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Biomedical subjects

C Brostjan

Publications and source records attributed to C Brostjan.

At least 19 recordsLinked to original sources
PubMed2003

Dendritic cell-based vaccination in solid cancer.

PURPOSE: Dendritic cell (DC)-based immunotherapy is rapidly emerging as a viable tool in cancer treatment. This approach has been used mostly in patients in the presence of defined tumor antigens such as melanoma. In this study, cancer patients with advanced disease that lacks defined tumor antigens were vaccinated with tumor lysate-pulsed DCs. PATIENTS AND METHODS: Twenty patients (pancreatic, hepatocellular, cholangiocellular, and medullary thyroid carcinoma) with stage IV disease were enrolled in the study. In 3-week intervals, freshly isolated autologous CD14 magnetic bead-selected monocytes were cultured in granulocyte-macrophage colony-stimulating factor and interleukin-4 to obtain immature DCs. These cells were pulsed with autologous tumor lysate and matured with tumor necrosis factor alpha. Mature DCs were applied into a groin lymph node, under ultrasound guidance. Adjuvant interleukin-2 (20,000 U/kg) was given subcutaneously daily, for 12 days, after each vaccination. Toxicity, tumor marker profile, immune response, and clinical response were determined. RESULTS: Vaccination was well tolerated. No physical signs of autoimmunity were detected. DC vaccination induced delayed-type hypersensitivity reactivity in 18 patients. Tumor marker responses were observed in eight patients. In addition, in three patients the generation of interferon gamma-positive T cells was induced during the vaccination. Objective changes in measurable lesions or tumor markers were evident in seven of 20 assessed patients. None of the patients was found to meet the criteria for partial or complete responses. CONCLUSION: These data indicate that vaccination with autologous tumor-pulsed DCs generated from peripheral blood is safe and can induce tumor-specific cellular cytotoxicity. Clinical responses are achievable, even in patients with advanced disease.

Adult↗
PubMed2002

Stimulation of autologous antitumor T-cell responses against medullary thyroid carcinoma using tumor lysate-pulsed dendritic cells.

Dendritic cells (DCs) have attracted wide interest because of their unique capacity to elicit primary and secondary antitumor responses. We have generated autologous tumor lysate-pulsed DCs from three patients with medullary thyroid carcinoma (MTC) and tested them for their ability to stimulate cytotoxic T-cell responses against autologous MTC tumor cells in vitro. The aim of our investigations was to evaluate the potential efficacy of DC-based immunotherapy in patients with MTC. DCs were generated from peripheral blood monocytes using GM-CSF and IL-4 (immature DCs) or GM-CSF, IL-4, and TNFalpha (mature DCs). Our results indicate that mature tumor lysate-pulsed DCs are able to elicit a human leukocyte antigen class I-restricted cytotoxic T-cell response against autologous MTC tumor cells, whereas immature tumor lysate-pulsed DCs do not stimulate significant antitumor activity. We feel that our data may be relevant for future clinical trials of active immunotherapy using tumor lysate-pulsed DCs in patients with MTC who have residual or distant disease after surgical treatment. The fact that mature DCs displayed a substantially higher capacity to stimulate autologous antitumor T-cell responses than immature DCs underlines the importance of a maturation step in immunotherapy protocols based on DCs.

Adult↗
PubMed2001

The centromeric part of the human natural killer (NK) receptor complex: lectin-like receptor genes expressed in NK, dendritic and endothelial cells.

The human natural killer (NK) receptor complex encompasses a region of about 2 Mb on the short arm of chromosome 12. It contains at least 18 lectin-like receptor genes, of which some are expressed in NK and NK/T cells and function as NK receptors. Close to the CD94 and NKG2 NK receptor genes in the centromeric part, a novel family of genes, expressed in myeloid, dendritic and/or endothelial cells, recently became evident. These genes encode a receptor for oxidized low density lipoprotein in endothelial cells and three other receptors potentially serving regulatory functions in dendritic cells. Although the overall structure of the human NK receptor complex is similar to the syntenic rodent regions, the centromeric part lacks the cluster of Ly49 genes. This supports the notion that recognition of MHC class Ia molecules has evolved separately in rodents and humans in the lectin-like Ly49 and the killer immunoglobulin-like receptors, respectively. In the telomeric part, other lectin-like genes expressed in different hematopoietic lineages are found. The receptors of the NK receptor complex apparently serve important functions in several leukocytes and in endothelial cells, and the exact role of these receptors, their ligands, and their distinct and co-ordinate regulation in different cell lineages warrants further investigation.

Animals↗
PubMed2000

The NKG2 natural killer cell receptor family: comparative analysis of promoter sequences.

The NKG2 receptor family is crucially involved in target cell recognition by natural killer cells and comprises several activating as well as inhibitory family members. We have established approximately 3 kilobases of upstream promoter sequences of the human NKG2-C, -E and -F genes and have carried out a comparative analysis with available NKG2-A sequences. We found extended regions of homology which contain numerous putative transcription factor binding sites conserved in the NKG2 genes. However, variation in Alu insertion among family members has led to promoter structures unique to the respective family members, which could contribute to differences in transcriptional initiation as well as gene-specific regulation.

Base Sequence↗
PubMed2000

Catecholamines up-regulate lipopolysaccharide-induced IL-6 production in human microvascular endothelial cells.

The catecholamine-mediated modulation of the cytokine network has primarily been demonstrated for leukocytes. Whereas catecholamines decrease the LPS-induced production of IL-6 by leukocytes, serum levels of IL-6 are dramatically increased by the catecholamine epinephrine in animal endotoxemia models. We now demonstrate that epinephrine as well as norepinephrine can induce IL-6 in an endothelial cell line (HMEC-1). Furthermore, these catecholamines could even potentiate the LPS-induced IL-6 protein production. The synergistic effect of catecholamines and LPS could be reproduced in primary human skin microvascular endothelial cells. The catecholamine-induced IL-6 stimulation is based on increased IL-6 mRNA levels. RNA stability assays revealed that this regulation is not a result of enhanced RNA stability and therefore is most likely due to an increased transcription. Treatment with cycloheximide indicated that new protein synthesis is not necessary for this transcriptional up-regulation of IL-6 mRNA. Preincubation with alpha and beta receptor antagonists showed that the effect is mediated by beta(1)- and beta(2)-adrenergic receptors. Thus, endothelial cells might be a possible source of increased IL-6 production observed in situations such as stress or septic shock, in which catecholamines are elevated due to endogenous production or exogenous application.

Adrenergic beta-Antagonists↗
PubMed1999

Linkage of the NKG2 and CD94 receptor genes to D12S77 in the human natural killer gene complex.

The human natural killer (NK) gene complex is located on the short arm of chromosome 12 and contains a number of genes encoding C-type lectin receptors important for natural killer cell function. Among these are CD94 and the five NKG2 genes. The CD94 protein associates with different NKG2 isoforms to heterodimeric receptors which function to inhibit or trigger cytotoxicity of NK cells depending on the NKG2 isoform. We selected two yeast artificial chromosome clones comprising approximately 1.5 Mb of the NK gene complex and established a contig of underlying P1-derived artificial chromosome clones containing all NKG2 and the CD94 genes. A detailed analysis shows that all six genes are found within a region of 100 to 200 kilobases proximal of the marker D12S77. The gene order established is D12S77 - CD94 - NKG2D - NKG2F - NKG2E - NKG2C - NKG2A. The NKG2 genes are of identical transcriptional orientation, whereas the CD94 gene is placed in opposite orientation. The tight genomic linkage of these genes and the identical orientation of the NKG2 genes suggest coordinate regulation of expression during the differentiation of natural killer cells.

Antigens, CD↗
PubMed1998

Membrane-associated lymphotoxin on natural killer cells activates endothelial cells via an NF-kappaB-dependent pathway.

BACKGROUND: Inhibition of complement in small animal models of xenotransplantation has demonstrated graft infiltration with natural killer (NK) cells and monocytes associated with endothelial cell (EC) activation. We have previously demonstrated that human NK cells activate porcine EC in vitro, which results in adhesion molecule expression and cytokine secretion. In this study, we used the NK cell line NK92 to define the molecular and cellular basis of NK cell-mediated EC activation. METHODS: EC were transfected with either reporter constructs containing the luciferase gene driven either by E-selectin or interleukin (IL)-8 promoters or a synthetic NF-kappaB-dependent promoter. In addition, a dominant-negative mutant tumor necrosis factor receptor I (TNFRI) expression vector was co-transfected in inhibition studies. Forty-eight hours after transfection, EC were stimulated with NK cells or NK cell membrane extracts for 7 hr and activation was measured by a luciferase assay. RESULTS: Co-culture of NK cells with transfected EC enhanced E-selectin, IL-8, and NF-kappaB-dependent promoter activity. NK cell membrane extracts retained the capacity to activate EC and induced nuclear translocation of NF-kappaB (p50 and p65). Western blotting of NK cell and membrane extracts detected the presence of Lymphotoxin-alpha (LTalpha) but not tumor necrosis factor-alpha. Furthermore, LTalpha was secreted in NK:EC co-cultures. Co-transfection with dominant-negative mutant TNFRI inhibited EC activation by NK cell membrane extracts and by NK cells by 80% and 47%, respectively. The same pattern of inhibition was observed using anti-human LT sera. CONCLUSIONS: Human NK cell membrane-bound LT signals across species via TNFRI, leading to NF-kappaB nuclear translocation and transcription of E-selectin and IL-8, which results in EC activation. The discrepancy in the degree of inhibition by membrane extracts and NK cells with mutant TNFRI suggests that additional pathways are utilized by NK cells to activate EC.

Animals↗
PubMed1998

The genomic organization of NKG2C, E, F, and D receptor genes in the human natural killer gene complex.

Interactions of natural killer cell receptors with their cognate ligands play a major role in regulating NK cell function. The NKG2 gene family encodes several highly similar proteins, which are known to form heterodimers with the CD94 receptor. These dimers play a role in the inhibition as well as the activation of NK cells. We have analyzed the gene structures of the NKG2C, D, E, and F genes, and determined their genomic organization. Restriction mapping and sequencing revealed the four genes to be closely linked to one another, and of the same transcriptional orientation. An exon duplication within the NKG2C and E genes was identified, although the duplicated version of this exon has not yet been found in mRNA sequences. The NKG2C, E, and F genes, despite being highly similar, are variable at their 3' ends. We show that NKG2C consists of six exons, whereas NKG2E has seven, and the splice acceptor site for the seventh exon occurs in an Alu repeat. NKG2F consists of only four exons and part of exon IV is in some cases spliced to the 5' end of the NKG2D transcript. NKG2D has only a low similarity to the other NKG2 genes.

Base Sequence↗
PubMed1997

Glucocorticoids inhibit E-selectin expression by targeting NF-kappaB and not ATF/c-Jun.

E-selectin, an adhesion molecule expressed on the surface of activated endothelial cells, is essential for leukocyte rolling on endothelium which leads to extravasation in the process of inflammation. Induction of E-selectin expression by proinflammatory stimuli such as TNF-alpha or LPS is reduced markedly in the presence of dexamethasone, a synthetic glucocorticoid and potent anti-inflammatory agent. We have investigated the molecular mechanism underlying dexamethasone-mediated E-selectin repression in porcine aortic endothelial cells. Reduced E-selectin protein expression is paralleled by a decrease in E-selectin mRNA and is based on changes in transcription rate. Analysis of the E-selectin promoter revealed that induction by proinflammatory stimuli as well as repression by dexamethasone are mediated by the same promoter region containing three closely spaced binding sites for nuclear factor (NF)-kappaB and an element, NF-ELAM-1 (endothelial leukocyte adhesion molecule-1), constitutively occupied by ATF and c-Jun. NF-ELAM-1 contributes to maximal promoter activity, but does not confer glucocorticoid inhibition, as demonstrated by site-directed mutagenesis. In contrast, transcription directed by the E-selectin NF-kappaB elements is reduced strongly in the presence of dexamethasone, thus identifying NF-kappaB as the primary target for glucocorticoid-mediated E-selectin repression.

Animals↗
PubMed1997

Inhibition of bovine endothelial cell activation in vitro by regulated expression of a transdominant inhibitor of NF-kappa B.

The activation of endothelial cells is a recurrent phenomenon linked to pathologic conditions such as inflammation, chronic arthritis, allo- and xenograft rejection. To inhibit endothelial cell activation we have constructed a transactivation-deficient derivative of the p65/RelA subunit of NF-kappa B, a transcription factor known to be crucial for the induction of adhesion molecules, cytokines and procoagulants in activated endothelial cells. This protein (p65RHD) comprises the Rel homology domain of the RelA subunit, retaining dimerization, DNA binding, and nuclear localization functions, but is deficient in transcriptional activation, and acts as a competitive inhibitor of NF-kappa B. Our data demonstrate that p65RHD is a potent and specific inhibitor of NF-kappa B-mediated induction of a number of genes, such as I kappa B alpha, IL-8, E-selectin, P-selectin, and tissue factor in endothelial cells. Furthermore, tetracycline-inducible expression of p65RHD in stably transfected primary endothelial cells inhibits the induction of gene expression equally well. This regulated system of gene expression provides the basis for a novel therapeutic approach to the pathologic effects of endothelial cell activation, especially in delayed xenograft rejection, by using transgenic animals as organ donors.

Animals↗
PubMed1996

The intron-exon structure of the porcine E-selectin-encoding gene.

We have cloned and sequenced the gene encoding porcine E-selectin. The gene comprises 12 exons and 11 introns. Two pseudoexons are contained within intron 4 and intron 6. These sequences are similar to the corresponding exons in the human E-selectin sequence; however, they are not present in the porcine E-selectin-encoding cDNA. Transcription starts at position -498 relative to the translation initiation site. The first ATG is located within exon 2. Translation stops in exon 11 leaving exon 12 untranslated in its entirety.

Amino Acid Sequence↗
PubMed1996

Glucocorticoid-mediated repression of NFkappaB activity in endothelial cells does not involve induction of IkappaBalpha synthesis.

Repression of NFkappaB-dependent gene expression is one of the major elements of immunosuppression by glucocorticoids. Protein-protein interactions between the glucocorticoid receptor and NFkappaB have been characterized and shown to be a possible mechanism of mutual inhibition of transactivation properties. More recently, glucocorticoid-mediated induction of IkappaBalpha, an inhibitor of NFkappaB, has been described in monocytes and lymphocytes; an increase in IkappaBalpha mRNA and protein resulted in inactivation and cytosolic retention of NFkappaB. Thus, rather than the physical interaction between the glucocorticoid receptor and NFkappaB, the up-regulation of IkappaBalpha was presented as the key element in immunosuppression by glucocorticoids. In contrast, we show that the IkappaBalpha pathway is not involved in glucocorticoid-mediated inhibition of NFkappaB activity in endothelial cells. Although transcriptional activation by NFkappaB was significantly reduced in the presence of glucocorticoids, we did not detect induction of IkappaBalpha protein that could prevent nuclear translocation of NFkappaB upon stimulation with lipopolysaccharide or tumor necrosis factor alpha. Furthermore, treatment with glucocorticoids did not seem to affect the transcription rate or mRNA stability of IkappaBalpha. We therefore conclude that, although induction of IkappaBalpha expression by glucocorticoids seems to be of importance in monocytes and lymphocytes, it cannot explain inhibition of NFkappaB-dependent gene expression in endothelial cells. Our results emphasize the relevance of physical interaction between the glucocorticoid receptor and NFkappaB in endothelial cells and thus in suppression of inflammation by glucocorticoids.

Animals↗
PubMed1996

A20 blocks endothelial cell activation through a NF-kappaB-dependent mechanism.

The A20 gene product is a novel zinc finger protein originally described as a tumor necrosis factor alpha (TNF)-inducible early response gene in human umbilical vein endothelial cells (HUVEC). Its described function is to block TNF-induced apoptosis in fibroblasts and B lymphocytes, but more recently it has also been shown to play a role in lymphoid cell maturation. The mechanism of action of A20 is unknown. The aim of our study was to assess the effect of A20 upon endothelial cell activation. By transfecting bovine aortic endothelial cells (BAEC) with A20 as well as reporter constructs consisting of the promoters of genes known to be up-regulated during endothelial cell activation, i.e. E-selectin, interleukin (IL)-8, tissue factor (TF), and inhibitor of nuclear factor kappaBalpha (IkappaBalpha), we demonstrate that A20 expression inhibits gene up-regulation associated with TNF, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), and hydrogen peroxide (H2O2)-induced endothelial cell (EC) activation. The mechanism of action of A20 is in part, or totally, due to the blockade of nuclear factor kappaB (NF-kappaB), as shown by its ability to suppress the activity of a NF-kappaB reporter. This effect is specific, as A20 does not block a noninducible, constitutively expressed reporter, Rous sarcoma virus-luciferase (RSV-LUC); nor does it block the c-Tat-inducible, NF-kappaB-independent reporter, human immunodeficiency virus-chloramphenicol acetyltransferase (HIV-CAT). How A20 blocks NF-kappaB is unclear, although we demonstrate that it does not affect p65 (RelA)-mediated gene transactivation. The inhibition of endothelial cell activation by A20 is a novel function for A20.

Animals↗
PubMed1995

Inhibition of NF-kappa B by pyrrolidine dithiocarbamate blocks endothelial cell activation.

Endothelial cell activation is achieved by the rapid, protein synthesis-independent induction of a characteristic set of genes. Because of the abundance of binding sites for the transcription factor NF-kappa B in the regulatory region of the aforementioned genes, we hypothesized that this factor might play a key role. Reactive oxygen intermediates act as second messengers in the activation of NF-kappa B. We have used the antioxidant pyrrolidine dithiocarbamate to analyze the effect of NF-kappa B inhibition on TNF alpha-induced EC activation in vitro. We show that pyrrolidine dithiocarbamate strongly reduces the TNF alpha-mediated induction of E-selectin, VCAM-1, ICAM-1, PAI-1, tissue factor, IL-8 and I kappa B-alpha. We present evidence identifying NF-kappa B as a central of EC activation. Therefore, this factor may represent a prime target for therapeutic intervention in pathologic conditions associated with EC activation such as allo- and xenograft rejection, atherosclerosis, ischemic reperfusion injury and vasculitis.

Animals↗