 was transcribed constitutively, but its production was detected only in D2SC/1 cells after treatment with granulocyte/macrophage colony-stimulating factor (GM-CSF). GM-CSF also promoted transcription and synthesis of interleukin (IL)-1 beta in CB1 cells that need pretreatment with GM-CSF to present major histocompatibility complex class II-restricted antigens efficiently in vitro. Lipopolysaccharide (LPS) up-regulated gene expression and induced release of tumor necrosis factor-alpha in both DC clones. In addition, LPS induced transcription of IL-1 alpha and both gene expression and synthesis of IL-1 beta in D2SC/1 cells. Interferon-gamma was ineffective in inducing cytokine gene expression, although it augmented the antigen-presentation capacity of DC, IL-4, IL-10 and IL-12 mRNA were not induced by any of the tested stimuli. The results suggest that DC have a limited cytokine gene expression pattern compared to macrophages and are heterogenous in some functional properties.

Animals↗
PubMed1994

Molecular cloning of a recombinant retrovirus carrying a mutated envAKR-mycMH2 fusion gene immortalizing cells of the monocytic-macrophage lineage.

The VN-11 recombinant retroviruses, originally generated by co-transfection of the avian MH2 and AKRv viral genomes, were molecularly cloned from an infected mouse cell line named N11. The analysis of the proviral genome sequence from one of these recombinants showed a possible envAKR-mycMH2 fusion. Point mutations were also found in this envAKR-mycMH2 gene. The cloned viral genome was co-transfected with the neo gene into the psi 2 packaging cell line. Selected clones were shown to transcribe the viral genome and supernatants from these cultures, containing C-type particles, were used to infect primary cultures from mouse lymphoid tissues and brain. Proliferating macrophages and microglial cell clones were obtained, indicating that various types of cells of the mouse monocytic-macrophage lineage can be immortalized in spite of the absence of selection or special growth conditions.

Animals↗
PubMed1993

Immortalized dendritic cell line fully competent in antigen presentation initiates primary T cell responses in vivo.

Dendritic cells (DC) can provide all the known costimulatory signals required for activation of unprimed T cells and are the most efficient and perhaps the critical antigen presenting cells in the induction of primary T cell-mediated immune responses. It is now shown that mouse cell lines with many of the features of DC can be generated using the MIB phi 2-N11 retroviral vector transducing a novel envAKR-mycMH2 fusion gene. The immortalized dendritic cell line (CB1) displays most of the morphologic, immunophenotypic, and functional attributes of DC, including constitutive expression of major histocompatibility complex (MHC) class II molecules, costimulatory molecules B7/BB1, heat stable antigen, intracellular adhesion molecule 1, and efficient antigen-presenting ability. Granulocyte/macrophage colony-stimulating factor (GM-CSF) proved to be effective in increasing MHC class II molecule expression and in enhancing presentation of native protein antigens. In comparison with macrophages, CB1 dendritic cells did not exhibit phagocytic and chemotactic activity in response to various stimuli and lipopolysaccharide activation was ineffective in inducing tumor necrosis factor alpha or interleukin 1 beta production. CB1 cells, pulsed with haptens in vitro and injected into naive mice were able to induce delayed-type hypersensitivity responses, further increased with pretreatment with GM-CSF, indicating that these cells may represent an immature, rather than a mature DC. The ability of CB1 to prime T cells in vivo could provide a tool to design novel immunization strategies.

Animals↗
PubMed1993

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.

Animals↗