Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats.

The human insulin-resistance syndromes, type 2 diabetes, obesity, combined hyperlipidaemia and essential hypertension, are complex disorders whose genetic basis is unknown. The spontaneously hypertensive rat (SHR) is insulin resistant and a model of these human syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridaemia and hypertension map to a single locus on rat chromosome 4. Here we combine use of cDNA microarrays, congenic mapping and radiation hybrid (RH) mapping to identify a defective SHR gene, Cd36 (also known as Fat, as it encodes fatty acid translocase), at the peak of linkage to these QTLs. SHR Cd36 cDNA contains multiple sequence variants, caused by unequal genomic recombination of a duplicated ancestral gene. The encoded protein product is undetectable in SHR adipocyte plasma membrane. Transgenic mice overexpressing Cd36 have reduced blood lipids. We conclude that Cd36 deficiency underlies insulin resistance, defective fatty acid metabolism and hypertriglyceridaemia in SHR and may be important in the pathogenesis of human insulin-resistance syndromes.

Hereditary C1q deficiency and systemic lupus erythematosus.

We describe a 27-year-old women with systemic lupus erythematosus, C1q deficiency and cytomegalovirus retinitis. She suffered from severe SLE, with cutaneous and CNS involvement, and died of CNS disease aged 28. Review of 29 other published cases of C1q deficiency shows that SLE in these patients is often severe (five with CNS disease, ten with glomerulonephritis). The results of autoantibody studies in this and another patient with C1q deficiency and SLE are presented--both patients had autoantibodies to the extractable nuclear antigens, Sm, RNP and Ro, and one patient had high titres of antibodies to dsDNA. One of the patients had previously been treated with fresh frozen plasma, and antibodies to C1q were present in his serum. Homozygous C1q deficiency is associated with a very high prevalence of severe SLE with the full panoply of autoantibodies characteristic of this disease.

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

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 C1qR(p), 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 C1qR(p). 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 lambda 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 C1qR(p) 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 lipopolysaccharide are 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-1A (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 Salm. minnesota, but not to a 'smooth' E. coli, or Strep. pyogenes. 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-1A in the presence of serum, nor the enhancement of complement-mediated binding of HA-1A:Salm. minnesota immune complexes to erythrocytes by antibiotic treatment. Binding of HA-1A to small bacterial fragments was, however, demonstrable after in vitro treatment with a beta-lactam antibiotic, which disrupts the bacterial cell wall, but not with gentamicin, an aminoglycoside antibiotic which blocks protein synthesis.

Immune complex processing in C1q-deficient mice.

Complement and Fcgamma 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.

Significance of anticardiolipin and anti-beta(2)-glycoprotein I antibodies in lupus nephritis.

OBJECTIVE: To investigate whether anticardiolipin (aCL) and anti-beta(2)-glycoprotein I (anti-beta(2)GPI) antibodies are associated with lupus nephritis (group II patients), and whether there are differences in the prevalence of these two autoantibodies between group II patients and patients with non-nephritis SLE (group I) and primary antiphospholipid syndrome (PAPS) patients (group III). METHODS: IgG and IgM aCL were measured in 31 patients and anti-beta(2)GPI in 30 patients with systemic lupus erythematosus (SLE) nephritis and 25 without SLE nephritis and in 36 PAPS patients by validated enzyme immunoassays. Relationships of anti-double-stranded DNA (anti-dsDNA) antibodies and antibodies to the collagenous region of C1q (anti-C1q) with SLE nephritis were also examined. RESULTS: The prevalence and levels were higher for aCL, but not for anti-beta(2)GPI, antibodies in group II than in group I patients. Absolute values of aCL and anti-beta(2)GPI in all three patient groups correlated with each other. The prevalences of aCL, anti-dsDNA and anti-C1q antibodies were significantly higher in group II than in group I and group III patients. CONCLUSION: The observations in this paper suggest that raised levels of aCL antibodies are associated with lupus nephritis. We were not able to demonstrate an association between anti-beta(2)GPI antibodies and kidney disease either in patients with lupus or in patients with primary antiphospholipid syndrome. In SLE, we demonstrated that the presence of anticardiolipin antibodies in conjunction with elevated levels of anti-dsDNA and anti-C1q antibodies is highly specific for glomerulonephritis in patients with lupus.

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 less than 0.001), and (ii) human Clq-CLR alone (rsp = 0.86, P = 0.001). Antibodies to Clq 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-Clq antibody level in MRL/lpr mice aged 5 months was 76 (35-142) EU, significantly higher than that at 3 months (U = 558, P less than 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 less than 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.

Clearance pathways of soluble immune complexes in the pig. Insights into the adaptive nature of antigen clearance in humans.

Efficient delivery of immune complexes (ICs) to the mononuclear phagocytic system, and subsequent IC processing, may prevent their potentially harmful effects in other tissues and may also be important in the development of humoral immune responses. In mice, rabbits, and primates, the liver and spleen are the main sites of IC clearance. It has been demonstrated previously that the pulmonary capillaries in the pig are lined with macrophages and that certain particulates, including bacteria, localize to this organ. In this study, we used gamma scintigraphy to explore the sites and kinetics of clearance of soluble IC comprising 123I-labeled hepatitis B surface Ag (HBsAg):porcine anti-HBsAg in the Large White pig. At t = 10 min after i.v. injection, 43 +/- 5% (mean +/- SE) IC localized in the lungs, and 36 +/- 6% counts in the liver. At t = 85 min, values were: lungs, 15 +/- 4% and liver, 29 +/- 2%. Findings were similar following intraarterial injection. Complement depletion resulted in more rapid initial IC clearance (t1/2 = 5 min), reduced lung uptake (23 +/- 3% at 10 min), and impaired IC catabolism. In normal animals, 5 to 7% injected IC bound to PBMCs, but no E binding was seen. A fall in PBMC numbers (46 to 59% of baseline), was observed following IC injection. These findings contrast with our previous observations using analogous IC in humans, in which we did not observe any change in peripheral blood leukocyte counts consequent upon complex processing, suggesting that in humans, Es may function as a buffering system for complement-bearing IC in the circulation, preventing their interaction with leukocytes bearing complement and FcR, and the potential activation of these cells.

A high-resolution radiation hybrid map of the proximal region of rat chromosome 4.

Radiation hybrid (RH) mapping has been used to produce genome maps in the human and mouse, but as yet the technique has been applied little to other species. We describe the use of RH mapping in the rat, using a newly available rat/hamster RH panel, to construct an RH map of the proximal part of rat Chromosome (Chr) 4. This region is of interest because quantitative trait loci (QTLs) for defective insulin and catecholamine action, hypertension, and dyslipidemia map to this region. The RH map includes 23 rat genes or microsatellites previously mapped to this part of Chr 4, one rat gene not previously mapped in the rat, and markers for four new genes, homologs of which map to the syntenic region of the mouse genome. The RH map integrates genetic markers previously mapped on several rat crosses, increases the resolution of existing maps, and may provide a suitable basis for physical map construction and gene identification in this chromosomal region. Our results demonstrate the utility of RH mapping in the rat genome and show that RH mapping can be used to localize, in the rat genome, the homologs of genes from other species such as the mouse. This will facilitate identification of candidate genes underlying QTLs on this chromosomal segment.