Activation of endothelial cells during insulin-dependent diabetes mellitus: a biochemical and morphological study.

Plasma membrane lipid dynamics and cellular morphology were evaluated in endothelial cells obtained from umbilical cords of five women affected by insulin-dependent diabetes mellitus (IDDM) and six healthy pregnant women of similar age and gestational age. Endothelial cells were prepared by an adaptation of the method of Jaffe et al. Membrane fluidity was studied by means of the steady-state fluorescence anisotropy (r) of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), a fluorescent probe specifically anchoring at the membrane surface. Fluid phase endocytosis was evaluated by the measurement of the changes in fluorescence intensity of TMA-DPH at various times, owing to the internalization of the fluorescent marker in endocytic vesicles. The morphological and morphometric studies were performed by means of transmission electron microscopy (TEM). Endothelial cells obtained from IDDM women showed: (a) increased fluidity of the superficial region of the plasma membrane; (b) a more active fluid phase endocytosis compared with cells from healthy women; (c) increase in mitochondrial area, Weibel-Palade bodies and rough reticulum with wide cisternae. No statistically significant correlation was found between metabolic control and membrane fluidity and endocytosis. All the observed modifications suggest the presence of endothelial cell activation with membrane reshaping during IDDM. These alterations might play a central role in the pathophysiology of atherosclerosis and microangiopathy associated with diabetes mellitus.

Stimulatory effect on bone formation exerted by a modified chitosan.

A novel modified chitosan carrying covalently linked imidazole groups (average molecular weight 700,000, degree of substitution 0.28, degree of acetylation 0.08) was used to stimulate bone formation in an animal model. Lesions (7 mm diameter) were surgically made in the femoral condyle of sheep and treated with the modified chitosan. Within 40 d after surgery, the neoformed tissue occluded the surgical hole and assumed a trabecular structure in the peripheral area of the lesion, while looking like a mineralization nodule in the central part in association with a fibrous component. In the control, no sign of osteoinduction or reparative process was observed and bone marrow was rich in adipocytes.

A biochemical-morphological study on microvillus plasma membrane development.

The microvillus plasma membrane of the human placental syncytiotrophoblast at term has been extensively studied, while little is known about the characteristics of its development. The aim of the present work was to compare functional and structural properties of this membrane at early and term gestational age. Ten normal term placentas (40 weeks) and ten placentas at 10 weeks of gestational age were studied. The Na+/K+-ATPase activity is significantly decreased in the syncytiotrophoblast plasma membrane obtained from term placentas as compared to the early ones, with significant variation of maximum velocity (Vmax). The microviscosity, evaluated by the P parameter of DPH and Sn parameters of 5- and 16-NS, is increased in the term placentas compared to the early placentas. This alteration is accompanied by an increased cholesterol to phospholipids ratio in term placentas, while there is a decreased unsaturated to saturated fatty acid ratio. As follows from morphological studies, an increased mean diameter in the E face was observed in the term placenta with respect to the early placenta. The distribution factor DF, which indicates the particle aggregation state, decreased in the E face in the term placenta as compared to the early one. The present biochemical morphological study shows that a deep modification of the membrane is at the basis of the syncytiotrophoblast plasma membrane development.

Platelet abnormalities in idiopathic myelofibrosis: functional, biochemical and immunomorphological correlations.

BACKGROUND: An extensive study of platelet function was performed on 18 consecutive patients affected by idiopathic myelofibrosis (IM). MATERIALS AND METHODS: Clinical hematological and morphofunctional parameters were studied in IM patients and control subjects. Platelet tests, ultrastructural data, immunocytochemical von Willebrand factor detection, freeze fracturing results and free cytosolic calcium level were evaluated. RESULTS: Bleeding time was frequently found to be prolonged, but it never reached levels which could give any cause for concern. Aggregation by ADP, collagen and epinephrine was always altered, sometimes profoundly; on the contrary, agglutination by ristocetin was almost always normal, albeit occasionally increased. Plasma beta-TG and PF4 levels were found to be elevated in 11 and 12 patients, respectively. This indicated an abnormal release from platelet alpha-granules. Depletion of alpha-granules was also confirmed by the intraplatelet von Willebrand factor (vWF) labelling with colloidal gold particles bound to polyclonal antibodies against human vWF. In fact: 1) the number of positive alpha-granules/microm2 and per single platelet was reduced; 2) the intensity of the immunocytochemical reaction for single positive alpha-granules and for each platelet was significantly reduced. Freeze-fracturing studies showed an increase in the number of intra-membrane particles (IMP) on the P face of the platelet membrane with respect to normal platelets preincubated with ADP. However, no differences in their distribution or diameter were observed. High concentrations of free cytosolic calcium were always found and Ca++ ATPase activity was increased. Conversely, Na+/K+ ATPase activity was always reduced. CONCLUSIONS: We can hypothesize that the platelet membrane is altered in IM, resulting in facilitated activation, even by subliminal stimuli, and that this continuous platelet activation ultimately leads to alpha-granule depletion.

Inhibition of poly(ADP-ribose) polymerization preserves the glutathione pool and reverses cytotoxicity in hydrogen peroxide-treated lymphocytes.

DNA damage caused by oxygen radicals activates poly(ADP-ribosyl) polymerase (pADPRP), a nuclear enzyme that utilizes NAD+ as substrate. It has been demonstrated that pharmacological inactivation of pADPRP rescues human lymphocytes damaged by oxygen radicals, but not those damaged by equitoxic doses of ionizing radiation. In the present paper we demonstrate that the NAD+ pool decreases after both damaging treatments and is preserved in a similar fashion by pADPRP inhibition. On the contrary, the ATP pool, cell energy charge and reduced thiols are decreased only by the administration of oxygen radicals, and are preserved if poly(ADP)ribosylation is inhibited. In fact, treatment with oxidant agents depletes the cell energy pools owing to the simultaneous demands of the glutathione (GSH)/NADPH cycle and pADPRP-driven NAD+ consumption, while in irradiated cells only the latter mechanism operates. We suggest that, when pADPRP is inhibited, enough energy is available for the preservation of cell thiols, thereby allowing oxidant-treated cells to survive and undergo mitosis. Thus, GSH and energy shortage appear to be the main cause of cell death in oxidant-injured cells.

Gingival fibroblasts "in vitro" and Down's syndrome.

Gingival fibroblast cultures from four patients with Down's Syndrome (DS) and periodontal disease were compared with four in vitro age-matched fibroblast cultures of handicapped subjects (ND) also affected by periodontitis. The extra copy of chromosome 21 could alter growth regulation and biochemical mechanisms, so we examined quantitatively some DS phenotypical aspects to detect possible differences from those of controls. The growth properties of gingival fibroblast cultures from DS patients were more elevated than their ND age-matched controls. There were no differences in plasma membrane polarization and in neutral endopeptidase activity. The succinate-cytochrome C reductase activity decreases in DS fibroblasts compared with ND. Our results outline the difficulties to in using fibroblast cultures as an in vitro system to study premature ageing Down's Syndrome.

Gingival fibroblasts "in vitro" and Down's Syndrome.

Gingival fibroblast cultures from four patients with Down's Syndrome (DS) and periodontal disease were compared with four in vitro age-matched fibroblast cultures of handicapped subjects (ND) also affected by periodontitis. The extra copy of cromosome 21 could alter growth regulation and biochemical mechanisms, so we examined quantitatively some DS phenotypical aspects to detect possible differences from those of controls. The growth properties of gingival fibroblast cultures from DS patients were more elevated than their ND age-matched controls. There were no differences in plasma membrane polarization and in neutral endopeptidase activity. The succinate-cytochrome C reductase activity decreases in DS fibroblasts compared with ND. Our results outline the difficulties to inusing fibroblast cultures as an in vitro system to study premature ageing Down's Syndrome.

Prevalence of the primitive megakaryocyte progenitors (BFU-meg) in adult human peripheral blood.

The in vitro growth of early (megakaryocyte burst-forming units, BFU-meg) and late (megakaryocyte colony-forming units, CFU-meg) megakaryocyte (meg) progenitors has been evaluated in normal adult human peripheral blood (PB). All the experiments were carried out using CD34+ cells, which were assayed in a serum-free fibrinclot assay. PB BFU-meg were morphologically characterized as plurifocal aggregates containing greater than 50 cells/colony, distinct from unifocal CFU-meg, in a limiting dilution assay. At variance with PB CFU-meg, PB BFU-meg were unaffected by the complement-mediated cytotoxicity with anti-HLA-DR. The optimal source of colony-stimulating activity for PB BFU-meg growth was recombinant human interleukin 3 (rhIL-3; 100 U/ml), which supported a significantly higher number of BFU-meg in comparison with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 200 U/ml, p = 0.043). Combinations of rhIL-3 (100 U/ml) plus rhGM-CSF (200 U/ml), rhIL-3 plus recombinant human interleukin 6 (rhIL-6; 100 U plus 100 U/ml) or rhIL-3 plus rhGM-CSF plus rhIL-6 (100 U plus 200 U/ml plus 100 U/ml) failed to further increase the number of PB BFU-meg with respect to rhIL-3 (100 U/ml) alone. Both PB BFU-meg and CFU-meg were markedly inhibited, in a dose-dependent fashion, by increasing doses of human purified transforming growth factor-beta 1 (TGF-beta 1) (from 0.001 to 10 ng/ml). Finally, the CFU-meg/BFU-meg ratio in PB (0.52) was significantly different from that of normal bone marrow (2.3), clearly indicating that adult human peripheral blood predominantly carries primitive megakaryocytic progenitors.

In vitro inhibition of interferon alpha-2a antiproliferative activity by antibodies developed during treatment for essential thrombocythaemia.

BACKGROUND: Various authors have reported the development of anti-interferon (IFN) antibodies following IFN-alpha treatment for haematological malignancies. So far the methods for detecting these antibodies have not considered the antiproliferative activity of this IFN, which is its most important property in anticancer therapy. METHODS: In this in vitro study we evaluated the ability of anti-IFN alpha-2a neutralising antibodies to inhibit the antiproliferative activity of IFN alpha-2a and lymphoblastoid IFN alpha using megakaryocyte colony growth as the revelatory system. These antibodies were detected in two patients affected by essential thrombocythaemia (ET) who lost their haematological response to IFN alpha-2a, but responded to a subsequent treatment with lymphoblastoid IFN alpha. RESULTS AND CONCLUSION: The results show that the inhibition of megakaryocyte colony growth induced by IFN alpha-2a was totally suppressed in the presence of the two ET patients' sera, whereas the inhibition induced by lymphoblastoid IFN alpha was not significantly affected. These in vitro data demonstrate the high specificity and activity of these antibodies on the antiproliferative effect of IFN alpha-2a.

Phorbol esters, but not the hormonal form of vitamin D, induce changes in protein kinase C during differentiation of human histiocytic lymphoma cell line (U-937).

Human histiocytic lymphoma cells (U-937) undergo similar differentiation when incubated with the phorbol ester 12-0-tetradecanoyl phorbol-13-acetate (TPA) and 1,25-dihydroxycholecalciferol. In this action, TPA somehow implicates calcium-sensitive and phospholipid-dependent protein kinase (protein kinase C), which is rapidly and significantly affected by this inducer. On the contrary, 1,25-dihydroxycholecalciferol in its differentiating action does not involve protein kinase C thus suggesting that the secosteroid induces monocytic differentiation possible through a different mechanism of that of phorbol ester.

D-ribose inhibits DNA repair synthesis in human lymphocytes.

D-ribose is cytotoxic for quiescent human lymphocytes and severely inhibits their PHA-induced proliferation at concentrations (25-50 mM) at which other simple sugars are ineffective. In order to explain these effects, DNA repair synthesis was evaluated in PHA-stimulated human lymphocytes treated with hydroxyurea and irradiated. D-ribose, in contrast to other reducing sugars, did not induce repair synthesis and therefore did not apparently damage DNA in a direct way, although it markedly inhibited gamma ray-induced repair. Taking into account that lymphocytes must rejoin physiologically-formed DNA strand breaks in order to enter the cell cycle, we suggest that D-ribose exerts its cytotoxic activity by interfering with metabolic pathways critical for the repair of DNA breaks.

1 alpha, 25-dihydroxycholecalciferol and human histiocytic lymphoma cell line (U-937): the presence of receptor and inhibition of proliferation.

Human histiocytic lymphoma cells (U-937) contain high affinity binding protein for 1 alpha, 25-dihydroxycholecalciferol. The Kd (0.2 nM) and sedimentation coefficient on sucrose gradients (3.7 S) are similar to those reported for 1 alpha, 25-dihydroxycholecalciferol receptor in other tissues. This secosteroid added to the culture was able to inhibit cell proliferation in a dose dependent manner. Differentiation-associated properties such as phagocytic ability, C3 rosette formation, positive reaction for alpha- naphtyl acetate esterase as well as electron microscopy examination indicate that 1 alpha, 25-dihydroxycholecalciferol induces in vitro monocyte-macrophage differentiation in this monoblast-like cell line.

In vitro culture and ultrastructure analysis of peripheral blood cells separated by discontinuous albumin density gradient during blast crisis of chronic granulocyte leukemia.

Peripheral blood from 4 patients with chronic granulocytic leukemia during blast crisis has been investigated. One case has been studied before and after treatment with Arabinosyl-Cytosine and 6-Thioguanine. The nucleated blood cells have been separated by a discontinuous density gradient. Cells were obtained from six different gradient fractions (F1-F6: density ranging from 1.052 to 1.078). 0.5 X 10(5) cells from each density fraction have been cultured in agar culture system to evaluate the granulocyte-monocyte committed stem cells (Colony Forming Units-granulocyte monocyte: CFU-GM). Cells recovered from the same density fractions have been studied by electron microscopy to evaluate the number of less differentiated cells. A quantitative correlation between plating efficiency and blast cells number was carried out. The results indicate that the highest recovery of both CFU-GM and blast cells is present in light density fractions (specific density below 1.063). However a discrepancy between blast cell frequency and granulocyte-monocyte colony formation in the same density fractions appears to be evident. In the patient studied before and after treatment it appears that only one out of two light density fractions (F1) responds to the antiblastic treatment.

[The distribution of peroxidase-positive granuloblastic precursors of normal human bone marrow separated in discontinuous albumin gradient density]. / La distribuzione dei precursori granuloblastici perossidasi-positivi del midollo umano normale separati in gradiente di densita' discontinuo di albumina.

Human normal marrow cells from 5 donors have been separated via discontinuous density gradient according to the Dicke's technique and the morphology of granulocytic cells recovered from density fractions has been examined; peroxidase negative myeloblasts are concentrated in low density fractions (specific density below 1.063), while granulated myeloblasts (peroxidase positive) are concentrated at densities ranging between 1.063 and 1.073; promyelocytes and myelocytes have been observed in all fractions although their maximal concentration is at density 1.073; metamyelocytes and polymorphonucleated cells are concentrated in the highest density fraction (density 1.078); some differentiated cells which are peroxidase negative have been observed; they concentrate at a density lower than that of peroxidase positive cells which look morphologically identical.

[Growth in vitro of human bone marrow after freezing in liquid nitrogen: analysis of a case]. / La crescita in vitro del midollo osseo umano dopo congelamento in azoto liquido: analisi di un caso.

Human marrow is frozen according to the Schafer's technique; CFU-C proliferation and differentiation is then evaluated in relation to the time of storage in liquid nitrogen and to the time of in vitro culture in agar system according to the Pike and Robinson's technique. The highest plating efficiency is observed at the twelfth day of culture while in fresh cell suspension the growth peaks at the eighth day.