In vitro induction of anti-thyroid microsomal antibody-secreting cells in peripheral blood mononuclear cells from normal subjects

Secretion of immunoglobulin G (IgG) and IgM antithyroid microsomal antibodies (AMA) was induced in vitro by coculturing non-T cells (B lymphocytes) and autologous CD4 (helper/inducer) cells from normal subjects stimulated with pokeweed mitogen (PWM) or a combination of human thyroid microsomal antigen (McAg) and Staphylococcus aureus Cowan I (SAC) strain. With PWM stimulation, AMA production was induced in more IgM-secreting cells (AMA-M) than IgG-secreting cells (AMA-G). However, McAg plus SAC stimulation resulted in similar numbers of AMA-G- and AMA-M-secreting cells. PWM induced a significantly greater number of both AMA-M (and generalized IgM)-secreting cells than did McAg plus SAC, while the number of AMA-G-secreting cells induced by the two stimuli were similar. There were no significant differences between autologous or allogeneic CD4 cells from normal subjects or patients with autoimmune thyroid disease (AITD) when cocultured with B cells from normal subjects in terms of helper activity in the induction of AMA-M- or IgM-secreting cells with PWM stimulation. However, with McAg plus SAC, CD4 cells from patients with AITD induced a significantly greater number of AMA-M-secreting cells than did autologous or allogeneic CD4 cells from normal subjects. There was no difference in helper activity between autologous and allogeneic normal CD4 cells in the induction of generalized IgM-secreting cells regardless of the stimulus used. Normal autologous or allogeneic CD8 (suppressor/cytotoxic) cells cocultured with normal B cells and autologous CD4 cells suppressed the induction of AMA-M-secreting cells by PWM stimulation. On the other hand, CD8 cells from patients with AITD suppressed the induction of AMA-M-secreting cells significantly less effectively. All CD8 cells suppressed the induction of IgM-secreting cells equally well. We conclude that 1) B lymphocytes from normal subjects are capable of producing autoantibodies in vitro in the presence of CD4 cells; 2) the helper activity of CD4 cells from patients with AITD to induce AMA-M secreting cells is greater than that of normal CD4 cells with thyroid antigen stimulation; and 3) this helper activity may be due to relatively impaired suppressor activity in thyroid antigen-specific CD8 cells from patients with AITD, whereas the immunoregulatory function of CD8 cells from normal subjects appears to play an important role in the maintenance of self-tolerance.     

HIV-1 viral DNA load in peripheral blood mononuclear cells from seroconverters and long-term infected individuals.

OBJECTIVE: To determine viral DNA load in peripheral blood mononuclear cells (PBMC) from HIV-1-infected individuals. DESIGN: HIV-1 copy numbers were determined using a quantitative polymerase chain reaction (PCR), the PCR-aided template titration assay (PATTY). PATTY utilizes an internal plasmid control DNA, which is amplified within the same tube and using the same primers as the PBMC target DNA. HIV-1 copy numbers were confirmed by limiting-dilution PCR analysis. RESULTS: PBMC viral load of 19 long-term (greater than 4 years) HIV-1-infected individuals ranged from 0.8 to 100 copies per 10(3) PBMC. Significantly higher copy numbers were found among p24-antigen-positive than among p24-antigen-negative individuals. In addition, the PBMC viral load of two HIV-1-infected individuals was monitored during the first 3 months after acute infection. For both patients, the HIV-1 copy numbers were shown to peak at the time of HIV-1-antibody seroconversion and decline subsequently (range, 0.6-10 copies per 10(3) PBMC). CONCLUSIONS: PATTY is a useful method for assessing the HIV-1 copy numbers in PBMC DNA. Viral DNA load peaks shortly after infection and reaches an individual specific level that is probably stable within a few months of infection. Viral DNA load in PBMC varies widely among long-term HIV-1-infected individuals.     

Release of proinflammatory cytokines by mitogen-stimulated peripheral blood mononuclear cells from critically ill multiple-trauma victims

This study investigated the alterations in circulating proinflammatory cytokines and cytokine production by peripheral blood mononuclear cells (PBMCs) in response to lipopolysaccharide (LPS) or phytohemagglutinin (PHA) after severe trauma. Plasma and PBMCs were collected from 17 severely injured trauma patients and 10 healthy subjects. Plasma was stored at -80 degrees C and analyzed for cytokines. Isolated PBMCs from each subject were stimulated with LPS or PHA and incubated at 5% CO2 for 24 hours. Supernatants were collected and analyzed for cytokines. There was no significant change in the plasma concentration of free TNF-alpha and IL-1beta between healthy subjects and trauma patients. Plasma IL-6, total TNF-alpha, and total IL-1beta were significantly increased in severely traumatized patients compared with healthy control subjects. PBMCs from trauma patients produced higher levels of TNF-alpha in response to LPS but it showed no significant change in IL-1beta and IL-6 production in response to PHA or LPS in comparison to PBMCs from control subjects. We conclude that severe trauma results in a significant increase in plasma proinflammatory cytokine IL-6. Free TNF-alpha and IL-1beta in plasma remain at levels comparable to those in uninjured controls, while plasma free IL-6 levels in trauma patients remain high. Serious injury is associated with an enhanced production of TNF-alpha by PBMCs stimulated with LPS.     

The pharmacokinetics of lamivudine phosphorylation in peripheral blood mononuclear cells from patients infected with HIV-1

OBJECTIVE: To assess the pharmacokinetics of lamivudine phosphorylation in peripheral blood mononuclear cells (PBMC) from patients infected with HIV-1. DESIGN: Single-center, open-label, randomized, two-period, cross-over study in 10 asymptomatic, antiretroviral-experienced, HIV-1-infected patients who had a CD4+ lymphocyte count of 200-500 x 10(6)/l and had received combination treatment with lamivudine 150 mg twice a day plus zidovudine 600 mg a day (divided into two or three doses) for > or = 16 weeks prior to study entry. METHODS: Patients were randomly assigned to receive lamivudine 150 mg twice a day or lamivudine 300 mg twice a day for 14 days, with at least a 48-h washout period between treatments. Serial blood samples were collected over 36 h for determination of lamivudine serum concentrations using liquid chromatography/mass spectrometry and intracellular phosphate PBMC concentrations using high performance liquid chromatography/radioimmunoassay methods. Pharmacokinetic parameters were calculated based on lamivudine and lamivudine anabolite concentration-time data. RESULTS: Intracellular pharmacokinetic parameters were highly variable between patients (coefficient of variations approximately 50%). The two regimens produced lamivudine-total phosphate (totP) values of a similar magnitude. Although the 300-mg regimen tended to produce higher lamivudine-monophosphate (MP) and -triphosphate (TP) values, differences from values produced by the 150-mg regimen were not statistically significant. As lamivudine diphosphate (DP) was the predominant anabolite, accounting for 50-55% of lamivudine-totP (compared with 30-35% for lamivudine-MP and 15-20% for lamivudine-TP), the conversion of lamivudine-DP to lamivudine-TP can be regarded as the rate-limiting step. The median lamivudine-TP intracellular half-life (t1/2) for the 150-mg and 300-mg regimens did not differ significantly (15.3 and 16.1 h, respectively). Serum lamivudine pharmacokinetic parameters were consistent with those observed in previous studies in HIV-1-infected patients. No apparent linear relationships were observed between lamivudine intracellular anabolite and serum data. CONCLUSIONS: The intracellular pharmacokinetics of lamivudine phosphorylation in PBMC from asymptomatic HIV-1-infected patients are highly variable and do not differ statistically between the 150- and 300-mg twice a day regimens. The variations in intracellular lamivudine-TP concentrations following these two lamivudine dosage regimens are unlikely to result in differences in clinical effect. This was confirmed by the results of a large phase III study in HIV-1-infected patients which showed no differences in HIV-1 RNA or CD4+ lymphocyte counts between the 150- and 300-mg lamivudine regimens in combination with zidovudine.     

A recombinant clone of HIV-1 preferentially transmitted in normal peripheral blood mononuclear cells

To study biologic properties associated with specific regions of HIV-1, a chimera, pHX-JY1, was constructed by exchanging the vif-env region of a Zairian molecular clone (JY1) with that of pHXB2gpt, a full-length biologically active proviral clone of North American origin. Virus was produced by transfection of permissive cells with parental and recombinant clones, and the biologic and molecular properties of these viruses were compared. Virus derived from pHXB2gpt infected phytohemagglutinin (PHA)-activated normal peripheral blood mononuclear cells (PBMC) and CD4+ leukemic T cell lines equally well. In contrast, virus derived from pHX-JY1 was transmitted slowly to both PBMC and cell lines, and the infectivity of pHX-JY1 virus was two orders of magnitude greater for PBMC than for T cell lines. All essential viral genes in the exchanged JY1 vif-env region were intact and functioned comparably to those of the parent clone in transfected COS-1 cells. The findings suggest differences in these regions of the HIV-1 genome may play an important role in differential cell tropism.     

Folic acid treatment reduces chemokine release from peripheral blood mononuclear cells in hyperhomocysteinemic subjects

Elevated plasma homocysteine concentration is an independent risk factor for cardiovascular disease. However, the mechanisms by which hyperhomocysteinemia induces vascular disease are uncertain. An early step in atherogenesis involves leukocyte migration into the arterial wall, a process regulated in part by chemokines. We hypothesized that homocysteine may exert its atherogenic effect in part through chemokine-mediated mechanisms, and in the present study, we examined the effects of folic acid supplementation for 6 weeks on chemokine levels in hyperhomocysteinemic individuals. Data showed the following: (1) Compared with control subjects, hyperhomocysteinemic subjects had elevated plasma levels of the CXC chemokines, epithelial neutrophil-activating peptide (ENA)-78 (P<0.05), and growth-regulated oncogene (GRO)alpha (P=0.088), and homocysteine was significantly correlated with ENA-78 and GROalpha. (2) During folic acid treatment, normalization of homocysteine levels was accompanied by a marked reduction in oxidized low density lipoprotein-stimulated release of CXC chemokines (ie, GROalpha, ENA-78, and interleukin-8) and CC chemokines (ie, monocyte chemoattractant peptide-1 and RANTES) in peripheral blood mononuclear cells from these individuals. (3) The oxidized low density lipoprotein-induced release of ENA-78 from peripheral blood mononuclear cells from control subjects was significantly reduced when cells were incubated in the presence of folic acid. These data may suggest that homocysteine exerts atherogenic effects in part by enhancing chemokine responses in cells involved in atherogenesis and that folic acid supplementation may downregulate these inflammatory responses.     

DNA amplification of HIV-1 in seropositive individuals and in seronegative at-risk individuals

We assessed the HIV-1 status of seropositive and seronegative at-risk individuals by the polymerase chain reaction. Fifty-four out of 55 HIV-1-seropositive samples scored positive. However, HIV-1 proviral DNA was not detected in 16 seronegative homosexuals, 20 seronegative polytransfused haemophiliacs and 20 seronegative thalassaemic children, 20 individuals with isolated and persistent anti-core antibodies and 74 seronegative blood donors. These data indicate that positive HIV-1 DNA is likely to be an exceptional phenomenon in HIV-seronegative people.     

Patient demographics and disease variables correlate with distinct cytokine patterns in mitogen-stimulated peripheral blood mononuclear cells from rheumatoid arthritis patients

There is paucity of literature on the association of peripheral blood cytokine patterns with patient demographics and disease variables in rheumatoid arthritis (RA). We test the hypothesis that there may be differences in peripheral blood levels of inflammatory cytokines in RA subjects according to various disease variables. In this case, we could identify peripheral blood cytokine markers that correlate with different disease variables. Forty-two seropositive RA patients were characterized according to the age at onset, gender, disease duration, severity, activity and ACR functional class. The production levels in mitogen-stimulated PBMCs of five pro-inflammatory cytokines (IFNγ, TNFα, TNFβ, IL-8, IL-18) and three anti-inflammatory cytokines (IL-4, IL-10, IL-13) were evaluated in these patients and in healthy controls. Several new findings emerge: (1) higher levels of IL-4 correlate with female gender, milder disease, non-erosive disease, and earlier age at onset; (2) higher levels of IL-10 correlate with the requirement of ≤2 DMARDs; (3) higher levels of IL-18 correlate with non-erosive disease and younger age at onset; (4) higher TNFβ levels correlate with older present age of patients; and (5) higher IL-8 levels correlate with established/late disease. There are several interesting differences in cytokine patterns with respect to age at onset, current age, disease severity, and the number of DMARDs the patients require.     

Characterization of HIV-1-specific antibodies and HIV-1-crossreactive antibodies to platelets in HIV-1-infected haemophiliac patients

Sera from HIV-1-infected haemophiliacs were examined for human immunodeficiency virus (HIV) specific antibodies and for platelet crossreactive antibodies. Using HIV sepharose 4B affinity columns for serum absorption, antibodies against various HIV antigens, including HIV lysate, HIV-p24 and HIV-gp120, were eluted either by low or by high pH buffer. The eluates were examined by ELISA for HIV specificity and by flow cytometry for platelet crossreactivity. Two types of HIV antibodies could be eluted, i.e. acid-sensitive and alkaline-sensitive antibodies. HIV antibodies were obtained in 26/29 acid eluates and in 25/29 of the alkaline eluates from HIV-lysate columns; 96% (25/26) of the acid-eluted antibodies were HIV-specific but 48% (12/25) of the alkaline-eluted antibodies also showed crossreactivity to platelets. Of the 20 alkaline-eluted HIV-p24 antibodies, 40% (8/20) reacted specifically with HIV-p24 and 60% (12/20) were platelet crossreactive. In contrast, of the alkaline-eluted HIV-gpl20 antibodies ( n =17), 88% (15/17) were HIV gpl20-specific and only 12% (2/17) were platelet crossreactive. Western blot analysis of platelets demonstrated that the anti-p24 antibodies recognized three bands with approximate molecular weights of 72 000 to 95 000. 69% of the serum antiplatelet antibodies showed platelet glycoprotein IIbIIIa specificity. Anti-HIV antibodies could be eluted from platelets. Hence, platelet crossreactive antibodies in HIV infection are primarily alkaline-sensitive and are associated predominantly with HIV p24 antibody; these antibodies may play a role in the immune thrombocytopenia of HIV-infected haemophiliacs.     

The in vitro production of antibodies to mitochondrial antigens by peripheral blood mononuclear cells from patients with primary biliary cirrhosis

Peripheral blood mononuclear cells from 7 patients with primary biliary cirrhosis and 7 healthy control subjects were studied for their ability to produce antibodies to mitochondrial antigens in vitro. Peripheral blood mononuclear cells were collected by lymphapheresis and cultured with or without pokeweed mitogen for 10 days. The culture supernatants were then tested for antibodies to mitochondrial antigens by both immunofluorescence microscopy and a microtiter ELISA. Peripheral blood mononuclear cells from 5 of 7 patients with primary biliary cirrhosis but from none of the healthy controls produced antibodies to mitochondrial antigens spontaneously (without pokeweed mitogen stimulation). In contrast, peripheral blood mononuclear cells from 6 of 7 patients with primary biliary cirrhosis and from 6 of 7 control subjects synthesized detectable levels of antibodies to mitochondrial antigens after stimulation with pokeweed mitogen. In general, peripheral blood mononuclear cells from the primary biliary cirrhosis patients produced higher titers of antibodies to mitochondrial antigens in culture than cells from healthy controls. Furthermore, the antibodies to mitochondrial antigens reactivity produced by peripheral blood mononuclear cells of primary biliary cirrhosis patients exhibited a specificity for the M2 mitochondrial antigen which is present on the inner membrane of mitochondrial cristae and which is closely associated with a mitochondrial ATPase activity. In contrast, the antibodies to mitochondrial antigens reactivity produced by peripheral blood mononuclear cells of healthy controls appeared to be directed at a broader range of mitochondrial antigens. These findings indicate that, inpatients with primary biliary cirrhosis, there is a marked expansion of B lymphocyte clones that produce an antibody to a specific mitochondrial antigen.     

Longitudinal monitoring of 2-long terminal repeat circles in peripheral blood mononuclear cells from patients with chronic HIV-1 infection

OBJECTIVE: To evaluate the potential use of 2-long terminal repeats (LTR) HIV circular DNA quantification for the monitoring of ongoing virus replication in treated HIV-1-infected patients. DESIGN AND METHODS: In a longitudinal setting, where the natural course of HIV-1 infection was in most cases disrupted by continuous or discontinuous antiviral therapy, 2-LTR circles of HIV-1 DNA were quantified in serial peripheral blood mononuclear cell samples, selected in retrospect from 16 patients with chronic HIV-1 infection, using quantitative real-time PCR. We compared variations of 2-LTR circle level with concomitant variations in plasma viral RNA level and with the frequency of productively infected cells and chromosome associated proviral DNA copy numbers in patient's peripheral blood mononuclear cells. RESULTS: Antiviral treatment led to a sharp decrease in plasma viraemia and infectious cell frequency. In contrast, we found that levels of proviral DNA and 2-LTR circles were significantly lower under treatment only when groups of specimens that were homogeneous, with respect both to plasma viraemia and infectious cell frequency, were compared. Moreover, during the time of undetectable plasma viraemia, scarcely any decline in proviral DNA or 2-LTR circle levels was observed. CONCLUSIONS: The low impact of antiviral treatment on 2-LTR circle levels in vivo, when plasma viraemia and infectious cell frequency both dramatically decline lead us to conclude that 2-LTR circles should not be used for the monitoring of recent viral replication in treated patients.     

Production of inflammatory molecules in peripheral blood mononuclear cells from severely glucose-6-phosphate dehydrogenase-deficient subjects

OBJECTIVE: We have previously demonstrated that Mediterranean glucose-6-phosphate dehydrogenase (G6PD)-deficient peripheral blood mononuclear cells (PBMC) respond to mitogenic stimuli with a reduced cholesterol synthesis and growth. In the present study, we have investigated the release of inflammatory molecules by PBMC following a mitogenic stimulus, as well as the transformation to foam cells of monocyte-derived macrophages from severely G6PD-deficient and normal subjects. METHODS AND RESULTS: PBMC from G6PD-deficient subjects produced interleukin (IL)-1beta and IL-6 to a lower extent compared with normal subjects. 5-Hydroxyeicosatetraenoic acid, a primary product of 5-lipoxygenase, was slightly decreased. Tumour necrosis factor-alpha and IL-1beta secretion was significantly reduced in monocyte-derived macrophages. No difference was found in IL-10 secretion, whereas transforming growth factor-beta was invariably found to be significantly higher in G6PD-deficient cells. In cells incubated with acetylated low-density lipoprotein, cholesterol esterification and its storage in lipid droplets were lower than in normal G6PD cells. CONCLUSIONS: We conclude that by reducing the secretion of inflammatory molecules by PBMC and increasing the secretion of transforming growth factor-beta and the capability of monocyte-derived macrophages to accumulate lipid droplets and convert into foam cells, G6PD deficiency may confer a partial protection against atherosclerosis leading to the reduced risk of cardiovascular diseases reported in G6PD-deficient subjects.     

Detection of pre-S1 proteins in peripheral blood mononuclear cells from patients with HBV infection.

The presence of pre-S1 proteins in peripheral blood mononuclear cell (PBMC) samples from 115 patients with different forms of hepatitis B virus (HBV) infection was investigated by Western blot. Among 67 chronic HBsAg carriers, HBV antigens were detected in the PBMC in 80% for HBsAg, 27% for HBc/e Ag and 34% for pre-S1 proteins. The detection of pre-S1 proteins in PBMC was significantly associated with the presence of serum markers of HBV replication (HBV DNA and/or DNA polymerase). In the group of 48 consecutive patients negative for serum HBsAg, but positive for anti-HBc with or without anti-HBs, HBsAg and pre-S1 proteins could be detected in PBMC. This finding was more frequent among anti-HIV-positive patients (77 and 23% of the cases, respectively) than in the negative ones (23 and 4% of the cases, respectively). The detection of HBV DNA and polyadenylated RNA in some of the PBMC samples positive for HBV proteins suggests that these proteins may be expressed in PBMC, especially during intense HBV replication. In patients negative for serum HBsAg, PBMC may constitute a reservoir of HBV.     

Electrophoretic distributions of human peripheral blood mononuclear white cells from normal subjects and from patients with acute lymphocytic leukemia.

The electrophoretic mobilities of mononuclear white cells from the peripheral blood of normal subjects and leukemic patients were measured. Analysis of both fresh and cryopreserved samples of each type showed that cryopreservation had no distinguishable effect on the electrophoretic distribution of either normal or leukemic cells. Mode mobilities (mobilities at which maximum intensities are observed) of cells from nine leukemic patients were 7-28% below the average mode mobility of normal cells. The standard deviation of the electrophoretic mobilities of normal cells was 2%. Normal cells had an asymmetric, often bimodal, electrophoretic distribution; leukemic cells consistently had a single symmetric distribution.     

Increased spontaneous osteoclastogenesis from peripheral blood mononuclear cells in phenylketonuria

Phenylketonuria (PKU) is commonly complicated by a progressive bone impairment of uncertain aetiology. The therapeutic phenylalanine (Phe)-restricted diet and the possible noxious effects of high plasma Phe concentrations on bone have previously been suggested as possible determinant factors. Since osteoclasts are involved in bone reabsorption, they could play a role in determining bone damage in PKU. The reported increased excretion of bone resorption markers in PKU patients is consistent with this hypothesis. Although different diseases characterized by bone loss have been related to increased spontaneous osteoclastogenesis from peripheral blood mononuclear cells (PBMCs), to date there is no evidence of increased osteoclast formation in PKU. In this study, we compared the spontaneous osteoclastogenesis from PBMCs in 20 patients affected by PKU with that observed in age- and sex-matched healthy subjects. Phenylketonuric patients showed the number of osteoclasts to be almost double that observed in controls (159.9 ± 79.5 and 87.8 ± 44.7, respectively; p = 0.001). Moreover, a strict direct correlation between the spontaneous osteoclastogenesis in PKU patients and the mean blood Phe concentrations in the preceding year was observed (r = 0.576; p = 0.010). An imbalance between bone formation and bone resorption might explain, at least in part, the pathogenesis of bone loss in this disease. These findings could provide new insights into the biological mechanisms underlying bone damage in PKU.     

Morphine promotes the growth of HIV-1 in human peripheral blood mononuclear cell cocultures

Because morphine has been shown to alter the function of human T lymphocytes and monocytes, we postulated that morphine would promote the growth of HIV-1 in these cells. To test this hypothesis, a coculture assay was used consisting of phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC) from normal donors and PBMC which had been infected with a viral isolate from an asymptomatic patient, HIV-1AT. The growth of HIV-1AT, as reflected by the concentration of p24 antigen in coculture supernatants, was markedly increased in cocultures that contained morphine. A bell-shaped dose-response curve was observed with three- to fourfold increased growth at a morphine concentration of 10(-12) M. Augmentation of HIV-1AT growth by morphine required an interaction with the PHA-activated donor PBMC. Furthermore, potentiation of HIV-1AT growth by morphine was stereospecific and was antagonized by naloxone and beta-funaltrexamine indicating involvement of an opiate receptor mechanism. These findings provide an additional explanation of how opiates could act as a cofactor in the pathogenesis of HIV-1 in intravenous drug users.