Previous studies have demonstrated that the expression of CD25 can distinguish CD25 ؊ latently infected cells from CD25 ؉ cells actively producing virus. Our studies were designed to characterize the nature and stability of the viral genome in CD25 ؊ quiescent HIV-1-infected cells and to determine whether these cells could be infected Using a model of acute in vitro infection, we have previously demonstrated that the expression of CD25 can distinguish latently infected cells (CD25 Ϫ ) from cells actively producing virus (CD25 ϩ ) (1) and that these two populations display significant differences with regard to the nature of the proviral genome. Thus, by combining the use of a potent anti-CD25 immunotoxin and sensitive immunofluorescence staining we have obtained highly purified resting cells. This is important because interpretations of viral latency have been confounded, in part, by the degree of purity of the resting cells. Indeed, partially reverse-transcribed DNAs (2, 3), full-length extrachromosomal DNA (4, 5), and stable integrated viral genomes (6) have been reported to occur in resting peripheral blood mononuclear cells (PBMCs). Although these differences can be explained in part by the different methodologies employed, they could also suggest the existence of several subsets of latently infected cells and possibly the contamination of resting cells with activated ones.Using our in vitro model of acute HIV-1 infection, the present studies were designed to characterize the nature and stability of the viral genome in quiescent HIV-1-infected CD25 Ϫ cells and to determine whether CD25 Ϫ cells can be infected de novo. The results of these analyses have implications regarding the origin of latently infected cells. MATERIALS AND METHODSVirus Strain and Standard Inoculum. HIV-1 JR-CSF is a clinical strain provided by the National Institutes of Health AIDS research and reference reagent program (7). The virus was cultured with phytohemagglutinin (PHA)-activated PBMCs for 6 days. The culture supernatant was purified on an Amicon concentrator (Centriprep 100) by centrifugation at 500 ϫ g at 4ЊC for 1 hr to remove cytokines and mitogens (M r Ͻ100 kD). This standard inoculum was adjusted to 10 times its original concentration, aliquoted, and stored at Ϫ80ЊC.Purification of CD4 ؉ CD25 ؊ Human Quiescent T Lymphocytes. Fresh PBMCs were obtained from normal healthy donors by centrifugation over Ficoll͞Hypaque and were cultured in RPMI 1640 supplemented with 5% autologous plasma [complete-medium (CM)] for 18 hr to remove peripheral blood macrophages by plastic adhesion. Adherent cells were used for additional experiments. The nonadherent cells were further cultured under the same conditions for 2 hr, and were treated with 5 mM leucine methyl ester in RPMI 1640 for 1 hr at 25ЊC to further deplete the macrophages and natural killer cells (8). CD4 ϩ T cells were purified by positive selection on anti-CD4-coated magnetic beads (Dynal, Great Neck, NY) according to the instructions of the manufacturer. By trypan blue...

The performance of a nested PCR-based assay (the RAPID BAP-MTB; AsiaGen, Taichung, Taiwan) and the BD ProbeTec ET (DTB) system (Becton Dickinson, Sparks, Md.) for detection of Mycobacterium tuberculosis was evaluated with 600 consecutive clinical samples. These samples, including 552 respiratory specimens and 48 nonrespiratory specimens, were collected from 333 patients treated at National Taiwan University Hospital from September to October 2003. The results of both assays were compared to the gold standard of combined culture results and clinical diagnosis. The overall sensitivity and specificity of the RAPID BAP-MTB assay for respiratory specimens were 66.7% and 97.2%, respectively, and for the DTB assay they were 56.7% and 95.3%, respectively. The positive and negative predictive values for the RAPID BAP-MTB were 74.1% and 96.0%, respectively, and for the DTB assay they were 59.6% and 94.7%, respectively. For smear-negative samples, the sensitivity of the RAPID BAP-MTB and DTB assays was 57.1% and 40.5%, respectively. The RAPID BAP-MTB assay produced 14 false-positive results in 14 samples, including one of the six samples yielding Mycobacterium abscessus, one of the six samples yielding Mycobacterium avium intracellulare, one sample from a patient with a history of pulmonary tuberculosis with complete treatment, and three samples from three patients with a previous diagnosis of tuberculosis who were under treatment at the time of specimen collection. Among the 48 nonrespiratory specimens, the RAPID BAP-MTB assay was positive in one biopsy sample from a patient with lumbar tuberculous spondylitis and one pus sample from a patient with tuberculous cervical lymphadenopathy. Our results showed that the RAPID BAP-MTB assay is better than the DTB assay for both respiratory specimens and nonrespiratory specimens. The overall time for processing this assay is only 5 h. In addition, its diagnostic accuracy in smear-negative samples is as high as in smear-positive samples.

Despite the success of highly active antiretroviral therapy (HAART) in lowering circulating HIV-1 to undetectable levels in most infected individuals, several studies have documented the presence of a small reservoir of latently infected cells in HAART patients, the majority of which are CD45RO؉ memory T cells. We previously have demonstrated that latently infected, replication-competent cells can be generated in vitro after eliminating CD25؉ cells with an immunotoxin (IT). The present study was designed to determine whether these latent cells could be eliminated by an anti-CD45RO IT. Our results indicate that the anti-CD45RO IT eliminates >99%, of either M-tropic or T-tropic virus produced by the latently infected cells after mitogen stimulation. This IT also appears to be as effective as the anti-CD25 IT in eliminating the activated, HIV-1-producing cells. In contrast, the anti-CD45RO IT does not kill CD45RA ؉ naive cells. Further studies using cells from HIV-1-infected individuals on HAART will be necessary to determine the potential clinical utility of this IT.

Herpesvirus saimiri (H. saimiri) is a highly oncogenic lymphotropic herpesvirus which can immortalize T lymphocytes and cause tumors in rabbits and New World monkeys. T cells infected with strain 484-77 of group C express four viral U-like small RNAs (HSUR1-4) and a 1.2-kb mRNA which encodes open reading frames ORF-1 and ORF-2. ORF-1 encodes a collagen-like oncoprotein. Deletion mutation analysis showed that ORF-1 and ORF-2 are essential for IL-2 independent growth of human T cells infected with H. saimiri. An earlier study also demonstrated that H. saimiri-immortalized cells carry functional IL-2 receptors. The work presented in this report investigated whether IL-2 and IL-4 is produced by H. saimiri-immortalized T lymphocytes. Both IL-2 mRNA and IL-4 mRNA were detected in various monkey T cells as well as human peripheral blood lymphocytes infected with wild-type H. saimiri. Secretion of IL-2 was suggested by cyclosporin A inhibition. IL-4 secretion by monkey T cell cultures was demonstrated by a bioassay and inhibition of bioactivity by an antibody to IL-4. The data also show that recombinant IL-4 stimulate H. saimiri-immortalized T cells; thus, IL-4 receptors are expressed. However, antibodies to human IL-4, IL-4 receptor, or soluble IL-4 receptor did not curtail growth of transformed cells. T cells infected with ORF-1 and ORF-2 deletion mutants expressed no detectable IL-2 mRNA. ORF-1, ORF-2, HSUR1, and HSUR2, were all essential for expression of IL-4 mRNA. These data are consistent with the hypothesis that H. saimiri-immortalized monkey and human T lymphocytes proliferate through autocrine secretion of IL-2 and that ORF-1, ORF-2, and HSUR sequences of the virus are involved in expression of lymphokines.