RESUMEN
Sepsis caused by Staphylococcus aureus is a major health problem worldwide. Better outcomes are achieved when rapid diagnosis and determination of methicillin susceptibility enable early optimization of antimicrobial therapy. Eight large clinical laboratories, seven from the United States and one from Scotland, evaluated the combination of the Staphylococcus QuickFISH BC and the new mecA XpressFISH assay (both AdvanDx, Woburn, MA, USA) for the detection of methicillin-resistant S. aureus in positive blood cultures. Blood cultures flagged as positive by automated blood culture instruments and demonstrating only Gram-positive cocci in clusters on Gram stain were tested by QuickFISH, a 20-min assay. If only S. aureus was detected, mecA XpressFISH testing followed. The recovered S. aureus isolates were tested by cefoxitin disk diffusion as the reference method. The QuickFISH assay results were concordant with the routine phenotypic testing methods of the testing laboratories in 1,211/1,221 (99.1%) samples and detected 488/491 S. aureus organisms (sensitivity, 99.4%; specificity, 99.6%). Approximately 60% of the samples (730) contained coagulase-negative staphylococci or nonstaphylococci as assessed by the QuickFISH assay and were not tested further. The 458 compliant samples positive exclusively for S. aureus by the QuickFISH assay were tested by the mecA XpressFISH assay, which detected 209 of 211 methicillin-resistant S. aureus organisms (sensitivity, 99.1%; specificity, 99.6%). The mecA XpressFISH assay also showed high reproducibility, with 534/540 tests performed by 6 operators over 5 days achieving reproducible results (98.9% agreement). The combination of the Staphylococcus QuickFISH BC and mecA XpressFISH assays is sensitive, specific, and reproducible for the detection of methicillin-resistant S. aureus and yields complete results in 2 h after the blood culture turns positive.
Asunto(s)
Sangre/microbiología , Hibridación Fluorescente in Situ/métodos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Sepsis/diagnóstico , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/microbiología , Técnicas Bacteriológicas/métodos , Humanos , Staphylococcus aureus Resistente a Meticilina/genética , Reproducibilidad de los Resultados , Escocia , Sensibilidad y Especificidad , Sepsis/microbiología , Estados UnidosRESUMEN
Our previous work showed that the nuclear scaffold (NS) protease is required for apoptosis of both thymocytes and chronic lymphocytic leukemic (CLL) lymphocytes. Because partial sequencing of one of the subunits of the NS protease revealed homology to the proteasome, we tested the effects of classical proteasome inhibitors on apoptosis in CLL cells. Here we report that proteasome inhibition caused high levels of DNA fragmentation in all patients analyzed, including those resistant to glucocorticoids or nucleoside analogs, in vitro. Proteasome inhibitor-induced DNA fragmentation was associated with activation of caspase/ICE family cysteine protease(s) and was blocked by the caspase antagonist, zVADfmk. Analysis of the biochemical mechanisms involved showed that proteasome inhibition resulted in mitochondrial dysregulation leading to the release of cytochrome c and a drop in mitochondrial transmembrane potential (triangle upPsi). These changes were associated with inhibition of NFkappaB, a proteasome-regulated transcription factor that has been implicated in the suppression of apoptosis in other systems. Together, our results suggest that drugs that target the proteasome might be capable of bypassing resistance to conventional chemotherapy in CLL.
Asunto(s)
Apoptosis/efectos de los fármacos , Cisteína Endopeptidasas/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Leucemia Linfocítica Crónica de Células B/patología , Complejos Multienzimáticos/efectos de los fármacos , Resistencia a Antineoplásicos , Glucocorticoides/farmacología , Glucocorticoides/uso terapéutico , Humanos , Membranas Intracelulares/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Activación de Linfocitos/efectos de los fármacos , Linfocitos/patología , Complejo de la Endopetidasa ProteasomalRESUMEN
Recent work has demonstrated that glucocorticoids, nucleoside analogues, and other cancer chemotherapeutics induce apoptosis in chronic lymphocytic leukemia (CLL) cells. In this study, we investigated the involvement of protease activation in these responses using selective peptide inhibitors of the interleukin-1beta converting enzyme (ICE)/caspase family and a Ca2+-activated protease we recently implicated in thymocyte apoptosis. Apoptosis was associated with proteolytic cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) and increased caspase protease activity, and cell-permeant caspase antagonists [zVAD(OMe)fmk and Boc-D(OBzl)cmk] blocked apoptosis in response to the glucocorticoid methylprednisolone or the nucleoside analogue fludarabine, indicating that caspase activation was required for these responses. However, a peptide-based inhibitor of the Ca2+-dependent lamin protease (zAPFcmk) also completely suppressed DNA fragmentation and the cleavage of lamin B1 . Strikingly, treatment of cells with zAPFcmk alone led to characteristic PARP cleavage, depletion of the precursor forms of two ICE family proteases (CPP32 and ICH-1), and phosphatidylserine exposure, suggesting that blockade of the lamin protease led to activation of the ICE family. Our results implicate the lamin protease as a target for Ca2+ during chemotherapy-induced apoptosis in CLL lymphocytes, and they identify a novel functional interaction between the protease and members of the ICE family.
Asunto(s)
Apoptosis/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Endopeptidasas/metabolismo , Glucocorticoides/farmacología , Leucemia Linfocítica Crónica de Células B/patología , Linfocitos/patología , Caspasa 1 , Activación Enzimática , Humanos , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/inmunología , Linfocitos/enzimología , Células Tumorales CultivadasRESUMEN
PURPOSE: To compare the efficacy and toxicity of fluconazole and amphotericin B in the treatment of hematogenous candidiasis in cancer patients. PATIENTS AND METHODS: A matched cohort study of cancer patients with hematogenous candidiasis was conducted. Forty-five patients with hematogenous candidiasis who received fluconazole (200 to 600 mg/day) in an open-label trial at the University of Texas M. D. Anderson Cancer Center, Houston, Texas, between February 1990 and June 1992 were matched to 45 patients treated with amphotericin B (0.3 to 1.2 mg/kg/day) for the same diagnosis. Criteria for matching included the following prognostic variables at the initiation of therapy: pneumonia, neutropenia (< 1,000 cells/mm3), number of positive blood cultures before therapy, infecting Candida species, underlying disease, and the simplified acute physiology score. Response and survival at 48 hours, after 5 days of therapy, and at the end of therapy, as well as toxicity rates were obtained. Other post hoc analyses were performed. Differences in outcomes were assessed by the McNemar, the sign, and the log rank tests. RESULTS: Patients were similar with respect to the matching criteria, age, sex, status of underlying disease, use of antibiotics and growth factors, duration of treatment, presence and removal of central venous catheters, disseminated disease, and concomitant infections. Response rates at 48 hours and 5 days were similar between the two study groups. Overall response rates at the end of therapy were 73% for patients treated with fluconazole and 71% for patients treated with amphotericin B (P = 0.78). There were no differences in survival rates or causes of death. Toxicity was observed in 9% of patients treated with fluconazole and in 67% of patients treated with amphotericin B (P < 0.0001). Toxic effects of amphotericin B included nephrotoxicity, hypokaliemia, and fever and chills. CONCLUSION: Fluconazole is effective and better tolerated than amphotericin B for the treatment of hematogenous candidiasis in cancer patients.