RESUMO
Dialysis with unmodified cellulose membranes is associated with such bioincompatibility phenomena as leukopenia, increased expression of adhesion molecules on leukocytes, and release of reactive oxygen species. Dialysis biocompatibility can be improved by modifications in the structure of the cellulose membrane to diminish leukocyte activation and/or protect against the released free oxygen radicals. Excebrane (Terumo Corp, Tokyo, Japan) is a vitamin E-modified cellulose membrane. In the present study, the effect of dialysis with Excebrane membranes on granulocyte and monocyte counts; CD11b, CD11c, and CD45 expression on the surface of granulocytes; and CD14 expression on monocytes was evaluated and compared with low-flux polysulfone membranes. Fifteen minutes after the start of dialysis, granulocytopenia and monocytopenia were more pronounced with the Excebrane membrane compared with polysulfone. The increase in basal expression of CD11b and CD45 on circulating granulocytes was more pronounced during dialysis with Excebrane than polysulfone membranes. Regarding the increased expression on in vitro stimulation with phorbol myristate acetate, blunted upregulation was obtained during dialysis using Excebrane membranes for CD11c and CD45 expression on granulocytes and CD14 expression on monocytes. In conclusion, such indices of membrane bioincompatibility as leukocyte counts and expression of leukocyte surface molecules show more profound alterations with Excebrane than the standard low-flux polysulfone membrane in both basal and in vitro activated states.
Assuntos
Membranas Artificiais , Diálise Renal/instrumentação , Materiais Biocompatíveis , Moléculas de Adesão Celular/imunologia , Celulose , Granulócitos/imunologia , Humanos , Contagem de Leucócitos , Leucopenia/diagnóstico , Leucopenia/etiologia , Monócitos/imunologia , Polímeros , Espécies Reativas de Oxigênio/imunologia , Diálise Renal/efeitos adversos , Sulfonas , Vitamina ERESUMO
Overall leukocyte counts decrease during certain forms of hemodialysis, but little information is available on the intradialytic evolution of phagocytic metabolic function, especially during dialysis with dialyzers not affecting the number of circulating phagocytes. This study evaluated the phagocytic capacity of granulocytes and monocytes to generate CO2 out of glucose under basic unchallenged conditions and after stimulation with latex or zymosan, before and after 15, 60 and 240 minutes of dialysis with reused cuprophan, AN69S, polysulphone, polymethylmethacrylate and hemophan hemodialyzers. Phagocytic metabolic function was assessed in whole blood on the basis of 14CO2-production from labelled glucose during the phagocytic process. There were no changes in basic unchallenged CO2-production with any of the dialyzers. Reactivity to latex and zymosan, expressed per number of phagocytes, showed no decrease, irrespective of the membrane type. For polymethylmethacrylate and reused cuprophan, a slight but significant increase in metabolic reactivity was observed in response to latex and zymosan. The test employed may give a screening picture of the phagocytic reaction to contact with dialyzers and membranes and thus of their degree of biocompatibility towards the phagocyte system.
Assuntos
Materiais Biocompatíveis , Fagócitos/fisiologia , Diálise Renal , Celulose/análogos & derivados , Glucose/metabolismo , Humanos , Látex/farmacologia , Contagem de Leucócitos , Membranas Artificiais , Metilmetacrilatos , Fagocitose , Polímeros , Distribuição Aleatória , Sulfonas , Zimosan/farmacologiaRESUMO
An index of the activation of NADPH-oxidase in the production of free radicals is CO2 production by the hexose monophosphate shunt of polymorphonuclear cells (PMN). 14CO2 production from glucose-1-C14 was studied as an indicator of PMN function after addition of latex or zymosan to predialysis whole blood, and correlated with the years spent on hemodialysis. In a series of 49 patients, a nadir in PMN metabolic activity, when compared with healthy controls, was reached in a subgroup treated for less than 24 months (n = 24, latex: 109.5 +/- 10.1 vs. 233.7 +/- 11.8; zymosan: 181.9 +/- 18.9 vs. 407.8 +/- 19.3 DPM/10(3) PMNs; p less than 0.01). In patients treated by dialysis for longer than 24 months, PMN metabolic function was significantly better, resulting in partial correction of the defect for both latex and zymosan. Phagocyte CO2 production in response to latex or zymosan correlated significantly with years of dialysis treatment (r = 0.63 and 0.56, respectively, n = 49; p less than 0.001). These findings may have implications for phagocyte reactivity towards infection, bioincompatible dialyzer membranes, and renal transplantation.
Assuntos
Metabolismo Energético/imunologia , Falência Renal Crônica/imunologia , Rins Artificiais , Neutrófilos/imunologia , Fagocitose/imunologia , Seguimentos , Humanos , Tolerância Imunológica/imunologia , Contagem de Leucócitos , Assistência de Longa DuraçãoRESUMO
The disappearance rate of endogenous renin from the circulating blood after bilateral nephrectomy was studied in 4 puppies aged 16-21 days. Mean half-times of the fast and slow component of the disappearance curves of renin were 9.15 min +/- 0.87 (SD) and 84.0 min +/- 16.8 (SD), respectively. These values are not different from the values reported for adult dogs in the literature. The metabolic degradation of renin is not different between young and adult dogs and does not contribute to the elevated plasma renin activity in the puppy.
Assuntos
Envelhecimento , Renina/sangue , Animais , Cães , Cinética , Taxa de Depuração Metabólica , NefrectomiaRESUMO
These studies were designed to explore the effects of ketanserin (K), a serotonergic S2-receptor blocker on glomerular filtration rate (GFR), renal plasma flow (RPF) and autoregulation of renal blood flow (RBF) in the normal, anesthetized rat. Two doses of ketanserin were used: a high dose that, in addition to its serotonin blocking effect, possessed alpha 1-adrenergic blocking capacities; and a low dose that acted only as a serotonin S2 blocking agent. The effects of the high dose were compared to the effects of phenotolamine. Both the high dose of K and phentolamine resulted in a similar fall of systemic blood pressure from 117 +/- 4 to 78 +/- 3 and from 121 +/- 4.5 to 76 +/- 5 mm Hg, respectively (P less than 0.01). Despite this fall, GFR and RPF remained unchanged from 2.36 +/- 0.16 +/- to 2.26 +/- 0.12 ml/min, and from 5.33 +/- 0.41 to 5.76 +/- 0.5 ml/min with K, while both parameters significantly decreased with phentolamine. A remarkable preservation of the autoregulation of RBF until a renal perfusion pressure (RPP) of 70 to 75 mm Hg was noted with K, but not with phentolamine or Ringer infusion. With the low dose of K, a significant rise in GFR and PAH clearance was noted, from 2.12 +/- 0.17 to 2.59 +/- 0.18 and from 4.81 +/- 0.35 to 5.66 +/- 0.48 ml/min, respectively (P less than 0.05). A similar preservation of autoregulation of RBF was observed. Our studies suggest that in the pressure ranges below normal autoregulation of RBF in the rat, serotonin blockade is associated with maintenance of both GFR and RBF.
Assuntos
Ketanserina/farmacologia , Circulação Renal/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Relação Dose-Resposta a Droga , Taxa de Filtração Glomerular/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Ketanserina/administração & dosagem , Masculino , Fentolamina/farmacologia , Ratos , Ratos EndogâmicosRESUMO
BACKGROUND: Citrate, used for the anticoagulation of the extracorporeal dialysis circuit, reduces ionized calcium by chelation and has been claimed to attenuate dialyser membrane bioincompatibility. Dialysis with complement-activating cuprophane membranes is associated with leukopenia which has been related to an increase in adhesion molecule expression on the surface of circulating leukocytes. METHODS: The effect of citrate anticoagulation on the expression of CD11b, CD11c and CD45 on the surface of granulocytes and CD14 on monocytes during haemodialysis with cuprophane membranes, was evaluated by flow cytometric analysis. A comparison of standard heparin vs citrate was performed in 14 chronic haemodialysis patients. During citrate anticoagulation a calcium-free dialysate was used and citrate was infused to obtain a concentration of 4.3 mmol/l blood. The unchallenged 'baseline state' expression of the surface molecules and the increase after ex vivo stimulation with phorbol 12-myristate 13-acetate (delta-PMA) or formyl-methionyl-leucyl-phenylalanine (delta-fMLP) was studied. RESULTS: With heparin, as well as with citrate, a sharp fall in granulocyte and monocyte count was observed after 15 min of dialysis, followed by a recovery at the end of the session. The expression of CD11b, CD11c and CD45 on granulocytes increased markedly during cuprophane dialysis with a peak at 15 min; there were no differences in response between heparin and citrate anticoagulation. Delta-PMA and delta-fMLP for CD45, CD11c and CD14 showed a decrease during cuprophane dialysis vs t0; again there were no differences between heparin and citrate. CONCLUSION: We conclude that the use of citrate was not associated with reduced leukocyte activation as measured by the expression of surface molecules during cuprophane dialysis and that no effect on dialysis leukocytopenia could be registered.
Assuntos
Anticoagulantes/farmacologia , Materiais Biocompatíveis/efeitos adversos , Celulose/análogos & derivados , Ácido Cítrico/farmacologia , Leucócitos/imunologia , Membranas Artificiais , Diálise Renal/efeitos adversos , Idoso , Idoso de 80 Anos ou mais , Celulose/efeitos adversos , Feminino , Granulócitos/imunologia , Humanos , Integrina alfaXbeta2/metabolismo , Rins Artificiais/efeitos adversos , Antígenos Comuns de Leucócito/metabolismo , Contagem de Leucócitos , Receptores de Lipopolissacarídeos/metabolismo , Antígeno de Macrófago 1/metabolismo , Masculino , Teste de Materiais , Pessoa de Meia-Idade , Monócitos/imunologia , Fatores de TempoRESUMO
Phagocytosis is an important part of the host defense against infection. Antibiotics can influence phagocytic function. In the present study, leukocyte metabolic response to phagocytic challenge by latex was assessed in relation to in vitro addition of cotrimoxazole, imipenem/cilastatin, cefodizime, dexamethasone (DXM), and/or cyclosporin A (CsA). Using latex particles as phagocytic challenge, glucose-1-14C utilization and 14CO2 production were measured by liquid scintillation counting. The phagocytic response was impaired by in vitro addition of DXM or CsA and this setup was used as an experimental model of immunodepression. The addition of co-trimoxazole to control samples (without DXM or CsA) depressed the response to latex challenge, whereas imipenem and cefodizime had a neutral effect. In the presence of DXM, co-trimoxazole induced a further decrease. The depressive effect of DXM was partially neutralized in the presence of cefodizime. With CsA depression, co-trimoxazole also induced a further decrease, imipenem had a neutral effect, while cefodizime partially restored the CsA suppressed reaction. Co-trimoxazole depressed the phagocytic response, imipenem had a neutral effect, whereas cefodizime restored the experimentally induced immunosuppression.
Assuntos
Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Ciclosporina/farmacologia , Dexametasona/farmacologia , Imunossupressores/farmacologia , Fagócitos/efeitos dos fármacos , Cefotaxima/análogos & derivados , Cefotaxima/farmacologia , Cefalosporinas/farmacologia , Cilastatina/farmacologia , Interações Medicamentosas , Feminino , Glucose/metabolismo , Humanos , Imipenem/farmacologia , Látex/imunologia , Masculino , Fagócitos/imunologia , Fagócitos/metabolismo , Inibidores de Proteases/farmacologia , Estatísticas não Paramétricas , Tienamicinas/farmacologia , Combinação Trimetoprima e Sulfametoxazol/farmacologiaRESUMO
Twenty-three stabilized chronic uremic patients with no active or recent infection were treated for 10 days with either cefodizime (5 x 2 g intravenously, n = 10) or cotrimoxazole (960 mg orally b.i.d., n = 8) in order to evaluate the effects on the depressed polymorphonuclear metabolic response to phagocytic challenge; a separate group of 5 patients received placebo. Ex vivo evaluation in whole blood of energy delivery to the phagocytosis-associated respiratory burst activity in response to latex and zymosan challenge was determined by measuring hexose-monophosphate shunt NAD(P)H-oxidase-related glycolytic activity. Cefodizime induced a statistically significant increase in the baseline-depressed glycolytic response for both latex and zymosan challenge, in contrast to cotrimoxazole and placebo. Depressed phagocytosis-related metabolic function in hemodialyzed patients was stimulated by cefodizime in recommended therapeutic doses but not by cotrimoxazole, the effect persisting for at least 2 weeks after the end of treatment.
Assuntos
Cefotaxima/análogos & derivados , Fagocitose/fisiologia , Diálise Renal , Explosão Respiratória/fisiologia , Combinação Trimetoprima e Sulfametoxazol/uso terapêutico , Uremia/fisiopatologia , Uremia/terapia , Adulto , Idoso , Cefotaxima/administração & dosagem , Cefotaxima/uso terapêutico , Doença Crônica , Relação Dose-Resposta a Droga , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Feminino , Glicólise/fisiologia , Humanos , Injeções Intravenosas , Látex/farmacologia , Masculino , Pessoa de Meia-Idade , NADH NADPH Oxirredutases/fisiologia , NADPH Oxidases , Neutrófilos/metabolismo , Fagócitos/metabolismo , Fagocitose/efeitos dos fármacos , Explosão Respiratória/efeitos dos fármacos , Fatores de Tempo , Combinação Trimetoprima e Sulfametoxazol/administração & dosagem , Zimosan/farmacologiaRESUMO
BACKGROUND: The use of radiographic contrast media in the setting of possible bowel ischemia and potential perforation is known to carry a risk of morbidity and mortality. However, studies of the effect of available contrast media on host immunological defense mechanisms are lacking. We have examined the effect of barium and of two water-soluble contrast agents of differing iodine concentration and osmolality, Conray 30 and Cysto Conray II, on leukocyte phagocytosis. MATERIALS AND METHODS: Blood samples were incubated with the contrast media alone (termed the "resting state"), and in combination with a standard phagocytic challenge (Zymosan polysaccharide extract) and with Staphylococcus epidermidis, Streptococcus faecalis and Escherichia coli, to determine the effect of contrast media upon leukocyte phagocytic response. Incubation with saline was used as control. In the case of barium, the "resting state" and standard challenge experiments were repeated at nine dilutions, ranging from 1:1 to 1:1000. The leukocyte phagocytic response was measured in two ways: CO2 generation (an index of metabolic activity) and chemiluminescence (an index of generation of reactive oxygen species and bacterial killing). RESULTS: Barium, at clinical dilutions, causes a significant increase of baseline "resting state" phagocytic activity, which in turn leads to significant blunting of subsequent response to phagocytic challenge and adversely affects the response to all bacteria tested. There is no baseline activation of leukocytes by the water-soluble media, although there was some inhibition (rather than activation) of leukocyte metabolic activity. The effect of the water-soluble media on bacteria was more complex (although inhibition is minor compared to barium). CONCLUSIONS: Our data demonstrate that barium is a significant activator of phagocytic cells, which results in deactivation of phagocytic response when challenged; these data serve to explain the enhanced adverse effect of barium in cases of fecal peritonitis.
Assuntos
Sulfato de Bário/farmacologia , Meios de Contraste/farmacologia , Iotalamato de Meglumina/farmacologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Humanos , Fagocitose/efeitos dos fármacosRESUMO
Renal function parameters and urinary thromboxane B2-excretion were studied in the rat in HgCl2-induced acute renal failure. Studies were performed before and 3 h after the inhibition of thromboxane synthesis alone, after HgCl2 alone, or after the combination of HgCl2 and thromboxane-synthesis inhibition. Thromboxane-synthesis inhibition alone by indomethacin (5 mg/kg i.v.), imidazole (25 and 50 mumol/kg per min i.v.) and dazoxiben (5 mg/kg i.v.) had no effect on glomerular filtration rate (GFR) or para-aminohippuric acid clearance (CPAH), whereas urinary thromboxane excretion was suppressed. Only the administration of the selective blockers imidazole and dazoxiben resulted in a marked increase in urinary volume (V) and fractional sodium excretion (FENa). HgCl2 alone (2 mg/kg i.v.) caused a decrease in GFR and CPAH with -38% (P less than 0.01), and urinary thromboxane B2 excretion increased from 20.3 +/- 1.5 to 30.6 +/- 2.6 pg/min. (P less than 0.01). The administration of indomethacin, imidazole (50 mumol/kg per min) and dazoxiben prevented the increase in thromboxane B2 excretion 3 h after HgCl2 to values of 3.3 +/- 1.2, 6.9 +/- 0.6 and 13.0 +/- 1.6 pg/min respectively (P less than 0.01 versus control for all values). Despite this, the decrease in GFR and CPAH after HgCl2 could not be prevented. A decrease of GFR with -44, -54, -57 and -32% and of CPAH with -37, -49, -57 and -27% were observed for indomethacin, imidazole 25 and 50 mumol/kg per min and dazoxiben respectively. This evolution was not significantly different from what was observed with mercury alone. Selective thromboxane synthesis inhibition resulted in a decrease of serum free ionised calcium.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Injúria Renal Aguda/induzido quimicamente , Cloreto de Mercúrio/toxicidade , Tromboxanos/biossíntese , Injúria Renal Aguda/fisiopatologia , Injúria Renal Aguda/urina , Animais , Cálcio/metabolismo , Taxa de Filtração Glomerular/efeitos dos fármacos , Imidazóis/farmacologia , Indometacina/farmacologia , Masculino , Natriurese/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Tromboxano-A Sintase/antagonistas & inibidores , Tromboxanos/urina , Ácido p-Aminoipúrico/metabolismoRESUMO
It is generally recognized that the uremic syndrome results in a depression of immune function, but the uremic solutes responsible remain largely unidentified. In this study, the effect of 18 known uremic retention solutes, including urea and creatinine, on hexose monophosphate shunt (HMS)-dependent glucose-1-C14 utilization (G1C-U), chemiluminescence production (CL-P) and flow cytometric parameters (FCP) of respiratory burst and phagocytosis were evaluated in granulocytes and/or monocytes. Among the compounds studied, only p-cresol depressed whole blood respiratory burst reactivity (G1C-U, CL-P) dose dependently at concentrations currently encountered in end-stage renal disease (ESRD) (P < 0.05 from 5 micrograms/ml on). The effect of p-cresol was enhanced by increasing incubation times from 10 to 120 minutes. HMS activity of isolated packed erythrocytes remained unaffected. FCP of respiratory burst activity (Bursttest, expressed as log fluorescence units, LFU) revealed a marked depression in the presence of p-cresol (from 700 +/- 167 to 291 +/- 128 LFU for granulocytes, from 278 +/- 102 to 146 +/- 52 LFU for monocytes, P < 0.01), whereas particle ingestion (Phagotest) remained unaffected. Cell-free myeloperoxidase activity was also markedly depressed in the presence of p-cresol. Polarity based HPLC-elution of a standard solution containing all the solutes studied, using a gradient from 100% formic acid to 100% methanol during 60 minutes, revealed elution of p-cresol after 46.6 minutes, pointing to its relative hydrophobicity. Conjugation of p-cresol to p-cresylsulfate anihilated the depressive effect of p-cresol on granulocyte function, and at the same time caused a shift in HPLC-elution pattern to a less lipophilic range.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Cresóis/metabolismo , Fagócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Uremia/metabolismo , Cromatografia Líquida de Alta Pressão , Cresóis/farmacologia , Relação Dose-Resposta a Droga , Humanos , Fagócitos/efeitos dos fármacos , Fatores de TempoRESUMO
BACKGROUND: Dialysis with complement-activating membranes is associated with leukopenia, which is related to an increased expression of adhesion molecules on leukocytes. Citrate chelates calcium and has been claimed to attenuate leukopenia. METHODS: In this study, the effects of citrate anticoagulation on leukocyte and granulocyte counts, complement activation, and the expression of CD11b, CD11c, and CD45 on the surface of granulocytes were evaluated during hemodialysis with unmodified cellulose membranes. Standard heparin was compared to citrate in three different schedules: citrate was infused to obtain a concentration of either 7 or 10 mmol/l blood. CaCl(2) was administered into the dialyzer outlet at 8. 25 mmol Ca(2+)/h (citrate 10 mmol/l) or at 11 mmol Ca(2+)/h (citrate 7 and 10 mmol/l) to reconstitute the calcium levels in the blood returning to the patient. RESULTS: The use of citrate at a high concentration (10 mmol/l) was associated with a blunted upregulation of CD11b, both at the inlet and at the outlet bloodline; for CD11c a reduced upregulation was observed on granulocytes harvested from the inlet bloodline. No effects of citrate were observed on leukopenia, granulocytopenia, or complement activation. A positive correlation between the decrease in systemic ionized Ca(2+) concentration and the increase in CD11b and CD11c expression was found. CONCLUSION: Citrate/CaCl(2) administration affects leukocyte adhesion molecule expression in a dose-dependent way; however, no significant effect could be demonstrated on leukopenia and complement activation.
Assuntos
Anticoagulantes/administração & dosagem , Citratos/administração & dosagem , Ativação do Complemento/efeitos dos fármacos , Granulócitos/química , Falência Renal Crônica/complicações , Leucopenia/tratamento farmacológico , Diálise Renal/métodos , Adulto , Idoso , Materiais Biocompatíveis , Cálcio/sangue , Celulose , Complemento C3a/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Feminino , Granulócitos/efeitos dos fármacos , Heparina/administração & dosagem , Humanos , Falência Renal Crônica/terapia , Antígenos Comuns de Leucócito/análise , Contagem de Leucócitos , Leucopenia/prevenção & controle , Antígeno de Macrófago 1/análise , Masculino , Membranas Artificiais , Pessoa de Meia-Idade , Sódio/sangueRESUMO
Previous studies from our laboratory have demonstrated that the activity of the hexose monophosphate shunt (HMS) pathway in phagocytosis-related respiratory burst is disturbed in end-stage renal disease. To determine whether uraemic solute retention is responsible for this defect the HMS-path was evaluated by measurements of glucose-1-C14 utilization and determination of 14CO2 production in polymorphonuclear cells (PMNLs), suspended in normal plasma or uraemic biological fluids. Normal PMNLs, while suspended in normal or uraemic plasma, were stimulated with either latex, zymosan or Staph. aureus; CO2 generation (measured as DPM/10(3) PMNL, normal versus uraemic plasma) was depressed in uraemic plasma in response to latex (from 43 +/- 5 to 20 +/- 3), zymosan (from 72 +/- 8 to 47 +/- 4) (P < 0.01), and Staph aureus (from 73 +/- 17 to 47 +/- 8 DPM/10(3) PMNL) (P < 0.05). The degree of inhibition was similar for each stimulus. To characterize the substances responsible for this defect we fractionated uraemic plasma ultrafiltrate by polarity-based semipreparative C18 reversed phase HPLC and found a decreased response to Staph. aureus in the presence of fraction 2 (from 102 +/- 13 to 23 +/- 10 DPM/10(3) PMNL, P < 0.05), and in fractions 8 and 11 (lowest value in fraction 8, 54 +/- 14 DPM/10(3) PMNL, P < 0.05 versus control). The pattern of HPLC elution on a gradient from 100% formiate (pH 4.0) to 100% methanol indicates that there are at least two chemically distinguishable groups of compounds, one hydrophilic (in fraction 2), and one lipophilic (in fractions 8 and 11). We conclude that uraemic biological fluids contain factors that inhibit HMS activity related to phagocytosis, and that at least two groups of components with different characteristics are involved.
Assuntos
Fatores Biológicos/farmacologia , Neutrófilos/fisiologia , Fagocitose/fisiologia , Uremia/sangue , Adulto , Fatores Biológicos/isolamento & purificação , Dióxido de Carbono/metabolismo , Cromatografia Líquida de Alta Pressão , Contagem de Colônia Microbiana , Feminino , Glucose/metabolismo , Humanos , Falência Renal Crônica/sangue , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Diálise Renal , Staphylococcus aureusRESUMO
The expression of CD14 on monocytes and CD45 on monocytes and granulocytes was evaluated during hemodialysis by flow cytometric analysis in the 'resting state' and after in vitro stimulation with phorbol myristate acetate (PMA). A comparison of complement activating cuprophane (CU) versus less complement activating polysulfone (PS) was undertaken. 'Resting state' CD45 expression on granulocytes increased markedly during CU dialysis compared to time 0, whereas this rise was only moderate with PS (CU vs. PS, p < 0.01). When considering the increase in expression upon PMA stimulation, a lower value was obtained during CU dialysis for both CD14 (monocytes at 60 and 240 min) and for CD45 (monocytes and granulocytes at 15 min). In conclusion, granulocytes in the 'resting state' expressed more CD45 on their cell membranes during CU dialysis, whereas CD14 and CD45 upregulation after ex vivo addition of PMA was blunted during CU dialysis.
Assuntos
Antígenos CD/sangue , Materiais Biocompatíveis , Celulose/análogos & derivados , Granulócitos/imunologia , Antígenos Comuns de Leucócito/sangue , Receptores de Lipopolissacarídeos/sangue , Monócitos/imunologia , Polímeros , Diálise Renal , Sulfonas , Idoso , Antígenos CD/biossíntese , Células Cultivadas , Feminino , Citometria de Fluxo , Granulócitos/efeitos dos fármacos , Humanos , Antígenos Comuns de Leucócito/biossíntese , Contagem de Leucócitos , Receptores de Lipopolissacarídeos/biossíntese , Masculino , Monócitos/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
BACKGROUND: It has been demonstrated that uraemic serum/ultrafiltrate inhibits cell-mediated immune response in vitro, and that it suppresses calcitriol synthesis and its biological actions. METHODS: In the present in vitro study, the effect of calcitriol, uraemic ultrafiltrate (UUF) and a combination of both on the human promyelocytic leukaemia cell line, HL-60, was studied by evaluating bromodeoxyuridine (BrdU) incorporation into the DNA, luminol-amplified chemiluminescence (CL) production, expression of CD14, and levels of vitamin D receptor mRNA (VDR mRNA) and CD14 mRNA. RESULTS: The ability of calcitriol to block cell proliferation (37.4+/-5.4 to 30.5+/-5.6% cells incorporating BrdU, P:<0.01) was neutralized when UUF was applied together with calcitriol (53.4+/-21.3% cells incorporating BrdU, P:<0.01 vs calcitriol alone). Similarly to what was observed for BrdU incorporation, the CL production of HL-60 cells was enhanced in the presence of calcitriol (20126+/-10154 to 61528+/-24021 cpm, P:<0.01), and was suppressed again in the presence of calcitriol and UUF (20916+/-12075 cpm, P:<0.01 vs calcitriol alone); finally UUF also inhibited the calcitriol-induced CD14 expression (71.1+/-11.2 to 54.9+/-17.7% CD14 positive cells, P:<0.05). On the other hand, the calcitriol-induced CD14 mRNA levels were not significantly different in the presence of calcitriol and UUF compared to calcitriol alone. This points to an inhibition by UUF at a post-transcriptional level. Similar data were found for VDR mRNA levels. UUF was fractionated by HPLC in four fractions, hydrophilic uraemic solutes being eluted first (F1) and hydrophobic solutes being eluted last (F4); fractions 1, 2 and 3 simultaneously affected both BrdU incorporation and CL production in a significant way. CONCLUSIONS: It is concluded that UUF contains factors that impair calcitriol-activated function of HL-60 cells. Hence, the differentiation and immune response of these promyelocytic leukaemia cells, as induced by the supplementation of calcitriol, is neutralized in the presence of uraemic biological fluids. This may be of relevance for the propensity to infection and malignancy of the uraemic patient.
Assuntos
Fatores Biológicos/farmacologia , Calcitriol/antagonistas & inibidores , Calcitriol/farmacologia , Uremia/metabolismo , Bromodesoxiuridina/metabolismo , Divisão Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Feminino , Células HL-60 , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Medições Luminescentes , Masculino , Pessoa de Meia-Idade , UltrafiltraçãoRESUMO
In this study, the factors in overnight dwell fluid (8 to 10 hr dwell) depressing granulocyte (GC) NAD(P)H-oxidase dependent radical species production are characterized. At present, most studies have essentially focused on fresh, unspent dialysate and on peritoneal macrophages. The response to Staphylococcus aureus (Staph A) was dose-dependently depressed for both GC CO2 production (from 91.3 +/- 8.4 to 9.0 +/- 1.5 dpm/10(3) GC, P < 0.01) and chemiluminescence (CL) (peak from 7.3 +/- 0.8 to 1.6 +/- 0.8 cps x 10(3)/GC, P < 0.01). Stimulation with formyl-methionine-leucine-phenylalanine (f-MLP), phorbol myristic acid (PMA), Staphylococcus epidermidis (Staph Epi), E. coli, latex and zymosan revealed a parallel depression, pointing to an intrinsic metabolic defect, rather than failure of particle ingestion. The addition of glucose to the normal cell medium to obtain the same concentration as in the CAPD effluent (2.9 +/- 0.3 mg/dl) depressed function but not to the same extent as the genuine PD effluent. Opsonization of Staph A and E. coli induced a partial correction. No effect of pH or osmolality was observed. HPLC fractionation of CAPD effluent on a polarity based gradient revealed an elution of depressive factors in hydrophobic fractions with a nadir in F7 and F12. Analysis of the elution pattern of various uremic solutes revealed elution in F12 of p-cresol, a solute with known inhibitory effect on GC function. These events may be related to recent peritonitis (CL in response to Staph A 0.3 +/- 0.1 in effluent of 6 patients with recent peritonitis versus 2.6 +/- 0.8 cps x 10(3)/GC in 12 patients without recent peritonitis (P < 0.01). We conclude that the GC response is depressed in the presence of CAPD effluent due to excess glucose, lack of opsonization, and uremic solutes of which p-cresol is one of the responsible compounds.