Low levels of nitric oxide (NO) in systemic sclerosis: inducible NO synthase production is decreased in cultured peripheral blood monocyte/macrophage cells.

OBJECTIVE: To investigate nitric oxide (NO) production and inducible NO synthase expression by cultured peripheral blood mononuclear cells (PBMC) in patients with systemic sclerosis (SSc). METHODS: Eighteen patients with SSc were compared with two control groups: 16 patients with rheumatoid arthritis (RA) and 23 patients with mechanical sciatica. Nitrate was determined by fluorimetry in plasma and by spectrophotometry in supernatants. Inducible NO synthase (iNOS) was detected in cultured PBMC by immunofluorescence, immunoblotting and flow cytometry with or without treatment of the cells with interleukin (IL) 1beta+ tumour necrosis factor alpha (TNF-alpha), IL-4 or interferon gamma (IFN-gamma) from day 1 to day 5. RESULTS: NO metabolite concentrations were lower in SSc patients (mean+/-s.e.m. 34.3+/-2.63 micromol/l) than in RA (48.3+/-2.82 micromol/l; P<0.02) and sciatica (43.3+/-5.24 micromol/l; P<0.03) patients. iNOS was detected in cultured monocytes in all three groups but induction occurred on day 1 in RA, day 2 in sciatica and only on day 3 in SSc, whatever the stimulus. CONCLUSIONS: The concentrations of NO metabolites are decreased in SSc patients and the metabolism of these compounds in PBMC is altered. Low levels of NO, a vasodilator, may be involved in vasospasm, which is critical in SSc. This may have therapeutic implications.

Frequency of cytokine-producing T cells in HIV-infected patients treated with stavudine, didanosine, and ritonavir.

To assess prospectively the influence of the control of viral replication on the frequency of cytokine-producing T cells, and to correlate these changes with immune activation, we conducted a 15-month follow-up study of IFN-gamma- and IL-2-producing CD4+ and CD8+ T cells at a single-cell level in 12 previously untreated patients receiving highly active antiretroviral therapy (HAART). At baseline we observed a strikingly high proportion of IFN-gamma-producing CD8+ T cells. The treatment-induced decrease in the proportion of IFN-gamma-producing CD8+ T cells ran parallel to the decrease in HLA-DR+ and CD38+CD8+ T cell subsets and was associated with the reduction in HIV RNA level. IL-2-producing cells were mainly CD4+. As a consequence of CD4+ T cell loss, the number of IL-2-producing CD4+ T cells was lower in patients than in control subjects (52 vs. 171 cells/microl), but the proportion of these cells was unchanged (22.4 vs. 19.3). During therapy the proportion of CD4+ IL-2-producing cells was initially stable and then fell markedly at month 5, followed by a gradual return to previous values. The reduction in viral load was associated with the fall in the proportion of CD4+ activated subsets. Intracellular cytokine assays are a new approach to the assessment of T cell function in HIV infection. Our results suggest that the functional capacity of CD4+ T cells is probably less severely altered than previously thought on the basis of conventional assays. CD8+ T cells exhibit an increased capacity to produce IFN-gamma that is associated with an increase in activation marker expression. These alterations decrease partially and in parallel under treatment.

[Antibacterial activity of urine after administration of ofloxacin for 5 days]. / Activité antibactérienne des urines après administration d'ofloxacine pendant 5 jours.

The antibacterial activity of ofloxacin was evaluated in urine over a period of 96 h after oral administration for 5 days of 200 mg twice a day in 12 healthy female volunteers. Bacteriostatic and bactericidal activity of urines were studied for five strains of enterobacterias recovered from urinary infections: two strains of Escherichia Coli Nal-S and Nal-R, two strains of Proteus mirabilis Nal-S and Nal-R, and one strain of Klebsiella pneumoniae Nal-S. Mean urinary concentrations of ofloxacin were very high during the first 12 h following last intake. They were still above 7 mg/l till the 48th hour and above 1.6 mg/l till the 72nd hour. Bactericidal activity of urine was present for 72 h in respect of four strains studied at that time; urine was not bactericidal as regards E. coli Nal-R. After 5 days of oral treatment with ofloxacin (200 mg b.i.d.), urine retains a bactericidal activity for at least 72 h against bacterial strains of urinary tract infections.

Prospective study of CD8+ lymphocyte activation in relation to viral load in HIV-infected patients with > or = 400 CD4+ lymphocytes per microliter.

To investigate the temporal relationship between CD8+ lymphocyte phenotypic alterations, the CD4+ T cell decline, and plasma HIV RNA levels during the natural history of HIV infection, 33 treatment-naive HIV-infected patients with > or =400 CD4+ cells/microl were studied prospectively for 3 years. During the study period, 20 patients remained untreated, and only 6 received more than 6 months of therapy. A significant relationship was found between changes in plasma HIV RNA and changes in the proportion of CD38+CD8+ cells. Conversely, the number of CD4+ T cells lost per year was strongly related to the increase in the proportion of CD28-CD8+ T cells. A strong relationship between mean yearly changes in CD4+ T cell numbers and changes in HIV RNA was also observed. CD4+ T cell changes were associated with changes in both viral load and CD8+ T cell activation. These results provide support for the use of both virologic and immunologic parameters for prognosis and management during HIV infection.

[Drugs for respiratory tropism and pregnancy]. / Médicaments à tropisme respiratoire et grossesse.

The pharmokinetics of drugs used to treat lung disease in pregnant women undergo changes due to the physiological variations induced by pregnancy. Dosage must therefore be adapted; increased doses are often required for the treatment of severe lung infections. Most drugs used for lung disease have a teratogenic potential and thus carry a risk for the fetus. Drugs used for asthma usually present little risk for the fetus. Administration by inhalation is particularly well adapted as it limits systemic diffusion. Excessively high doses can however lead to neonatal toxicity. Penicillins, cephalosporins and erythromycin have been shown to be well tolerated and are the choice antibiotics. Aminoglycosides require careful monitoring due to the risk of renal and auditory toxicity. Fluoroquinolones, sulfamides and tetracyclines should be avoided. Available data on recent compounds such as the new macrolides (azithromycin, clarithromycin) are too limited for recommending their use during pregnancy. In case of resistant tuberculosis, it is sometimes necessary to prescribe a second choice anti-tuberculosis drug with known or suspected fetal toxicity.

Changes in blood CD8+ lymphocyte activation status and plasma HIV RNA levels during antiretroviral therapy.

OBJECTIVE: To analyse the relationship between CD8+ lymphocyte phenotype alterations and plasma HIV RNA levels in HIV-infected patients treated with the zidovudine-didanosine combination. METHODS: A total of 30 HIV-infected patients who had never received antiretroviral therapy and who were starting treatment with a combination of zidovudine and didanosine were prospectively studied. Multiparameter flow cytometric analysis of CD8+ lymphocytes and plasma HIV RNA determination were performed on day 0, day 15 and monthly from months 1 to 6. RESULTS: Patients were divided into three categories according to the time-course of plasma HIV RNA levels. In 14 patients, an early and sustained fall in plasma HIV RNA to below the detection limit (500 copies/ml) was observed; in 10 patients, the fall was transient; in six patients, plasma HIV RNA was always detectable (non-responders). The mean CD4+ lymphocyte gain was 120 x 10(6)/l at month 6 in sustained and transient responders, and 55 x 10(6)/l in non-responders. A significant fall in the proportion of CD8+ lymphocytes with an activated phenotype was observed only in the two groups of responders, and was higher in the sustained responders (CD38+HLA-DR+, -56.8%; CD38+CD45RO+, -54.0%; HLA-DR+CD45RO+, -48.4%; CD38+CD28-, -47.3%). CONCLUSION: A fall in the proportion of activated CD8+ lymphocytes is associated with the disappearance of HIV RNA from plasma during antiretroviral therapy. Undetectable plasma HIV RNA is not associated with a return to normal CD8+ lymphocyte activation status after 6 months of treatment, suggesting that viral replication persists in lymphoid tissues.

Cytofluorometric analysis of chondrotoxicity of fluoroquinolone antimicrobial agents.

To better understand quinolone-related arthropathy, we conceived an experimental ex vivo model using cell cultures of articular chondrocytes issued from pretreated New Zealand White rabbits (NZW). Juvenile (4- to 5-week-old) NZW were orally dosed with ofloxacin or pefloxacin (300 mg/kg of body weight for 1 day) or with pefloxacin (300 mg/kg for 7 days). Adult (5-month-old) NZW were treated with pefloxacin (300 mg/kg for 1 day). Chondrocytes were enzymatically recovered from cartilage and were analyzed by cytofluorometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and dihydrorhodamine 123 (DHR), reflecting cellular respiratory-burst activity, and rhodamine 123 (Rh123) and 10-N-nonyl-acridine orange (NAO), specific for the mitochondrial activity and mass, respectively. A significant increase in the respiratory burst was detected by DCFH-DA and DHR in all treated groups of young animals, compared with untreated control groups. No significant increase of respiratory burst was noted in older treated rabbits. The 7-day treatment resulted in a decrease in mitochondrial uptake of Rh123 and an increase in NAO uptake. Fluoroquinolone arthrotoxicity seems to involve in its early phase the respiratory burst of immature articular chondrocytes.

The significance of activation markers on CD8 lymphocytes in human immunodeficiency syndrome: staging and prognostic value.

The objective of this prospective cohort study was to evaluate the expression of activation markers on CD8 lymphocytes at various clinical stages of HIV infection and to determine the value of these markers in identifying patients likely to have rapidly progressive disease. One hundred and three HIV+ patients, divided into four disease stages, and 34 seronegative controls were evaluated at study entry using flow cytometric immunophenotyping. The HIV patients were followed clinically for disease progression during the following 2 years. CD8 cell numbers and percentage of lymphocytes are increased after HIV infection. Expression of the CD38, HLA-DR and CD57 markers on CD8 cells was significantly increased in asymptomatic HIV-infected patients when compared with controls, as was the CD8 cell population which did not coexpress Leu-8. These activation markers were observed to be further increased in patient groups with more clinically advanced infection. The percentage of CD38 on CD8 cells emerged not only as a discriminator of disease severity, but was a strong predictor of progression in asymptomatic, lymphadenopathy and ARC patients. Given the utility of activation markers on CD8 lymphocytes in staging disease and predicting clinical outcome, the measurement of these parameters should be considered in the monitoring and management of HIV patients.

T activation marker evaluation in ARC patients treated with AZT. Comparison with CD4+ lymphocyte count in non-progressors and progressors towards AIDS.

Reductions in the percentage and absolute number of CD4+ lymphocytes, as well as abnormally high levels of activated peripheral T lymphocytes (CD3+ HLA-DR+ phenotype) and an increased proportion of CD8+ cells coexpressing the CD57 surface antigen (involved in natural killer activity) have been reported in HIV infection and associated with disease progression. We prospectively measured these subsets of lymphocytes in 34 patients with advanced AIDS-related complex (ARC) treated with azidothymidine (AZT). Peripheral blood lymphocyte phenotyping was performed before treatment, then at weeks 12 and 24. A striking fall in the proportion of activated T lymphocytes from baseline was observed (P less than 0.001) at week 24. In contrast, the percentage of CD4+ cells showed a slight and transient rise at week 12 (P less than 0.05). No modification in levels of CD8+ or CD8+ CD57+ cells was detected during the study. Of the 34 patients, 11 developed AIDS, and 23 remained AIDS-free during 51 weeks of follow-up. Similar patterns of change in CD4+ and HLA-DR+ CD3+ lymphocytes were found in the AIDS progressors and nonprogressors. Likewise, HIV p24 antigenaemia showed parallel decreases in both groups of patients. Although changes in CD4+ cells, p24 antigenaemia and HLA-DR-reactive T lymphocytes were not predictive of clinical outcome, large differences existed between the two groups prior to the initiation of therapy. The short-term onset of AIDS was associated with lower CD4+ cell numbers, higher levels of serum p24 antigen and a greater proportion of activated T lymphocytes. Our results suggest that the possible interest of T lymphocyte activation markers, in conjunction with conventional phenotyping, should be investigated further.

Effect of zinc on the immune status of zinc-depleted AIDS related complex patients.

Zinc deficiency has been recently reported in AIDS patients to be associated with a decrease in T helper cells. The effect of zinc supplementation was evaluated in 5 ARC patients and 5 control subjects using lymphocyte subset counts, blast transformation and chemiluminescence of polymorphonuclear leukocytes. Zinc was analysed by atomic absorption spectrophotometry. T-cell subsets were quantified by cytofluorometry. Chemiluminescence of PMN was measured by phagocytosis of opsonized zymosan. The ARC patients had significantly lower zinc concentrations in the RBC prior to supplementation (p < 0.05). This level increased following the administration of zinc gluconate. This increase was accompanied by (i) an increase in HLA.Dr + cells with no CD4 CD8 ratio alteration; (ii) a stimulation of lymphocyte transformation and PMN chemiluminescence particularly after 15 days' high zinc supplementation. Such a reconstitution in specific and non specific immune functions in ARC patients may warrant further investigation.

Genetic differences in response to pulmonary cytochrome P-450 inducers and oxygen toxicity.

The effects of cytochrome P-450 inducers on O2 toxicity were studied in mice. We first examined three cytochrome P-450 inducers, which differ by their specific tissue affinity: phenobarbital sodium (PB), essentially active in the liver, and 3-methylcholanthrene (3-MC) and beta-naphthoflavone (BNF), which are also active in the lung. Both BNF and 3-MC increased the survival rate and significantly decreased pulmonary edema (pulmonary water and wet-to-dry weight ratio) in C57BL/6J mice exposed to hyperoxia (O2 greater than or equal to 95%), whereas PB had no protective effect. In the second part of this study, we compared the action of BNF in two strains of mice. In one (C57BL/6J), cytochrome P-450 can be induced by aromatic hydrocarbons, whereas in the other (DBA/2J) cytochrome P-450 is not inducible by these compounds. Protection against O2 toxicity was assessed in terms of lethality and pulmonary edema and of lung lipid peroxidation (assessed by measuring malondialdehyde). BNF only protected against O2 toxicity in the inducible strain. This protective effect of BNF on O2 toxicity in C57BL/6J mice was associated mainly with a large increase in the components of the cytochrome P-450 system (cytochrome P-450 and cytochrome b5) in the lung. The activity of pulmonary superoxide dismutase was also slightly increased, but the enhancement was not statistically significant. In contrast, in DBA/2J mice neither the components of the cytochrome P-450 system nor the activity of superoxide dismutase showed any increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-lactam antibiotics and human lymphocyte function: the in vitro effect on blastogenesis, lymphokine production and suppressor cell functions.

The effects of penicillin, cephalothin and cefoxitin on lymphokine production and mitogen stimulation of human peripheral blood lymphocytes and suppressor cell functions in man were studied with concentrations achievable in serum. Penicillin (50 and 100 micrograms/ml) and cephalosporins (50 and 100 micrograms/ml) directly stimulated production of the lymphokine leukocyte aggregating factor (LAgF) by unstimulated lymphocytes and enhanced mitogen-stimulated production of this lymphokine. Neither beta-lactam derivative interfered directly with neutrophil aggregation. Using the same concentration, penicillin did not suppress but had either no effect on or slightly increased the unstimulated and mitogen-stimulated lymphocyte transformation responses, whereas cephalosporins significantly suppressed both responses. Suppression of blastogenesis by the latter could be mediated through suppressor cell enhancement as indicated by the significant suppressed lymphocyte transformation observed while using the supernatant of cephalosporin-stimulated and cultured lymphocytes. This suppressive activity was more pronounced with cephalothin than with cefoxitin while it was not observed with penicillin which rather stimulated thymidine uptake. These findings suggest that enhancement of lymphokine production and suppression of lymphocyte transformation by cephalosporins are not contradictory and reflect a stimulating effect on two different lymphocyte subsets. The enhancement of suppressor function might account for the lower incidence of late hypersensitivity reactions observed in patients treated with the cephalosporins compared with those treated with penicillins.

Activated human lymphocytes secrete a soluble factor that aggregates leukocytes.

Human polymorphonuclear leukocytes can be activated by various inflammatory stimuli to display increased cell aggregation which is potentially an important pathogenetic mechanism. This study describes a soluble factor produced by concanavalian A-stimulated lymphocytes that causes human leukocytes to aggregate. This factor could be assayed quantitatively by measuring the light absorbance of polynuclear leukocyte suspension using a spectrophotometer. The lymphokine involved, namely the leukocyte aggregating factor (LAgF) was released by non pulse exposure to the mitogen for up to 72 hr with a maximum at 48 hr. LAgF was characterized by Sephadex gel filtration, chromatofocusing, enzymatic and chemical treatment. Sephadex G 100 gel filtration showed LAgF activity in a molecular range of 40,000-65,000. Chromatofocusing of culture supernatant showed LAgF in a single broad peak (4.8-5.4) with a maximum activity at pI 5.2. Human LAgF was heat sensitive, inactivated by treatment with chymotrypsin, and not affected by neuraminidase. Activity was partially recovered from the supernatant after protein precipitation with 1 M perchloric acid and not destroyed by 0.02 M sodium periodate. These findings characterize LAgF as a protein. These data suggest that LAgF is not different from leukocyte inhibiting factor by virtue of its size and physiological properties.

Diethyldithiocarbamate provides partial protection against pulmonary and lymphoid oxygen toxicity.

Prolonged exposure of C57B16 mice to pure O2 at 1 ATA induced pulmonary edema associated with involution of lymphoid system and depressed immunity. The consequences of these toxic events were evaluated by 1) mortality rate, 2) determination of pulmonary water, 3) thymic and splenic cellularity, and 4) humoral (primary antibodies) and cellular (mitogenic) immune responses. Pretreatment of mice with 125 mg kg-1 of diethyldithiocarbamate (DDC) several days before exposure to O2 resulted in 1) an increase in animal survival (92-100% vs. 59% O2 controls), 2) a reduction in pulmonary edema, 3) partial stabilization of thymus and spleen lymphocyte populations, and 4) restoration of the humoral response (specific antibodies appeared earlier than in O2 control animals) and improvement of the mitogenic proliferative response of the spleen cells after hyperoxia. None of these effects were observed when DDC treatment coincided with the beginning of exposure. Our results indicated that DDC protects mice from both pulmonary and lymphoid hyperoxic injury, but only in a partial manner. It is suggested that the mechanism of this antioxidative property is indirect.

Evaluation of cellular immune response during chronic schistosomiasis in humans by the leukocyte aggregation test and the leukocyte migration inhibition test.

Cellular immune response was evaluated in 31 patients with chronic Schistosoma haematobium and Schistosoma mansoni infections and in 15 healthy normal persons by using S. mansoni soluble worm and egg antigens. Although the leukocyte migration inhibition test demonstrated false-positive reactions, the specificity of the leukocyte aggregation test was confirmed by the negativity of all of the controls. Among the patients, only 10% were positive for the leukocyte aggregation test. This low cellular reactivity was in contrast to markedly elevated specific humoral response determined by an enzyme-linked immunosorbent assay for immunoglobulin G and paper allergosorbent test for immunoglobulin E with soluble worm antigen. These results confirm that the cellular immune reactivity to schistosome antigen as demonstrated by the leukocyte aggregation test is either minimal or absent in chronically infected patients.

Humoral immunodepression following acute NO2 exposure in normal and adrenalectomized mice.

The effects following 20 ppm NO2 exposure on humoral immunity were investigated in C57Bl/6 mice after 48, 72, and 96 h exposure. Both spleen plaque-forming cell (PFC) responses and serum hemagglutinins (HA) using sheep red blood cells (SRBC) as antigen were studied. Splenic and thymic weight and cellularity decreased on acute exposure to NO2. PFC were markedly depressed after 48 h exposure and continued to decrease as exposure time was lengthened. HA titers were also depressed. The same significant suppression of PFC and HA titers was observed in adrenalectomized mice after 96 h NO2 exposure. The depression of humoral immunity in NO2-exposed mice was independent of stress-induced endogenous steroids.

[Mitogenic responses of splenic lymphoid cells of rats exposed in vitro to normobaric oxygen]. / Réponses mitogéniques des cellules lymphoïdes spléniques de Rat exposées in vitro à l'oxygène normobare.

Rat spleen cells were cultivated in 1 ATA pure oxygen. The mitogenic responses with Con A were evaluated at different time of exposure (12 to 72 hrs). There was a stimulation of the cells after 12 hrs of exposure, which diminished at 18 hrs, in spite of the fact that the viability of the cells remained unchanged till 24 hrs of exposure. The kinetics of the mitogenic response of the splenic lymphocytes exposed to oxygen is biphasic as has been observed after irradiation.

Diagnosis of penicillin allergy. An evaluation of the leucocyte aggregation test in man.

A study was made to establish the value of the leucocyte aggregation test (LAT) in drug allergy using penicillin antigen. The antigen-induced human peripheral blood leucocyte aggregation was measured quantitatively. The results obtained have been compared with the leucocyte migration inhibition test (LMIT) in patients with or without delayed penicillin allergy. Among forty-four penicillin-allergic subjects and thirty-six control subjects, LAT was found positive in respectively 70.5 and 30.5% (P less than 0.001) whereas LMIT was found positive in respectively 56.8 and 50% of the patients. These results were confirmed by multiple correspondence analysis (MCA), using a computer. Furthermore, this method, enables a more comprehensive and reliable interpretation of the tests, by the help of various quantitative and qualitative criteria. It is concluded that LAT shows more discrimination than the LMIT in distinguishing a penicillin-allergic population from a non-allergic one. In addition, LAT offers great technical advantages over the LMIT for the diagnosis of drug allergy.