In vivo modulation of rat complement activities by infusion of anti-H antibodies

Regulation of the activity of the alternative pathway of complement occurs by the plasma proteins I and H. H is able not only to prevent formation of the amplification convertase C3bBb but also to cause the decay-dissociation of Bb from C3bBb. The present studies have investigated the role of H in vivo. Affinity-purified goat (Fab')2-anti H was infused intravenously in rats, and its effect on C4, C3 and CH50 activities was assessed. In addition, H, C3, C4 and B were quantitated by immunochemical methods. H depletion was dependent on the dose of anti-H, and maximal consumption in vivo occurred between 5 and 15 min. A maximum of 51% and 77% hemolytic C3-consumption was seen after 15 min with 3.8 and 8.4 mg anti-H, respectively. The injection of 6.3 mg affinity-purified goat IgG anti-rat H caused 96.8 +/- 0.7% and 85.0 +/- 1.4% consumption of CH50 and C3 hemolytic activity, respectively. Using this dose of IgG anti-rat H, it was found that the clearance in rats of rat erythrocytes sensitized with 8000 molecules of guinea pig IgG2 per E was impaired as compared to the clearance of these intermediates in control rats injected with a comparable dose of normal goat IgG. The results indicate that H functions as a potent regulator of C in vivo and that infusion of anti-H can be used as a method to achieve depletion of circulating complement components.     

In vivo activation of complement by IgA in a rat model.

In this study we investigated the capacity of rat IgA to activate complement (C) in vivo in a rat model. Rat monomeric (m-), dimeric (d-) and polymeric (p-) IgA MoAbs were injected intravenously and assessed for deposition of C3 and C4 on IgA. By ELISA it was shown that both d- and p-IgA bound C3 whereas no binding of C3 by m-IgA was observed. Polymeric IgA was more efficient in binding of C3 as compared with d-IgA. However, in haemolytic assays no consistent decrease of plasma complement levels was observed except for dimeric IgA which induced a marginal consumption of AP50. When rats were pre-treated with cobra venom factor (CVF) to deplete C3, no C3 deposition was found on m-, d- or p-IgA. Neither m- nor d- or p-IgA was able to bind C4 in vivo. In agreement with the results described above, large sized polymeric IgA was shown to be taken up by Kupffer cells (KC) together with C3. No C3 was detected when rats were depleted of C using CVF. Taken together, the experimental data suggest that d- and p-IgA are able to activate C via the alternative pathway in vivo.     

In vivo complement activation and binding of C3 to encapsulated Cryptococcus neoformans.

Tissues from mice infected with Cryptococcus neoformans were examined by immunofluorescence to determine the extent of deposition of complement component C3 on encapsulated cryptococci. The relative percentages of cryptococci in each tissue having readily visible C3 were greatest for liver and lung tissues, with the kidney tissue having the next highest percentage and the spleen having the lowest percentage. Binding of C3 fragments to cryptococci in brain tissue was essentially absent.     

乙型肝炎病毒在体内外抑制补体C3和C4表达的研究

目的 探讨乙型肝炎病毒(HBV)对补体C3和C4表达的影响,并探讨其调节机制.方法 采用基因芯片筛选HepG2和HepG2.2.15细胞的差异表达基因,免疫比浊法检测HBV患者和健康对照者补体C3和C4血清学水平,将HBV感染性克隆pHBV1.3转染HepG2细胞,RT-PCR和Western blot法检测C3和C4表达水平的变化.结果 补体C3和C4 mRNA在HepG2.2.15细胞中水平降低;C3和C4在慢性乙型肝炎患者和肝癌患者的血清学水平明显低于健康对照者(P＜0.05);HBV能够在mRNA和蛋白水平下调C3和C4的表达.结论 HBV能够在体内外抑制补体C3和C4的表达.     

In vitro biosynthesis of complement factor I by human endothelial cells.

We have studied the secretion of the complement regulatory protein factor I by human umbilical vein endothelial cells (HUVEC). Northern and Western blot analysis and biosynthetic labeling experiments indicate that HUVEC secrete factor I at very low levels in basal conditions and that this secretion is significantly enhanced by interferon-gamma. Analysis of the proteolytic inactivation of C3b by HUVEC supernatants show that factor I is secreted in a functional form and can promote the specific proteolytic inactivation of C3b to iC3b. Together with previous studies establishing the secretion of complement factor H by HUVEC, this work demonstrates that the endothelial cell is able to secrete in its environment two complement regulatory proteins, factor I and factor H, which can mediate the degradation of C3b to iC3b. The secretion of factor I by HUVEC provides a useful in vitro model to analyze the modulation of this secretion and may be relevant to the local deposition of iC3b at the surface of the endothelium during the inflammatory reaction.     

Activation of the complement system by radiographic contrast media: studies in vivo and in vitro.

The effect of radiographic contrast media (RCM) on the complement system was studied in vivo and in vitro. In 65 patients undergoing intravenous pyelography, plasma was obtained before and 3, 5, 10, 20, 30, and 60 min postinfusion. Nine patients experienced immediate generalized reactions (IGR) post-RCM infusions. Six patients developed urticaria, 1 developed angioedema, and 2 developed nausea and hypotension. A significant decrease in CH50 occurred in 29 patients, including 6 with IGR. Split products of C3 were detected in 24 of these patients. Six patients without change in CH50 had detectable C3 split products. There was a statistically significant decrease in hemolytically active C3 and factor B, but not C1q or C4 in the group with decreased CH50 when compared to the group without CH50 change. All patients had normal levels of C1 esterase inhibitor, C3aI (serum carboxypeptidase B), C3bI, and βIH. Neither anti-RCM antibodies nor immune complexes were detected in any patient's plasma. In vitro, decreased CH50 and hemolytically active C4, C3, properdin, and factor B were found with incubation of plasma with RCM. These changes occurred independent of the buffer used (EDTA/EGTA). Finally, C3 split products were detected in the reaction mixtures. These data indicate that RCM is associated with decreased hemolytic complement activity, both in vivo and in vitro. Furthermore, the in vitro changes occurred without the presence of calcium or magnesium, indicating that complement activation by RCM may occur in a nonsequential manner.     

C3b and factor H: key components of the complement system

In all three complement pathways, the central molecule is C3, which, upon activation cleavage, forms the major opsonin C3b – the key component of complement. C3b is also essential for propagation of the complement cascade to the stage of the lytic terminal complement complexes. In order to prevent damage to self cells and tissues and restrict overconsumption of the complement components, C3b molecules need to be controlled by factor H. Defect in C3 functions leads to compromised microbial defence and increased susceptibility to certain autoimmune diseases. Deficiency of factor H, or a functional defect in its N terminus, often leads to membranoproliferative glomerulonephritis and complement depletion, owing to continuous overconsumption of C3. Defect in the factor H C terminus leads to a dramatically increased risk of atypical hemolytic uremic syndrome. In addition, recently, a polymorphism in the middle part of factor H (Y402H) has been shown to be the major risk factor for the most common cause of blindness in the industrialized world: age-related macular degeneration. In future, analysis of patient samples for defects in these key complement components may prove useful in diagnosis of these diseases and new therapeutic targets will certainly be the aim for use in the recently recognized factor H-related diseases.     

In vitro and in vivo modulation of cholinergic muscarinic receptors in rat lymphocytes and brain by cholinergic agents

A binding site for 3H-quinuclidinyl benzylate (QNB) has been identified in rat lymphocytes which has the characteristics of a cholinergic muscarinic receptor (Costa, L. G., Kaylor, G. & Murphy, S. D. (1988). Muscarinic cholinergic binding sites on rat lymphocytes. Immunopharmacology, 16, 139-149.) Here we show that prolonged exposures to cholinergic compounds in vitro and in vivo modulate muscarinic receptor binding in lymphocytes as well as in brain tissue. Exposure of rat splenic lymphocytes in vitro to oxotremorine caused a time- and concentration-dependent decrease in the density of 3H-QNB binding sites. This decrease occurred only when incubation with oxotremorine was carried out at 37 degrees C and not at 0-4 degrees C, suggesting that it was not an artifact due to residual, unwashed, oxotremorine. The effect of oxotremorine was mimicked by two other cholinergic agonists, acetylcholine and carbachol, and was antagonized by atropine, which, when present alone, caused an increase in 3H-QNB binding. In vivo exposures to oxotremorine or atropine (both at 20 mg/kg/day for 14 days via an ALZA minipump) caused a significant decrease (20-30%) and increase (13-30%), respectively, of 3H-QNB binding in various brain areas as well as circulating lymphocytes. Repeated administrations of the organophosphorus insecticide disulfoton (2 mg/kg/day for 14 days, i.p.) caused significant reductions (59-88%) of acetylcholinesterase activity in brain, lymphocytes, plasma and red blood cells, as well as a 23-39% decrease of 3H-QNB binding in brain areas and circulating lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo clearance studies of the terminal fluid-phase complement complex in rabbits.

The present study was directed at obtaining information on the in vivo elimination rate of SC5b-9, the terminal fluid-phase product of complement activation. A sandwich ELISA based on the use of mono- and polyclonal antibodies was constructed that permitted quantitation of rabbit SC5b-9 in plasma. Rabbit serum was activated with inulin in vitro to generate SC5b-9, and the activated serum was applied intravenously in normal and C6-deficient rabbits. Elimination of SC5b-9 in normal rabbits was rapid, half-life in the range of 30-50 min. No differences were noted between the clearance of homologous rabbit and heterologous human SC5b-9, SC5b-9 concentrations returned to basal levels 2-3 h after application. Plasma of C6-deficient rabbits contained no SC5b-9 and these animals displayed an even more effective clearance capacity for the complex. Quantitative considerations indicated that basal plasma SC5b-9 levels in healthy animals result from a spontaneous turnover rate of approximately 0.2% of C5-C9 components per h. When multiple doses of SC5b-9 were injected in sequence, the same half-life and total elimination time were found as with single-dose experiments. The results demonstrate the existence of an effective clearance mechanism for SC5b-9, consistent with recent findings that SC5b-9 plasma levels are very low not only in healthy adults, but also in the majority of patients with complement-consuming diseases.     

In vivo expression of the intermediate filament peripherin in rat motoneurons: modulation by inhibitory and stimulatory signals

Peripherin is a type III intermediate filament which, in contrast to the neurofilaments, is strongly up-regulated after nerve injury. Although peripherin expression is stimulated in vitro by neurotrophins and cytokines, little is known about its in vivo regulation. In this report, we show that the in vivo down-regulation of peripherin expression to normal levels during regeneration closely correlates with target reconnection in rat facial motoneurons. Prevention of reconnection, by transection and suture, results in the persistence of strong peripherin expression for prolonged periods of up to 11months. This contrasts with the modulation of the p75 low-affinity neurotrophin receptor, whose expression returns to normal even in the absence of reconnection. We further demonstrate that blockade of the axonal transport in non-injured motoneurons increases the expression of peripherin. Blockade of the axonal transport simultaneously to, or after injury of, facial motoneurons does not abolish the axotomy-induced peripherin up-regulation.These data demonstrate that the in vivo expression of peripherin is normally restrained by a distal retrogradely transported inhibitory signal. Thus, peripherin up-regulation results primarily from a lack of supply in this factor. Our results show that stimulatory factors released at the injury site are not required for the initial up-regulation and maintenance of high peripherin expression. However, they appear to enhance this increase during the acute post-lesion phase. Peripherin expression is thus finely tuned by both glial cell-derived stimulatory and distal inhibitory signals that reflect neuron-target interactions.     

Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis

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Ellipsometric studies in vitro on kinetics of rat complement activation.

The role of complement activation may be important during the early interactions between implantable materials and blood and during the acute inflammatory phase, but it is not well understood. This applies especially to rats that are extensively used in in vivo animal models for materials and surface testing. Features of the kinetics of rat complement activation were studied and compared with human complement by the ellipsometry and antibody techniques. The results indicate that the rat classical pathway is rapidly activated, but it is not as fast as the human system. The activation of the alternative pathway was observed within 5 min in the rat system and within 15 min for the human. Thus, the observations indicate substantial differences in the kinetics between the two species. This may influence the choice of the rat experimental model and the tissue response to materials during in vivo conditions.     

Inhibition of classical C5 convertase in the complement system by factor H

This paper described the influence of factor H on the haemolytic activity of the classical C5 convertase. Factor H showed little effect on the interaction of C5 with EAC1,4b,2a,3b cells bearing low numbers of C3b sites, but displayed the inhibitory effect on the interaction of C5 with the intermediate cells bearing high numbers of C3b sites. The higher the number of C3b sites on the cells, the greater the degree of the inhibition by factor H. The inhibition by factor H was accompanied by the inhibition of consumption of C5 from the fluid phase, indicating that factor H inhibits the activity of C5 convertase, not the binding of activated C5 to the cells.     

Initiation of complement activation following oxidative stress. In vitro and in vivo observations

Ischemia and reperfusion of organs/tissues induce a state of inflammation that can lead to tissue injury. Focus on development of effective therapeutics based on sound pre-clinical work and the role of leukocytes in models of human disease has not lead to a successful clinical trial for anti-leukocyte technologies. For the past >30 years, it has been known that complement activation plays a role in the inflammation and tissue injury associated with ischemia/reperfusion (I/R) injury. In the last 10 years, several complement inhibitors have made their way from the bench to bedside. Will a complement inhibitor eventually be approved for clinical treatment of I/R type diseases? What pathway(s) are involved in I/R injury, and what role do they play? What specific complement components are needed for resolution of inflammation and what components need to be inhibited to decrease tissue injury? This short review will focus on the current state of the art knowledge about complement, complement pathways, complement components and several promising clinical biologics that inhibit complement activation. This review is not a complete review of complement in ischemia/reperfusion injury, but it raises important questions about the role of complement, its pathways and the current knowledge in the area of ischemia/reperfusion injury.     

In vitro and in vivo study of the expression vector encoding vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is an angiogenic cytokine with potential therapeutic applications in human diseases. It is a mitogen primarily for endothelial cells. The transfer of the cDNA encoding VEGF to ischemic tissues, which cannot be revascularized otherwise, represents a novel and promising approach to the treatment of vascular disorders. In this work the VEGF165 cDNA was cloned into the expression vector pSecTag2B. The activity of the construct was studied in cell culture as well as in vivo. Western blotting study showed that the cells transfected with the vector secreted significantly higher amounts of VEGF to the culture medium than the non-transfected cells. In vivo study revealed an increased number of new vessels in animals injected with vector encoding VEGF as compared with empty plasmid. Also, tumor cells transfected with the VEGF plasmid exhibited extensive vascularization.