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Low dosages of interleukin-1 protect mice against lethal cerebral malaria
SECOND
505
WORKSHOP
ON CYTOKINES
/
157
508
ADMINISTPATIONOF rHu'INF APHPSE 1STUDY. M. Scherinqa, J.N.M. IJzermns, Erasmus University Rotterdam,
INTOLIVERICETASTASFS: J.Jeekel and R.L. !Che Netherkmds.
m
at;
A phase 1 study was initiated to determine the effects of a single injection of rerxkinant hmwan lNF, administered intialesionally into liver rretastases of patients with therapy-resistant mali-ties. Iacal iniection of TNF was acccmlished by ultrasound guided puncture.*A dose escalating schedule was applied, stxting with a dosage of lOOug/ptient (n=3). At present ten patients have been treated; the higkst dose given is 25Oug. Nine out of ten patients have been found to experienoe chills. Fever, nausea and vomiting were less frequently observed (60-70%). These effects were not dose-dependent. Hy@ension was not observed. Except for an increase in alkaline phosphatase, no significant alterations in liver functions wsre noted. Renal function, h-tologic and jnnunolcgic paramters (NK-cells) remained unchanged. Compared to results of other phase 1 studies, in which lNF was given iv., it can be concluded that the intralesional route of administration is clearly correlated with less serious side-effects. Thus, this method offers new perspectives to enhance the antiproliferative efficacy of TNF.
506
PRODUCTION FACTOR BY DIFFERENT
INTERNATIONAL
THE EFFECT OF FISH OIL ENRICHED DIET ON RESISTANCE TO GRAM-NEGATIVE INFECTION IN MICE. W.L. Blok. M.T.E. Voeels and J.W.M. van der Meer University Hospital Nijmegen, Nijmegen, The Netherlands. A diet rich in n-3 polyunsaturated fatty acids (fish-oil enriched diet) has been shown to induce a significant decrease in interleukin-1 (IL-l) and tumor necrosis factor (TNF) production. Since it is not clear whether production of IL-I and TNF in response to infection is beneficial or harmful to the organism, we investigated the effect of diets with different fatty acid compositions on survival of mice during lethal Gramnegative infection. During 6 weeks, 25g female Swiss mice were fed standard lab chow with daily supplementation of either 0.2 ml of Mepatrin@ (34% eicosapentanoic acid and docosahexaenoic acid), 0.2 ml corn oil or 0.2 ml palm oil by instillation in the stomach. After this period, mice were injected with approximately 105cfu of Klebsiella oneumoniae (ATCC43816) in the thigh muscle, and survival was scored during 48 h. Although survival in the fish oil group was slightly better than in the other groups the differences were not statistically significant. Thus, fish oil supplementation does not appear to be harmful in this rapidly fatal infection.
509
OF INTERLEUKIN-1 AND MONOCYTE POPULATIONS METHODS. m.M .: “y
TUMOR NECROSIS ISOLATED BY I Meer. H.G.G, de ker and P. de Mulder, University Hospital Nijmegen, The Netherlands. The amounts of interleukin-1 (IL-l) and tumor necrosis factor (TNF) produced by monocytes in vitro is critically dependent on the conditions of cell separation and culture. Contamination with endotoxin and other microbial products will lead to background stimulation. We have investigated whether the production and secretion of these cytokines is different when the cells are isolated by Ficoll Hypaque (FH), Percoll (P) or Percoll and elutriation (PE) centrifugation and cultured for 24 h. Monocytes and lymphocytes isolated with FH or P did produce very low concentrations of immunoreactive IL-la, IL-18 and TNF, as did monocytes separated by PE. When stimulated with LPS (50 &ml), the production of these cytokines by 0.5 x lo6 monocytes was in the nanogram range, and approximately equal for the different methods of isolation. However, experiments so far show that PE-isolated monocytes that remain non-adherent in culture produce most of the IL-18, whereas IL-la and TNF are produced in equal amounts by adherent and non-adherent monocytes.
INTERLEUKIN-1 AND TUMOR NECROSIS FACTOR DURING MIGRAINE ATTACKS. J.J. . . van Hilten. J.W.M. va der Meer. J. va der Ven-JoneekmP P. Demacker. M.I? Ferrari Univerzty Hospital Leiden, University Hospital Nijmegen, The Netherlands. During attacks, migraine patients feel chilly, complain of cold extremities and anorexia and tend to sleep. Since these symptoms could be induced by cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-l) , we measured plasma concentrations of TNF, IL-la and extractable IL-18, as well as the production of these cytokines in whole blood cultures with or without lipopolysaccharide, during and between attacks in 20 migraine patients. Rectal temperatures were also measured in these patients. Plasma concentrations of IL-la, IL-18, and TNF, and unstimulated and stimulated production of these cytokines in vitro did not differ between attacks and attack-free episodes. Unexpectedly, body temperatures were lower during the attacks than between attacks (t=2.47, ~~0.05). From these data we conclude, that it is unlikely that systemic cytokine responses are responsible for the altered thermoregulation during migraine attacks. Supported by the Dutch Migraine Society.
507
510
LOW DOSAGES OF INTERLEUKIN-1 PROTECT MICE AGAINST LETHAL CEREBRAL MALARIA. U.A.J. Curf& J.W.M. van de Meer. R. Sauerwein a d W.M.C. Eline, Catholic University of Nijmegen, Nijmegen, the Netherlands. A single injection of a low dose of interleukin-1 (IL-l) protects neutropenic mice from lethal Gram-negative or candidal infections [PNAS 1988;85:1620; Eur J Immunol 1988;18:11431. We investigated, whether IL-1 would provide protection in murine malaria. C57Bl mice were injected i.v. with 1000 erythrocytes parasitized by mod’um &I&$ In this model, death occurs early in the second wick, and coincides with a collapse of thermoregulation and morphological features of cerebral malaria [Clin Exp Immunol 1989;75:1361. Although a single i.p. injection of 80 ng human recombinant IL-la, 3 days after infection had a protective effect, optimal protection against hypothermia and death was obtained with daily injections of IL1 from day 0 - 5 after infection. If IL-1 was administered shortly before the development of the cerebral syndrome, i.e., on day 8 or 9, there was no protection. Mice that received IL-1 for 6 days had a significantly lower degree of parasitemia compared to controls. After day 12 the parasitemia became as high as that of control mice at day 7, but a decrease in body temperature and cerebral lesions did not occur. Further studies are being performed to elucidate the mechanism of protection.
DEVELOPMENT IMMUNOASSAY
OF A SENSITIVE AND SPECIFIC RADIOFOR MOUSE INTERLEUKIN-la . M.T.E. ne&ikz P. Demacker. A. Shaw and University Hospital Nijmegen, Nijmegen, The Netherlands and Glaxo, Geneva, Switzerland. The radioimmunoassay (RIA) for human IL-la, developed by Lonnemann et al (Lymphokine Res 1988,7:75) does not crossreact with mouse IL-la. To be able to measure IL-la in body fluids of mice and in supernatants and cell lysates of murine macrophages, we decided to develop an RIA for mouse IL-a. An antiserum against recombinant (r) mouse interleukin-la (IL-la) was produced in rabbits. There was no crossreactivity with mouse rIL-16 and human rIL-la, rIL-18, and rTNFa. Mouse rIL-la was labeled with 125-I using the chloramin T method and purified over G56; the specific activity was 5.3 pCi/pg. The pyrogenicity of IL-la appeared not to be affected by the labeling procedure. For the RIA the standards and samples were incubated with the IL-la antiserum and the tracer for 3 days before sheep anti-rabbit antiserum was added and the immune complexes were precipitated. The detection limit for murine IL-la appeared to be approximately 300 pg/ml.
505
WORKSHOP
ON CYTOKINES
/
157
508
ADMINISTPATIONOF rHu'INF APHPSE 1STUDY. M. Scherinqa, J.N.M. IJzermns, Erasmus University Rotterdam,
INTOLIVERICETASTASFS: J.Jeekel and R.L. !Che Netherkmds.
m
at;
A phase 1 study was initiated to determine the effects of a single injection of rerxkinant hmwan lNF, administered intialesionally into liver rretastases of patients with therapy-resistant mali-ties. Iacal iniection of TNF was acccmlished by ultrasound guided puncture.*A dose escalating schedule was applied, stxting with a dosage of lOOug/ptient (n=3). At present ten patients have been treated; the higkst dose given is 25Oug. Nine out of ten patients have been found to experienoe chills. Fever, nausea and vomiting were less frequently observed (60-70%). These effects were not dose-dependent. Hy@ension was not observed. Except for an increase in alkaline phosphatase, no significant alterations in liver functions wsre noted. Renal function, h-tologic and jnnunolcgic paramters (NK-cells) remained unchanged. Compared to results of other phase 1 studies, in which lNF was given iv., it can be concluded that the intralesional route of administration is clearly correlated with less serious side-effects. Thus, this method offers new perspectives to enhance the antiproliferative efficacy of TNF.
506
PRODUCTION FACTOR BY DIFFERENT
INTERNATIONAL
THE EFFECT OF FISH OIL ENRICHED DIET ON RESISTANCE TO GRAM-NEGATIVE INFECTION IN MICE. W.L. Blok. M.T.E. Voeels and J.W.M. van der Meer University Hospital Nijmegen, Nijmegen, The Netherlands. A diet rich in n-3 polyunsaturated fatty acids (fish-oil enriched diet) has been shown to induce a significant decrease in interleukin-1 (IL-l) and tumor necrosis factor (TNF) production. Since it is not clear whether production of IL-I and TNF in response to infection is beneficial or harmful to the organism, we investigated the effect of diets with different fatty acid compositions on survival of mice during lethal Gramnegative infection. During 6 weeks, 25g female Swiss mice were fed standard lab chow with daily supplementation of either 0.2 ml of Mepatrin@ (34% eicosapentanoic acid and docosahexaenoic acid), 0.2 ml corn oil or 0.2 ml palm oil by instillation in the stomach. After this period, mice were injected with approximately 105cfu of Klebsiella oneumoniae (ATCC43816) in the thigh muscle, and survival was scored during 48 h. Although survival in the fish oil group was slightly better than in the other groups the differences were not statistically significant. Thus, fish oil supplementation does not appear to be harmful in this rapidly fatal infection.
509
OF INTERLEUKIN-1 AND MONOCYTE POPULATIONS METHODS. m.M .: “y
TUMOR NECROSIS ISOLATED BY I Meer. H.G.G, de ker and P. de Mulder, University Hospital Nijmegen, The Netherlands. The amounts of interleukin-1 (IL-l) and tumor necrosis factor (TNF) produced by monocytes in vitro is critically dependent on the conditions of cell separation and culture. Contamination with endotoxin and other microbial products will lead to background stimulation. We have investigated whether the production and secretion of these cytokines is different when the cells are isolated by Ficoll Hypaque (FH), Percoll (P) or Percoll and elutriation (PE) centrifugation and cultured for 24 h. Monocytes and lymphocytes isolated with FH or P did produce very low concentrations of immunoreactive IL-la, IL-18 and TNF, as did monocytes separated by PE. When stimulated with LPS (50 &ml), the production of these cytokines by 0.5 x lo6 monocytes was in the nanogram range, and approximately equal for the different methods of isolation. However, experiments so far show that PE-isolated monocytes that remain non-adherent in culture produce most of the IL-18, whereas IL-la and TNF are produced in equal amounts by adherent and non-adherent monocytes.
INTERLEUKIN-1 AND TUMOR NECROSIS FACTOR DURING MIGRAINE ATTACKS. J.J. . . van Hilten. J.W.M. va der Meer. J. va der Ven-JoneekmP P. Demacker. M.I? Ferrari Univerzty Hospital Leiden, University Hospital Nijmegen, The Netherlands. During attacks, migraine patients feel chilly, complain of cold extremities and anorexia and tend to sleep. Since these symptoms could be induced by cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-l) , we measured plasma concentrations of TNF, IL-la and extractable IL-18, as well as the production of these cytokines in whole blood cultures with or without lipopolysaccharide, during and between attacks in 20 migraine patients. Rectal temperatures were also measured in these patients. Plasma concentrations of IL-la, IL-18, and TNF, and unstimulated and stimulated production of these cytokines in vitro did not differ between attacks and attack-free episodes. Unexpectedly, body temperatures were lower during the attacks than between attacks (t=2.47, ~~0.05). From these data we conclude, that it is unlikely that systemic cytokine responses are responsible for the altered thermoregulation during migraine attacks. Supported by the Dutch Migraine Society.
507
510
LOW DOSAGES OF INTERLEUKIN-1 PROTECT MICE AGAINST LETHAL CEREBRAL MALARIA. U.A.J. Curf& J.W.M. van de Meer. R. Sauerwein a d W.M.C. Eline, Catholic University of Nijmegen, Nijmegen, the Netherlands. A single injection of a low dose of interleukin-1 (IL-l) protects neutropenic mice from lethal Gram-negative or candidal infections [PNAS 1988;85:1620; Eur J Immunol 1988;18:11431. We investigated, whether IL-1 would provide protection in murine malaria. C57Bl mice were injected i.v. with 1000 erythrocytes parasitized by mod’um &I&$ In this model, death occurs early in the second wick, and coincides with a collapse of thermoregulation and morphological features of cerebral malaria [Clin Exp Immunol 1989;75:1361. Although a single i.p. injection of 80 ng human recombinant IL-la, 3 days after infection had a protective effect, optimal protection against hypothermia and death was obtained with daily injections of IL1 from day 0 - 5 after infection. If IL-1 was administered shortly before the development of the cerebral syndrome, i.e., on day 8 or 9, there was no protection. Mice that received IL-1 for 6 days had a significantly lower degree of parasitemia compared to controls. After day 12 the parasitemia became as high as that of control mice at day 7, but a decrease in body temperature and cerebral lesions did not occur. Further studies are being performed to elucidate the mechanism of protection.
DEVELOPMENT IMMUNOASSAY
OF A SENSITIVE AND SPECIFIC RADIOFOR MOUSE INTERLEUKIN-la . M.T.E. ne&ikz P. Demacker. A. Shaw and University Hospital Nijmegen, Nijmegen, The Netherlands and Glaxo, Geneva, Switzerland. The radioimmunoassay (RIA) for human IL-la, developed by Lonnemann et al (Lymphokine Res 1988,7:75) does not crossreact with mouse IL-la. To be able to measure IL-la in body fluids of mice and in supernatants and cell lysates of murine macrophages, we decided to develop an RIA for mouse IL-a. An antiserum against recombinant (r) mouse interleukin-la (IL-la) was produced in rabbits. There was no crossreactivity with mouse rIL-16 and human rIL-la, rIL-18, and rTNFa. Mouse rIL-la was labeled with 125-I using the chloramin T method and purified over G56; the specific activity was 5.3 pCi/pg. The pyrogenicity of IL-la appeared not to be affected by the labeling procedure. For the RIA the standards and samples were incubated with the IL-la antiserum and the tracer for 3 days before sheep anti-rabbit antiserum was added and the immune complexes were precipitated. The detection limit for murine IL-la appeared to be approximately 300 pg/ml.