Morpho-functional changes in human tendon tissue.

Insertion tissue biopsies of right arm common extensor tendons from 11 patients with chronic lateral epicondylitis were processed for light and electron microscopy. The subjects were aged between 38 and 54 years (only one was 25). The specimens showed a variety of structural changes such as biochemical and spatial alteration of collagen, hyaline degeneration, loss of tenocytes, fibrocartilage metaplasia, calcifying processes, neovascularization and vessel wall modifications. Tissue alterations were evident in limited zones of the tendon fibrocartilage in which the surgical resection was generally visible. The areas where the degenerative processes were localized, were restricted and in spatial contiguity with morphologically normal ones. The observed cases presented histological and electron microscopic findings that characterize lateral epicondylitis as a degenerative phenomenon involving all tendon components.

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.

Lineage-related susceptibility of human hemopoietic cell lines to apoptosis.

Apoptosis plays a fundamental role in shaping normal hematopoiesis. We have investigated the relationship existing between susceptibility to apoptosis and lineage commitment in hemopoietic cells. The presence and degree of apoptosis were investigated in myeloid (HL-60 and K562), T (Jurkat and MOLT-4), and B (CESS and Raji) lymphoid cell lines by using a variety of techniques-transmission electron and light microscopy, flow cytometry and DNA gel electrophoresis. The major achievement of this study is that hematopoietic cells respond to different chemical (staurosporin, tiazofurin, camptothecin) and physical (hyperthermia or hypothermia) stimuli by apoptosis in a lineage-related way. Moreover, with respect to the methods used to detect apoptosis, a strong correlation was observed between the presence of the hypodiploid peak determined by flow cytometry and the DNA laddering evaluated by gel electrophoresis, but both techniques failed to demonstrate the presence of apoptosis in some cases. We conclude that cells of different hematopoietic lineages mostly show a lineage-related behaviour in their apoptotic response to different stimuli, suggesting that the lineage commitment and the stage of differentiation can confer different sensitivities to specific apoptotic stimuli. Moreover, morphological techniques still represent the most reliable approach to detect apoptosis in hemopoietic cells.

Age-dependent decreases of NK cell phosphoinositide turnover during spontaneous but not Fc-mediated cytolytic activity.

The progressive increase in the number of peripheral NK cells found in the elderly does not correlate with a corresponding increase in lytic activity. On the contrary, a decreased function of circulating NK cells purified from old subjects was observed on a per cell basis. Most of the studies on NK cells have focused on late events such as lytic activity. In view of this, little is currently known about the modification of the early signalling pathways of NK cells in elderly people. This study investigated whether the modification of NK lytic activity could be related to differences in the metabolic pattern of activation of these cells in the elderly. NK cells were negatively purified by immunomagnetic depletion from the peripheral blood of selected old and young healthy subjects. Hydrolysis of inositol phospholipids was measured following incubation with K562 target cells and/or CD16 mAb for different times. Our data show that there is a pronounced age-related decrease in the ability to generate total inositol monophosphates and, particularly, inositol trisphosphates by NK cells following K562 stimulation (spontaneous cytolytic activity) together with an attenuated and delayed hydrolysis of phosphatidylinositol bisphosphate, while phosphoinositide turnover is preserved following Fc triggering (antibody-dependent cell-mediated cytotoxicity). These results confirm that, also in old subjects, different biochemical pathways of activation are involved in NK cells when target or antibody-mediated triggering occurs and may aid the development of experimental and therapeutic strategies to counteract declines in cell mediated immune functions associated to advancing age.

Kinetics of in vitro natural killer activity against K562 cells as detected by flow cytometry.

Natural killer (NK) cells bind to K562 tumor target cells in vitro and kill them. The binding and cytotoxic activities of NK cells are tightly related to each other: degranulation of the cytotoxic effector is the basis for target cell damage and a consequence of effector-target recognition and binding. However, the two phases of NK activity, binding and killing, have always been measured separately by various methodologies and under different experimental conditions, because of the lack of a comprehensive methodology able to measure both of them at one time. Here we describe the simultaneous measurement of the binding and killing activities against K562 of resting and cytokine (IL-2 or IL-12)-stimulated NK cells by flow cytometry. NK, K562 and conjugates can be identified and measured by flow cytometry on the basis of NK mAb staining and target cells autofluorescence (Binding Plot). Within each population of the binding plot, killed targets can be identified and measured by their scatter characteristics (Cytotoxicity Plot). We show that i) the conjugate formation is enhanced in cytokine-stimulated cells, even at relatively short co-incubation times; ii) the conjugate release is also accelerated by cytokines; iii) the conjugate release is always quicker than the induction of the morphological changes in the target cell that generate its modified scattering properties.

Apoptosis induced by NK cells is modulated by the NK-active cytokines IL-2 and IL-12.

NK cells kill sensitive targets via exocytosis of cytoplasmic granules containing membrane damaging perforins and DNA damaging granzymes. Therefore, the target cell can either die by necrosis or by apoptosis. A third, non-secretory, mechanism of killing mediated by Fas-FasL interaction can be used by activated NK cells to kill Fas+ targets. Here, we have studied the modulation exerted by two NK active cytokines, IL-2 and IL-12, on the apoptosis-inducing activity of NK cells in NK-sensitive Fas- K562 and NK-insensitive Fas+ Raji cells. Our results show that apoptosis is preferentially induced at low target:effector ratios. Resting NK cells virtually do not induce apoptosis in target cells while IL-2 stimulation endows NK cells with the capacity of inducing apoptosis and IL-12 further enhances this effect against both targets. Finally, we demonstrate that cytokine-stimulated NK cells use both granzyme and Fas-FasL pathways of apoptosis induction.

Different approaches to the study of apoptosis.

The morphological features of cell undergoing programmed cell death is well known and has been widely described in a number of experimental models with a variety of apoptotic triggering agents. Despite the similar cell behaviour, underlying molecular events seem variable and only partially understood. A multiple approach appears crucial to better clarify the phenomenon. The first technique, DNA gel electrophoresis, allows the identification of fragmented DNA and has been long considered the hallmark of apoptosis. Different patterns of DNA cleavage, which can be identified by conventional or "pulsed-field gel" electrophoresis, are presented and discussed. "In situ" labelling methods are also described both with terminal deoxynucleotidyl transferase and DNA polymerase I, aimed at the study of the distribution of DNA cleavage areas. Flow cytometry is also proposed and different technical approaches, based on different laser utilizations, are discussed. Ultrastructural analysis, allowing the study of apoptotic cell details, is finally considered.

Standardization of a micro-cytotoxicity assay for human natural killer cell lytic activity.

Cytotoxicity assays are widely used to evaluate the functional activity of NK and T cells against tumour target cells and the release of radioactive sodium chromate from labelled target cells is still the most commonly used marker of target lysis in culture supernatants. We describe here the standardization of a micro-cytotoxicity test in which the number of cytolytic effector and tumour target cells have been decreased by a factor of 10. The release obtained by 500 tumour target cells was compared with the release obtained by 5000 target cells in the standard cytotoxicity assay for target:effector cell ratios from 1:1 to 1:100. Both gamma and beta emissions of the 51Cr isotope were evaluated to determine the assay release. The results obtained by the micro-cytotoxicity assay (500 target cells) were comparable to those of the standard assay (5000 target cells) and 51Cr release evaluation using the gamma counter was the most sensitive method of determining lytic activity using 500 tumour target cells. beta counter evaluation using solid phase scintillation was found to be a reproducible alternative method, even if the lytic curves cannot be compared with those obtained using the traditional method.

Commercial serum-free media: hybridoma growth and monoclonal antibody production.

Various growth factors, hormones and proteins present in serum are presumed to be responsible for its growth-stimulating activity in culture media. However, synthetic or serum substitute media supporting cell growth are advantageous when it is necessary to standardize culture conditions, particularly when cell products are used. In this study we have evaluated and compared the effects of some commercially available serum substitute media (10% NU Serum, 10% BMS, 2% Ultroser HY, 1% ITS + Premix, 1% Nutridoma, Ultradoma, FEB100, DCCM1 and DCCM2) on growth and immunoglobulin production in different hybridoma cell lines. Six different hybrids were studied: OKT3, OKT4 and OKT8 (producing monoclonal antibodies against CD3, CD4 and CD8 molecules), HB43 and HB57 (producing monoclonal antibodies against human IgG and IgM), and CRL 8019 (producing monoclonal antibodies against high-molecular weight CEA). Several parameters were evaluated, such as viability, doubling time, DNA synthesis, monoclonal antibody production and cell cycle under different culture conditions. Since not all of the hybridomas grew equally well in the same serum substitute media, one synthetic medium cannot be used for all the lines. We found that the serum-free media that best supported hybridoma growth were Nutridoma, DCCM1, DCCM2, NU Serum and FEB100.

Membrane modifications in the course of hepatocyte isolation.

A transmission E/M, scanning E/M and freeze fracture ultrastructural study has been performed on the rat hepatocyte in the course of isolation from the liver parenchyma. The cell submicroscopic aspect indicates a good morpho-functional preservation from the liver perfusion to the final stages of cell isolation. The freeze fracture membrane analysis evidentiates the constant presence of gap junctions and tight junctions, characterized by particular structural alterations, probably due to progressive functional uncoupling. The persistence of these cell differentiations until complete cell isolation may be considered a further morphological expression of the maintenance of the differentiated stage of the hepatocyte. Fragments of membranes from adjacent cells, still adherent to isolated hepatocyte surfaces, can also be occasionally detected by freeze-fracture techniques.

[Pericardial effusions as the initial manifestation of extracardiac tumors. A discussion of 2 cases and a review of the literature]. / I versamenti pericardici come prima manifestazione di tumori extracardiaci. Discussione su due casi e revisione della letteratura.

The authors describe two cases of cardiac tamponade that were the first manifestation of an extracardiac tumor; they stress that this type of presentation described in the literature as an extremely rare occurrence, has become more frequent in recent years as a result of both a higher prevalence in absolute terms and of a more reliable detection thanks to the availability of more sensitive and more practicable diagnostic techniques. Having briefly described the pathophysiology of pericardial effusions with special reference to those of neoplastic origin, the authors detail the most sensitive tools for a correct diagnosis.

Age-associated changes in CD8+ and CD16+ cell reactivity: clonal analysis.

A cloning technique was used to estimate the frequency of proliferating T cell precursors, the growth capacity of clone-forming cells and the functional activity of clones established in vitro from peripheral blood lymphocytes of young and old people. The mean frequency of proliferating precursors was lower in the elderly as was the proliferative capacity of CD8+ clones. In contrast, CD4+ and CD16+ clones showed a proliferation similar to that obtained from young subjects. When the clones were examined for their functional activity, CD4+ clones from both groups failed to show any cytolytic activity, while CD8+ clones exerted cytolysis against K562 and in antibody-dependent cell-mediated cytotoxicity but this function was reduced in clones derived from old subjects. Similarly, CD16+ clones from the elderly showed a decreased activity at some effector-to-target cell ratios. We conclude that the impaired functional activity (T or NK-dependent) found in the peripheral blood of aged subjects persists after in vitro selection when these cells are analysed at clonal level.

A morphological study of membrane lesions during natural killer-mediated lysis.

Early and late events occurring during cell-mediated cytolysis have been examined ultrastructurally using transmission, scanning and freeze-fracture electron microscopy. K562 erythroleukemic cells were used as target for human natural killer lymphocytes, a subpopulation involved in host defense against tumor, virus infected and allogeneic cells. Cell binding between target and effector is already evident after 15 min, but membrane lesions and target hydration appear after 30 min incubation. At 60 min K562 cells appear swollen and microvilli-deprived. Progressive membrane disruption is also evident and the underlying cytoplasmic material is clearly exposed after 120 min. Submicroscopic membrane lesions appear to be early events in comparison with 51Cr (isotope) release from damage cells.

[Efficacy and tolerability of captopril-hydrochlorothiazide vs amiloride-hydrochlorothiazide combination in mild to moderate arterial hypertension]. / Efficacia e tollerabilità dell'associazione captopril-idroclorotiazide vs amiloride-idroclorotiazide nell'ipertensione arteriosa medio-lieve.

Forty patients, mean age 56.87 yrs. with light or moderate essential arterial hypertension were randomized double-blind into two subgroups of 20 subjects each, and submitted to daily combined drug treatment with either captopril 50 mg + hydrochlorothiazide 25 mg (group A) or amiloride 5 mg + hydrochlorothiazide 50 mg (group B). Patients were monitored after the washout period and after 4 and 8 weeks of treatment approximately 20-24 after the last dose. The following parameters were studied: blood pressure, heart rate, body weight, untoward side effects. Standard laboratory tests were performed in all patients after washout and at the end of the 8-week treatment period. Both combinations significantly reduced pressure values but the captopril-hydrochlorothiazide combination reduced blood pressure more readily and proved more effective in reducing diastolic values. There were no dropouts due to subjective side effects which were of little relevance and were equally distributed among the two groups. As for laboratory data, patients taking the captopril-hydrochlorothiazide combination had a statistically significant increase in blood glucose. Neither combination induced significant changes in the other parameters, especially as far as potassemia was concerned.

Evaluation of NK-to-target cell binding and evidence for T cell conjugates by flow cytometry.

A flow cytometric methodology was set up to assess the binding capability of peripheral blood NK and T cells to the K562 tumor cell line. Differential side scatter characteristics between effectors and targets were used to analyze conjugated and unconjugated cells. The previous labeling of NK and T cells with anti-Leu 11c and anti-Leu 4 monoclonal antibodies, allowed the distinction between unconjugated non-fluorescent and conjugated fluorescent targets and the percentual evaluation of bound anti-Leu 11c(+) and anti-Leu 4(+) cells.

A high yield technique for freeze-fracturing of small fractions of isolated cells.

A simple, high-yield technique for the freeze-fracturing of small amounts of isolated cells is described. A drop of cells fixed in suspension is deposited on a polylysine-treated coverslip, forming a monolayer through electrostatic forces. After cryoprotection, the coverslip is inverted on a gold carrier covered with Vinol and then frozen in liquid nitrogen. The monolayer will be fractured by advancing the knife under the coverslip. Large areas of cell surface can be exposed despite their low number, such as that obtainable after cell sorting by flow cytometry.

Natural killer function in flow cytometry. I. Evaluation of NK lytic activity on K562 cell line.

Flow cytometry has been employed to establish an NK assay using the K562 target cell line. These cells show a perpendicular light scatter (PLS) characteristically different from lymphocytes. Other physical parameters, such as forward light scatter (FLS), do not discriminate between the two populations, since dead K562 cells display similar FLS characteristics as effector cells. Killed cells are stained with propidium iodide and followed by flow analysis. It was advisable to add the dye in the medium so that, as long as the target cells are killed they will also be stained. Moreover the flow cytometric and trypan blue evaluation of target cell death rate showed a stronger correlation than did either test with the conventional 51Cr release assay, the first two methods both being based on the same biological mechanism.