Biomedical subjects

L D Falo

Publications and source records attributed to L D Falo.

31 records · Page 2Linked to original sources

PubMed1996

DNA-based immunization by in vivo transfection of dendritic cells.

Delivery of antigen in a manner that induces effective, antigen-specific immunity is a critical challenge in vaccine design. Optimal antigen presentation is mediated by professional antigen-presenting cells (APCs) capable of taking up, processing and presenting antigen to T cells in the context of costimulatory signals required for T-cell activation. Developing immunization strategies to optimize antigen presentation by dendritic cells, the most potent APCs, is a rational approach to vaccine design. Here we show that cutaneous genetic immunization with naked DNA results in potent, antigen-specific, cytotoxic T lymphocyte-mediated protective tumor immunity. This method of immunization results in the transfection of skin-derived dendritic cells, which localize in the draining lymph nodes. These observations provide a basis for further development of DNA-based vaccines and demonstrate the feasibility of genetically engineering dendritic cells in vivo.

Amino Acid Sequence↗

PubMed1995

Targeting antigen into the phagocytic pathway in vivo induces protective tumour immunity.

Cytotoxic T lymphocytes (CTLs) kill neoplastic or virally infected cells after recognizing on their surface antigenic peptides bound to major histocompatibility complex class I molecules. These peptides are derived from antigens that are degraded in the cytosol of the affected cell. Because exogenous proteins cannot enter the cytosol, immunizations with killed pathogens or their proteins do not generally elicit CTLs. However, antigens that are internalized into phagocytic cells can enter the cytosol and be processed for class I presentation. Here we show that immunization with a purified antigen on an avidly phagocytized particle primes CTLs, which in turn protect animals from subsequent challenge with tumours transfected with the antigen gene. Interestingly, these animals also become immune to other antigens expressed by the tumour. This approach could be exploited to develop tumour and viral vaccines.

Amino Acid Sequence↗

PubMed1995

Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity.

Dendritic cells, the most potent 'professional' antigen-presenting cells, hold promise for improving the immunotherapy of cancer. In three different well-characterized tumour models, naive mice injected with bone marrow-derived dendritic cells prepulsed with tumour-associated peptides previously characterized as being recognized by class I major histocompatibility complex-restricted cytotoxic T lymphocytes, developed a specific T-lymphocyte response and were protected against a subsequent lethal tumour challenge. Moreover, in the C3 sarcoma and the 3LL lung carcinoma murine models, treatment of animals bearing established macroscopic tumours (up to 1 cm2 in size) with tumour peptide-pulsed dendritic cells resulted in sustained tumour regression and tumour-free status in more than 80% of cases. These results support the clinical use of tumour peptide-pulsed dendritic cells as components in developing effective cancer vaccines and therapies.

Animals↗

PubMed1994

Expression and function of the costimulatory molecule B7 on murine Langerhans cells: evidence for an alternative CTLA-4 ligand.

We have previously shown, through transfection experiments, that the murine B7 (mB7) molecule, a ligand for the CD28 and CTLA-4 receptors, is a sufficient costimulatory signal for the antigen-specific and major histocompatibility complex (MHC)-restricted activation of murine CD4+ T lymphocytes. In addition to mB7, another ligand with affinity for CTLA-4 has been described on spleen cells. Here we report our studies on the expression and function of these molecules on murine Langerhans cells (LC). Both anti-mB7 monoclonal antibody (mAb) 16-10A1 and human CTLA4Ig (hCTLA4Ig), a chimeric fusion protein consisting of the extracellular domain of human CLTA-4 and the constant domain of human IgG1, detected antigen(s) on cultured but not freshly isolated LC. Preincubation of cultured LC with anti-mB7 mAb did not significantly affect binding of hCTLA4Ig to these cells. This result demonstrate the existence of at least one other ligand for the CLTA-4 receptor on cultured LC. Functional studies revealed that the costimulatory activity of LC was inhibited better by hCTLA4Ig than by the anti-mB7 mAb. This differential effect was seen in the case of both alloreactive and antigen-specific, syngeneic T cell responses. These findings suggest that the non-mB7-ligand for CTLA-4 is functional and participates in the induction of immune responses by LC. Importantly, even synergistic combinations of anti-mB7 mAb and hCTLA4Ig did not inhibit completely the activity of LC. These findings therefore raise the possibility that LC express other costimulatory ligands besides mB7 and related family members.

Abatacept↗

PubMed1994

Evolution of a naevus spilus.

The evolution of a naevus spilus is documented with serial photographs over a 12-year period. The lesion was remarkable for increasing numbers of macules and papules within the background of the café-au-lait macule. Biopsy of a new papule showed lentiginous melanocytic hyperplasia without atypia. We advise photography and periodic follow-up of large, clinically atypical or changing naevi spili. Indications for considering biopsy of naevi spili, irrespective of size, include atypical clinical features or change in one of the hyperpigmented macules or papules, eventuating in an atypical clinical appearance.

Adult↗

PubMed1992

Serum proteases alter the antigenicity of peptides presented by class I major histocompatibility complex molecules.

Any effect of serum on the antigenicity of peptides is potentially relevant to their use as immunogens in vivo. Here we demonstrate that serum contains distinct proteases that can increase or decrease the antigenicity of peptides. By using a functional assay, we show that a serum component other than beta 2-microglobulin enhances the presentation of ovalbumin peptides produced by cyanogen bromide cleavage. Three features of this serum activity implicate proteolysis: it is temperature dependent, it results in increased antigenicity in a low molecular weight peptide fraction, and it is inhibited by the protease inhibitor leupeptin. Conversely, presentation of the synthetic peptide OVA-(257-264) is inhibited by serum. This inhibition is unaffected by leupeptin but is blocked by bestatin, a protease inhibitor with distinct substrate specificities. Implications for peptide-based vaccine design and immunotherapy are discussed.

Animals↗

PubMed1988

Effects of in vivo monoclonal anti-I-A antibody treatment in neonatal mice on intrathymic and peripheral class II antigen expression.

Intrathymic, Ia-bearing antigen-presenting cells (APC) are believed to play an important role in the development of a mature, functional T-cell repertoire. We used chronic in vivo treatment of neonatal mice with anti-I-A monoclonal Ab (MAb) to examine the expression of I-A and I-E antigens on intrathymic and peripheral APC. Three weeks after continuous treatment with anti-I-A MAb, FACS analysis of unfractionated spleen cells revealed a 75-90% reduction in the number of I-A bearing cells. Splenic antigen-presenting capacity measured by the ability of unseparated or density gradient-enriched APC to stimulate I-A- or I-E-reactive T-cell hybridomas was also greatly reduced. In contrast to the expression of I-A and I-E molecules in the splenic APC, anti-I-A MAb treatment resulted in decreased thymic APC I-A expression without significant changes in I-E as measured by FACS analysis. This was confirmed in functional studies in which allo-I-A- or auto-I-A-reactive T-cell hybridomas could not be stimulated by treated thymic APC. Unlike splenic APC, anti-I-A-treated thymic APC did not differ significantly from normals in their ability to stimulate allo-I-E-reactive T hybridomas. This lack of linkage or comodulation of I-A and I-E expression on thymic but not splenic APC may allow us to study the role of I-A molecules and I-E molecules on the development and expansion of functional, mature T-cell repertoires.

Animals↗

PubMed1987

Cerulenin is a potent inhibitor of antigen processing by antigen-presenting cells.

Cerulenin is an antibiotic that inhibits eukaryotic lipid and sterol synthesis and blocks lipid modification of proteins. The effect of cerulenin on the ability of accessory cells to present antigen to T cells was investigated. This antibiotic strongly inhibits the ability of accessory cells to present antigen to murine T-T hybrids. This effect is observed for multiple distinct antigens including L-glutamic acid60-L-alanine30-L-tyrosine10, bovine insulin, L-glutamic acid56-L-lysine35-L-phenylalanine9, and ovalbumen. Presentation by both macrophage and B lymphoblastoid cell lines is inhibited. The ability to effectively pulse these cells with antigen is inhibited but not the ability of these same cells to present antigen that they have previously processed. Furthermore, this inhibition is selective as it can occur without significant inhibition of the antigen-presenting cell protein or DNA synthesis. Cerulenin does not inhibit antigen uptake or catabolism as assessed with labeled antigen. By these criteria this drug is shown to interfere with an antigen-processing step. The ability of cerulenin to block processing was compared with other known inhibitors. Although cerulenin was effective with all antigens tested, at least one inhibitor was not. Taken together, these results suggest that the effect of cerulenin may define a distinct step in antigen processing and provides evidence that some other processing events are not universally required. The ability of cerulenin to interfere with antigen processing is discussed in the context of the known actions of this antibiotic and events of antigen processing and presentation.

Ammonium Chloride↗

PubMed1987

Characterization of antigen association with accessory cells: specific removal of processed antigens from the cell surface by phospholipases.

To characterize the basis for the cell surface association of processed antigen with the antigen-presenting cell (APC) we analyzed its sensitivity to enzymatic digestion. Antigen-exposed APC that are treated with phospholipase and then immediately fixed lose their ability to stimulate antigen-plus-Ia-specific T-T hybridomas. This effect is seen with highly purified phospholipase A2 and phospholipase C. In addition it is observed with three distinct antigens--ovalbumin, bovine insulin, and poly(LGlu56LLys35LPhe9) [(GluLysPhe)n]. The effect of phospholipases is highly specific. Identically treated APC are equivalent to controls in their ability to stimulate alloreactive hybridomas specific for precisely the same Ia molecule that is corecognized by antigen-plus-Ia-specific hybrids. Furthermore, the antigen-presenting function of enzyme-treated, fixed APC can be reconstituted by the addition of exogenous in vitro processed or "processing independent" antigens. In parallel studies 125I-labeled avidin was shown to specifically bind to APC that were previously exposed and allowed to process biotin-insulin. Biotin-insulin-exposed APC that are pretreated with phospholipase bind significantly less 125I-labeled avidin than do untreated, exposed APC. Identical enzyme treatment does not reduce the binding of avidin to a biotinylated antibody already bound to class II major histocompatibility complex molecules of APC. At least some of the biotin-insulin surface sites are immunologically relevant, because the presentation of processed biotin-insulin by fixed APC is blocked by avidin. This effect is specific. Avidin binding to biotin-insulin-exposed APC does not inhibit allospecific stimulation nor the presentation of unconjugated insulin. These studies demonstrate that phospholipase effectively removes processed cell surface antigen.

Animals↗

PubMed1986

Phospholipase treatment of accessory cells that have been exposed to antigen selectively inhibits antigen-specific Ia-restricted, but not allospecific, stimulation of T lymphocytes.

The corecognition of antigen and class II major histocompatibility complex (MHC) molecules (Ia molecules) by the T-cell receptor is a cell surface event. Before antigen is recognized, it must be taken up, processed, and displayed on the surface of an Ia-bearing accessory cell (antigen-presenting cell, APC). The exact nature of antigen processing and the subsequent associations of antigen with the APC plasma membrane, Ia molecules, and/or the T-cell receptor are not well defined. To further analyze these events, we have characterized the processing and presentation of the soluble polypeptide antigen bovine insulin. We found that this antigen requires APC-dependent processing, as evidenced by the inability of metabolically inactivated APCs to present native antigen to antigen plus Ia-specific T-T hybridomas. The ability of the same APCs to present antigen after uptake and processing showed that this antigen subsequently becomes stably associated with the APC plasma membrane. To characterize the basis for this association, we analyzed its sensitivity to enzymatic digestion. APCs exposed to antigen, treated with phospholipase A2, and then immediately fixed lost the ability to stimulate bovine insulin plus I-Ad-specific hybridomas. In contrast, the ability of these same APCs to stimulate I-Ad allospecific hybridomas was unaffected. This effect of phospholipase is not mimicked by the broadly active protease Pronase, nor is there evidence for contaminating proteases in the phospholipase preparation. These results suggest that one consequence of antigen processing may be an antigen-lipid association that contributes to the anchoring of antigen to the APC membrane. The implications of this model are discussed.

Antigen-Presenting Cells↗

PubMed1985

Analysis of antigen presentation by metabolically inactive accessory cells and their isolated membranes.

Several amino acid copolymers are potent immunogens under the control of major histocompatibility complex (MHC)-encoded Ir genes. We have further characterized their accessory-cell-dependent, MHC-restricted presentation to T lymphocytes. We initially characterized their processing requirements by investigating the ability of paraformaldehyde-fixed antigen-presenting cells (APC) to present these copolymers. Fixed APC can present poly(Glu56Lys35Phe9) and poly(Glu60Ala30Tyr10) provided that they have been incubated with antigen prior to fixation. The inability of these same fixed preparations to present soluble antigen indicates a fixation-sensitive antigen-processing step. In contrast, the antigens poly(Glu55Lys35Leu10) and poly(Glu55Lys35Tyr10) can be presented by APC fixed before antigen exposure. This differential requirement for antigen processing was exploited to analyze the events of antigen presentation in two related systems. First, the ability of isolated APC membranes to process and present antigen was assessed. APC membranes can present the antigens poly(GluLysLeu) and poly(GluLysTyr) in a specific and MHC-restricted manner. However, the isolated membranes fail to present either poly(GluLysPhe) or poly(GluAlaTyr), suggesting that such preparations can present but not process antigen. Second, the distinct properties of the various copolymers were used with fixed APC to test the effects of antigen processing on the phenomenon of antigen competition. APC that had processed poly(GluLysPhe) or poly(GluAlaTyr) were subsequently fixed and used to present antigen in the presence or absence of various antagonists. Under these conditions, poly(GluLysLeu) and poly(Glu50Tyr50) could effect specific inhibition, clearly indicating that antigen competition occurs distal to and does not require antigen processing. In contrast, native antigen with an absolute processing requirement is not capable of competing with preprocessed antigen on fixed APC. Taken together, these results suggest that processing is important for the molecular interactions between the copolymer antigens and the APC cell surface that are relevant to both antigen presentation and competitive inhibition.

Antigen-Presenting Cells↗

PubMedDate not supplied

Systemic production of IL-12 by naked DNA mediated gene transfer: toxicity and attenuation of transgene expression in vivo.

BACKGROUND: IL-12 is a potent antitumor cytokine for cancer gene therapy. Previously, we demonstrated that single systemic administration of naked DNA (encoding IL-12) could serve as a good model for in vivo evaluation of the antitumor effect of a candidate gene (unpublished data). In the present study, we propose that this gene delivery method could be a very useful model for in vivo evaluation of the toxicity of a given therapeutic gene (using IL-12 as an example). By comparing the toxicities and the effects of initial IL-12 administration on subsequent transgene expression, both IL-12 gene delivery and recombinant murine IL-12 protein (rmIL-12) administration showed similar toxicity profiles. METHODS: Naked DNA encoding murine IL-12 (mIL-12) was delivered into mice by systemic administration. Toxicity profiles of mice treated with DNA or rmIL-12 were compared. RESULTS: Systemic administration of naked DNA encoding mIL-12 resulted in very similar toxicity as rmIL-12 with respect to liver enzyme, hematological and immunological profiles. Repeated injection of mIL-12 gene did not recover a high level of mIL-12 production as the first injection. Moreover, initial mIL-12 administration resulted in inhibition of subsequent reporter gene expression with both viral and non-viral promoters (CMV, human alpha-antitrypsin or chicken beta-actin promoter). This transgene inhibition effect was entirely mediated by IFN-gamma as the transgene expression was fully recovered in IFN-gamma knockout mice. CONCLUSIONS: Systemic IL-12 therapy, with either a protein or gene therapy approach, resulted in comparable liver and systemic toxicities. Refractoriness of mIL-12 production by subsequent administration of mIL-12 gene was observed. The transgene attenuation effect of IL-12 pre-dosing (either by IL-12 or rmIL-12), mediated by IFN-gamma, provided important insights for the design of IL-12 combination gene therapy and the improvement of gene vectors for IL-12 therapy. The present results show that simple injection of naked DNA could serve as a good model for in vivo evaluation of the toxicity of a candidate therapeutic gene.

Animals↗

PubMedDate not supplied

Targeting the skin for genetic immunization.

One of the most promising applications of recent advances in gene therapy is the development of immunization strategies based on the delivery of antigen-encoding DNA. DNA-based vaccination, also referred to as genetic vaccination or polynucleotide vaccination, offers considerable promise for improvement over existing immunization strategies, and the skin offers unique potential as a target tissue for genetic vaccines. The expression of genetically introduced antigens in a cutaneous microenvironment rich in both professional antigen-presenting cells and accessory cells, which are capable of producing immunostimulatory cytokines, has the potential to overcome the historical limitations of vaccinology and immunotherapy. Though the precise molecular mechanisms of genetic immunization remain unclear, a general working model of the events through which antigen-encoding plasmids introduced into the skin initiate an immune response can be constructed. The finding that Langerhans cells can be transfected in vivo raises the exciting possibility that these migrating professional antigen-presenting cells can be genetically engineered in vivo. By designing strategies to codeliver genes encoding antigens with genes encoding immunoregulatory molecules to the same antigen-presenting cell, it may be possible to either induce or suppress antigen-specific immune responses in the host. Though many aspects of the biology of cutaneous DNA immunization remain unknown, the skin appears to offer unique potential for the application of advances in gene therapy to vaccination and genetic engineering of the immune response.

Animals↗