RESUMO
Whether cancer is maintained by a small number of stem cells or is composed of proliferating cells with approximate phenotypic equivalency is a central question in cancer biology. In the stem cell hypothesis, relapse after treatment may occur by failure to eradicate cancer stem cells. Chronic myeloid leukaemia (CML) is quintessential to this hypothesis. CML is a myeloproliferative disorder that results from dysregulated tyrosine kinase activity of the fusion oncoprotein BCR-ABL. During the chronic phase, this sole genetic abnormality (chromosomal translocation Ph(+): t(9;22)(q34;q11)) at the stem cell level causes increased proliferation of myeloid cells without loss of their capacity to differentiate. Without treatment, most patients progress to the blast phase when additional oncogenic mutations result in a fatal acute leukaemia made of proliferating immature cells. Imatinib mesylate and other tyrosine kinase inhibitors (TKIs) that target the kinase activity of BCR-ABL have improved patient survival markedly. However, fewer than 10% of patients reach the stage of complete molecular response (CMR), defined as the point when BCR-ABL transcripts become undetectable in blood cells. Failure to reach CMR results from the inability of TKIs to eradicate quiescent CML leukaemia stem cells (LSCs). Here we show that the residual CML LSC pool can be gradually purged by the glitazones, antidiabetic drugs that are agonists of peroxisome proliferator-activated receptor-γ (PPARγ). We found that activation of PPARγ by the glitazones decreases expression of STAT5 and its downstream targets HIF2α and CITED2, which are key guardians of the quiescence and stemness of CML LSCs. When pioglitazone was given temporarily to three CML patients in chronic residual disease in spite of continuous treatment with imatinib, all of them achieved sustained CMR, up to 4.7 years after withdrawal of pioglitazone. This suggests that clinically relevant cancer eradication may become a generally attainable goal by combination therapy that erodes the cancer stem cell pool.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzamidas/administração & dosagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , PPAR gama/agonistas , Piperazinas/administração & dosagem , Pirimidinas/administração & dosagem , Tiazolidinedionas/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , PPAR gama/metabolismo , Pioglitazona , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT5/metabolismo , Tiazolidinedionas/farmacologia , Tiazolidinedionas/uso terapêutico , Transativadores/metabolismoRESUMO
BACKGROUND: We recently reported that peroxisome proliferator-activated receptor γ agonists target chronic myeloid leukemia (CML) quiescent stem cells in vitro by decreasing transcription of STAT5. Here in the ACTIM phase 2 clinical trial, we asked whether pioglitazone add-on therapy to imatinib would impact CML residual disease, as assessed by BCR-ABL1 transcript quantification. METHODS: CML patients were eligible if treated with imatinib for at least 2 years at a stable daily dose, having yielded major molecular response (MMR) but not having achieved molecular response 4.5 (MR4.5 ) defined by BCR-ABL1/ABL1IS RNA levels ≤ 0.0032%. After inclusion, patients started pioglitazone at a dosage of 30 to 45 mg/day in addition to imatinib. The primary objective was to evaluate the cumulative incidence of patients having progressed from MMR to MR4.5 over 12 months. RESULTS: Twenty-four patients were included (age range, 24-79 years). No pharmacological interaction was observed between the drugs. The main adverse events were weight gain in 12 patients and a mean decrease of 0.4 g/dL in hemoglobin concentration. The cumulative incidence of MR4.5 was 56% (95% confidence interval, 37%-76%) by 12 months, despite a wide range of therapy duration (1.9-15.5 months), and 88% of 17 evaluable patients who were still on imatinib reached MR4.5 by 48 months. The cumulative incidence of MMR to MR4.5 spontaneous conversions over 12 months was estimated to be 23% with imatinib alone in a parallel cohort of patients. CONCLUSION: Pioglitazone in combination with imatinib was well tolerated and yielded a favorable 56% rate. These results provide a proof of concept needing confirmation within a randomized clinical trial (EudraCT 2009-011675-79). Cancer 2017;123:1791-1799. © 2016 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
Assuntos
Antineoplásicos/uso terapêutico , Hipoglicemiantes/uso terapêutico , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Tiazolidinedionas/uso terapêutico , Adulto , Idoso , Quimioterapia Combinada , Feminino , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Masculino , Pessoa de Meia-Idade , Pioglitazona , RNA Mensageiro/metabolismo , Adulto JovemRESUMO
In the past decade, the treatment of chronic myeloid leukemia (CML) has undergone a drastic evolution. The discovery and success of imatinib and second-generation tyrosine kinase inhibitors have substantially increased the outcome for CML patients. The next step in medical and scientific research is to better understand the malignancy so as to eventually find a cure to eliminate all leukemic cells from patients. One of the key issues is about the resistance of the leukemic stem cells to tyrosine kinase inhibitors. Here, we briefly describe our current studies on CML resistance, and leukemic stem cell modeling and characterization.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , História do Século XXI , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologiaRESUMO
The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors.
Assuntos
Adipogenia/efeitos dos fármacos , Azepinas/farmacologia , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Histona Acetiltransferases/antagonistas & inibidores , Lisina/metabolismo , Triazóis/farmacologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Proteínas Cromossômicas não Histona/metabolismo , Regulação para Baixo/efeitos dos fármacos , Histona Acetiltransferases/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Fatores de TranscriçãoRESUMO
Infection of primates by HIV-1 and SIV induces multiple hematological abnormalities of central hematopoietic origin. Although these defects greatly contribute to the pathophysiology of HIV-1 infection, the molecular basis for altered BM function remains unknown. Here we show that when cynomolgus macaques were infected with SIV, the multipotent potential of their hematopoietic progenitor cells was lost, and this correlated with downregulation of STAT5A and STAT5B expression. However, forced expression of STAT5B entirely rescued the multipotent potential of the hematopoietic progenitor cells. In addition, an accessory viral protein required for efficient SIV and HIV replication and pathogenicity, "Negative factor" (Nef), was essential for SIV-mediated impairment of the multipotent potential of hematopoietic progenitors ex vivo and in vivo. This newly uncovered property of Nef was both conserved between HIV-1 and SIV strains and entirely dependent upon the presence of PPARgamma in targeted cells. Further, PPARgamma agonists mimicked Nef activity by inhibiting STAT5A and STAT5B expression and hampering the functionality of hematopoietic progenitors both ex vivo and in vivo. These findings have extended the role of Nef in the pathogenicity of HIV-1 and SIV and reveal a pivotal role for the PPARgamma/STAT5 pathway in the regulation of early hematopoiesis. This study may provide a basis for investigating the potential therapeutic benefits of PPARgamma antagonists in both patients with AIDS and individuals with hematopoietic disorders.
Assuntos
Produtos do Gene nef/metabolismo , HIV-1/metabolismo , Hematopoese/fisiologia , PPAR gama/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/fisiologia , Vírus da Imunodeficiência Símia/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Produtos do Gene nef/genética , HIV-1/genética , Doenças Hematológicas/metabolismo , Doenças Hematológicas/fisiopatologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Células K562 , Macaca fascicularis , Masculino , Dados de Sequência Molecular , PPAR gama/genética , Fator de Transcrição STAT5/genética , Vírus da Imunodeficiência Símia/genéticaRESUMO
Myelofibrosis (MF) is a non-BCR-ABL myeloproliferative neoplasm associated with poor outcomes. Current treatment has little effect on the natural history of the disease. MF results from complex interactions between (a) the malignant clone, (b) an inflammatory context, and (c) remodeling of the bone marrow (BM) microenvironment. Each of these points is a potential target of PPARγ activation. Here, we demonstrated the therapeutic potential of PPARγ agonists in resolving MF in 3 mouse models. We showed that PPARγ agonists reduce myeloproliferation, modulate inflammation, and protect the BM stroma in vitro and ex vivo. Activation of PPARγ constitutes a relevant therapeutic target in MF, and our data support the possibility of using PPARγ agonists in clinical practice.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Proteínas de Neoplasias/agonistas , Neoplasias Experimentais/tratamento farmacológico , PPAR gama/agonistas , Mielofibrose Primária/tratamento farmacológico , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Modelos Animais de Doenças , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , PPAR gama/genética , PPAR gama/metabolismo , Mielofibrose Primária/genética , Mielofibrose Primária/metabolismo , Mielofibrose Primária/patologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genéticaRESUMO
BACKGROUND: Prolonged, altered hematopoietic reconstitution is commonly observed in patients undergoing myeloablative conditioning and bone marrow and/or mobilized peripheral blood-derived stem cell transplantation. We studied the reconstitution of myeloid and lymphoid compartments after the transplantation of autologous CD34+ bone marrow cells following gamma irradiation in cynomolgus macaques. RESULTS: The bone marrow cells were first transduced ex vivo with a lentiviral vector encoding eGFP, with a mean efficiency of 72% +/- 4%. The vector used was derived from the simian immunodeficiency lentivirus SIVmac251, VSV-g pseudotyped and encoded eGFP under the control of the phosphoglycerate kinase promoter. After myeloid differentiation, GFP was detected in colony-forming cells (37% +/- 10%). A previous study showed that transduction rates did not differ significantly between colony-forming cells and immature cells capable of initiating long-term cultures, indicating that progenitor cells and highly immature hematopoietic cells were transduced with similar efficiency. Blood cells producingeGFP were detected as early as three days after transplantation, and eGFP-producing granulocyte and mononuclear cells persisted for more than one year in the periphery. CONCLUSION: The transplantation of CD34+ bone marrow cells had beneficial effects for the ex vivo proliferation and differentiation of hematopoietic progenitors, favoring reconstitution of the T- and B-lymphocyte, thrombocyte and red blood cell compartments.
Assuntos
Células da Medula Óssea/efeitos da radiação , Transplante de Medula Óssea , Raios gama , Células-Tronco Hematopoéticas/metabolismo , Linfócitos/citologia , Macaca fascicularis , Células Mieloides/citologia , Animais , Antígenos CD34/análise , Antígenos CD34/genética , Diferenciação Celular , Macaca , Transplante AutólogoRESUMO
PURPOSE: More than half of patients with chronic-phase chronic myelogenous leukemia (CP-CML) in complete molecular response (CMR) experience molecular relapse after imatinib discontinuation. We investigated loss of major molecular response (MMR) as a criterion for resuming therapy. PATIENTS AND METHODS: A multicenter observational study (A-STIM [According to Stop Imatinib]) evaluating MMR persistence was conducted in 80 patients with CP-CML who had stopped imatinib after prolonged CMR. RESULTS: Median time from imatinib initiation to discontinuation was 79 months (range, 30 to 145 months);median duration of CMR before imatinib discontinuation was 41 months (range, 24 to 96 months); median follow-up after discontinuation was 31 months (range, 8 to 92 months). Twenty-nine patients (36%) lost MMR after a median of 4 months off therapy (range, 2 to 17 months). Cumulative incidence of MMR loss was estimated as 35% (95% CI, 25% to 46%) at 12 months and 36% (95% CI, 26% to 47%) at 24 months, whereas probability of losing CMR was higher. Fluctuation of BCR-ABL transcript levels below the MMR threshold (≥ two consecutive positive values) was observed in 31% of patients after imatinib discontinuation. Treatment-free remission was estimated as 64% (95% CI, 54% to 75%) at 12 and 24 months and 61% (95% CI, 51% to 73%) at 36 months. Median to time to second CMR was estimated as 7.3 months in re-treated patients. CONCLUSION: Loss of MMR is a practical and safe criterion for restarting therapy in patients with CML with prolonged CMR.
Assuntos
Antineoplásicos/administração & dosagem , Benzamidas/administração & dosagem , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/efeitos adversos , Benzamidas/efeitos adversos , Intervalo Livre de Doença , Esquema de Medicação , Feminino , França , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Estimativa de Kaplan-Meier , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Piperazinas/efeitos adversos , Inibidores de Proteínas Quinases/efeitos adversos , Pirimidinas/efeitos adversos , Recidiva , Indução de Remissão , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Adulto JovemAssuntos
Antígenos CD34/imunologia , Produtos do Gene nef/fisiologia , PPAR gama/fisiologia , Síndrome de Imunodeficiência Adquirida dos Símios/fisiopatologia , Animais , Regulação da Expressão Gênica , Macaca , Fator de Transcrição STAT5/genética , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologiaRESUMO
Although clinical benefits have been reported in several human hematopoietic gene therapy trials, a remaining important goal is the transition to nonmyeloablative pretransplantation conditioning to decrease toxicity. Previous attempts at reduced intensity conditioning in nonhuman primates have resulted in only temporary vector marking of autologous blood cells or their persistence at low levels, well below the thresholds for clinical efficacy. In addition, we reasoned that lentiviral vector particles displaying cytokines at their surface have the potential to preserve stem cell fitness better than current ex vivo transduction protocols, which involve exposure to cytokine overstimulation. Here we show that the classically nonmyeloablative agent fludarabine (30 mg/m(2)/day for 3 days) together with low-level total body irradiation (2 Gy) and the use of a stem cell factor-displaying simian immunodeficiency virus-based vector, resulted in sustained, single-copy vector marking of autologous blood cells in two macaques over 3 years posttransplantation at levels averaging 1% of all lineages. This percentage is within the range of anticipated efficacy levels for hemophilia and related diseases and forms a basis for further improvement.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Vírus da Imunodeficiência Símia/genética , Fator de Células-Tronco/genética , Condicionamento Pré-Transplante , Animais , Antígenos CD34/metabolismo , Quimerismo , Terapia Genética , Vetores Genéticos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HEK293 , Hemofilia A/terapia , Humanos , Contagem de Linfócitos , Macaca fascicularis , Masculino , Agonistas Mieloablativos/administração & dosagem , Radiossensibilizantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transdução Genética , Transplante Autólogo , Vidarabina/administração & dosagem , Vidarabina/análogos & derivados , Irradiação Corporal TotalAssuntos
Antineoplásicos/administração & dosagem , Benzamidas/administração & dosagem , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/administração & dosagem , Feminino , Humanos , MasculinoRESUMO
Experimental infection of macaques with pathogenic strains of simian immunodeficiency virus (SIV) represents one of the most relevant animal models for studying HIV pathogenesis. In this study, we demonstrated a significant decrease in the generation of CD4+ T cells from bone marrow (BM) CD34+ progenitors in macaques infected with SIVmac251. This decrease appears to result from changes in the clonogenic potential of BM progenitors of both the myeloid and lymphoid lineages. We also demonstrated a significant decrease in the numbers of the most immature long-term culture-initiating cells (LTC-ICs). Hematopoietic failure occurred as early as primary infection, in the absence of CD34+ BM cell infection and was not related to plasma viral load. No major change was observed in the phenotype of BM CD34+ cells from infected macaques, including apoptosis markers such as annexin V staining and BcL-2 expression, but a significantly higher that normal proportion of cells were in the G0/G1 phase. This is the first demonstration that failure of BM hematopoiesis results in impaired T-cell production, which may contribute to the disruption of T-lymphocyte homeostasis characteristic of pathogenic lentiviral infections in primates.
Assuntos
Linfócitos T CD4-Positivos , Linfopoese , Macaca fascicularis/sangue , Doenças dos Macacos/sangue , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Vírus da Imunodeficiência Símia , Animais , Anexina A5/sangue , Antígenos CD34 , Apoptose , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Fase G1 , Regulação da Expressão Gênica , Genes bcl-2 , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/virologia , Homeostase , Macaca fascicularis/virologia , Masculino , Doenças dos Macacos/patologia , Doenças dos Macacos/virologia , Mielopoese , Fase de Repouso do Ciclo Celular , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Carga ViralRESUMO
A novel membrane protein has been identified in the course of screening for differentially expressed cDNAs in human embryonic hematopoietic sites. This 37- to 38-kDa molecule, designated KLIP-1 (killer lineage protein), consisting of 350 amino acids and containing five transmembrane domains, is encoded by the 5093-bp KLIP-1 gene, composed of nine exons and located on chromosome 6 (6p21.1-6p21.2). We found the KLIP-1 protein to be expressed by nucleated hematopoietic cells, from early embryonic hematopoietic stem cells through mature adult blood lymphoid lineages, either as membrane or as cytoplasmic molecules. In day-30/32 human embryo sections, KLIP-1 protein expression is restricted to circulating hematopoietic cells at hematopoiesis sites. Membrane KLIP-1 is expressed by fetal and adult GP-A(+) erythroblasts, the fetal liver CD34(+) subset, fetal spleen, and adult bone marrow CD56(+) NK and CD19(+) B cells. Among mature blood cells, surface KLIP-1 expression is restricted to CD56(+) NK cells, indicating KLIP-1 to be a novel marker of this population. Altogether, these results indicate that membrane export of KLIP-1 antigen is developmentally and ontogenetically regulated. The high degree of conservation of the KLIP-1 protein sequence among mammals strongly suggests that it plays an important role during hematopoiesis and may exercise similar functions in human and mouse blood cells. The KLIP-1 molecule may therefore constitute a powerful tool for improving knowledge of both human hematopoiesis and NK cell ontogeny and immune functions.
Assuntos
Linhagem da Célula , Embrião de Mamíferos/citologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Proteínas de Membrana/fisiologia , Adulto , Sequência de Aminoácidos , Animais , Antígenos CD34 , Biomarcadores , Mapeamento Cromossômico , Embrião de Mamíferos/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Distribuição TecidualRESUMO
Recent efforts to design an human immunodeficiency virus type 1 (HIV-1) vaccine candidate have focused on means of eliciting anti-viral T-cell responses. We tried to improve the immunogenicity of DNA vaccines by designing hybrid DNA constructs encoding hepatitis B surface antigen (HBsAg) fused to antigenic domains of simian/human immunodeficiency virus (SHIV 89.6P). Immunisation with hybrid DNA induced both effector and long-lasting precursor T-cells. Following boosting with a recombinant modified vaccinia Ankara (rMVA) producing full-length SIV and HIV antigens, it appeared that priming with hybrid DNA had increased virus-specific T-cell responses in terms of both the number of virus-specific IFN-gamma-secreting T-cells and virus-specific lymphoproliferation. After intrarectal challenge with SHIV 89.6P, immunised animals demonstrated early control of SHIV 89.6P replication and stable CD4+ T-cell counts.