The telomerase activity and the senescence profile of cultured breast fibroblasts from normal human interstitial and malignant stromal tissue were studied in comparison with their proliferation and ...differentiation pattern. Fibroblasts were grown either in the presence or absence of a conditioned medium (CM) obtained from cultures of the oestrogen receptor-positive breast cancer MCF-7 cell line. At different passages (from the 2nd up to the 48th), fibroblasts were examined for the telomerase activity by the Telomerase Repeats Amplification Protocol (TRAP) assay, for proliferation profile by Ki-67 antigen expression, and the myofibroblast or smooth muscle cell-like differentiation pattern by immunofluorescence with monoclonal antibodies specific for smooth muscle markers. Serial passages of fibroblasts from normal or tumour breast reveal that the relationship between the levels of telomerase activity and phenotypic/proliferation profile changes with cell subcultivation in a different manner in the two cell populations. The fibroblasts from normal tissue completed 12 passages in a CM-independent way prior to senescence whereas fibroblasts from tumour stroma senescence were attained after 48 passages. These cells showed a marked decrease of telomerase activity, growth rate and smooth muscle alpha-actin expressing myofibroblasts after the 32nd passage. CM treatment of this fibroblast population induces a decline in the myofibroblast content, which precedes the changes in telomerase activity. Passaged fibroblasts from normal breast tissue can be converted to myofibroblasts upon CM treatment whereas those from tumour stroma were CM-insensitive. Taken together our data suggest that a heterogeneous fibroblast population with different life span is activated/recruited in the breast interstitium and poses the problem of a unique activation/recruitment of fibroblasts in neoplastic conditions.
Monoclonal antibodies are one of the most important products of biotechnology and laboratories and companies all over the world are pursuing their large-scale production. Herein we report a protocol ...for hybridoma cell cultivation over small glass cylinders inside a 3 liter bioreactor vessel which leads to the production and purification--in order of grams--of one MAb intended for human therapeutic use. This protocol proved to be simple, reproducible and cost effective. Three trials are reported: the first two using conventionally serum-supplemented medium culture and producing 3.15 and 2.1 g of purified MAb in 30 and 21 days respectively, and the third one using serum-free medium culture and producing 6 g of purified MAb in 36 days. We have ascertained the stability of the hybridoma by its cloning directly in serum-free medium. The downstream processing of the serum-free trial was done in a single step, concentrating large volumes of supernatant while simultaneously purifying the antibody.
Two groups of patients with anatomically proved acute myocardial infarction were compared in order to study specificity and sensitivity of the ECG criteria previously described in clinical and ...experimental right ventricular infarction ( RVI ). Group 1 included 21 patients with left inferior infarction and with a variable degree of right ventricular involvement; group 2 included nine patients with myocardial infarction confined to the left inferior wall. In both groups the presence of ST elevation (at least 0.05 mV) and the morphology of the QRS complex in V4R , V3R, and V1 were assessed in ECGs performed at the time of admission. Also, in order to evaluate the morphology of the ST segment and QRS complex in right precordial leads in normal subjects, an ECG with 12 standard and four right precordial leads ( V6R to V3R) was performed in 82 subjects (group 3) without clinical and ECG evidence of heart disease. Our data reveal that in normal subjects an rS pattern is always present in V3R and frequently (91%) in V4R . On the contrary, the presence of QS or QR complexes in both V4R and V3R are specific markers of right ventricular necrosis (specificity 100%; sensitivity 78%). The presence of injury and necrosis waves in V4R or V4R to V3R during inferior infarction is a useful diagnostic criterion in that it insures a highly specific diagnosis of acute RVI in the great majority (76 and 71%, respectively) of the cases with autopsy evidence of right ventricular involvement.
Teamwork training has been targeted as a primary method for addressing safety and effectiveness issues in healthcare. This paper presents a systematic and event-based method for developing scenarios ...and accompanying performance measurement tools for simulation-based team training in healthcare. The approach described here, SMARTER-Team, is adapted from a recently advanced methodology for using SBT to train and assess the ACGME core competencies in Emergency Medicine residency programs. SMARTER-Team focuses on the development of teamwork competencies and can be used to develop SBT for teams across specialty areas.
