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Recent attention has highlighted the importance of oral microbiota in human health and disease, e.g., in Parkinson's disease, notably using shotgun metagenomics. One key aspect for efficient shotgun metagenomic analysis relies on optimal microbial sampling and DNA extraction, generally implementing commercial solutions developed to improve sample collection and preservation, and provide high DNA quality and quantity for downstream analysis. As metagenomic studies are today performed on a large number of samples, the next evolution to increase study throughput is with DNA extraction automation. In this study, we proposed a semi-automated DNA extraction protocol for human salivary samples collected with a commercial kit, and compared the outcomes with the DNA extraction recommended by the manufacturer. While similar DNA yields were observed between the protocols, our semi-automated DNA protocol generated significantly higher DNA fragment sizes. Moreover, we showed that the oral microbiome composition was equivalent between DNA extraction methods, even at the species level. This study demonstrates that our semi-automated protocol is suitable for shotgun metagenomic analysis, while allowing for improved sample treatment logistics with reduced technical variability and without compromising the structure of the oral microbiome.
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ADN , Microbiota , Humanos , Análisis de Secuencia de ADN/métodos , ARN Ribosómico 16S/genética , ADN/genética , ADN/química , Microbiota/genética , MetagenomaRESUMEN
BACKGROUND: Emerging evidence supports the role of cell-derived microparticles (MPs) in the pathophysiology of acute coronary syndrome (ACS). OBJECTIVES: To explore the relationship between coronary and systemic MP levels, investigate the correlation between MPs, inflammatory markers and Troponin T in patients with ACS. METHODS: Thirty seven patients with ACS scheduled for percutaneous coronary interventions (PCI) were studied. Eleven patients with stable angina (SA) were included as a control group. AnnexinV+MPs (AnV+MPs) and activated platelet-monocyte aggregates (PMA) from right atrium (RA) and culprit coronary artery (CO) distal to culprit lesion were measured using flow cytometry. High sensitivity C-reactive protein (CRP), Interleukin - 6 (IL-6), tumour necrosis factor - α (TNF-α), serum amyloid A (SAA) and Troponin T were assayed. RESULTS: Total and cell specific AnV+MP expression were higher in the ACS group in both the CO and RA, with greater levels detected in the CO. Platelet activation showed positive correlation with Troponin-T and platelet MP in both CO and RA of the ACS group (r=0.4 for both; p=0.04 & p=0.03 respectively). Inflammatory markers levels did not differ between the ACS and SA patients. CONCLUSIONS: Elevated coronary and systemic MP levels and positive correlation of platelet activation with Troponin-T and platelet MPs suggest a pathogenic role for MPs in ACS.
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Síndrome Coronario Agudo/sangre , Micropartículas Derivadas de Células/metabolismo , Inflamación/metabolismo , Activación Plaquetaria/fisiología , Síndrome Coronario Agudo/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
UNLABELLED: Human Natural Killer (NK) cells require at least two signals to trigger tumor cell lysis. Absence of ligands providing either signal 1 or 2 provides NK resistance. We manufactured a lysate of a tumour cell line which provides signal 1 to resting NK cells without signal 2. The tumor-primed NK cells (TpNK) lyse NK resistant Acute Myeloid Leukemia (AML) blasts expressing signal 2 ligands. We conducted a clinical trial to determine the toxicity of TpNK cell infusions from haploidentical donors. 15 patients with high risk AML were screened, 13 enrolled and 7 patients treated. The remaining 6 either failed to respond to re-induction chemotherapy or the donor refused to undergo peripheral blood apheresis. The conditioning consisted of fludarabine and total body irradiation. This was the first UK trial of a cell therapy regulated as a medicine. The complexity of Good Clinical Practice compliance was underestimated and led to failures requiring retrospective independent data review. The lessons learned are an important aspect of this report. There was no evidence of infusional toxicity. Profound myelosuppression was seen in the majority (median neutrophil recovery day 55). At six months follow-up, three patients treated in Complete Remission (CR) remained in remission, one patient infused in Partial Remission had achieved CR1, two had relapsed and one had died. One year post-treatment one patient remained in CR. Four patients remained in CR after treatment for longer than their most recent previous CR. During the 2 year follow-up six of seven patients died; median overall survival was 400 days post infusion (range 141910). This is the first clinical trial of an NK therapy in the absence of IL-2 or other cytokine support. The HLA-mismatched NK cells survived and expanded in vivo without on-going host immunosuppression and appeared to exert an anti-leukemia effect in 4/7 patients treated. TRIAL REGISTRATION: ISRCTN trial registry ISRCTN11950134.
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Células Asesinas Naturales/inmunología , Leucemia Mieloide Aguda/terapia , Adulto , Anciano , Trasplante de Células/efectos adversos , Femenino , Humanos , Leucemia Mieloide Aguda/inmunología , Masculino , Persona de Mediana Edad , Monitoreo Fisiológico , Acondicionamiento Pretrasplante , Trasplante Homólogo , Reino Unido , Adulto JovenRESUMEN
Decellularized (acellular) scaffolds, composed of natural extracellular matrix, form the basis of an emerging generation of tissue-engineered organ and tissue replacements capable of transforming healthcare. Prime requirements for allogeneic, or xenogeneic, decellularized scaffolds are biocompatibility and absence of rejection. The humoral immune response to decellularized scaffolds has been well documented, but there is a lack of data on the cell-mediated immune response toward them in vitro and in vivo. Skeletal muscle scaffolds were decellularized, characterized in vitro, and xenotransplanted. The cellular immune response toward scaffolds was evaluated by immunohistochemistry and quantified stereologically. T-cell proliferation and cytokines, as assessed by flow cytometry using carboxy-fluorescein diacetate succinimidyl ester dye and cytometric bead array, formed an in vitro surrogate marker and correlate of the in vivo host immune response toward the scaffold. Decellularized scaffolds were free of major histocompatibility complex class I and II antigens and were found to exert anti-inflammatory and immunosuppressive effects, as evidenced by delayed biodegradation time in vivo; reduced sensitized T-cell proliferative activity in vitro; reduced IL-2, IFN-γ, and raised IL-10 levels in cell-culture supernatants; polarization of the macrophage response in vivo toward an M2 phenotype; and improved survival of donor-derived xenogeneic cells at 2 and 4 wk in vivo. Decellularized scaffolds polarize host responses away from a classical TH1-proinflammatory profile and appear to down-regulate T-cell xeno responses and TH1 effector function by inducing a state of peripheral T-cell hyporesponsiveness. These results have substantial implications for the future clinical application of tissue-engineered therapies.
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Músculo Esquelético/inmunología , Andamios del Tejido , Trasplante Heterólogo , Animales , Proliferación Celular , Citocinas/inmunología , Regulación hacia Abajo , Matriz Extracelular , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Macrófagos/inmunología , Músculo Esquelético/citología , ConejosRESUMEN
Huntington's disease (HD) is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin (HTT) gene. Both central and peripheral innate immune activation have been described as features of the disease. Isolated human HD monocytes have been shown to produce more cytokines upon LPS stimulation compared to control monocytes. Understanding alterations in the signalling cascades responsible and activated by this increase in pro-inflammatory cytokine production is crucial in understanding the molecular basis of this phenomenon. Here we investigated the signalling cascade most commonly activated by pro-inflammatory cytokines such as IL-6 - the JAK/STAT signalling cascade. Using flow cytometry, we show that one out of three key transcription factors activated by JAK/STAT signalling is altered in primary human HD innate immune cells, suggesting that this pathway may only play a minor, additive role in the immune cell dysfunction in HD.
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BACKGROUND: Platelet-monocyte complex (PMC) formation is a marker of in vivo platelet activation and may be readily measured by flow cytometry. Due to the high frequency of free platelets relative to monocytes and PMCs, false-positive identification through coincidence remains a significant technical problem.To overcome this problem, we evaluated the use of a doublet-discriminator strategy (DDM) to allow faster sample acquisition whilst significantly reducing aberrant coincidence. METHODS: Fourteen healthy volunteers and 20 patients with coronary artery disease (CAD) gave arterial and/or peripheral venous blood samples (NaCit). Whole blood was labelled in duplicate with anti-CD61 and anti-CD14 using a standard lyse/wash protocol. One of each paired sample was serially diluted before analysis; the second was analyzed at full concentration but using FL1-width to exclude co-incident platelet and monocyte events. Control experiments were performed with ex vivo thrombin activated samples. RESULTS: With the DDM use PMC frequencies in the peripheral blood of healthy individuals and in CAD patients fell significantly [6.27% ± 1.77 (mean ± sd) to 2.57% ± 0.99 (P = 0.02)] and from 16.04% (± 11.26) to 7.66% (± 5.18) (P < 0.01), respectively. DDM use significantly reduced the percentage of PMCs in the ex vivo thrombin activated samples (P < 0.05). CONCLUSIONS: Use of DDM effectively reduces the coincidence and enumerates true PMC in the samples of normal individuals and in patients with CAD and in ex vivo thrombin activated samples.
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Plaquetas/citología , Citometría de Flujo/métodos , Monocitos/citología , Adulto , Anciano , Biomarcadores/sangre , Recuento de Células/métodos , Enfermedad de la Arteria Coronaria/sangre , Análisis Discriminante , Femenino , Humanos , Integrina beta3/análisis , Receptores de Lipopolisacáridos/análisis , Masculino , Persona de Mediana Edad , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/fisiología , Reproducibilidad de los Resultados , Trombina/farmacologíaRESUMEN
Resting human NK cells require a two-stage activation process that we have previously described as "priming" and "triggering." NK-sensitive tumor cells provide both priming and triggering signals. NK-resistant tumors evade lysis, mostly by failure to prime; however, we recently reported a tumor cell line (CTV-1) that primes resting NK cells but fails to trigger lysis. In this article, we report two additional leukemia cell lines that prime NK cells but are resistant to lysis. Tumor-mediated NK priming is via CD2 binding to a ligand within CD15 on the tumor cell. NK-resistant RAJI cells became susceptible to NK lysis following transfection and expression of CD15. Blockade of CD15 on K562 cells or on CD15(+) RAJI cells significantly inhibited lysis, as did blockade of CD2 on resting NK cells. NK priming via CD2 induced CD16 shedding, releasing CD3ζ to the CD2, leading to its phosphorylation and the subsequent phosphorylation of linker for activation of T cells and STAT-5 and synthesis of IFN-γ. Blockade of C-type lectin receptors significantly suppressed the tumor-mediated priming of NK cells, whereas blockade of Ig-superfamily-like receptors had no effect at the NK-priming stage. Tumor priming of resting NK cells was irrespective of HLA expression, and blockade of HLA-killer Ig-like receptor interactions did not influence the incidence or degree of priming. However, CD15-CD2 interactions were critical for NK priming and were required, even in the absence of HLA-mediated NK inhibition. Tumor-mediated priming led to a sustained primed state, and the activated NK cells retained the ability to lyse NK-resistant tumors, even after cryopreservation.
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Antígenos CD2/metabolismo , Citotoxicidad Inmunológica , Fucosiltransferasas/fisiología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Antígeno Lewis X/fisiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Receptores KIR/fisiología , Fase de Descanso del Ciclo Celular/inmunología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Linaje de la Célula/inmunología , Técnicas de Cocultivo , Resistencia a la Enfermedad , Fucosiltransferasas/metabolismo , Humanos , Células Asesinas Naturales/citología , Leucemia Monocítica Aguda/inmunología , Leucemia Monocítica Aguda/metabolismo , Leucemia Monocítica Aguda/patología , Leucemia Mielomonocítica Aguda/inmunología , Leucemia Mielomonocítica Aguda/metabolismo , Leucemia Mielomonocítica Aguda/patología , Antígeno Lewis X/metabolismo , Ligandos , Activación de Linfocitos/inmunología , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/metabolismo , Linfoma no Hodgkin/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Unión Proteica/inmunología , Transducción de Señal/inmunologíaRESUMEN
Natural killer (NK) cells are cytotoxic lymphocytes able to kill tumor cells and virus-infected cells. Human-resting NK cells can be activated by co-culture with NK-resistant CTV-1a cells. These tumor-activated cells (TaNKs) are cytotoxic to a range of NK-resistant tumor cells in vitro. This potential, however, has not been explored in multiple myeloma (MM). In this study, we demonstrate that TaNK cells from 21 MM patients lyse a variety of myeloma targets, including primary isolates of autologous and allogeneic CD138+ myeloma cells whilst sparing CD138-ve bone marrow cells. Myeloma patients' TaNK-induced lysis of the U266 cell line was significantly higher compared to normal controls (median-specific lysis 79.1% vs. 69.5%) (P = 0.003). In addition, TaNKs induced substantial lysis of autologous and allogeneic CD138+ myeloma cells (median-specific lysis 52.5% and 37.4%, respectively). The percentage of specific lysis did not correlate with important disease characteristics (ISS, age, and high-risk molecular abnormalities) or with the disease status and antimyeloma treatment, including novel agents and dexamethasone. In conclusion, tumor-primed NK cells are able to induce substantial lysis of myeloma targets including autologous and allogeneic CD138+ myeloma plasma cells and could be an additional therapeutic approach in MM, particularly in the era of novel agents.
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Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Mieloma Múltiple/patología , Células Plasmáticas/patología , Adulto , Anciano , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Línea Celular Tumoral , Técnicas de Cocultivo , Citotoxicidad Inmunológica/efectos de los fármacos , Dexametasona/farmacología , Dexametasona/uso terapéutico , Progresión de la Enfermedad , Humanos , Inmunofenotipificación , Células Asesinas Naturales/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Persona de Mediana Edad , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/inmunología , Mieloma Múltiple/metabolismo , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Sindecano-1/metabolismo , Células Tumorales CultivadasRESUMEN
We studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 microM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation.
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Antineoplásicos/farmacocinética , Apoptosis/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Sulfonamidas/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Anciano , Anciano de 80 o más Años , Anexina A5/metabolismo , Caspasa 3/metabolismo , Citocromos c/metabolismo , Citosol/metabolismo , Citosol/patología , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Mitocondrias/patología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Linfocitos T/metabolismo , Linfocitos T/patología , Factores de Tiempo , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
NK cells are defined as those cells that lyse tumor cells without priming. In this study, we show that the preincubation of resting human NK cells with the leukemia cell CTV-1 primes NK cells to lyse NK-resistant cell lines, primary leukemias, and solid tumors even when HLA-matched, allogeneic or autologous. The primed NK cells remained nonresponsive to HLA-C matched and mismatched normal mononuclear cells from multiple donors. CD69, a known NK trigger receptor, was shown to be the predominant trigger on the tumor-primed NK cells because lysis was blocked with the rCD69 protein. The lack of lytic activity against normal hemopoietic cells implied that the ligand for CD69 is tumor restricted, and this was confirmed by experiments using fluorochrome labeled rCD69. It has been recently shown that resting NK cells require prior stimulation with IL-2 before triggering by all known NK-triggering ligands. In this study, we show that a tumor cell can provide the NK priming signal independently of IL-2. These data provide evidence for two NK evasion strategies for tumor cells, namely the prevention of priming (type1 evasion) and failure to trigger (type 2 evasion). Most NK-resistant cell lines are type 1 and fail to prime resting NK cells but are lysed by IL-2-primed NK cells. In contrast, CTV-1 cells prime resting NK cells but fail to trigger (type 2), and coincubation with CTV-1 primes for triggering by type 1 NK-resistant tumor cells. These tumor-activated NK cells lyse a broad spectrum of tumor cells with a degree of specificity never previously reported.
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Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Células Asesinas Naturales/inmunología , Leucemia/inmunología , Neoplasias/inmunología , Subgrupos de Linfocitos T/inmunología , Adulto , Anciano , Comunicación Celular , Técnicas de Cocultivo , Reactividad Cruzada , Femenino , Humanos , Células Asesinas Naturales/efectos de los fármacos , Lectinas Tipo C , Ligandos , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/farmacología , Subgrupos de Linfocitos T/efectos de los fármacos , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
It has been previously shown that the subset of human natural killer (NK) cells which express CD8 in a homodimeric alpha/alpha form are more cytotoxic than their CD8- counterparts but the mechanisms behind this differential cytolytic activity remained unknown. Target cell lysis by CD8- NK cells is associated with high levels of effector cell apoptosis, which is in contrast to the significantly lower levels found in the CD8alpha+ cells after lysis of the same targets. We report that cross-linking of the CD8alpha chains on NK cells induces rapid rises in intracellular Ca2+ and increased expression of CD69 at the cell surface by initiating the influx of extracellular Ca2+ ions. We demonstrate that secretion of cytolytic enzymes initiates NK-cell apoptosis from which CD8alpha+ NK cells are protected by an influx of exogenous calcium following ligation of CD8 on the NK-cell surface. This ligation is through interaction with fellow NK cells in the cell conjugate and can occur when the target cells lack major histocompatibility complex (MHC) Class I expression. Protection from apoptosis is blocked by preincubation of the NK cells with anti-MHC Class I antibody. Thus, in contrast to the CD8- subset, CD8alpha+ NK cells are capable of sequential lysis of multiple target cells.
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Apoptosis/inmunología , Antígenos CD8/inmunología , Citotoxicidad Inmunológica/inmunología , Células Asesinas Naturales/inmunología , Antígenos CD8/sangre , Moléculas de Adhesión Celular/sangre , Granzimas , Antígenos HLA/inmunología , Humanos , Inmunofenotipificación , Glicoproteínas de Membrana/sangre , Perforina , Proteínas Citotóxicas Formadoras de Poros , Receptores Inmunológicos/sangre , Receptores Inmunológicos/inmunología , Receptores KIR , Serina Endopeptidasas/sangre , Transducción de Señal/inmunología , Células Tumorales CultivadasRESUMEN
The alpha-helical amphipathic peptide D-(KLAKLAK)2 is toxic to eukaryotic cells if internalized by a suitable targeting mechanism. We have targeted this peptide to malignant hemopoietic cells via conjugation to monoclonal antibodies, which recognize lineage-specific cell surface molecules. An anti-CD19/peptide conjugate efficiently killed 3/3 B lymphoid lines. However, an anti-CD33/peptide conjugate was cytotoxic to only one of three CD33-positive myeloid leukemia lines. The IC50 towards susceptible lines were in the low nanomolar range. Conjugates were highly selective and did not kill cells that did not express the appropriate cell surface cognate of the antibody moiety. Anti-CD19/peptide conjugates efficiently killed cells from patients with chronic lymphocytic leukemia but anti-CD33/peptide reagents were less effective against fresh acute myeloid leukemia cells. We therefore suggest that amphipathic peptides may be of value as targeted therapeutic agents for the treatment of a subset of hematologic malignancies.
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Apoptosis/efectos de los fármacos , Neoplasias Hematológicas/tratamiento farmacológico , Inmunotoxinas/farmacología , Péptidos/administración & dosificación , Enfermedad Aguda , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Antígenos CD/inmunología , Antígenos CD19/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Linfocitos B/efectos de los fármacos , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Hematológicas/inmunología , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/inmunología , Linfoma/tratamiento farmacológico , Linfoma/inmunología , Lectina 3 Similar a Ig de Unión al Ácido SiálicoRESUMEN
We studied the actions of geldanamycin (GA) and herbimycin A (HMA), inhibitors of the chaperone proteins Hsp90 and GRP94, on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses, T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA, but not HMA, showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase, a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL.