No abstract

Although proteases of the caspase family are essential mediators of apoptosis in nucleated cells, in anucleate cells their presence and potential functions are almost completely unknown. Human erythrocytes are a major cell population that does not contain a cell nucleus or other organelles. However, during senescence they undergo certain morphological alterations resembling apoptosis. In the present study, we found that mature erythrocytes contain considerable amounts of caspase-3 and -8, whereas essential components of the mitochondrial apoptotic cascade such as caspase-9, Apaf-1 and cytochrome c were missing. Strikingly, although caspases of erythrocytes were functionally active in vitro, they failed to become activated in intact erythrocytes either during prolonged storage or in response to various proapoptotic stimuli. Following an increase of cytosolic calcium, instead the cysteine protease calpain but not caspases became activated and mediated fodrin cleavage and other morphological alterations such as cell shrinkage. Our results therefore suggest that erythrocytes do not have a functional death system. In addition, because of the presence of procaspases and the absence of a cell nucleus and mitochondria erythrocytes may be an attractive system to dissect the role of certain apoptosis-regulatory pathways. Cell Death and Differentiation (2001) 8, 1197 ± 1206.

Intercellular communication among certain cell types can occur via ATP secretion, which leads to stimulation of nucleotide receptors on target cells. In epithelial cells, however, intercellular communication is thought to occur instead via gap junctions. Here [Arg8]vasopressin, phenylephrine, and propidium iodide were from Sigma; fluo-3/AM was from Molecular Probes; suramin was from Biomol (Plymouth Meeting, PA); and indodicarbocyanine (Cy5) was from Biological Detection Systems (Pittsburgh). All other chemicals were of the highest quality commercially available.Preparation of Isolated Hepatocytes. Isolated rat hepatocytes were prepared in the Hepatocyte Isolation Core of the Yale Liver Center as described (5, 12). Briefly, rat livers were perfused with Hanks' A medium and then with Hanks' B medium containing 0.05% collagenase (Boehringer Mannheim) and 0.8 unit trypsin inhibitor (Sigma)/U tryptic activity.Livers were then excised, minced, and passed through serial nylon mesh filters, and the resultant cells were washed. These cells were suspended at a concentration of 7.5 x 105 cells per ml in Leibovitz L-15 medium (GIBCO) containing 10% fetal calf serum, 50 units penicillin, and 50 mg streptomycin/ml, and plated onto glass coverslips. Cells were incubated at 37°C and used 2-6 h after plating. When prepared in this fashion, "40% of isolated hepatocytes were not in contact with other hepatocytes, while '60% were in aggregates of two or more. Cell viability by trypan blue exclusion was measured 2 h after plating and exceeded 90%.Preparation of Isolated Bile Duct Units. Bile duct units, which are polarized and physiologically intact segments of bile duct epithelia, also were prepared in the Hepatocyte Isolation Core of the Yale Liver Center as described (13). After livers were perfused with Hanks' B medium as described above, the portal tissue residue was mechanically separated from parenchymal tissue first by shaking and then by forcing the tissue through a syringe to dissociate the remaining hepatocytes. The tissue was then minced in solution C, which contained a-MEM supplemented -with 0.066% collagenase, 0.033% Pronase, 0.006% DNase, 3% fetal calf serum, 0.1% bovine serum albumin, and penicillin/streptomycin at 100,000 units per 100 mg/liter. The minced tissue was then shaken at 37°C for 30 min, minced again, and sequentially filtered. Fragments remaining on the filters were digested for an additional 30 min Abbreviations: [Ca2+]i, cytosolic calcium; Cy5, indodicarbocyanine.

Both caspase-1-and caspase-3-like activities are required for Fas-mediated apoptosis. However, the role of caspase-1 and caspase-3 in mediating Fas-induced cell death is not clear. We assessed the contributions of these caspases to Fas signaling in hepatocyte cell death in vitro. Although wild-type, caspase-1 ؊/؊ , and caspase-3 ؊/؊ hepatocytes were killed at a similar rate when cocultured with FasL expressing NIH 3T3 cells, caspase-3 ؊/؊ hepatocytes displayed drastically different morphological changes as well as significantly delayed DNA fragmentation. For both wild-type and caspase-1 ؊/؊ apoptotic hepatocytes, typical apoptotic features such as cytoplasmic blebbing and nuclear fragmentation were seen within 6 hr, but neither event was observed for caspase-3 ؊/؊ hepatocytes. We extended these studies to thymocytes and found that apoptotic caspase-3 ؊/؊ thymocytes exhibited similar ''abnormal'' morphological changes and delayed DNA fragmentation observed in hepatocytes. Furthermore, the cleavage of various caspase substrates implicated in mediating apoptotic events, including gelsolin, fodrin, laminB, and DFF45͞ICAD, was delayed or absent. The altered cleavage of these key substrates is likely responsible for the aberrant apoptosis observed in both hepatocytes and thymocytes deficient in caspase-3.

BackgroundData on the performance of Mycobacterium-tuberculosis-specific interferon-(IFN)-γ release assays (IGRAs) in the serial testing of health care workers (HCWs) is limited. The objective of the present study was to determine the frequency of IGRA conversions and reversions and to identify predictors of persistent IGRA positivity among serially tested German HCWs in the absence of recent extensive tuberculosis (TB) exposure.MethodsIn this observational cohort-study HCWs were prospectively recruited within occupational safety and health measures and underwent a tuberculin skin test (TST) and the IGRA QuantiFERON®-TB Gold In-Tube (QFT-GIT) at baseline. The QFT-GIT was repeated 18 weeks later in the median. QFT-GIT conversions (and reversions) were defined as baseline IFN-γ < 0.35 IU/ml and follow-up IFN-γ ≥ 0.35 IU/ml (and vice versa). Predictors of persistently positive QFT-GIT results were calculated by logistic regression analysis.ResultsIn total, 18 (9.9%) and 15 (8.2%) of 182 analyzed HCWs were QFT-GIT-positive at baseline and at follow-up, respectively. We observed a strong overall agreement between baseline and follow-up QFT-GIT results (κ = 0.70). Reversions (6/18, 33.3%) occurred more frequently than conversions (3/162, 1.9%). Age and positive prior and recent TST results independently predicted persistent QFT-GIT positivity. Furthermore, the chance of having persistently positive QFT-GIT results raised about 3% with each additional 0.1 IU/ml increase in the baseline IFN-γ response (adjusted odds ratio 1.03, 95% confidence interval 1.01-1.04). No active TB cases were detected within an observational period of more than two years.ConclusionsThe QFT-GIT's utility for the application in serial testing was limited by a substantial proportion of reversions. This shortcoming could be overcome by the implementation of a borderline zone for the interpretation of QFT-GIT results. However, further studies are needed to clearly define the within-subject variability of the QFT-GIT and to confirm that increasing age, concordantly positive TST results, and the extend of baseline IFN-γ responses may predict the persistence of QFT-GIT positivity over time in serially tested HCWs with only a low or medium TB screening risk in a TB low-incidence setting.