Rapid cytomegalovirus pp65 antigenemia assay by direct erythrocyte lysis and immunofluorescence staining

A rapid cytomegalovirus (CMV) pp65 antigenemia assay with direct erythrocyte lysis (DL) with 0.8% NH4Cl, followed by indirect immunofluorescence staining (IF), was evaluated with 82 blood samples from renal transplant recipients, and the results were compared to those of the conventional antigenemia assay with dextran sedimentation and two-cycle alkaline phosphatase, anti-alkaline phosphatase staining (DS-APAAP). The DL-IF modification gave a higher leukocyte yield compared to DS-APAAP (75.4 versus 54.9%; P < 0.05), with similar leukocyte viability rates of >95%. The DL-IF methodology involved fewer technical steps, and the assay time was shortened from 5 h to less than 3 h. Nineteen of the 82 samples concordantly tested positive for pp65 antigenemia by both assays, and the readings showed a good correlation (r = 0.996; P < 0.01). No discordant results were observed. We conclude that the CMV pp65 antigenemia assay by this novel DL-IF modification is technically simpler, cheaper, and less time-consuming but yields results comparable to those of the conventional DS-APAAP assay. The shortened assay time and increased capacity to handle more samples confer distinct advantages in the rapid diagnosis and prompt treatment of CMV disease in immunosuppressed patients.     

Comparison of PCR and pp65 antigenemia assay with quantitative shell vial culture for detection of cytomegalovirus in blood leukocytes from solid-organ transplant recipients

This study compared PCR and an assay for cytomegalovirus (CMV) pp65 antigenemia (CMV-vue; INCSTAR Corp.) with a quantitative shell vial culture (QSVC) technique for the detection of CMV in serial blood specimens from 46 solid-organ transplant recipients. In a comparison based on 535 specimens tested by PCR and QSVC, CMV was detected by PCR in 41 and by QSVC in 37 of 43 recipients at risk of CMV infection. The mean number of days after transplantation of initial detection of CMV was 29.9 for PCR and 34.0 for QSVC (P = 0.01). The antigenemia assay was performed on 395 specimens, including 304 of those also tested by PCR. In these specimens, CMV was detected by the antigenemia assay, QSVC, and PCR in 30, 32, and 35 (respectively) of 38 patients at risk, with no statistically significant difference in the time to detection. Each of the assays detected CMV in similar proportions of patients with and without clinically significant CMV infection. PCR stayed positive longer after transplantation than the other assays but frequently returned to negative when more than 6 months had elapsed after transplantation. The antigenemia assay and PCR stayed positive longer after institution of antiviral therapy than QSVC. PCR can provide highly sensitive detection of CMV viremia, but a PCR assay for CMV is not yet available in kit form. The pp65 antigenemia assay and shell vial culture are quantifiable and comparable in sensitivity. Either is recommended for rapid detection of CMV in blood specimens from solid-organ transplant recipients.     

Novel DNA assay for cytomegalovirus detection: comparison with conventional culture and pp65 antigenemia assay

We compared conventional cytomegalovirus (CMV) isolation, rapid viral culture, a CMV pp65 antigenemia assay, and a novel CMV DNA hybrid capture system (HCS). A total of 309 blood samples from individuals in different risk groups were assessed by at least two of the methods mentioned above. Leukocytes were recovered either after centrifugation in Leucosep tubes containing 1.080 Ficoll for pp65 assay or after simple differential lysis steps for DNA detection. HCS was based on DNA hybridization with a CMV RNA probe and its capture by antibodies to DNA-RNA hybrids. The CMV pp65 lower matrix protein was detected by fluorescence with c10-c11 monoclonal antibody in formalin-fixed leukocytes. Concordant results were observed for 92.9, 78.3, and 82.7% of the patients when comparing (i) viral culture and the pp65 antigenemia assay, (ii) viral culture and HCS, and (iii) the pp65 antigenemia assay and HCS, respectively. Discordant results were observed between a positive HCS result and negative culture and/or pp65 results. These results were associated with relatively low DNA levels (< 20 pg/10(6) cells) and positive viruria. In conclusion, the pp65 antigenemia assay is a rapid and reliable method of detecting CMV and is preferable to culture, but the Murex HCS appears to be more sensitive for CMV detection.     

In vitro generation of human cytomegalovirus pp65 antigenemia, viremia, and leukoDNAemia

Immunocompromised patients with disseminated human cytomegalovirus (HCMV) infection have circulating PMN carrying HCMV pp65 (antigenemia), infectious virus (viremia), and viral DNA (leukoDNAemia). Because HCMV does not fully replicate in PMN, it is generally hypothesized that virions and viral materials are taken up by phagocytosis from fully permissive HCMV-infected endothelial cells. However, no experimental evidence has ever been provided for these PMN-endothelium interactions. PMN from 11 donors were cocultured with endothelial cells infected with an endothelium-adapted HCMV strain and with human fibroblasts infected with low-passaged clinical and laboratory-adapted HCMV strains. pp65-positive PMN were detected after coculture with both HCMV-infected endothelial and fibroblast cells, provided that wild and not laboratory-adapted strains were used. In addition, cocultured PMN carried infectious virus as demonstrated by virus isolation and presence of complete virus particles by electron microscopy. Moreover, high levels of viral DNA were consistently detected by quantitative PCR in cocultured PMN. Thus, we have generated in vitro the three most important viral parameters detected in patients with disseminated HCMV infection (antigenemia, viremia, and leukoDNAemia). The failure of laboratory-adapted HCMV strain to induce this phenomenon demonstrates that important modifications have occurred in attenuated viral strains affecting basic biological functions.     

Quantification of cytomegalovirus DNA in peripheral blood leukocytes by a branched-DNA signal amplification assay

Quantification of cytomegalovirus (CMV) DNA in blood may aid in the identification of patients at highest risk for developing CMV disease, the evaluation of new therapeutics, and the prompt recognition of drug-resistant CMV strains. A branched-DNA (bDNA) assay was developed for the reliable quantification of CMV DNA in peripheral blood leukocytes. The bDNA assay allowed for the highly specific and reproducible quantification of CMV DNA in clinical specimens. Furthermore, the bDNA assay was at least as sensitive as culture techniques and displayed a nearly 3 log10 dynamic range in quantification. Changes in CMV DNA levels measured by the bDNA assay in a human immunodeficiency virus-positive patient undergoing therapy were consistent with CMV culture, antigen, and genotype results and correlated with disease progression and resistance markers. The bDNA assay for the quantification of CMV DNA may provide a useful tool that can be used to aid physicians in monitoring disease progression, evaluating therapeutic regimens, and recognizing viral resistance and drug failure.     

Murine cytomegalovirus DNA in peripheral blood of latently infected mice is detectable only in monocytes and polymorphonuclear leukocytes

Cytomegalovirus (CMV), as do other herpesviruses, establishes a lifelong latent infection in its natural host. While in immunologically intact hosts most CMV infections are subclinical, clinical disease follows severe immunosuppression and immunodeficiency. In these situations CMV may produce serious life-threatening disease, and virus reactivated from the latent state is often responsible. Essential to understanding this virus and its pathogenesis is the need to define particular tissue and cell types harboring viral DNA. We searched for viral DNA and RNA in subpopulations of blood cells from mice latently infected with murine CMV by using differential centrifugation and fluorescent antibody cell sorting followed by polymerase chain reaction analysis. Following intravenous inoculation, the viral DNA was found to be present in the buffy coat at and after 21 days postinfection, and both granulocytes and peripheral blood mononuclear leukocytes (PBML) were reservoirs. Further analysis of the PBML fraction by separation into Mac-1+ and Mac-1- cells revealed that monocytes harbored the DNA while lymphocytes were not sites of persistence. We conclude that in buffy coat of latently infected mice the viral DNA is present only in cells of the myeloid lineage. The relationship of this DNA to the latent infection is discussed.     

IFN-gamma is produced by polymorphonuclear neutrophils in human uterine endometrium and by cultured peripheral blood polymorphonuclear neutrophils

Cytokines present in the human uterus play an important role both in modulating immune responses to infectious challenge and in the establishment and maintenance of pregnancy. In particular, successful implantation and pregnancy is thought to require the establishment of a Th2 environment, while Th1 cytokines are associated with pregnancy loss and infertility. On the other hand, a Th1 response appears to be required for the resolution of acute infection. Using novel confocal microscopic analysis of fresh sections of human tissue, we have investigated the production of IFN-gamma, a Th1 cytokine, in human endometria. Extracellular IFN-gamma, mostly associated with matrix components, was located immediately beneath the luminal epithelium and along the glandular epithelium proximal to the lumen. As evidenced by intracellular staining, IFN-gamma is produced by both stromal cells and intraepithelial lymphocytes through all stages of the menstrual cycle. Surprisingly, the stromal cell containing intracellular IFN-gamma was identified as a polymorphonuclear neutrophil on the basis of its reactivity with a panel of mAbs and its nuclear morphology. We further found that polymorphonuclear neutrophils isolated from normal donors produce IFN-gamma in response to stimulation with LPS, IL-12, and TNF-alpha. Taken together, these findings suggest that polymorphonuclear neutrophils are capable of producing IFN-gamma both in vitro and in vivo, indicating that their role in shaping immune responses may be more extensive than previously thought. Furthermore, these studies strongly suggest that polymorphonuclear neutrophils play an important role in determining immune responsiveness within the female reproductive tract.     

Activation of human polymorphonuclear leukocytes by products derived from the peroxidation of human red blood cell membranes

Oxidation of red blood cell (RBC) ghost preparations initiated by tert-butyl hydroperoxide (tBuOOH) was employed to explore the formation of lipid products derived from endogenous phospholipids that specifically expressed biological activity toward the human polymorphonuclear leukocyte (PMN). Common measure of lipid peroxidation, thiobarbituric acid-reactive substances (TBARS) and the increased absorbance at 235 nm consistent with the formation of conjugated dienes, was observed following a 90-min incubation of RBC ghosts with tBuOOH. Saponification of phospholipids and separation of the resultant fatty acids by RP-HPLC permitted direct mass spectrometric analysis of oxidized fatty acids. Individual HPLC fractions were assayed for their ability to increase intracellular free calcium ion concentrations in human PMN to guide structural investigations. Two fractions were found to contain biologically active components, and tandem mass spectrometric analysis of the abundant ions observed in these fractions resulted in the characterization of several oxidized polyunsaturated fatty acids derived from arachidonic and linoleic acids. The major components in these fractions included 5-hydroxyeicosatetraenoic acid (5-HETE) and 5-hydroperoxyeicosatetraenoic acid (5-HpETE). The dose-dependent increases in intracellular calcium in the neutrophil using synthetic 5(rac)-HETE, 5(rac)-HpETE, and 5-oxo-ETE were found to have EC50's of 250, 6, and 3 nM, respectively. The quantity of 5-oxygenated arachidonate components present in oxidized RBC was consistent with the observed biological response elicited by fractions A and B. This study suggests that 5-HETE and 5-HpETE are abundant products of lipid peroxidation of cellular membranes and that these racemic products possess significant biological activity. Such compounds could play important roles as mediators of the cellular response to toxicologic stimuli that generate free radical species.     

Use of the cytomegalovirus antigenemia (CMV-Ag) assay for the detection of CMV in the blood of AIDS patients

Direct specimen testing was performed on 186 peripheral blood specimens to identify the presence of antigen to cytomegalovirus (viz., the cytomegalovirus antigenemia (CMV-Ag) assay). Confirmatory testing was performed using the shell vial indirect immunofluorescence assay (SVA-IFA), the indirect immunoperoxidase assay (TC-IPA), and conventional tube culture isolation (TC-CPE). The primary reagent for the CMV-Ag assay consisted of anti-CMV monoclonal antibody directed against the internal matrix structural phosphoprotein (1C3; Clonatec-Biosoft, France). The 72-kDa early nuclear antigen (Dupont) was utilized in the SVA-IFA and the TC-IPA. All test systems received an equal number of polymorphonuclear leukocytes in the inoculum. CMV was detected and isolated from 30% (55/186) of the specimens evaluated by either one or a combination of the tests. Detection and (or) isolation of CMV from blood by the CMV-Ag assay, SV-IFA, TC-IPA, and TC-CPE occurred at a rate of 17 (31/186), 12 (22/186), 16 (29/186), and 26% (49/186). Three of 55 positive specimens were identified only by the CMV-Ag assay; each patient in question, however, had at least one previous CMV isolate. No significant differences in sensitivity occurred between the CMV-Ag assay, the SVA-IFA, or the TC-IPA. However, TC-CPE including the blind passage of all negative tube cultures yielded a significantly larger number of positive blood specimens than either of the rapid detection methodologies. The CMV-Ag assay encompasses the benefits of a nonculture system, is simple to perform and easy to read, permits a same-day diagnosis, and requires less reagents than the routinely used SVA-IFA or TC-IPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of culture and the antigenemia assay for detection of cytomegalovirus in blood specimens submitted to a reference laboratory

We compared the antigenemia assay (AA) with tandem shell vial cultures (SVCs) and tube cultures (TCs) for detection of cytomegalovirus (CMV) in 343 blood specimens. For 249 specimens, the AA was performed in duplicate with two different commercially available monoclonal antibody reagents (Biotest Diagnostic Corporation and Argene Biosoft). Specimens considered true positives were positive in either culture system or both AAs. Only specimens which were negative in both cultures and positive in a single AA were tested retrospectively with a CMV PCR assay. CMV recovery rates were also calculated to determine if increased specimen age resulted in decreased positivity. CMV recovery rates for the AA and the combination of both cultures were 20.0 and 5.0% at 3 to 18 h, 20.2 and 14.0% at 18 to 35 h, 12.5 and 7.8% at 36 to 52 h, and 18.8 and 6.3% at 64 to 75 h, respectively. The sensitivities and specificities of the Biotest AA, the Argene AA, SVC, and TC were 84.4 and 100.0, 100.0 and 99.6, 44.4 and 100.0, and 46.0 and 100.0%, respectively. The AA was significantly more sensitive than either culture method alone and was also more sensitive than the two culture methods used in tandem (the tandem culture sensitivity was 63.5%); the Argene AA identified more positives than the Biotest AA.     

Stress-associated modulation of proto-oncogene expression in human peripheral blood leukocytes.

Changes in the cellular immune response associated with psychological stress were studied by using an academic stress model with medical students. The authors examined the expression of 2 proto-oncogenes, c-myc and c-myb, in peripheral blood leukocytes (PBLs) obtained from medical students at the time of examinations and at a baseline period approximately 1 mo prior to the examinations. The level of messenger ribonucleic acid (mRNA) expression of both proto-oncogenes was significantly lower in PBLs obtained during examinations than in those from the baseline period. In addition, a significant decrease in the level of mRNA to the glucocorticoid receptor and gamma interferon was also found in the same preparations. The decrease in mRNA content of c-myc, c-myb, the glucocorticoid receptor, and gamma interferon in PBLs obtained from Ss during examinations is consistent with data from previous studies using the same model that have demonstrated a down-regulation of T-lymphocyte activation and proliferation response to mitogens. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Functional activity of blood polymorphonuclear leukocytes as an oxidative stress biomarker in human subjects

In the present work, we studied the role of polymorphonuclear leukocytes (PMN) in aged individuals and coronary heart disease (CHD)-bearing patients, two physiopathological processes associated with overproduction of reactive oxygen species (ROS). The effects of antioxidant supplementation on the functional activity of PMN from CHD patients were also determined. The function of PMNs was evaluated by measuring of phagocytosis, killing activity, and ROS production. Luminol amplified chemiluminescence (CL) was used to estimate ROS production by stimulated PMNs. Total cholesterol and the LDL-cholesterol fraction from CHD patients were found to be higher than those recommended, returning to normal levels after antioxidant therapy. PMN CL of CHD patients was found to be higher than the associated control groups. Antioxidant therapy administrated to CHD patients lead to an increase in the killing activity accompanied by a decrease in PMN CL of these subjects. The study also showed that killing activity of PMN from human subjects over 60 years was significantly lower than the activity measured in younger subjects. PMN CL produced after stimulation was found to be positively correlated with the increasing age of human subjects (r=.946, p < .01).     

Comparison between 2 monoclonal antibodies (clones 95/12 and 1C3 + AYm-1) for the detection of pp65 antigenemia associated with human cytomegalovirus

BACKGROUND: A prospective parallel and blind comparative study was carried out to evaluate the diagnostic efficacy of two available anti-pp65 monoclonal antibodies (clone 95/12 and the pool 1C3 + AYM-1) for the cytomegalovirus (CMV) antigenemia assay. MATERIAL AND METHODS: We carried out a comparative study of 107 blood samples from immunodepressed patients (renal transplant and AIDS patients) with suspected disseminated infection by CMV. The PMNLs were obtained using the method of sedimentation in saline dextran. Slides were stained by an indirect immunofluorescence assay with two commercially available monoclonal antibodies. RESULTS: Of the 107 blood samples studied 33 (30.8%) had a positive antigenemia test. The clone 95/12 detected 30 (90.9%) samples and the pool 31 (93.9%), no statistically significant difference was observed in the sensitivity of two reagents (p = 0.42). The values of the mean CMV-positive cell count obtained with the clone 95/12 was 60.6 vs 61.9 with the monoclonal pool (p = 0.026). CONCLUSIONS: No significant difference was detected between the two commercial monoclonal antibodies. However the pool detected a slightly superior CMV-positive cell count.     

Down-regulation of CXCR2 expression on human polymorphonuclear leukocytes by TNF-alpha

TNF-alpha is implicated in the initiation of cytokine cascades in various inflammatory settings. To assess the interactions of multiple cytokines at the level of inflammatory effector cells, we examined the effects of TNF-alpha on the expression of two IL-8Rs (CXCR1 and CXCR2) on polymorphonuclear leukocytes (PMNs). TNF-alpha decreased the surface expression of CXCR2 in a dose- and time-dependent manner. In contrast, CXCR1 expression was not affected by TNF-alpha. The release of CXCR2 into the supernatant of TNF-alpha-treated PMNs was detected by immunoblotting and immuno-slot-blot analyses, suggesting that the down-regulation of CXCR2 was caused mainly by shedding from the cell surface. The CXCR2 down-regulation was inhibited by PMSF and aprotinin, supporting the hypothesis that the shedding was mediated by serine protease(s). The intracellular Ca2+ mobilization and chemotaxis in response to IL-8 were suppressed by the pretreatment of PMNs with TNF-alpha, indicating that the decrease in CXCR2 was reflected in the decreased functional responses to IL-8. In contrast, the O2- release, which is mediated by CXCR1, was not suppressed by TNF-alpha. The treatment of whole blood with TNF-alpha also caused a significant reduction in CXCR2 and markedly suppressed intracellular Ca2+ mobilization and chemotaxis in response to IL-8, while enhancing the O2- release. These findings suggest that TNF-alpha down-regulates CXCR2 expression on PMNs and modulates IL-8-induced biologic responses, leading to the intravascular retention of PMNs with an enhanced production of reactive oxygen metabolites.     

Opsonin requirements for phagocytosis of Enterococcus spp. by human polymorphonuclear leukocytes

BACKGROUND: Interaction of Enterococcus spp. and host defense mechanisms is not well known. Opsonic requirements of Enterococcus faecalis and Enterococcus faecium to be phagocyted by human polymorphonuclear leukocytes (PMN) were evaluated. METHODS: Twenty strains (10 E. faecalis and 10 E. faecium) were studied. Phagocytosis was determined by a radiometric assay. Bacterial cells were labelled with 3H-adenine and opsonized with: a) 10% of human pool sera (HPS); b) 10% of decomplemented HPS, and c) albumin and fibronectin. RESULTS: Phagocytosis of Enterococcus spp. by PMN in the presence of HPS was significantly higher than that in the absence of opsonins. The phagocytosis of E. faecium was higher than that of E. faecalis. A strain-dependent effect of complement in the phagocytosis of Enterococcus spp. was observed. Neither albumin nor fibronectin showed an opsonic activity on Enterococcus spp. CONCLUSIONS: A great heterogeneity in the opsonic requirements of Enterococcus spp. was observed. Serum opsonins show a critical role in the phagocytosis of E. faecalis and E. faecium by PMN, this effect being more relevant with E. faecium. A strain-dependent opsonic activity of complement was observed.     

Simultaneous flow cytometric measurement of Streptococcus suis phagocytosis by polymorphonuclear and mononuclear blood leukocytes

A simple flow cytometric method was used to study simultaneously the phagocytosis of Streptococcus suis serotype 2 by polymorphonuclear and mononuclear blood leukocytes from swine and humans. Using this method with a bacteria-to-leukocytes ratio of 10:1 and after 60 min of incubation, 80.2 +/- 2.8% of swine granulocytes and 77.0 +/- 2.8% of swine monocytes were shown to contain FITC-labelled S. suis serotype 2 strain 735. Using the same strain, FITC-labelled bacteria were found in 95.5 +/- 3.2% of human granulocytes and in 92.8 +/- 3.6% of human monocytes. The phagocytosis rates of avirulent and virulent strains of S. suis were not significantly different.     

The effect of verapamil on resting oxygen metabolism of human peripheral blood polymorphonuclear granulocytes

It was found that 0.051 mmol/l concentration of verapamil inhibits the resting (spontaneous) oxygenic metabolism of the human peripheral blood polymorphonuclear granulocytes. In experiments in vitro it was expressed by the statistically significant lower capacity to reduction of nitroblue tetrazolium (NBT) by the granulocytes incubated with the verapamil solution in comparison with the same capacity without the verapamil solution.