Activation mechanism of the complement system

Complement is an activation system designed for the elimination of pathogens and is activated in three ways, i.e. the classical, alternative and newly discovered lectin pathways. The classical pathway is initiated by the binding of C1 to immune complexes, whereas the alternative pathway is activated by pathogens themselves without involvement of immune complexes. Upon binding of MBL (mannose-binding lectin) to certain carbohydrates on pathogens, the lectin pathway is activated by two C1r/C1s-like serine proteases termed MASP-1 and MASP-2, which are associated with MBL. As a result, C4, C2 and C3 are activated. The lectin pathway plays a crucial role in innate immunity both in vertebrates and in invertebrates. The mechanism underlying lectin pathway activation remains unsolved. From an evolutional point of view, the classical and lectin pathways are closely related.     

Activation of the complement system in dilated cardiomyopathy

The levels of anaphylatoxins (C3a, C5a) in blood, components of complements C3 and C4, antibodies to cardiolipin, IgG, IgA, IgM, IgE and circulating immune complexes were measured in 21 patients with dilated and hypertrophic cardiomyopathy (DCMP and HCMP) and in 11 donors. The mean level of C3a in DCMP patients (572 +/- 55 ng/ml) was significantly higher than in HCMP patients (344 +/- 30 ng/ml) and in donors (294 +/- 43 ng/ml) (p less than 0.001). On comparison of C5a concentrations in DCMP patients (2.2 +/- 0.52 ng/ml), HCMP patients (3.3 +/- 1.2 ng/ml) and in donors (1.6 +/- 0.82 ng/ml) no significant differences were found (p greater than 0.05). It has been established that in the group of DCMP patients with thromboembolic complications, the concentrations of C3a (736 +/- 95 ng/ml) were significantly higher than in those without such complications (334 +/- 14 ng/ml) (p less than 0.001). The data obtained permit discussing the role of anaphylatoxins in the development of thromboembolic complications in DCMP patients.     

Plasma porphyrins in the porphyrias.

BACKGROUND: As an aid in the diagnosis and management of porphyria we have developed a method to fractionate and quantify plasma porphyrins and have evaluated its use in various porphyrias. METHODS: We used HPLC with fluorometric detection to measure plasma concentrations of uroporphyrin I and III, heptacarboxyl III, hexacarboxyl III, pentacarboxyl III, and coproporphyrin I and III. We studied 245 healthy subjects, 32 patients with classical porphyria cutanea tarda (PCT), 12 patients with PCT of renal failure, 13 patients with renal failure, 8 patients with pseudoporphyria of renal failure, 3 patients with acute intermittent porphyria, 5 patients with variegate porphyria, 5 patients with hereditary coproporphyria, and 4 patients with erythropoietic protoporphyria. RESULTS: Between-run CVs were 5.4-13%. The recoveries of porphyrins added to plasma were 71-114% except for protoporphyrin, which could not be reliably measured with this technique. Plasma porphyrin patterns clearly identified PCT, and its clinical sensitivity equaled that of urine porphyrin fractionation. The patterns also allowed differentiation of PCT of renal failure from pseudoporphyria of renal failure. CONCLUSIONS: The assay of plasma porphyrins identifies patients with PCT and appears particularly useful for differentiating PCT of renal failure from pseudoporphyria of renal failure.     

Anesthesia and the porphyrias.

A simplified classification of the porphyrias is given which is thought to be advantageous to the anesthesiologist in determining those patients who are predisposed to acute attacks. These acute attacks may be precipitated by the administration of barbiturates, but may also be spontaneous. The current theory for the precipitation of the acute attack is described, with the probable mechanism being a decrease in uroporphyrinogen synthetase levels and the resultant interference in heme production. Increased formation of cytochrome P-450 with barbiturates also produces increased levels of delta aminolevulinic acid, which may be a cause of the acute attack. The significance in anesthesia and suggested means of anesthetic management are discussed.     

Porphyrins, porphyrin metabolism and porphyrias. II. Diagnosis and monitoring in the acute porphyrias

The acute porphyrias constitute a group of metabolic disorders engaging enzymes in the haem synthetic chain and generally following dominant inheritance patterns. Some gene carriers are vulnerable to a range of exogenous and endogenous factors, which may trigger neuropsychiatric symptoms. Early diagnosis is of prime importance since it makes way for counselling with the aim to block the development of acute, as well as late, disease. The medical and psycho-social consequences of a porphyria diagnosis are considerable and the freedom for maldiagnosis correspondingly small. The strain imposed upon the diagnostic process makes management in specialized laboratories necessary. Inadvertent handling of the diagnostic procedures in laboratories lacking in knowledge, experience and technical competence is repeatedly the reason for harmful underdiagnosis and overdiagnosis. Gene diagnosis of the carrier condition, principally within reach in all types of acute porphyria, is of incomparable versatility and accuracy. However, despite recent great achievements in the molecular biology of porphyric disease, genomic procedures cannot replace biochemical methods in monitoring the activity and progress of the disease, or the effects of therapy. The classical methods are also useful when it comes to screening for the associated disease states. In these tasks, professional handling of the methods and skillful interpretation of the results are of paramount importance. Knowledge of the limitations and pitfalls of the procedures is a guard against maldiagnosis, which may be fatal. In the article the main diagnostic challenges are discussed; the strategy for early detection of the gene carrier state, the recognition and surveillance of the acute porphyric crisis, the evaluation of subacute/subchronic symptoms, the differential diagnoses of the cutaneous porphyrias and the monitoring of late complications.     

Porphyrins,porphyrin metabolism and porphyrias. II. Diagnosis and monitoring in the acute porphyrias

The acute porphyrias constitute a group of metabolic disorders engaging enzymes in the haem synthetic chain and generally following dominant inheritance patterns. Some gene carriers are vulnerable to a range of exogenous and endogenous factors, which may trigger neuropsychiatric symptoms. Early diagnosis is of prime importance since it makes way for counselling with the aim to block the development of acute, as well as late, disease. The medical and psycho-social consequences of a porphyria diagnosis are considerable and the freedom for maldiagnosis correspondingly small. The strain imposed upon the diagnostic process makes management in specialized laboratories necessary. Inadvertent handling of the diagnostic procedures in laboratories lacking in knowledge, experience and technical competence is repeatedly the reason for harmful underdiagnosis and overdiagnosis. Gene diagnosis of the carrier condition, principally within reach in all types of acute porphyria, is of incomparable versatility and accuracy. However, despite recent great achievements in the molecular biology of porphyric disease, genomic procedures cannot replace biochemical methods in monitoring the activity and progress of the disease, or the effects of therapy. The classical methods are also useful when it comes to screening for the associated disease states. In these tasks, professional handling of the methods and skilful interpretation of the results are of paramount importance. Knowledge of the limitations and pitfalls of the procedures is a guard against maldiagnosis, which may be fatal. In the article the main diagnostic challenges are discussed; the strategy for early detection of the gene carrier state, the recognition and surveillance of the acute porphyric crisis, the evaluation of subacute/subchronic symptoms, the differential diagnoses of the cutaneous porphyrias and the monitoring of late complications.     

Activation of kinin-prekallikrein system after infusion of factor VIII concentrates in hemophilic patients

Possible changes in the kinin-prekallikrein system after infusion of factor VIII concentrate were studied in seven patients with severe hemophilia. The functional and immunological activities of factors XI and XII, prekallikrein, high molecular weight kininogen, and C-1-esterase inhibitor were measured before and at 0.5, 2, 6, and 12 hours after infusion of 50 U/kg of factor VIII concentrate. A significant decrease (P less than 0.05) in the functional and immunological activities of prekallikrein was observed at 0.5 and 2 hours after factor VIII administration. The other proteins did not change. The results suggest that the kinin system is activated after infusion of high doses of factor VIII concentrate, although clinical evidence of such activation by these changes was not detected.     

Activation of terminal components of complement in patients with Guillain-Barré syndrome and other demyelinating neuropathies.

In the present study, the role of antiperipheral nerve myelin antibody (anti-PNM Ab) in demyelination by generating the terminal attack complex (C5b-9) of complement was explored in patients with Guillain-Barré syndrome (GBS) and other demyelinating neuropathies. The presence in serum of SC5b-9, an inactive C5b-9 containing S protein, was assessed quantitatively by enzyme-linked immunosorbent assay using an antibody (Ab) to neoantigens expressed on C9 when complexed with C5b-8 or after tubular polymerization. SC5b-9 was detected in all 19 GBS, four patients with paraprotein-associated neuropathy and five of six patients with chronic recurrent polyneuritis. No SC5b-9 was detected in 10 normal controls. Kinetic studies from six GBS patients showed the highest values of SC5b-9 on the 3rd to 5th d of admission; in contrast, the anti-PNM Ab were highest on the day of admission. Anti-PNM Ab fell rapidly to very low levels by the 15th to 20th d. SC5b-9 declined with similar kinetics to undetectable levels by the 30th d. Levels of Ab and SC5b-9 did not quantitatively correlate with soluble immune complexes in these patients' serum. Membrane-bound C5b-9 was also detected by immunohistochemistry in the peripheral nerves from a GBS patient. These results, which show a relationship between levels of complement-fixing anti-PNM Ab and the tissue-damaging C5b-9 complex, suggest that peripheral nerve myelin may serve as the target for Ab-mediated complement attack.     

Activation of the complement system by different autologous transfusion devices: an in vitro study

BACKGROUND: The aim of the present investigation was to study whether autologous transfusion devices activate the complement system and whether complement-activated blood is more vulnerable to further activation during processing. STUDY DESIGN AND METHODS: Forty-eight blood units were randomized to be processed by one of three different salvage systems: Group 1 underwent whole blood filtration (hemofiltration) (n=16); Group 2 underwent continuous processing, saline washing, and centrifugation (CATS, Fresenius AG ) (n=16); and Group 3 underwent saline washing and centrifugation (Cell-Saver, Haemonetics Corp.) (n=16). Eight blood units for each system were activated with cobra venom factor (CVF) at a concentration of 0.2 U per mL whole blood before processing. C activation was studied by determinations of C4d, Bb, C3a, and SC5b-9. Samples were drawn from whole blood, processed blood, and the waste bags. RESULTS: The concentrations of Bb, C3a, and SC5b-9 in whole blood after activation with CVF were significantly elevated compared to blood that was not activated (p < 0.01). Processed blood from hemofiltration contained significantly higher levels of complement-split products than techniques that use washing and centrifugation. The concentrations of SC5b-9 in blood processed by hemofiltration were higher in the experiments with CVF activation (p < 0.05). CONCLUSION: The tested autologous transfusion systems did not themselves activate the complement system, and complement-activated blood was not more vulnerable to further activation during processing. A blood-salvaging technique that used washing and centrifugation reduced elevated concentrations of complement-split products, whereas hemofiltration did not.     

Activation of the alternative pathway of complement in childhood acute lymphoblastic leukemia.

Sequential studies in children with acute lymphoblastic leukemia have demonstrated that at diagnosis or relapse there is defective utilization of complement by the alternative pathway. Thus, the sera of 17/18 patients fail to completely consume C3 to C9 when incubated with zymosan or cobra venom factor (CoF). This underutilization is due to a specific inhibitor of C3 activation which has been partially isolated. By remission, the inhibotor disappears and the CoF and zymosan assays return to normal. In addition, serum levels of C3 and factor B are elevated at the time of diagnosis or relapse but fall to below 3 S.D. from the mean in nearly 60 per cent of the cases during induction therapy. Similarly, serum C4 levels which are normal at diagnosis fall to less that 3 S.D. from the mean in 7/12 cases during treatment. Low C3 levels correlate well with factor B values, suggesting that if C3 to C9 are utilized after the inhibitor has been eliminated, such utilization occurs primarily through the alternative pathway. Presumably, as illustrated by the low C4 levels, this activity is mediated by the ampliciation loop of the alternative pathway involving classical pathway generation of C3b.     

Interactions of the platelets in paroxysmal nocturnal hemoglobinuria with complement. Relationship to defects in the regulation of complement and to platelet survival in vivo.

The blood cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) have abnormal interactions with complement. The activity of the alternative pathway C3 convertase on the platelets of 9 out of 19 patients with PNH was elevated. 10 patients had C3 convertase activity within the normal range even though 80-95% of their platelets lacked the complement regulatory protein decay accelerating factor (DAF) that is absent from the affected blood cells in PNH. PNH and normal platelets released factor H when C3 was bound to their surfaces. This may account for the apparent regulation of C3 convertase activity on platelets that lack DAF. The abnormal uptake of the membrane attack complex of complement by PNH III erythrocytes was not seen in PNH platelets. 111Indium-labeled platelet survival times were normal in five of eight patients, which suggests that the lack of the membrane attack complex defect results in normal platelet survival in PNH.     

Change of complement system predicts the outcome of patients with severe thermal injury

To establish the clinical relevance of the changes in the complement system in patients with thermal injury, we studied 20 patients who had third-degree burns on more than 60% of total body surface area. Their levels of the C3, C4, soluble C5b-9, and functional hemolytic activities of total (CH50) and alternative (AH50) complement pathways were sequentially measured for 2 weeks after thermal injury. All patients showed low C3 levels initially but increased C3 levels in the following days. The increasing trend of C3 levels was prominent in survivors but transient and diminished in nonsurvivors. The change of levels of C3, CH50, and AH50 was closely associated with one another, and their chronological trends related to the survival of patients (P =.0060,.0064 and.0066, respectively). The recovery of C3, AH50, and CH50 to normal or supranormal level during the early treatment period relates to the survival of patients with thermal injury. The failure of recovery of the complement system indicates a poor prognosis for patients and the monitoring of complement system might be beneficial in the care of patients with thermal injury.     

Activation of human complement by immunoglobulin G antigranulocyte antibody.

The ability of antigranulocyte antibody to fix the third component of complement (C3) to the granulocyte surface was investigated by an assay that quantitates the binding of monoclonal anti-C3 antibody to paraformaldehyde-fixed cells preincubated with Felty's syndrome serum in the presence of human complement. The sera from 7 of 13 patients with Felty's syndrome bound two to three times as much C3 to granulocytes as sera from patients with uncomplicated rheumatoid arthritis. The complement-activating ability of Felty's syndrome serum seemed to reside in the monomeric IgG-containing serum fraction. For those sera capable of activating complement, the amount of C3 fixed to granulocytes was proportional to the amount of granulocyte-binding IgG present in the serum. Thus, complement fixation appeared to be a consequence of the binding of antigranulocyte antibody to the cell surface. These studies suggest a role for complement-mediated injury in the pathophysiology of immune granulocytopenia, as has been demonstrated for immune hemolytic anemia and immune thrombocytopenia.     

Activation of the kallikrein system in hyperbetalipoproteinemia.

In 30 patients with hyperlipoproteinemia (19 type II and 11 type IV), the role of the intrinsic coagulation pathway was evaluated by assays of preK, Kl's, and Hageman factor (factor XII). Analysis of the plasma of type II patients suggested activation of the intrinsic pathway characterized by a 40% decrease in preK (p less than 0.01) and a 50% decrease Kl (p less than 0.01); in contrast, analysis of the plasma of type IV patients showed no activation of the intrinsic pathway. In type II patients C-1INH was present in normal concentrations as measured by immunochemical techniques. The findings of normal levels of C-1INH by immunoassay, in association with altered electrophoretic mobility, are suggestive of formation of a kallikrein-C-1INH complex in vivo.     

Erythropoietic protoporphyria. Photoactivation of the complement system.

The complement system was analysed in 14 asymptomatic patients with erythropoietic protoporphyria. In the majority of the sera studied the levels of complement components C1, C4, C2, and C3 were within the normal range. Upon ultraviolet light (330--460 nm) irradiation of the serum samples in vitro, a marked decrease in total hemolytic activity accompanied by reduction of C1, C4, C2, and C3 levels was observed. The loss of total hemolytic activity can be directly correlated with the levels of protoporphyrin (PP) and similar changes can be obtained in normal serum upon addition of PP followedf by ultraviolet light irradiation. It is postulated that after irradiation the excited PP develops the capacity to activate the complement sequence with the production of cleavage products, which may contribute to the skin changes observed in these patients upon sun exposure.     

Activation of the alternative pathway of complement by antiserum to factor B.

Monospecific rabbit and goat antisera to human complement proteins and human immunoglobulins were tested for their ability to activate the alternative complement pathway. This activation was detected by two methods where classical pathway activation was blocked with EGTA and alternative pathway activation was promoted with added magnesium ions. These two methods consisted of lysis of GSHE and conversion of factor B into split products. C1q-depleted serum was used in a third assay system. Only antiserum to human factor B was able to activate the alternative pathway in the various systems used. None of the other anticomplement sera showed such activity. When antiserum to factor B was fractionated by ammonium sulfate and column chromatography, activation of the alternative pathway was found in the IgG fraction, and this activity was completely removed by absorption with purified factor B but not with other purified complement components.     

Enzyme heterogeneity in the porphyrias

The use of immunological methods for measuring enzyme mass has identified several varieties of porphyria in which the reduction of porphyrin enzyme activity is not accompanied by a corresponding change in the enzyme mass. Currently, acute intermittent porphyria and hepatoerythropoietic porphyria have exhibited this phenomenon. In porphyria cutanea tarda, it has recently been shown that the pattern of enzyme deficiency in erythrocytic and nonerythrocytic tissues does not strictly follow the inheritance pattern (familial and sporadic) previously described. Also, contrary to previous dogma, some cases of type 1 porphyria cutanea tarda appear to be positive for cross reactive immunological material (CRIM).     

高强度聚焦超声体外辐照兔股动脉后的病理学观察

目的 探讨高强度聚焦超声(HIFU)体外辐照兔股动脉后血管的破坏情况。方法HIFU的工作频率为0．8MHz，焦距135mm，焦点平均强度为8000w／cm^2。30只兔随机分为三组，采用弹力纤维维多利亚蓝和丽春红S组织化学染色方法，观察HIFU辐照兔股动脉后即日、3d和7d的病理学变化。结果HIFU辐照后，各实验组均可见兔股动脉血管内皮细胞脱落、消失，管腔有微小血栓形成；平滑肌细胞出现核固缩，细胞排列紊乱；血管弹力纤维出现崩溃和断裂等现象。结论HIFU体外辐照可以造成兔股动脉血管损伤，并导致血流动力学改变。