Evaluation of Opisthorchis viverrini calreticulin for potential host modulation.

The multifunctional calreticulin (CALR) was identified as a major calcium-binding protein of the endoplasmic reticulum before being recognized as a chaperone in the same place. Only later were activities of calreticulin outside the endoplasmic reticulum described that for example affect cell proliferation and the innate immune system. In the present work we have investigated those extracellular activities of CALR from the cancerogenic human liver fluke Opisthorchis viverrini (OvCALR), as they might be important in host/parasite interaction. We first demonstrate that OvCALR is released from the parasite and stimulates a specific humoral immune response. Recombinant OvCALR is then shown to suppress proliferation of primary endothelial cells, their motility and sprouting activities. The potential of OvCALR to interfere with the complement system is established, firstly by demonstrating its direct binding to C1q and, secondly by suppression of hemolysis of sensitized red blood cells. These findings suggest that OvCALR is an important parasite antigen that could modulate diverse host functions and support parasite survival.

Distinct in vitro Complement Activation by Various Intravenous Iron Preparations.

BACKGROUND: Intravenous (IV) iron preparations are widely used in the treatment of anemia in patients undergoing hemodialysis (HD). All IV iron preparations carry a risk of causing hypersensitivity reactions. However, the pathophysiological mechanism is poorly understood. We hypothesize that a relevant number of these reactions are mediated by complement activation, resulting in a pseudo-anaphylactic clinical picture known as complement activation-related pseudo allergy (CARPA). METHODS: First, the in-vitro complement-activating capacity was determined for 5 commonly used IV iron preparations using functional complement assays for the 3 pathways. Additionally, the preparations were tested in an ex-vivo model using the whole blood of healthy volunteers and HD patients. Lastly, in-vivo complement activation was tested for one preparation in HD patients. RESULTS: In the in-vitro assays, iron dextran, and ferric carboxymaltose caused complement activation, which was only possible under alternative pathway conditions. Iron sucrose may interact with complement proteins, but did not activate complement in-vitro. In the ex-vivo assay, iron dextran significantly induced complement activation in the blood of healthy volunteers and HD patients. Furthermore, in the ex-vivo assay, ferric carboxymaltose and iron sucrose only caused significant complement activation in the blood of HD patients. No in-vitro or ex-vivo complement activation was found for ferumoxytol and iron isomaltoside. IV iron therapy with ferric carboxymaltose in HD patients did not lead to significant in-vivo complement activation. CONCLUSION: This study provides evidence that iron dextran and ferric carboxymaltose have complement-activating capacities in-vitro, and hypersensitivity reactions to these drugs could be CARPA-mediated.

Uncoupling complement C1s activation from C1q binding in apoptotic cell phagocytosis and immunosuppressive capacity.

Complement activation contributes to inflammation in many diseases, yet it also supports physiologic apoptotic cells (AC) clearance and its downstream immunosuppressive effects. The roles of individual complement components in AC phagocytosis have been difficult to dissect with artificially depleted sera. Using human in vitro systems and the novel antibody complement C1s inhibitor TNT003, we uncoupled the role of the enzymatic activation of the classical pathway from the opsonizing role of C1q in mediating a) the phagocytosis of early and late AC, and b) the immunosuppressive capacity of early AC. We found that C1s inhibition had a small impact on the physiologic clearance of early AC, leaving their immunosuppressive properties entirely unaffected, while mainly inhibiting the phagocytosis of late apoptotic/secondary necrotic cells. Our data suggest that C1s inhibition may represent a valuable therapeutic strategy to control classical pathway activation without causing significant AC accumulation in diseases without defects in AC phagocytosis.

Anticomplement monoterpenoid glucosides from the root bark of Paeonia suffruticosa.

Six new (1-6) and 19 known monoterpenoid glucosides were isolated from the root bark of Paeonia suffruticosa. The monoterpenoid glucosides 1, 2, 7, 10-19, and 22 exhibited anticomplement effects with CH50 and AP50 values ranging from 0.14 to 2.67 mM and 0.25 to 3.67 mM, respectively. In a mechanistic study, suffrupaeoniflorin A (1) interacted with C1q, C3, C5, and C9, while galloylpaeoniflorin (12) and galloyloxypaeoniflorin (19) acted on C1q, C3, and C5 components in the complement activation cascade.

Targeting gC1qR domains for therapy against infection and inflammation.

Abstract The receptor for the globular heads of C1q, gC1qR/p33, is a widely expressed cellular protein, which binds to diverse ligands including plasma proteins, cellular proteins, and microbial ligands. In addition to C1q, gC1qR also binds high molecular weight kininogen (HK), which also has two other cell surface sites, namely, cytokeratin 1 and urokinase plasminogen activator receptor (uPAR). On endothelial cells (ECs), the three molecules form two closely associated bimolecular complexes of gC1qR/cytokeratin 1 and uPAR/cytokeratin 1. However, by virtue of its high affinity for HK, gC1qR plays a central role in the assembly of the kallikrein-kinin system, leading to the generation of bradykinin (BK). BK in turn is largely responsible for the vascular leakage and associated inflammation seen in angioedema patients. Therefore, blockade of gC1qR by inhibitory peptides or antibodies may not only prevent the generation of BK but also reduce Clq-induced or microbial-ligand-induced inflammatory responses. Employing synthetic peptides and gClqR deletion mutants, we confirmed previously predicted sites for C1q (residues 75-96) and HK (residues 204-218) and identified additional sites for both C1q and HK (residues 190-202), for C1q (residues 144-162), and for HIV-1 gp41 (residues 174-180). With the exception of residues 75-96, which is located in the alphaA coiled-coil N-terminal segment, most of the identified residues form part of the highly charged loops connecting the various beta-strands in the crystal structure. Taken together, the data support the notion that gC1qR could serve as a novel molecular target for the design of antibody-based and/or peptide-based therapy to attenuate acute and/or chronic inflammation associated with vascular leakage and infection.

Distinct phases of blood gene expression pattern through tuberculosis treatment reflect modulation of the humoral immune response.

BACKGROUND: Accurate assessment of treatment efficacy would facilitate clinical trials of new antituberculosis drugs. We hypothesized that early alterations in peripheral immunity could be measured by gene expression profiling in tuberculosis patients undergoing successful conventional combination treatment. METHODS: Ex vivo blood samples from 27 pulmonary tuberculosis patients were assayed at diagnosis and during treatment. RNA was processed and hybridized to Affymetrix GeneChips, to determine expression of over 47,000 transcripts. RESULTS: There were significant ≥ 2-fold changes in expression of >4000 genes during treatment. Rapid, large-scale changes were detected, with down-regulated expression of 1261 genes within the first week, including inflammatory markers such as complement components C1q and C2. This was followed by slower changes in expression of different networks of genes, including a later increase in expression of B-cell markers, transcription factors, and signaling molecules. CONCLUSIONS: The fast initial down-regulation of expression of inflammatory mediators coincided with rapid killing of actively dividing bacilli, whereas slower delayed changes occurred as drugs acted on dormant bacilli and coincided with lung pathology resolution. Measurement of biosignatures during clinical trials of new drugs could be useful predictors of rapid bactericidal or sterilizing drug activity, and would expedite the licensing of new treatment regimens.

Evidence for a novel chemotactic C1q domain-containing factor in the leech nerve cord.

In vertebrates, central nervous system (CNS) protection is dependent on many immune cells including microglial cells. Indeed, activated microglial cells are involved in neuroinflammation mechanisms by interacting with numerous immune factors. Unlike vertebrates, some lophotrochozoan invertebrates can fully repair their CNS following injury. In the medicinal leech Hirudo medicinalis, the recruitment of microglial cells at the lesion site is essential for sprouting of injured axons. Interestingly, a new molecule homologous to vertebrate C1q was characterized in leech, named HmC1q (for H. medicinalis) and detected in neurons and glial cells. In chemotaxis assays, leech microglial cells were demonstrated to respond to human C1q. The chemotactic activity was reduced when microglia was preincubated with signaling pathway inhibitors (Pertussis Toxin or wortmannin) or anti-human gC1qR antibody suggesting the involvement of gC1qR in C1q-mediated migration in leech. Assays using cells preincubated with NO chelator (cPTIO) showed that C1q-mediated migration was associated to NO production. Of interest, by using anti-HmC1q antibodies, HmC1q released in the culture medium was shown to exhibit a similar chemotactic effect on microglial cells as human C1q. In summary, we have identified, for the first time, a molecule homologous to mammalian C1q in leech CNS. Its chemoattractant activity on microglia highlights a new investigation field leading to better understand leech CNS repair mechanisms.

Inflammatory myositis complicating hypocomplementemic urticarial vasculitis despite on-going immunosuppression.

We present a patient with previously diagnosed hypocomplementemic urticarial vasculitis syndrome, with skin, lung, and renal involvement, who presented with congestive cardiac failure. During the course of her hospitalization, she was also found to have profound proximal muscle weakness in both upper and lower limbs associated with raised creatinine kinase levels. A muscle biopsy was performed, which demonstrated evidence of an inflammatory myositis with vasculitis, which had returned despite on-going immunosuppression. This occurrence of a new autoimmune disease may well be an example of the "waste disposal" hypothesis.

Unique toxicities and resistance mechanisms associated with monoclonal antibody therapy.

Anti-CD20 therapy has had a truly dramatic impact on treatment and outcome of patients with follicular lymphoma. Unfortunately, the majority of responses to single-agent rituximab are incomplete, and all patients with follicular lymphoma will experience disease progression at some point following rituximab therapy. Rituximab has multiple mechanisms of inducing in vivo cytotoxicity, including antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, direct apoptotic signaling, and possible vaccinal effects. The cellular microenvironment within follicular lymphoma has a profound impact on which mechanism is dominant, and confers resistance in many situations. Both tumor-associated and host-associated factors also contribute to rituximab resistance. There are multiple potential approaches to overcoming rituximab resistance, including rational biologic combination immunotherapy, engineered antibodies, and radioimmunoconjugates. Improved ability to overcome resistance will require further elucidation of critical signaling pathways involved in rituximab induced cytotoxicity and a comprehensive understanding of interactions between its multiple mechanisms of action.

Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagents.

Despite the success of anti-CD20 monoclonal antibody (mAb) in the treatment of lymphoma, there remains considerable uncertainty about their mechanism(s) of action. Here, we show that certain of these reagents (rituximab and 1F5), which redistribute CD20 into membrane rafts, are bound efficiently by C1q, deposit C3b, and result in complement-dependent cytotoxicity (CDC). This activity is important in vivo, because complement depletion using cobra venom factor (CVF) markedly reduced the efficacy of rituximab and 1F5 in 2 lymphoma xenograft models. However, complement depletion had no effect on the potent therapeutic activity of B1, a mAb that does not redistribute CD20 into membrane rafts, bind C1q, or cause efficient CDC. Equivalent immunotherapy also occurred in the presence or absence of natural killer (NK) cells. Perhaps most surprising was the observation that F(ab')2 fragments of B1 but not 1F5 were able to provide substantial immunotherapy, indicating that non-Fc-dependent mechanisms are involved with B1. In accordance with this, B1 was shown to induce much higher levels of apoptosis than rituximab and 1F5. Thus, although complement is important for the action of rituximab and 1F5, this is not so for B1, which more likely functions through its ability to signal apoptosis.

[The mechanism of inhibition of the activation of the complement first component by polyanions and polycations ]. / Mekhanizm ingibirovaniia polianionami i polikationami aktivatsii pervogo komponenta komplementa.

Polyethyleneimine (PEI, 50 kDa) and polymethacrylic acid (PMA, 200 kDa) were shown to inhibit the lysis of sheep erythrocytes induced by the guinea pig complement. They twofold suppress the hemolysis at the concentrations of 0.47 and 0.89 microgram/ml, respectively. The inhibitory effect on the binding of the C1q subunit of human complement to the sensitized sheep erythrocytes (EA) was found to depend on the component of the reaction with which the inhibitors were preliminarily incubated. When an inhibitor, C1q, and EA were simultaneously incubated, the inhibition constants for PEI and PMA were 17 +/- 6 and 8.1 +/- 0.1 micrograms/ml, respectively. The preincubation of EA with PEI and the subsequent washing out of the inhibitor resulted in the inhibition constant of 22 +/- 3 micrograms/ml. No inhibitory effect was observed after a similar preincubation of EA with PMA. No inhibition was also detected when the inhibitors were added after the formation of the C1q complex with antibodies. These observations suggest that the binding of antibodies to cationic PEI prevents the C1q-antibody complex formation, while the binding of anionic PMA to the active site of C1q impedes the interaction of this subunit with immunoglobulins. Moreover, within the range of concentrations studied, the studied inhibitors did not affect the subsequent C1q binding to the C1r and C1s enzymes.

Induction of the complement component C1qB in brain of transgenic mice with neuronal overexpression of human cyclooxygenase-2.

We report that overexpression of human (h) cyclooxygenase-2 h(COX-2) in the brain of a transgenic mouse line leads to selective induction of endogenous complement component C1qB expression in neurons. No detectable induction of the C3 and C4 complement components in the brain was found. Chronic treatment of mice with the selective COX-2 inhibitor nimesulide reduced the hCOX-2-mediated induction of hippocampal C1qB mRNA expression. The data suggest that neuronal COX-2 expression may influence inflammatory responses in the brain, in part through modulation of complement gene expression. Because there is extensive evidence that C1q and other complement components are involved in Alzheimer's disease (AD) neurodegeneration, this study advances our understanding of the apparent benefits of COX-2 inhibition in AD.

Inflammatory repertoire of Alzheimer's disease and nondemented elderly microglia in vitro.

We have previously developed and characterized isolated microglia and astrocyte cultures from rapid (<4 h) brain autopsies of Alzheimer's disease (AD) and nondemented elderly control (ND) patients. In the present study, we evaluate the inflammatory repertoire of AD and ND microglia cultured from white matter (corpus callosum) and gray matter (superior frontal gyrus) with respect to three major proinflammatory cytokines, three chemokines, a classical pathway complement component, a scavenger cell growth factor, and a reactive nitrogen intermediate. Significant, dose-dependent increases in the production of pro-interleukin-1beta (pro-IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory peptide-1alpha (MIP-1alpha), IL-8, and macrophage colony-stimulating factor (M-CSF) were observed after exposure to pre-aggregated amyloid beta peptide (1-42) (Abeta1-42). Across constitutive and Abeta-stimulated conditions, secretion of complement component C1q, a reactive nitrogen intermediate, and M-CSF was significantly higher in AD compared with ND microglia. Taken together with previous in situ hybridization findings, these results demonstrate unequivocally that elderly human microglia provide a brain endogenous source for a wide range of inflammatory mediators.

Transforming growth factor-beta1 induces transforming growth factor-beta1 and transforming growth factor-beta receptor messenger RNAs and reduces complement C1qB messenger RNA in rat brain microglia.

Transforming growth factor-beta1 is a multifunctional peptide with increased expression during Alzheimer's disease and other neurodegenerative conditions which involve inflammatory mechanisms. We examined the autoregulation of transforming growth factor-beta1 and transforming growth factor-beta receptors and the effects of transforming growth factor-beta1 on complement C1q in brains of adult Fischer 344 male rats and in primary glial cultures. Perforant path transection by entorhinal cortex lesioning was used as a model for the hippocampal deafferentation of Alzheimer's disease. In the hippocampus ipsilateral to the lesion, transforming growth factor-beta1 peptide was increased >100-fold; the messenger RNAs encoding transforming growth factor-beta1, transforming growth factor-beta type I and type II receptors were also increased, but to a smaller degree. In this acute lesion paradigm, microglia are the main cell type containing transforming growth factor-beta1, transforming growth factor-beta type I and II receptor messenger RNAs, shown by immunocytochemistry in combination with in situ hybridization. Autoregulation of the transforming growth factor-beta1 system was examined by intraventricular infusion of transforming growth factor-beta1 peptide, which increased hippocampal transforming growth factor-beta1 messenger RNA levels in a dose-dependent fashion. Similarly, transforming growth factor-beta1 increased levels of transforming growth factor-beta1 messenger RNA and transforming growth factor-beta type II receptor messenger RNA (IC(50), 5pM) and increased release of transforming growth factor-beta1 peptide from primary microglia cultures. Interactions of transforming growth factor-beta1 with complement system gene expression are also indicated, because transforming growth factor-beta1 decreased C1qB messenger RNA in the cortex and hippocampus, after intraventricular infusion, and in cultured glia. These indications of autocrine regulation of transforming growth factor-beta1 in the rodent brain support a major role of microglia in neural activities of transforming growth factor-beta1 and give a new link between transforming growth factor-beta1 and the complement system. The auto-induction of the transforming growth factor-beta1 system has implications for transgenic mice that overexpress transforming growth factor-beta1 in brain cells and for its potential role in amyloidogenesis.

A small molecular weight factor in aqueous humor acts on C1q to prevent antibody-dependent complement activation.

PURPOSE: Aqueous humor inhibits activation of the classic complement pathway; however, the mechanism of this inhibition is unknown. We have examined at the molecular level potential factors responsible for the inhibition, and we have tried to determine where in the complement pathway the inhibition takes place. METHODS: Fresh rabbit aqueous humor was size fractionated by centrifuge concentrators and by size exclusion column chromatography, and each fraction was assayed for inhibition of the classic complement pathway in a standard CH50 hemolytic assay. Fractions with inhibitory activity were assayed for protein and the presence of ascorbic acid and were subjected to heat treatment. To identify where in the pathway the inhibitor(s) function, the expression of activated complement components bound to the surface of antibody-coated erythrocytes was analyzed by flow cytometry using fluorescein isothiocyanate-labeled antibodies to specific complement components. In addition, hemolytic assays were performed for the function of individual complement components. RESULTS: The most potent inhibition of the classic pathway was in a fraction of aqueous humor of less than 1.3 kDa. The inhibitory activity in the fraction was unassociated with detectable protein or ascorbic acid, and it remained present after heat treatment. The functional analysis through flow cytometry and hemolytic assays for individual complement components showed that the inhibitor in the less than 1.3-kDa fraction caused a blockade in the complement pathway at the level of C1q. CONCLUSIONS: The aqueous humor contains a unique potent anticomplementary factor that has a molecular weight less than 1.3 kDa. This heat-stable inhibitory factor inhibits the classic pathway at the level of C1q. These results imply that within the eye the complement pathway is inhibited at the earliest steps of its initiation. Such inhibition would prevent production of complement products that mediate inflammation and chemotaxis of inflammatory cells. Therefore, as part of the adaptation of immune privilege, the ocular microenvironment is protected from inflammation induced by antigen-antibody complexes.

Complement activation by cross-linked truncated and chimeric full-length beta-amyloid.

The activation of complement by beta-amyloid (A beta) has been implicated in the local inflammatory response in Alzheimer's disease. To assess the structural parameters required for this activation, beta-sheet-containing fibrils of A beta1-28 were induced by low pH and then chemically cross-linked to constrain the beta-sheet conformation. Chimeric A beta peptides with a substituted C-terminal sequence derived from two different transmembrane proteins were also assessed for the ability to form fibrils rich in beta-sheet structure and to activate complement. Both the cross-linked A beta1-28 and the chimeric A beta peptides were strong activators of the classical complement pathway. These results suggest that the C-terminal residues (29-42) of A beta facilitate fibril assembly required for complement activation but do not contain the interaction sites required for complement activation, further supporting the hypothesis that C1q binds to the N-terminal hydrophilic domain of A beta, and that a fibrillar beta-sheet-rich conformation is required for effective binding and activation of C1.

Short-term kinetics of the humoral anti-C1q response in SLE using the ELISPOT method: fast decline in production in response to steroids.

Twenty four systemic lupus erythaematosus patients and 17 patients with other diagnoses were investigated regarding the presence of cells producing C1q reactive antibodies in peripheral blood mononuclear cells using the ELISPOT technique. These results were then compared with parallel serum levels of C1q reactive antibodies. Current production of anti-C1q was almost entirely confined to the systemic lupus erythaematosus group. Longitudinal analysis of anti-C1q ELISPOT positive patients showed rapid changes in the number of anti-C1q producing cells, but only slowly changing serum levels of the corresponding antibodies in response to glucocorticoids. In one systemic lupus erythaematosus patient prednisolone treatment had a selective effect on this autoantibody production, as the production of anti-C1q spot forming cells rapidly dropped to zero, at the same time as the number of total spot-forming cells showed only less change. In another patient, self-limiting connective tissue disease was associated with temporal occurrence of IgM anti-C1q. We believe, from these data, that the ELISPOT method for determination of current antibody production may be of particular value in longitudinal evaluation of disease course and therapeutic effects in systemic lupus erythaematosus and other rheumatic diseases.

Effect of Pseudomonas aeruginosa elastase and alkaline protease on serum complement and isolated components C1q and C3.

The present study was undertaken to examine and compare the direct effect of two Pseudomonas enzymes, elastase and alkaline protease, on the serum hemolytic complement as a whole, and on the two recognition molecules of complement, C1q and C3 in particular. The results of our study show that incubation of serum with 0-50 micrograms/ml elastase or protease (60 min, 37 degrees C) resulted in a dose-dependent depletion of hemolytic complement with the protease being 3-4 times more efficient than elastase. Incubation of highly purified C3 (20 hr, 37 degrees C) with protease (2% w/w) resulted in the conversion of the 190-kDa molecule to a 120-kDa fragment. When analyzed by SDS-PAGE under reducing conditions, the 120-kDa piece yielded three distinct bands: an intact 75-kDa beta-chain and two alpha-chain pieces of approximately 41- and 26-kDa. NH2-terminal end sequence analysis localized the 26-kDa fragment within the cysteine-rich 41-kDa, COOH-terminal piece. This in turn suggests that the 70-kDa fragment which is not accounted for on SDS-PAGE is derived from the NH2-terminal end of the alpha-chain molecule which is completely degraded into small fragments. While the degradation pattern obtained with elastase is similar to that of protease, the latter enzyme was found to be more efficient. Exposure of C1q (0-5 hr, 37 degrees C) to protease or elastase on the other hand appears to reveal preferential sensitivity of the 28-kDa A-chain and 24-kDa C-chain, of the C1q molecule, with the protease being more potent than the elastase. Since both C1q and physiologic fragments of C3 (C3b, iC3b, and C3dg) are important opsonins of varying efficiencies, degradation of these molecules by Pseudomonas enzymes may, in part, facilitate the survival and proliferation of the organism in plasma. Furthermore, degradation of the key recognition molecules of complement, C1q and C3, would enhance the virulence of this organism by aborting complement-mediated bacterial killing. In addition the results imply that during Pseudomonas bacteremia, PaAP may be a much more destructive enzyme than PaE with regards to C3 and C1q but combined, the synergistic effect may overwhelm not only the proteins of the complement system, but other proteins of the humoral immune defense system as well.

[The immunomodulating properties of a macro glycopeptide isolated from serum. II. The activation of macrophages and polymorphonuclear cells]. / Imunomodulirashti svoistva na gliukomakropeptid, izoliran ot surovatka. II. Aktivirane na makrofagi i polimorfonukleari.

The stimulating properties of three fractions of a glucomacropeptide consisting of different carbohydrate and peptide components were studied. The phagocytic activity of polymorphonuclears was increased on the 24th hour and of macrophages on the 3rd day after application of the preparations to mice. In vitro fraction 3 stimulates the lysozyme activity and the synthesis of proteins and Clq of the macrophages complement. These properties of the cells are collaborated by the established ultrastructural changes. The different composition of the fractions determines their immunostimulating effect.