Discrimination of apoptotic cells in flow cytometry using trypan blue and FDA.
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Apoptosis↗
Biomedical subjects
F Luchetti
Publications and source records attributed to F Luchetti.
Interaction between the mu-agonist dermorphin and the delta-agonist [D-Ala2, Glu4]deltorphin in supraspinal antinociception and delta-opioid receptor binding.
1. In rats, the interaction between the mu-opioid agonist dermorphin and the delta-opioid agonist [D-Ala2, Glu4]deltorphin was studied in binding experiments to delta-opioid receptors and in the antinociceptive test to radiant heat. 2. When injected i.c.v., doses of [D-Ala2, Glu4]deltorphin higher than 20 nmol produced antinociception in the rat tail-flick test to radiant heat. Lower doses were inactive. None of the doses tested elicited the maximum achievable response. This partial antinociception was accomplished with an in vivo occupancy of more than 97% of brain delta-opioid receptors and of 17% of mu-opioid receptors. Naloxone (0.1 mg kg-1, s.c.), and naloxonazine (10 mg kg-1, i.v., 24 h before), but not the selective delta-opioid antagonist naltrindole, antagonized the antinociception. 3. In vitro competitive inhibition studies in rat brain membranes showed that [D-Ala2, Glu4]deltorphin displaced [3H]-naltrindole from two delta-binding sites of high and low affinity. The addition of 100 microM Gpp[NH]p produced a three fold increase in the [D-Ala2, Glu4]deltorphin Ki value for both binding sites. The addition of 10 nM dermorphin increased the Ki value of the delta-agonist for the high affinity site five times. When Gpp[NH]p was added to the incubation medium together with 10 nM dermorphin, the high affinity Ki of the delta-agonist increased 15 times. 4. Co-administration into the rat brain ventricles of subanalgesic doses of dermorphin and [D-Ala2, Glu4]deltorphin resulted in synergistic antinociceptive responses. 5. Pretreatment with naloxone or with the non-equilibrium mu-antagonists naloxonazine and beta-funaltrexamine completely abolished the antinociceptive response of the mu-delta agonist combinations. 6. Pretreatment with the delta-opioid antagonists naltrindole and DALCE reduced the antinociceptive response of the dermorphin-[D-Ala2, Glu4]deltorphin combinations to a value near that observed after the mu-agonist alone. At the dosage used, naltrindole occupied more than 98% of brain delta-opioid receptors without affecting mu-opioid-receptors. 7. These data suggest that in the rat tail-flick test to radiant heat, mu- and delta-opioid agonists co-operate positively in evoking an antinociceptive response. Although interactions between different opioid pathways cannot be excluded, in vitro binding results indicate that this co-operative antinociception is probably mediated by co-activation of the delta-opioid receptors at the cellular level by the mu- and delta-agonist.
Amino Acid Sequence↗
Disappearance rate of serum calcitonin after total thyroidectomy for medullary thyroid carcinoma.
We studied the half-life of serum calcitonin (CT) in patients subjected to total thyroidectomy for medullary thyroid carcinoma (MTC). One patient showed a rapid serum CT component with a half-life of 3 hours and a slow component with a half-life of 30 hours; in another case only the 30-hour component was found. By chromatography of tumor extracts, we found that all the immunoreactive CT had a molecular weight of 3,600. After surgery, normalization of serum CT was achieved within 15 days in 4 patients, at 3 months and at 6 months in 2 other patients, while 1 patient never normalized. Normalization of serum CT after surgery is not an index of definitive cure in MTC, as demonstrated by one patient who relapsed 3 months after normalization of serum CT. However, as a general rule, patients who reach undetectable serum CT levels soon after surgery, are those having the best prognosis.
Adult↗
Autologous bone marrow purging with LAK cells.
In this study we will demonstrate that LAK cells, in vitro, can lyse hematologic neoplastic cells with a minor toxicity of the staminal autologous marrow cells. In fact, after bone marrow and LAK co-culture at a ratio of 1/1 for 8 hours, the inhibition on the GEMM colonies resulted to be 20% less compared to the untreated marrow. These data made LAK an inviting agent for marrow purging in autologous bone marrow transplantation.
Bone Marrow Purging↗
Apoptotic pathways depend on the target enzymatic activity and not on the triggering agent.
Molt-4 human leukemia cells were triggered to apoptosis by various agents with different mechanisms of action. Staurosporine, a protein kinase C (PKC) inhibitor; camptothecin, a topoisomerase I blocking drug; and tiazofurin, an inhibitor of inosine 5'-phosphate dehydrogenase (IMPDH), were used. Ultrastructural analysis showed morphologic changes characteristic of apoptosis that were very similar for all three agents. Nevertheless, DNA oligonucleosomic fragmentation was not detectable by agarose gel electrophoresis. However, a genomic DNA cleavage appeared after pulse-field gel electrophoresis (PFGE) in cells treated with these agents for 24 h. Furthermore, in situ nick translation (NT) showed a finely spotted nuclear labelling in staurosporine-treated cells and a compact fluorescence after camptothecin incubation. In tiazofurin-treated cells an intermediate pattern was found. Therefore, apoptotic agents with different mechanisms of action induced the formation of large genomic DNA fragments and very similar ultrastructural changes. Therefore, both phenomena and the closely related apoptosis progression depend on target cell machinery and not on the triggering agent.
Apoptosis↗
Limiting dilution analysis of IL-2 producing cells: I. Studies of normal human peripheral blood.
BACKGROUND: After marrow transplantation, the interaction of helper T lymphocytes from the donor with the patient alloantigens leads to cellular activation and release of IL-2 as initial events of the graft versus host reaction. A method for assessing the size of the pool containing allospecific helper T cells capable of producing IL-2 could be applied in the selection of better donors for marrow transplantation. MATERIAL AND METHODS: PBMC are added to replicate sets of wells each containing various amounts of EBV-LCL cells and PHA. After culture for some days the supernatant is removed from each well and added to IL-2 dependent CTLL-2 line. The proliferation of the CTLL-2 line is assessed by pulse labeling with 3H-thimidine. The precursor frequency of cell capable of producing IL-2 per ml/blood is estimated from the minimum X2 regression of the function of non-responding wells plotted as logarithmic function of the number of PBMC added per well. RESULTS: Approximately 30-40% of PBMC are found to produce IL-2 under the following conditions in culture: the optimal PHA concentration is 1.25 micrograms/ml, the optimal number of stimulator EBV-LCL cells is 1 x 10(3) and 3 days of culture are required. CONCLUSION: Here we report a rapid and quantitative technique of limiting dilution analysis that can estimate the frequency of peripheral blood mononuclear cells capable of secreting interleukin-2 following interaction with specific alloantigen.
Cell Line, Transformed↗
The onset of CML in blastic crisis: molecular features.
The lack of distinguishing characteristics between lymphoid blastic crisis (BC) of Philadelphia (Ph)+ chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) remains an exciting dilemma. Indeed, the genetic defect of approximately half of Ph+ ALL patients is identical to that identified in CML. Here we report the case of one patient admitted with immunological and molecular patterns indicative of Ph+ ALL. The patient was brought into complete remission by chemotherapy and was transplanted with an HLA identical sibling donor, but relapsed a few months later with immunological and molecular evidence of BC of CML, displaying myeloid markers and lacking lymphoid antigens and immunoglobulin heavy chain (IgH) rearrangement. This suggest a CML case with an initial BC without a previous chronic phase or supervening on a subclinical CML.
Adult↗