Pharmacokinetics of zidovudine phosphorylation in peripheral blood mononuclear cells from patients infected with human immunodeficiency virus.

As part of an effort towards optimization of dosing of zidovudine (ZDV), formation and elimination of total phosphorylated ZDV (ZDVPt) in peripheral blood mononuclear cells were examined in 21 asymptomatic human immunodeficiency virus-infected patients during their first 24 weeks of therapy (AIDS Clinical Trials Group Protocol 161). Intracellular concentrations of ZDVPt were measured with a previously described and validated radioimmunoassay technique. Although ZDV phosphorylation occurred readily upon initiation of therapy, it declined with time; the area under the concentration-time curve (AUC) at week 4 (mean +/- standard deviation, 3.41 +/- 0.93 pmol.h/10(6) cells) was significantly greater than that at week 24 (2.19 +/- 1.10 pmol.h/10(6) cells). Plasma ZDV AUC did not change with time and did not correlate with ZDVPt AUC. In dose-response experiments (20 to 100 mg orally), phosphorylation did not proportionally increase with increasing plasma ZDV concentrations. Similarly, compared with a single dose, two doses of ZDV over an 8-h period resulted in little ZDVPt increase in cells relative to increase in plasma ZDV concentrations. The half-life of intracellular ZDVPt was twice that of plasma ZDV (4 versus 2 h), suggesting that an every-8-h dosing regimen is justifiable. These findings suggest that metabolism of ZDV to its active intracellular forms may be saturable in some patients, is poorly correlated with plasma concentrations, and diminishes over time. These findings have implications for future development and management of anti-human immunodeficiency virus nucleoside therapy.     

Infection of human peripheral blood mononuclear cells by varicella-zoster virus

The infection of human peripheral blood leukocytes by varicella-zoster virus (VZV) was studied using an infectious center assay, indirect immunofluorescence and electron microscopy. Subsets of freshly isolated leukocytes were prepared, including granulocytes, mononuclear cells from Ficoll-Hypaque gradients, lymphocytes, and glass-adherent monocytes. When each of these populations was inoculated with VZV (MOI = 0.1), there was no evidence of effective infection. However, when monocytes were cultured in vitro for 7 days, they differentiated into macrophages that were productively infected with VZV. Peak percentages of infectious macrophages were detected 8-24 h after inoculation (mean 17.5%; range 10.2-30.4%). Using indirect immunofluorescence, viral antigens were detected in the cytoplasm and at the nuclear membranes of infected macrophages between 24 and 72 h after infection. Electron microscopy demonstrated the appearance of viral particles in the nucleus by 24 h. Large numbers of virions, often collected in tubules or vacuoles, were present in the cytoplasm at 48 h. The difference between the infection of fresh monocytes and cultured macrophages by VZV might reflect differences in their metabolic or differentiation state. The possible significance of these observations to VZV infection of immunocompromised hosts is discussed.     

Prevalence of TT virus infection and viral integration in human hepatocellular carcinoma]

In 1997, Nishizawa et al cloned a novel DNA virus designated as TT virus (TTV) from a patient with post-transfusion hepatitis and this virus is being thought to be a new hepatitis virus. Approximately 5 to 10% of hepatocellular carcinomas (HCCs) in Japan occurs in hepatitis B virus-negative and hepatitis C virus-negative (NBNC) patients. In order to study the possible role of TTV in hepatocarcinogenesis, we studied the prevalence of the TTV DNA in liver tissue of HCC patients. As a result, TTV was shown not to be specific for NBNC HCC and TTV integration into host DNA was not detected in any HCC patient by Southern blotting.     

Interleukin-2 and neopterin-induced neopterin release from peripheral blood mononuclear cells

Stimulation of peripheral blood mononuclear cells (PBMC) by the mitogenic lectins, phytohaemagglutinin (PHA) and concanavalin A (conA), the lymphokine gamma-interferon (gamma-IFN) and interleukin-2 (IL-2) and the pterin neopterin, caused an increased release of neopterin from those cells, with peak levels after 7 days of stimulation. In contrast to gamma-IFN, IL-2 and neopterin failed to induce neopterin release from purified macrophages. IL-2- and neopterin-induced release of neopterin from PBMC is not dependent on proliferation and is partially inhibited by the addition of anti gamma-IFN or anti IL-2 receptor. Neopterin autoinductive production can explain the amplificated neopterin release during activation of the cellular-mediated immune response (CMI), in spite of the decrease in the T helper cell subsets, which are the main gamma-IFN producers.     

Prevalence of TT virus in patients with fulminant hepatic failure in Japan]

A novel virus(TTV) was isolated from patients with post-transfusion hepatitis of unknown etiology. We studied the prevalence of TTV in 26 patients with fulminant hepatic failure. Serum samples at admission from seven(27%) of the 26 patients were positive for TTV. TTV was also detected in 29(27%) of the 106 healthy blood donors. There were no differences in the positive rate between patients with fulminant hepatic failure and healthy blood donors. There were no differences in clinical findings, or duration from onset to coma in patients with and without TTV. However, the outcome of the patients with TTV was significant worse than those without. TTV may not cause severe hepatitis such as fulminant hepatic failure.     

In vitro effects of Epstein-Barr virus on peripheral blood mononuclear cells from patients with rheumatoid arthritis and normal subjects

Peripheral blood mononuclear cells from 10 patients with rheumatoid arthritis and 9 control subjects were cultured in vitro for 30 days with and without infection by Epstein-Barr virus. All cultures showed polyclonal stimulation of B cells as indicated by rising levels of IgM in the culture supernates, reaching maximal at 18-24 days, and with no quantitative or kinetic difference between the RA and control cells. IgM anti-IgG was also produced in both groups and maximally at 18-24 days, but in greater quantity by the RA lymphocytes. The anti-IgG made by the RA lymphocytes was more easily absorbed by solid phase IgG than was the anti-IgG made by the normal lymphocytes and thus was judged to be of higher affinity. RA lymphocytes uninfected with EBV had higher transformation scores than did the normal controls and developed spontaneously into permanent cell lines in six instances.     

Proinflammatory lipoxygenase products from peripheral mononuclear cells in patients with rheumatoid arthritis

The formation of 5-lipoxygenase products (5-hydroxyeicosatetraenoic acid [5-HETE], leukotriene B4 [LTB4], and leukotriene C4 [LTC4]) by polymorphonuclear and mononuclear leukocytes isolated from peripheral blood of patients with rheumatoid arthritis was evaluated and compared with the data obtained from a group of control subjects. Although the levels of arachidonic acid metabolites via 5-lipoxygenase pathway by stimulated polymorphonuclear cells were comparable between patients and controls, mononuclear leukocytes from patients synthesized, when stimulated, significantly greater amounts of 5-HETE, LTB4, and LTC4 than did cells isolated from normal subjects. In addition, the release of superoxide anion, stimulated by either a particulate or a soluble stimulus, was increased in mononuclear cells from patients. The enhanced capacity of peripheral mononuclear leukocytes isolated from patients with rheumatoid arthritis to generate proinflammatory metabolites of arachidonic acid and oxygenated species with bactericidal and tissue-damaging properties may contribute to the pathogenesis of this complex disease.     

非透析终末期尿毒症患者血清对外周血单个核细胞IL-18分泌的影响

目的探讨非透析终末期尿毒症(ESRD)患者混合血清及不同分子质量血清对外周血单个核细胞(PBMCs)、白细胞介素18(IL-18)分泌的影响。方法分离非透析ESRD患者混合血清,采用超滤离心装置分离非透析ESRD患者不同分子质量血清,同时用Ficoll密度梯度离心分离14例非透析ESRD患者PBMCs,将非透析ESRD患者不同分子质量的血清与非透析ESRD患者PBMCs,共同培养72h,用酶联免疫吸附分析(ELISA)检测试验对象的血清和培养上清液中IL-18浓度。结果 (1)非透析ESRD患者外周血IL-18含量显著高于对照组,差异有统计学意义P0.001);(2)非透析ESRD患者大分子质量血清刺激同源PBMCs分泌IL-18较高,差异有统计学意义(P0.05)。结论分子质量大于10000U的尿毒症毒素是刺激PBMCs引起IL-18分泌增加的主要毒素,提示大分子质量尿毒症毒素可能是导致非透析ESRD患者微炎性反应状态的独立危险因素。     

Comparative analysis of anti-human immunodeficiency virus type 1 activities of dideoxynucleoside analogs in resting and activated peripheral blood mononuclear cells.

We determined the anti-human immunodeficiency virus type 1 (anti-HIV-1) activities of various dideoxy-nucleoside analogs by using phytohemagglutinin-activated peripheral blood mononuclear cells (PHA-PBMs) and resting PBMs (R-PBMs) as target cells. The comparative order of anti-HIV-1 activity in PHA-PBMs was azidothymidine (AZT) > dideoxycytidine (ddC) > dideoxythymidinene (d4T) > dideoxyinosine (ddI) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) > 2'-beta-fluoro-dideoxyadenosine (F-ara-ddA), while that in R-PBMs was ddC > ddI, PMEA, and F-ara-ddA, >> AZT and d4T. A pronucleotide, bis-(S-acetylthioethanol)phosphotriester-ddAMP, which bypasses the anabolic monophosphorylation step for the intracellular delivery of ddAMP, was highly active both in PHA-PBMs and R-PBMs. These data may have basic and clinical relevance in the design of anti-HIV chemotherapy, particularly combination chemotherapy with dideoxynucleosides, and in the development of active pronucleotides.     

In vitro generation of antigen-specific hemolytic plaque-forming cells from human peripheral blood mononuclear cells

We have described a culture and assay system for the sensitization of human peripheral blood mononuclear cells with a T cell-dependent antigen, sheep erythrocytes, in the absence of nonspecific stimulatory agents and with the subsequent generation of macroscopic hemolytic plaques. We have shown that the antibody produced by the plaque-forming cells generated in this culture system is specific for the sensitizing antigen, and that the plaques created are not false plaques because their formation is inhibited by cycloheximide. The success of this system can be attributed to several critical factors including large numbers of peripheral blood mononuclear cells (5 x 10(6) culture), a prolonged period of incubation (10-11 d), continuous rocking during the entire period of incubation, culturing in large (35-mm) flat-bottomed culture dishes in the presence of human plasma, and the appropriate antigen concentration (5 x 10(6) sheep erythrocytes/culture). Furthermore, the generation of macroscopic hemolytic plaques requires plaquing sensitized peripheral blood mononuclear cells in target cell monolayers fixed in an agarose matrix with an incubation period of 2-3 h. We have further shown that the antigen-specific response measured by this system is dependent on adherent cells and T lymphocytes. At least one population of the helper T cells is sensitive to 2,000 rad irradiation. This system is simple, sensitive, and should serve as an effective tool for the analysis of cellular interactions involved in the generation of human antigen-specific plaque-forming cells, the genetic control the human immune response, and the pathophysiology of altered immunoregulation in disease.     

Role of interleukin-18 in peripheral blood mononuclear cells infected with human herpes virus type 6

OBJECTIVE: Interleukin 18 (IL-18) production represents a critical step in the polarization of the Th1 immune response. Human herpes virus type 6 (HHV-6) possesses a peculiar tropism for immunocompetent cells. To understand the relationships among immunocompetent cells, HHV-6 and cytokines, the role of IL-18 during infection of peripheral blood mononuclear cells (PBMC) with HHV-6 was evaluated. METHODS: PBMC were obtained from healthy HHV-6-seronegative donors, after centrifugation of heparinized venous blood over a Ficoll-Hypaque gradient. Supernatants from PBMC were analyzed for the presence of cytokines. To study the effects of exogenous recombinant human (rh) IL-18 on HHV-6 replication, the number of cells expressing viral antigens and the amount of extracellular virus were analysed. RESULTS: No basal production of IL-18 was found in supernatants of unstimulated PBMC. Appreciable amounts of the cytokine were produced by lipopolysaccharide (LPS)-stimulated PBMC. HHV-6 infection of LPS-treated PBMC downregulated IL-18 production. It was found that the addition of rhIL-18 to HHV-6-infected PBMC downregulated the percentage of antigen-positive cells and the release of extracellular virus. CONCLUSION: Impairment of IL-18 release, which is involved in the induction of antiviral cytokines, such as interferon-gamma, could represent a strategy of the virus to evade the immune response of the host.     

Quantitation of intracellular triphosphate of emtricitabine in peripheral blood mononuclear cells from human immunodeficiency virus-infected patients.

An analytical methodology combining solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) was developed to quantitate the intracellular active 5'-triphosphate (TP) of beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine (emtricitabine) (FTC) in human peripheral blood mononuclear cells (PBMCs). The FTC nucleotides, including 5'-mono-, di-, and triphosphates, were successively resolved on an anion-exchange SPE cartridge by applying a gradient of potassium chloride. The FTC-TP was subsequently digested to release the parent nucleoside that was finally analyzed by HPLC with UV detection (HPLC-UV). Validation of the methodology was performed by using PBMCs from healthy donors exposed to an isotopic solution of [(3)H]FTC with known specific activity, leading to the formation of intracellular FTC-TP that was quantitated by an anion-exchange HPLC method with radioactive detection. These levels of FTC-TP served as reference values and were used to validate the data obtained by HPLC-UV. The assay had a limit of quantitation of 4. 0 pmol of FTC-TP (amount on column from approximately 10(7) cells). Intra-assay precision (coefficient of variation percentage of repeated measurement) and accuracy (percentage deviation of the nominal reference value), estimated by using quality control samples at 16.2, 60.7, and 121.5 pmol, ranged from 1.3 to 3.3% and -1.0 to 4. 8%, respectively. Interassay precision and accuracy varied from 3.0 to 10.2% and from 2.5 to 6.7%, respectively. This methodology was successfully applied to the determination of FTC-TP in PBMCs of patients infected with human immunodeficiency virus after oral administration of various dosing regimens of FTC monotherapy.     

Enzymatic imbalance in peripheral blood mononuclear cells isolated from individuals with anti-HIV antibodies

Plasma membrane-bound 5'-nucleotidase (5'-NT), gamma-glutamyltransferase (gamma-GT) and soluble deoxynucleotidyltransferase (TdT) were studied in peripheral blood cells (PBMN) of 35 individuals, 26 male and 9 female, with circulating anti-HIV antibodies. Twenty-six were drug abusers, 2 were drug abusers and homosexuals and 4 were homosexuals. Three did not fall into any risk group. The surface immunologic phenotype of cells stained with the fluorescent monoclonal antibodies Leu 5, Leu 3, Leu 2, Leu 12, Leu M3, Leu M1, anti-CALLA and anti-HLA-DR was delineated by flow cytometry. While the gamma-GT activity did not change, the lymphocyte 5'-NT activity was significantly less than normal in anti-HIV positive individuals and in anti-HIV negative drug abusers. TdT activity was detectable in 14 anti-HIV positive patients (40%), who did not have clinical AIDS. Of 8 patients with AIDS, 3 had a low level of TdT activity but 5 had cells completely devoid of TdT and 5'-NT activity. 5'-nucleotidase activity and the frequency of Leu 2 suppressor antigen bearing cells were the only independent variables that correlated with AIDS incidence.     

Detection of TTV in peripheral blood mononuclear cells of intravenous drug users.

In order to further elucidate the tropism of the novel hepatitis TT virus (TTV) we investigated 22 intravenous drug users (IVDU) for the presence of viral DNA in their peripheral blood mononuclear cells (PBMC) by means of seminested polymerase chain reaction using a set of primers specific for the conserved region of its genome. We detected TTV DNA in 63% of those individuals who had previously been found positive for the agent in their serum, whereas the three remaining ones not displaying TTV DNA in their serum and hence, serving as controls also proved negative in their PBMC. The results presented here further support earlier findings by other authors and their conclusion as to the virus employing a parenteral route of transmission. Further investigation will be required in order to clarify the mechanism of viral infection.