Cytotoxicity of mouse peritoneal cells after alloimmunization]

CBA Mice were immunized by two intraperitoneal injections of 30 X 10(6) DBA/2 or C57BL/6 spleen cells at days--12 and--2. Peritoneal cell population was obtained at day zero by washing the peritoneal cavity of Mice. Adherent cells were then separated using a 2 hrs. incubation in "Falcon" plates followed by washing. This macrophage-rich peritoneal cell population was found nonspecifically cytotoxic against 51Cr labeled tumoral target cells: P815 X DBA/2 mastocytoma cells, EL4 X C57BL/L lymphoma cells and spontaneous lymphoma AKR cells (same H--2k as CBA). This adherent peritoneal cell cytoxicity was demonstrated after 24 hrs. incubation with the target cells. It was found in nonspecific combination as well as when using target cells syngeneic to the donor. These findings suggest that adherent peritoneal cell cytotoxicity could be at least partly due to macrophages and result from factor (s) released by sensitized lymphocytes in vivo in the same way as has been previously demonstrated in vitro.     

Serglycin – Structure and biology

Serglycin is a proteoglycan found in hematopoietic cells and endothelial cells. It has important functions related to formation of several types of storage granules. In connective tissue mast cells the covalently attached glycosaminoglycan is heparin, whereas mucosal mast cells and activated macrophages contain oversulfated chondroitin sulfate (type E). In mast cells, serglycin interact with histamine, chymase, tryptase and carboxypeptidase, in neutrophils with elastase, in cytotoxic T cells with granzyme B, in endothelial cells with tissue-type plasminogen activator and in macrophages with tumor necrosis factor-α. Serglycin is important for the retention of key inflammatory mediators inside storage granules and secretory vesicles. Serglycin can further modulate the activities of partner molecules in different ways after secretion from activated immune cells, through protection, transport, activation and interactions with substrates or target cells. Serglycin is a proteoglycan with important roles in inflammatory reactions. Received 2 October 2007; received after revision 7 November 2007; accepted 12 November 2007     

Resistance of in vivo-selected spontaneously transformed cells and Rous sarcoma virus-transformed cells to macrophage-mediated cytotoxicity.

The cytotoxic activity (CTA) of activated peritoneal macrophages (MP) on variant lines of Syrian hamster embryo (HE) cells of differing malignant characteristics was studied. The target cells were a line of low-malignant cells resulting from spontaneous transformation of HE cells in vitro (STHE strain), and malignant variants selected from them in vivo (STHE-LM-4, STHE-LM-8, and STHE-75/18 strains). In addition, we used cells of the HET-SR-1 strain; these are HE cells transformed in vitro by a tumorigenic Rous sarcoma virus (Schmidt-Ruppin strain, RSV-SR), or the TU-SR strain induced by RSV-SR in vivo. Thioglycollate-elicited peritoneal MP from Syrian hamsters were activated in vitro with bacterial levan, LPS or MDP and used as effector cells. MP-mediated cytolysis was determined by means of a 42-h radioactivity release assay with 3H-thymidine-labeled target cells. We found that only the parental STHE cells were susceptible towards fully-activated MP-mediated CTA. All three of the in vivo-selected malignant variants of the STHE cell sublines, as well as the tumorigenic RSV-SR transformants, were resistant to cytolysis by activated MP. Non-activated thioglycollate-elicited MP did not lyse any of the tumor cells studied.     

Resistance of in vivo-selected spontaneously transformed cells and Rous sarcoma virus-transformed cells to macrophage-mediated cytotoxicity

The cytotoxic activity (CTA) of activated peritoneal macrophages (MP) on variant lines of Syrian hamster embryo (HE) cells of differing malignant characteristics was studied. The target cells were a line of low-malignant cells resulting from spontaneous transformation of HE cells in vitro (STHE strain), and malignant variants selected from them in vivo (STHE-LM-4, STHE-LM-8, and STHE-75/18 strains). In addition, we used cells of the HET-SR-1 strain; these are HE cells transformed in vitro by a tumorigenic Rous sarcoma virus (Schmidt-Ruppin strain, RSV-SR), or the TU-SR strain induced by RSV-SR in vivo. Thioglycollate-elicited peritoneal MP from Syrian hamsters were activated in vitro with bacterial levan, LPS or MDP and used as effector cells. MP-mediated cytolysis was determined by means of a 42-h radioactivity release assay with³H-thymidine-labeled target cells. We found that only the parental STHE cells were susceptible towards fully-activated MP-mediated CTA. All three of the in vivo-selected malignant variants of the STHE cell sublines, as well as the tumorigenic RSV-SR transformants, were resistant to cytolysis by activated MP. Non-activated thioglycollate-elicited MP did not lyse any of the tumor cells studied.     

Phospholipase C activation via a GTP-binding protein in tumoral islet cells stimulated by carbamylcholine

Summary Carbamylcholine and GTP act synergistically in stimulating the production of [³H]inositol-1-phosphate by digitonized tumoral islet cells (RINm5F line) prelabeled with myo-[2-³H(N)]inositol. The response to these two agents is similar to that evoked by GTPS. These findings suggest that a GTP-binding regulatory protein couples the occupancy of muscarinic receptors to activation of phospholipase C in pancreatic islet cells.This work was supported by grants from the Belgian Foundation for Scientific Medical Research.     

Phospholipase C activation via a GTP-binding protein in tumoral islet cells stimulated by carbamylcholine

Carbamylcholine and GTP act synergistically in stimulating the production of [3H]inositol-1-phosphate by digitonized tumoral islet cells (RINm5F line) prelabeled with myo-[2-3H(N)]inositol. The response to these two agents is similar to that evoked by GTP gamma S. These findings suggest that a GTP-binding regulatory protein couples the occupancy of muscarinic receptors to activation of phospholipase C in pancreatic islet cells.     

Granzyme B is recovered by natural killer cells via clathrin-dependent endocytosis

When they recognize a target cell, natural killer (NK) cells mount an attack to kill the target by exerting their cytotoxicity via the exocytosis of cytotoxic granules. Although the details of this process (which includes the movement of cytotoxic granules in the immune synapse and their fusion with the plasma membrane, releasing granzymes and perforin into the synaptic cleft) are relatively better understood, the post-exocytosis regulation of the process is still largely unknown. Here we show that a clathrin-dependent endocytosis stimulated by target cell occurs in NK92 cell line, which is closely correlated with granzyme B recovery. Inhibition of the endocytosis significantly attenuates the cytotoxicity of NK92 cells. The NK cell recovery of its released effector molecules, in turn, suggests that endocytosis may well play a key role in the post exocytosis regulation of immune cells.     

The pathogenesis of cardiac fibrosis

Cardiac fibrosis is characterized by net accumulation of extracellular matrix proteins in the cardiac interstitium, and contributes to both systolic and diastolic dysfunction in many cardiac pathophysiologic conditions. This review discusses the cellular effectors and molecular pathways implicated in the pathogenesis of cardiac fibrosis. Although activated myofibroblasts are the main effector cells in the fibrotic heart, monocytes/macrophages, lymphocytes, mast cells, vascular cells and cardiomyocytes may also contribute to the fibrotic response by secreting key fibrogenic mediators. Inflammatory cytokines and chemokines, reactive oxygen species, mast cell-derived proteases, endothelin-1, the renin/angiotensin/aldosterone system, matricellular proteins, and growth factors (such as TGF-β and PDGF) are some of the best-studied mediators implicated in cardiac fibrosis. Both experimental and clinical evidence suggests that cardiac fibrotic alterations may be reversible. Understanding the mechanisms responsible for initiation, progression, and resolution of cardiac fibrosis is crucial to design anti-fibrotic treatment strategies for patients with heart disease.     

The peritoneal “soil” for a cancerous “seed”: a comprehensive review of the pathogenesis of intraperitoneal cancer metastases

Various types of tumors, particularly those originating from the ovary and gastrointestinal tract, display a strong predilection for the peritoneal cavity as the site of metastasis. The intraperitoneal spread of a malignancy is orchestrated by a reciprocal interplay between invading cancer cells and resident normal peritoneal cells. In this review, we address the current state-of-art regarding colonization of the peritoneal cavity by ovarian, colorectal, pancreatic, and gastric tumors. Particular attention is paid to the pro-tumoral role of various kinds of peritoneal cells, including mesothelial cells, fibroblasts, adipocytes, macrophages, the vascular endothelium, and hospicells. Anatomo-histological considerations on the pro-metastatic environment of the peritoneal cavity are presented in the broader context of organ-specific development of distal metastases in accordance with Paget’s “seed and soil” theory of tumorigenesis. The activity of normal peritoneal cells during pivotal elements of cancer progression, i.e., adhesion, migration, invasion, proliferation, EMT, and angiogenesis, is discussed from the perspective of well-defined general knowledge on a hospitable tumor microenvironment created by the cellular elements of reactive stroma, such as cancer-associated fibroblasts and macrophages. Finally, the paper addresses the unique features of the peritoneal cavity that predispose this body compartment to be a niche for cancer metastases, presents issues that are topics of an ongoing debate, and points to areas that still require further in-depth investigations.     

Anthrax lethal toxin: a weapon of multisystem destruction

Lethal toxin (LT) is a major virulence factor secreted by anthrax bacteria. It is composed of two proteins, PA (protective antigen) and LF (lethal factor). PA transports the LF inside the cell, where LF, a zinc-dependent metalloprotease cleaves the mitogen activated protein kinase kinase (MAPKK) enzymes of the mitogen activated protein kinase (MAPK) signaling pathway, thereby impairing their function. This disruption of the MAPK pathway, which serves essential functions such as proliferation, survival and inflammation in all cell types, results in multisystem dysfunction in the host. The inactivation of the MAPK pathway in both macrophages and dendritic cells leads to inhibition of proinflammatory cytokine secretion, downregulation of costimulatory molecules such as CD80 and CD86, and ineffective T cell priming. The net result is an impaired innate and adaptive immune response. Endothelial cells of the vascular system undergo apoptosis upon LT exposure, also likely due to inactivation of the MAPK pathway. The activity of various hormone receptors such as glucocorticoids, progesterone and estrogen is also blocked, due to inhibition of p38 MAPK phosphorylation, thus affecting the bodys response to stress. The present review summarizes the various disarming effects of Bacillus anthracis through the use of a single weapon, the lethal toxin.Received 12 June 2004; received after revision 13 July 2004; accepted 28 July 2004     

Long-term production and culture of clones of human T-lymphocytes

Culture of blood T lymphocytes collected from normal individuals and cancer patients were carried out in presence of T cell growth factor (TCGF); these cultures presented cytotoxic activity directed against different targets (lectin activated cells, autologous cancer cells, antibody coated cells and K 562). In order to study separately the different effector subpopulations, isolation of single cultured cells were performed with the help of a micropipette under microscope and monoclonal cultures were carried out in presence of TCGF. In the preliminary cytotoxic assays performed in the clones: (1) a marked activity directed against lectin targets was observed in many clones and (2) an important N K activity was exhibited by the clone 45 B9 (65% of the tested cells lysed human lymphoma K 562 cells).     

Iron deposits in the chronically inflamed central nervous system and contributes to neurodegeneration

Neurodegenerative disorders are characterized by the presence of inflammation in areas with neuronal cell death and a regional increase in iron that exceeds what occurs during normal aging. The inflammatory process accompanying the neuronal degeneration involves glial cells of the central nervous system (CNS) and monocytes of the circulation that migrate into the CNS while transforming into phagocytic macrophages. This review outlines the possible mechanisms responsible for deposition of iron in neurodegenerative disorders with a main emphasis on how iron-containing monocytes may migrate into the CNS, transform into macrophages, and die out subsequently to their phagocytosis of damaged and dying neuronal cells. The dying macrophages may in turn release their iron, which enters the pool of labile iron to catalytically promote formation of free-radical-mediated stress and oxidative damage to adjacent cells, including neurons. Healthy neurons may also chronically acquire iron from the extracellular space as another principle mechanism for oxidative stress-mediated damage. Pharmacological handling of monocyte migration into the CNS combined with chelators that neutralize the effects of extracellular iron occurring due to the release from dying macrophages as well as intraneuronal chelation may denote good possibilities for reducing the deleterious consequences of iron deposition in the CNS.     

Cell-mediated cytotoxicity by natural killer and killer cells, lipid peroxidation and glutathione

In the course of spontaneous cell-mediated cytotoxicity (SCMC) and antibody-dependent cell-mediated cytotoxicity (ADCC) with human peripheral lymphocytes as effector cells, no lipid peroxidation occurred as measured by the production of ethane and thiobarbituric acid-reactive material. Furthermore, impairment of major cellular defense systems of target cells (K562 cells for SCMC, Chang liver cells for ADCC), by decreasing their glutathione content, had no effect on either lipid peroxidation or the cytotoxic response. These findings indicate that peroxidative damage is not a mechanism of NK and K cell-mediated cytotoxicity.     

Ontogenic development of peptide hormones in the mammalian fetal pancreas

Summary The ontogeny of insulin, glucagon, PP and somatostatin in the mammalian fetal pancreas has been examined in recent years largely by immunocytochemistry and in some instances by radioimmunoassay. Complete ontogenic data are available only for the rat, human pig and sheep. Figure 3 compares the time of appearance of the endocrine cell-types within the fetal pancreas when the periods of gestation of the four species are converted to a uniform scale. The striking ontogenic difference in the rat probably reflects the immaturity of the rodent fetus at birth compared with the human, pig and sheep. In the fetal pancreas, differences in cell number of glucagon and PP cells in the dorsal and ventral lobes become apparent from an early gestational period. Factors responsible for the functional and structural maturation of the fetal pancreatic endocrine cells and the processes involved in pancreatic organogenesis are poorly understood. Studies in these areas would have clinical implications since it may be possible in the future to employ agents for selective replication of fetal -cells for transplantation in patients with Type I diabetes, bearing in mind that such cells must have the capacity to respond to normal stimuli and repressors when transplanted. The presence of the other islet cell-types may be obligatory for these appropriate responses. This would require a more complete knowledge of those factors which produce the normal selectivity of the four hormonal cell-types.     

S-adenosyl-L-homocysteine: A non-cytotoxic hypomethylating agent

The cytotoxic effect caused by the hypomethylating agent S-adenosyl-L-homocysteine (SAH) was compared with that of two drugs commonly used to induce DNA hypomethylation, 5-azacytidine and 5-aza-2-deocycytidine. Two in vitro cytotoxicity tests, the tetrazolium MTT assay and the intracellular lactate dehydrogenase (LDH) activity test, suggest that SAH induces hypomethylation without causing any cytotoxic effect. We propose the use of SAH as a non-cytotoxic agent which may be more suitable for inducing experimental DNA hypomethylation.     

Bactericidal activity againstPseudomonas aeruginosa is acquired by cultured human monocyte-derived macrophages after uptake of myeloperoxidase

Myeloperoxidase (MPO) is an enzyme located within polymorphonuclear neutrophils capable of producting cytotoxic oxidant species that are particularly active against bacteria with polysaccharide capsules.Pseudomonas aeruginosa (10⁶ bacteria per 1 ml) are killed within 1 h in vitro by a MPO/H₂O₂/Cl^– system (48 mU=132 ng of MPO). The question arose as to whether human macrophages would acquire cytotoxic activity when loaded with this enzyme. Monocytes were therefore isolated from human blood and cultured for up to ten days to induce maturation to macrophages. These cells lost endogenous MPO within five days while H₂O₂ production in response to stimulation by phorbol myristate acetate (10^–6M) decreased to 23% within ten days. On the other hand, their capacity to take up exogenous MPO increased fourfold from day three to day ten. Human macrophages cultured from eight days (when both H₂O₂ production and MPO uptake were sufficient) were therefore used to study the effects of MPO uptake on cytocidal activity againstPseudomonas aeruginosa. After a 1 h MPO loading period, macrophages (5×10⁵ cells per ml) were incubated in the presence of bacteria (0.5 to 2×10⁶ bacteria per ml) for 2 h at 37°C. At a bacteria/macrophage ratio of 1, only 34.8±7.0% of bacteria survived (compared to killing by non-loaded macrophages), while 74.4±9.3% survived at a ratio of 4. From these results, we conclude that loading macrophages with exogenous MPO could enhance their microbicidal activity, suggesting a potentially useful therapeutic application.     

Introduction: p53 – the first twenty years

The p53 protein was discovered 20 years ago, as a cellular protein tightly bound to the large T oncoprotein of the SV40 DNA tumour virus. Since then, research on p53 has developed in many exciting and sometimes unexpected directions. p53 is now known to be the product of a major tumour suppressor gene that is the most common target for genetic alterations in human cancer. The nonmutated wild-type p53 protein (wtp53) is often found within cells in a latent state and is activated in response to various intracellular and extracellular signals. Activation involves an increase in overall p53 protein levels, as well as qualitative changes in the protein. Upon activation, wtp53 can induce a variety of cellular responses, most notable among which are cell cycle arrest and apoptosis. To a great extent, these effects are mediated by the ability of p53 to activate specific target genes. In addition, the p53 protein itself possesses biochemical functions which may facilitate DNA repair as well as apoptosis. The role of p53 in normal development and particularly in carcinogenesis has been elucidated in depth through the use of mouse model systems. The insights provided by p53 research over the years are now beginning to be utilized towards better diagnosis, prognosis and treatment of cancer.