[Assessment of clinical competence of medical school graduates in Italy with standardized patients. The opinion of the examinees]. / Sperimentazione in Italia del metodo del paziente standardizzato per la valutazione della competenza clinica. L'opinione dei neolaureati.

An international collaborative project for the evaluation of clinical competence at the end of the Medical School curriculum using the ECFMG-CSA (Educational Commission for Foreign Medical Graduates--Clinical Skills Assessment) prototype was started in Italy in April 1996. Faculty representatives from Italian Medical Schools and experts from the ECFMG in Philadelphia participated in the Project. The CSA consists of integrated clinical encounters with 10 standardized patients during which the examinee is asked to obtain a focused history, perform a relevant physical examination and communicate initial diagnoses and management plan to the Standardized Patient (SP). The SP then completes checklists that are scored by Faculty members. The project was concluded in Spring 1998 and a total of 173 new graduates were examined. The data elaborated by the primary site in Chieti University will be available in the Fall 1998 by the ECFMG in Philadelphia. This preliminary communication reports the opinions of the examinees on the ECFMG-CSA, contained in the questionnaires administered after the test. Most of the examinees considered this new methodology as a valid tool for the assessment of clinical competence, especially history-taking and interpersonal skills and stated that the SP simulations were realistic. The 72% of examinees indicated that the Medical School curriculum does not adequately prepare for the CSA examination. Lastly, 68% was in favour of including the SP in the Medical Licensing Examination.

Effect of desferrioxamine and hydroxypyridones on hemopoietic progenitors and neuroectodermal tumor cells.

The iron chelator desferrioxamine (DFO) has been shown to inhibit the proliferation of hemopoietic progenitors and several tumor cell lines. We have compared the in viro hemopoietic inhibitory effect of desferrioxamine (DFO) and hydroxypyridones (HPOs) on hemopoietic progenitors and two human neuroectodermal (NE) tumor cell lines, NB 100 and SKNMC. Both DFO and HPOs showed a direct dose-related inhibitory effect on BFU-E and CFU-GM obtained from purified human non-T MNAC (T-lymphocyte-depleted nonadherent mononuclear cells) and CD34+ cells. DFO and HPOs displayed both an inhibitory and a cytotoxic effect on NE cell lines. We calculated the ratio between NE cell and hemopoietic cell growth inhibition for a range of concentrations of chelators. DFO showed the most satisfactory ratio. This suggests that DFO is still the most preferable chelating agent for the treatment of neuroblastoma, since it combines the highest antineuroblastoma effect with the lowest hematopoietic toxicity.

Interleukin 3 stimulates proliferation and triggers endothelial-leukocyte adhesion molecule 1 gene activation of human endothelial cells.

Proliferation and functional activation of endothelial cells within a tissue site of inflammation are regulated by humoral factors released by cells, such as T lymphocytes and monocytes, infiltrating the perivascular space. In the present study we investigated the effects of interleukin 3 (IL-3), an activated T lymphocyte-derived cytokine, on cultured human umbilical vein endothelial cells (HUVEC). Proliferative activity, evaluated both by estimation of the fraction of cells in the S phase and by direct cell count demonstrated that IL-3, at the dose of 25 ng/ml, enhances more than threefold both DNA synthesis and cell proliferation above baseline control conditions. Binding studies with radioiodinated ligand demonstrated that HUVEC constitutively express a smaller number of IL-3 binding sites (approximately 99 binding sites per cell, with an apparent Kd of 149 pM). Accordingly, molecular analysis showed the presence of transcripts for both alpha and beta subunits of the IL-3 receptor. Functional activation of endothelial cells was evaluated by the expression of the endothelial-leukocyte adhesion molecule 1 (ELAM-1) transcript and by leukocyte adhesion. The ELAM-1 gene transcript was clearly detectable 4 h after IL-3 addition and started to decrease after 12 h. Moreover, IL-3-induced ELAM-1 transcription was followed by enhanced adhesion of neutrophils and CD4+ T cells to HUVEC. The findings that IL-3 can stimulate both proliferation and functional activation of endothelial cells suggest that this cytokine can be involved in sustaining the process of chronic inflammation.

Modulation of in vitro myelopoiesis by LGL: different effects on early and late progenitor cells.

We describe here the modulatory activity of human peripheral blood natural killer (NK) cells on the growth and differentiation of myeloid progenitor cells at different stages of maturation. NK-enriched cell fractions containing 54 to 75% large granular lymphocytes (LGL) and displaying high levels of NK activity significantly inhibited the growth of late (7 day) granulocyte-macrophage colony-forming cells (CFU-GM) from about 50% of normal human bone marrow samples. However, the same fractions strongly enhanced the growth of early (14 day) stem cells from peripheral blood. Enhancing activity on early CFU-GM from blood was greater in highly purified NK cell preparations containing 96% LGL than in NK-depleted T cell preparations from the same donors. Analogous to the results when using the NK-enriched fractions, the NK-purified preparations inhibited late CFU-GM and stimulated the early ones. We conclude from these observations that human LGL have a modulatory effect on myelopoiesis depending on the maturation stage of the progenitor cell.

Insulin requirement of human leukemic cell lines.

The growth characteristics of human leukemic cell lines in serum supplemented medium and in serum free medium with and without the addition of insulin were investigated. No relation was found between the insulin binding capacity of the cells and their hormone-dependence for growth.

Subpopulation heterogeneity in human acute myeloid leukemia determined by monoclonal antibodies.

The leukemic population in 63 patients with acute myeloid leukemia (AML) was studied with 15 monoclonal antibodies that detect lineage-related and stage-related antigens on normal hemopoietic cells. Indirect immunofluorescence and fluorescence-activated cell sorting showed that subpopulations of leukemic cells reacted with some or all antibodies, but the percentage of cells reacting with a single antibody varied widely among patients. The composite antigenic phenotype of the various cases, as determined by immunofluorescence assay, did not correlate with the French-American-British morphological classification. Furthermore, some cells in each case failed to express any antigen normally expressed on myelomonocytic precursors from the level of the early CFU-GM to the mature granulocyte or monocyte. In double-fluorescence experiments, the individual cells expressed none, one, or both antigens. These results demonstrate that there is considerable subpopulation heterogeneity in AML. This heterogeneity may considerably limit or complicate the use of monoclonal antibodies for diagnosis, prognosis, and treatment of acute nonlymphocytic leukemia (ANLL).

Molecular mechanisms of human hemoglobin switching: selective undermethylation and expression of globin genes in embryonic, fetal, and adult erythroblasts.

The globin chain synthetic pattern and the extent of DNA methylation within embryonic, fetal, and adult beta-like globin gene domains were evaluated in greater than or equal to 90% purified human erythroblasts from yolk sacs and fetal livers in the 6- to 12-wk gestational period as well as from adult marrows. The 6-wk erythroblasts produce essentially embryonic epsilon chains, whereas the 12-wk erythroblasts synthesize largely fetal gamma globin and the adult marrow erythroblasts synthesize almost exclusively adult beta chains. In all phases of ontogenic development, a strong correlation exists between DNA hypomethylation in the close flanking sequences of globin genes and their expression. These results suggest that modulation of the methylation pattern may represent a key mechanism for regulating expression of human globin genes during embryonic leads to fetal and fetal leads to adult Hb switches in humans. In ontogenic development this mechanism might in turn correlate with a gradual modification of chromatin structure in the non-alpha gene cluster, thus leading to a 5' leads to 3' activation of globin genes in a balanced fashion.

Antigenically distinct subpopulations of myeloid progenitor cells (CFU-GM) in human peripheral blood and marrow.

Two types of progenitor cells of the human granulocytic and monocytic lineages (CFU-GM) can be distinguished by using mouse monoclonal antibodies against human hemopoietic cells. Type 1 CFU-GM contribute all of the peripheral blood CFU-GM as well as a small fraction of bone marrow CFU-GM and express surface antigens recognized by "anti-lymphomonocytic" monoclonal antibodies S3-13 and S17-25 but not the antigens recognized by R1B19 and WGHS-29-1 (two monoclonal antibodies that react with all the cells of the granulocytic lineage). Type 2 CFU-GM are present only in the marrow and react with S3-13, R1B19, and WGHS-29-1. Partial reactivity with S17-25 was observed only in the complement-dependent cytotoxicity test. In vitro culture of type 1 CFU-GM in liquid medium in the presence of granulocyte-macrophage colony-stimulatory factor (GM-CSF) generates colony-forming cells that have the surface phenotype of type 2 CFU-GM. This finding supports the idea of two different stages of maturation of myelomonocytic progenitor cells represented by type 1 and type 2 CFU-GM.

Induction of differentiation of human myeloid leukemias: surface changes probed with monoclonal antibodies.

The surface changes occurring in three acute myeloid leukemia cell lines (HL60, ML3, and KG1) induced to differentiate by a variety of agents (dimethylsulfoxide, retinoic acid, 12-O-tetradecanoylphorbol-13-acetate, and factors present in lymphocyte conditioned medium) were probed using monoclonal antibodies that are differentiation stage- and lineage-specific. In all cases, the differentiated phenotype was defective and varied with the inducing agent and the cell line used. HL60 proved to be the most sensitive to the effect of the inducers. Retinoic acid was better than DMSO, and TPA was better than the medium factors in the ability to induce granulocytic and monocytic differentiation, respectively, in HL60 cells. These findings indicate that the differentiation block in acute myeloid leukemias is heterogeneous and that each cell line has different phenotypic characteristics that are responsible for the extent of differentiation obtained with a given inducer. These results also suggest that the extent of the differentiation response in vitro may be improved by the use of more suitable inducers for each specific leukemic line.

Induction of differentiation of human myeloid leukemias by phorbol diesters: phenotypic changes and mode of action.

Treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) of acute myeloblastic leukemia cells halts proliferation and induces expression of monocyte/macrophage markers. Surface characteristics of leukemic HL60 cells, as defined using a panel of monoclonal antibodies, were found to be similar to those of normal human promyelocytes. TPA treatment, however, induced a phenotype that, unlike normal monocytes, contained several myeloid-specific markers and lacked several monocyte-specific markers. TPA treatment of HL60 cells causes the rapid disappearance of the transferrin receptor from the cell surface. Because transferrin is essential for HL60 cell proliferation in culture, the disappearance of this receptor is followed by an irreversible accumulation of the cells in the G1 phase of the cell cycle. The TPA-induced arrest of cell proliferation suggests the potential of this agent in experimentally treating myeloblastic leukemias.

Induction of differentiation of HL-60 cells by dimethyl sulfoxide: evidence for a stochastic model not linked to the cell division cycle.

Induction of differentiation of human promyelocytic leukemia cell line HL-60 by dimethyl sulfoxide (Me2SO) was analyzed to determine the relationship between exposure time of the inducer and cell cycle. A minimum incubation time of 12 hr with Me2SO was required in order to induce differentiation in a small but significant proportion of cells. These expressed differentiation markers (morphology, phagocytosis, and nitroblue tetrazolium reduction) up to 12 hr after Me2SO was removed from the medium. For periods beyond 12 hr and as long as 120 hr of contact of HL-60 cells with the inducing agent, a linear rise in the percentage of differentiated cells was observed. The sensitivity to Me2SO of HL-60 cells synchronized by double thymidine block was examined and was found to be similar to that of nonsynchronized cells. The effect of Me2SO was not altered when incubated with cells at different phases of the cell cycle. Finally, even nonproliferating cells were sensitive to the inducing effect of Me2SO. The data are consistent with a stochastic model of induction to differentiation without having any linkage to the cell cycle.

Kinetics of phytohemagglutinin stimulated lymphocytes in chronic lymphocytic leukemia.

The DNA synthesis pattern and several kinetic parameters of in vitro PHA stimulated normal and CLL lymphocytes were determined. The DNA synthesis peak of CLL lymphocytes occurred 2-3 days later than that of normal lymphocytes. The generation time, estimated by the labeled mitoses method, was found to be 28 hr and 20 hr for CLL and normal lymphocytes respectively. This difference was mainly due to longer S and G1 periods. It was also shown that both CLL and normal lymphocytes divide several times. These data were confirmed by the chromatid labeling pattern and by the halving of the grains and the double labeling techniques. By combining continuous and pulse labeling the growth fraction of CLL lymphocytes was found to be progressively increasing, because of the recruitment of new cells in cycle, from the third day of culture. Therefore the delayed peak of DNA synthesis of CLL lymphocytes was caused by a longer cell cycle and by a longer pre-replicative phase.

Persisting production of out-of-cycle blasts during methotrexate therapy in acute leukaemia.

Kinetics of the blast populations in two cases of acute lymphoblastic leukaemia during treatment with methotrexate have been studied in vivo. Beside a striking cytocidal effect on blasts arrested in the S phase and a synchronization and a slowing of those in the G2 phase, the most important finding was the persistence of a flux of drug-affected cells from the proliferation of the non-proliferating compartment. Some of these non-proliferating cells were able to re-enter subsequently the mitotic cycle. This fact can explain why a cell-cycle-specific drug fails to eradicate completely acute leukaemic cells in man.

L-1210 and human acute leukemia kinetics as related to therapy.

Comparative considerations are made between human acute leukemia (AL) and mouse transplantable L-1210 leukemia. The main kinetic parameters, such as the growth fraction (GF), growth rate, and cell cycle times, of both human and mouse diseases, are compared. The striking differences in cell kinetics and in response to treatment may be viewed as depending on different leukemogenesis mechanisms. Therefore, some improvement in human AL chemotherapy is considered possible both by researching a more rational employment of cytostatic drugs, and studying other animal models quite similar to the human disease, such as AKR leukemia.