Photofrin binds to procaspase-3 and mediates photodynamic treatment-triggered methionine oxidation and inactivation of procaspase-3.

Diverse death phenotypes of cancer cells can be induced by Photofrin-mediated photodynamic therapy (PDT), which has a decisive role in eliciting a tumor-specific immunity for long-term tumor control. However, the mechanism(s) underlying this diversity remain elusive. Caspase-3 is a critical factor in determining cell death phenotypes in many physiological settings. Here, we report that Photofrin-PDT can modify and inactivate procaspase-3 in cancer cells. In cells exposed to an external apoptotic trigger, high-dose Photofrin-PDT pretreatment blocked the proteolytic activation of procaspase-3 by its upstream caspase. We generated and purified recombinant procaspase-3-D(3)A (a mutant without autolysis/autoactivation activity) to explore the underlying mechanism(s). Photofrin could bind directly to procaspase-3-D(3)A, and Photofrin-PDT-triggered inactivation and modification of procaspase-3-D(3)A was seen in vitro. Mass spectrometry-based quantitative analysis for post-translational modifications using both (16)O/(18)O- and (14)N/(15)N-labeling strategies revealed that Photofrin-PDT triggered a significant oxidation of procaspase-3-D(3)A (mainly on Met-27, -39 and -44) in a Photofrin dose-dependent manner, whereas the active site Cys-163 remained largely unmodified. Site-directed mutagenesis experiments further showed that Met-44 has an important role in procaspase-3 activation. Collectively, our results reveal that Met oxidation is a novel mechanism for the Photofrin-PDT-mediated inactivation of procaspase-3, potentially explaining at least some of the complicated cell death phenotypes triggered by PDT.

Peripheral blood eosinophilia in association with sarcoidosis.

OBJECTIVES: To review retrospectively our experience with peripheral blood eosinophilia (PBE) in sarcoidosis and to analyze histologically lung biopsy specimens for the presence of lung tissue eosinophils. PATIENTS AND METHODS: We reviewed 140 cases of sarcoidosis diagnosed between May 1975 and January 1998. Ninety-five patients (66.3% women; 70.5% African American; mean age, 35.9 years) met the inclusion criteria. Transbronchial biopsy specimens from 82 patients were divided into 4 morphologic compartments: parenchyma, bronchial wall, parenchymal granulomas, and bronchial wall granulomas. Within compartments, up to 10 high-power fields were scored semiquantitatively for eosinophils, from 0 (none) to 4+ (numerous). RESULTS: Thirty-nine patients (41%) had PBE. Four had PBE greater than 10%. The highest eosinophil count (21%) occurred in 1 patient. Sixty-five (79%) of 82 patients had no or few (1+) eosinophils in lung tissue; 17 patients had eosinophils scored as 2+ or higher. There was no correlation between peripheral blood eosinophil count and presence of eosinophils in transbronchial biopsy specimens. Eosinophils were least conspicuous in parenchyma but evenly distributed in bronchial wall and parenchymal and bronchial wall granulomas. CONCLUSIONS: Peripheral blood eosinophilia occurs frequently in sarcoidosis. However, there appears to be no association between peripheral blood eosinophil count and presence of lung tissue eosinophils. Whether eosinophils participate in the pathogenesis of sarcoidosis requires further study.

Evaluation of a proposed panel of cardiac markers for the diagnosis of acute myocardial infarction in patients with atraumatic chest pain.

OBJECTIVE: The purpose of this study was to evaluate retrospectively the efficacy of a proposed panel of three cardiac markers (myoglobin, creatine kinase-MB mass [CK-MB], and cardiac troponin I) in the diagnosis of acute myocardial infarction (AMI) in patients with atraumatic chest pain. DESIGN: A total of 110 patients admitted for the evaluation of atraumatic chest pain were examined. Forty-one of these patients were diagnosed with AMI. RESULTS: Five of the 41 patients with AMI had abnormally elevated myoglobin levels, whereas values of CK-MB and/or cardiac troponin I remained negative. Creatine kinase-MB mass alone had a sensitivity of 92.7%, a specificity of 89.9%, a positive predictive value of 84.4%, and a negative predictive value of 95.0% for the diagnosis of AMI. Cardiac troponin I alone had a sensitivity of 90.2%, a specificity of 95.7%, a positive predictive value of 92.5%, and a negative predictive value of 94.3% for the diagnosis of AMI. Cardiac troponin I is a more specific marker for the diagnosis of AMI than CK-MB, particularly in patients with chronic renal failure who are evaluated for chest pain. The combination of CK-MB and cardiac troponin I increased the sensitivity to 100% and the negative predictive value to 100% and had a specificity of 88.4% and a positive predictive value of 83.7%. The panel was diagnostic for all patients with AMI within 12 hours after admission. CONCLUSIONS: Our preliminary results indicate that this panel is highly effective for evaluation of AMI in patients with atraumatic chest pain. Elevated myoglobin levels were useful in detecting patients at high risk for AMI who initially were not detected with other markers. The combination of CK-MB and cardiac troponin I provided much higher sensitivity and had a much higher negative predictive value for the evaluation of AMI than cardiac troponin I or CK-MB alone. The 100% negative predictive value is particularly important because it indicates that patients with negative CK-MB and cardiac troponin I values 12 hours after admission have a negligible likelihood of AMI.

Histologic, microbiologic, and clinical correlates of the diagnosis of sarcoidosis by transbronchial biopsy.

OBJECTIVE: To determine the frequency of positive microbiologic cultures in patients with epithelioid granulomas and negative histochemical stains for microorganisms in transbronchial biopsy specimens. Secondary objectives were to compare the histologic features of sarcoidosis with those of infectious granulomas and to assess the reliability of histology in establishing the diagnosis of sarcoidosis. DESIGN: Retrospective study. Specific histologic features of transbronchial biopsy specimens were correlated with clinical and microbiologic data, final diagnosis, and an estimate of the probability, on admission, that the patient had sarcoidosis. SETTING: A large, urban, tertiary-care, university-affiliated hospital. PATIENTS: Ninety-two adult patients in whom epithelioid granulomas, negative for microorganisms on Ziehl-Neelsen and Gomori methemaine silver stain, were found in transbronchial biopsy specimens. Patients were identified through a search of surgical pathology files from 1975 to 1987. RESULTS: Ten patients (10.9%) had mycobacterial or fungal granulomas, while 82 had sarcoidosis. In all patients with a high clinical probability of sarcoidosis, the diagnosis was confirmed. Transbronchial biopsy specimens from patients with infectious granulomas had fewer granulomas (2.0 +/- 1.7 (SD) versus 7.1 +/- 6.6; P<.01), which involved a smaller proportion of lung tissue per case (9.5 +/- 10.0% versus 26.6 +/- 24.0%; P<.01). Sarcoid granulomas often exhibited Schaumann bodies (69.5% versus 10%; P<.01). Necrosis tended to predominate in infectious granulomas (19.5 versus 40%; not significant). CONCLUSIONS: Numerous granulomas, Schaumann bodies, and a high clinical probability of sarcoidosis are significantly associated with that diagnosis. Necrosis does not exclude sarcoidosis. Clinicopathologic assessment of transbronchial biopsy specimens is useful in predicting the final diagnosis of sarcoidosis but does not obviate the need for microbiologic cultures, which were positive in 10.9% of patients in this study.