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Diluted whole blood (WB) culturing may be the most appropriate milieu in which to study cytokine production in vitro. We tested TNFalpha and IL-6 production using small volumes of WB (25 microl) from HIV-1 positive patients with a one-step procedure that combines WB stimulation with LPS, PHA and cytokine measurement. We studied 49 patients without secondary infection or at distance of secondary infection staged according to the 1993 classification of the CDC and 12 healthy seronegative subjects. Heparinized blood from 5 control subjects had been collected sequentially during a period of 5 months. The individual variations of TNFalpha and IL-6 production were limited for all these individuals. In 1 out of 20 CDC group A patients, 6 out of 17 CDC group B patients and 3 out of 12 CDC group C patients, we obtained higher values of TNFalpha than the mean + 2 S.D. of the control group. In 3 out of 20 CDC group A patients, 1 out of 17 CDC group B patients without AIDS and 5 out of 12 CDC group C patients, the TNFalpha values were lower than the mean - 2 S.D. of the control group. Low IL-6 values were obtained in 1 out of 20 CDC group A patients and 1 out of 17 CDC group B patients and 3 out of 12 CDC group C patients. There was no correlation between TNFalpha production in vitro and plasma level of TNFalpha. We found no correlation between the levels of cytokines and monocyte count or between the levels of cytokines and CD4 T-cell count in peripheral blood. Our data point out a disarray in TNFalpha and IL-6 production by WB from HIV-1 infected patients. The relationship between the disarray of cytokine production and cytopathogenicity of HIV-1 isolates in the P4 cell line was investigated in this study. We found a correlation between the high level of TNFalpha produced by WB and the phenotype of HIV-1 isolates isolated from patients. The one-stage procedure used in this work is of potential value to investigate the activation status of cells for monitoring HIV-1 positive individuals and predicting HIV-1 phenotype.

Culture techniques for isolation of HIV-1 from small amounts of whole blood (WB) treated with anticoagulant have been reported and gave results identical to those of culture of separated peripheral blood mononuclear cells. Some authors obtained much higher isolation rates when EDTA was used instead of heparin. We compared two previously described techniques for cultivation of HIV-1 from WB of adult HIV+ patients staged according to the CDC classification. In addition, we assessed the influence of the type of anticoagulant used for the collection of blood in viral replication in cell cultures from whole blood. Small volumes of WB treated with either heparin or EDTA were cocultivated with phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PHA-PBMC) from healthy donors. We used two procedures for WB culture: procedure I, based on the culture of 250 microliters of WB with 1 x 10(6) PHA-PBMC from donors; and procedure II based on the culture of 500 microliters of WB with 4 x 10(6) PHA-PBMC from donors. The cocultures were placed in 24-well plates and incubated for as long as 28 days in medium containing interleukin 2 (IL-2). Twice weekly half of the medium was replaced with fresh medium. In procedure II, one million fresh PHA-PBMC from donors was added on the 7th day of culture. The culture supernatant was assayed for the presence of HIV-1 p24 antigen in an enzyme immunoassay. The kinetics of HIV-1 replication in cultures of WB from 7 AIDS patients were similar using procedures I and II. In 8 HIV+ patients the isolation rate was higher with heparin- than with EDTA-treated samples. The isolation rate was higher in AIDS patients (n = 8) than in others with both methods. In stage IV patients without AIDS (n = 8) we failed to isolate HIV-1 in 1 patient with procedure I, whereas we succeeded with procedure II. In stage II, HIV-I was isolated in 1 of 4 patients with both methods. HIV was isolated in cultures of WB from patients receiving zidovudine or related nucleoside analogues and in cultures of WB from untreated patients. HIV-1 could not be isolated from WB of patients with more than 400 CD4+ T lymphocytes in their peripheral blood (n = 4); however, it was isolated from 14 of 16 patients with less than 400 CD4+ T lymphocytes. Our results suggest that procedure II is more sensitive than procedure I and that heparin is better than EDTA for collecting WB. We showed that the rate of HIV-1 isolation from WB increased in advanced-stage patients. Further studies are needed to define the clinical applications of WB culture.

The pathogenesis of CVB3-induced chronic myocarditis remains unknown. Activated monocytes and macrophages may maintain ongoing inflammation during a persistent CVB3 infection and possibly represent the major mechanism leading to chronic myocarditis. We decided to study the activation status of cells by studying TNF alpha secretion in vitro using whole blood culture in CVB3-induced murine chronic myocarditis. Seven DBA/2 +/+ mice and 18 NMRI nu/nu mice were inoculated intraperitoneally with 5x10(5) pfu of CVB3, and mice were mock-infected. Thirty-one days post-infection, all mice were sacrificed, blood samples were obtained from the heart, and the heart was removed. Enteroviral genomic detection by RT-PCR, virus isolation and histological analysis of heart samples were performed. Heparinized whole blood (25 microliters) was cultured for 4 hr and 24 hr in sterile 96 well-plate containing 225 microliters RPMI in the presence or the absence of activators (LPS + PHA). The TNF alpha levels in the whole blood from mock-infected DBA/2 (n = 4) and NMRI nu/nu mice (n = 5) were not different. A moderate increase of TNF alpha was observed in three out of five DBA/2 mice with negative CVB3 that had no histological abnormalities in myocardium. An increased level of TNF alpha was found in the sole DBA/2 mouse with positive CVB3 detection and chronic myocarditis. An increased level of TNF alpha was found in one out of nine NMRI nu/nu mice with positive CVB3 detection and chronic myocarditis and in one out of seven mice with positive CVB3 detection exempt of lesions in myocardium. In other infected mice, the level of TNF alpha was normal. Enteroviral genome was not detected in the blood from infected mice at 31 days post-infection. The increased TNF alpha level in some mice may be designed for a beneficial inflammatory and immune response, however, an exaggerated release may be associated with an adverse effect. The normal TNF alpha level in whole blood cultures from mice with chronic myocarditis does not exclude enhanced cytokine production at infected loci such as myocardial tissue. This is the first report to use whole blood cultures to study the production of cytokines in virus-induced disease in a small animal model.

Correlations between the in vitro biological properties of HIV strains isolated from patients and the prognosis of their disease have been reported. We developed a technique to study the phenotype of HIV strains isolated from patients. We used the P4 cell line, derived from HeLa cells, which has been transfected with receptor CD4 gene. HIV laboratory strain (HIVLAI) and peripheral blood leukocytes (PBLs) from donors infected with HIVLAI induce syncytium in P4 cell cultures in vitro. The presence of reporter gene (LacZ gene) under the control of the HIV-1 long terminal repeat (LTR) in these cells allows colorimetric visualization of syncytia in the cytoplasm using a beta-galactosidase (beta gal) assay in the presence of X-gal. We cocultivated 1 x 10(6) patient PBLs with 2 x 10(6) normal PHA-activated normal PBLs for 4 days in the presence of IL-2 in 24-well plates. Half of the medium was replaced twice a week and PHA-activated normal PBLs were added every 7 days. HIV-1 was isolated from cocultured PBLs of 18 patients with advanced-stage HIV infection as assessed by the production of HIV p24 detected with a commercially available HIV-1 p24 ELISA. Supernatant and 10(5) cells were collected twice a week from cocultured PBLs and were added to P4 cells in 96-well microtiter plates. The cultures were observed every day for 3 days and then the beta gal assay was performed. We did not observe any effect with cells and supernatant from 8 patients, harvested from cultures incubated for as long as 28 days. The phenotype of these isolates was called NC (noncytopathic). In cells from 2 patients, we obtained blue multinucleated giant cells; the phenotype of these strains was called SI (syncytium inducing). In cultures from 8 other patients, we obtained the death of P4 cells without syncytium formation, and the phenotype of these strains was called CI (cell-killing inducing). In every case, the cytopathic effect of HIV-1 isolates could be detected with cocultured PBLs collected as early as day 4 of culture. Cocultured PBLs from 13 healthy controls did not alter the P4 cells. We displayed the replication of CI strains of HIV-1, but not the one of NC strains in P4 cell line. Our micromethod allowed the detection of cytopathic effects of HIV isolates. Further investigations should define the clinical applications of this method.

Soluble forms of the two molecular species of the cell surface TNF receptors (sTNFR p55 and sTNFR p75) can reduce the activity of TNF alpha but they may also enhance its function by stabilizing the active TNF alpha oligomer. Considering the pathophysiological importance of sTNFR p75 for the regulation of the bioavailability of TNF alpha in the body, we determined the serum levels of sTNFR p75 and TNF alpha in 45 children and 28 adults with laboratory-confirmed dengue infection by using immunoassays. The serum samples were obtained from day 1 to day 15 after the onset of the disease during the 1989-1990 outbreak of dengue-3 in Tahiti, French Polynesia. The patients were clinically classified as having dengue hemorrhagic fever (DHF) and graded according to the criteria of the World Health Organization (WHO) into four grades from less severe (grade I) to severe (grade IV). The sera of both children and adult patients of all severity grades contained higher levels of sTNFR p75 than the sera of control subjects. Although high levels of TNF alpha were also detected in children and adults among grade I, II, III and IV patients, we found no correlation between sTNFR p75 and TNF alpha. We observed in adults a moderate elevation of sTNFR p75 and TNF alpha in sera compared with that observed in children. The raised levels of immunoreactive sTNFR p75 and TNF alpha in all clinical groups of dengue-infected patients strongly indicate activation of the TNF alpha system during dengue infection. The balance between sTNFR p75 and TNF alpha may be altered in dengue infection. Further investigations are needed to understand the role of sTNFR p75 and TNF alpha in the pathogenesis of DHF and to improve the management of dengue infection.

We investigated whether HIV-1 can regulate tumor necrosis factor receptor (TNFR) expression in SupT-1, a CD4+ T-cell line. The cells were infected with HIV-1 containing 1,000 cpm RT activity, as early as day 3 after infection and all along the culture the supernatant level of core protein p24 was > 250 pg/ml, and on days 6 and 9 after infection, p24 was found in 10% of the cells as determined by indirect immunofluorescence assay. The cells were growing without loss of viability. The study of TNFR expression was based on a microassay for measurement of binding of 125I-TNF alpha to cells, in which free and cell-bound ligand separation was performed by centrifugation through oil. Scatchard analysis of TNF alpha binding on days 6 and 9 after infection revealed a 90% increase in the expression of high-affinity membrane receptors in HIV+SupT-1 culture compared with uninfected cells (mean +/- S.D. = 501 +/- 148.5 vs. 263 +/- 77.8 receptors/cell, n = 9, P < 0.001) with no change in dissociation constants (mean +/- S.D. = 4.36 +/- 1.06 vs. 4.00 +/- 1.12 x 10(-10) M).

The level of sCD23 produced in the course of human immunodeficiency virus (HIV) infection was measured in patients grouped according to the Centers for Disease Control by using an immunoradiometric assay. Soluble CD23 was evaluated in supernatants of peripheral blood mononuclear cell (PBMC) (10(6) cells/ml) stimulated by phytohemagglutinin (PHA). Compared with healthy controls (m +/- S.D. = 1.0 +/- 0.34 U/ml, n = 7), higher values were observed in some of the patients of group II (asymptomatic) (m +/- S.D. = 2 +/- 1.33, n = 9) and some of the patients of group IV (AIDS) (m +/- S.D. = 1.3 +/- 1.40, n = 8). Those results prompted us to compare the plasma levels of sCD23 in group II and group IV HIV-infected patients and in healthy individuals. Soluble CD23 plasma levels in healthy patients (n = 42) ranged from 0 to 1.5 U/ml (m +/- S.D. = 0.9 +/- 0.33), in group II patients (n = 17) from 0 to 3 U/ml (m +/- S.D. = 0.92 +/- 0.83) and in group IV patients (n = 73) from 0 to 2.9 U/ml (m +/- S.D. = 1.15 +/- 0.71). The differences between the patients and the healthy individuals were not statistically significant but individual sCD23 values higher than 2 U/ml were obtained in 6% of the group II patients and 16.7% of the group IV patients. Increased values of sCD23 were obtained in plasma from patients with secondary infectious diseases (groups IV-Cl and IV-C2) and from patients without secondary infectious diseases (group II, group IV-A and group IV-B).(ABSTRACT TRUNCATED AT 250 WORDS)