Peter Norsworthy

Murine CD93 (C1qRp) Contributes to the Removal of Apoptotic Cells In Vivo but Is Not Required for C1q-Mediated Enhancement of Phagocytosis

Human CD93 (known as C1qRp) has been shown to be a phagocytic receptor involved in the in vitro C1q-dependent enhancement of phagocytosis. However, binding of CD93 to C1q and its function remain controversial. In this study, we have generated CD93-deficient mice (CD93−/−) to investigate its biological role(s). The CD93−/− mice were viable and showed no gross abnormalities in their development. Thioglycolate-elicited peritoneal macrophages deficient in CD93 showed a similar enhancement in complement- and FcγR-dependent uptake of RBC to the wild-type macrophages when plated on C1q-coated surfaces suggesting that the lack of this receptor had no effect on these C1q-mediated events. There was no impairment in either complement- or FcγR-dependent phagocytic assays in vivo. By contrast, the CD93−/− mice had a significant phagocytic defect in the clearance of apoptotic cells in vivo (human Jurkat T cells and murine thymocytes: p = 0.0006 and p = 0.0079, respectively) compared with strain-matched controls. However, in vitro, the CD93−/− macrophages showed similar engulfment of apoptotic cells to wild-type macrophages. Furthermore, no supporting evidence for a role of CD93 as an adhesion molecule was found using intravital microscopy or analyzing peritoneal cell recruitment in response to three different inflammatory stimuli (thioglycolate, zymosan A, and IL-1β). Thus, our findings indicate that murine CD93 is expressed on the peritoneal macrophage, especially on thioglycolate-elicited cells, but does not appear to play a key role in C1q-mediated enhancement of phagocytosis or in the intercellular adhesion events tested. However, our results suggest that it may contribute to the in vivo clearance of dying cells.

Overrepresentation of the FCγ receptor type IIA R131/R131 genotype in caucasoid systemic lupus erythematosus patients with autoantibodies to C1q and glomerulonephritis

OBJECTIVE To test the hypothesis that there is an association between the Fcgamma receptor type IIA (FcgammaRIIA)-H/R131 polymorphism and autoantibodies to the collagenous region (CLR) of C1q in patients with systemic lupus erythematosus (SLE). METHODS One hundred ninety-five Caucasoid lupus patients were studied. Anti-C1q(CLR) antibodies in serum were measured by enzyme-linked immunosorbent assay (ELISA) and FcgammaRIIA genotype analysis was performed by polymerase chain reaction. Immunoglobulin subclass of the autoantibodies was measured by ELISA. RESULTS Fifty-six patients were anti-C1q antibody positive, and Ig subclass analysis indicated a predominance of IgG2 anti-C1q antibodies. Analysis of the SLE population as a whole revealed no significant difference in the allele frequencies of R131 and H131 compared with controls. There was, however, a significantly increased frequency of the R131 allele both in the anti-C1q-positive subgroup of patients (chi2 = 7.66, P<0.01) and in the 71 patients with nephritis (chi2 = 7.76, P< 0.01), compared with controls. CONCLUSION FcgammaRIIA-R131 constitutes a heritable susceptibility factor for the development of SLE with manifestations in the kidney in Caucasoid patients. The close associations demonstrated between this FcgammaRII variant, antibodies to C1q(CLR), and glomerulonephritis may be due to a failure of clearance of the potentially pathogenic IgG2 autoantibody.

Immune complex processing in C1q-deficient mice

Complement and Fcγ receptors are known to mediate the processing of immune complexes (IC), and abnormalities in these mechanisms may predispose to the development of lupus. We explored the processing of IC in mice deficient in complement component C1q. 125I‐labelled IC comprising Hepatitis B surface antigen (HBsAg)/human anti‐HBsAg (HBsAg/Ab) were injected intravenously and the sites of IC clearance determined by direct counting of organ uptake at various time points. The liver and spleen were the main sites of IC uptake in all mice. The splenic uptake of IC was significantly reduced in the C1q‐deficient mice compared with the control mice. C1q‐deficient mice also exhibited an initial accelerated hepatic uptake of IC similar to that seen in human subjects with hypocomplementaemia. The hepatic localization of IC at later time points was similar in both groups of mice. These data in mice are consistent with previous observations in humans that confirm that the classical pathway of complement plays an important role in the appropriate processing of IC in vivo.

Autoantibodies to the collagenous region of C1q occur in three strains of lupus‐prone mice

We have developed an ELISA to measure murine autoantibodies to the collagenous region (CLR) of C1q, using the whole human C1q molecule as the solid‐phase ligand, in the presence of 1 m NaCl. The assay was validated by testing positive sera from 20 mice using purified mouse C1q, and from 10 mice using purified human C1q‐CLR, as the solid‐phase ligands. There were highly significant correlations between results obtained with human C1q (whole molecule) and: (i) mouse C1q (rSp = 0.73, P < 0.001), and (ii) human C1q‐CLR alone (rSp = 0.86, P = 0.001). Antibodies to C1q were measured in 53 MRL/lpr, 17 BXSB and 25 NZB/W lupus‐prone mice. Median (range) anti‐C1q (CLR) antibody levels in MRL/lpr, BXSB, and NZB/W autoimmune mice aged 3 months were 22 (16–66), 21 (17–39) and 19 (15–27) EU, respectively. The median anti‐C1q antibody level in MRL/lpr mice aged 5 months was 76 (35–142) EU, significantly higher than that at 3 months (U = 558, P < 0.0005). Median anti‐C1q antibody level in NZB/W mice at 8 months was 37 (13–74) EU and in BXSB mice at 11 months was 62 (31–231) EU, significantly higher than corresponding values at 3 months (U = 326, and U = 4, P < 0.001, respectively). This is the first demonstration of anti‐C1q (CLR) antibodies in NZB/W and BXSB mice. The pathologic significance and the potential utility of these antibodies for monitoring disease in lupus‐prone mice are under evaluation.

Hepatitis C in Egypt – past, present, and future

Hepatitis C viral infection is endemic in Egypt with the highest prevalence rate in the world. It is widely accepted that the implementation of mass population antischistosomal treatment involving administration of tartar emetic injections (from 1950s to 1980s) led to widespread infection. What is less well known, however, is that these schemes were implemented by the Egyptian Ministry of Health on the advice of the World Health Organization. There has been a spectrum of treatments to target the public health disaster represented by the hepatitis C problem in Egypt: from the use of PEGylated interferon to the recent use of direct acting antiviral drugs. Some new treatments have shown >90% efficacy. However, cost is a key barrier to access these new medicines. This is coupled with a growing population, limited resources, and a lack of infection control practices which means Egypt still faces significant disease control issues today.

Cloning of the mouse homolog of the 126-kDa human C1q/MBL/SP-A receptor, C1qR(p).

Abstract. Binding of C1q to cell surfaces has been shown to mediate a number of biological activities including enhancement of phagocytosis and stimulation of superoxide production. Several C1q binding proteins have been proposed as candidate receptors for these functions. The 126-kDa human C1q membrane receptor, termed C1qRp, has recently been cloned. This molecule is believed to play a role in the enhancement of phagocytosis in monocytes and macrophages, and its expression has been shown to be restricted to cells of the myeloid lineage, endothelial cells, and platelets. Here we report the isolation and genomic characterization of the murine homolog of C1qRp. Degenerate oligonucleotide primers based on the published human sequence were used to amplify a region of the murine homolog spanning from the carbohydrate recognition domain to the fourth epidermal growth factor (EGF) domain. This fragment was used as a probe to isolate the murine gene from a 129/Sv genomic λ library. The predicted primary protein sequence displayed 68.1% identity with the human homolog. All the major structural domains were conserved between the two molecules. The coding sequence of the murine gene was contained within two exons separated by a small intron of approximately 250 bp. The structure of the human gene was found to be similar, with the position of the intron conserved. Cloning of the murine C1qRp will facilitate further investigation of the physiological function of this molecule.

The anti-lipid A antibody HA-1A binds to rough Gram-negative bacteria, fixes complement and facilitates binding to erythrocyte CR1 (CD35)

MoAbs to bacterial cell wall Iipopolysaccharidc arc currently under evaluation for the treatment of Gram‐negative sepsis. The mode of action of these reagents remains poorly understood. In this study we examined the ability of radiolabelled HA‐IA (an IgM anti‐lipid A MoAb) to bind in vitro to Salmonella minnesota (Re 595), Escherichia coli, and Streptococcus pyogenes. HA‐1A was able to bind specifically to the “rough” mutant Stilm. minnesota. but not to a ‘smooth’E. coli, or Strep, pvogenes. Binding to Salm. minnesota led to complement fixation which resulted in bacterial adherence to erythrocyte CR1, suggesting a possible mechanism whereby the antibody might enhance clearance of bacteria by facilitating delivery to the fixed mononuclear phagocytic system. We were not able to demonstrate the formation of immune complexes between free lipopolysaccharide and HA‐IA in the presence of serum, nor the enhancement of complement‐mediated binding of HA‐1 A:Salm. minnesota immune complexes to erythrocytes by antibiotic treatment. Binding of HA‐IA to small bacterial fragments was, however, demonstrable after in vitro treatment with a β‐lactam antibiotic, which disrupts the bacterial cell wall, but not with gentamicin, an aminoglycoside antibiotic which blocks protein synthesis.

Familial clustering of non-nuclear autoantibodies and C3 and C4 complement components in systemic lupus erythematosus.

OBJECTIVE To determine whether key features of systemic lupus erythematosus (SLE), namely, production of non-nuclear antibodies (anti-C1q and anticardiolipin antibodies [aCL]) and depletion of complement components C3 and C4, aggregate in families. In addition, we examined relationships between anti-C1q and C3 and C4 levels. METHODS The study cohort comprised 1,037 predominantly white (82%) nuclear families in which at least 1 member had SLE. Associations of antibody measurements between probands and their unaffected siblings were examined using parametric and nonparametric analyses, along with associations between unaffected siblings and their parents. The heritability of anti-C1q, C3, and C4 was estimated, and interdependencies between these factors were examined in a regression model accounting for the family structure of the data set. RESULTS We demonstrated associations between siblings for anti-C1q (odds ratio [OR] 3.74, 95% confidence interval [95% CI] 2.65, 5.28) and IgG and IgM aCL (OR 4.08, 95% CI 1.83, 5.13 and OR 2.06, 95% CI 1.46, 2.91, respectively) and, for anti-C1q, association between unaffected parents and their unaffected offspring (OR 4.34, 95% CI 2.16, 8.72). We also demonstrated significant heritability of anti-C1q, C3, and C4 (approximately 45%). Anti-C1q was negatively associated with C3 and C4 in SLE probands but not in their healthy relatives. CONCLUSION Non-nuclear antibodies and C3 and C4 cluster within the families of SLE probands, suggesting that specific autoantibody formation is partly genetically determined, even if the total genetic effect in unaffected relatives is insufficient to cause disease. Anti-C1q antibodies accelerate C3 and C4 depletion in patients with SLE but have no effect in the absence of disease.

Regulation of B cell tolerance by 129‐derived chromosome 1 loci in C57BL/6 mice

Objective Systemic lupus erythematosus is a multifactorial disease with a strong genetic component. Previous studies have shown that a 129-derived chromosome 1 interval (Sle16) on the C57BL/6 (B6) background is sufficient to induce humoral autoimmunity. The aim of the present study was to elucidate the mechanisms by which this locus contributes to the loss of peripheral tolerance. Methods Anti–single-stranded DNA (anti-ssDNA)–knockin transgenic mice (VH3H9R/Vκ8R and VH3H9R) were crossed with a B6 congenic line named B6.129chr1b that carries the Sle16 locus. A parallel study of a gene-targeted animal, whose mutated gene is located within the 129chr1b interval on chromosome 1, was also performed. Results The combination of VH3H9R/Vκ8R with the 129chr1b interval resulted in impaired B cell anergy, and transgenic IgM and IgG anti-ssDNA antibodies were found in the circulation. The presence of IgG2aa anti-ssDNA and IgMa anti-Sm antibodies in sera indicated that the autoreactive transgenic B cells underwent class switching and epitope spreading. The 129chr1b locus appeared to have a dominant effect, since transgenic antibodies were also detected in mice carrying a single allele. The gene-targeted animals showed a similar phenotype. Conclusion The presence of a single 129chr1b locus on the B6 background impaired B cell anergy, prevented deletion of anti-DNA transgenic B cells, and induced receptor revision. The findings of this study also emphasize that the autoimmune phenotype observed in mice with targeted genes located on chromosome 1 may simply arise from epistatic interactions between the 129 and B6 parental strains.

Developing a Bayesian adaptive design for a phase I clinical trial: a case study for a novel HIV treatment

The design of phase I studies is often challenging, because of limited evidence to inform study protocols. Adaptive designs are now well established in cancer but much less so in other clinical areas. A phase I study to assess the safety, pharmacokinetic profile and antiretroviral efficacy of C34‐PEG4‐Chol, a novel peptide fusion inhibitor for the treatment of HIV infection, has been set up with Medical Research Council funding. During the study workup, Bayesian adaptive designs based on the continual reassessment method were compared with a more standard rule‐based design, with the aim of choosing a design that would maximise the scientific information gained from the study. The process of specifying and evaluating the design options was time consuming and required the active involvement of all members of the trials protocol development team. However, the effort was worthwhile as the originally proposed rule‐based design has been replaced by a more efficient Bayesian adaptive design. While the outcome to be modelled, design details and evaluation criteria are trial specific, the principles behind their selection are general. This case study illustrates the steps required to establish a design in a novel context.