- Email: [email protected]
Activation of peritoneal macrophages by gram + bacterium Bacillus firmus
Nitric oxide in infectious diseases
440
25 June 1997 - Poster presentations
Forum lounges
09:00-18:30/12:00-14:00
P.4.10
Nitric oxide in infectious diseases
P.4.10.01
Nitric oxide (NO) productlon In patients with urinary tract infecttons and various renal diseases
J. Morovic-Vergles ‘, F. Culo * . ’ Department of htemal Medicine, Clinical Hospital Dubmva, Zagreb, Croatia, ‘Department of Physiology, School of Medicine, Univebfy of Zagreb, salata 3, 10 000 Zagreb, Cmatia
Reeultaz The results obtained are showed in the table. Normal-rat glomeruli in culture (3.09CYml)were incubated wkh different concentrations of the above mentioned compounds and the NOs- was quantified in the supematants. Only those glomeruli incubated with PAN 10-3M, showed a signiftcative reducti6n of N4- concentration (9-6.65 PM), which increased in the presence of L-Arg 20 mM and of SOD 156 U/ml (1.73 and ~0.8 M, mspecttvety). Conclusions: The results showed that both the 02- and NO- are involved in this model of kidney tissue damage, most possibly due to the unbalance exlstlng between the synthesis and the interaction of such radicals. In addition, treatment with the antioxidant agent Verapamil practically abrogated both proteinuria and the interstiial nephrftis occurrfng in this disease.
P.4.10.03
Introduction:Nitric oxide (NO) is important defense molecule secreted by macrophages when encountered by bacteria, intracellular parasites and tumor cells. Various stimuli (BCG, LPS, interferon-y, TNF-cr, IL-l) induce secretion of NO by mouse macrophages and endothelial cells. However, its secretion under similar conditions by human macrophages is not so well proven. In this study we analyzed the production of NO in uropoietic system in patients with urtnary tract infections, as well as in patients with various renal diseases. Materials and Methods:The study included 157 patients hospitalized at nephrologic department and 15 healthy volunteers. Eighty three patients had bacterial urfnary tract infections (UTI) and the rest (74) had vartous renal diseases. The production of NO was determined by measudng nitrite (NOs-) concentration in urine. In parallel, the concentration of nitrates (NOs-) was also measured. The concentration of nitrite was determined by a calorimetric method, using Gtiess reagent. One hundred ~1of urine was mixed with the same volume of Grfess reagent in 96-well plates and the absorbance was in microplate optical reader at 650 nm. Standard curve for NO was obtained by using sodium nitrite as astandard. Nitrate concentration was determined indirectly by their reduction with elementary zinc into nitrite. Standard laboratory examinations of urine and blood was performed in all patients. Rwum Nitrite (NO*-) could not be detected in urine of 15 healthy volunteers. However, nitrite could be detected in 54 out of 157 (34.4%) patients with renal diseases. Out of 63 patients with UTI, NO was detected in 34 patients (41%). In most patients with UTI the causative agent was E. coli (39 patients). NO could be detected in 16 or 46% of the latter patients. NO was positive in urine in variable proportions of patients with different renal disease, including hypoplasttc kidneys, lypus nephritis etc. Nitrates were found in significant amounts in urine of all patients and healthy controls, and its concentration did not differ significantly between them or between dkferent groups of patients. There was not signtficant correlation between level of nitrites or nitrates in urine and ESR, number of leukocytes or erythrocytes, level of proteinuria and blood pressure. Conoluslon: NO could be detected in urine of about one third of patients hospitalized at nephrologic department. Most of them have urinary tract infection. However, only 41% of patients with UTI have detectable NO in urine.
P.4.10.02 -
Role of nitric oxide and superoxlde in experimental nephrosis
A. Martin, E. Escudero, J. Pascual, R. Femandez-Espino, F. Mampaso. Ramdn y Cajal Hospital, Madrid, Spain
Introduction:We have studied the role of nitric oxide (NO-) and superoxide (02-) in rats with severe proteinuria a acute interstitial nephrttls after aminonucleoside of puromycin (PAN) administration. Materialsand Methods: A total number of 69 Sprague-Dawley rats (149-166 g) were divided into 6 groups. Animals from Groups 1 to 7 received a single dosis i.p (IO mgIl9r-Jg b.w.) of PAN. Group 2, also received three injections Lp. of 4.500 U/l 96 g b.w. of SOD. Groups 3 and 4, were also treated with 50 m@l of L-NAME and 1.25 mg/l of L-Arginine (L-Arg), respectively. Groups 5 and 6, received the same treatment that Groups 3 and 4 as well as the same dosis of SOD. Group 7, in addition was treated with 0.7 @ of Verapamil. Group 6 (control), only received normal saline. Onne samples were collected and pmteinuria (ma/24 hours) and nitrite concentration (Non-, @M/24 hours), were quantified. The animals were sacrificed on day 14th of the experiment and the kidneys processed for the interstitial leukocytes cells infiltrate evaluation. Pmt. (day 7) Control PAN PAN PAN PAN PAN PAN PAN
+ + + + + +
3fl SOD L-NAME L-NAME + SOD L-Arginine L-Arginine + SOD VeraDamil
244551 235f60 369f93 146fl7 296f37 174 f 33 32fl5
Pmt. (day 14) 4f0.5 lOQf35 133fl2 117*30 46f12 190f15 74f21 43f23
Interst. InfRrate
N02-
(day 14)
(day 0)
+++ f+ ++f ++ +++ ++ +I-
434*50 13Of25 lSoof244 356 f 65 15oof320 977f46 650f96 6Oof57
N02(day 5) 422 f 42 153f47 73k25 41 f 16 4f2 74f25 91 f55 330f66
Nitric oxide production by peritoneal macrophages induced by pentapeptldedissaccharlde from B. divaf/csfum and some of its derivatives
T. Balog, M. Bogunovic, B. VraneSiC,I. HrSak. “R. So#koviic”tnstitute, and Institute of Immunology. loooD Zagreb, Cmatia
Intmductlon:Low mdecular weight bacterial peptidoslycans like muramyl peptides are immunomodulators usually acting indirectly through activation of macmphages. They are generally less toxic but in the same time less potent than larger bacterial cell wall fragments. Therefore, many derivatives of muramyl peptides have been constructed with the aim to improve their acttvlty. Here we present the data of the influence of one dissacchartde-pentapeptkte from Bt-evibacterium divaricatum (PGM), its typophilic (Na-tauroyi PGM) and metalo derivative (Fe-PGM), as well as two adamantyldipeptlde stereolsomeres (AdTPl and AdTP2) on NO production by mouse peritoneal macrophages. Materials and Methods: PGM, Na-laumil-PGM and Fe-PGM are the pmducts by “Pliva” (Zagreb, Croatia). AoTPl and AdTPP are from lmunoloski Zavod (Zagreb, Croatia). Mouse peritoneal cells were harvested from 3 month old CBA mice and incubated with various concentrations of PGM, their derivatives, LPS, IFN-y -alone or in combination. The amount of nitrite in the supematants of stimulated macmphages was measured by Griess reaction. NMMA (Sigma) and anti-TNF-a antibodies (Genzyme) were used to prove the specfficity of the results obtained. Results: PGM and its Na-laumil derivative alone increased the productlon of NO by peritoneal macrophages, however less than LPS did. Fe-PGM and adamantyl dipeptides alone were ineffective. In combination wtth IFN both adamantyl dipeptides, but not PGM, expressed synergistic effect on the NO production (up to the level of LPS stimulatory effect). In combination with LPS only one stereoisomere of adamantytdipepttde (AdTF’2) showed synergistic stimulatory effect. Conclusion: Among all PGM derivatives tested no one was per se better stimulator of macrophages NO production than was native PGM. Only AdTPP was able to synergize with IFN-y and LPS in boosting macmphage production of NO. P.4.10.04
Activation of peritoneal macrophages by gram + bacterium Bacillus ffmws
L. TuEkova, Z. Zldek ‘, L. Pmkesovs 2, M. Mara, H. Tlaskalova. tnstttute of Micmbiolog~ Prague, Czech RepuWic,’ Institute of Phannecology Czech Acad. Sci.. Prague, Czech Republic, 2 Ist Faculty of Medicine, Charles Uniwsity Prague, Czech Republic Intmduction: Bacillusfirmus,a non-toxic Gram+ bacterium of external envlmnment, has distinct immunostimulatory properties: it is a strong potyclonal activator of B lymphocytes in mouse and humans and increases the resistance of experimental animals to infections and tumors. It is suggested that macrophages participate in these anttinfectious and anthumor activities. In the present study we tried to determine NO and cytokine production after in vitro stimulation of mouse macmphages by B. firmus. Material and Methods: The effect of 8. fimx~son mouse NO Synthase (type II NOS) and cytokine production by peritoneal macrophages was followed in tissue culture supematants using Gdess reagent, for determination of nitrites, and cytokine spectfic ELISA methods. Reauftaz While B. finnus itself does not activate NO production, a combination of S. finnus and IFNy very efficiently stimulate NO secretion by peritoneal cells (36+l99%). The effect is dose dependent and is statisticatly signfflcant even in concentration of bacteria as low as 10 pg/ml. B. firmosalone does not influence IFNy secretion by peritoneal cells, but significantly stimulate productfon of TNF 01and IL-IO, which were found to participate in NOS induction. The role of these two cytokines is analyzed by inhibition studies with cytokine specific mAbs. Furthermore, in our study we did not find any synergism of B. firmus wlth other bacterial NOS activators of both Gram+ and Gram- origin. Conclusions: The macmphage activating effect of 6. finnus is consistent with its ability to increase resistance to bacteriai infections.
440
25 June 1997 - Poster presentations
Forum lounges
09:00-18:30/12:00-14:00
P.4.10
Nitric oxide in infectious diseases
P.4.10.01
Nitric oxide (NO) productlon In patients with urinary tract infecttons and various renal diseases
J. Morovic-Vergles ‘, F. Culo * . ’ Department of htemal Medicine, Clinical Hospital Dubmva, Zagreb, Croatia, ‘Department of Physiology, School of Medicine, Univebfy of Zagreb, salata 3, 10 000 Zagreb, Cmatia
Reeultaz The results obtained are showed in the table. Normal-rat glomeruli in culture (3.09CYml)were incubated wkh different concentrations of the above mentioned compounds and the NOs- was quantified in the supematants. Only those glomeruli incubated with PAN 10-3M, showed a signiftcative reducti6n of N4- concentration (9-6.65 PM), which increased in the presence of L-Arg 20 mM and of SOD 156 U/ml (1.73 and ~0.8 M, mspecttvety). Conclusions: The results showed that both the 02- and NO- are involved in this model of kidney tissue damage, most possibly due to the unbalance exlstlng between the synthesis and the interaction of such radicals. In addition, treatment with the antioxidant agent Verapamil practically abrogated both proteinuria and the interstiial nephrftis occurrfng in this disease.
P.4.10.03
Introduction:Nitric oxide (NO) is important defense molecule secreted by macrophages when encountered by bacteria, intracellular parasites and tumor cells. Various stimuli (BCG, LPS, interferon-y, TNF-cr, IL-l) induce secretion of NO by mouse macrophages and endothelial cells. However, its secretion under similar conditions by human macrophages is not so well proven. In this study we analyzed the production of NO in uropoietic system in patients with urtnary tract infections, as well as in patients with various renal diseases. Materials and Methods:The study included 157 patients hospitalized at nephrologic department and 15 healthy volunteers. Eighty three patients had bacterial urfnary tract infections (UTI) and the rest (74) had vartous renal diseases. The production of NO was determined by measudng nitrite (NOs-) concentration in urine. In parallel, the concentration of nitrates (NOs-) was also measured. The concentration of nitrite was determined by a calorimetric method, using Gtiess reagent. One hundred ~1of urine was mixed with the same volume of Grfess reagent in 96-well plates and the absorbance was in microplate optical reader at 650 nm. Standard curve for NO was obtained by using sodium nitrite as astandard. Nitrate concentration was determined indirectly by their reduction with elementary zinc into nitrite. Standard laboratory examinations of urine and blood was performed in all patients. Rwum Nitrite (NO*-) could not be detected in urine of 15 healthy volunteers. However, nitrite could be detected in 54 out of 157 (34.4%) patients with renal diseases. Out of 63 patients with UTI, NO was detected in 34 patients (41%). In most patients with UTI the causative agent was E. coli (39 patients). NO could be detected in 16 or 46% of the latter patients. NO was positive in urine in variable proportions of patients with different renal disease, including hypoplasttc kidneys, lypus nephritis etc. Nitrates were found in significant amounts in urine of all patients and healthy controls, and its concentration did not differ significantly between them or between dkferent groups of patients. There was not signtficant correlation between level of nitrites or nitrates in urine and ESR, number of leukocytes or erythrocytes, level of proteinuria and blood pressure. Conoluslon: NO could be detected in urine of about one third of patients hospitalized at nephrologic department. Most of them have urinary tract infection. However, only 41% of patients with UTI have detectable NO in urine.
P.4.10.02 -
Role of nitric oxide and superoxlde in experimental nephrosis
A. Martin, E. Escudero, J. Pascual, R. Femandez-Espino, F. Mampaso. Ramdn y Cajal Hospital, Madrid, Spain
Introduction:We have studied the role of nitric oxide (NO-) and superoxide (02-) in rats with severe proteinuria a acute interstitial nephrttls after aminonucleoside of puromycin (PAN) administration. Materialsand Methods: A total number of 69 Sprague-Dawley rats (149-166 g) were divided into 6 groups. Animals from Groups 1 to 7 received a single dosis i.p (IO mgIl9r-Jg b.w.) of PAN. Group 2, also received three injections Lp. of 4.500 U/l 96 g b.w. of SOD. Groups 3 and 4, were also treated with 50 m@l of L-NAME and 1.25 mg/l of L-Arginine (L-Arg), respectively. Groups 5 and 6, received the same treatment that Groups 3 and 4 as well as the same dosis of SOD. Group 7, in addition was treated with 0.7 @ of Verapamil. Group 6 (control), only received normal saline. Onne samples were collected and pmteinuria (ma/24 hours) and nitrite concentration (Non-, @M/24 hours), were quantified. The animals were sacrificed on day 14th of the experiment and the kidneys processed for the interstitial leukocytes cells infiltrate evaluation. Pmt. (day 7) Control PAN PAN PAN PAN PAN PAN PAN
+ + + + + +
3fl SOD L-NAME L-NAME + SOD L-Arginine L-Arginine + SOD VeraDamil
244551 235f60 369f93 146fl7 296f37 174 f 33 32fl5
Pmt. (day 14) 4f0.5 lOQf35 133fl2 117*30 46f12 190f15 74f21 43f23
Interst. InfRrate
N02-
(day 14)
(day 0)
+++ f+ ++f ++ +++ ++ +I-
434*50 13Of25 lSoof244 356 f 65 15oof320 977f46 650f96 6Oof57
N02(day 5) 422 f 42 153f47 73k25 41 f 16 4f2 74f25 91 f55 330f66
Nitric oxide production by peritoneal macrophages induced by pentapeptldedissaccharlde from B. divaf/csfum and some of its derivatives
T. Balog, M. Bogunovic, B. VraneSiC,I. HrSak. “R. So#koviic”tnstitute, and Institute of Immunology. loooD Zagreb, Cmatia
Intmductlon:Low mdecular weight bacterial peptidoslycans like muramyl peptides are immunomodulators usually acting indirectly through activation of macmphages. They are generally less toxic but in the same time less potent than larger bacterial cell wall fragments. Therefore, many derivatives of muramyl peptides have been constructed with the aim to improve their acttvlty. Here we present the data of the influence of one dissacchartde-pentapeptkte from Bt-evibacterium divaricatum (PGM), its typophilic (Na-tauroyi PGM) and metalo derivative (Fe-PGM), as well as two adamantyldipeptlde stereolsomeres (AdTPl and AdTP2) on NO production by mouse peritoneal macrophages. Materials and Methods: PGM, Na-laumil-PGM and Fe-PGM are the pmducts by “Pliva” (Zagreb, Croatia). AoTPl and AdTPP are from lmunoloski Zavod (Zagreb, Croatia). Mouse peritoneal cells were harvested from 3 month old CBA mice and incubated with various concentrations of PGM, their derivatives, LPS, IFN-y -alone or in combination. The amount of nitrite in the supematants of stimulated macmphages was measured by Griess reaction. NMMA (Sigma) and anti-TNF-a antibodies (Genzyme) were used to prove the specfficity of the results obtained. Results: PGM and its Na-laumil derivative alone increased the productlon of NO by peritoneal macrophages, however less than LPS did. Fe-PGM and adamantyl dipeptides alone were ineffective. In combination wtth IFN both adamantyl dipeptides, but not PGM, expressed synergistic effect on the NO production (up to the level of LPS stimulatory effect). In combination with LPS only one stereoisomere of adamantytdipepttde (AdTF’2) showed synergistic stimulatory effect. Conclusion: Among all PGM derivatives tested no one was per se better stimulator of macrophages NO production than was native PGM. Only AdTPP was able to synergize with IFN-y and LPS in boosting macmphage production of NO. P.4.10.04
Activation of peritoneal macrophages by gram + bacterium Bacillus ffmws
L. TuEkova, Z. Zldek ‘, L. Pmkesovs 2, M. Mara, H. Tlaskalova. tnstttute of Micmbiolog~ Prague, Czech RepuWic,’ Institute of Phannecology Czech Acad. Sci.. Prague, Czech Republic, 2 Ist Faculty of Medicine, Charles Uniwsity Prague, Czech Republic Intmduction: Bacillusfirmus,a non-toxic Gram+ bacterium of external envlmnment, has distinct immunostimulatory properties: it is a strong potyclonal activator of B lymphocytes in mouse and humans and increases the resistance of experimental animals to infections and tumors. It is suggested that macrophages participate in these anttinfectious and anthumor activities. In the present study we tried to determine NO and cytokine production after in vitro stimulation of mouse macmphages by B. firmus. Material and Methods: The effect of 8. fimx~son mouse NO Synthase (type II NOS) and cytokine production by peritoneal macrophages was followed in tissue culture supematants using Gdess reagent, for determination of nitrites, and cytokine spectfic ELISA methods. Reauftaz While B. finnus itself does not activate NO production, a combination of S. finnus and IFNy very efficiently stimulate NO secretion by peritoneal cells (36+l99%). The effect is dose dependent and is statisticatly signfflcant even in concentration of bacteria as low as 10 pg/ml. B. firmosalone does not influence IFNy secretion by peritoneal cells, but significantly stimulate productfon of TNF 01and IL-IO, which were found to participate in NOS induction. The role of these two cytokines is analyzed by inhibition studies with cytokine specific mAbs. Furthermore, in our study we did not find any synergism of B. firmus wlth other bacterial NOS activators of both Gram+ and Gram- origin. Conclusions: The macmphage activating effect of 6. finnus is consistent with its ability to increase resistance to bacteriai infections.