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H D Flad

Publications and source records attributed to H D Flad.

At least 73 records · Page 4Linked to original sources
PubMed1996

Alterations in structure and cellular localization of molecular forms of DP IV/CD26 during T cell activation.

Dipeptidyl peptidase IV (DP IV, CD26), known as an activation marker of T lymphocytes, is a proline-specific protease thought to be involved in the regulation of the immune response. The physiological role of dipeptidyl peptidase IV in the immune system and the molecular events of lymphocyte activation mediated by this enzyme are only partly established. Former results suggested the occurrence of different molecular forms of DP IV in distinct human sources. As yet it has been unknown whether DP IV from human hematopoietic cells also appears in different forms and whether similar structural modifications are involved in functional processes of the regulation of the immune response. Here we describe that lymphocytic DP IV/CD26 occurs in various molecular forms and that some of them are associated with the activation process. In cell lysates of mitogen-activated lymphocytes at least 5 enzymatically active DP IV forms and up to 11 immunoreactive molecular forms of this enzyme with isoelectric points between pH 3.5 and 5.9 were discernible. Corresponding analyses of soluble and membrane cell fractions of human lymphocytes showed significant differences in the staining pattern of molecular DP IV structures. After mitogenic stimulation a special molecular form of DP IV arises in the membrane, which was originated either from the soluble part of the cell (translocation) or represents a new synthesized form. Particularly, changes of molecular DP IV forms after mitogenic stimulation strongly suggest that special forms/epitopes of this enzyme are directly involved in the process of lymphocyte activation and growth. Importantly, different monoclonal DP IV antibodies partly define different molecular forms of DP IV. Moreover, the pattern of immunostaining and enzymatic staining (Gly-Pro-beta-methoxynaphthylamide) also reveals drastic differences. These data strongly suggest a direct relationship between the expression/recognition of special DP IV epitopes and the contradictory functional effects of monoclonal DP IV antibodies found by us and other groups.

Cells, Cultured↗
PubMed1996

The use of reverse transcription polymerase chain reaction to analyse large numbers of mRNA species from a single cell.

A PCR method is described for determining the expression of multiple heterogeneous mRNAs from single cells. The total mRNA pool of a single selected cell is subjected to reverse transcription and subsequent tailing with poly(dA). This cDNA is preamplified by a sequence non-specific PCR protocol using oligo(dT)-containing primers. The single cell cDNA library obtained permits the analysis of virtually unlimited numbers of mRNA species per cell using sequence-specific PCR. This procedure of multiple mRNA analysis enables phenotyping of any cell for its mRNA composition and could be used to study the cytokine mRNA expression of individual human T cells ex vivo. The method should greatly facilitate the analysis of combinatorial expression of known genes in any cell.

Base Sequence↗
PubMed1996

TNF-alpha renders human neutrophils responsive to platelet factor 4. Comparison of PF-4 and IL-8 reveals different activity profiles of the two chemokines.

Platelet factor 4 (PF-4), like IL-8, is a member of the chemokine superfamily of proinflammatory cytokines. However, although the capacity of IL-8 to stimulate functions in neutrophils is well established, reports on PF-4 are still contradictory. In the present study, we have prepared highly purified PF-4 and examined its ability to induce chemotaxis, degranulation, adhesion to gelatin and plasma proteins, and changes in intracellular calcium levels. Even over a broad range of concentrations, PF-4 alone was unable to induce functional changes in PMN. However, neutrophils pre- or co-incubated with physiologically relevant concentrations of TNF-alpha responded to PF-4 by the selective mobilization of the secondary granule marker lactoferrin but not of the primary granule marker elastase. Contrary to IL-8, PMN did not require pretreatment with cytochalasin B for PF-4-induced exocytosis of lactoferrin. The synergistic effect of PF-4 with TNF-alpha was not a priming phenomenon because the cooperative response remained unchanged even when TNF-alpha was added 5 min after the chemokine. In contrast, TNF-alpha-treated PMN did not respond to PF-4 by chemotaxis or by an increase of intracellular calcium levels, and no competition of PF-4 for IL-8 receptors was observed. Our results suggest a mechanism as well as a biologic role of PF-4 in the regulation of neutrophil function, which is different from that of IL-8 and other alpha-chemokines.

Biomarkers↗
PubMed1996

Nitric oxide synthase: expression of the endothelial, Ca2+/calmodulin-dependent isoform in human B and T lymphocytes.

Nitric oxide (NO) is a pleiotropic mediator of a variety of cellular processes such as vasorelaxation, neurotransmission, and cytotoxicity. We studied the expression of the human endothelial, Ca2+/calmodulin-dependent NO synthase (NOS) isoform (ecNOS) in highly purified human lymphocytes from peripheral blood and tonsillar T cells. ecNOS mRNA was detected in IgD+ or IgD- B cells, peripheral blood and tonsillar T cells. Upon stimulation, ecNOS mRNA expression decreased in all these lymphocyte subpopulations. Germinal center T cells and follicular dendritic cells did not express ecNOS mRNA either in an unstimulated or in a stimulated state. ecNOS expression by human lymphocytes was further substantiated by its mRNA detection in lymphoid cell lines such as Raji, Daudi, and Jurkat. By the use of a specific monoclonal antibody, ecNOS was shown to be present in T cells from peripheral blood and in various germinal center cells of frozen tonsillar sections. These data are the first to demonstrate the expression of the endothelial ecNOS at mRNA and protein level in human lymphocytes.

B-Lymphocytes↗
PubMed1996

Human follicular dendritic cells and vascular cells produce interleukin-7: a potential role for interleukin-7 in the germinal center reaction.

Interleukin-7 (IL-7) has long been known as a potent growth factor in lymphocyte development. However, recent data obtained in vitro revealed additional functions for this cytokine, e.g. IL-7 influences the generation of cytotoxic T cells and NK cells, and in higher concentrations, activates monocytes in a manner similar to bacterial lipopolysaccharide. Furthermore, human tonsillar B cells are able to proliferate upon stimulation by IL-7 and cross-linking of the B cell receptor. Considering the latter role of IL-7 for B cell proliferation, we investigated which cells of the human immune system express IL-7 in vivo. mRNA expression of tonsillar cells was analyzed using the reverse transcription polymerase chain reaction, and protein expression was assessed by immunohistochemical staining of frozen sections. Among a variety of different immune cell types isolated from human tonsils, only follicular dendritic cells (FDC) expressed specific IL-7 products, whereas B and T cells were consistently negative. Immunohistochemical staining of tonsillar sections revealed the expression of immunoreactive IL-7 protein on FDC, and a pronounced expression could also be detected in oral mucosa and vascular endothelial cells. For the latter, we could also demonstrate mRNA expression in primary cultured cells. In light of the previous finding that IL-7 can act as a co-stimulus for the induction of proliferation in tonsillar B cells, these data suggest a role of IL-7 in the germinal center reaction. It is tempting to speculate that FDC may function as regulatory cells in B cell development in the tonsil, as epithelial nurse cells are thought to govern T cell development in the thymus.

B-Lymphocytes↗
PubMed1996

Fc gamma receptor I (CD64)-negative human monocytes are potent accessory cells in viral antigen-induced T cell activation and exhibit high IFN-alpha-producing capacity.

Blood monocytes represent a heterogeneous cell population with respect to phenotype and function. We have previously described that a minor subset of Fc gamma receptor I-negative (CD64-) monocytes, comprising < 10% of all monocytes, exhibits a significantly higher accessory capacity in allogeneic or purified protein derivative (PPD) of tuberculin-induced T cell activation than the majority of CD64-expressing (CD64+) monocytes. CD64- monocytes were also found to represent the major source of Newcastle disease virus (NDV)-induced interferon (IFN)-alpha within human monocytes. In the present study we demonstrate that CD64- monocytes are also the main producers of IFN-alpha in response to Herpes simplex virus type 1 (HSV-1) or influenza (type A) antigens. The virus-induced IFN-alpha release by monocytes, but mainly that by CD64- monocytes, can be further increased by co-culture with autologous T cells, which alone do not produce significant amounts of IFN-alpha in response to virus. In addition, CD64- and CD64+ monocytes also differ in their accessory capacity in virus-induced T cell responses. CD64- monocytes exposed to influenza antigens induced higher IFN-gamma release and proliferation by the responding autologous T cells than virus-exposed CD64+ monocytes. In virus-stimulated monocyte/T cell co-cultures, CD64- monocytes also induced larger size cell clusters than CD64+ monocytes, indicating direct cell-to-cell interaction with a higher number of T cells, which represent the main component of these clusters.

Antigen-Presenting Cells↗
PubMed1996

Differential expression and function of CD80 (B7-1) and CD86 (B7-2) on human peripheral blood monocytes.

The interaction of CD28 with its ligands is important for T-cell activation. Recent studies demonstrated the existence of at least two ligands on accessory cells, CD80 (B7-1) and CD86 (B7-2). In this study we demonstrate that, although CD80 and CD86 are both expressed on monocytes, they seem to have different functions. Freshly isolated monocytes express CD86 but are CD80-negative. CD80 expression is weakly induced after 6-8 hr of in vitro culture and is enhanced by stimulation. CD86 expression is enhanced faster than CD80 expression and reaches the peak level after 4-6 hr in stimulated cells. Reverse transcription-polymerase chain reaction studies demonstrate that freshly isolated monocytes contain no CD80-mRNA. The mRNA of CD80 is induced after 4-6 hr of culture, which matches with the expression of the protein. Inhibition studies using different antibodies against both molecules and the fusion protein CTLA4Ig show that only anti-CD80 and CTLA4Ig could partially inhibit antigen-specific (tuberculin) and polyclonal (anti-CD3) lymphoproliferation and interferon-gamma (IFN-gamma) secretion of T cells cocultured with autologous monocytes. IFN-gamma secretion was more sensitive to blocking costimulation than proliferation. The antibody BB-1 did not inhibit proliferation and cytokine secretion, nor did the anti-CD86 clone IT2.2. CTLA4Ig, which binds both CD80 and CD86, has the same inhibitory capacity as the anti-CD80 antibody tested. From those findings we conclude that human monocytes use CD80 as a costimulatory ligand for CD28 and utilize other costimulatory mechanisms besides those mediated via molecules of the B7 family.

Antigens, CD↗
PubMed1996

Apoptosis of monocytes and prolonged survival of granulocytes as a result of phagocytosis of bacteria.

Monocytes and granulocytes were incubated with suspensions of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, or Salmonella enteritidis and, after being washed free of bacteria, cultured for up to 48 h. Every few hours, samples of cultured cells were taken for DNA isolation. Monocytes which phagocytosed bacteria showed features of apoptotic cells, as determined by light microscopy and DNA fragmentation detected by gel electrophoresis. The phenomenon was observed 2 to 4 h after phagocytosis, in contrast, control monocytes did not show signs of apoptosis until 48 h of culture. Intact control granulocytes spontaneously became apoptotic after 12 h of culture. In contrast, degradation of DNA in cells exposed to bacteria was delayed by 12 to 24 h. In conclusion, our observation suggests that granulocytes and monocytes react differently to phagocytosis of bacteria.

Apoptosis↗
PubMed1996

Effect of in vitro infection of human monocytes with low numbers of Mycobacterium tuberculosis bacteria on monocyte apoptosis.

The effect of Mycobacterium tuberculosis H37Rv on spontaneous apoptosis of human monocytes from healthy donors in vitro was investigated. Infection with low numbers of viable M. tuberculosis bacteria prevented spontaneously occurring apoptosis in monocytes. Our data suggest that apoptosis of monocytes appears to be negatively associated with the presence of tumor necrosis factor alpha.

Apoptosis↗
PubMed1996

Assessment of mycobacterial DNA in cells and tissues of mycobacterial and sarcoid lesions.

In this study we applied a polymerase chain reaction (PCR) assay for the detection and species-specific identification of mycobacteria to samples from patients with sarcoidosis and mycobacterial infections and from control patients. The PCR-technique is based on the amplification of mycobacterial DNA coding for 16S rRNA, which is present in all mycobacterial species, and on the additional sequencing of the PCR fragment to determine the species. Mycobacterial DNA could be detected in lung tissues and bronchoalveolar lavage cells from cases of tuberculosis and infections with atypical mycobacteria. On the other hand, mycobacterial DNA was amplified only in lung tissue from one patient with sarcoidosis. Twenty-three samples from patients with sarcoidosis were negative for mycobacterial DNA. From our results we conclude that the granulomatous lesions in sarcoidosis may not be due to mycobacterial infections.

Base Sequence↗
PubMed1996

Molecular characterization of the gene locus of the human cell proliferation-associated nuclear protein defined by monoclonal antibody Ki-67.

The human antigen defined by the monoclonal antibody Ki-67, the 'Ki-67 protein', is an ubiquitously expressed human nuclear protein strictly associated with cell proliferation and is widely used in routine pathology as a 'proliferation marker' to measure the growth fraction in human tumours. In immunoblots of proteins from proliferating cells, Ki-67 detects two bands with the apparent molecular weights of 345 and 395 kDa. Recently we reported on the cloning and sequencing of the complete cDNA of the Ki-67 protein. We found two isoforms of cDNA with full lengths of 11.4 and 12.5 kb, respectively, likely formed by the alternative splicing of exon 7. The remarkable exon 13 at the 'centre' of this gene contains 16 homologous segments of 366 bp (Ki-67 repeats), each including a highly conserved new motif of 66 bp (Ki-67 motif). Computer analyses confirmed that the cDNAs encode for a new class of nuclear proteins. The complete gene locus of the Ki-67 protein, comprising a 74 bp 5' region and a 264 bp 3' region, has been sequenced and aligned to a continuous sequence of 29,965 bp length located on chromosome 10q25-ter. The gene is organized in 15 exons with sizes from 67 to 6845 bp and in 14 introns with sizes from 87 to 3569 bp. Three introns contain homologue copies of 'Alu-repeats'. Interestingly, the introns flanking the alternative spliced exon 7 are free of any consensus donor and acceptor splicing signal. All other intron-exon transitions contained a potential branch site. The complete 5' region including the first two exons represents a CpG-rich island. We found the transcription initiation site in exon two adjacent to the consensus sequence of a cap site. Upstream of this cap site no TATA- or CCAAT-box could be located, but downstream we found two remarkable directly repeated elements of 24 bp lengths each containing a TATA box in inverse orientation.

Amino Acid Sequence↗
PubMed1995

Lipopolysaccharide-induced change of phosphorylation of two cytosolic proteins in human monocytes is prevented by inhibitors of ADP-ribosylation.

Interaction of LPS with human monocytes causes altered phosphate labeling of cytosolic proteins of 36 kDa and 38 kDa (p36/38). This property, determined by in vitro studies, is shared by other monocyte activators. Phosphorylated p36/38 are distinct from p38, 42-kDa, and 44-kDa isoforms of mitogen-activated protein kinases expressed in monocytes. Occupation of LPS binding sites by a LPS antagonist, the synthetic tetraacylated bisphosphate precursor of Escherichia coli lipid A (also known as compound 406, lipid IVa, or precursor Ia), prevents LPS-induced changes in the phosphate labeling of the two proteins. Abs against CD14 inhibit protein phosphorylation induced by low concentrations of LPS (10 ng/ml), whereas at high concentrations (1 microgram/ml), the Abs fail to prevent phosphorylation. In addition to phosphorylation, ADP-ribosylation of proteins has been implicated in a number of biologic processes. Here we show that inhibitors of ADP-ribosylation, namely meta-iodobenzylguanidine and nicotinamide, inhibit LPS-initiated altered phosphorylation of p36/38. This loss of phosphate labeling of p36/38 is accompanied by an inhibition of TNF-alpha and Il-6 mRNA and protein production. The synthesis of IL-1 is not affected. This suggests that the inhibitors interfere with specific steps in IL-6 and TNF-alpha production, which are not required for IL-1 synthesis. Taken together, the data indicate that ADP-ribosylation may be involved in LPS-induced alteration of the phosphorylation state of two cytosolic proteins (p36/38) and that these proteins modulate cellular processes leading to TNF-alpha and IL-6 release.

3-Iodobenzylguanidine↗
PubMed1995

Limited and defined truncation at the C terminus enhances receptor binding and degranulation activity of the neutrophil-activating peptide 2 (NAP-2). Comparison of native and recombinant NAP-2 variants.

We have previously described a C-terminally truncated variant of the chemokine neutrophil-activating peptide 2 (NAP-2) that exhibited higher neutrophil-stimulating capacity than the full-size polypeptide. To investigate the impact of the NAP-2 C terminus on biological activity and receptor binding, we have now purified the novel molecule to homogeneity. Furthermore, we have cloned, expressed in Escherichia coli, and purified full-size recombinant NAP-2 (rNAP-2-(1-70)) and a series of C-terminally deleted variants (rNAP-2-(1-69) to rNAP-2-(1-64)). Biochemical and immunochemical analyses revealed that the natural NAP-2 variant was structurally identical to the rNAP-2-(1-66) isoform. As compared with their respective native and recombinant full-size counterparts, both molecules exhibited approximately 3-4-fold enhanced potency in the induction of neutrophil degranulation as well as 3-fold enhanced binding affinity for specific receptors on these cells. All other variants were considerably less active. The natural occurrence of a NAP-2 variant truncated by exactly four residues at the C terminus suggests that limited and defined proteolysis at this site plays a role in the regulation of the biological function of the chemokine.

Amino Acid Sequence↗
PubMed1995

IL-8 specifically binds to endothelial but not to smooth muscle cells.

The proinflammatory cytokine and potent chemoattractant IL-8 is involved in regulation of infectious or inflammatory processes. Human vascular endothelial cells (EC) and smooth muscle cells (SMC) probably contribute to these responses by recognition and/or production of rIL-8. We demonstrate here in competitive binding studies with radiolabeled rIL-8 that EC and fibroblasts, but not SMC, specifically bind IL-8 with low affinity. The binding was not saturated by ligand concentrations up to 80 nM 125I-rIL-8. Unlabeled neutrophil-activating peptide-2 competed the binding of 125I-rIL-8, although less potently than unlabeled rIL-8, as reported previously for polymorphonuclear neutrophils. In contrast, connective tissue-activating peptide III, platelet factor 4, or lysozyme did not reduce binding of 125I-rIL-8 to EC or fibroblasts. In accordance with these binding studies, EC and fibroblasts, but not SMC, expressed human IL-8 receptor type I mRNA. Neither cell type expressed mRNA for IL-8 receptor type II. Stimulation with IL-1 alpha or LPS did not alter the results obtained in PCR or binding studies. Although SMC did not express specific binding sites for IL-8, Western blot experiments showed that IL-1 alpha-, TNF-, or LPS-stimulated SMC released two major immunoreactive isoforms of IL-8 in a time- and dose-dependent manner. The m.w. were similar to IL-8 isoforms released by EC or mononuclear cells. The differential capacity of EC and SMC to produce IL-8 and express IL-8 binding sites indicates that vascular cell-derived IL-8 may contribute to differential regulation of infectious and inflammatory responses in the vessel wall.

Base Sequence↗
PubMed1995

An improved method for the species-specific assessment of mycobacteria in routinely formalin-fixed and paraffin-embedded tissues.

A polymerase chain reaction (PCR) assay for the rapid and species-specific diagnosis of mycobacterial infections in paraffin-embedded clinical specimens was developed using oligonucleotide primers to amplify a fragment of the DNA coding for the ribosomal 16S RNA of mycobacteria. The oligonucleotide primers amplified DNA from all 14 species of mycobacteria tested. By means of a reamplification protocol, as few as one to two mycobacteria could be detected in the presence of human DNA. The method of DNA isolation and amplification was applied on sections of routinely formalin-fixed and paraffin-embedded tissues. PCR for the beta-actin gene served as a control for successful DNA isolation. Mycobacterial DNA could be detected in cases of mycobacterial infections. The mycobacterial species was determined by additional sequencing of the PCR fragment. This PCR method may be a powerful tool for the diagnosis of mycobacterial infections from histopathological material and for the assessment of those mycobacteria that cannot readily be cultured, such as Mycobacterium leprae.

Base Sequence↗
PubMed1995

The human germinal centre cells, follicular dendritic cells and germinal centre T cells produce B cell-stimulating cytokines.

Cytokine gene expression was investigated in the germinal centre constituents of follicular dendritic cells and germinal centre T cells and compared to the mRNA expression of nongerminal centre tonsillar cells. Cells were isolated from human tonsils by preenrichment with MACS and subsequent FACS sorting. Cytokine gene expression was investigated by intronspanning RT-PCR for IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IFN-alpha, IFN-beta, IFN-gamma, and TNF-alpha. Frequency of cytokine-producing cells and the cytokine production pattern of single cells were determined by single cell PCR. Furthermore, cytokine protein expression was investigated by immunohistology. Using these methods, we found a strong production of IL-1 beta mRNA and protein in a small percentage of FDC. Germinal centre T cells showed production of IL-2, IL-4, IL-10, IFN-gamma, and TNF-alpha mRNA. The mRNAs of these cytokines could also be detected at the single cell level; as they were produced in a high percentage of germinal centre T cells, whereas immunohistological staining was negative, we conclude that a high percentage of germinal centre T lymphocytes produce IL-2, IL-4, IL-10, IFN-gamma, and TNF-alpha in low concentrations in contrast to T cells outside the germinal centre, in which strong cytokine production in few cells was shown earlier.

B-Lymphocytes↗