Pediatric Atypical Hemolytic Uremic Syndrome Advances.

Atypical hemolytic uremic syndrome (aHUS) is a rare disorder characterized by dysregulation of the alternate pathway. The diagnosis of aHUS is one of exclusion, which complicates its early detection and corresponding intervention to mitigate its high rate of mortality and associated morbidity. Heterozygous mutations in complement regulatory proteins linked to aHUS are not always phenotypically active, and may require a particular trigger for the disease to manifest. This list of triggers continues to expand as more data is aggregated, particularly centered around COVID-19 and pediatric vaccinations. Novel genetic mutations continue to be identified though advancements in technology as well as greater access to cohorts of interest, as in diacylglycerol kinase epsilon (DGKE). DGKE mutations associated with aHUS are the first non-complement regulatory proteins associated with the disease, drastically changing the established framework. Additional markers that are less understood, but continue to be acknowledged, include the unique autoantibodies to complement factor H and complement factor I which are pathogenic drivers in aHUS. Interventional therapeutics have undergone the most advancements, as pharmacokinetic and pharmacodynamic properties are modified as needed in addition to their as biosimilar counterparts. As data continues to be gathered in this field, future advancements will optimally decrease the mortality and morbidity of this disease in children.

Functional Analysis of Variants in Complement Factor I Identified in Age-Related Macular Degeneration and Atypical Hemolytic Uremic Syndrome.

Complement factor I (FI) is a central inhibitor of the complement system, and impaired FI function increases complement activation, contributing to diseases such as age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (aHUS). Genetic variation in complement factor I (CFI) has been identified in both AMD and aHUS, with more than half of these variants leading to reduced FI secretion levels. For many of the variants with normal FI secretion, however, functional implications are not yet known. Here we studied 11 rare missense variants, with FI secretion levels comparable to wildtype, but a predicted damaging effects based on the Combined Annotation Dependent Depletion (CADD) score. Three variants (p.Pro50Ala, p.Arg339Gln, and p.Ser570Thr) were analyzed in plasma and serum samples of carriers affected by AMD. All 11 variants (nine for the first time in this study) were recombinantly expressed and the ability to degrade C3b was studied with the C3b degradation assay. The amount of degradation was determined by measuring the degradation product iC3b with ELISA. Eight of 11 (73%) mutant proteins (p.Pro50Ala, p.Arg339Gln, p.Ile340Thr, p.Gly342Glu, p.Gly349Arg, p.Arg474Gln, p.Gly487Cys, and p.Gly512Ser) showed significantly impaired C3b degradation, and were therefore classified as likely pathogenic. Our data indicate that genetic variants in CFI with a CADD score >20 are likely to affect FI function, and that monitoring iC3b in a degradation assay is a useful tool to establish the pathogenicity of CFI variants in functional studies.

Anti-complement factor I antibody associated atypical hemolytic uremic syndrome - A new insight for future perspective!

Atypical hemolytic uremic syndrome (aHUS) is caused mainly by complement dysregulation. Although various defects in the complement system explaining pathophysiology have been described in recent years, the etiology still remains unclear in about thirty percent of cases. In exploring other causes, similar to anti- complement factor H (anti-CFH) antibody associated HUS, we hypothesized that anti-complement factor I (anti-CFI) antibody could play a role in aHUS. Further, we tried to describe the clinical profile and outcome of those with high anti CFI antibody titers. Eleven of thirty five children (31 %) diagnosed with aHUS from July 2017 to December 2018 had high IgG anti-CFI antibody titers. Median age was 10 months (6, 33) with no sex difference. Thirty-six percent (4/11) had nephrotic-range proteinuria. C3 was low in 8 children (72.7 %) with mean C3 (68.1 ±â€¯14.7 mg/dL). Plasmapheresis was done in 2 children who promptly responded, suggesting the possible role of anti-CFI antibody in pathogenesis of aHUS in these patients. Further studies examining role of anti-CFI antibodies in aHUS is warranted with longitudinal and genetic studies.

The trypsin-like serine protease domain of Paralichthys olivaceus complement factor I regulates complement activation and inhibits bacterial growth.

In mammals, complement factor I (CFI) is a serine protease in serum and plays a pivotal role in the regulation of complement activation. In the presence of cofactor, CFI cleaves C3b to iC3b, and further degrades iC3b to C3c and C3d. In teleost, the function of CFI is poorly understood. In this study, we examined the immunological property of CFI from Japanese flounder (Paralichthys olivaceus) (PoCFI), a teleost species with important economic value. PoCFI is composed of 597 amino acid residues and possesses a trypsin-like serine protease (Tryp) domain. We found that PoCFI expressions occurred in nine different tissues and were upregulated by bacterial challenge. Recombinant PoCFI-Tryp (rPoCFI-Tryp) inhibited complement activation and degraded C3b in serum. rPoCFI-Tryp exhibited apparent binding capacities to a board-spectrum of bacteria and inhibited bacterial growth. These results provide the first evidence to indicate that CFI in teleost negatively regulates complement activation via degradation C3b, and probably plays a role in host immune defense against bacterial infection.

Understanding the Role of Anti-PEG Antibodies in the Complement Activation by Doxil in Vitro.

Infusion reactions (IRs) are common immune-mediated side effects in patients treated with a variety of drug products, including, but not limited to, nanotechnology formulations. The mechanism of IRs is not fully understood. One of the best studied mechanisms of IRs to nanomedicines is the complement activation. However, it is largely unknown why some patients develop reactions to nanomedicines while others do not, and why some nanoparticles are more reactogenic than others. One of the theories is that the pre-existing anti-polyethylene glycol (PEG) antibodies initiate the complement activation and IRs in patients. In this study, we investigated this hypothesis in the case of PEGylated liposomal doxorubicin (Doxil), which, when used in a clinical setting, is known to induce IRs; referred to as complement activation-related pseudoallergy (CARPA) in sensitive individuals. We conducted the study in vitro using plasma derived from C57BL/6 mice and twenty human donor volunteers. We used mouse plasma to test a library of well-characterized mouse monoclonal antibodies with different specificity and affinity to PEG as it relates to the complement activation by Doxil. We determined the levels of pre-existing polyclonal antibodies that bind to PEG, methoxy-PEG, and PEGylated liposomes in human plasma, and we also assessed complement activation by Doxil and concentrations of complement inhibitory factors H and I in these human plasma specimens. The affinity, specificity, and other characteristics of the human polyclonal antibodies are not known at this time. Our data demonstrate that under in vitro conditions, some anti-PEG antibodies contribute to the complement activation by Doxil. Such contribution, however, needs to be considered in the context of other factors, including, but not limited to, antibody class, type, clonality, epitope specificity, affinity, and titer. In addition, our data contribute to the knowledge base used to understand and improve nanomedicine safety.

Geographic distribution of rare variants associated with age-related macular degeneration.

Purpose: A recent genome-wide association study by the International Age-related Macular Degeneration Genomics Consortium (IAMDGC) identified seven rare variants that are individually associated with age-related macular degeneration (AMD), the most common cause of vision loss in the elderly. In literature, several of these rare variants have been reported with different frequencies and odds ratios across populations of Europe and North America. Here, we aim to describe the representation of these seven AMD-associated rare variants in different geographic regions based on 24 AMD studies. Methods: We explored the occurrence of seven rare variants independently associated with AMD (CFH rs121913059 (p.Arg1210Cys), CFI rs141853578 (p.Gly119Arg), C3 rs147859257 (p.Lys155Gln), and C9 rs34882957 (p.Pro167Ser)) and three non-coding variants in or near the CFH gene (rs148553336, rs35292876, and rs191281603) in 24 AMD case-control studies. We studied the difference in distribution, interaction, and effect size for each of the rare variants based on the minor allele frequency within the different geographic regions. Results: We demonstrate that two rare AMD-associated variants in the CFH gene (rs121913059 [p.Arg1210Cys] and rs35292876) deviate in frequency among different geographic regions (p=0.004 and p=0.001, respectively). The risk estimates of each of the seven rare variants were comparable across the geographic regions. Conclusions: The results emphasize the importance of identifying population-specific rare variants, for example, by performing sequencing studies in case-control studies of various populations, because their identification may have implications for diagnostic screening and personalized treatment.

Patients with hypertension-associated thrombotic microangiopathy may present with complement abnormalities.

Thrombotic microangiopathy (TMA) is a pattern of endothelial damage that can be found in association with diverse clinical conditions such as malignant hypertension. Although the pathophysiological mechanisms differ, accumulating evidence links complement dysregulation to various TMA syndromes and in particular the atypical hemolytic uremic syndrome. Here, we evaluated the role of complement in nine consecutive patients with biopsy-proven renal TMA attributed to severe hypertension. Profound hematologic symptoms of TMA were uncommon. In six out of nine patients, we found mutations C3 in three, CFI in one, CD46 in one, and/or CFH in two patients either with or without the risk CFH-H3 haplotype in four patients. Elevated levels of the soluble C5b-9 and renal deposits of C3c and C5b-9 along the vasculature and/or glomerular capillary wall, confirmed complement activation in vivo. In contrast to patients without genetic defects, patients with complement defects invariably progressed to end-stage renal disease, and disease recurrence after kidney transplantation seems common. Thus, a subset of patients with hypertension-associated TMA falls within the spectrum of complement-mediated TMA, the prognosis of which is poor. Hence, testing for genetic complement abnormalities is warranted in patients with severe hypertension and TMA on renal biopsy to adopt suitable treatment options and prophylactic measures.

Chromosomal rearrangement-A rare cause of complement factor I associated atypical haemolytic uraemic syndrome.

Chromosomal rearrangements affecting the genes encoding complement factor H and the factor H related proteins have been described in aHUS patients. To date such disorders have not been described in other aHUS associated genes. We describe here a heterozygous 875,324bp deletion encompassing the gene (CFI) encoding complement factor I and ten other genes. The index case presented with aHUS and did not recover renal function. No abnormalities were detected on Sanger sequencing of CFI but a low factor I level led to a multiplex ligation-dependent probe amplification assay being undertaken. This showed a complete heterozygous deletion of CFI. The extent of the deletion and the breakpoint was defined. In the Newcastle aHUS cohort we have identified and report here 32 different CFI variants in 56 patients but to date this is the only deletion that we have identified. This finding although rare does suggest that screening for chromosomal rearrangements affecting CFI should be undertaken in all aHUS patients particularly if the factor I level is unexplainably low.

Further studies of the down-regulation by Factor I of the C3b feedback cycle using endotoxin as a soluble activator and red cells as a source of CR1 on sera of different complotype.

In this paper we have extended our earlier studies of the action of increasing Factor I concentration on complement activation by using a soluble activator, lipopolysaccharide (LPS) endotoxin, and using human erythrocytes as a source of CR1 - the co-factor needed for the final clip of iC3b to C3dg by Factor I. Using this more physiological system, the results show that we can predict that a quite modest increase in Factor I concentration - 22 µg/ml of extra Factor I - will convert the activity of the highest risk sera to those of the lowest risk. Preliminary experiments have been performed with erythrocytes allotyped for CR1 number. While we have not been able to perform an adequate study of their co-factor activities in our assays, preliminary experiments suggest that when Factor I levels are increased the difference produced by different allotypes of red cells is largely overcome. This suggests that in patients with paroxysmal nocturnal haemoglobinuria (PNH) treated with eculizumab, additional treatment with Factor I may be very useful in reducing the need for blood transfusion. We have also explored the age-related allele frequency for the two polymorphisms of Factor H and the polymorphism of C3. In our population, unlike the 1975 study, we found no age variation in the allele frequency in these polymorphisms. This may, however, reflect that the Cambridge BioResource volunteers do not include many very young or very elderly patients, and in general comprise a population not greatly at risk of death from infectious disease.

Complement factor I from flatfish half-smooth tongue (Cynoglossus semilaevis) exhibited anti-microbial activities.

Complement factor I (Cfi) is a soluble serine protease which plays a crucial role in the modulation of complement cascades. In the presence of substrate modulating cofactors (such as complement factor H, C4bp, CR1, etc), Cfi cleaves and inactivates C3b and C4b, thereby controlling the complement-mediated processes. In this study, we sequenced and characterized Cfi gene from Cynoglossus Semilaevis (designated as CsCfi) for the first time. The full-length cDNA of CsCfi was 2230 bp in length, including a 98 bp 5'-untranslated region (UTR), a 164 bp 3'-UTR and a 1968 bp open reading frame (ORF). It encoded a polypeptide of 656 amino acids, with a molecular mass of 72.28 kDa and an isoelectric point of 7.71. A signal peptide was defined at N-terminus, resulting in a 626-residue mature protein. Multiple sequence alignment revealed that Cfi proteins were well conserved with the typical modular architecture and identical active sites throughout the vertebrates, which suggested the conserved function of Cfi. Phylogenetic analysis indicated that CsCfi and the homologous Cfi sequences from teleosts clustered into a clade, separating from another clade from the cartilaginous fish and other vertebrates. Tissue expression profile analysis by quantitative real-time PCR (qRT-PCR) showed that CsCfi mRNA constitutively expressed in all tested tissues, with the predominant expression in liver and the lowest in stomach. Temporal expression levels of CsCfi after challenging with Vibrio anguillarum showed different expression patterns in intestine, spleen, skin, blood, head kidney and liver. The recombinant CsCfi (rCsCfi) protein showed broad-spectrum antimicrobial activities against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and Shewanella putrefaciens. The research revealed that CsCfi plays an important role in C. Semilaevis immunity.

Overactivation of Complement Alternative Pathway in Postpartum Atypical Hemolytic Uremic Syndrome Patients with Renal Involvement.

PROBLEM: Postpartum atypical hemolytic uremic syndrome (aHUS) is a life-threatening syndrome with unclear pathogenesis. The current study aimed to investigate the clinical and pathological features, complement activation status, and the genetic variations in a Chinese cohort of patients with renal biopsy-proven postpartum aHUS. METHOD OF STUDY: Five patients with postpartum aHUS were recruited. Renal biopsy specimens were examined and scored. Plasma levels of complements were detected, and coding sequences of complement regulators were screened. Anti-CFH/CFI autoantibodies were further detected. RESULTS: Patients with postpartum aHUS patients presented with severe clinical manifestations and renal involvement. The renal biopsies of the five patients showed typical features of thrombotic microangiopathies. The levels of the following complement components, C4d, Bb, C3a, C5a, and SC5b-9, were significantly elevated in patients with postpartum aHUS compared with normal non-pregnant controls. The plasma levels of CFH and CFI significantly decreased in patients with postpartum aHUS compared with normal pregnant women. Three CFH single nucleotide polymorphisms (SNPs) were identified in the five patients. Two patients presented with CFH autoantibodies. CONCLUSION: Postpartum aHUS is a clinical syndrome with severe renal damage. Genetic deficiencies and autoantibodies of CFH may lead to alternative pathway overactivation and participated in the pathogenesis of postpartum aHUS.

Anti-factor H autoantibodies in C3 glomerulopathies and in atypical hemolytic uremic syndrome: one target, two diseases.

Autoantibodies targeting factor H (FH), which is a main alternative complement pathway regulatory protein, have been well characterized in atypical hemolytic uremic syndrome (aHUS) but have been less well described in association with alternative pathway-mediated glomerulopathies (GP). In this study, we studied 17 patients presenting with GP who were positive for anti-FH IgG. Clinical data were collected and biological characteristics were compared with those of patients presenting with anti-FH Ab-associated aHUS. In contrast to the aHUS patients, the GP patients had no circulating FH-containing immune complexes, and their anti-FH IgG had a weaker affinity for FH. Functional studies demonstrated that these Abs induced no perturbations in FH cell surface protection or the binding of FH to its ligand. However, anti-FH IgG samples isolated from three patients were able to affect the factor I cofactor activity of FH. Epitope mapping identified the N-terminal domain of FH as the major binding site for GP patient IgG. No homozygous deletions of the CFHR1 and CFHR3 genes, which are frequently associated with the anti-FH Ab in aHUS patients, were found in the GP patients. Finally, anti-FH Abs were frequently associated with the presence of C3 nephritic factor in child GP patients and with monoclonal gammopathy in adult GP patients, who frequently showed Ig Lchain restriction during reactivity against factor H. These data provide deeper insights into the pathophysiological differences between aHUS and GP, demonstrating heterogeneity of anti-FH IgG.

Complement activation in diseases presenting with thrombotic microangiopathy.

The complement system contains a great deal of biological "energy". This is demonstrated by the atypical hemolytic uremic syndrome (aHUS), which is a thrombotic microangiopathy (TMA) characterized by endothelial and blood cell damage and thrombotic vascular occlusions. Kidneys and often also other organs (brain, lungs and gastrointestinal tract) are affected. A principal pathophysiological feature in aHUS is a complement attack against endothelial cells and blood cells. This leads to platelet activation and aggregation, hemolysis, prothrombotic and inflammatory changes. The attacks can be triggered by infections, pregnancy, drugs or trauma. Complement-mediated aHUS is distinct from bacterial shiga-toxin (produced e.g. by E. coli O:157 or O:104 serotypes) induced "typical" HUS, thrombotic thrombocytopenic purpura (TTP) associated with ADAMTS13 (an adamalysin enzyme) dysfunction and from a recently described disease related to mutations in intracellular diacylglycerol kinase ε (DGKE). Mutations in proteins that regulate complement (factor H, factor I, MCP/CD46, thrombomodulin) or promote (C3, factor B) amplification of its alternative pathway or anti-factor H antibodies predispose to aHUS. The fundamental defect in aHUS is an excessive complement attack against cellular surfaces. This can be due to 1) an inability to regulate complement on self cell surfaces, 2) hyperactive C3 convertases or 3) complement activation and coagulation promoting changes on cell surfaces. The most common genetic cause is in factor H, where aHUS mutations disrupt its ability to recognize protective polyanions on surfaces where C3b has become attached. Most TMAs are thus characterized by misdirected complement activation affecting endothelial cell and platelet integrity.

Factor I autoantibodies in patients with atypical hemolytic uremic syndrome: disease-associated or an epiphenomenon?

BACKGROUND AND OBJECTIVES: Atypical hemolytic uremic syndrome is a disease associated with mutations in the genes encoding the complement regulators factors H and I. In addition, factor H autoantibodies have been reported in ∼10% of patients with atypical hemolytic uremic syndrome. This study searched for the presence of factor I autoantibodies in atypical hemolytic uremic syndrome. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This study screened 175 atypical hemolytic uremic syndrome patients for factor I autoantibodies using ELISA with confirmatory Western blotting. Functional studies using purified immunoglobulin from one patient were subsequently undertaken. RESULTS: Factor I autoantibodies were detected in three patients. In one patient with a high titer of autoantibody, the titer was tracked over time and was found to have no association with disease activity. This study found evidence of an immune complex of antibody and factor I in this patient, but purified IgG, isolated from current serum samples, had only a minor effect on fluid phase and cell surface complement regulation. Genetic analysis of the three patients with factor I autoantibodies revealed that they had two copies of the genes encoding factor H-related proteins 1 and 3 and therefore, did not have a deletion commonly associated with factor H autoantibodies in atypical hemolytic uremic syndrome. Two patients, however, had functionally significant mutations in complement factor H. CONCLUSIONS: These findings reinforce the concept of multiple concurrent risk factors being associated with atypical hemolytic uremic syndrome but question whether autoantibodies per se predispose to atypical hemolytic uremic syndrome.

Association of factor H autoantibodies with deletions of CFHR1, CFHR3, CFHR4, and with mutations in CFH, CFI, CD46, and C3 in patients with atypical hemolytic uremic syndrome.

Factor H autoantibodies have been reported in approximately 10% of patients with atypical hemolytic uremic syndrome (aHUS) and are associated with deficiency of factor H-related proteins 1 and 3. In this study we examined the prevalence of factor H autoantibodies in the Newcastle cohort of aHUS patients, determined whether the presence of such autoantibodies is always associated with deficiency of factor H-related proteins 1 and 3, and examined whether such patients have additional susceptibility factors and/or mutations in the genes encoding complement regulator/activators. We screened 142 patients with aHUS and found factor H autoantibodies in 13 individuals (age 1-11 years). The presence of the autoantibodies was confirmed by Western blotting. By using multiplex ligation-dependent probe amplification we measured complement factor H-related (CFHR)1 and CFHR3 copy number. In 10 of the 13 patients there were 0 copies of CFHR1, and in 3 patients there were 2. In 3 of the patients with 0 copies of CFHR1 there was 1 copy of CFHR3, and these individuals exhibited a novel deletion incorporating CFHR1 and CFHR4. In 5 patients mutations were identified: 1 in CFH, 1 in CFI, 1 in CD46, and 2 in C3. The latter observation emphasizes that multiple concurrent factors may be necessary in individual patients for disease manifestation.

Complement in human diseases: Lessons from complement deficiencies.

Complement deficient cases reported in the second half of the last century have been of great help in defining the role of complement in host defence. Surveys of the deficient individuals have been instrumental in the recognition of the clinical consequences of the deficiencies. This review focuses on the analysis of the diseases associated with the deficiencies of the various components and regulators of the complement system and their therapeutic implications. The diagnostic approach leading to the identification of the deficiency is discussed here as a multistep process that starts with the screening assays and proceeds in specialized laboratories with the characterization of the defect at the molecular level. The organization of a registry of complement deficiencies is presented as a means to collect the cases identified in and outside Europe with the aim to promote joint projects on treatment and prevention of diseases associated with defective complement function.

Analysis of the serine protease function of porcine factor I produced by liver cells for xenotransplantation.

The use of a bioartificial liver with pig liver cells in the treatment of fulminant hepatic failure has already begun as a clinical trial in several countries. Therefore, studies on plasma complement regulatory proteins of the pig are necessary, because the liver produces them. Complement factor I is a serine protease that cleaves C3b and C4b. The DNA sequences of factor I have been reported in many species, with the noted exception of pigs. In this study, porcine factor I was cloned and an open reading frame of 1794 base pairs were identified. The predicted amino acid sequence maintained a relatively high homology compared to those of other mammals, especially in the serine protease (SP) region. The cell membrane-bound forms of the porcine and the human SP domain of factor I were constructed. Amelioration of complement-mediated cell lysis by these molecules was then tested, using several kinds of sera and Chinese hamster ovary (CHO) cell transfectants. Both molecules had a suppressing effect on pig, human and dog complements, indicating little species-specificity. Further studies of other plasma complement regulatory proteins produced from pig liver cells will need to be considered as the primary feature of the pig bioartificial liver.

The tick-over theory revisited: formation and regulation of the soluble alternative complement C3 convertase (C3(H2O)Bb).

The molecular interactions between the components of the C3 convertase of the alternative pathway (AP) of complement and its regulators, in both surface-bound and fluid-phase form, are still incompletely understood. The fact that the AP convertase is labile makes studies difficult to perform. According to the so called tick-over theory, hydrolyzed C3, called C3(H(2)O), forms the initial convertase in fluid phase together with factor B. In the present study, we have applied western blot analysis and ELISA together with fluorescence resonance energy transfer (FRET) to study the formation of the fluid-phase AP convertases C3(H(2)O)Bb and C3bBb and their regulation by factor H and factor I at specific time points and, with FRET, in real time. In our hands, factor B showed a higher affinity for C3(H(2)O) than for C3b, although in both cases it was readily activated to Bb. However, the convertase activity of C3bBb was approximately twice that of C3(H(2)O)Bb, as monitored by the generation of C3a. But in contrast, the C3(H(2)O)Bb convertase was more resistant to inactivation by factor H and factor I than was the C3bBb convertase. Under conditions that totally inactivated C3bBb, C3(H(2)O)Bb still retained approximately 25% of its initial activity.

Recurrent infections in partial complement factor I deficiency: evaluation of three generations of a Brazilian family.

We report here on the evaluation of a factor I-deficient Brazilian family (three generations, 39 members) with strong consanguinity. The complete factor I-deficient patients (n = 3) presented recurrent respiratory infections, skin infections and meningitis; one of them died after sepsis. They presented an impaired total haemolytic activity (CH50), low C3, low factor H and undetectable C3dg/C3d. Partial factor I deficiency was detected in 16 family members (normal low cut-off value was 25 microg/ml). Respiratory infections were the most common clinical occurrence among partial factor I-deficient relatives. Two of them were submitted to nephrectomy following recurrent urinary tract infections. An additional two heterozygous relatives presented with arthritis and rheumatic fever. Apparently, patients with partial factor I deficiency are also at higher risk for recurrent infections. Vaccination against capsulated bacteria and the eventual use of prophylactic antibiotics should be considered individually in this patient group.

A complement receptor for opsonized immune complexes on erythrocytes from Oncorhynchus mykiss but not Ictalarus punctatus.

Immune complexes activate the classical pathway of complement resulting in the covalent deposition of fragments of the third (C3b) and fourth (C4b) components of complement, thus opsonizing the complexes for uptake by CD35 found on human erythrocytes. The complexes are then transported to and cleared from the circulation by the reticuloendothelial system. It has been shown that rainbow trout can remove immune complexes from the circulation in a complement-dependent manner similar to that found in the human. However, the cell or cell types involved have not been identified. The purpose of this study was to investigate whether a complement-dependent immune adherence receptor is expressed on erythrocytes from the rainbow trout (Oncorhynchus mykiss) and the channel catfish (Ictalarus punctatus). Coating fluorescent microparticles with BSA, and then binding them to anti-BSA created an artificial immune complex that was incubated with normal fish serum, normal human serum or EDTA-treated serum. The complement-coated immune complexes were then incubated with either fish or human erythrocytes and analyzed for binding by flow cytometry and further visualized by fluorescence microscopy. Our results indicate that erythrocytes from rainbow trout are capable of binding immune complexes when pretreated with serum from either the trout or human, but not when pretreated with serum containing EDTA. By contrast, erythrocytes from the channel catfish did not bind immune complexes pretreated with autologous or human serum. These data suggest that differences exist in receptor distribution between two closely related species of fish, and a potentially homologous relationship in receptor expression, and possibility function, exist between two highly divergent species.