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Biomedical subjects

G L Costa

Publications and source records attributed to G L Costa.

11 recordsLinked to original sources
PubMed1994

Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction.

We have developed a facile procedure for rapid PCR-based site-directed mutagenesis of double-stranded DNA. Increasing the initial template concentration and decreasing the PCR cycles to 5-10 allows us to reduce the rate of undesired second-site mutations and dramatically increase the time savings. Following PCR, DpnI treatment is used to select against parental DNA molecules. The DpnI (target sequence 5'-Gm6ATC) is specific for methylated and hemimethylated DNA and is used to digest parental DNA and select for mutation-containing amplified DNA. DNA isolated from almost all common Escherichia coli strains is Dam methylated and therefore susceptible to DpnI digestion. Pfu DNA polymerase is used, prior to intramolecular ligation of the linear template, to remove any bases extended onto the 3' ends of the PCR product by Taq DNA polymerase. The recircularized vector DNA incorporating the desired mutations is transformed into E. coli. This method can be used independently of any host strain and vector.

Base Sequence↗
PubMed1994

Cloning and analysis of PCR-generated DNA fragments.

Methods are presented for the improved yield and analysis of blunt-ended cloning of PCR-generated DNA fragments. We show that Pfu DNA polymerase polishing of Taq DNA polymerase-generated fragments increases the yield and efficiency of cloning. Using a triple primer set consisting of two outside, asymmetrically distanced primers and one fragment-specific primer, both the presence and orientation of cloned inserts can be determined. Application of these methods allows the generation and cloning of a fragment in 1 day and the analysis of putative clones the next, thereby saving a substantial amount of both time and effort.

Base Sequence↗
PubMed1993

Suppression of monocyte function and differential regulation of IL-1 and IL-1ra by IL-4 contribute to resolution of experimental arthritis.

IL-4 has diverse effects on hematopoietic cells, including the ability to suppress certain mononuclear cell functions. To evaluate the effect of IL-4 on the evolution of acute and chronic arthritis, murine recombinant IL-4 was administered systemically to animals receiving an arthropathic dose of group A streptococcal cell wall fragments. Daily treatment with IL-4 had a minimal effect on the acute phase, but significantly suppressed the chronic, destructive phase. By 4 wk after initiation of disease, the articular index of IL-4-treated animals was reduced > 60% (articular index = 4 +/- 0.9) compared with the untreated rats (11.5 +/- 0.48, p < 0.001). A substantial decrease in the influx of inflammatory cells and virtual elimination of pannus and erosions occurred after IL-4 therapy. Associated with the reduced accumulation of mononuclear leukocytes was a decrease in their proinflammatory functions including cytokine production and reactive oxygen intermediate metabolism. These observations are consistent with the selective effects of IL-4 on phagocytic cell function demonstrated in vitro. Furthermore, IL-4 induced gene expression for IL-1ra, a protein that antagonizes the action of IL-1 by binding to the IL-1 receptor without agonist activity. Through an expanding spectrum of effects on monocyte-macrophage phenotypic and functional parameters, IL-4 is emerging as an important inhibitor of cell-mediated immune responses and pathogenic processes.

Animals↗
PubMed1993

Transforming growth factor-beta mediates IL-1-dependent induction of IL-1 receptor antagonist.

Transforming growth factor-beta (TGF-beta) is a potent immunomodulatory molecule that promotes inflammation through recruitment of monocytes and induction of IL-1 and other cytokines. These proinflammatory processes may be modulated by the ability of TGF-beta to induce de novo synthesis and secretion of an IL-1 receptor antagonist (IL-1ra) that binds to and blocks IL-1 receptors. In this study, we show that the addition of TGF-beta to human peripheral blood monocytes induced the sequential transcription of the 1.8-kb mRNA for IL-1 beta and for IL-1ra. The expression of detectable mRNA and synthesis of IL-1 beta peptide routinely preceded that for IL-1ra, suggesting possible dependency of IL-1ra generation on IL-1 beta. Antibody to IL-1 blocked TGF-beta induction of IL-1ra mRNA expression demonstrating an unique IL-1-dependent induction of its own antagonist. Confirmatory evidence that IL-1 participates in the generation of IL-1ra was obtained when exogenously added IL-1 induced and, IL-1 receptor antagonist blocked, IL-1ra transcription. Thus, these data suggest that TGF-beta, after release at a site of inflammation, induces synthesis of IL-1 that participates in the initial cytokine cascade central to an inflammatory response, and then triggers the generation of its own natural inhibitor by autocrine or paracrine pathways. This TGF-beta-induced IL-1-dependent induction of IL-1ra may provide a negative feedback loop, thereby promoting the resolution of an inflammatory response.

Animals↗
PubMed1993

Interleukin (IL) 4 differentially regulates monocyte IL-1 family gene expression and synthesis in vitro and in vivo.

Interleukin (IL) 4 is a multifunctional T cell-derived cytokine that inhibits cytokine production and certain effector functions in human monocytes, while enhancing others. We show that IL-4 may contribute to the downregulation and resolution of an inflammatory response by selectively promoting expression of the IL-1 receptor antagonist (IL-1ra) that blocks the action of IL-1. IL-1ra specifically binds to the IL-1 receptor without initiating signal transduction. Peripheral blood monocytes obtained from cancer patients, before and immediately after a regimen of IL-4 immunotherapy, were examined for IL-1ra gene expression. After IL-4 therapy, monocytes from the patients showed a marked increase in IL-1ra mRNA. This selective induction of IL-1ra mRNA in circulating monocytes was reflected by significantly enhanced serum levels of IL-1ra (p < 0.01) during IL-4 therapy, which declined after IL-4 treatment. In vitro analysis of IL-4 regulation of monocytes from normal individuals revealed a dose-dependent induction of IL-1ra mRNA within 2-4 h after stimulation without a concomitant effect on the expression of IL-1 mRNA. Increased IL-1ra mRNA was not due to RNA stabilization, but occurred at the level of transcription. In the presence of LPS, IL-4 not only augmented IL-1ra levels, but markedly inhibited LPS-induced IL-1 mRNA expression. The selective upregulation of IL-1ra by resting or activated monocytes, coupled with inhibition of IL-1 production by activated monocytes, as we demonstrate both in vitro and in vivo, suggests that IL-4 may prove clinically useful as a systemic antiinflammatory agent.

Blotting, Northern↗
PubMed1993

Reversal of acute and chronic synovial inflammation by anti-transforming growth factor beta.

Transforming growth factor beta (TGF-beta) induces leukocyte recruitment and activation, events central to an inflammatory response. In this study, we demonstrate that antagonism of TGF-beta with a neutralizing antibody not only blocks inflammatory cell accumulation, but also tissue pathology in an experimental model of chronic erosive polyarthritis. Intraarticular injection of monoclonal antibody 1D11.16, which inhibits both TGF-beta 1 and TGF-beta 2 bioactivity, into animals receiving an arthropathic dose of bacterial cell walls significantly inhibits arthritis. Inhibition was observed with a single injection of 50 micrograms antibody, and a 1-mg injection blocked acute inflammation > 75% compared with the contralateral joints injected with an irrelevant isotype control antibody (MOPC21) as quantitated by an articular index (AI = 0.93 +/- 0.23 for 1D11.16, and AI = 4.0 +/- 0 on day 4; p < 0.001). Moreover, suppression of the acute arthritis achieved with a single injection of antibody was sustained into the chronic, destructive phase of the disease (on day 18, AI = 0.93 +/- 0.07 vs. AI = 2.6 +/- 0.5; p < 0.01). The decreased inflammatory index associated with anti-TGF-beta treatment was consistent with histopathologic and radiologic evidence of a therapeutic response. These data implicate TGF-beta as a profound agonist not only in the early events responsible for synovial inflammation, but also in the chronicity of streptococcal cell wall fragment-induced inflammation culminating in destructive pathology. Interrupting the cycle of leukocyte recruitment and activation with TGF-beta antagonists may provide a mechanism for resolution of chronic destructive lesions.

Acute Disease↗
PubMed1993

Role of transforming growth factor beta in the pathophysiology of chronic inflammation.

Transforming growth factor beta (TGF-beta), a cytokine identified in acute and chronic inflammatory sites, mediates leukocyte recruitment and activation essential to the development of such lesions. Released by platelets upon aggregation and by leukocytes stimulated with bacterial products or inflammatory mediators, TGF-beta has potent chemotactic activity for blood neutrophils, monocytes, and lymphocytes. By augmenting integrin expression, TGF-beta facilitates leukocyte adhesion to the vessel wall and extracellular matrix at the site of inflammation. Once within the inflammatory site, mononuclear cells are stimulated by TGF-beta to release cytokines important in the network of molecules regulating the host response to microorganisms and immunologic challenge. Thus, bacteria and their products, in addition to directly recruiting and activating leukocytes at sites of infection, indirectly influence these events through the induction of cytokines such as TGF-beta. By antagonizing the activity of TGF-beta with neutralizing antibodies, a causal relationship between this cytokine, inflammation, and pathogenesis has been demonstrated. Administration of anti-TGF-beta to sites of chronic destructive inflammation not only blocked leukocyte recruitment and activation, but also inhibited the subsequent destruction of bone and cartilage characteristics of such lesions.

Bacterial Physiological Phenomena↗