Carbohydrate interference of complement-dependent cell lysis

Summary The antibody-mediated cytotoxicity of three autoreactive sera, an allogeneic hyperimmune serum and a xenogeneic hyperimmune serum was abrogated by the presence of either glucosamine, galactosamine, lactulose or lactose. This inhibition could be overcome in a dose-dependent fashion by increasing the amount of complement in the cytotoxicity assay, but not by increasing the amount of antibody. Furthermore, the inhibition was specific for these sugars in that isomers and N-acetylated derivatives were not inhibitory. The results suggest that these sugars directly blocked events of the complement cascade.     

Etude de l'activité anticomplémentaire du sérum décomplémenté de rats hypo-, normo- et hyperglycémiques

Summary The anticomplementary activity of decomplemented rat serum was determined using a technique of inhibition of guinea-pig complement in a 50% hemolysis system. The anticomplementary activity of the serum varies inversely to the glycemic state of the rats, being highest in the serum from hypo, and lowest in that of hyperglycemic animals.     

Verhütung der Hitzekoagulation von Serumproteinen durch Zucker

Summary Sixteen individuals of different types of sugars have been investigated as to their ability of inhibiting the visible heat coagulation of serum. When bovine serum was diluted with an equal amount of water and maintained at 70° C during half an hour, the following sugars were able to prevent coagulation in a minimum concentration of 5% per volume:l-arabinose,d-ribose,l-ascorbic acid, and digitoxose.     

Destruction of virus infected cells by neutrophils and complement

The paper describes an antibody independent mechanism of cytotoxicity whereby virus infected but not uninfected cells are destroyed by the combined presence of neutrophils and complement.     

Fenretinide induces mitochondrial ROS and inhibits the mitochondrial respiratory chain in neuroblastoma

Fenretinide induces apoptosis in neuroblastoma by induction of reactive oxygen species (ROS). In this study, we investigated the role of mitochondria in fenretinide-induced cytotoxicity and ROS production in six neuroblastoma cell lines. ROS induction by fenretinide was of mitochondrial origin, demonstrated by detection of superoxide with MitoSOX, the scavenging effect of the mitochondrial antioxidant MitoQ and reduced ROS production in cells without a functional mitochondrial respiratory chain (Rho zero cells). In digitonin-permeabilized cells, a fenretinide concentration-dependent decrease in ATP synthesis and substrate oxidation was observed, reflecting inhibition of the mitochondrial respiratory chain. However, inhibition of the mitochondrial respiratory chain was not required for ROS production. Co-incubation of fenretinide with inhibitors of different complexes of the respiratory chain suggested that fenretinide-induced ROS production occurred via complex II. The cytotoxicity of fenretinide was exerted through the generation of mitochondrial ROS and, at higher concentrations, also through inhibition of the mitochondrial respiratory chain.     

Structural alterations in rat kidney proximal tubules perfused with fresh autologous serum

Two min of intraluminal perfusion of the rat proximal tubules with autologous serum induced marked ultrastructural alterations including extensive cytoplasmic vesiculation due to swelling of rough endoplasmic reticulum cisternae and occasional extrusion of nuclei and cytoplasm into the lumen. Within 4 min pronounced vesiculation of mitochondria was observed. These findings are consistent with the notion that serum-induced inhibition of proximal tubular fluid absorption is due to cell lysis, presumably mediated by complement activation.     

Structural alterations in rat kidney proximal tubules perfused with fresh autologous serum

Summary Two min of intraluminal perfusion of the rat proximal tubules with autologous serum induced marked ultrastructural alterations including extensive cytoplasmic vesiculation due to swelling of rough endoplasmic reticulum cisternae and occasional extrusion of nuclei and cytoplasm into the lumen. Within 4 min pronounced vesiculation of mitochondria was observed. These findings are consistent with the notion that serum-induced inhibition of proximal tubular fluid absorption is due to cell lysis, presumably mediated by complement activation.Acknowledgment. This study was supported by NIH grants AM16258, AM27857, a New York Health Council grant, and a Veterans Administration Research grant.     

Destruction of virus infected cells by neutrophils and complement

Summary The paper describes an antibody independent mechanism of cytotoxicity whereby virus infected but not uninfected cells are destroyed by the combined presence of neutrophils and complement.Supported by A.I. 14981 and by the MRC of Canada.Acknowledgment. We thank C.J. Wust for invaluable discussions.     

Production of monoclonal anti-Schistosoma mansoni antibodies. Preliminary study of their biological activities]

Monoclonal antibodies against Schistosoma mansoni have been produced by fusion of splenic lymphocytes from S. mansoni infected Rats and P3-X63-Ag8 BALB/c cells. In vitro and in vivo studies of the biological activities of these antibodies have led to the identification of IgE antibodies with a high reaginic activity and antibodies which in a complement dependent or eosinophil dependent system were shown to have a marked cytotoxicity for schistosomula in vitro. This methodology seems to open new perspectives for the study of antibody function in immunity against parasites as well as for the isolation of the corresponding target antigens.     

Rethinking synchronization of mammalian cells for cell cycle analysis

An analysis of different classes of forced or batch synchronization methods reveals why these methods, in theory, do not produce synchronized cultures. Cells may be aligned for a particular property after specific treatments, but these aligned cells do not correspond to any particular cell age during the normal cell cycle. The experimental methods analyzed are those that arrest cells with a G1 phase amount of DNA, those that inhibit DNA synthesis, and those that arrest cells at mitosis. Release of arrested cells from inhibition does not produce cells reflecting cells during the normal division cycle. Thus, cells produced by batch or forcing methods are not experimental models for analysis of the normal cell cycle.     

Drug susceptibility of PCA in WBB6F1-W/Wv mice

Our previous study revealed that passive cutaneous anaphylaxis (PCA) can be produced in congenitally mast cell-deficient WBB6F1-W/W^v (abbreviated as W/W^v) mice on sensitization with undiluted or slightly diluted allogeneic and xenogeneic antisera but not on sensitization with allogeneic monoclonal immunoglobulin (Ig)E and IgG1 antibodies regardless of the antibody concentration [1]. In view of these findings, the present study was conducted to characterize PCA in this strain from its drug susceptibilities using mast cell-bearing WBB6F1-+/+ (abbreviated as +/+) and B6D2F1 mice as references. PCA in W/W^v mice mediated by a low dilution (1  4) of hyperimmune serum to bovine serum albumin of the B6D2F1 mouse origin was markedly suppressed by CV-6209, an antagonist of platelet-activating factor (PAF), but not by antihistamines such as cyproheptadine and oxatomide. In contrast, PCA in +/+ and B6D2F1 mice mediated by a high dilution (1  128) of the anti-serum (virtually by IgG1 antibody) was nearly completely suppressed by antihistamines but not by CV-6209. A remarkable difference between PCA in W/W^v and reference mice was also observed in the susceptibility to monoclonal anti mouse granulocyte (Gr-1) antibody PCA in W/W^v mice was potently suppressed by the 1- to 3-day pretreatment with this antibody but that in references was not at all. Putting these present results together with the previous finding that anti-granulocyte antibody greatly reduces circulatory Gr-1⁺ leukocytes, 1 to 3 days after the treatment [2], it is highly probable that PCA in W/W^v mice mediated by some antibody isotypes other than IgE and IgG1 is produced by PAF mainly released from Gr-1⁺ cells, while IgG1 antibody-mediated PCA in mast cell-bearing reference mice is evoked by histamine derived from mast cells. PCA homologous to that in W/W^v mice could also be produced in the reference mice on sensitization with undiluted or slightly diluted antiserum, when generalized blueing due to excess IgG1 antibody was removed by the oxatomide treatment be fore the antigen challenge. Received 10 December 1997; received after revision 2 February 1998; accepted 23 February 1998     

Celiac disease IgA modulates vascular permeability in vitro through the activity of transglutaminase 2 and RhoA

Celiac disease is characterized by the presence of specific autoantibodies targeted against transglutaminase 2 (TG2) in untreated patients’ serum and at their production site in the small-bowel mucosa below the basement membrane and around the blood vessels. As these autoantibodies have biological activity in vitro, such as inhibition of angiogenesis, we studied if they might also modulate the endothelial barrier function. Our results show that celiac disease patient autoantibodies increase endothelial permeability for macromolecules, and enhance the binding of lymphocytes to the endothelium and their transendothelial migration when compared to control antibodies in an endothelial cell-based in vitro model. We also demonstrate that these effects are mediated by increased activities of TG2 and RhoA. Since the small bowel mucosal endothelium serves as a “gatekeeper” in inflammatory processes, the disease-specific autoantibodies targeted against TG2 could thus contribute to the pathogenic cascade of celiac disease by increasing blood vessel permeability.     

Characterization of the partial inhibition, by deficiency in serum factors, of multiplication of SV3T3 transformed fibroblasts]

Partial inhibition, by serum starvation, of multiplication of SV3T3 transformed fibroblasts was studied by cultivating cells in a medium containing 1% of serum. Two different and successive states are described: a first step, with a rate of multiplication for 24 hrs, which is lowered (2 instead of 3 in a normal medium containing 10% of serum) but which remains constant (i.e. exponential multiplication), the observed inhibition being reversed by normal medium; a second step, where the rate of multiplication for 24 hrs, decreases very fast, and which is not reversible.     

Glucosamine enhanced sperm-egg binding but inhibited sperm-egg fusion in mouse

In order to study the sperm-egg recognition mechanism on the surface of the plasma membrane, zonae were removed from mouse eggs by exposure to acidic conditions. Sperm binding to denuded eggs was then observed in the presence of various sugars. Among several carbohydrates tested, only glucosamine (GlcN) was found to increase the number of sperm bound to eggs while inhibiting sperm-egg fusion. The inhibition was reversible; when denuded eggs were transferred to a GlcN free medium, a high rate of polyspermy was observed.     

Lymphopenia and lymphocyte transformation in alcoholics.

The basis of peripheral blood lymphopenia observed in patients with chronic alcoholism and liver disease was investigated by examining the effect of sera of these patients on in vitro transformation of normal human peripheral blood lymphocytes. A positive correlation was demonstrated between the serum inhibition of phytohaemagglutinin- and pokeweed mitogen-induced transformation and the degree of lymphopenia. Thus serum factors may contribute to the observed lymphopenia by inhibiting lymphocyte production in vivo.     

Effects of gamma irradiation on dermal equivalents in vitro

Summary Dermal equivalents (DE), collagen lattices, were produced in vitro and used as a model for studying the possible role of a pure population of fibroblasts in post-radiotherapeutic dermal fibrosis. Single doses of gamma irradiation induced a partial inhibition of the collagen lattice retraction and of protein synthesis. The collagen production was less inhibited than was synthesis of non-collagen protein, which resulted in an increase of the relative amount of collagen synthesized by irradiated fibroblasts. These data suggest that gamma irradiation might be able to select some fibroblast clones able to produce increasing amounts of collagen. This selection process could be involved in the development of tissue fibrosis after therapeutic radiation.     

Effects of gamma irradiation on dermal equivalents in vitro

Dermal equivalents (DE), collagen lattices, were produced in vitro and used as a model for studying the possible role of a pure population of fibroblasts in post-radiotherapeutic dermal fibrosis. Single doses of gamma irradiation induced a partial inhibition of the collagen lattice retraction and of protein synthesis. The collagen production was less inhibited than was synthesis of non-collagen protein, which resulted in an increase of the relative amount of collagen synthesized by irradiated fibroblasts. These data suggest that gamma irradiation might be able to select some fibroblast clones able to produce increasing amounts of collagen. This selection process could be involved in the development of tissue fibrosis after therapeutic radiation.     

Role of serum components in the binding and phagocytosis of oxidatively damaged erythrocytes by autologous mouse macrophages

To investigate the role of autologous serum components in the recognition of damaged cells by macrophages, we examined the binding and phagocytosis of damage oxidatively damaged red blood cells with Cu2+ and ascorbate (oxRBCs) by autologous resident mouse peritoneal macrophages. The binding of oxRBCs by macrophages was independent of the presence of serum. However, phagocytosis by macrophages increased with serum concentration, and macrophages showed little ingestion of oxRBCs in a serum-free medium. Macrophages neither bound nor appreciably ingested native RBCs (before oxidation) in either the absence or presence of autologous serum. Mouse macrophages ingested significantly more native as well as oxRBCs in the presence of heat-inactivated fetal calf serum than in the presence of heat-inactivated mouse serum. Pretreated oxRBCs with normal serum were rarely ingested by macrophages in a serum-free medium. Phagocytosis of oxRBCs was significantly inhibited by depletion of IgG or calcium from serum, by heat inactivation of complement, or by antiserum against mouse C3. These results demonstrate that serum components such as IgG, C3, and calcium are involved in phagocytosis of oxRBCs by autologous macrophages.     

The complement system in age-related macular degeneration

Age-related macular degeneration (AMD) is a chronic and progressive degenerative disease of the retina, which culminates in blindness and affects mainly the elderly population. AMD pathogenesis and pathophysiology are incredibly complex due to the structural and cellular complexity of the retina, and the variety of risk factors and molecular mechanisms that contribute to disease onset and progression. AMD is driven by a combination of genetic predisposition, natural ageing changes and lifestyle factors, such as smoking or nutritional intake. The mechanism by which these risk factors interact and converge towards AMD are not fully understood and therefore drug discovery is challenging, where no therapeutic attempt has been fully effective thus far. Genetic and molecular studies have identified the complement system as an important player in AMD. Indeed, many of the genetic risk variants cluster in genes of the alternative pathway of the complement system and complement activation products are elevated in AMD patients. Nevertheless, attempts in treating AMD via complement regulators have not yet been successful, suggesting a level of complexity that could not be predicted only from a genetic point of view. In this review, we will explore the role of complement system in AMD development and in the main molecular and cellular features of AMD, including complement activation itself, inflammation, ECM stability, energy metabolism and oxidative stress.

     

视神经脊髓炎与水通道蛋白4的进展研究

水通道蛋白4(AQP4)是 NMO IgG(视神经脊髓炎特异性抗体)的靶抗原.水通道蛋白4抗体(AQP4抗体)通过破坏血脑屏障、参与抗原激活星形胶质细胞的经典补体途径等方法导致中枢神经炎症及细胞毒性而致病.AQP4抗体阳性有助于明确NMO诊断并指导临床治疗用药,从而减少 NMO复发频率及延缓残疾