[Correlation between availability of GM progenitors and endogenous CSA production in neutropenia]. / Correlazione tra disponibilità di progenitori-GM e produzione endogena di CSA nelle neutropenie.

We studied some cases with granulopoiesis disorders, owing to different causes, all showing peripheral neutropenia. Can these cases benefit by the growth factors G-CSF administration? To answer this question we assayed the bone marrow CFU-GM growth of the neutropenic patients with standard source of CSA and employing as CSA source the autologous peripheral blood mononucleated cells (APBMN). We proposed ourselves to investigate the granulopoiesis regulation referring to the relation between number of GM-progenitor cells and levels of CSA. The bone marrow CFU-GM cultures were performed by Pike and Robinson's double layer agar technique. For the evaluation of growth we applied the criterion of the dishes topographic lecture according to Ghizzi and De Caro. We plotted the experimental data both as absolute count of observed aggregates (tPP) and as dynamic patterns of in vitro aggregate proliferation, by differentiating the early proliferative events (AC-A and AC-B) from the later ones (AC-C). We observed cases showing growths higher under CSA standard source than under autologous source (CAA) and vice versa. In other cases there are not noteworthy differences. To estimate these growth patterns and to interpret the experimental data we correlated the autologous stimulus capacity to the CFU-GM absolute counts. Most cases of neutropenia showed inverse correlation between the two indexes. We consider a reduced stimulus capacity may be fit when the CFU-GM growth is high. We retain this capacity of modulating the CSA production as index of kept regulation of the granulopoiesis and, then, these cases can spontaneously evolve towards the neutropenia resolution.

[In vitro sensitivity of accessory hematopoietic cells from neutropenic subjects to the activity of human gamma-globulin]. / Sensibilità in vitro delle cellule accessorie emopoietiche di soggetti neutropenici all'azione delle gamma-globuline umane.

We studied eight patients all showing neutropenia: drug-induced isolated neutropenic failure (2 cases), immune cell-mediated neutropenia (2 case), severe bone marrow hypoplasia (2 cases), dysmyelopoietic syndrome (1 case), cyclic neutropenia (1 case). Aim of the study was to assay the effect on CSA production of the pretreatment of autologous peripheral blood mononucleated cells (APBMN) with immunoglobulins (Ig). The CFU-GM growth were tested in different experimental conditions: with standard source of CSA, with autologous source, with autologous source modified by means of the preincubation of the autologous cells with Ig. The bone marrow CFU-GM culture was performed by Pike and Robinson's double layer agar technique. The experimental data were pointed out with topographic lecture of the dishes repeated at two times so as to obtain besides the aggregates global growth their dynamic classes too. We observed that the APBMN cells pretreatment with Ig modulates CSA production: represses it in cases showing high production and increases it in those showing low production. The effect of Ig modulation appears clearer on the GM-progenitor proliferating late (AC-C) than on those proliferating early (AC-A). This effect turned out to be particularly evident in a case with immune cell-mediated neutropenia and in both cases with drug-neutropenia. We retain that the monomers Ig could block Fc receptors and modulate their expression on macrophagic cells, conditioning in this way the CSA production. The parenteral administration of intact Ig (in vivo treatment of the APBMN cells) appears therefore helpful both in cases with CSA defect and in cases with excess of CSA production.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effect of bisphosphonate on the in vitro proliferation dynamics of CFU-GM]. / Azione del bisfosfonato sulla dinamica proliferativa in vitro delle CFU-GM.

Bisphosphonates, potent inhibitors of osteoclast-mediated bone resorption, affect different ways of the intracellular metabolism of the osteoclasts. Likewise other cell lines are affected by bisphosphonate action, among these the macrophagic cells. So we assayed the dichloromethylene diphosphonate (clodronate, Cl2MBP) effect on the in vitro proliferation of granulocytic-macrophage progenitor cells (GM-progenitors). The bone marrow progenitor cells of four healthy volunteers were plated in double layer agar by Pike and Robinson's technique, with the addition of clodronate concentrations rising from 0.004 till 4.0 mMol. For each experimental point we evaluated: 1) the global growth of the proliferating GM-progenitors (tPP) deduced from the number of the counted cellular aggregates (CA); 2) the mean size (m.s.) of the CA; 3) the total proliferative capacity (TPC), deduced from the aggregate mean size per the number of the CA; 4) the contribution to the proliferative events by each subsets of proliferating GM-progenitors (early proliferating GM-progenitors, forming the CA present at the 7th day of the in vitro culture; later proliferating GM-progenitors, appearing only at the 12th day). The dose-effect curve showed decrease both of the tPP and TPC for increasing concentrations of clodronate up to a complete growth inhibition at the concentration 4.23 mMol of clodronate. Concerning the m.s. of the in culture growth aggregates, in three of the four cases it appears unvaried up to the Cl2MBP concentration 0.4 mMol. The trends of dose-effect curves have been analysed both for colonies (CA-P4) and for clusters (CA-P1, -P2, -P3 of our classification).(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics and pharmacodynamic effects of a novel prodrug of N-methyldopamine at single dose in healthy volunteers.

Kinetics and cardiovascular effects of SIM2055 (CAS 103878-96-2), the 4-O-phosphate of N-methyldopamine (epinine), at single dose by the oral route were studied in 9 healthy adult volunteers (6 women and 3 men) ranging in age from 22 to 39 years. SIM2055 was administered at increasing doses (from 100 to 300 mg). Plasma concentrations of free epinine were not detectable after the 100 mg dose, but were measurable after the 200 mg and 300 mg doses. Pharmacokinetic data suggest that in man SIM2055 is promptly absorbed, quickly hydrolysed to epinine, metabolized to homovanillic acid and 3,4-dihydroxy-phenylacetic acid, conjugated with sulphuric acid and excreted in large amounts into urine. The 24-h urinary recovery of the 3 metabolites considered together, expressed as a percentage of the dose of SIM2055 administered, was 78 +/- 6 with the 100 mg dose, 66 +/- 11 with the 200 mg dose and 55 +/- 6 with the 300 mg dose (mean +/- S. D.). SIM2055 was well tolerated by all subjects. After its administration, the heart rate and the systolic and diastolic blood pressure presented no systemic or clinically significant variations. No substantial changes were observed with the ECG parameters. No subject reported gastric or systemic effects during the period of observation.

[Activity of natural fatty acids on erythrocyte osmotic resistance]. / Sull'azione di acidi grassi naturali sulle resistenze osmotiche eritrocitarie.

The aim of the present study was to assess the influence on the functional characteristics of the erythrocyte membrane of adding in vitro different natural fatty acids to blood taken from normal subjects. Blood samples were collected without stasis from healthy volunteers, anticoagulated with heparin or EDTA and incubated at 37 degrees C for 60 min with the different fatty acids at concentrations ranging between 1 x 10(-4) and 3 x 10(-2) molar. Two ml of blood were used for each test. The treated blood was added to graded solutions of NaCl (0.90-0.20 g/dl) at a ratio of 100 microliters/5 ml of solution. The suspensions were kept at room temperature and centrifuged for 10 min at 2000 g, in order to accelerate the sedimentation of the erythrocytes which had not been broken down and so as to obtain a clear supernatant for spectrophotometry of the dissolved haemoglobin. Readings were compared with those obtained from blood samples which had been completely haemolyzed by suspension in distilled water. Results obtained with the blood samples prepared with the fatty acids were compared with control samples from the same donor, also incubated at 37 degrees C for 60 min. Preincubation of the erythrocyte with butyric or caproic or oleic acid at concentrations ranging between 5 and 20 millimolar, provoked a clear deterioration of the osmotic resistances of the erythrocytes, in proportion to the concentration of the fatty acid used. The osmotic insult was systematically more effective in those samples anticoagulated with EDTA than those treated with heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

[In vitro activity of folic acid on the proliferation dynamics of human bone marrow CFU-GM in propyphenazone-induced granulocytopenia]. / L'azione in vitro dell'acido folico sulla dinamica proliferativa delle CFU-GM midollari umane nella granulocitopenia da propifenazone.

The in vitro action of folic acid was tested on the proliferation of bone marrow granulocyte-macrophage progenitor cells from a patient with drug-induced (propyphenazone) neutropenia in remission 20 days after the drug had been suspended. Various bone marrow cultures were prepared with standard stimulant, adding, respectively: folic acid, propyphenazone and both folic acid and propyphenazone together. Growth was tested on day 7, 12 and 19 of incubation. Under baseline culture with standard stimulant, CFU-GM growth was characterized by successive proliferation waves of various entity: the first on day 7 was very high, the second, on day 12 was rather low, and the third, on day 19 was intermediate. This behaviour is different from what is usually observed in normal subjects in steady-state, whose first (AC-A+AC-B) and second (AC-C) proliferation period are of similar entity. The prevalence of the first proliferation period in our case is interpreted as the result of a renewed granulocytopoietic activity after drug-induced bone marrow suppression. This indicates a maintained integrity of the negative-feedback mechanism of homeostatic regulation on granulocytopoietic activity. The sole addition of propyphenazone on the in vitro bone marrow cell cultures of our patient produced a reduction of those CFU-GM that had grown during the first period whereas the growth during the second and third period remained unvaried. Thus the growth peak in cultures treated with propyphenazone occurred on day 19, which seems to correspond with the necessary time for a spontaneous remission from neutropenia, clinically observed to be 20 days after suspension of the propyphenazone-containing drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferation and degeneration of circulating CFU-GM in 15 adult normal subjects. Can any ontogenetic relationship be deduced from these data?

We studied, in 15 normal adults, the "in vitro" proliferation and differentiation of circulating CFU-GM, in order to assess their implication in the processes that regulate the dynamic equilibrium of granulopoiesis, analogous to bone marrow CFU-GM, and to deduce by their growth behaviour the ontogenetic relationship between CFU-GM subpopulations in the circulating and bone marrow compartments respectively. We found that "in vitro" proliferation of circulating CFU-GM predominates over their degeneration. We believe circulating CFU-GM and bone marrow CFU-GM are not implicated in granulopoiesis regulation in the same manner, and that circulating CFU-GM are more immature than bone marrow CFU-GM when taking proliferation and GM-CSF response into account. One cannot ignore the hypothesis that cells that "in vivo" are quiescent, are recruited "in vitro" with GM-CSF. Finally we would like to draw attention to the parallelism between CFU-GM classification in types 1 and 2 using monoclonal antibodies to track surface antigens, and our classification obtained by using a new mathematical model that takes the "birth" and "dead" of cellular aggregates into account.

[Sinus bradycardia in chronic neuroglycopenic syndrome. A case of "short intestine" syndrome]. / La bradicardia sinusale nelle sindromi neuroglicopeniche croniche. Osservazioni in un caso di sindrome da "intestino corto".

The clinical pattern of the chronic hypoglycemic disorders is different from that of the acute disorders. While in acute hypoglycemic syndromes tachycardia usually occurs, in chronic hypoglycemic disorders there is, even if seldom, sinusal bradycardia. The bradycardia occurs after months with glycemia under 60 mg/dl and may be correlated to the neuroglycopenic state. It is easily reversible when plasma glucose concentration approaches the normal range again. Recently we observed sinusal bradycardia set up during the chronic hypoglycemia state, lasting from more than 5 months, in a man submitted to subtotal intestinal resection, owing to wide extensive eosinophilic granuloma. The bradycardia disappeared quickly after the correction of the hypoglycemia, when the patient was submitted to parenteral hyperalimentation with a silicone rubber catheter in the superior vena cava. We suggest to set the bradycardia among the markers of undernutritional syndromes beside the known anthropometric, biochemical and immunological signs.

Growth of bone marrow CFU-GM in a case of cyclical neutropenia. Preliminary report.

We studied bone marrow CFU-GM growth behaviour of a 9-year-old male child with cyclic neutropenia. The cultures were performed on day 0 and on day 13 of cyclic oscillation, in order to study some correspondences between CFU-GM culture parameters and the phases of a whole cyclic oscillation "in vivo". We explored the CFU-GM growth under three different conditions of GM-CSA production: a) standard source of CSA; b) endogenous GM-CSA assay; c) GM-CSA-gamma-globulin assay. At both observation times the endogenous GM-CSA assay produced more aggregates than the baseline culture. The GM-CSA-gamma-globulin assay partly corrected the growth increase, produced by endogenous assay. At time 0, at the nadir of peripheral blood neutrophils, there was a balance between the number of aggregates, appeared early in culture and early degenerated, and those appeared late. From progenitor cells culture performed on day 13 of cycle, a week before the zenith of neutrophils in vivo, we obtained an increase in aggregates, which appeared late. The values of CFU-GM grown from the culture performed on day 13 reached higher levels than the ones performed on day 0. The CFU-GM growth behaviour shows that in our case with cyclic neutropenia there is no defect in progenitor cells, while on the contrary there is an increase in CSA production.

[Effect of testosterone on in vitro proliferation of bone marrow granulocyte-macrophage progenitor cells (CFU-GM). I. Observations in normal subjects]. / L'azione del testosterone sulla proliferazione in vitro dei progenitori granulocito-macrofagici (CFU-GM) midollari. Nota I. Osservazioni su soggetti normali.

A hypothetical mechanism whereby testosterone propionate (Tp) seems to stimulate the granulopoiesis has been studied. We cultured in agar human bone marrow cells harvested 24 h before and after parenteral administration of 100 mg Tp to 4 volunteer subjects having no hematologic disorders. The dynamic features of the proliferating progenitor cells (growth of the CFU-GM) were studied in agar cultures according to an original method of repeated topographic scoring of dishes. In this way we obtained a classification of the newformed cellular aggregates (CA) based on their size and on their appearance time in vitro. The results were analysed according to the following dynamic classification of the CFU-GM: AC-A = CA observed on day 7 of culture and degenerate on day 12; AC-B = CA present on both days 7 and 12, distinct as B-persistent (CA persistent with the same size in the interval between 7th and 12th day of incubation) and as B-progressed (CA progressed to a wider size in the same interval); AC-C = CA appeared ex novo at the 12th day of culture. After acute Tp pre-treatment we observed, in the first period of culture (observations made on day 7), a decrease of the number of the small size CA (P1 = 5-10 cells), while the number of the wide size CA (P2 = 11-30 cells; P3 = 31-50 cells; P4 = more than 50 cells per CA) was increased. The number of the AC-A was unmodified.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effect of testosterone on the in vitro proliferation of bone marrow granulocyte-macrophage cells (CFU-GM). II. Observations in hypogonadal subjects]. / L'azione del testosterone sulla proliferazione in vitro dei progenitori granulocito-macrofagici (CFU-GM) midollari. Nota II. Osservazioni in soggetti ipogonadici.

CFU-GM cultures in agar double layers were performed from bone marrow unfractionated cells of four subjects with hypogonadism, before and 24 hours after acute administration of 100 mg of testosterone propionate (Tp). Cell aggregates (CA) of CFU-GM were classified according to their sizes (P1 = 3-10 cells; P2 = 11-30; P3 = 31-50; P4 = over 50 cells) and according to their appearance time in culture (CA-A: appearing at the 7th day; CA-C: appearing only at the 12th day). The total of proliferating progenitors (tPP) also embraces CA present, in the same microscopic field, on both scoring times (CA-B). In all hypogonadal men studied, the treatment with Tp yielded increase of tPP (on the average of 65%) and increase of the total number of cells appeared in culture (TCP, increase on the average of 82%) calculated as product [CA number] x [average size of CA]. These results are in agreement with those already observed by us in CFU-GM cultures of normal subjects. Yet it is interesting to note that, while in normal subjects the exogenous testosterone effect expresses itself with a higher increase of CFU-GM number in the second interval of culture (CA-C), in hypogonadal men the CFU-GM number increase occurs mostly in the first period of the culture (CA-A).(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics and bioavailability of oral acetylcysteine in healthy volunteers.

The plasma pharmacokinetics of oral acetylcysteine(N-acetylcysteine, NAC) after the administration of single 600 mg and repeated 200 mg doses and the relative bioavailability of the two regimens were studied in 12 adult subjects. On two different occasions in a cross-over, balanced fashion the subjects were administered orally either a single dose of NAC 600 mg as effervescent tablets or 4 repeated doses of NAC as granules in sachets at the regimen of 200 mg t.i.d. Venous blood samples were obtained just before dosing and 20, 40 min, 1, 1.5, 2, 3, 4, 6, 8, 10 and 12 h after the administration of NAC 600 mg; with the 1st, the 2nd and the 4th doses of NAC 200 mg samples were taken just before dosing and after 20, 40 min, 1, 1.5, 2, 3, 4, 6 and 8 h, the last sampling after the 1st dose being the one before the 2nd dose. A detailed description of the assaying methods of NAC is given in the text. As indexes of bioavailability Cmax' tmax and AUC of NAC plasma concentrations were considered and MRT was taken as an estimate of its persistence in plasma. NAC was quickly absorbed without any significant difference in tmax among the doses. With the 600 mg dose Cmax' AUC and MRT were greater than with a single 200 mg dose; after summing up the values of these parameters for the 200 mg doses no significant differences were observed in comparison to the single 600 mg dose in Cmax and AUC, while MRT resulted significantly higher.(ABSTRACT TRUNCATED AT 250 WORDS)

[Biochemical diagnosis of insulinoma: correlations between blood glucose, blood insulin and the titer of plasma non-esterified fatty acids during fasting and during various diagnostic tests]. / La diagnosi biochimica di insulinoma: correlazioni tra glicemia, insulinemia e tasso degli acidi grassi non esterificati del plasma nel corso del digiuno e di alcuni tests diagnostici.

The difficulty of clinical and biochemical diagnosis of insulinoma lies in the extreme variability of both clinical symptoms and glycaemia/insulinaemia levels, so that neither parameter is a totally reliable diagnostic indicator. The possibility of introducing a third parameter, the non-esterified fatty acid (FFA) level, to enhance the reliability of insulinoma diagnosis was therefore investigated. The behaviour of glucose, insulin and plasmatic FFA levels and the correlation of the three parameters were studied in 4 patients whose suspected insulinomas were later surgically confirmed. The tests applied were as follows: 24 hour fast followed by endovenous glucose, oral glucose administration, tolbutamide stimulus test, adrenaline and glucagon tests. The results revealed anomalies in insulinaemia and glycaemia behaviour such as have already, in part, been described in organic hyperinsulinism, e.g. the discrepancy between insulin and glucose levels after prolonged fasting. It was also clear that the parameters examined still leave much room for uncertainly in the biochemical diagnosis of insulinomas. Numerous anomalies were also observed in the behaviour of plasmatic FFA. For example lipid mobilisation was often reduced in conditions that normally stimulate it (fasting, the administration of catecholamines). Equally the prolonged blockage of lipid mobilisation was encountered in the presence of factors that normally reduce lipolysis (endovenous glucose). On the basis of these results it is suggested that a combined assessment of glucose and plasmatic FFA levels may be a more sensitive diagnostic indicator of insulinoma than the insulin/glucose ratio commonly reported in the literature. The mechanism behind the lipid mobilisation anomalies of insulinomas is also discussed in the light of its apparent connection with the glucose/insulin interaction characteristic of the condition.