Biomedical subjects

H D Flad

Publications and source records attributed to H D Flad.

At least 91 records · Page 5Linked to original sources

PubMed1995

Follicular dendritic cells productively infected with immunodeficiency viruses transmit infection to T cells.

Lymphoid organs have been proposed to function as the major reservoir for the human immunodeficiency virus type 1 (HIV-1). Within lymphatic tissues germinal centers represent foci of rapidly proliferating B cells governed by the interaction between B and T cells and follicular dendritic cells (FDC). Accumulating evidence suggests an important role of FDC in the pathophysiology of the acquired immunodeficiency syndrome. Direct proof for the infectibility of FDC with HIV-1 has been lacking until recently when we were able to demonstrate a CD4-independent infection of FDC in vitro. Here we report that in vitro HIV-1-infected human FDC do not only contain proviral DNA, but also produce the virus, and transmit the infection to T cells. Furthermore, electron microscopical studies on ex vivo isolated FDC from simian immunodeficiency virus (SIV)-infected rhesus monkeys revealed typical virus budding. In addition, FDC from SIV-infected rhesus monkeys transmitted the infection to T cells in vitro. Due to this central role within the immune response FDC may serve as preferential targets for HIV both by trapping of virions on their surfaces and by productive infection. During disease FDC become productively infected and may, thus, be regarded as crucial elements in viral dissemination.

Animals↗

PubMed1995

The induction of bacillus-Calmette-Guérin-activated killer cells requires the presence of monocytes and T-helper type-1 cells.

Previously we have described the induction of MHC-unrestricted killer cells against bladder tumour cells by bacillus Calmette-Guérin (BCG), termed BCG-activated killer (BAK) cells. In the present paper we deal with the accessory-cell requirement for the activation of BAK cells. We show that monocytes are required for activating BAK cells, since no cytotoxicity can be induced in the absence of monocytes. Therefore, these phagocytes may represent the first step during the activation cascade of BAK cells. Furthermore, the presence of CD4+ T cells was essential for generating BAK cells: depleting peripheral blood mononuclear cells of CD4+ cells prior to stimulation with BCG abolished the cytotoxicity against bladder tumour cells. Experiments with monoclonal antibodies (mAb) neutralizing the activity of either interleukin-2 (IL-2) or interferon gamma (IFN gamma) underlined the importance of these cytokines: both mAb blocked the induction of BAK cells. Since both cytokines are related to the so-called Th1 pattern of T cells, we consider the second step of the generation of BAK cells as follows: monocytes presenting antigens of BCG trigger Th1-like cells in a preferred manner. These Th1-like T cells secrete IL-2 and IFN gamma and, thus, activate the BAK effector cells. Since CD4+ cells are dominant in the cells infiltrating the bladder wall after intravesical instillation of BCG in vivo, we postulate an important role for the Th1 subpopulation. We further postulate that the occurrence of macrophages in this infiltrate seems to be significant in the maintenance of the relapse-free state of the patient.

CD4-Positive T-Lymphocytes↗

PubMed1995

Elimination of monocytes from cultures activated with recall antigens.

Recent data provide evidence that antigen-specific CD4+ T-cell clones or antigen-activated T-cell lines can kill antigen-presenting cells (APC). We focused our studies on monocytes acting as APC in cultures of T cells freshly isolated from peripheral blood. The presence of monocytes in culture was monitored by their ability to emit light during phagocytosis of latex particles (latex-induced chemiluminescence). Using this approach as well as flow cytometry, evidence is presented that monocytes are eliminated from cultures with T cells activated with recall antigens (PPD or TT). The mechanism of monocyte elimination involved apoptosis as judged from in situ detection of DNA strand breaks by the terminal deoxynucleotidyl transferase assay. The antigen- but not lectin-dependent monocyte elimination was MHC-restricted and mediated by CD4+ T lymphocytes. This finding supports the hypothesis that elimination of APC is a general phenomenon during T-cell activation and may represent an important immunoregulatory mechanism.

Antigen-Presenting Cells↗

PubMed1995

Presence of mRNA for interferon-gamma (IFN-gamma) in blood mononuclear cells is associated with an active stage I sarcoidosis.

Peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage (BAL) cells obtained from sarcoidosis patients were investigated for the presence of mRNA for IFN-gamma. RNA was extracted from freshly obtained cells. In 12 cases PBMC were studied and in seven cases BAL cells were also available for concomitant investigation. In eight patients mRNA for IFN-gamma was present in blood and was significantly associated with stage I disease (P = 0.030). By comparison, BAL cells were less frequently positive for IFN-gamma transcripts (P = 0.039). Only one patient had simultaneous expression of IFN-gamma gene in blood and BAL cells. All other patients including four positive in blood were negative in BAL. The presence of IFN-gamma transcripts in PBMC was significantly associated with the capacity of these cells to generate IFN-gamma in unstimulated cultures. PBMC from patients showing IFN-gamma mRNA in blood were frequently high IFN-gamma producers in anti-CD3 MoAb-stimulated cultures.

Adult↗

PubMed1995

Endotoxin activates human vascular smooth muscle cells despite lack of expression of CD14 mRNA or endogenous membrane CD14.

During infection or inflammation, cells of the blood vessel wall, such as endothelial cells (EC) and smooth muscle cells (SMC), contribute to the regulation of the immune response by production of cytokines or expression of adhesion molecules. Little is known about the mechanism(s) involved in the stimulation of vascular cells by endotoxin (lipopolysaccharide [LPS]). As reported previously, LPS antagonists reduce LPS-induced cytokine production or adhesion in vitro specifically, suggesting a specific LPS recognition mechanism. We thus investigated the role of CD14 for stimulation of vascular SMC by LPS. Complement-fixing antibodies directed against CD14 (LeuM3, RoMo I, or Mo2) lysed monocytes but failed to mediate lysis of EC or SMC, indicating the lack of endogenous membrane CD14 in vascular cells. In addition, we did not detect expression of CD14 protein on EC and SMC in cell sorting analysis or cell immunoassay experiments. These observations are in line with our finding that a CD14 probe did not hybridize with mRNA or EC or SMC in Northern (RNA) blot experiments, although it hybridized well with monocyte-derived mRNA. We obtained the same results with the much more sensitive reverse transcription-PCR. Since the vascular SMC did not express endogenous CD14, we investigated the role of human serum-derived soluble CD14 (sCD14) for activation of SMC by LPS. In medium containing human serum, anti-CD14 antibodies inhibited activation of SMC by LPS. In contrast, the same antibodies did not inhibit activation of cells cultured in medium containing fetal calf serum. SMC cultured in sCD14-depleted medium responded 1,000-fold less to LPS than cells cultured in presence of sCD14. Reconstitution of sCD14-depleted serum or supplementation of serum-free medium with recombinant CD14 restored the capacity of the cells to respond to LPS. These results show that specific activation of vascular SMC by LPS does not involve binding to endogenous membrane CD14, but that the activation of vascular SMC by LPS is mediated to a great extent by serum-derived sCD14.

Antigens, CD↗

PubMed1995

Binding of lipopolysaccharide (LPS) to an 80-kilodalton membrane protein of human cells is mediated by soluble CD14 and LPS-binding protein.

Activation of cells by bacterial lipopolysaccharide (LPS) plays a key role in the pathogenesis of gram-negative septic shock. The 55-kDa glycoprotein CD14 is known to bind LPS and initiate cell activation. However, there must be additional LPS receptors because CD14 is linked by a glycosylphosphatidyl inositol anchor to the cell membrane and therefore unable to perform transmembrane signalling. Searching for potential LPS receptors, we investigated the binding of LPS to membrane proteins of the human monocytic cell line Mono-Mac-6. Membrane proteins were electrophoretically separated under reducing conditions, transferred to nitrocellulose, and exposed to LPS, which was visualized with anti-LPS antibody. Smooth- and rough-type LPS, as well as free lipid A, bound to a variety of proteins in the absence of serum. However, in the presence of serum, additional or preferential binding to a protein of approximately 80-kDa was observed. Experiments with differently acylated lipid A structures showed that the synthetic tetraacyl compound 406 was still able to bind, whereas no binding was detected with the bisacyl compound 606. The 80-kDa membrane protein was also detected on human peripheral blood monocytes and endothelial cells. The serum factors mediating the binding of lipid A to the 80-kDa membrane protein were identified as soluble CD14 and LPS-binding protein. From these results, we conclude that this 80-kDa protein is a candidate for the hypothetical molecule for LPS and/or LPS-CD14 recognition and signal transduction.

Acute-Phase Proteins↗

PubMed1995

Glycosphingolipids from Sphingomonas paucimobilis induce monokine production in human mononuclear cells.

Glycosphingolipids (GSL) isolated from the gram-negative lipopolysaccharide (LPS)-free bacterium Sphingomonas paucimobilis have remarkable structural similarities with LPS and its hydrophobic part, termed lipid A. Like LPS, but in contrast to the structurally related ceramides and cerebrosides, GSL contain an alpha-linked, negatively charged pyranosidic glycosyl component adjacent to the lipid portion and are capable of forming membranes. Because of these similarities, it was of interest to investigate whether these GSL are also able to induce monokine production in human mononuclear cells (MNC). Our results show that a GSL containing four sugar residues (GSL-4A) induced the release of tumor necrosis factor, interleukin-6, and interleukin-1 in MNC, whereas GSL-1, containing only one glycosyl residue, was inactive. A minimal concentration of 1 microgram of GSL-4A per ml was necessary to induce monokine production in MNC, whereas LPS was as active at a 10,000-fold-lower concentration (0.1 ng/ml). Both GSL-4A-induced monokine production and LPS-induced monokine production were reduced by the bactericidal/permeability-increasing protein and GSL-1. In contrast to LPS, GSL-4A-induced monokine release could be inhibited neither by an anti-CD14 monoclonal antibody nor by lipid A partial structures. We therefore conclude that at the receptor level, different mechanisms are involved in the LPS- and GSL-4A-induced monokine release.

Antigens, CD↗

PubMed1995

CD26 expression in leprosy and other granulomatous diseases correlates with the production of interferon-gamma.

BACKGROUND: Leprosy represents a spectrum of clinical manifestations that reflect the immune response to antigens of Mycobacterium leprae. The tuberculoid form of leprosy, which is characterized by an organized development of granulomas, has recently been correlated with a Th1-like immune response. The lepromatous form of leprosy, with a characteristic lack of cellular immunity, has been correlated with a Th2-like immune response to mycobacterial antigens. Dipeptidylpeptidase IV (CD26) is an ectopeptidase that is expressed in various tissues; in the hemopoietic system, it is predominantly expressed by T cells. EXPERIMENTAL DESIGN: We stained frozen sections of skin biopsies obtained from patients with different forms of leprosy, sarcoidosis, and Piringer's lymphadenitis. Sections were stained for interferon-gamma (IFN-gamma) and CD26 with the alkaline phosphatase anti-alkaline phosphatase technique and in two-color stainings by immunofluorescence. RESULTS: We found strong signals for IFN-gamma and for CD26 in all investigated cases of tuberculoid leprosy. In contrast, in all biopsies taken from patients with lepromatous leprosy, we found no or very weak signals for these antigens. By immunofluorescence double-labeling, we could show that IFN-gamma and CD26 were expressed by the identical cell population. We confirmed this correlation of CD26 expression and IFN-gamma production in other granulomatous inflammatory reactions such as sarcoidosis and Piringer's lymphadenitis. CONCLUSIONS: From our results, we conclude that a high expression of CD26 may be suggestive of Th1-like immune reactions.

Dipeptidyl Peptidase 4↗

PubMed1995

Antibody-induced modulation of CD26 surface expression.

The ability of different anti-CD26 monoclonal antibodies to modulate the expression of CD26 on human T lymphocytes was investigated. By means of a new non-radioactive method using fluorescein isothiocyanate (FITC)-labelled and unlabelled anti-CD26 monoclonal antibodies and flow cytometry, we measured the internalization and re-expression of CD26 on freshly isolated resting human T lymphocytes. The modulation of CD26 surface expression takes place in primarily CD26+ as well as in CD26- T lymphocytes, indicating the presence of an intracellular CD26 pool. In fact, with two different anti-CD26 monoclonal antibodies (Ta1 and M5) intracellular CD26 was detected out of which newly expressed CD26 might have originated. This intracellular CD26 pool appears to be maintained by continuous translation of CD26 mRNA.

Antibodies, Monoclonal↗

PubMed1995

Preactivation and phenotype of monocytes have no influence on their elimination from culture by activated T lymphocytes.

T lymphocytes can kill antigen-presenting cells (APC) in the presence of antigen or lectin. The subject of this study was to investigate whether the state of activation or phenotype of monocytes, influence their susceptibility to killing by T cells activated with pokeweed mitogen (PWM) or anti-CD3 monoclonal antibody. The data are presented which show that monocytes activation with cytokines (IFN-gamma, IL-4, or IL-2), PPD, phorbol ester or phagocytic stimulus, have no influence on monocyte susceptibility to killing by T lymphocytes. Furthermore, flow cytometry data suggest that monocytes eliminated from culture have no characteristic phenotype. In conclusion, our data indicate that elimination of monocytes by activated T lymphocytes does not depend on the state of activation of monocytes.

Cell Death↗

PubMed1995

Cell proliferation-associated nuclear antigen defined by antibody Ki-67: a new kind of cell cycle-maintaining proteins.

A decade of studies on the human nuclear antigen defined by monoclonal antibody Ki-67 (the "Ki-67 protein") has made it abundantly clear that this structure is strictly associated with human cell proliferation and that the expression of this protein can be used to assess the growth fraction of a given cell population. Until recently the Ki-67 protein was described as a nonhistone protein that is highly susceptible to protease treatment. We have isolated and sequenced cDNAs encoding for this antigen and found two isoforms of the full length cDNA of 11.5 and 12.5 kb, respectively, sequence and structure of which are thus far unique. The gene encoding the Ki-67 protein is organized in 15 exons and is localized on chromosome 10. The center of this gene is formed by an extraordinary 6845 bp exon containing 16 successively repeated homologous segments of 366 bp ("Ki-67 repeats"), each containing a highly conserved new motif of 66 bp ("Ki-67 motif"). The deduced peptide sequence of this central exon possess 10 ProGluSerThr (PEST) motifs which are associated with high turnover proteins such as other cell cycle-related proteins, oncogenes and transcription factors, etc. Like the latter proteins the Ki-67 antigen plays a pivotal role in maintaining cell proliferation because Ki-67 protein antisense oligonucleotides significantly inhibit 3H-thymidine incorporation in permanent human tumor cell lines in a dose-dependent manner.

Amino Acid Sequence↗

PubMed1995

Cytokines in mycobacterial infections: in vitro and ex vivo studies.

Different species of mycobacteria differ in their capacity to induce the production of tumor necrosis factor-alpha (TNF-alpha) by human monocytes in vitro. Whereas M. tuberculosis is a potent inducer of TNF-alpha, M. leprae is much less potent. TNF-alpha production is found to be associated with the availability of H2O2 generated by activated monocytes, as superoxide enhancing H2O2 concentration increases and catalase degrading H2O2 decreases TNF-alpha production. Furthermore, M. kansasii with high intrinsic catalase induce less TNF-alpha than mycobacteria with low intrinsic catalase. In vitro infection of monocytes with M. tuberculosis leads to an impairment of the antigen-presenting capacity, as determined by a reduction of antigen-induced T cell proliferation and interferon gamma (IFN-gamma) production. Of crucial importance is this impairment is the M. tuberculosis-induced down-modulation of MHC class II antigens. The role of TNF-alpha in vivo is reflected in patients with various forms of leprosy. In skin lesions of lepromatous leprosy patients TNF-alpha, interleukin 1 beta (IL-1 beta), and INF-gamma production are found to be rare, whereas these cytokines are well expressed in skin lesions of patients with tuberculoid leprosy. After multidrug chemotherapy an increase of local cytokine production is found. Taken together, these findings suggest that components of mycobacteria may interfere with local cell-mediated immune reactions in vivo. The molecular mechanisms involved in these local responses need to be defined.

Antigen-Presenting Cells↗

PubMed1994

Connective tissue-activating peptide III desensitizes chemokine receptors on neutrophils. Requirement for proteolytic formation of the neutrophil-activating peptide 2.

The connective tissue-activating peptide III (CTAP-III), which is released from activated platelets, represents an inactive precursor of the chemokine neutrophil-activating peptide 2 (NAP-2). Leukocytes and leukocyte-derived proteases have been found to convert CTAP-III into NAP-2 by proteolytic cleavage at the N terminus. We demonstrate here that rapid and efficient formation of NAP-2 is mediated by neutrophil granulocytes (PMN) but not by monocytes or lymphocytes. However, as seen in a degranulation assay, neutrophils processing CTAP-III did not become activated by the generated NAP-2 and even exhibited decreased responsiveness to high doses of NAP-2 or IL-8, but not to FMLP. The desensitizing effect, being maximal already after 5 min of preincubation with CTAP-III, was not mediated through binding of the precursor to specific receptors but correlated with the rapid down-modulation of common NAP-2/IL-8 high affinity binding sites. A similar functional and receptor desensitization was observed in PMN pre-exposed to nonstimulatory doses of NAP-2. Specific inhibition of the CTAP-III-cleaving enzyme by the serine protease inhibitor aprotinin abrogated the CTAP-III, but not the NAP-2-mediated effects. Desensitization of PMN by CTAP-III was due to NAP-2 generated by proteolytic truncation of CTAP-III. Our results suggest that CTAP-III may regulate PMN activation by protecting processing cells from premature activation.

Blood Coagulation Factors↗

PubMed1994

Endotoxin and lipid A stimulate proliferation of human T cells in the presence of autologous monocytes.

In this paper we describe a new activity of LPS and partial structures: the induction of DNA synthesis and lymphokine production of human T lymphocytes. The LPS-induced T cell proliferation is dose dependent and requires 100 to 10,000 ng/ml of LPS or synthetic lipid A (compound 506) for optimal stimulation. In contrast, the synthetic lipid A precursor Ia (compound 406) is not active but rather antagonizes LPS-induced proliferation. The proliferation is accompanied by the expression of mRNA for the Th1 cell-derived lymphokines IFN-gamma and IL-2, but not for the Th2 lymphokines IL-4, IL-5, or IL-10. Highly enriched T lymphocyte preparations with less than 0.1% monocytes are not stimulated by LPS, showing that monocytes are required for T cell proliferation. Reconstitution experiments show that only monocytes, but not B lymphocytes, are able to support induction of DNA synthesis. Separating LPS-stimulated monocytes from T lymphocytes by a membrane, permeable for cytokines but not for cells, abolishes T cell proliferation. Fixation of monocytes with paraformaldehyde also abrogates their accessory function for T cells. If the monocytes are preincubated for 2 h at 37 degrees C with LPS and then washed, they still are able to induce T cell proliferation in the absence of additional LPS. Our results indicate that human T cells respond in a monocyte-supported manner to LPS exposure by proliferation and lymphokine production. We hypothesize that this reactivity of T lymphocytes to LPS may be of clinical relevance.

Adult↗

PubMed1994

Neutrophil-activating peptides NAP-2 and IL-8 bind to the same sites on neutrophils but interact in different ways. Discrepancies in binding affinities, receptor densities, and biologic effects.

IL-8 and the neutrophil-activating peptide 2 (NAP-2) are members of the chemokine family of host defense cytokines. Although IL-8 was shown to interact with two different high affinity receptors on polymorphonuclear neutrophil granulocytes (PMN), direct demonstration of specific binding sites for NAP-2 is difficult, because the NAP-2 molecule lacks iodinable side chains. Here we present a modified labeling procedure for the chemokine that does not affect its biologic activity. The 125I-labeled NAP-2 specifically bound to PMN with two different affinities (KD = 0.65 and 22.4 nM). We observed complete cross-competition of unlabeled IL-8 with 125I-labeled-NAP-2 and of unlabeled NAP-2 with 125I-labeled IL-8, indicating the absence of monospecific binding sites for either chemokine. However, in contrast to former work by others, the total number of accessible sites was considerably lower for NAP-2 (13,000/cell) than for IL-8 (59,000/cell). In addition, PMN prepared from heparinized blood expressed significantly more receptors for NAP-2 than cells prepared from citrated blood, whereas receptor numbers for IL-8 were unchanged. Desensitization experiments suggested a regulatory role for the NAP-2 high affinity site. Short-term priming of PMN with a nonstimulatory dose of NAP-2 (or MGSA) but not with IL-8 led to drastic down-regulation of the subsequent degranulation response, challenged by higher dosages of NAP-2, MGSA, or IL-8. Reduced functional responsiveness of cells correlated with the rapid down-regulation and internalization of NAP-2 and IL-8 high affinity binding sites. Thus, our data indicate that chemokines could mediate by individual modes of interaction with common receptor's different biologic functions.

Binding Sites↗

PubMed1994

Nitric oxide synthase: mRNA expression of different isoforms in human monocytes/macrophages.

To detect mRNA expression of nitric oxide synthase (NOS) isoforms in human monocytes/macrophages reverse transcription polymerase chain reaction (RT-PCR) was used. mRNA was isolated from stimulated or unstimulated monocytes/macrophages and RT-PCR was performed using oligonucleotide primers derived from mRNA sequences of either human endothelial constitutive (c) or human hepatocyte inducible (i) NOS. RT-PCR of mRNA isolated from resting monocytes and macrophages resulted in the amplification of a cNOS specific mRNA fragment. When the cells were stimulated with lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) prior to mRNA extraction, RT-PCR yielded an iNOS-specific amplification product. Whereas the activation of both cell types was accompanied by expression of iNOS mRNA, the cNOS signal seemed to be diminished upon immunostimulation. Not only in purified human monocytes but also in the human monocytoid cell lines MonoMac 6, THP-1, and U937 cNOS mRNA was detected. The data clearly demonstrate the presence of iNOS and cNOS mRNA in human monocytes/macrophages and provide the necessary tools to investigate the regulation of NO synthesis in these cell populations.

Amino Acid Oxidoreductases↗

PubMed1994

HIV-1 envelope protein gp120 affects phenotype and function of monocytes in vitro.

We investigated the effect of human immunodeficiency virus type 1 (HIV-1) recombinant gp120 (rec.gp120) on phenotype and function of cultured monocytes. Rec.gp120 significantly reduced the accessory function of monocytes to stimulate autologous lymphocytes with anti-CD3, the Fc receptor-mediated chemiluminescence of monocytes, and the expression of CD4 and Fc receptor I/II, while the expression of the monocyte marker CD14 and major histocompatibility complex class I and II was not influenced. According to these phenotypic results, preincubation of monocytes with rec.gp120 depressed anti-CD3 antibody-induced T cell stimulation and Fc receptor-mediated phagocytosis as determined by chemiluminescence. Interferon-gamma release of lymphocytes induced by purified protein derivative of tuberculin was enhanced by gp120. These effects of isolated gp120 on monocyte immune functions in vitro might contribute to the understanding of the pathophysiology of HIV-1 infection in vivo.

Antigen Presentation↗

PubMed1994

Specific binding of bacillus Calmette-Guérin to urothelial tumor cells in vitro.

Intravesical immunotherapy with bacillus Calmette-Guérin (BCG) against recurrences of superficial bladder cancer and carcinoma in situ is a highly effective regimen in urology. Despite intensive efforts to clarify the immunological mechanisms of the most successful immunotherapy known today, the cellular mechanism of its antitumor activity remains unknown. In our approach to elucidate the way of action of intravesical BCG, we applied an in vitro adhesion assay to investigate the interaction of radiolabeled BCG with urothelial bladder-tumor cells. We demonstrated a BCG dose-dependent binding to bladder-tumor cell lines derived from tumors of different gradings. The binding of BCG is apparently specific, since competition experiments showed an inhibition by nonradioactive BCG but not by Escherichia coli. We also found that there was no difference between the binding of living or heat-killed mycobacteria. Control experiments showed only a low affinity of BCG for fibroblasts, smooth-muscle cells, and endothelial cells in comparison with the tumor cells. Furthermore, we investigated the role of fibronectin as an adhesion molecule that is also present in the bladder wall. We demonstrated that BCG was capable of binding to fibronectin-coated surfaces in a dose-dependent manner. However, competitive binding assays failed to reveal an inhibition of the binding of BCG to bladder-tumor cells by anti-fibronectin. Furthermore, binding was not influenced by soluble fibronectin. These data suggest that the in vitro attachment of BCG to bladder-tumor cells appears not to be mediated by fibronectin. In electron microscope studies an adhesion of BCG to bladder-tumor cells was observed after an incubation period of ony 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

BCG Vaccine↗