Up-regulation of nitric oxide production by interferon-gamma in cultured peritoneal macrophages from patients with cirrhosis.

We previously described a long-lasting overproduction of nitric oxide (NO) in cirrhotic patients with spontaneous bacterial peritonitis. The aim of the present study was to investigate the presence of the inducible NO pathway in peritoneal macrophages. Ascitic fluids were collected from 29 patients with cirrhosis, aged between 35 and 82 years. Peritoneal macrophages were isolated and cultured in the presence or absence of 1 microg/ml lipopolysaccharide and/or 500 units/ml interferon-gamma (IFN-gamma) for 6 days. NO production was measured as nitrate+nitrite (NO(x)), inducible NO synthase (iNOS) protein expression was analysed by immunocytochemistry and Western blot analysis using a specific anti-(human iNOS) antibody, and the catalytic activity of NOS was revealed by cytochemical staining for NADPH-dependent diaphorase. Cultured macrophages spontaneously released small amounts of NO(x) [median (10-90th percentile) of 18 separate experiments: 3.3 (0-8) micromol/l]. Addition of lipopolysaccharide alone or in combination with IFN-gamma to the culture medium did not change the levels of NO(x), while IFN-gamma alone dramatically increased NO production [13.4 (3.5-28.3) micromol/l; P<0.001]. Macrophages were stimulated by IFN-gamma to a greater extent in patients with recent spontaneous bacterial peritonitis (n=13) than in those in a stable clinical condition (n=18) [19.8 (10.5-30.1) and 10.0 (3.2-14.5) micromol/l respectively; P<0.001]. Macrophages freshly isolated or stimulated with IFN-gamma expressed iNOS protein, as shown by Western blot and immunocytochemical analysis, and stained for NADPH diaphorase. Our findings demonstrate the presence of iNOS protein in peritoneal macrophages from cirrhotic patients. The role of IFN-gamma appears to be a determinant for the up-regulation of NO production, particularly under conditions of infection. Therefore peritoneal macrophages producing large amounts of NO at the site of infection may contribute to maintaining splanchnic vasodilation in these patients.

Analysis of nitrite and nitrate in biological fluids by capillary electrophoresis.

OBJECTIVES: We report a method for determining nitrite and nitrate in biological fluids by capillary electrophoresis. METHODS: A Waters capillary electrophoresis system was used with a filter for detection at 214 nm. After dilution with distilled water, the sample was loaded hydrostatically onto a 60 cm x 100 microm capillary and electrophoresed at 15 kV in 15 mmol/L sulfate buffer, pH 8.0, containing 2.5% electroosmotic flow modifier. RESULTS: The retention times for nitrite and nitrate were 3.9 +/- 0.8 and 4.0 +/- 0.8 min, respectively. The detection limit was 10 micromol/L for serum nitrate. The recovery was 93-115% for nitrite and 92-106% for nitrate. The within-day and between-day coefficients of variation were lower than 3.3% and 5.0%, respectively, for two pools with normal (28 micromol/L) and high (87 micromol/L) nitrate concentration. A comparison with the nitrate reductase method gave a correlation coefficient of 0.982. CONCLUSION: Capillary electrophoresis provides many advantages, namely low cost, small sample and buffer requirements, rapidity, which makes its use particularly suitable for clinical laboratories.

Lack of a nitric-oxide response during the course of Leishmania infantum infection in the golden hamster (Mesocricetus auratus), with or without treatment with liposomal amphotericin B.

Liver and spleen volumes and serum concentrations of nitrate (the end-product of NO in vivo), albumin, gamma-globulin, protein, creatine and urea were measured during the course of progressive infections with Leishmania infantum MON-1 (MHOM/PR/93/CRE29) in 10 Syrian golden hamsters. Each hamster was infected by intraperitoneal injection with 4 x 10(7) promastigotes. Five of the infected animals were treated, with 6 mg liposomal amphotericin B (L-AmB)/kg given by intracardiac injection, on day 107 post-infection (p.i.). Compared with those in the uninfected hamsters used as controls, the liver volumes in the infected animals became significantly enlarged by day 40 p.i. (38% larger than the controls; P < 0.001) whereas significant enlargement of the spleen was first detected on day 72. Each infected animal had detectable serum levels of antileishmanial antibodies on day 72. There were significant elevations in gamma-globulin concentration as early as day 40 (P < 0.05) but significant falls in albumin concentrations were only detected from day 107 (P < 0.001). Nitrate, creatinine and urea concentrations remained unchanged during the course of infection, even after L-AmB treatment. Serum nitrate levels were not enhanced by L. infantum infection nor by the L-AmB treatment which induced a 98.2% decrease in parasite burden. The lack of NO production in visceral leishmaniasis, with or without L-AmB treatment, points to the unresponsiveness of inducible nitric oxide synthase in this rodent model.

Serum and tissue nitrate levels in murine visceral leishmaniasis correlate with parasite load but not with host protection.

Nitrate levels were measured in serum and in organs from Lshs BALB/c and Lshr C3H/HeN mice during the acute phase (30 d) of infection by Leishmania donovani strain LV9. Serum nitrate levels increased rapidly in BALB/c mice from a baseline level (17 +/- 4 mumol/L) to a plateau (504 +/- 129 mumol/L) at 24 d and correlated with parasite loads in the liver (r = 0.817, P < 0.01) and in the spleen (r = 0.854, P < 0.001). Liver and spleen nitrate contents were enhanced 2.7-fold and 22.8-fold, respectively, with respect to uninfected controls (2692 +/- 249 vs. 992 +/- 231 nmol, P < 0.02 and 20 +/- 1 vs. 456 +/- 43 nmol, P < 0.02). In contrast, serum nitrate increased to a lesser extent in C3H/HeN mice, from 31 +/- 5 mumol/L to 86 +/- 5 mumol/L at 20 d. Liver nitrate content did not differ significantly between infected and control mice (1093 +/- 83 vs. 867 +/- 104 nmol), whereas the former had a higher spleen nitrate content (145 +/- 22 vs. 40 +/- 2 nmol, P < 0.02). Our findings indicate that production of NO by the susceptible BALB/c strain exceeded that of the resistant C3H/HeN strain during the acute stage of infection by L. donovani. Tissue NO overproduction in organs infected by L. donovani was related to the progression of parasitic disease and contributed to high nitrate serum levels. It would be very interesting to extend this investigation to human disease with the aim of evaluating serum nitrate as a marker of parasite load in the follow-up of patients suffering from visceral leishmaniasis.

Long-lasting NO overproduction in cirrhotic patients with spontaneous bacterial peritonitis.

Nitric oxide production was studied in cirrhotic patients with spontaneous bacterial peritonitis (SBP) or with other infections. We followed up on the time course of serum nitrate levels in 51 hospitalized patients aged between 34 and 81 years. Four groups were defined: patients with SBP (group 1, n = 14), patients with bacteremia (group 2, n = 11), patients with urinary tract infection (group 3, n = 11) and patients in a stable clinical condition (group 4, n = 20). The four groups did not differ in terms of Pugh score (11 +/- 1, 10 +/- 1, 11 +/- 1, and 10 +/- 1, respectively). Serum nitrate levels averaged 31 +/- 2 micromol/L in group 4 (84 samples). On the day results of cytobacteriological examination were positive, mean serum nitrate levels were 75 +/- 17, 63 +/- 9, and 36 +/- 9 micromol/L, respectively, in groups 1 (17 cases), 2 (11 cases), and 3 (11 cases) (P < .001). The maximum nitrate values recorded during follow-up were higher in groups 1 (149 +/- 15 micromol/L) and 2 (112 +/- 11 micromol/L) than in group 3 (66 +/- 7 micromol/L; P < .001 and < .01, respectively). These maximum values were recorded in all groups approximately 2 weeks after the infection was diagnosed. The mean duration of NO overproduction, as defined by nitrate level (3)90 micromol/L, was 15 +/- 3 days in group 1 and 5 +/- 1 day in group 2. When the nitrate concentration was studied in serum and ascitic fluid sampled on the same day, it was found to be higher in ascitic fluid than in serum in eight cases of SBP in the period preceding the peak serum nitrate concentration (100 +/- 17 vs. 63 +/- 14 micromol/L; P < .001). Our data indicate that SBP in cirrhotic patients led to a long-lasting increased local production of NO. This overproduction may contribute to maintaining splanchnic vasodilation and thus worsen the hyperkinetic state in these patients.