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.     

Standardization of the human cytomegalovirus antigenemia assay by means of in vitro-generated pp65-positive peripheral blood polymorphonuclear leukocytes

We generated in vitro human cytomegalovirus (HCMV) pp65-positive polymorphonuclear leukocytes (PMN) resembling those detected in vivo, following cocultivation of PMN from healthy donors and wild-type HCMV-infected endothelial cells or fibroblasts. After purification, PMN are suitable for preparation of cytospots which can be used for the antigenemia assay. Cytospin preparations containing a predetermined number of in vitro-generated pp65-positive PMN were used to test some of the major parameters involved in performing the antigenemia assay. The results showed or confirmed that (i) formalin fixation followed by permeabilization is the best fixation procedure developed to date, (ii) the test performance levels provided by different pools of pp65-specific monoclonal antibodies may be significantly different, and (iii) long-term storage (for an unlimited time) is best achieved by keeping fixed slides at -80 degreesC, whereas short-term storage (for up to 1 month) is best achieved by keeping unfixed slides at room temperature. This finding signifies that slides can be shipped all over the world at room temperature. In conclusion, the newly developed procedure for in vitro generation of pp65-positive PMN will provide the basis for standardization of the HCMV antigenemia assay and development of quality control programs.     

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.     

Cytophotometric studies of DNA contents in peripheral blood leukocytes in patients with infectious mononucleosis

Within 12 years, 4000 patients with hepatic lesions, were examined by the author. Examinations included: clinical, laboratory, biopsy, together with radioactive Rose-bengal 131I. RBR test prooved to be more sensitive than known laboratory liver function tests. In diagnosis of chronic hepatitis and cirrhosis, histological examinations remain to be decisive, though biopsy is not always recommended from view point of prognosis. The isotope procedure has a great advantage besides to its sensitive registration of liver cells functions, it offers good informations about flowness of bile tract. The bladder diagrams increase by action of milk-chocolate meals (24 hours), after which, urine analysis, estimation of blood activity and resting-liver activity can be of value. Harm due to radiation in RBR test is a minimum, so it can be repeated several times in order to follow progression of liver diseases. RBR examination is recommended for all big hospitals.     

Uptake of fluoroquinolones in human monocytes isolated from peripheral blood

The aim of this study was to develop a technique for separating monocytic cells in suspension from peripheral blood to measure the intracellular penetration of three fluoroquinolones (ofloxacin, ciprofloxacin and sparfloxacin). Mononucleated cells were isolated from the blood on a density gradient with lymphoprep and purified by a specific technique of adhesion and disadhesion on fibronectin. The monocytes were obtained in suspension with 76.8% purity and 97.9% viability. This was a convenient form for measurement of intracellular accumulation by use of the velocity-centrifugation technique. Intra-monocytic penetration of ciprofloxacin, ofloxacin and sparfloxacin was measured at equilibrium after 30-min incubation in the presence of 16 microg mL(-1) antibiotic. The results revealed low intra-monocytic accumulation of ciprofloxacin (intracellular-extracellular = 1.76) and ofloxacin (intracellular-extracellular = 1.42). The penetration of sparfloxacin was significantly higher (intracellular-extracellular = 2.4). This study confirms the important differences between human immunocompetent cells in terms of their ability to concentrate quinolones. It also underlines the importance of monocyte-macrophage cellular differentiation as a determinant of antibiotic penetration.     

Murine cytomegalovirus replication in the lungs of athymic BALB/c nude mice

Uridine diphosphoglucose (UDPG) is a precursor of uridine that can be used as a rescuing agent from 5-fluorouracil (5FU) toxicity. Four doses of UDPG (2000 mg/kg i.p. or p.o. at 2, 6, 24, and 30 h after 5FU bolus) allowed the escalation of a weekly bolus of 5FU from 100 mg/kg (5FU100) to 150 mg/kg (5FU150) in healthy and tumor-bearing BALB/c, C57/BI, and CD8F1 (BALB/c x DBA/8) mice. 5FU150 without rescuing agents is not tolerated by the animals. When followed by UDPG, on the contrary, it is possible to increase the dose of 5FU even when it is modulated by leucovorin. Toxicity was the same for 5FU100 and 5FU150 + UDPG, and the nadir values (expressed as a percentage of pretreatment values) were 83 and 85% for weight, 45 and 45% for hematocrit, and 45 and 61% for leukocytes, respectively. Platelets were not affected by treatment. A protective effect was also shown for the gastrointestinal tract. The enzymes thymidine kinase, maltase, and sucrase were measured in the intestinal mucosa at different times after 5FU treatment with or without UDPG rescue. Even if the nadir values in enzyme activities were similar in mice receiving or not receiving UDPG, the pattern of recovery showed that cell repopulation was more rapid in the group treated with UDPG. 5FU150 + UDPG had enhanced antitumor activity against CD8F1 mammary carcinoma and against the resistant tumor Colon 26 (tumor doubling time 1.9 days for controls, 8.5 days for 5FU100, 13.7 days for 5FU150 + UDPG, and 15.9 days for 5FU150 + leucovorin + UDPG). We demonstrated that UDPG administered at 2, 24, and 30 h after 5FU100 does not reduce the antitumor activity of 5FU in two sensitive tumors (Colon 38 and Colon 26-10). In conclusion, UDPG is a promising rescuing agent for 5FU; it reduces the toxic side effects and increases the therapeutic index.     

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).     

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.     

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)     

Simultaneous flow cytometric measurement of phagocytotic and oxidative burst activity of polymorphonuclear leukocytes in whole bovine blood

Polymorphonuclear neutrophils (PMN) are important in host defence against bacterial infection in the bovine mammary gland and techniques are needed to evaluate their functional activities. A rapid and sensitive two-color flow cytometric method for simultaneous measurement of phagocytosis rate and oxidative burst activity of bovine PMN in small blood samples is described. The method utilizes the oxidation of intracellular dihydrorhodamine 123 to green fluorescent rhodamine 123 by oxidative burst products that were generated by incubating the PMNs with red fluorescent propidium iodide labeled Staphylococcus aureus. Phagocytosis and oxidative burst both increased with time of incubation and with increasing bacteria concentration. A 20 min phagocytosis time and a ratio of 25 bacteria to one cell were considered optimal for assaying bovine blood PMN activity. To further evaluate the proposed two-color flow cytometric method, blood samples were treated with factors known to interfere with phagocytosis and oxidative burst metabolism of human neutrophils. Incubation of whole bovine blood with cytochalasin B at 10 micrograms ml-1 resulted in an approximate 70% decrease in the phagocytosis rate with a concommitant decrease in oxidative burst activity. Staurosporine (2 micrograms ml-1) inhibited bovine neutrophil oxidative burst formation for 95% while the phagocytosis was unaffected. PMNs in whole blood samples of ten cows differed significantly in their ability to phagocytose Staphylococcus aureus and to produce reactive oxygen products. However, only a weak correlation was detected between the burst activity:phagocytosis ratio of ten individual cows as indicated by the ROD/PI index.     

The crucial role of polymorphonuclear leukocytes in resistance to Salmonella dublin infections in genetically susceptible and resistant mice

Macrophages are considered to be the mediators of resistance to extra-intestinal Salmonella infections. Nevertheless, the initial cellular response to Salmonella infections consists primarily of polymorphonuclear leukocytes (PMN). To determine whether PMN serve an important function for the infected host, we made mice neutropenic with the rat mAb to RB6-8C5 and infected them i.v. with approximately 10(3) Salmonella dublin or an isogenic derivative that lacks the virulence plasmid (LD842). We infected BALB/c mice, which have a point mutation in the macrophage-expressed gene Nramp1 that makes them susceptible to Salmonella, and BALB/c.D2 congenic mice, which have the wild-type Nramp1 gene that makes them resistant to Salmonella. Both mouse strains were resistant to LD842, and neutropenia made only the BALB/c strain susceptible to this infection. Neutropenic congenic mice, however, were susceptible only to wild-type S. dublin (plasmid+). These results show a complex interplay between plasmid-virulence genes in Salmonella, host macrophages, and PMN. Mice with normal macrophages need PMN to defend against nontyphoid Salmonella that carry a virulence plasmid but not against Salmonella without virulence plasmids. Mice with a mutant Nramp1 gene need PMN to defend against all Salmonella, even those that lack virulence plasmids. These results, plus the evidence that PMN kill Salmonella efficiently in vitro, suggest that Salmonella have adapted to grow inside macrophages where they are relatively sheltered from PMN. The adaptations that allow Salmonella to survive in macrophages do not protect them from PMN.     

Phagocytosis-enhancing effect of lactoferrin on bovine peripheral blood monocytes in vitro and in vivo

The effect of lactoferrin (LF) on in vitro and in vivo phagocytic ability of bovine blood monocytes was studied. It was demonstrated that bovine LF enhanced in vitro phagocytosis of bacteria and ovine erythrocyte-antibody complexes and increased intracellular killing of Staphylococcus albus. Monocytes of colostrum deprived calves, which were intravenously injected with LF, also exhibited elevated phagocytic properties.     

Early effects of ganciclovir therapy on the quantity of cytomegalovirus DNA in leukocytes of immunocompromised patients

The management of patients with autoimmune haemolytic anaemia of warm type (AIHA) is often problematic. Recently, pulsed high-dose dexamethasone (HDD) has been shown to be effective in the treatment of autoimmune thrombocytopenic purpura (AITP). In this study we treated seven patients with AIHA with HDD. The regimen recommended for treatment of refractory AITP (40 mg dexamethasone for 4 d at the beginning of each 28 d cycle) was employed in almost all cases. Prior to dexamethasone administration, haemolysis was decompensated in all seven patients. HDD was well tolerated and led to an improvement of haemolysis in all cases.