Modulation of the production of reactive oxygen species (ROS) by cAMP-elevating agents in granulocytes from diabetic patients: an Akt/PKB-dependent phenomenon.

BACKGROUND: Granulocytes from healthy subjects and from patients suffering from diabetes mellitus present differences in reactivity to stimulation with cyclic nucleotide-elevating agents. The production of reactive oxygen species (ROS) is inhibited in cells from non-diabetic subjects following such stimulation, but activated through a PKA-independent signaling pathway in granulocytes from type 1 and type 2 diabetic patients. The aim of the present study was to understand better the changes in signaling mechanisms induced by the disease. METHODS: ROS production in granulocytes from healthy subjects and from type 1 and type 2 diabetic patients was measured using a luminol-dependent chemiluminescence assay. Granulocytes were stimulated by the addition of the cAMP-elevating agent dibutyryl cAMP. In some experiments, granulocytes were pre-treated with an inhibitor of PKA or Akt/PKB prior to cAMP stimulation. RESULTS: Intracellular elevation of cAMP induced a PKA-dependent and Akt/PKB-independent inhibition of ROS production in granulocytes from healthy subjects, but a significant activation in cells from both type 1 and type 2 diabetic patients. Most significantly, activation of ROS generation in cells from diabetic patients was shown to be Akt/PKB-dependent and PKA-independent. CONCLUSIONS: These results suggest that chronic hyperglycaemia could induce metabolic adaptation in cAMP-related signaling mechanisms. Epac (exchange protein directly activated by cAMP) is a novel cAMP receptor besides PKA involved in different signaling pathways. The cAMP-stimulated inverse ROS response in granulocytes from type 1 and type 2 diabetic patients may be due to a change in signaling pathways from cAMP/PKA to cAMP/Epac/Akt/PKB. These preliminary results require further studies in order to evaluate their consequences on innate immunity and pathogenesis of diabetes mellitus.

Comparative clinical and ultrasound study of egg-negative and egg-positive individuals from Schistosoma mansoni low morbidity endemic areas, and hospitalized patients with hepatosplenic disease.

Two hundred and twenty three subjects from a Schistosoma mansoni low morbidity endemic area and nine hospitalized hepatosplenic patients were submitted to stool test and clinical examination and abdomen ultrasound assessments. According to stool examination and ultrasound results, they were grouped as follows: G1 -- 63 Schistosoma mansoni egg-negative individuals; G2 -- 141 egg-positive patients and without evidence of periportal fibrosis; G3 -- 19 egg-positive patients with periportal echogenicity (3-6 mm); and G4 -- 9 hepatosplenic patients with periportal echogenicity (> 6 mm). Hepatomegaly detected by physical examination of the abdomen evaluated in the midclavicular line was verified in G1, G2 and G3, respectively, in 11.1, 12.1 and 26.3%. In G1, G2 and G3, periportal thickening occurred only in schistosomal patients (8.5%). Mild pathological alterations in patients that cannot yet be detected by clinical examination were detectable in the liver by ultrasound and can be due to fibrosis. The degree of mild periportal fibrosis was diminished in 57.9% of patients 12 months after treatment of schistosomiasis with oxamniquine. At ultrasonography, the mean liver left lobe measurement of G3 was larger than that of G1, and that of G4 larger than that of G1 and G2. The mean size of the spleen of G4 was significantly larger than that of the other three groups, and that of G3 larger than that of G1 and G2.

Modulation of the reactive oxygen species (ROS) generation mediated by cyclic AMP-elevating agents or Interleukin 10 in granulocytes from type 2 diabetic patients (NIDDM): a PKA-independent phenomenon.

UNLABELLED: SUMMARY-BACKGROUND: The present study investigates the hypothesis that cells from ill patients and from healthy subjects may have different reactivity under metabolic stimulation as a consequence of an disease-induced metabolic adaptation. METHODS: Granulocytes either from healthy subjects or from type II-Non Insulin Dependent Diabetes Mellitus (NIDDM) patients were compared in their capacities to generate Reactive Oxygen Species (ROS). The ROS generation was comparatively determined in a chemiluminescence assay, luminol-dependent, after cell incubation in the presence of either cyclic AMP - elevating agents or Interleukin 10. In some experiments the cells were pretreated with H89 compound (a PKA inhibitor) or with diphenylene iodonium (DPI), a NADPH-oxidase inhibitor. RESULTS: Our results showed an increased ROS generation in granulocytes from diabetic patients in absence of cyclic AMP-elevating agents or IL-10. In the presence of cyclic AMP-elevating agents was observed an inverse metabolic response in granulocytes from diabetic patients in comparison to cells from healthy subjects. The granulocytes were pre-incubated in the presence of cyclic AMP-elevating agents--amminophylline (AMF) or dibutyryl cyclic AMP (dbcAMP)--or interleukin 10 (IL-10). The AMF, dbcAMP and IL-10 inhibited ROS production by granulocytes from healthy subjects. By contrast, AMF and dbcAMP activated cells from diabetic patients while IL-10 had no effect. The inhibition of ROS induced by AMF, dbcAMP or IL-10 was promptly abolished by the pretreatment of the cells with either PKA H89 inhibitor or NADPH-oxidase inhibitor (DPI) in granulocytes from healthy subjects. In relation to the granulocytes from type 2 diabetics patients, the activation of ROS generation mediated by AMF and dbcAMP was fully abolished by NADPH-oxidase DPI-inhibitor, but not by PKA H89 inhibitor. CONCLUSIONS: Our present results reinforce the hypothesis that cells from ill patients (type II diabetic) when compared to cells from healthy subjects have different reactivity under metabolic stimulation. ROS production by human granulocytes was modulated by cyclic AMP elevating agents and IL-10. The inhibition of the ROS production in cells from healthy subjects was PKA-dependent while the activation in granulocytes from patients was PKA-independent. This inverse metabolic response, in cells from patients, suggests the use of an alternative metabolic pathway PKA-independent, possible cAMP/Epac/PKB-dependent. The correlation between activation of ROS production in granulocytes from diabetic patients and pathogenesis of diabetes can be suggested, however, further and extensive studies are needed for demonstrating this suggestion.

Effect in vitro of cyclic nucleotides-elevating agents on nitric oxide production by human granulocytes from type 2-diabetic patients.

BACKGROUND: The present study was designed to investigate the hypothesis that cells from ill patients and from healthy subjects may have different reactivities under metabolic stimulation. METHODS: The study was performed with granulocytes from non-diabetic subjects and from type II -Non Insulin Dependent Diabetes mellitus (NIDDM) patients. The nitric oxide (NO) generation was comparatively determined by the nitrite concentration (micromolar of nitrite) after cell incubation in the presence of cyclic nucleotide-elevating agents. RESULTS: Our results showed an inverse reactivity for granulocytes from diabetic patients when compared to non-diabetic subjects. Granulocytes were incubated in the presence of drugs that elevate the intracellular level of cyclic AMP aminophylline (AMF), dibutyryl cyclic AMP (dbcAMP)], cyclic GMP [8.Br. cyclic GMP(8.Br.cGMP) or levamisole (LEV)]. The cyclic AMP-elevating agents (AMF and d bcAMP) inhibited NO production by granulocytes from non-diabetic subjects and activated cells from diabetic patients. By contrast, cyclic GMP-elevating agents (8.Br.cGMP and LEV) activated cells from non-diabetic subjects and inhibited granulocytes from diabetic patients. The activation of NO generation by cyclic nucleotides was blocked by pretreatment of granulocytes with L-NAME. CONCLUSION: The authors describe for the first time that both cyclic AMP and cyclic GMP were able to modulate nitric oxide production in human granulocytes and that cell reactivity in ill patients (diabetic) showed altered and inverse response in comparison to granulocytes from healthy subjects. This inverse reactivity possibly reflects a disease-induced adapted metabolic response. The consequences of this altered metabolic response on host defense and inflammation may be speculated, but further experiments are needed to confirm this hypothesis.

Resistance to oxamnique of a Schistosoma mansoni strain isolated from patient submitted to repeated treatments

Uma cepa de Schistosoma mansoni (R) foi isolada de paciente previamente submetido a quatro tratamentos com o oxamniquine e a um outro praziquantel. Os resultados obtidos com o teste quimioterapeutico, usando oxcamniquine em camundongos infectados com as cepas R1 e Le (padrao) mostraram resistencia evidente a droga em vermes de cepa R1. Assim, com a dose de 250 mg/kg de oxamniquine, todos os camundongos (17) dos 17 camundongos infectados com a cepa R1 apresentaram vermes sobreviventes...

Resistance to oxamniquine of a Schistosoma mansoni strain isolated from patient submitted to repeated treatments.

A strain of Schistosoma mansoni (R1) was isolated from patient previously submitted to four treatments with oxamniquine, and to another one with praziquantel. The results obtained with chemotherapeutic test, by using oxamniquine in mice infected with the strains R1 and LE (standard), showed an evident resistance to the drug in worms of the strain R1. Thus, at the dose of 250 mg/kg oxamniquine, all mice (17) infected with the LE strain did not show surviving worms, whereas 12 out of 17 mice infected with the R1 strain presented surviving worms. At the dose of 200 mg/kg, the LE strain showed recovery rates of 1.06% and 20.58%, whereas the R1 strain presented 18.57% and 61.14%, for male and female worms, respectively. At the dose of 100 mg/kg, the recovery of male worms was 2.6% for the LE strain, and 29.9% for the R1 strain. At the same dose, the recovery of females did not show statistically significant differences between the two strains (LE = 76.38%, R1 = 79.12%). Praziquantel showed similar antischistosomal activity against both studied strains, when administered at the dose of 500 mg/kg.

Effect of gamma interferon and interleukin 10 on phosphoinositol turnover by human neutrophils in vitro.

We evaluated the levels of inositolmono-(IP1), di(IP2), tri-(IP3) and tetraphosphates (IP4) in human neutrophils (N) stimulated with gamma interferon (IFN-gamma) (200 microliters from a pool of cell culture supernatant obtained from 1 x 10(7) PHA-primed peripheral blood mononuclear cells (30-60 min at 37 degrees C, 5% CO2)) in the presence of in the absence of interleukin 10 (IL-10) (10 micrograms/10 microliters). The results, reported as mean +/- SEM cpm, showed that IFN-gamma induced a significant increase only in the IP3 level (N + medium = 1,413 +/- 172 and N + IFN-gamma = 8,875 +/- 832). However, this activation mediated by IFN-gamma was blocked partially in the presence of IL-10 (N + IFN-gamma + IL-10 = 2,430 +/- 239) (P < 0.05). Interleukin 10 alone did not induce significant alterations in the content of IP1 (1,203 +/- 123), IP2 (1,880 +/- 163), IP3 (938 +/- 102) or IP4 (2,403 +/- 345) when compared to the respective controls in the absence of IL-10 (IP1 = 1,625 +/- 132; IP2 = 1,343 +/- 149; P3 = 1,413 +/- 172 and IP4 = 3,281 +/- 234). We also demonstrated the inhibitory effect of IL-10 of chemoluminescence generation by human neutrophils during phagocytosis of opsonized particles (OZ). Chemoluminescence generation was enhanced by IFN-gamma (N = OZ = 42.8 +/- 3.9 and N + OZ + IFN-gamma = 66.5 +/- 4.3) and this effect was reduced by IL-10 (N + OZ + IFN-gamma + IL-10 = 37.6 +/- 5.1). These data suggest that IL-10 modulates the neutrophil response and may be important for the development of new treatments of inflammatory injury.

Effect of gamma interferon and interleukin 10 on phosphoinositol turnover by human neutrophils in vitro

We evaluated the levels of inositolmono-(IP1), di-(IP2), tri- (IP3) and tetraphosphates (IP4) in human neutrophils (N) stimulated with gamma interferon (IFN-gamma) (200 mul from a pool of cell culture supernatant obtained from 1 x 10(7) PHA-primed peripheral blood mononuclear cells (30-60 min at 37 degrees Celsius, 5 per cent CO2) in the presence or in the absence of interleukin 10 (IL-10) (10 mug/10mul). The results, reported as mean + SEM cpm, showed that IFN-gamma induced a significant increase only in the IP3 level (N + medium = 1.413 + 172 and N + IFN-gamma = 8,875 + 832). However, this activation mediated by IFN-gamma was blocked partially in the presence of IL-10 (N + IFN-gamma + IL-10 = 2,430 + 239) (P<0.05). Interleukin 10 alone did not induce significant alterations in the content of IP1 (1,203 + 123), IP2 (1,880 + 163), IP3 (938 + 102) or IP4 (2,403 + 345) when compared to the respective controls in the absence of IL-10 (IP1 = 1,625 + 132; IP2 = 1,343 + 149; IP3 = 1,413 + 172 and IP4 = 3,281 + 234). We also demonstrated the inhibitory effect of IL-10 on chemoluminescence generation by human neutrophils during phagocytosis of opsonized particles (OZ). Chemoluminescence generation was enhanced by IFN-gamma (N + OZ = 42.8 + 3.9 and N + OZ + IFN-gamma = 66.5 + 4.3) and this effect was reduced by IL-10 (N + OZ + IFN-gamma + IL-10 = 37.6 + 5.1). These data suggest that IL-10 modulates the neutrophil response and may be important for the development of new treatments of inflammatory injury.

Colorimetric leukocyte adherence inhibition assay: a new auxiliary technique for the diagnosis of schistosomiasis.

We describe a modification of the leukocyte adherence inhibition assay (LAI) in which we propose the use of 3-(4,5-dimethylthiazole-2-yl)-2-5-diphenyltetrazolium bromide (MTT) dye which is taken up and reduced by mitochondria. The method was tested by screening peripheral blood leukocytes from Schistosoma mansoni-infected patients. Peripheral blood leukocytes from patients (N = 21) but not from the blood of normal subjects (N = 10) failed to adhere to glass in the presence of soluble adult worm antigenic preparation (SWAP). The non-adherence index (NAI) values for schistosomiasis patients were in the range of 11.0 to 72.3 (mean +/- SEM = 29.3 +/- 4.3), whereas the values for normal subjects were -56.0 to +2.0 (-25.9 +/- 7.6) and those for treated patients -59.6 to +4.0 (-19.3 +/- 5.8). Our results show that the colorimetric LAI assay can be used as an auxiliary test for the diagnosis of schistosomiasis.

A microcalorimetric assay to measure the reactivity of antigen-stimulated mononuclear cells from Schistosoma mansoni-infected patients.

The reactivity of mononuclear cells (2 x 10(6)/ml minimum Eagle's medium, MEM) from normal subjects and from Schistosoma mansoni-infected patients was evaluated by microcalorimetry. The results which are reported as heat production (mcal for 2 x 10(6) cells in 3600 s), were 2,087 +/- 21.3 and 2,497.0 +/- 21.3 for mononuclear cells from infected patients (N = 8) under stimulation with S. mansoni soluble egg antigen (SEA) and soluble adult worm antigenic preparation (SWAP), respectively. The values for cells from normal subjects (N = 8) were 13.7 +/- 1.1 and 29.3 +/- 3.2 in the presence of the same antigens. Pre-treatment of mononuclear cells from patients with 1 mM aminophylline (a cAMP phosphodiesterase inhibitor) totally abolished heat production. Cell viability (greater than 95%) was not changed after the measurement. The microcalorimetric assay described here measures the cellular metabolic activity and we feel justified in suggesting this technique as an auxiliary diagnosis of schistosomiasis. Given the sensitivity, precision and accuracy of this microcalorimetric assay, we feel it can be used for the diagnosis of disease conditions for which a reliable diagnostic method is required.

Correlation between the lack of mononuclear cell cytotoxicity against schistosomula in vitro and inositolphosphate turnover in patients with schistosomiasis.

A comparison was made of the reactivity of mononuclear cells from normal subjects and from S. mansoni-infected patients. The following parameters were evaluated: 1) ability of mononuclear cells to kill schistosoma in the presence of complement; 2) [3H]-inositol incorporation into phosphatidylinositol (PI) and the rate of inositolphosphates (IPx) released. Cells from normal subjects, but not from S. mansoni-infected patients, were able to kill schistosomula in vitro. A decrease in inositolpolyphosphates (IPx) was observed for phytohemagglutinin (PHA)-stimulated mononuclear cells from infected patients when compared with mononuclear cells from normal subjects after 24 h of incubation. The results suggest that the reactivity of mononuclear cells from infected patients is altered under conditions of nonspecific stimulation with PHA when compared with normal cells.