Summary. Cytomegalovirus (CMV) immediate-early (IE)antigen within the nucleus of cells was detected by flow cytometry in peripheral blood obtained from five bone marrow transplant recipients. In patients with an unexplained fever, CMV antigens were detected in monocytes or lymphocytes. On the other hand, in patients with CMV pneumonia, CMV antigens were detected in polymorphonuclear leucocytes (PMNLs). We suggest that the detection of CMV antigen in monocytes or lymphocytes may be related to CMV activation or reactivation, and the positive results in PMNLs indicate that the patient has a CMV-associated disease (CMV pneumonia). Our method may be useful in monitoring CMV activation or reactivation, and analysing the mechanisms of CMV infection.Keywords: cytomegalovirus, flow cytometry, antigenaemia, bone marrow transplantation.Cytomegalovirus (CMV) induced interstitial pneumonia sometimes leads to the death of a patient with severe immunodeficiency. Drugs such as ganciclovir (Stoffel et al, 1988) and foscarnet (Ringden et al, 1985) are available for treatment of CMV infection, but early diagnosis is essential for early treatment. Progress in molecular biology has led to the development of the polymerase chain reaction (PCR) (Saiki et al, 1985;Sjobring et al, 1990) which can detect CMV sequences with high sensitivity (Honda et al, 1994;Einsele et al, 1995;Urushibara et al, 1995). Moreover, monitoring the expression of CMV antigen in peripheral blood leucocytes, so-called CMV antigenaemia (The et al, 1992; Bacigalupo et al, 1995; Takenaka et al, 1997) has proved to be a sensitive, specific and rapid technique for the early diagnosis of CMV infection. Based on the importance of detecting CMV activation and initiation of treatment for CMV infection before occurrence of the disease, we developed a new method of detecting CMV antigen in peripheral blood using a flow cytometer (FCM) and monitored the development of CMV infection using this new method. MATERIALS AND METHODS Materials.Human fetal fibroblasts (MRC-5 cell strain) infected with CMV strain AD169 were used as a positive control for CMV-infected cells. Peripheral blood samples were obtained from five allogeneic bone marrow transplant (allo-BMT) recipients and from 10 healthy donors (eight were sero-positive for CMV and two were sero-negative). Each blood sample was collected into an evacuated tube containing EDTA-2K.Cell fixation. Red blood cells in 1 ml of whole blood were haemolysed for 8 min at room temperature in 9 ml of lysisreagent (0 . 83% NH 4 Cl), and the remaining cells were washed with phosphate-buffered saline (PBS buffer; 2 . 7 mM KCl, 1 . 2 mM KH 2 PO 4 , 138 mM NaCl, 8 . 1 mM NaH 2 PO 4 , 1% fetal calf serum, pH 7 . 3). The cells in the pellet were resuspended and fixed in 4% paraformaldehyde for 10 min at 4ЊC. The suspension was centrifuged for 8 min at 200 g. Then the pellet was resuspended in surfactant (0 . 3% NP-40) for after-fixation of the cells and incubated at room temperature for 5 min. The suspension was centrifuged for 10 min at 200 g...
In the present study, serologic data were compared with data obtained by capillary PCR to establish the efficacy of capillary PCR for the determination of Mycoplasma infection in samples obtained from throat swabs, bronchoalveolar lavage fluids (BALF), and sputum of patients with Mycoplasma pneumonia. We performed PCR analysis for Mycoplasma DNA on a total of 325 samples from 197 patients with community-acquired pneumonia and in whom Mycoplasma pneumonia was suspected. There were 68 PCR-positive specimens. Review of the differences in PCR positivity rates based on the site of specimen collection showed the highest rate of detection (28.6%) from throat swabs. From among the 31 patients with significantly elevated titers of serum Mycoplasmaantibodies, the PCR results were positive for 25 patients. Thus, capillary PCR had a sensitivity of 80.6% (25 of 31). Five of the six false-negative results were from throat swab specimens. Moreover, testing (PCR) had been performed only once for these five patients with false-negative results. From among the PCR-positive findings from BALF specimens, there were no false-positive results. BALF specimens were very useful, except for the technical procedures and increased patient burden required to obtain these specimens. We suggest that the use of throat swab specimens in capillary PCR is much more suitable for diagnosing Mycoplasma pneumonia in routine clinical practice; however, careful throat swab specimen collection and an increase in the number of times that the PCR is performed are necessary to reduce the rate of false-negative results.
Summary.We used flow cytometry to identify the presence of intracellular cytokines (cytoflow) and analyse the production of IL-4 in peripheral blood from AIDS patients who have practically no CD4 þ T cells. We found that IL-4 was produced by CD3 ¹ CD4 ¹ CD8 ¹ CD56 ¹ CD19 ¹ CD14 ¹ cells and CD3 þ CD8 dull-positive cells in AIDS patients. Moreover, CD3 ¹ CD4 ¹ CD8 ¹ CD56 ¹ CD19 ¹ CD14 ¹ cells had helper activity for immunoglobulin synthesis. These findings indicate that instead of CD4 þ T helper cells, C3 ¹ CD4 ¹ CD8 ¹ CD56 ¹ CD19 ¹ CD14 ¹ cells and CD3 þ CD8 dull-positive cells may be an important source of IL-4 in a variety of immune responses for AIDS patients.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.