Classical Complement Activity Research Articles

Alternative Complement Pathway Inhibition by Lampalizumab

Lampalizumab, an antigen-binding fragment of a humanized monoclonal antibody directed against complement factor D (CFD), is designed to treat geographic atrophy (GA) secondary to age-related macular degeneration. Given the lack of clinical efficacy observed in patients with GA in the phase III Chroma/Spectri trials, we investigated the impact of lampalizumab on the complement system invivo. We developed 6 novel assays to measure changes in complement pathway activities in aqueous humor samples collected from patients enrolled in these trials. Chroma/Spectri were double-masked, sham-controlled, 96-week trials. Aqueous humor samples from 97 patients with bilateral GA across all groups (i.e., intravitreous lampalizumab 10 mg every 6 weeks, every 4 weeks, or corresponding sham procedures) were tested. Novel antibody capture assays were developed on the Simoa platform for complement factor B (CFB), the Bb fragment of CFB, intact complement component 3 (C3), processed C3, intact complement component 4 (C4), and processed C4. The ratio of processed vs. intact complement factors (i.e., complement activity) in aqueous humor were assessed. Patients treated with either of the lampalizumab regimens demonstrated an increase in CFD level at week 24 compared with baseline, along with a corresponding median reduction in the Bb:CFB ratio of 41% to 43%. There were no strong correlations between lampalizumab concentrations in aqueous humor and change in CFD levels or Bb:CFB ratio over time. No change in downstream C3 processing was observed with lampalizumab treatment. Additionally, there was no change in C4 processing. The collection of aqueous humor samples from patients in Chroma and Spectri trials provided key insights on the effects of lampalizumab, a novel complement inhibitor, on local ocular complement activation. Lampalizumab inhibited the alternative complement pathway in the eyes of patients with GA; however, this did not translate into a measurable reduction in either classical or total complement activity, based on absence of changes in C4 and C3 processing, respectively. Proprietary or commercial disclosure may be found after the references.

Complement system changes in blood in Parkinson's disease and progressive Supranuclear Palsy/Corticobasal Syndrome

Parkinson's Disease (PD) is diagnosed clinically, and early PD is often challenging to differentiate from atypical parkinsonian disorders such as the Four-repeat (4R-) Tauopathies Progressive Supranuclear Palsy and Corticobasal Syndrome. Diagnostic biomarkers are needed, and proteomic studies have suggested that the plasma complement system is altered in PD, but validation studies are lacking.In this study, plasma from 148 individuals (PD, 4R-Tauopathies, and healthy controls (HC)) were used to quantify 12 complement proteins with immunoassays, and CH50 classical pathway complement activity was quantified in sera from further 78 individuals (PD and HC).Complement factors C1q and C3 in plasma were lower in individuals with 4R-Tauopathies (ANOVA, p = 0.0041, p = 0.0057 respectively) compared to both PD and HC. None of the complement proteins were altered between PD and HC, however a few proteins correlated with clinical parameters within the PD group. Notably, levels of C3 correlated with non-motor symptoms in female patients. Classical pathway complement activity was not altered in PD serum, but did correlate with mental fatigue.In conclusion, individuals with 4R-Tauopathies showed lower plasma C1q and C3 compared PD and HC. Neither complement levels nor CH50 activity were significantly altered in PD versus HC but may associate with PD symptom severity.

Associations between serum levels of C3, C4, and total classical complement activity in COVID-19 patients at the time of admission and clinical outcome

In the present study, we investigated the association between complement system status at the time of admission and clinical outcomes in COVID-19 patients. This single-center study was carried out with sixty-one adult patients with COVID-19 who were hospitalized at Imam Hassan Hospital of North Khorasan University of Medical Sciences (Bojnurd, Iran) with less than three days passage since onset of COVID-19 symptoms. Twenty-three healthy volunteers with demographic features similar to the patient group (matched by age and gender) were included in the study as a control group. Patient information including demographic information, demographic data, clinical characteristics, and clinical outcomes were obtained from electronic medical records. Of 61 hospitalized patients with COVID-19, 28 (47.54%) were female, and the average age was 48.78.8 years. The healthy control group included 23 cases (11 (47.8%) female, 12 (52.1%) males, mean age 46.44.4 years). Twenty-one of the 61 patients (34.4%) were admitted to the ICU, and sixteen of them (26.2%) died. Thirty-three (54.10%) patients with COVID-19 were hospitalized for less than 7 days, and 28 (45.90%) of them were hospitalized for 7 days. Our results show that length of hospital stay in the no-ICU group was significantly lower than the ICU admission or death groups (6.490.24 vs. 8.851.59 and 10.531.80, p = 0.0002). The levels of C3, C4, and CH50 were determined through the immunoturbidimetric method and single-radial-haemolysis plates, respectively, on serum samples obtained from patients at the time of admission or those in the control group. Our results indicate that C3, C4 and CH50 levels were markedly lower in COVID-19 patients than in the control group. We also found that complement parameter levels in COVID-19 patients who died or were admitted to ICU were significantly lower than in non-ICU COVID-19 patients. In general, it seems that serum level of C3, C4, and CH50 at admission may predict disease progression or adverse clinical outcome in COVID-19 patients.

Monoclonal antibodies effectively potentiate complement activation and phagocytosis of Staphylococcus epidermidis in neonatal human plasma.

Central line associated bloodstream infections (CLABSI) with Staphylococcus epidermidis are a major cause of morbidity in neonates, who have an increased risk of infection because of their immature immune system. As especially preterm neonates suffer from antibody deficiency, clinical studies into preventive therapies have thus far focused on antibody supplementation with pooled intravenous immunoglobulins from healthy donors (IVIG) but with little success. Here we study the potential of monoclonal antibodies (mAbs) against S. epidermidis to induce phagocytic killing by human neutrophils. Nine different mAbs recognizing Staphylococcal surface components were cloned and expressed as human IgG1s. In binding assays, clones rF1, CR5133 and CR6453 showed the strongest binding to S. epidermidis ATCC14990 and CR5133 and CR6453 bound the majority of clinical isolates from neonatal sepsis (19 out of 20). To study the immune-activating potential of rF1, CR5133 and CR6453, bacteria were opsonized with mAbs in the presence or absence of complement. We observed that activation of the complement system is essential to induce efficient phagocytosis of S. epidermidis. Complement activation and phagocytic killing could be enhanced by Fc-mutations that improve IgG1 hexamerization on cellular surfaces. Finally, we studied the ability of the mAbs to activate complement in r-Hirudin neonatal plasma conditions. We show that classical pathway complement activity in plasma isolated from neonatal cord blood is comparable to adult levels. Furthermore, mAbs could greatly enhance phagocytosis of S. epidermidis in neonatal plasma. Altogether, our findings provide insights that are crucial for optimizing anti-S. epidermidis mAbs as prophylactic agents for neonatal CLABSI.

Activation of Intracellular Complement in Lungs of Patients With Severe COVID-19 Disease Decreases T-Cell Activity in the Lungs.

A novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), arose late in 2019, with disease pathology ranging from asymptomatic to severe respiratory distress with multi-organ failure requiring mechanical ventilator support. It has been found that SARS-CoV-2 infection drives intracellular complement activation in lung cells that tracks with disease severity. However, the cellular and molecular mechanisms responsible remain unclear. To shed light on the potential mechanisms, we examined publicly available RNA-Sequencing data using CIBERSORTx and conducted a Ingenuity Pathway Analysis to address this knowledge gap. In complement to these findings, we used bioinformatics tools to analyze publicly available RNA sequencing data and found that upregulation of complement may be leading to a downregulation of T-cell activity in lungs of severe COVID-19 patients. Thus, targeting treatments aimed at the modulation of classical complement and T-cell activity may help alleviate the proinflammatory effects of COVID-19, reduce lung pathology, and increase the survival of COVID-19 patients.

Investigational RNAi Therapeutic Targeting C5 Is Efficacious in Pre-clinical Models of Myasthenia Gravis

Complement-mediated damage to the neuromuscular junction (NMJ) is a key mechanism of pathology in myasthenia gravis (MG), and therapeutics inhibiting complement have shown evidence of efficacy in the treatment of MG. In this study, we describe the development of a subcutaneously administered N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C5 component of complement that silences C5 expression in the liver (ALN-CC5). Treatment of wild-type rodents with ALN-CC5 resulted in robust and durable suppression of liver C5 expression. Dose-dependent serum C5 suppression was observed in non-human primates, with a lowering of serum C5 of up to 97.5% and the concomitant inhibition of serum complement activity. C5 silencing was efficacious in ameliorating disease symptoms in two standard rat models of MG, demonstrating the key role of circulating C5 in pathology at the NMJ. Improvement in disease activity scores and NMJ pathology was observed at intermediate levels of complement activity inhibition, suggesting that complete ablation of complement activity may not be required for efficacy in MG. The pre-clinical studies of ALN-CC5 and efficacy of C5 silencing in rat models of MG support further clinical development of ALN-CC5 as a potential therapeutic for the treatment of MG and other complement-mediated disorders.

A microplate assay to measure classical and alternative complement activity.

We developed and validated a kinetic microplate hemolytic assay (HA) to quantify classical and alternative complement activity in a single dilution of human plasma or serum. The assay is based on monitoring hemolysis of sensitized sheep (or uncoated rabbit) red blood cells by means of a 96-well microplate reader. The activity of the calibrator was evaluated by reference to 200 healthy adults. The conversion of 50% hemolysis time into a percentage of activity was obtained using a calibration curve plotted daily. The linearity of the assay as well as interference (by hemolysis, bilrubinemia and lipemia) was assessed for classical pathway (CP). The within-day and the between-day precision was satisfactory regarding the performance of commercially available liposome immunoassay (LIA) and ELISA. Patients with hereditary or acquired complement deficiencies were detected (activity was measured <30%). We also provided a reference range obtained from 200 blood donors. The agreement of CP evaluated on samples from 48 patients was 94% with LIA and 87.5% with ELISA. The sensitivity of our assay was better than that of LIA, and the cost was lower than either LIA or ELISA. In addition, this assay was less time consuming than previously reported HAs. This assay allows the simultaneous measurement of 36 samples in duplicate per run of a 96-well plate. The use of a daily calibration curve allows standardization of the method and leads to good reproducibility. The same technique was also adapted for the quantification of alternative pathway (AP) activity.

The effects of increasing dietary levels of soy protein concentrate (SPC) on the immune responses and disease resistance (furunculosis) of vaccinated and non-vaccinated Atlantic salmon (Salmo salar L.) parr

Juvenile salmon, with an initial weight of 9 g, were fed three experimental diets, formulated to replace 35 (SPC35), 58 (SPC58) and 80 (SPC80) of high quality fishmeal (FM) with soy protein concentrate (SPC) in quadruplicate tanks. Higher dietary SPC inclusion was combined with increased supplementation of methionine, lysine, threonine and phosphorus. The experiment was carried out for 177 days. On day 92 salmon in each tank were bulk weighed. Post weighing eighty salmon from each tank were redistributed in two sets of 12 tanks. Salmon from the first set of tanks were vaccinated, while the second group was injected with phosphate buffer saline (PBS). Salmon were sampled on day 92 (pre-vaccination), day 94 (2 days post vaccination [dpv]/PBS injection [dpPBSinj]) and day 154 (62 dpv/dpPBSinj) of the trial for the assessment of their immune responses, prior to the performance of salmon bulk weights for each tank. On day 154, fish from each tank were again bulk weighed and then seventeen salmon per tank were redistributed in two sets of twelve tanks and intra-peritoneally infected with Aeromonas salmonicida. At Day 154, SPC80 demonstrated lower performance (weight gain, specific growth rate and thermal growth coefficient and feed conversion ratio) compared to SPC35 salmon. Reduced classical and total complement activities for salmon fed diets with over 58% of protein from SPC, were demonstrated prior to vaccination. Reduced alternative complement activity was detected for both SPC58 and SPC80 salmon at 2 dpv and for the SPC80 group at 62 dpv. Total and classical complement activities demonstrated no differences among the dietary groups after vaccination. Numerical increases in classical complement activity were apparent upon increased dietary SPC levels. Increased phagocytic activity (% phagocytosis and phagocytic index) was exhibited for the SPC58 group compared to SPC35 salmon at 62 dpPBSinj. No differences in serum lysozyme activity, total IgM, specific antibodies, protein, glucose and HKM respiratory burst were detected among the dietary groups at any timepoint or state. Mortalities as a result of the experimental infection only occurred in PBS-injected fish. No differences in mortality levels were demonstrated among the dietary groups. SPC58 diet supported both good growth and health in juvenile Atlantic salmon while SPC80 diet did not compromise salmon’ immunity or resistance to intraperitoneally inflicted furunculosis.

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

Blood-feeding insects inject potent salivary components including complement inhibitors into their host’s skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.

Influence of β-Glucan (Macrogard®) on Innate Immunity of Carp Fry

Abstract β-glucan (Macrogard®) was administrated to enhance the immunity and growth of Cyprinus carpio fry. The whole body homogenate of fish sampled with one week intervals during 2nd - 6th weeks post hatching was assayed for the total protein content, lysozyme activity, α-2-macroglobulin content, classical complement pathway activation, and weight gain. After the 3rd week of experimental feeding, the total protein content and the classical complement activity of fish fed β-glucan supplemented diet were higher than controls. Significantly higher lysozyme activity and α-2-macroglobulin levels were noted in group fed β-glucan at the 2nd and 3rd week of diet application. It was demonstrated that β-glucan enriched feeding increased the immunity of common carp at the earliest stages of their development.

Across-line SNP association study for (innate) immune and behavioral traits in laying hens.

BackgroundAn association study between single nucleotide polymorphism markers (SNP) and (innate and adaptive) immune parameters but also feather condition score on the back, rump and belly of laying hens was performed. The immune parameters measured in blood samples were natural and acquired antibody titers and complement activity. Feather condition score as a measure of feather damage was determined, this parameter is closely related to feather pecking behavior in hens housed in groups.The aim of the study was to detect associations between genetic markers and immune parameters and feather condition score across nine lines of laying hens, focusing on the feather peckers as well as on the victims of feather pecking.MethodsA novel approach based on across-line analysis and testing of the SNP-by-line interaction was performed.ResultsIn total 59 significant associations between SNP and immune traits were detected. Previously identified QTL were confirmed and new associations of genes regulating immune function identified. The IL17A gene (chromosome 3) influences natural and acquired antibody titers and activation of classical and alternative complement pathways. The major histocompatibility complex on chromosome 16 showed significant association with natural and acquired antibody titers and classical complement activity. The IL12B and IRF1 genes on chromosome 13 were associated with natural antibody titers.The direct effect of the genotype of an individual on its feather condition and the associative effect of the genotype of the cage mates on the individual’s feather condition were analyzed. The direct genetic effect can be described as the susceptibility to be pecked at, and the associative genetic effect as the propensity to perform feather pecking. Eleven significant associations were detected for the direct effect, and 81 for the associative effect. The serotonin receptor 2C (HTR2C) on chromosome 4 was highlighted in both analyses.ConclusionsOur results confirmed previously identified QTL and identified new associations of genes regulating immune function. The results for feather condition score supports existing evidence of involvement of the serotonergic system in feather pecking in laying hens. Immune regulatory genes were found to be associated to feather condition score, revealing relationships between the immune system and behavior.

Across‐line SNP association study of innate and adaptive immune response in laying hens

The aim of the present study was to detect quantitative trait loci (QTL) for innate and adaptive immunity in laying hens. For this purpose, the associations between 1022 single nucleotide polymorphism (SNP) markers and immune traits were studied in 583 hens from nine different layer lines. Immune traits were natural antibodies for keyhole limpet haemocyanin (KLH) and lipopolysaccharide (LPS) at 20, 40 and 65 weeks, acquired antibodies to the vaccinal virus of Newcastle disease at 20 weeks, and complement activity measured on sheep and bovine red blood cells at 20, 40 and 65 weeks. We adopted a novel approach based on across-line analysis and testing of the SNP-by-line interaction. Among lines, linkage disequilibrium is conserved at shorter distances than in individual lines; therefore, SNPs significantly associated with immune traits across lines are expected to be near the functional mutations. In the analysis, the SNPs that had a significant across-line effect but did not show significant SNP-by-line interaction were identified to test whether the association was consistent in the individual lines. Ultimately, 59 significant associations between SNPs and immune traits were detected. Our results confirmed some previously identified QTL and identified new QTL potentially involved in the immune function. We found evidence for a role of IL17A (chromosome 3) in natural and acquired antibody titres and in the classical and alternative pathways of complement activation. The major histocompatibility genes on chromosome 16 showed significant association with natural and acquired antibody titres and classical complement activity. The IL12B gene on chromosome 13 was associated with natural antibody titres.

TheStreptococcuspneumoniaeCapsule Inhibits Complement Activity and Neutrophil Phagocytosis by Multiple Mechanisms

The Streptococcus pneumoniae capsule is vital for virulence and may inhibit complement activity and phagocytosis. However, there are only limited data on the mechanisms by which the capsule affects complement and the consequences for S. pneumoniae interactions with phagocytes. Using unencapsulated serotype 2 and 4 S. pneumoniae mutants, we have confirmed that the capsule has several effects on complement activity. The capsule impaired bacterial opsonization with C3b/iC3b by both the alternative and classical complement pathways and also inhibited conversion of C3b bound to the bacterial surface to iC3b. There was increased binding of the classical pathway mediators immunoglobulin G (IgG) and C-reactive protein (CRP) to unencapsulated S. pneumoniae, indicating that the capsule could inhibit classical pathway complement activity by masking antibody recognition of subcapsular antigens, as well as by inhibiting CRP binding. Cleavage of serum IgG by the enzyme IdeS reduced C3b/iC3b deposition on all of the strains, but there were still marked increases in C3b/iC3b deposition on unencapsulated TIGR4 and D39 strains compared to encapsulated strains, suggesting that the capsule inhibits both IgG-mediated and IgG-independent complement activity against S. pneumoniae. Unencapsulated strains were more susceptible to neutrophil phagocytosis after incubation in normal serum, normal serum treated with IdeS, complement-deficient serum, and complement-deficient serum treated with IdeS or in buffer alone, suggesting that the capsule inhibits phagocytosis mediated by Fcgamma receptors, complement receptors, and nonopsonic receptors. Overall, these data show that the S. pneumoniae capsule affects multiple aspects of complement- and neutrophil-mediated immunity, resulting in a profound inhibition of opsonophagocytosis.

Effects of a natural parasitical infection (Lernanthropus kroyeri) on the immune system of European sea bass, Dicentrarchus labrax L

The immune response of European sea bass, Dicentrarchus labrax, to a natural infection by the copepod parasite Lernanthropus kroyeri was evaluated for the first time in vivo. The results clearly demonstrated the triggering of the fish immune system by the parasite. Lysozyme activity and alternative complement pathway were involved in the early action against the parasitical infection, whilst classical complement and respiratory burst (RB) activity took over in the later stages of infection. It was hypothesized that the levels of alternative and classical complement and RB stimulation indexes may determine the resistance capacity of the fish to the parasite. It is not clear how parasites can survive despite the strong immunological arsenal deployed by the fish. The continual increase of prevalence and severity of parasite infection suggested that the parasite's mechanism of evasion of the immune system was extremely successful. The contrasting decrease in the negative effects of parasites on the fish health (such as gills anaemia) suggested that an equilibrium between the parasites and their hosts was reached in chronic infection. These dynamic interactions between parasites and fish hosts were probably the main determinant of host specificity.

Classical and alternative pathways complement activity in patients with Post-Traumatic stress disorder

Classical and alternative pathways complement activity in patients with Post-Traumatic stress disorder

Natural Humoral Immune Competence and Survival in Layers

The relation between survival and levels of humoral components of innate (and specific) immune competence of laying hens was investigated in a population of 1,063 laying hens from 12 purebred layer lines. Natural immune competence of the chickens was studied by measuring levels of natural antibodies (NAb) binding to keyhole limpet hemocyanin (KLH) or lipopolysaccharide (LPS), respectively, and hemolytic (classical and alternative) complement activity at 20, 40, and 65 wk of age. In addition, levels of antibodies binding a Newcastle disease vaccine strain as a measure of specific immunity were investigated at 20 wk of age. A distinction could be made between lines showing high or low immune competence with respect to NAb, complement activity, and specific antibodies. Within lines, significant correlations were found for each of the innate parameters among the 3 ages. The innate and specific parameters were, however, not correlated with each other. Based on the limited data set, it was not possible to draw conclusions on line differences for innate or specific immune competence in relation to survival. However, regardless of line, low levels of NAb binding to KLH or high levels of NAb binding to LPS were detected in chickens that did not survive the laying period. The major difference between the responses of NAb binding to KLH or LPS was that the chickens probably did not encounter KLH, which suggests a reflection of the capacity to respond, whereas the chickens most probably did encounter LPS, which suggests a reflection of the active status of the innate humoral immune system. In conclusion, we propose that levels (KLH) and activation (LPS) of components of natural antibodies are indicative for the probability that chickens survive a laying period.

Properdin deficiency in a boy with fulminant meningococcal septic shock

Bacterial meningitis is a rare presentation for congenital immunodeficiency, but meningococcal invasive diseases and meningitis have been associated with late complement component deficiencies and properdin deficiency. A 5-y-old boy of non-consanguineous parents was admitted to our hospital with meningococcal septic shock. He had previously been suffering from recurrent respiratory infections. His 13-y-old brother had also been treated for meningococcal meningitis when he was 7 y old. Immunological studies, done after recovery, on the patient and his two brothers revealed normal immunoglobulin, IgG subclasses, C3, C4 and CH50 levels. Haemolytic activity of the alternative complement pathway could not be detected, and properdin concentrations were <0.01 mg/l in serum samples from the patient and his brothers. The patient and family members received quadrivalent polysaccharide meningococcal vaccine. The patient was discharged on penicillin prophylaxis, and he remained healthy during the ensuing year. Our findings stress that measurement of the haemolytic activity of the alternative complement pathway in addition to classical pathway haemolytic complement activity should be performed in patients with meningococcal disease to reveal various forms of complement deficiency. This is particularly important when there is a family history, or recurrences or infection due to uncommon serogroups. Deficient individuals and affected family members might be protected from infection by vaccination.

Subtly Impaired Humoral Immunity Predisposes to Frequently Recurring Genital Herpes Simplex Virus Type 2 Infection and Herpetic Neuralgia

Immunogenetic factors predisposing to recurrent genital herpes remain poorly characterized. In a prospective case-control study, 52 consecutive patients with frequently recurring outbreaks of genital herpes were compared with 80 herpes simplex virus (HSV)-seropositive (types 1 and 2) and 70 HSV-seronegative control subjects. Immunoglobulins (Igs), type-specific anti-HSV-2 IgG and IgG subclass antibodies against glycoprotein G, levels of C3 and C4, and classical pathway hemolytic complement activity were measured, and IgG1 and IgG3 allotyping; C4 immunophenotyping; C4* real-time polymerase chain reaction (PCR) genotyping; and HLA-A*, B*, and DR* typing were performed. The G3m(g),G1m(a/a(x)) haplotype was more frequent in patients than in HSV-seronegative control subjects (P=.047). Compared with all control subjects, low levels of total IgG1 (odds ratio [OR], 4.9 [95% confidence interval {CI}, 2.0-12.5]; P=.001) and IgG3 (OR 3.6 [95% CI 1.7-7.8]; P=.001), but not of anti-HSV-2 antibodies, were associated with recurrences. Levels of complement were lowest in patients. The C4* null type was negatively associated with neuralgia (OR, 0.2 [95% CI, 0.06-0.81]; P=.022). Low levels of antibody-dependent cellular cytotoxicity-mediating IgG1 and IgG3 antibodies, partly dependent of allotype, may predispose to recurrent genital herpes. Antibodies produced by T helper type 1 responses, potentially against an unknown epitope, appear to be relevant in recurrences. In patients, C4* deficiencies are associated with protection from herpetic neuralgias, possibly through reduced inflammation.

Classical pathway complement activity in Familial Mediterranean fever

Objectives: The present study emphasizes the important role of the immune reactions in the pathogenesis of Familial Mediterranean fever. In the present study, the total hemolytic activity of the complement and the activities of individual complement components, C1, C2, C3, and C4, were determined in the blood serum of 32 patients with FMF and 28 healthy subjects. Design and methods: Hemolytic assay was applied, measuring THAC and individual complement components' activities. The patients were divided into 3 groups upon the regularity of colchicine therapy: patients receiving regular, irregular and not receiving colchicine treatment for at least 1 year. Results: No significant changes in the hemolytic activities of the C1, C2, C3, and C4 complement components were found between the healthy subjects and those FMF patients, who were receiving regular colchicine treatment. Conclusions: Our data obtained have raised a number of important questions relevant to FMF pathogenesis and once again confirms the efficiency of regular colchicine treatment.

Endothelial cell protection and complement inhibition in xenotransplantation: a novel in vitro model using whole blood

Studying the interactions between xenoreactive antibodies, complement and coagulation factors with the endothelium in hyperacute and acute vascular rejection usually necessitates the use of in vivo models. Conventional in vitro or ex vivo systems require either serum, plasma or anti-coagulated whole blood, making analysis of coagulation-mediated effects difficult. Here a novel in vitro microcarrier-based system for the study of endothelial cell (EC) activation and damage, using non-anticoagulated whole blood is described. Once established, the model was used to study the effect of the characterized complement- and coagulation inhibitor dextran sulfate (DXS, MW 5000) for its EC protective properties in a xenotransplantation setting. Porcine aortic endothelial cells (PAEC), grown to confluence on microcarrier beads, were incubated with non-anticoagulated whole human blood until coagulation occurred or for a maximum of 90 min. PAEC-beads were either pre- or co-incubated with DXS. Phosphate buffered saline (PBS) experiments served as controls. Fluid phase and surface activation markers for complement and coagulation were analyzed as well as binding of DXS to PAEC-beads. Co- as well as pre-incubation of DXS, followed by washing of the beads, significantly prolonged time to coagulation from 39 +/- 12 min (PBS control) to 74 +/- 23 and 77 +/- 20 min, respectively (P < 0.005 vs. PBS). DXS treatment attenuated surface deposition of C1q, C4b/c, C3b/c and C5b-9 without affecting IgG or IgM deposition. Endothelial integrity, expressed by positivity for von Willebrand Factor, was maintained longer with DXS treatment. Compared with PBS controls, both pre- and co-incubation with DXS significantly prolonged activated partial thromboplastin time (>300 s, P < 0.05) and reduced production of thrombin-antithrombin complexes and fibrinopeptide A. Whilst DXS co-incubation completely blocked classical pathway complement activity (CH50 test) DXS pre-incubation or PBS control experiments showed no inhibition. DXS bound to PAEC-beads as visualized using fluorescein-labeled DXS. This novel in vitro microcarrier model can be used to study EC damage and the complex interactions with whole blood as well as screen ''endothelial protective'' substances in a xenotransplantation setting. DXS provides EC protection in this in vitro setting, attenuating damage of ECs as seen in hyperacute xenograft rejection.