EC workshop on human somatic gene therapy. Commission of the European Communities Directorate--General XII for Science, Research and Development, 23-24 September 1993.
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Europe↗
Biomedical subjects
A Meager
Publications and source records attributed to A Meager.
5th International Congress on Tumour Necrosis Factor and Related Cytokines: Scientific Advances and their Medical Applications.
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Animals↗
Hepatic expression of interferon-alpha in chronic hepatitis B virus infection.
Hepatic expression of interferon-alpha (IFN-alpha) was examined by immunohistochemistry in 90 Chinese patients (M/F 67:23, age: 14-69) with a spectrum of hepatitis B virus (HBV)-related chronic liver diseases. Immunoreactive IFN-alpha was detected in sinusoidal cells in 79 patients (88%) and in mononuclear cells in 59 patients (65.6%). Patients with active liver diseases (chronic active hepatitis, active cirrhosis, N = 55) had a higher level of IFN-alpha expression compared to patients with inactive histology (N = 35; sinusoidal cells, P < 0.01; mononuclear cells, P < 0.01). Cytoplasmic HBsAg, nuclear HBcAg, and cytoplasmic HBcAg were detected in 79 (88%), 42 (47%), and 23 (27%) patients respectively. Expression of IFN-alpha in mononuclear cells correlated with the expression of cytoplasmic HBcAg (P < 0.05) but not with nuclear HBcAg or cytoplasmic HBsAg. When the patients were divided into four different phases according to the natural history of chronic HBV infection, patients in the active liver disease phase had higher IFN-alpha expression compared to the immunotolerant and late phase patients (P < 0.01). Using double immunohistochemical staining, both IFN-alpha and cytoplasmic HBcAg were frequently detected near inflammatory infiltrates but no correlation existed between the hepatic expression of HBsAg and IFN-alpha. These data indicate that IFN-alpha is expressed in the liver in HBV-related active liver diseases and that the reported suboptimal production of IFN-alpha by PBMC in HBV-related chronic active liver diseases may be due to a redistribution of the IFN-alpha-producing mononuclear cells into the liver, the site of inflammation.
Adolescent↗
TNF receptor fusion proteins are effective inhibitors of TNF-mediated cytotoxicity on human KYM-1D4 rhabdomyosarcoma cells.
KYM-1D4 cells are a subline derived from a human rhabdomyosarcoma which are highly sensitive to TNF-mediated cytotoxicity. They were selected for this study because they express human TNF-R and are therefore a more relevant target for comparing the potential therapeutic value of human TNF-inhibitory agents than the usual murine cell lines. Two recombinant soluble TNF-R-IgG fusion proteins, one containing p55 TNR-R, the other containing p75 TNF-R, and a recombinant monomeric soluble p55 TNF-R were all found to block the cytotoxicity generated by human TNF-alpha and LT as well as also murine TNF. The p55 TNF-R-IgG fusion protein (p55-sf2) was the most effective of the antagonists tested, requiring an equimolar, (based on a monomeric configuration of TNF-alpha) or a 3-fold higher (based on a trimeric configuration of TNF-alpha) molar concentration to inhibit the cytotoxicity mediated TNF-alpha by 50%. p55-sf2 was also as effective at inhibiting the cytotoxicity mediated by LT or murine TNF in the KYM-1D4 assay. In contrast, the monomeric soluble p55 TNF-R was the least effective inhibitor, requiring a > 4000-fold higher molar concentration than p55-sf2 to achieve a similar degree of protection. The fusion proteins, particularly p55-sf2, may be useful as human therapeutic agents, as at low concentrations they can prevent both TNF-alpha-mediated and LT-mediated effects on human cells. As TNF-R-IgG fusion proteins also block the action of murine TNF in vitro, they may also be useful in the investigation of murine models of human inflammatory disease.
Animals↗
Human antibodies to insulin in diabetes.
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Autoantibodies↗
The role of tumour necrosis factor-alpha and IL-1 in polymorphonuclear leucocyte and T lymphocyte recruitment to joint inflammation in adjuvant arthritis.
The mediators involved in leucocyte recruitment to joints during arthritis are not fully defined, but two important proinflammatory cytokines, IL-1 and tumour necrosis factor-alpha (TNF-alpha), are produced in joints in rheumatoid arthritis (RA). We investigated in the rat adjuvant arthritis model whether endogenous IL-1 and TNF-alpha contribute to joint inflammation and polymorphonuclear leucocyte (PMNL) and T lymphocyte infiltration. The migration of 51Cr-labelled rat blood PMNL and 111In-labelled T lymphocytes to the joints of rats with adjuvant arthritis was measured along with plasma protein extravasation, which was quantified using 125I-labelled human albumin. Rats with active arthritis of 5 days' duration received i.p. non-immune serum, polyclonal neutralizing anti-serum to rat TNF-alpha, antiserum to IL-1 alpha and IL-1 beta, or both anti-TNF plus anti-IL-1 for 5 days. Treatment with anti-IL-1 alpha and IL-1 beta did not affect plasma protein extravasation, or PMNL or T lymphocyte accumulation in the joints (i.e. talar joint, hind paws, and tail) despite the fact that this treatment inhibited 80-90% of the PMNL migration into dermal sites injected with IL-1 alpha or IL-1 beta. In contrast, anti-TNF-alpha treatment significantly improved clinical scores, decreased plasma protein extravasation by 60-80%, inhibited PMNL accumulation by 40-50% and decreased T lymphocyte accumulation by 30-50%. Treatment with anti-IL-1, together with anti-TNF-alpha, significantly potentiated the inhibition of T lymphocyte accumulation observed with anti-TNF-alpha alone. These results indicate that endogenous TNF-alpha production may play an important role in the inflammatory changes and leucocyte recruitment in this experimental model of human arthritis, while IL-1 may have a less important role in leucocyte recruitment to these joints.
Animals↗
Application of an immortalized human endothelial cell line to the leucocyte:endothelial adherence assay.
This report shows that an immortalized endothelial cell line (EA.hy 926) is able to substitute for secondary cultures of human umbilical vein endothelial cells (HUVEC) in the leucocyte:endothelial adherence assay. Enriched preparations of blood polymorphonuclear leucocytes, monocytes and resting and activated lymphocytes exhibited similar adherence characteristics to HUVEC and the EA.hy 926 cells. Cytokines such as tumour necrosis factor (TNF) act on endothelial cells to increase their adhesiveness for leucocytes and in this study there was no difference between TNF-treated HUVEC and EA.hy 926 cells in supporting the enhanced binding of leucocytes. The adherence promoting effect of TNF-treated EA.hy 926 cells appears to be dependent upon their endothelial properties since TNF treatment of A549 cells, the permanent human cell line used to generate the hybrid EA.hy 926 cells did not augment lymphocyte attachment. Monoclonal antibodies against CD11a and CD18 inhibited the binding of lymphocytes to untreated and TNF-treated HUVEC and EA.hy 926 cells and ICAM-1 expression was increased on both monolayers following treatment with TNF. The availability of a hybrid endothelial cell line whose adhesive properties are similar to those of recently isolated endothelial cells should benefit the study of factors that govern leucocyte-endothelial cell interactions and be advantageous to the longitudinal investigation of leucocyte adherence under static conditions.
Antigens, CD↗
Protein kinase C-dependent phosphorylation is involved in resistance to tumour necrosis factor-alpha-induced cytotoxicity in a human monocytoid cell line.
To investigate the mechanism underlying resistance to tumour necrosis factor-alpha (TNF alpha)-induced cytotoxicity, we have developed a human hybrid cell line, designated A10, derived from the fusion of human U-937 monocytoid cells and human monocytes, which expressed large numbers of TNF alpha receptors and yet remained highly resistant to TNF alpha. However, in the presence of the protein kinase C (PKC) inhibitors RO-31-7549 or RO-31-8220 (donated by Roche), these cells became sensitive to TNF alpha-induced cytotoxicity, suggesting that PKC activity is required for protective mechanisms. On investigation of protein phosphorylation in TNF alpha-stimulated permeabilized A10 cells, a rapid increase in serine/threonine phosphorylation of phosphoproteins of molecular masses 130, 90, 80, 65 and 42 kDa was found. Subsequently, we found a similar pattern of increased phosphorylation following stimulation of A10 cells with mezerein, a phorbol ester derivative which activates PKC, a serine/threonine kinase. The theory that activation of PKC was responsible for increased phosphorylation was confirmed by a dose-dependent inhibition of the TNF alpha-induced protein phosphorylation by the PKC inhibitors RO-31-7549 and RO-31-8220. The possible link between the TNF alpha-stimulated early protein phosphorylation events and the maintenance of protective mechanisms against TNF alpha-induced cytotoxicity is discussed.
Cell Survival↗
Effects of macrophage-colony stimulating factor on human monocytes: induction of expression of urokinase-type plasminogen activator, but not of secreted prostaglandin E2, interleukin-6, interleukin-1, or tumor necrosis factor-alpha.
It is often assumed that macrophage-colony stimulating factor (M-CSF) or CSF-1, as well as granulocyte macrophage-CSF (GM-CSF), can induce inflammatory mediator production by monocytes/macrophages. We demonstrate with elutriation-purified human monocytes that, in contrast to lipopolysaccharide, recombinant human CSF-1 does not induce secretion of prostaglandin E2, interleukin-6 (IL-6), IL-1 beta, or tumor necrosis factor alpha, as measured by immunoassay; however, increased urokinase-type plasminogen activator (u-PA) activity in cell lysates and mRNA was observed. Similar results were obtained when the monocytes were treated with recombinant human GM-CSF. Such increased u-PA expression may contribute to the function of CSF-1 at sites of inflammation.
Cells, Cultured↗
Reduction of tumor necrosis factor alpha and interleukin-1 beta levels in human synovial tissue by interleukin-4 and glucocorticoid.
The effects of recombinant human interleukin-4 (IL-4) and the glucocorticoid, dexamethasone, on tumor necrosis factor alpha (TNF alpha) and interleukin-1 beta (IL-1 beta) levels in cultures of rheumatoid and osteoarthritic synovial tissue were studied. Low concentrations of IL-4 and dexamethasone suppressed the levels of both cytokines in the supernatants of both types of tissue after stimulation with lipopolysaccharide (LPS); the IL-1 beta and TNF alpha levels were measured by ELISA. It is suggested that it is the monocyte/macrophage in the synovial tissues that is responsive to the inhibitors. It is proposed that glucocorticoids may act on synovial tissue in this manner in vivo and IL-4 may do so if administered intraarticularly.
Arthritis, Rheumatoid↗
Development of resistance to tumour necrosis factor (TNF alpha) in KYM-1 cells involves both TNF receptors.
In this study, we investigated the type of TNF receptor expressed by the human rhabdomyosarcoma cell line KYM-1 and related TNF-sensitive sublines and showed that the majority (> 90%) of TNF receptors were type II (p75) receptors with only a relatively small number (< 10%) of type I (p55) receptors. Selection with TNF alpha led to the isolation of KYM-1 related TNF-resistant cell lines of which three cloned lines showed a near to total loss of type II TNF receptors. To investigate further the role of each type of TNF receptor in the regulation of TNF-mediated cytotoxicity and the development of TNF resistance, we used receptor-specific antibodies and antisera, able to compete with ligand binding to the respective receptor molecules, but representing efficient agonists via crosslinking of receptors. We found that in KYM-1 and related TNF-sensitive sublines both type I and type II TNF receptors can be functional on their own, as selective cross-linking of each receptor subset lead to cytolysis. However, investigation of three TNF-resistant KYM-1 related cell lines by selective receptor stimulation via cross-linking indicated different mechanisms underlied the development of TNF-resistant for each receptor type. Our data indicate that resistance to TNF-mediated cytotoxicity in KYM-1 related cell lines can be developed both by a selective loss of type II receptor expression and the selective loss of type I receptor function without reduction in type I receptor numbers.
Antibodies, Monoclonal↗
Modulation of antibody-mediated glomerular injury in vivo by interleukin-6.
We have shown previously that pretreatment with small doses of bacterial lipopolysaccharide (LPS), human recombinant interleukin-1 beta (hrIL-1 beta) and human recombinant tumor necrosis factor-alpha (hrTNF) increase injury in the heterologous phase of nephrotoxic nephritis (NTN). All three pretreatments induce synthesis of interleukin-6 (IL-6) which in some systems down-regulates synthesis of IL-1 and TNF. We have now investigated the influence of IL-6 on injury in both heterologous and autologous phases of NTN in rats. Injection of hrIL-6 in doses sufficient to induce hepatic synthesis of acute phase proteins (assessed by plasma alpha 2-macroglobulin concentration) had no effect on glomerular injury in the heterologous phase of NTN (albuminuria in NTAb alone 9 +/- 6; LPS/NTAb 34 +/- 10 and IL-6/NTAb 2 +/- 1 mg/24 hr, P < 0.001, Wilcoxon test). In contrast, IL-6 pretreatment partially abrogated the effect of LPS on albumin excretion (NTAb 4 +/- 2; LPS/NTAb 85 +/- 11 and IL-6/LPS/NTAb 32 +/- 6 mg/24 hr, P < 0.002), percentage of glomerular capillary thrombi (3 +/- 1%; 39 +/- 8%; and 6 +/- 1%, P < 0.001) and glomerular neutrophil infiltrate (29 +/- 3; 58 +/- 5; and 34 +/- 2 neutrophils/50 glomeruli in section, P < 0.001, respectively) at 24 hours. The effect of IL-6 was also evident four hours after induction of nephritis and was associated with a marked reduction in glomerular concentration of mRNA for IL-1 beta and TNF, without change in that of tubulin. Serum TNF concentrations were also significantly reduced at four hours in IL-6 treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
High levels of interferon alpha in the sera of children with dengue virus infection.
We measured the levels of interferon alpha (IFN alpha) in the sera of Thai children hospitalized with dengue hemorrhagic fever (DHF) or dengue fever (DF) to examine the role of IFN alpha in dengue virus infections of humans. The percentage of patients who had detectable levels of IFN alpha (> or = 3 U/ml) was higher in patients with DHF (80%, P < 0.001) and in patients with DF (60%, P < 0.001) than in healthy Thai children (7%). The levels of IFN alpha were higher in patients with DHF and in patients with DF on the first few days after the onset of fever than in healthy Thai children. The average levels of IFN alpha in patients with DHF were high two days before defervescence, decreasing gradually until the day of defervescence. There was a subset of patients with DHF who had increasing levels of IFN alpha after defervescence. However, the levels of IFN alpha in patients with DF were not high after fever subsided. The levels of IFN alpha were not different among children with DHF grades 1, 2 and 3. Among patients with DHF, T lymphocytes were activated to a higher degree in high IFN alpha producers than in low IFN alpha producers. These results indicate that similarly high levels of IFN alpha are produced in vivo during the acute stages of DHF and DF, and that high levels of IFN alpha remain after fever subsides in some patients with DHF, but not in patients with DF.
Acute Disease↗
TR60 and TR80 tumor necrosis factor (TNF)-receptors can independently mediate cytolysis.
The human rhabdomyosarcoma cell line KYM-1 coexpresses high numbers of both tumor necrosis factor-alpha (TNF) receptors, TR60 and TR80, and is highly sensitive to TNF-induced cytotoxicity. We show here that each receptor type can mediate cytotoxicity on its own on selective stimulation. This was achieved using receptor specific antibodies, able to compete with ligand binding to the respective receptor molecules. Thus, the TR60-specific monoclonal antibody H398 was strongly agonistic, i.e., cytotoxic, in the presence of a secondary cross-linking immunoglobulin. Selective stimulation of TR80 by antibody-mediated receptor crosslinking also induced strong cytotoxicity in KYM-1 cells. Interestingly, when both receptor subsets were stimulated in parallel by limited receptor cross-linking, additive cytolytic effects were observed. In each case the respective Fab fragments showed no agonistic activity. When the natural ligand TNF was used to induce cytolysis, blocking studies with receptor specific Fab fragments indicated that the main cytotoxic effect of TNF is mediated via TR60, although these receptors represent only about 8% of the total TNF receptor number.
Antibodies, Monoclonal↗
Interactions of tumor necrosis factor with granulocyte-macrophage colony-stimulating factor and other cytokines in the regulation of dendritic cell growth in vitro from early bipotent CD34+ progenitors in human bone marrow.
Colonies of CD1a+ HLA-DR+/DQ+ CD4+ cells with the functional and some of the structural attributes of Langerhans cells are observed in human bone marrow cultures in semi-solid media and are assumed to be the progeny of an early progenitor, the dendritic/Langerhans cell CFU (CFU-DL). The cytokine-regulated growth of these cells has been studied using a chemically defined serum-free system to culture both unfractionated and highly enriched bone marrow progenitor cell populations. Although unfractionated cell growth was optimal in serum replete cultures with PHA-stimulated leukocyte-conditioned medium (PHA-LCM) suboptimal proliferation of CFU-DL was observed in serum even in the absence of PHA-LCM. No colonies were observed under serum-free conditions when granulocyte-macrophage CSF (GM-CSF), IL-3, granulocyte CSF (G-CSF), and macrophage CSF (M-CSF) were present at levels optimal for granulocyte colony-forming unit (CFU-G) and macrophage colony-forming unit (CFU-M) growth. Addition of IL-1 alpha to these cytokines stimulated a small number of CFU-DL. However, in the presence of GM-CSF and IL-3, TNF-alpha or TNF-beta (5 U/ml) were both highly effective in promoting growth up to 82% of optimal and CFU-G growth was also enhanced at these concentrations. TNF was only active during the first 3 days of culture and higher concentrations of TNF-alpha but not TNF-beta were inhibitory for both CFU-DL and CFU-G. CD34+ cell-enriched populations were also enriched for both myeloid progenitors (CFU-G + CFU-M) and CFU-DL to 36- and 48-fold, respectively, and single cell cultures of CD34+ cells yielded single colonies containing both CD1a+ dendritic cells and CD1a- macrophages. Thus dendritic/Langerhans progenitors in the bone marrow expresses CD34, have a capacity for both macrophage and dendritic cell differentiation, and depend on hemopoietic growth factors and TNF for their further development in vitro.
Antigens, CD↗
Fourth International Congress on Tumour Necrosis Factor and Related Cytokines. Meeting at Koningshof, Veldhoven, The Netherlands 2-6 May 1992.
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Animals↗
Characterization and partial purification of a novel cytotoxic lymphokine (factor 2) produced by a human B cell line (Karpas 160).
A subclone (160b) of the human B cell (Karpas 160) was shown to produce a novel cytotoxic lymphokine [Factor 2 (F2)] in addition to tumour necrosis factor alpha (TNF-alpha) and beta (TNF-beta). F2 was found to have selective toxicity to numerous human tumour cell lines, particularly the erythroleukaemic cell line K562, whereas TNF-alpha/beta were not cytotoxic to these cells, even at relatively high concentrations. Our studies have shown that F2 activity in crude preparations is heterogeneous both in its molecular weight, isoelectric point (pI) and hydrophobicity, which depends not only on the source of F2 cytotoxicity but also on the conditions of methods for its production. Our studies also showed that F2 was separable from TNF by DE52, S-300 gel filtration and Rotofor isoelectric focusing. F2 was partially purified up to 1000-fold by two procedures. The major active form, as assessed by gel filtration was of mol. wt of 45-67 kDa. On SDS-PAGE, F2 activity was recovered mainly from two regions of the gels corresponding to 10-14 kDa and 60-70 kDa. Antibodies of human TNF-alpha, TNF-beta, IFN-alpha, IFN-gamma, and TGF-beta failed to prevent F2-mediated cytotoxicity to K562. F2 activity was not inhibited by mannose-6-PO4 or mouse mAb to rat granule content, both of which have been reported to block human natural killer cytotoxic factor. Our studies indicated that F2 is likely to be a distinct human cytokine with selective cytotoxic activity against tumour cells.
B-Lymphocytes↗