Multicenter external quality assessment of molecular methods for detection of human herpesvirus 6.

The purpose of this study was to evaluate the performance of laboratories for the detection and quantification of human herpesvirus 6 (HHV-6) by an external quality assessment (EQA) evaluation. The HHV-6 EQA panel consisted of eight samples containing various concentrations of HHV-6 type A (strain GS) or type B (strain Z29), two samples containing other herpesviruses (i.e., human cytomegalovirus [HCMV] and Epstein-Barr virus [EBV]), and two HHV-6-negative samples. Panel samples were prepared in human plasma, heat inactivated, and lyophilized. Panel distribution, data management, and analysis were coordinated by Quality Control for Molecular Diagnostics (QCMD), Glasgow, United Kingdom. Fifty-one laboratories participated and submitted 57 data sets. Eleven (19.3%) data sets were generated using conventional in-house assays, 11 (19.3%) data sets using commercial real-time PCR assays, and 35 (61.4%) data sets using in-house real-time PCR assays. The presence of HHV-6 DNA at viral loads exceeding 6,000 copies/ml was detected by all participants, and over 80% of the participants still reported correct qualitative results for the sample containing just over 200 copies/ml. The false-positivity rate was 1.8% for both the negative samples and the samples containing HCMV or EBV DNA. The majority (23/33; 69.7%) of quantitative data sets were generated using in-house real-time PCR assays. The standard deviations of the geometric means of the samples ranged from 0.5 to 0.7 log(10). The results of this first international EQA demonstrate encouraging analytical sensitivity for the detection of HHV-6-DNA in human plasma, although we observed extensive interlaboratory variation of quantitative HHV-6 DNA results. Standardization needs to be improved to allow further elucidation of the clinical significance of HHV-6 loads.

Role for oxygen radicals in self-sustained HIV-1 replication in monocyte-derived macrophages: enhanced HIV-1 replication by N-acetyl-L-cysteine.

N-acetyl-L-cysteine (NAC) has been proposed as a therapeutic agent for AIDS patients because it reduces human immunodeficiency virus type 1 (HIV-1) replication in stimulated T cells. However, NAC and glutathione enhanced acute HIV-1 replication in monocyte-derived macrophages. Buthionine sulfoximine did not affect NAC-mediated enhanced HIV-1 replication, indicating that the NAC-mediated effects are glutathione-independent. Superoxide dismutase and the hydroxyl radical scavengers dimethylthiourea and thiourea, but not urea, inhibited acute HIV-1 replication in macrophages. NAC reduced ferricytochrome c and increased dose-dependently Fe(III)-citrate and Fe(III)-EDTA-catalyzed hydroxyl radical formation in a system using glucose and glucose oxidase. Dimethylthiourea and thiourea, but not urea and superoxide dismutase, dose-dependently inhibited NAC-mediated enhancement of HIV-1 replication. These data suggest that oxygen radicals play an important role in self-sustained HIV-1 replication in macrophages and that oxygen radical scavengers other than NAC should be considered as therapeutic agents for AIDS patients.

N-acetyl-L-cysteine-induced up-regulation of HIV-1 gene expression in monocyte-derived macrophages correlates with increased NF-kappaB DNA binding activity.

Nuclear factor kappaB (NF-kappaB) is an important cellular regulator of human immunodeficiency virus (HIV) gene expression. In T cells, N-acetyl-L-cysteine (NAC) inhibits the induction of NF-kappaB and transcription of HIV-1. However, NAC up-regulates HIV-1 replication in monocyte-derived macrophages (MDM). In this study we demonstrate that NAC treatment of MDM transfected with a chloramphenicol acetyltransferase (CAT) construct under transcriptional control of the HIV-1 long terminal repeat resulted in an up-regulation of CAT activity. Furthermore, MDM transfected with a HIV-1-NF-kappaB-CAT construct also produced increased CAT activity after NAC treatment. In addition, electrophoretic mobility shift assays revealed that nuclei of NAC-treated MDM contained increased binding activity to wild-type, but not mutant, kappaB oligonucleotides. Components of the binding activity were identified with antibodies as the NF-kappaB subunits p50 and p65. These data indicate that NAC-induced enhancement of HIV-1 replication in MDM is regulated at the level of viral gene expression and mediated by NF-kappaB.

Antibodies and complement enhance binding and uptake of HIV-1 by human monocytes.

OBJECTIVE: To characterize antibody- and complement-mediated binding and uptake of HIV-1 by human monocytes. DESIGN: The first step in the infection of the monocyte by HIV-1 is binding of the virus to the susceptible cell. Procedures were designed to assess the influence of anti-HIV-1 antibodies and complement on this binding, and to study the process of internalization following binding. METHODS: Human monocytes were incubated with fluorescein-labelled purified HTLV-IIIB virions and human sera with high-titre anti-HIV-1 antibodies and/or complement. Binding and uptake of virus by the monocytes was measured as fluorescence per cell by flow cytometry. RESULTS: Binding of purified HIV-1 to monocytes was increased by complement and, to a lesser extent, by anti-HIV-1 antibodies. Uptake of HIV-1 bound to the monocyte appeared to be mediated by antibodies and was increased further by the presence of complement. Complement alone, however, resulted in the uptake of only a small part of the bound virus. CONCLUSIONS: Complement significantly increases the binding of HIV-1 to human monocytes, and a combination of antibodies and complement efficiently mediates uptake of HIV-1 by monocytes.

Intracellular pathways involved in TNF-alpha and superoxide anion release by Abeta(1-42)-stimulated primary human macrophages.

In this study, the intracellular signal transduction pathways leading to the production of TNF-alpha and superoxide anions by amyloid-beta-stimulated primary human monocyte-derived macrophages was investigated. Using Western blotting and specific inhibitors it is shown that both ERK 1/2 and p38 MAPK signal transduction pathways as well as PKC are involved in the amyloid-beta-stimulated superoxide anion production. In contrast, only ERK 1/2 MAPK seems to be involved in TNF-alpha production: questioning the connection between PKC and ERK 1/2 activation. Our results suggest the use of ERK 1/2 MAPK inhibitors in the prevention of macrophage activation in the context of Alzheimer's disease.

The amino-terminus of the amyloid-beta protein is critical for the cellular binding and consequent activation of the respiratory burst of human macrophages.

Here, we show that amyloid-beta (Abeta) is capable to prime and activate the respiratory burst of human macrophages. Previously, the N-terminus of Abeta(1-42) has been shown to contain a cell binding domain that is implicated in eliciting neuropathogenic microglia in vitro. To evaluate the role of this domain in the Abeta(1-42)-induced respiratory burst activity, the effect of Abeta subfragments on the Abeta(1-42)-induced superoxide release were studied. On the basis of the antagonistic properties of Abeta(1-16), it is concluded that the N-terminal region of Abeta is critical for the cellular binding and consequent activation of the respiratory burst of human phagocytes.

Reaction of the vaginal flora to ornidazol in patients with cervicitis.

The cervical and high vaginal flora of 76 patients with cervicitis were studied before and after therapy with Ornidazol by quantitative culture methods. Lactobacilli were the predominant organisms, but Peptostreptococci, Bacteroides and Trichomonas were encountered in 17, respectively 32 and 81% of all specimens. During and after therapy Trichomonas disappeared completely, the bacterial flora normalized and became comparable to that of healthy women with incidences for Bacteroides of 8-13% and Peptostreptococci of 4-5%. The in vitro susceptibility (MIC and MBC) of 50 strains of Bacteroides to Ornidazol was determined by a broth dilution method and an agar plate technique. The MIC varied from 0.07 to 10 microgram/ml. All strains were susceptible to 10 microgram/ml. There was a slight variation in resistance between the various species tested. B. fragilis was less susceptible to Ornidazol than other Bacteroides species. Within the species B. fragilis the subspecies thetaiotaomicron and 'other' were most susceptible, spp. fragilis and spp. distasonis least.

Intracellular polysaccharide of Bacteroides fragilis.

Formation of iodophilic polysaccharide (IPS) from glucose was demonstrated in 27 strains of Bacteroides fragilis. Synthesis was dependent on the glucose concentration of the medium, the pH and the growth phase. When glucose was in short supply the cellular polysaccharide was degraded rapidly at pH 4.5 to 6.5 and fatty acids accumulated in the medium. Storage of IPS was not responsible for the low carbon recoveries observed in fermentation balance studies. In electron micrographs of thin sections, the IPS was observed as cytoplasmic granules dispersed throughout the whole cell. After extraction and purification the IPS was characterized as a glycogen.

Composition and ecology of the human intestinal flora.

Longitudinal quantitative cultures of fecal flora of 20 newborns, 4 older babies and 10 healthy adults were carried out to study the composition and development of the intestinal flora. In all newborns the same sequence of colonization was observed. The numbers of aerobic and anaerobic bacteria fluctuated and reached finally numbers of 10(10)/g wet weight. In adults the flora was in balance with 10(5)-10(7) aerobic and 10(10)-10(11) anaerobic bacteria/g wet weight. Interaction experiments in vitro showed growth inhibition of Bacteroides fragilis by all intestinal species isolated. Bifidobacteria were not inhibited. The assumption was made that this type of interaction could be one of the mechanisms involved in the intestinal micro-ecology. Three of the Bacteroides fragilis strains tested were able to grow on "natural intestinal substrates" as gastric mucin, glycogen and a variety of plant polysaccharides. Acetic, lactic, propionic and succinic acids were detected as fermentation products.

Effect of iron on neonatal gut flora during the first three months of life.

To study the effect of milk supplemented with iron on neonatal gut flora, faecal specimens of ten infants receiving breast milk, six receiving a cow-milk preparation supplemented with iron (5 mg/l) and seven receiving the same product without iron supplement (iron concentration less than 0.5 mg/l) were examined during the first 12 weeks of life. In breast-fed infants bifidobacteria was predominant, counts of Escherichia coli were low, and other bacteria were rarely present. Infants receiving fortified cow-milk preparation had high counts of Escherichia coli, counts and isolation frequency of bifidobacteria were low and other bacteria were frequently isolated. In those on unfortified cow-milk preparation isolation frequency of Escherichia coli, bifidobacteria and bacteroides was comparable with that in breast-fed infants; however, counts of Escherichia coli were high. It is concluded that the faecal flora of infants fed unfortified cow-milk preparation acquires characteristics of that found in breast-fed infants.

Antibody mediated enhancement of HIV-1 infection of an EBV transformed B cell line is CD4 dependent.

Low levels of anti-viral antibodies may facilitate virus infection of Fc-receptor bearing cells. For human immunodeficiency virus (HIV) it has been reported that antibodies can enhance infection of phagocytic cells. We show that HIV-1 can infect an Epstein-Barr virus transformed B cell line and that low levels of anti-HIV antibodies enhance infection. The enhanced infection was characterized by an increase in viral DNA and increased HIV p24 protein production. Detection of cell surface antigen expression of CD4, the receptor for HIV, Fc-receptor type II for IgG, but not of type I and III could be demonstrated by immunofluorescence cytometry. The enhancement was abrogated when infection was performed in presence of a monoclonal antibody directed against CD4. Based on these results we conclude that antibody mediated enhancement of HIV-1 infection can also occur in non-phagocytic cells in a CD4 dependent manner and that IgG Fc-receptors other than types I or III are involved in this process.

Monocytes modulate enhancement of HIV-1 replication by Mycobacterium tuberculosis.

To investigate the effects of Mycobacterium tuberculosis on HIV-1 replication, peripheral blood mononuclear cells (PBMC) of bacille Calmette-Guerin (BCG)-vaccinated donors and non-BCG-vaccinated donors were infected in vitro with a lymphotropic isolate of HIV-1 and cultured in the presence of purified protein derivative (PPD). Addition of PPD resulted in enhanced HIV-1 replication and lymphoproliferation in BCG-vaccinated donor PBMC, while PPD had no such effects in control PBMC. HIV-1 replication increased even more when monocytes were removed from PBMC, while lymphoproliferation was decreased. High percentages of monocytes were associated with a decreased HIV-1 replication and proliferation that could not be reversed by addition of antibodies against the cytokines IL-1, transforming growth factor-beta (TGF-beta) or indomethacin. PPD stimulates PBMC to release IL-10, a cytokine known to down-regulate proliferation and HIV-1 replication. PPD-induced effects on proliferation as well as HIV-1 replication could be partially blocked by adding a monoclonal antibody against MHC class II molecules, suggesting that part of the mechanism of PPD-induced enhancement is T memory cell activation.

Effect of iron on neonatal gut flora during the first week of life.

Faecal specimens from 23 infants during the first week of life were compared. Ten infants received breast milk, six received cow-milk preparation supplemented with iron (+/- 5 mg/l) and seven unfortified cow-milk preparation (iron concentration less than 0.5 mg/l). Those on breast milk had low faecal pH, high counts of bifidobacteria and low counts of Enterobacteriaceae, bacteroides and clostridia. Infants receiving fortified cow-milk preparation had a high faecal pH and high counts of Enterobacteriaceae and putrefactive bacteria such as bacteroides and clostridia. Counts of bifidobacteria were also high. In those on unfortified cow-milk preparation a slow rise was observed in counts of Enterobacteriaceae followed by an increase in counts and isolation frequency of bifidobacteria: the latter was still rising on day 7. It is concluded that a low iron content in standard preparations of cow's milk enhances resistance of the neonatal gut to colonization.

Phagocytic function of monocyte-derived macrophages is not affected by human immunodeficiency virus type 1 infection.

The immunopathogenesis of human immunodeficiency virus (HIV) infection is characterized by the failure to control opportunistic infections. Here, the direct effect of HIV on macrophage phagocytic function was studied. HIV-1-infected monocyte-derived macrophages expressed as many Fc gamma and complement receptors as did control macrophages. The function of these receptors was not affected by HIV-1 infection since binding and internalization of opsonized Escherichia coli and Staphylococcus aureus were not impaired. Production of reactive oxygen species induced by stimulation of the HIV-1-infected macrophages with opsonized E. coli, zymosan, or PMA was intact. HIV-1-infected macrophages killed opsonized E. coli and Candida albicans as effectively as did control macrophages. These results, therefore, do not support the hypothesis that HIV-1 infection of macrophages causes phagocytic dysfunction and suggest that HIV-induced abnormalities outside the mononuclear phagocyte system may lead to the inability to control opportunistic pathogens.

Mannoproteins of Cryptococcus neoformans induce proliferative response in human peripheral blood mononuclear cells (PBMC) and enhance HIV-1 replication.

To investigate the possible role of Cryptococcus neoformans var. neoformans in HIV disease progression, and to identify the responsible cryptococcal components, an in vitro cell culture model was set up to study the C. neoformans-induced enhancement of HIV replication in HIV-1-infected PBMC. Similar to whole C. neoformans, cell-wall membrane fraction and mannoproteins induced proliferation of PBMC and enhancement of lymphotropic HIV replication in HIV-infected PBMC, while galactoxylomannan did not. MoAbs capable of interfering with MHC class II-mediated antigen presentation prevented the induction of cell proliferation by whole C. neoformans or cryptococcal mannoproteins. MoAb binding to adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) also inhibited C. neoformans-induced cell proliferation. In addition, anti-MHC class II MoAb inhibited the enhancement of HIV replication by C. neoformans. The results suggest that: (i) C. neoformans may accelerate HIV disease progression by stimulation of HIV replication through MHC class II-mediated antigen presentation; and (ii) cryptococcal mannoprotein may be one of the responsible components. The ability to enhance HIV replication in PBMC in vitro is not unique for C. neoformans. However, this is the first report to study in detail a yeast-induced enhancement of HIV replication in PBMC.

Hydroxyurea interferes with antigen-dependent T-cell activation in vitro.

BACKGROUND: Hydroxyurea is believed to inhibit human immunodeficiency virus type 1 (HIV-1) in HIV disease by decreasing the amount of intracellular deoxynucleotides needed for viral replication. A plasma concentration of 400 micromol L-1 is tolerated in oncological diseases. The present study focused on the possible interference of hydroxyurea with antigen-dependent T-cell activation as an alternative explanation for inhibiting HIV replication in vivo. METHODS: The effect of hydroxyurea on common antigen-induced cell proliferation was studied in peripheral blood mononuclear cells (PBMC) in vitro. RESULTS: Hydroxyurea inhibited Candida albicans-induced cell proliferation at a low concentration (1 micromol L-1), while at least 10 micromol L-1 was required to block HIV-1 replication in phytohaemagglutinin (PHA)-stimulated PBMC. CONCLUSION: Hydroxyurea inhibits antigen-induced lymphoproliferation in vitro at a concentration at which it does not inhibit PHA-induced HIV replication. Hydroxyurea may inhibit HIV-1 in CD4+ T cells in vivo not only by decreasing the amount of intracellular deoxynucleotides, but more specifically by interfering with antigen-dependent T-cell activation, thereby causing a reduction in the number of HIV target cells.

Activation of human macrophages by amyloid-beta is attenuated by astrocytes.

In Alzheimer's disease, neuritic amyloid-beta plaques along with surrounding activated microglia and astrocytes are thought to play an important role in the inflammatory events leading to neurodegeneration. Studies have indicated that amyloid-beta can be directly neurotoxic by activating these glial cells to produce oxygen radicals and proinflammatory cytokines. This report shows that, using primary human monocyte-derived macrophages as model cells for microglia, amyloid-beta(1-42) stimulate these macrophages to the production of superoxide anions and TNF-alpha. In contrast, astrocytes do not produce both inflammatory mediators when stimulated with amyloid-beta(1-42). In cocultures with astrocytes and amyloid-beta(1-42)-stimulated macrophages, decreased levels of both superoxide anion and TNF-alpha were detected. These decreased levels of potential neurotoxins were due to binding of amyloid-beta(1-42) to astrocytes since FACScan analysis demonstrated binding of FITC-labeled amyloid-beta(1-42) to astrocytoma cells and pretreatment of astrocytes with amyloid-beta(1-16) prevented the decrease of superoxide anion in cocultures of human astrocytes and amyloid-beta(1-42)-stimulated macrophages. To elucidate an intracellular pathway involved in TNF-alpha secretion, the activation state of NF-kappaB was investigated in macrophages and astrocytoma cells after amyloid-beta(1-42) treatment. Interestingly, although activation of NF-kappaB could not be detected in amyloid-beta-stimulated macrophages, it was readily detected in astrocytoma cells. These results not only demonstrate that amyloid-beta stimulation of astrocytes and macrophages result in different intracellular pathway activation but also indicate that astrocytes attenuate the immune response of macrophages to amyloid-beta(1-42) by interfering with amyloid-beta(1-42) binding to macrophages.

Induction of HIV-1 IIIb neutralizing antibodies in BALB/c mice by a chimaeric peptide consisting of a T-helper cell epitope of Semliki Forest virus and a B-cell epitope of HIV.

A colinearly synthesized peptide consisting of a H-2d restricted T-helper cell epitope of Semliki Forest virus (SFV) and triple repeats of sequence GPGRAF, derived from the V3 domain of HIV-1 strains, was used to immunize BALB/c (H-2d) mice. Pepscan analysis of sera from peptide-immunized mice revealed that the chimaeric peptide GREKFTIRPHYGKEIGPGRAFGPGRAFGPGRAF contains three distinct antibody-reactive sequences GREKFTIR, PHYGKEI and GPGRAF. The chimaeric peptide evoked HIV-1 IIIb neutralizing antibodies in serum as measured in vitro by reduction of syncytia formation and reduction of p24 production as well. So, the T-helper cell epitope of SFV provided help to a small linear neutralization epitope of HIV-1 strains. Interestingly, the T-helper cell epitope alone might induce antibodies cross-reactive with HIV-1 IIIb specific peptide GPGRAFVTIGK which shows some homology (residues underlined) with the antibody-reactive sequence GREKTIR of SFV.

Activation and cell cycle antigens in CD4+ and CD8+ T cells correlate with plasma human immunodeficiency virus (HIV-1) RNA level in HIV-1 infection.

The relationship between T cell activation and human immunodeficiency virus type 1 (HIV-1) replication was studied in HIV-infected subjects, 20 with and 10 without anti-HIV treatment. Expression of Ki-67 proliferation-associated antigen was increased in CD4+ and CD8+ T cells and correlated with HLA-DR. In subjects without anti-HIV treatment, the plasma HIV-1 RNA level correlated with HLA-DR in CD4+ T cells, with Ki-67 in CD8+ T cells, and with expression of CD38 in both T cell subsets. A proportion of treated subjects had increased T cell activation despite 4 months of highly active antiretroviral treatment (HAART). In subjects receiving HAART, a high percentage of HLA-DR+ CD4+ T cells was associated with signs of opportunistic infections. This work supports the concept that, in the natural course of HIV-1 infection, HIV replication itself leads to general T cell activation and that opportunistic infections generate additional CD4+ T cell activation and HIV replication.