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H D Flad

Publications and source records attributed to H D Flad.

At least 145 records · Page 8Linked to original sources
PubMed1991

Cellular cytotoxicity of human natural killer cells and lymphokine-activated killer cells against bladder carcinoma cell lines.

The cytotoxicity of natural killer (NK) and lymphokine activated killer (LAK) cells against two human bladder tumor cell lines (BT-A and BT-B) was investigated using a fluorometric assay by labeling tumor cell DNA with Hoechst dye No. 33342. Our results demonstrate that BT-A and BT-B cells have low sensitivity to the cytotoxic activity of mononuclear cells (MNC) and NK cells. Cytotoxicity of MNC or NK cells against both tumor cell lines is enhanced during co-culture of the effector cells with the target cells, which suggests that BT-A and BT-B cells provide the signals which could activate MNC to exert cytotoxicity. In contrast to NK cells, IL-2-generated LAK cells showed profound cytotoxicity to BT-A and BT-B within 24 h. In addition to cellular cytotoxicity to bladder tumor cells, we also tested the effect of recombinant interleukin 1 beta (rIL-1 beta), recombinant tumor necrosis factor (rTNF), and the supernatants of co-culture of MNC or LAK cells with bladder tumor cells. The results show no cytotoxic or growth-promoting activity of rIL-1, rTNF, or the crude culture supernatants on bladder tumor cells. We found that LAK cells, but not macrophages or NK cells, may play a major role in cellular cytotoxicity against the two bladder tumor cell lines tested. From this finding we conclude that activation of LAK cells may be one important mechanism induced by adjuvant bacillus Calmette-Guérin (BCG) therapy leading to effective prevention of urothelial bladder carcinoma reappearance.

Animals↗
PubMed1991

Enhancement of monocyte antimycobacterial activity by diethyldithiocarbamate (DTC).

Diethyldithiocarbamate (DTC) has been recently reported to significantly reduce the incidence of opportunistic infections in HIV-infected patients. The present study addresses the question whether DTC is capable of stimulating antimycobacterial activity of mononuclear phagocytes. We found that peripheral blood mononuclear cells (PBMC) of healthy subjects preincubated in vitro with 100-1000 ng/ml of DTC and thereafter infected with Mycobacterium tuberculosis H37Rv or Mycobacterium avium-intracellulare complex exhibited an enhanced antimycobacterial activity compared with control-incubated cells as assessed by the determination of mycobacterial colony-forming units. In subsequent experiments monocytes from healthy volunteers injected with 5 mg/kg body weight of DTC were tested ex vivo for antimycobacterial activity at various periods of time after injection. Injection of DTC resulted in a significant enhancement of antimycobacterial activity which was most evident 24 h after DTC injection. We conclude that DTC stimulates the antimicrobial function of mononuclear phagocytes both in vitro and in vivo. These results may explain the favourable clinical course observed in HIV-infected patients treated with DTC and may serve as a basis for treatment with DTC in patients with drug-resistant atypical mycobacteriosis.

Blood Bactericidal Activity↗
PubMed1991

Neutrophils can generate their activator neutrophil-activating peptide 2 by proteolytic cleavage of platelet-derived connective tissue-activating peptide III.

We have investigated the conditions that lead to the generation of the neutrophil-activating peptide 2 (NAP-2) from its precursor, the platelet-derived connective tissue-activating peptide III (CTAP-III). Lysed platelets were found to contain predominantly CTAP-III in the cytosolic fraction, but further truncated derivatives, among these NAP-2, occurred tightly bound to the membrane fraction of fresh platelets. NAP-2 biological activity, as measured by the induction of enzyme release in human neutrophils [polymorphonuclear leukocytes (PMN)] was released by stimulated platelets to a low degree. Much higher activities were formed in the presence of peripheral blood leukocytes. Coincubation of CTAP-III with PMN resulted in the almost complete conversion of the precursor to NAP-2, as did incubation of CTAP-III with PMN-conditioned medium. In both situations, the generation of NAP-2 could be prevented by serine-protease inhibitor phenylmethylsulfonyl fluoride but not by inhibitors specific for Ca(2+)-dependent or thiol proteases. From several PMN-derived proteases tested, only cathepsin G had the capacity to cleave CTAP-III into NAP-2 with high specificity and in a relatively short period of time (30 min). Our data indicate that NAP-2, released by platelets in small quantities, could cause PMN to enter into a positive feedback cycle by initiating the secretion of serine proteases, which in turn could convert platelet-derived CTAP-III into biologically active NAP-2.

Amino Acid Sequence↗
PubMed1991

Expression of CD26 (dipeptidyl peptidase IV) on resting and activated human T-lymphocytes.

CD26 is an activation antigen which is expressed on the surface of human T-lymphocytes. It has been characterized to be the dipeptidyl peptidase IV (DPP IV). Considerable amounts of CD26 are already present on resting T-lymphocytes. The expression of CD26 is enhanced by T-cell mitogens or antigens. A correlation of CD26 expression and of enhanced enzymatic activity was observed after T-cell activation. Our data indicate that not only the immunoreactivity, but also the enzymatic activity of CD26 are detectable on the cell surface. In addition, de novo expression of CD26 was demonstrated on CD26-negative T-cells after mitogenic or antigenic stimulation. CD26 expression is initiated during the G1 phase of the cell cycle. The expression occurs nearly simultaneously with HLA-DR, but later than CD25. Similar to CD25 and HLA-DR, CD26 is not a permanent marker on the surface of T-lymphocytes, but is down-regulated after 7 days of culture. When testing the influence of interleukin 1, interleukin 2, tumour necrosis factor, and interferon-gamma on the expression of CD26, no effect was found on unstimulated or on mitogen-stimulated T-lymphocytes. The binding of two different monoclonal antibodies against CD26 (anti-DPP IV and anti-Tal) to resting and activated T-lymphocytes revealed a different pattern of immunoreactivity. Resting T-lymphocytes reacted stronger with anti-DPP IV than with anti-Tal. However, binding of the two monoclonal antibodies to T-cell blasts did not show significant differences. These data indicate that CD26 may be expressed in differently modulated configurations on the surface of T-cells, which may be associated with a distinct status of activation and/or function.

Amino Acid Sequence↗
PubMed1991

Inhibition of endotoxin-induced interleukin-6 production by synthetic lipid A partial structures in human peripheral blood mononuclear cells.

The effect of two synthetic lipid A partial structures, compound 406 (or LA-14-PP, identical in structure to the lipid A precursor, known as Ia or IVa) and compound 401 (lipid X), on the in vitro modulation of endotoxin (lipopolysaccharide)-induced interleukin-6 production by human blood mononuclear cells was investigated. Lipopolysaccharide of Salmonella abortus equi and synthetic Escherichia coli-type lipid A (compound 506, or LA-15-PP) had potent interleukin-6-inducing capacities. The maximum release of interleukin-6 was found after stimulation with 1 to 10 ng of lipopolysaccharide or 10 to 100 ng of synthetic E. coli-type lipid A per ml. Both synthetic lipid A partial structures (compounds 406 and 401) failed to induce interleukin-6 release. However, they inhibited lipopolysaccharide- or lipid A-induced interleukin-6 production in a dose-dependent manner. Inhibition was found not only in mononuclear cells but also in purified monocytes and was not due to a shift in the kinetics of cytokine production. Suppression was manifested in the early stage of interleukin-6 production. Inhibition was also found in the presence of recombinant gamma interferon, indicating that compound 406 and recombinant gamma interferon act in different, independent pathways. Our data, therefore, indicate that the inhibition of interleukin-6 production by lipid A partial structures may help elucidate the mechanism of interaction of the lipid A component of lipopolysaccharide with immune cells in the inflammatory reaction during gram-negative infection.

Dose-Response Relationship, Drug↗
PubMed1991

Neutrophil-activating polypeptides IL-8 and NAP-2 induce identical signal transduction pathways in the regulation of lysosomal enzyme release.

Interleukin-8 (IL-8/NAP-1), the neutrophil-activating peptide 2 (NAP-2), and formyl-peptides (fMLP) have been described as potent stimulators for human neutrophils (PMN). We have compared the mechanism of signal transduction induced by these factors during neutrophil activation (elastase release), by using activators and inhibitors and by direct measurement of the enzymatic activity of kinases. Moreover, costimulation kinetics of the combined factors were analyzed. Our results show that each of these stimulators induces elevated levels of cAMP, indicating the activation of adenylate-cyclase. Further results obtained with the kinase inhibitor H-7 and the cAMP analogue dibutyryl-cAMP (dbcAMP) gave evidence that cAMP-dependent kinases are involved in the down-regulation of the activation process. Degranulation could not be prevented by the inhibitor W-7, nor did the treatment of cells with calcium ionophore (A23187) lead to elevation of intracellular calcium levels. Both phenomena exclude the participation of calcium calmodulin-dependent kinases. Further results obtained with the novel protein kinase C (PKC) inhibitor BM 41440 as well as by direct measurement of PKC enzyme activity demonstrated the involvement of PKC in fMLP-mediated stimulation but not with IL-8/NAP-1 or NAP-2. Analysis of costimulation experiments conducted with these three factors and TPA confirmed these results and gave evidence that fMLP activates two different signaling pathways, one of which is PKC dependent, while the other is not. Moreover, our data indicate that NAP-2, IL-8/NAP-1, and fMLP use identical PKC-independent transduction mechanisms.

Amino Acid Sequence↗
PubMed1991

Differential inhibition of IL-1 beta activities and receptor binding by monoclonal antibodies mapping within a discrete region of the protein.

The importance of the region in position 148-192 for the biological activities and receptor-binding capacity of the human IL-1 beta protein has been assessed by the use of mAbs. Four mAbs have been used, which recognize different epitopes within the 148-192 region. None of the mAbs could inhibit binding of IL-1 beta to IL-1RI (expressed on T cells and fibroblasts), suggesting that the 148-192 region does not contain IL-1RI binding sites. Conversely, mAbs Vhp20 (recognizing the fragment 166-169) and BRhD2 (directed to an epitope in the sequence 177-186) recognize sites partially involved in binding to IL-1RII (expressed on B cells, macrophages, and PMN). Only mAbs BRhD2 and FIB 1 (which recognizes an epitope in the sequence 174-186) can inhibit IL-1 beta-induced thymocyte proliferation, whereas all four can inhibit the adjuvant capacity of IL-1 beta in vivo. It is concluded that the region 148-192 encompasses domains important for T cell activation but not for binding to the IL-1RI on T cells, others involved in immunostimulation in vivo, and others important for binding to IL-1RII, although not directly involved in it.

Amino Acid Sequence↗
PubMed1991

Bacterial endotoxin: molecular relationships between structure and activity.

The significance of endotoxins in bacterial infection and their role as bacterial surface antigens (O antigens) have stimulated investigations into their chemical nature and the mechanisms of their biologic action during the last few decades. This article summarizes some of the recent results and emphasizes structure-activity relationships.

Animals↗
PubMed1991

Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67.

The monoclonal antibody Ki-67 detects a human nuclear antigen that is present in proliferating cells, but absent in quiescent cells. The aim of this study was to characterize the Ki-67 antigen by means of immunobiochemical and molecular biology techniques. Enzymatic digestion experiments showed that this antigen is highly susceptible to protease treatment, and the antigen cannot be extracted by 0.1 normal HCl, indicating that Ki-67 antigen is a nonhistone protein. Immunoblot analysis of cell lysates with Ki-67 showed a double band with apparent molecular weights of 395 kd and 345 kd, regardless of whether the gels were run under reducing or nonreducing conditions. It is noteworthy that these bands were exclusively detectable in lysates prepared from proliferating cells, whereas they were absent in lysates obtained from quiescent cells. These immunobiochemical data are further substantiated by our molecular cloning approaches. By means of immunocloning with Ki-67, the authors isolated and sequenced several cDNA fragments from lambda gt11 libraries. A 1095-bp fragment gave a strong hybridization signal at 7.5 to 9.5 kb in Northern blot analysis with RNA prepared from proliferating cells, whereas it was negative with RNA prepared from quiescent cells. This cDNA fragment could be bacterially expressed, and in subsequent immunoblot analysis Ki-67 reacted exclusively with those fusion proteins that were derived from bacteria containing the insert in the right reading frame.

Antibodies, Monoclonal↗
PubMed1991

Induction and modulation of interleukin 1 production by defined partial structures of lipopolysaccharide.

Bacterial lipopolysaccharides (LPS) consist of a hydrophilic heteropolysaccharide component and a covalently linked hydrophobic lipid portion termed lipid A. Lipid A is responsible for most of the toxic and immunoregulatory effects of LPS. In studying the structure-function relationship of lipid A partial structures we now report that certain synthetic analogues, with themselves being biologically "inactive", exert immunomodulating effects on LPS-induced Interleukin 1 production in vitro.

Dose-Response Relationship, Drug↗
PubMed1990

Inhibition of HIV progression by dithiocarb. German DTC Study Group.

60 patients with HIV-1 infection in Walter Reed stages 2-4 were randomised to treatment with intravenous or oral dithiocarb (diethyldithiocarbamate, DTC) or placebo for 24 weeks in a paired double-blind design. 55 patients were evaluable at the end of the study: no patient who had received DTC but 6 placebo patients had AIDS, a significant difference. Significantly delayed disease progression was observed in the intravenous DTC group compared with its matching placebo. The benefit in the oral DTC group was not statistically significant. During an 18-month follow-up 3 deaths occurred in the original placebo groups, whereas no patient who had initially received DTC died. A significant delay in progression to AIDS was observed in the DTC groups.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Lipid A, the immunostimulatory principle of lipopolysaccharides?

Lipid A has been found to be an inducer of IL-1 with similar potency as LPS. Partial structures of lipid A or core oligosaccharides are less active or not active at all. We propose that lipid A is the structure responsible for induction of immunostimulatory or immunoregulatory properties of LPS.

Adjuvants, Immunologic↗
PubMed1990

Detection of urinary TNF, IL 1, and IL 2 after local BCG immunotherapy for bladder carcinoma.

Intravesical application of bacillus Calmette-Guérin (BCG) is highly active against recurrences of superficial urothelial bladder carcinoma. In an attempt to monitor the immunological effects of this therapy, we analyzed the urine of patients following the sixth intravessical instillation, to show the presence of the monokines TNF and IL 1 and the lymphokine IL 2. Within 24 hours following the instillation, all (n = 10) patients exhibited a strong increase in urinary cytokine secretion, which was significantly different from the control group (n = 10), with respect to TNF L929 biological assay (P less than 0.01), TNF sandwich-ELISA (P less than 0.01), IL 2 CTL 6 biological assay (P less than 0.05), IL 2 sandwich-ELISA (P less than 0.005), and IL 1 sandwich-ELISA (P less than 0.05), but not to the IL 1 fibroblast biological assay. The maximum urinary secretion varied between individual patients and different cytokines, but was generally found within 2 to 8 hr after the instillation. A persistent high urinary activity was demonstrated in BCG-treated patients for IL 2 in sandwich-ELISA. These results reflect the local inflammatory response to BCG and suggest an immunomodulatory mode of action against urothelial carcinoma cells. Elucidation of the possible role of each urinary cytokine against this cancer warrants further investigations.

BCG Vaccine↗
PubMed1990

Immunoenzymatic assessment of interferon-gamma in Hodgkin and Sternberg-Reed cells.

The typical histological picture seen in Hodgkin's disease is consistent with the release of cytokines and other active mediators by the malignant cells, i.e., Hodgkin and Sternberg-Reed cells. Since interferon-gamma is regarded as an important regulator of the cytokine cascade, we have undertaken an immunohistological assessment of this mediator in Hodgkin's disease tissue biopsies. In approximately 50% of the cases investigated we found Hodgkin and Sternberg-Reed cells to be positive with antibodies against interferon-gamma. These in situ findings were substantiated by immunostaining of Hodgkin's disease-derived cell lines L428 and L540. L540 was consistently positive, whereas L428 was negative. It is noteworthy that L428 exhibit a B-cell pheno- and genotype, whereas L540 is of T-cell origin. These data are consistent with theories that propose that cytokine production by tumour cells is central to the pathogenesis of Hodgkin's lymphoma.

Antibodies, Monoclonal↗
PubMed1990

Effects of local bacillus Calmette-Guerin therapy in patients with bladder carcinoma on immunocompetent cells of the bladder wall.

The antitumoral effects of intravesical bacillus Calmette-Guerin against recurrent superficial urothelial bladder cancer seem to be linked to immunological effector mechanisms. To characterize further the local cellular response in the bladder wall of patients receiving intravesical bacillus Calmette-Guerin bladder biopsies were examined before and immediately after a 6-week course of bacillus Calmette-Guerin and for up to 1 year at 3-month intervals thereafter. The results showed a marked infiltration after bacillus Calmette-Guerin therapy predominantly of the suburothelial tissue with mononuclear cells, which expressed activation markers and accumulated to follicle-like granulomatous structures. With regard to T lymphocyte subsets the ratio of T helper-to-T suppressor cells in the bladder wall increased from 0.5 to nearly 2. This phenomenon persisted even in biopsies 1 year after initial treatment. In some probes activated mononuclear cells invaded the urothelium, emanating from suburothelial granulomas. It is tempting to assume that these changes within the local immunocompetent cells are related to the therapeutic effect of bacillus Calmette-Guerin against bladder cancer.

Administration, Intravesical↗
PubMed1990

Elevations of cytokines interleukin-1, interleukin-2 and tumor necrosis factor in the urine of patients after intravesical bacillus Calmette-Guerin immunotherapy.

In an attempt to elucidate further the immunological mechanisms responsible for the effectiveness of intravesical bacillus Calmette-Guerin in the therapy of superficial urothelial bladder cancer, a prospective study was performed in which the urine of patients was examined before and after 6 intravesical instillations of bacillus Calmette-Guerin for the presence of the cytokines interleukin-1, interleukin-2 and tumor necrosis factor-alpha. Biological assays such as specific sandwich enzyme-linked immunosorbent assays were used for the analysis of each cytokine. Urinary titers of interleukin-1, interleukin-2 and tumor necrosis factor increased significantly after bacillus Calmette-Guerin instillation but showed inter-individual differences. The maximum of secretion into the urine was seen between 4 and 8 hours after the instillation, and titers returned to baseline values within 24 hours. The differences in 24-hour secretion between the bacillus Calmette-Guerin-treated (10 patients) and the control (10) groups were significant with respect to all cytokines as tested in both assays each, except for the interleukin-1 biological assay. These results reflect the strong inflammatory response in the bladder wall to bacillus Calmette-Guerin, in which the urinary secretion of the detected cytokines may be associated with the local tumor control.

BCG Vaccine↗
PubMed1990

CD26 antigen is a surface dipeptidyl peptidase IV (DPPIV) as characterized by monoclonal antibodies clone TII-19-4-7 and 4EL1C7.

In this study we investigated the binding of three different monoclonal antibodies (MoAb), TII 19-4-7, 4EL1C7, and B1.19.2, which are clustered in CD26 to the ectoenzyme dipeptidyl peptidase IV (DPP IV) and to T lymphocytes. We found that all three MoAb bind to both unstimulated and mitogen-stimulated T lymphocytes. Further results indicated an inconsistency within the CD26-clustered MoAb: TII 19-4-7 and 4EL1C7, but not B1.19.2, recognized DPP IV on the surface o T lymphocytes and immobilized on solid-phase ELISA or Western blot. There was competition of binding to DPP IV between TII 19-4-7 and 4EL1C7. From these results we conclude that CD26 antigen is represented by the ectoenzyme DPP IV. TII 19-4-7 and 4EL1C7 recognize the same or partly identical epitopes on DPP IV, whereas B1.19.2 recognizes a different antigen. TII 19-4-7 and 4EL1C7, but not B1.19.2, should be clustered in CD26.

Antibodies, Monoclonal↗