Declining levels of rescued lymphoproliferative response to human cytomegalovirus (HCMV) in AIDS patients with or without HCMV disease following long-term HAART.

OBJECTIVE: To investigate the lymphoproliferative response (LPR) to human cytomegalovirus (HCMV) in two groups of AIDS patients undergoing long-term highly active antiretroviral therapy (HAART): group 1 ( n = 22) with nadir CD4(+) cell count <50/microl and no HCMV disease; group 2 ( n = 16) with <50/microl CD4(+) T-cell count and HCMV disease. All patients had previously undergone antiretroviral monotherapy or dual therapy before initiating HAART. STUDY DESIGN AND METHODS: The two groups of patients were tested prospectively for CD4(+) T cell count, HIV RNA load, HCMV viremia, and LPR to HCMV at baseline, and then after 3 and 4 years of HAART. A control group of 13 recently diagnosed treatment-naive AIDS patients with CD4(+) T-cell counts <100/microl was also investigated. RESULTS: No LPR to HCMV was found in any of the treatment-naive patients nor in any patient of the two groups examined at baseline, when HCMV viremia was 13.6% in the patient group without disease and 87.5% in the group with disease ( p <.0001). After 3 years of HAART, the frequency of patients who recovered an LPR to HCMV was not significantly different (81.8% in the group without HCMV disease, and 68.7% in the group with HCMV disease), whereas, compared with baseline, the HIV load decreased and the CD4(+) T-cell count increased significantly and to a comparable extent in the two groups of patients. In addition, the frequency of patients with HCMV viremia, although reduced, became comparable in both groups. After 4 years of HAART, the frequency of responders to HCMV without and with HCMV disease dropped to comparable levels (50.0 vs. 56.3%, respectively) in association with high median CD4(+) T-cell counts and low median HIV RNA plasma levels. In parallel, the frequency of patients with HCMV viremia did not change significantly. In addition, after between 3 and 4 years of HAART, although the frequency of stable responders and nonresponders remained unchanged (50%) in both groups, most of the remaining patients showed declining levels of responsiveness to HCMV. Although some patients from both groups were found to have CD4(+) T-cell counts >150/microl in the absence of LPR to HCMV, thus suggesting dissociation of specific and nonspecific immune reconstitution, a significant correlation was found between CD4(+) T-cell count and LPR to HCMV (r = 0.44; p <.001). From a clinical standpoint, anti-HCMV therapy could be safely discontinued in 8 patients with HCMV retinitis showing CD4(+) T-cell counts >150/microl, recovery of HCMV LPR, and no HCMV viremia. CONCLUSIONS: Declining levels of the previously recovered LPR to HCMV are often observed after long-term HAART. However, because the role of LPR in the evolution of HCMV infection and disease during HAART remains to be defined, the clinical impact of the declining LPR to HCMV must still be clarified in long-term prospective studies.

Comparative analysis of human cytomegalovirus-specific CD4(+) T-cell frequency and lymphoproliferative response in human immunodeficiency virus-positive patients.

Evaluation of human cytomegalovirus (HCMV)-specific T-helper immunity could contribute in optimizing anti-HCMV therapy in human immunodeficiency virus (HIV)-infected patients. Testin the lymphoproliferative response (LPR) is the standard technique used to evaluate T-helper response, but its use in the routine diagnostic laboratory setting can be problematic. The most promising new alternative technique is the determination of HCMV-specific CD4(+) T-cell frequency by flow cytometry detection of intracellular cytokine production after short-term antigen-specific activation of peripheral blood mononuclear cells. HCMV-specific LPR and CD4(+) T-cell frequency were compared in a group of 78 blood samples from 65 HIV-infected patients. The results showed concordance in 80.7% of samples. In addition, comparative analysis of sequential blood samples from 13 HIV-infected patients showed that while in about half of patients the T-helper HCMV-specific immune response remained stable during highly active antiretroviral therapy (HAART), in the other half declining levels of the HCMV-specific CD4(+)-mediated immune response were determined by either one or both assays. In conclusion, our data suggest that the determination of HCMV-specific CD4(+) T-cell frequency can be considered a valuable alternative to the LPR test for the detection of HCMV-specific T-helper response in HIV-infected patients. It could facilitate wider screening of anti-HCMV T-helper activity in HIV-infected patients, with potential benefits for clinicians in deciding strategies of discontinuation or maintenance of anti-HCMV therapy.

Human cytomegalovirus replicates abortively in polymorphonuclear leukocytes after transfer from infected endothelial cells via transient microfusion events.

Using a recently developed model for in vitro generation of pp65-positive polymorphonuclear leukocytes (PMNLs), we demonstrated that PMNLs from immunocompetent subjects may harbor both infectious human cytomegalovirus (HCMV) and viral products (pp65, p72, DNA, and immediate-early [IE] and pp67 late mRNAs) as early as 60 min after coculture with human umbilical vein endothelial cells (HUVEC) or human embryonic lung fibroblasts (HELF) infected with a clinical HCMV isolate (VR6110) or other wild-type strains. The number of PMNLs positive for each viral parameter increased with coculture time. Using HELF infected with laboratory-adapted HCMV strains, only very small amounts of viral DNA and IE and late mRNAs were detected in PMNLs. A cellular mRNA, the vascular cell adhesion molecule-1 mRNA, which is abundantly present in both infected and uninfected HUVEC, was detected in much larger amounts in PMNLs cocultured with VR6110-infected cells than in controls. Coculture of PMNLs with VR6110-infected permissive cells in the presence or absence of RNA, protein, and viral DNA synthesis inhibitors showed that only IE genes were transcribed in PMNLs during coculture. Synthesis of IE transcripts in PMNLs was also supported by the finding that only the copy number of IE mRNA (and not the DNA or the pp67 mRNA) per infected PMNL increased markedly with time, and the pp67 to IE mRNA copy number ratio changed from greater than 10 in infected HUVEC to less than 1 in cocultured PMNLs. Fluorescent probe transfer experiments and electron microscopy studies indicated that transfer of infectious virus and viral products from infected cells to PMNLs is likely to be mediated by microfusion events induced by wild-type strains only. In addition, HCMV pp65 and p72 were both shown to localize in the nucleus of the same PMNLs by double immunostaining. Two different mechanisms may explain the virus presence in PMNLs: (i) one major mechanism consists of transitory microfusion events (induced by wild-type strains only) of HUVEC or HELF and PMNLs with transfer of viable virus and biologically active viral material to PMNLs; and (ii) one minor mechanism, i.e., endocytosis, occurs with both wild-type and laboratory strains and leads to the acquisition of very small amounts of viral nucleic acids. In conclusion, HCMV replicates abortively in PMNLs, and wild-type strains and their products (as well as cellular metabolites and fluorescent dyes) are transferred to PMNLs, thus providing evidence for a potential mechanism of HCMV dissemination in vivo.

A complex translocation (5;7) in a patient with acute nonlymphocytic leukemia evolved from a myelodysplastic syndrome.

Complete or partial monosomy for the long arms of chromosomes 5 or 7 or both is frequently observed in therapy-related myelodysplastic syndromes and acute nonlymphocytic leukemia. Sporadic cases have been reported in which partial monosomy is due to unbalanced translocations. The patient described herein carries one such rearrangement. 46,XY,t(1;2) (q32;p23),del(5)(q13),der(7)(5qter-->5q22::7p15-->7 q21:),del(12)(p12), resulting in partial monosomy for the long arms of chromosomes 5 and 7 and in partial monosomy for the short arm of chromosome 7.

Detection and monitoring of trisomy 8 by fluorescence in situ hybridization in acute myeloid leukemia: a multicentric study.

BACKGROUND AND OBJECTIVE: The role of fluorescence in situ hybridization (FISH) in the detection and monitoring of trisomy 8 (+8) in acute myelogenous leukemia (AML) has not been defined exactly. This multicentric study was performed in order to: i) analyze the sensitivity of interphase FISH with respect to conventional chromosome analysis (CCA) in detecting +8; ii) compare the results of FISH and CCA in the quantitation of the frequency of +8-positive cells; iii) analyze the possible role of FISH in the cytogenetic follow-up of patients with +8. DESIGN AND METHODS: One hundred and ninety-eight nonconsecutive patients with a diagnosis of AML seen at five centers over a 3-year period were studied by CCA and FISH with a chromosome 8-specific centromeric probe. Two hundred interphase cells were scored in each test and the cut-off for the recognition of +8 was set at 3%. An irrelevant pericentromeric probe was used as negative control in those cases with an apparently normal karyotype and trisomy 8 in interphase cells. FISH studies were conducted at diagnosis and, in 14 cases with +8, on 1.5 occasions during follow-up. RESULTS: Karyotype aberrations were seen in 121 cases (61.1%), with +8 being present in 38 of them (16 as the sole aberration). Interphase FISH detected +8 in 37/38 cases; in a patient with 1/10 metaphases with +8, 2.3% interphase cells with 3 signals were seen. Fourteen additional cases with occult +8 were detected by FISH, which showed 4-22% interphase cells with three signals; 6 patients had an abnormal karyotype without +8, 3 had a normal karyotype, 5 had no analyzable mitoses. In 24 cases with > 15 analyzable metaphases, percent variations between CCA and FISH in the estimation of the size of the trisomic clone ranged between 0.4% and 51%, median value 22%. Underestimation of the percent of trisomy 8 by FISH occurred in all 10 cases with > 90% +8 metaphases. In 7/14 cases investigated sequentially, FISH detected 5-35% trisomic cells in the BM after induction therapy (4 CR, 3 PR); 4 cases relapsed with +8 at 8-15 months. The absence of +8 in remission marrows was documented in the remaining 7 cases, 4 of which relapsed at 20-32 months. INTERPRETATION AND CONCLUSIONS: It is concluded that FISH was a valuable method in this multicentric study since it showed greater sensitivity than CCA in detecting minor clones with +8, in patients with both normal and abnormal karyotypes. The role of FISH in the cytogenetic follow-up of trisomies in AML patients may be promising.