Alternative Pathway Of Complement System Research Articles

#2890 Role of alternative pathway of complement system in membranous nephropathy: a study from STARMEN clinical trials

Abstract Background and Aims Membranous nephropathy (MN) is an antibody-mediated autoimmune disease characterized by glomerular immune complexes containing complement components. A recent animal model of PLA2r antibody-induced MN induced has demonstrated the crucial role of complement pathway, although is still unclear which complement pathway is most involved in its pathogenesis This study aimed to explore the role of alternative pathway of complement cascade in a cohort of patients from STARMEN clinical trial. Method We analyzed longitudinal plasma samples from 51 patients from STARMENT cohort stored in a biobank. Samples were stored at enrollment and longitudinally throughout the follow up at 3, 6, 9, 12, 18 and 24 months. We measured the circulating plasma levels of C3, FB, FH, FHR1, FHR2 and FHR5 in every time. Results Fifty-two patients with a mean age of 56 ± 10 years and 60% male were included. 44% were treated with tacrolimus+Rituximab and 56% with ponticelli regimen. The basal creatinine was 0.9 ± 0.3 mg/dl, median proteinuria/day was 8.8 g/day (IQR 5.7-11.6 gr/day) and serum albumin of 2.6 g/dl (IQR 2.3-2.9 g/dl). All patients were followed-up for two years. Thirty-four patients achieved complete remission (CR), 11 patients partial remission (PR) and 15 did not respond (including relapse). During active disease, the mean concentration of FH, FHR-1 and FHR-2 were significantly increased compared with remission status (complete or partial). We did not find different in other factors: FB or FHR-5. Moreover, a positive correlation was observed between FH and FB with C3. There were no differences according to treatment arm. PLA2r antibodies tested positive in 68%. The median of baseline anti-PLA2R title levels was 80 RU/ml (IQR 43-141). Circulating plasma levels of FHR1 at baseline were significantly higher in PLA2r testing negative vs positive patients (199 ± 108 vs 249 ± 41 µg/ml, p = 0.021). This significant difference was present when remission was achieved (205 ± 97 vs 264 ± 49 µg/ml, p = 0.023). Similar tendency was observed for FB. Conclusion These data provide evidence of complement system activation through the alternative pathway in membranous nephropathy. We know that different subtypes of IgG are behind the different antibodies-induced MN induced, and that every subtype active different complement pathway. The results of this study lead to speculate that not all the antigens that induced MN active complement pathway in the same way.

Atypical hemolytic-uremic syndrome - A case series from a tertiary care hospital from Eastern India

Atypical hemolytic–uremic syndrome (aHUS) is a form of thrombotic microangiopathy that occurs due to dysregulation of alternate pathway of complement system, which progressively causes systemic complications, end-stage renal disease, and death. As prognosis is poor compared to typical hemolytic–uremic syndrome, early diagnosis and treatment is crucial for favorable outcome. We came across seven patients of aHUS in our pediatric intensive care unit in the last 5 years. Plasma exchange (PE) along with immunosupressives was used for treatment. First child who did not receive PE died. Rest six patients underwent PE and attained hematological remission; however, one later on progressed to chronic kidney disease and expired. All others are on regular follow-up and doing well. A high index of suspicion is required to diagnose aHUS. Early PE can give a better prognosis.

Local Activation of the Alternative Pathway of Complement System in Mycotic Keratitis Patient Tear.

Aspergillus flavus and Fusarium solani are the predominant causative agents of mycotic keratitis in the tropical part of the world. Tear proteins play a major role in the innate immune response against these fungal infections as has been shown by the presence of complement proteins and neutrophil extracellular trap proteins in keratitis patients tear. In this study, we established the presence of the components of the alternate pathway of complement system and their functional state in the tear film of mycotic keratitis patients. The complement proteins namely, C3 and CFH were found only in the open-eye tear of patients but not in control individuals. In vitro analysis showed binding of purified C3b and CFH to fungal spores, which confirmed that the spores can provide a foreign surface for forming the complement complex. Analysis of spore bound tear proteins by mass spectrometry exhibited the presence of known proteins of the alternate pathway complement cascade in keratitis patient tear. Hemolytic assay using rabbit RBC confirmed the presence of a functional alternate pathway of complement cascade in the tear proteome of the patients. The presence of negative regulators, CFH and CFI, in the patient tear indicate that the complement activity is tightly regulated during fungal infection. Mass spectrometry data show vitronectin and clusterin, two known inhibitors of the membrane attack complex only in the patient tear. These data demonstrate the activation of the alternate pathway of complement cascade during the early stages of infection. Interestingly, the production of multiple negative regulators of complement cascade implies the pathogen can effectively evade the host complement system during infection.

Mathematical Modelling of Alternative Pathway of Complement System.

The complement system (CS) is an integral part of innate immunity and can be activated via three different pathways. The alternative pathway (AP) has a central role in the function of the CS. The AP of complement system is implicated in several human disease pathologies. In the absence of triggers, the AP exists in a time-invariant resting state (physiological steady state). It is capable of rapid, potent and transient activation response upon challenge with a trigger. Previous models of AP have focused on the activation response. In order to understand the molecular machinery necessary for AP activation and regulation of a physiological steady state, we built parsimonious AP models using experimentally supported kinetic parameters. The models further allowed us to test quantitative roles played by negative and positive regulators of the pathway in order to test hypotheses regarding their mechanisms of action, thus providing more insight into the complex regulation of AP.

Study of the complement activation by amyloid aggregates of smooth muscle titin in vitro

ABSTRACTThe giant muscle protein, titin, is the third most abundant protein in muscle (after myosin and actin). It was shown previously that smooth muscle titin (SMT) with a molecular mass of 500 kDa can form in vitro amorphous amyloid aggregates in two conditions: in solution of low ionic strength (0.15 M Glycine-KOH, pH 7.0) (SMT(Gly) aggregates) and in solution with ionic strength in the physiological range (0.2 M KCl, 20 mM imidazole, pH 7.2–7.4) (SMT(KCl) aggregates). Such aggregation in vivo, which may play a pathological or functional role, is not excluded. In view of the fact that some pathological amyloids can activate the classical and alternative pathways of complement system, we investigated the binding of complement component C1q and C3b to smooth muscle titin amyloid aggregates. The binding of С1q and C3b to SMT aggregates was not observed with ELISA assay. Since SMT aggregates do not activate the complement system, they are hardly implicated in the inflammatory process caused by muscle damage in amyloidoses.Abbreviations: SMT: smooth muscle titin; SMT(KCl) aggregates: SMT aggregates in solution containing 0.2 M KCl, 10 mM imidazole, pH 7.0; SMT(Gly) aggregates: SMT aggregates in solution containing 0.15 M glycine-KOH, pH 7.2-7.4; MAC: membrane attack complex; DLS: dynamic light scattering; NHS: Normal Human Serum

Supercritical Extracts from Arctium lappa as a Potential Inhibitor for the Activation of Complement System

Abstract Arctium lappa is a perennial species of the Asteraceae family originally from Europe and Asia. Considered a weed species in the southern region of Brazil, it is popularly used as a natural anti-inflammatory. The complement system is an important component of the innate immune response. However, its exacerbated activation can lead to harmful conditions like autoimmune and inflammatory disorders. Plants that inhibit the activation of complement can be a promising tool in the treatment of inflammatory diseases. Here, we evaluated the effect of A. lappa leaves extracts on the activation of the classical and alternative pathways of complement system. Two extracts were obtained under supercritical conditions using scCO2 with ethanol as cosolvent, at 313.15K, 15 MPa (E1) and 25 MPa (E2). Classical and alternative activation were evaluated using complement fixation test. Different concentrations of A. lappa extracts E1 and E2 showed an inhibitory effect on both complement pathways, and heparin was used as control. The IC50 of E1, E2, and heparin were 28.26, 20.12 and 92.54 µg/mL for classical and 26.12, 27.70, 27.78 µg/mL for the alternative pathway. Results demonstrate that A. lappa is a promising complementary treatment for diseases associated with complement activation.

Screening of Complement Stimulating Activity of Lindernia madayiparense Extracts

This study evaluates the influence of different extracts of newly identified Lindernia madayiparense from Linderniaceae on complement system. The similar species under this family are used in China and India to prevent and cure diverse ailments and to rejuvenate the immune system without any marketing standards. Various extracts of plant were evaluated through complement mediated hemolytic assay. Two plant extracts shows complement modulating property against alternative pathway of complement system, i.e. ethanol extract and decocted extract. These fractions showed significant complement stimulating activity compared to positive control used.

Effect of some essential oils on phagocytosis and complement system activity.

The aim of the present study was to investigate the in vitro activity of 15 essential oils, 4 essential oil fractions, and 3 pure compounds (thymol, carvacrol, and eugenol) on phagocytosis by human neutrophils and on complement system. Samples were characterized by GC and GC-MS. Most of the oils (nutmeg, clove, niaouli, tea tree, bay laurel, lemon, red thyme, ginger), nutmeg terpenes, eugenol, and carvacrol showed mild to moderate inhibition of phagocytosis (25-40% inhibition at doses ranging from 40 to 60 μg/mL); highest inhibitory activity was found for thymol (72% at 56 μg/mL), whereas the mixture of bornyl and isobornyl acetates showed a mild stimulating activity (21% at 56 μg/mL). All samples were inactive in the alternative pathway of complement system, whereas on classical pathway, clove oil, eugenol, palmarosa oil, red thyme oil, tarragon oil, and carvacrol showed the highest activity, with IC50 values ranging from 65 to 78 μg/mL.

Levels of Urinary Complement Factor H in Patients with IgA Nephropathy are Closely Associated with Disease Activity

Factor H plays a key inhibitory role in control of the activation of alternative pathway of complement system. The aim of the study was to investigate the predictive value of factor H as a biomarker of renal injury in IgA nephropathy (IgAN). Urine factor H concentration from 202 patients was measured and compared with that of 60 healthy volunteers. Forty-eight patients fulfilled Haas-I or II (group 1), 60 fulfilled Haas-III (group 2) and 94 fulfilled Haas-IV or V (group 3). Co-deposition of factor H and C3b in kidneys were investigated using confocal microscope. The levels of urinary factor H, when expressed as a ratio of urinary creatinine, were significantly higher in groups 3 than group 1 and 2, also significantly higher in group 2 than group 1. In addition, the levels of urinary factor H were significantly higher in those with factor H deposition in the kidney than those without deposition. The levels of urinary factor H may be a useful biomarker to evaluate kidney injury in IgAN.

Anticomplementary principles of a Chinese multiherb remedy for the treatment and prevention of SARS

Aim of the study To elucidate the anticomplementary principles of a Chinese multiherb remedy used for the treatment and prevention of SARS, which contains five ingredients of Herba Houttuyniae, Flos Chrysanthemi Indici, Herba Artemisiae Scopariae, Herba Eupatorii and Fructus Tsaoko. Materials and methods The anticomplementary activity was evaluated from hemolytic assays through the classical pathway (CP) and the alternative pathway (AP) of complement system in vitro. Compounds were isolated using bioactivity-guided fractionation and tested in vitro for their complement-inhibiting properties on the CP and AP. HPLC-DAD-ESI-MS was used to assign the isolated compounds in the five ingredient herbs. Results 15 compounds, including chlorogenic acid ( 1), rutin ( 2), hyperoside ( 3), p-hydroxyacephenone ( 4), scopoletin ( 5), quercitrin ( 6) (3 R,4 R,6 S)-3,6-dihydroxy-1-menthene ( 7), acaciin ( 8), scoparone ( 9), luteolin ( 10), quercetin ( 11), apigenin ( 12), acacetin ( 13), aristolactam ( 14), and apigenin-7,4′-dimethyl ether ( 15) were isolated and identified. Nine flavonoids ( 2, 3, 6, 8, 10– 13, 15) were found to show inhibitory effects towards the CP and AP of complement system, luteolin ( 10) was the most potent with the CH 50 and AP 50 values of 0.19 and 0.17 mM. The bioactive flavonoids were mainly derived from Herba Houttuyniae, Flos Chrysanthemi Indici and Herba Artemisiae Scopariae. Conclusions A Chinese multiherb remedy used for the treatment and prevention of SARS has robust inhibitory effect on complement system through the CP and AP, and its anticomplementary activity could be attributed to the flavonoids present in some ingredient herbs. Luteolin could be a potential anticomplementary agent.

Effects of triterpenoids from Pueraria lobata on immunohemolysis: beta-D-glucuronic acid plays an active role in anticomplementary activity in vitro.

The anticomplementary properties of kaikasaponin III (4) and soyasaponin I (8) from Pueraria lobata and their hydrolytic analogs were investigated in vitro. Diglycosidic saponins [kaikasaponin I (3), soyasaponin III (7)] showed most potent anticomplementary activities, followed by monoglycosidic saponins [soyasapogenol B monoglucuronide (6), sophoradiol monoglucuronide (2)] and triglycosidic saponins [soyasaponin I (8), kaikasaponin III (4)], whereas sophoradiol (1) and soyasapogenol B (5) showed enhancement of hemolysis under the presence of serum on the classical pathway of complement system. But all of them showed very weak or no anticomplementary activities on the alternative pathway of complement system. The anticomplementary activity of the saponins was influenced by the nature of glucuronic acid, where the free acid forms (-COOH) showed much more potent activity than the sodium salt forms (-COO-Na+) or methyl ester forms (-COOCH3), and the reduced forms (-CH2OH) decreased the activity significantly.

Immunoglobulins and complement in migraine.

In 54 patients with migraine and 70 persons comprising the control group, the total complement level (CH50) was evaluated together with its C3 and C4 fraction level and the level of IgG, IgA and IgM immunoglobulins. It was found that the average C3 fraction level was significantly decreased, while the C4 fraction level and total complement activity remained in the normal range. The immunoglobulin level did not show any statistically significant alterations except for the IgA level, which was lowered in migraine patients. On this basis, i.e. lowering of the C3 fraction level with normal C4 fraction level and total complement activity and lack of elevation of IgA, it is felt that the alternative pathway of complement system may be activated in migraine.

The activation mechanism of the alternative pathway of the human complement system by the immune precipitate formed with F(ab′) 2 of rabbit IgG antibody: The generation of C3- and C5-cleaving enzymes of the immune precipitate

Immune precipitate (F), formed between egg albumin and the F(ab′) 2 fragment of rabbit anti-egg albumin, activated the alternative pathway of the complement system in human serum. On incubation with serum, F combined with several factors in serum (X) to form a complex, FX, which was capable on inactivating purified C3 and C5. FX having maximal activity was obtained by incubation of F with serum for 10–20 min (T max). T max depended on the amount of F added to serum and a increased amound of F shortened the T max/ When FX was incubated at 37°C, its activities decayed as a first order reaction and were completely lost in 120 min. FX regenerated from decayed FX (FX d ) by the addition of both B and D . FX also lost activity by treatment with anti-B or anti-C3. These findings indicated that F(ab′) 2 activated the alternative pathway of the complement system in human serum by the formation of the properdin system enzymes on F.

Activation of the Alternative Complement Pathway by Water-Insoluble Glucans of Streptococcus Mutans: The Relation between Their Chemical Structures and Activating Potencies

Water insoluble, sticky glucan was synthesized by reacting glucosyltransferase obtained from the sulture filtrate of cariogenic Streptococcus mutans OMZ 176 on sucrose. This glucan (OMZ 176 glucan), that consisted of the backbone chains of consecutive alpha-1, 3 glucosidic linkages (65%) and the side chain of alpha-1, 6 glucosidic bonds (35%), showed an ability to activate the alternative pathway of the complment system in human serum. Water soluble glucans synthesized from the reaction of glycosyltransferases of S. mutans strain AHT, FA-I, and Ingbritt with sucrose, that have a high content of alpha-1,6 and a low content of alpha-1, 3 glucosidic linkages, were less effective to activate the complement system. Sephadex (G-25) and Dextran (T-2,000 and T-40), that consisted exclusively of alpha-1, 6 glucosidic linkages, did not significantly activate the complement system. Controlled Smith degradation products of OMZ 176 glucan, composed of the alpha-1, 3 linked glucose backbone chain alone, lost its activity. From these results it was concluded that both backbone chain of alpha-1, 3 glucose units and branched alpha-1, 6 glucose units were essential for the OMZ 176 glucan to activate the alternative pathway of the human complement system.