The rare C9 P167S risk variant for age-related macular degeneration increases polymerization of the terminal component of the complement cascade.

Age-related macular degeneration (AMD) is a complex neurodegenerative eye disease with behavioral and genetic etiology and is the leading cause of irreversible vision loss among elderly Caucasians. Functionally significant genetic variants in the alternative pathway of complement have been strongly linked to disease. More recently, a rare variant in the terminal pathway of complement has been associated with increased risk, Complement component 9 (C9) P167S. To assess the functional consequence of this variant, C9 levels were measured in two independent cohorts of AMD patients. In both cohorts, it was demonstrated that the P167S variant was associated with low C9 plasma levels. Further analysis showed that patients with advanced AMD had elevated sC5b-9 compared to those with non-advanced AMD, although this was not associated with the P167S polymorphism. Electron microscopy of membrane attack complexes (MACs) generated using recombinantly produced wild type or P167S C9 demonstrated identical MAC ring structures. In functional assays, the P167S variant displayed a higher propensity to polymerize and a small increase in its ability to induce hemolysis of sheep erythrocytes when added to C9-depleted serum. The demonstration that this C9 P167S AMD risk polymorphism displays increased polymerization and functional activity provides a rationale for the gene therapy trials of sCD59 to inhibit the terminal pathway of complement in AMD that are underway.

Ensilicated tetanus antigen retains immunogenicity: in vivo study and time-resolved SAXS characterization.

Our recently developed ensilication approach can physically stabilize proteins in silica without use of a pre-formed particle matrix. Stabilisation is done by tailor fitting individual proteins with a silica coat using a modified sol-gel process. Biopharmaceuticals, e.g. liquid-formulated vaccines with adjuvants, frequently have poor thermal stability; heating and/or freezing impairs their potency. As a result, there is an increase in the prevalence of vaccine-preventable diseases in low-income countries even when there are means to combat them. One of the root causes lies in the problematic vaccine 'cold chain' distribution. We believe that ensilication can improve vaccine availability by enabling transportation without refrigeration. Here, we show that ensilication stabilizes tetanus toxin C fragment (TTCF), a component of the tetanus toxoid present in the diphtheria, tetanus and pertussis (DTP) vaccine. Experimental in vivo immunization data show that the ensilicated material can be stored, transported at ambient temperatures, and even heat-treated without compromising the immunogenic properties of TTCF. To further our understanding of the ensilication process and its protective effect on proteins, we have also studied the formation of TTCF-silica nanoparticles via time-resolved Small Angle X-ray Scattering (SAXS). Our results reveal ensilication to be a staged diffusion-limited cluster aggregation (DLCA) type reaction. An early stage (tens of seconds) in which individual proteins are coated with silica is followed by a subsequent stage (several minutes) in which the protein-containing silica nanoparticles aggregate into larger clusters. Our results suggest that we could utilize this technology for vaccines, therapeutics or other biopharmaceuticals that are not compatible with lyophilization.

Thermal resilience of ensilicated lysozyme via calorimetric and in vivo analysis.

Ensilication is a novel method of protein thermal stabilisation using silica. It uses a modified sol-gel process which tailor fits a protective silica shell around the solvent accessible protein surface. This, electrostatically attached, shell has been found to protect the protein against thermal influences and retains its native structure and function after release. Here, we report the calorimetric analysis of an ensilicated model protein, hen egg-white lysozyme (HEWL) under several ensilication conditions. DSC, TGA-DTA-MS, CD, were used to determine unfolding temperatures of native, released and ensilicated lysozyme to verify the thermal resilience of the ensilicated material. Our findings indicate that ensilication protects against thermal fluctuations even at low concentrations of silica used for ensilication. Secondly, the thermal stabilisation is comparable to lyophilisation, and in some cases is even greater than lyophilisation. Additionally, we performed a mouse in vivo study using lysozyme to demonstrate the antigenic retention over long-term storage. The results suggest that protein is confined within the ensilicated material, and thus is unable to unfold and denature but is still functional after long-term storage.

The role of ADAMTS-13 activity and complement mutational analysis in differentiating acute thrombotic microangiopathies.

UNLABELLED: ESSENTIALS: Molecular diagnostics has improved the differentiation of acute thrombotic microangiopathys (TMAs). Atypical hemolytic uremic syndrome may have features mimicking thrombotic thrombocytopenic purpura. We identified novel complement mutations and a high incidence of CD46, with favorable long term outcomes. Complement mutation analysis in TMA where the diagnosis is unclear and ADAMTS-13 activity is >10%. BACKGROUND: Differentiation of acute thrombotic microangiopathy (TMA) at presentation has historically been dependent on clinical parameters. Confirmation of thrombotic thrombocytopenic purpura (TTP) is increasingly reliant on demonstrating deficient ADAMTS-13 activity. The identification of alternative complement pathway abnormalities in atypical hemolytic uremic syndrome (aHUS), along with the proven efficacy of terminal complement inhibitors in treatment, has increased the need for rapid differentiation of TTP from aHUS. OBJECTIVES: We describe the clinical phenotype and nature of complement mutations in a cohort of aHUS patients referred as acute TMAs. PATIENTS/METHODS: Fourteen consecutive aHUS patients were screened for mutations in C3, CD46, CFH, CFI, and CFB, as well as factor H (FH) antibodies. All aHUS patients had ADAMTS-13 activity > 10%. RESULTS: Of 14 aHUS patients, 11 (79%) had platelet counts < 30 × 10(9) /L during the acute phase. Median presenting creatinine level was 295 µmol L(-1) , while five (36%) of 14 presented with a serum creatinine level < 200 µmol L(-1) . Alternative complement pathway mutations were detected in 9 (64%) of 14 patients, including CD46 mutations in five (36%) of 14 patients. Patients were identified with novel mutations in CFB and C3 that have not been previously reported. CONCLUSIONS: We demonstrate that diagnostic differentiation based on platelet count and renal function is insufficient to predict an underlying complement mutation in some aHUS cases. Specifically, we demonstrate a high frequency of functionally significant CD46 mutations which may mimic TTP. ADAMTS-13 activity > 10% in a patient with a TMA should necessitate genetic screening for complement abnormalities.

A novel method for direct measurement of complement convertases activity in human serum.

Complement convertases are enzymatic complexes that play a central role in sustaining and amplification of the complement cascade. Impairment of complement function leads directly or indirectly to pathological conditions, including higher infection rate, kidney diseases, autoimmune- or neurodegenerative diseases and ischaemia-reperfusion injury. An assay for direct measurement of activity of the convertases in patient sera is not available. Existing assays testing convertase function are based on purified complement components and, thus, convertase formation occurs under non-physiological conditions. We designed a new assay, in which C5 blocking compounds enabled separation of the complement cascade into two phases: the first ending at the stage of C5 convertases and the second ending with membrane attack complex formation. The use of rabbit erythrocytes or antibody-sensitized sheep erythrocytes as the platforms for convertase formation enabled easy readout based on measurement of haemolysis. Thus, properties of patient sera could be studied directly regarding convertase activity and membrane attack complex formation. Another advantage of this assay was the possibility to screen for host factors such as C3 nephritic factor and other anti-complement autoantibodies, or gain-of-function mutations, which prolong the half-life of complement convertases. Herein, we present proof of concept, detailed description and validation of this novel assay.

Postpartum aHUS secondary to a genetic abnormality in factor H acquired through liver transplantation.

We report here a young female who underwent a successful deceased donor liver transplant for hepatic vein thrombosis. Five years after transplantation she developed postpartum atypical hemolytic uremic syndrome (aHUS). She did not recover renal function. Mutation screening of complement genes in her DNA did not show any abnormality. Mutation screening of DNA available from the donor showed a nonsense CFH mutation leading to factor H deficiency. Genotyping of the patient showed that she was homozygous for an aHUS CD46 at-risk haplotype. In this individual, the development of aHUS has been facilitated by the combination of a trigger (pregnancy), an acquired rare genetic variant (CFH mutation) and a common susceptibility factor (CD46 haplotype).

Impact of compound heterozygous complement factor H mutations on development of atypical hemolytic uremic syndrome-A pedigree revisited.

Atypical hemolytic uremic syndrome (aHUS) is associated with mutations in the gene CFH encoding the complement regulator factor H (CFH). We previously reported a family, in which three individuals had partial CFH deficiency but only one was affected by aHUS. We have investigated this family further to show that the partial CFH deficiency is associated with a heterozygous CFH mutation (c.2768T>G, p.Tyr899Asp). We used the polymorphic CFH variant p.His402Tyr to track expression of p.Tyr899Asp, and found that this mutant was expressed in minimal quantities in serum. In the one affected individual we found a second CFH mutation (c.3581G>A, p.Gly1194Asp) on the other allele which was expressed normally. We showed that this mutant, which has been described previously in aHUS, has impaired regulation of cell surface complement activation. The affected individual in this family is therefore a compound heterozygote for two functionally significant CFH mutations. Two individuals (mother and male sib) in the pedigree carried only c.2768T>G, p.Tyr899Asp and one (father) carried only c.3581G>A, p.Gly1194Asp, and all three were asymptomatic. Thus, further investigation of this family has enabled us to clarify the genotype-phenotype correlation.

Expression of human complement receptor 2 (CR2, CD21) in Cr2-/- mice restores humoral immune function.

Complement receptor type 2 (CR2, CD21) is expressed by both human and murine B cells and has been demonstrated to play a pivotal role in the humoral immune response. We have reconstituted Cr2-/- mice with an 80-kb human genomic fragment (designated P1-5) containing the full-length human CR2 (hCR2) gene. Transfection of P1-5 into the mouse A20 B cell line confirmed that it would direct expression of the hCR2 protein in mouse B cells. Immunoprecipitation analysis in these cells revealed that hCR2 coassociates with mouse CD19. After creation of transgenic mice using P1-5, we found significant expression of hCR2 on peripheral blood and splenic B cells by flow cytometric analysis. RT-PCR analysis of tissues and purified cell populations from transgene-positive mice revealed that hCR2 expression was restricted to B cells and the spleen in a pattern that matches mouse CR2. To rigorously assess the functional capabilities of hCR2, the transgene was bred onto Cr2-/- mice, which have a notable defect in response to SRBC Ag. We found that Cr2-/- mice expressing hCR2 had a substantial restoration of the humoral immune response to SRBC as compared with nontransgenic Cr2-/- littermate controls. Overall, this study suggests that hCR2 is able to substitute for mouse CR2 in the murine immune system. Therefore, hCR2-transgenic mice offer a valuable model system to further examine immunologic roles as well as structure-function relationships important for hCR2 function in primary cells in vivo.

Mechanisms of complement resistance induced by non-lethal complement attack and by growth arrest.

Non-lethal complement (C) attack on K562 cells has been shown to induce a transient resistance to lethal amounts of C. We have previously shown that incubation of K562 with phorbol 12-myristate 13-acetate (PMA) caused an increase in both CD59 expression and resistance to C killing and we were interested to examine whether non-lethal C attack caused a similar effect. We here demonstrate that expression of the C inhibitors decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59 was unaltered on K562 after non-lethal C attack and that neutralization of these inhibitors with specific blocking antibodies did not reverse the induced resistance. In an effort to understand the mechanisms of resistance we searched for other conditions that might induce C resistance in K562 cells. Growth-arrested cells showed a similar degree of resistance to C killing. The levels of DAF and MCP on these cells were unaltered whereas expression of CD59 was markedly reduced. Non-lethal C attack on these growth-arrested cells induced a further increase in resistance to C killing, suggesting that the mechanisms of resistance were not identical. Indeed, resistance of non-lethally attacked cells was completely lost within 8 hr of attack whereas resistance of growth-arrested cells was detectable for up to 48 hr after returning to cell cycle. These data demonstrate that C resistance induced by two distinct strategies is not mediated by the known membrane C inhibitors. Resistance may be a result of the expression of a novel inhibitor or due to metabolic depletion, a likely common consequence of non-lethal C attack and induction of growth arrest, implying that cells take an active role in C-mediated killing.

Molecular cloning, chromosomal localization, expression, and functional characterization of the mouse analogue of human CD59.

We have previously described the isolation and cloning of the rat analogue of the human complement inhibitor CD59 (hCD59). Using the rat CD59 (rCD59) coding region as probe, we have isolated positive cDNAs from a mouse kidney cDNA library. Sequence analysis of these clones indicated that they contained an open reading frame encoding a 124 amino acid protein. Comparisons with the known sequences of hCD59 and rCD59 suggested that the clones contained a full-length cDNA encoding the mouse analogue of CD59 (mCD59). The cDNA encoded a 81-bp 5'-flanking region, a 23 amino acid NH2-signal peptide, a 101 amino acid coding region including putative N-glycosylation sites and a glycosyl phosphatidylinositol (GPI) anchoring signal, and approximately 0.8 kb 3'-untranslated flanking region. Reverse transcriptase PCR revealed the presence of mCD59 mRNA in all mouse tissues examined. The gene for mCD59 was mapped by fluorescence in situ hybridization to the E2-E4 region of mouse chromosome 2, a region that includes areas syntenous with the location of the human CD59 gene on chromosome 11p13. Expression of mCD59 in a CD59-negative human cell line conferred protection against lysis by complement from rodent, human, and several other species, confirming that mCD59 was the functional analogue of hCD59 and that function was not species restricted. The expressed protein was glycosyl phosphatidylinositol anchored as demonstrated by its partial removal from U937 cells on treatment with phosphatidylinositol-specific phospholipase C. Abs raised against the expressed protein demonstrated the presence of mCD59 on all mouse blood cell types and on several mouse cell lines and neutralized function of mCD59 on mouse E and expressed on U937. Western blot analysis revealed that both expressed and endogenous mCD59 had a molecular mass of 22 to 24 kDa.

Regulation of CD59 expression on K562 cells: effects of phorbol myristate acetate, cross-linking antibody and non-lethal complement attack.

CD59 is the major membrane attack complex of complement (MAC) inhibiting protein on human cells. Its regulation is therefore an important factor in determining the fate of cells at sites of complement activation. We have chosen the K562 erythroleukaemia cell line as a model for studies of the regulation of CD59 expression, because it has previously been reported that phorbol 12-myristate 13-acetate (PMA) caused a 15-fold up-regulation of CD59 mRNA in these cells, implying a substantial capacity for CD59 synthesis. However, no assessment of CD59 protein expression was made in these studies. We show here that surface expression of CD59, as assessed by flow cytometry, was increased four-fold over a 16-hr incubation with PMA, whereas surface expression of decay-accelerating factor (DAF) (CD55) and membrane cofactor protein (MCP) (CD46) was not altered. The newly expressed CD59 was functionally active and anchored through glycosyl-phosphatidylinositol (GPI). Increased expression was dependent upon de novo protein synthesis. CD59 released into cell supernatant was also increased seven-fold by PMA, this 'secreted' CD59 retained its GPI anchor. Non-lethal complement attack did not alter CD59 expression but antibody cross-linking of CD59 caused a rapid loss of the CD59-antibody complexes. However, CD59 was quickly restored to pre-attack levels. This rapid restoration was not dependent upon protein synthesis, suggesting release from preformed stores.