Semiquinone glucoside derivative provides protection against γ-radiation by modulation of immune response in murine model.

Present study was undertaken to evaluate radioprotective and immunomodulatory activities of a novel semiquinone glucoside derivative (SQGD) isolated from Bacillus sp. INM-1 in C57 BL/6 mice. Whole body survival study was performed to evaluate in vivo radioprotective efficacy of SQGD. To observe effect of SQGD on immunostimulation, Circulatory cytokine (i.e., interleukin-2 (IL-2), IFN-γ, IL-10, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and macrophage colony stimulating factor (M-CSF) expression was analyzed in serum of irradiated and SQGD treated mice at different time intervals using ELISA assay. Results of the present investigation indicated that SQGD pre-treatment (-2 h) to lethally irradiated mice provide ∼ 83% whole body survival compared with irradiated mice where no survival was observed at 30(th) post irradiation day. Significant (p < 0.05) induction in IL-2 and IFN-γ expression was observed at all tested time intervals with SQGD pre-treated irradiated mice as compared with irradiated mice alone. However, sharp increase in IL-10 expression was observed in irradiated mice which were found to be subsidized in irradiated mice pre-treated with SQGD. Similarly, significant (p < 0.05%) induction in G-CSF, M-CSF and GM-CSF expression was observed in irradiated mice treated with SQGD as compared with irradiated control mice at tested time intervals. In conclusion, SQGD pre-treatment to irradiated mice enhanced expression of IL-12 and IFN-γ while down-regulated IL-10 expression and thus modulates cytoprotective pro-inflammatory TH1 type immune response in irradiated mice. Further, SQGD pre-treatment to irradiated mice accelerate G-CSF, GM-CSF and M-CSF expression suggesting improved haematopoiesis and enhanced cellular immune response in immuno-compromised irradiated mice that may contribute to in vivo radiation protection.

Semiquinone derivative isolated from Bacillus sp. INM-1 protects cellular antioxidant enzymes from γ-radiation-induced renal toxicity.

This study was focused to evaluate protection of indigenous antioxidant system of mice against gamma radiation-induced oxidative stress using a semiquinone (SQGD)-rich fraction isolated from Bacillus sp. INM-1. Male C57bl/6 mice were administered SQGD (50 mg/kgb.w.i.p.) 2 h before irradiation (10 Gy) and modulation in antioxidant enzymes activities was estimated at different time intervals and compared with irradiated mice which were not pretreated by SQGD. Compared to untreated controls, SQGD pretreatment significantly (p < 0.05) accelerates superoxide dismutase, catalase, GSH, and glutathione-S-transferase activities. Similarly, significant (p < 0.05) increase in the expression of superoxide dismutase, catalase, GSH, and glutathione-S-transferase was observed in irradiated mice pretreated by SQGD, compared to only irradiated groups. Total antioxidant status equivalent to trolox was estimated in renal tissue of the mice after SQGD administration. Significant ABTS(+) radical formation was observed in H2O2-treated kidney homogenate, due to oxidative stress in the tissue. However, significant decrease in the levels of ABTS(+) radical was observed in kidney homogenate of the mice pretreated with SQGD. Therefore, it can be concluded that SQGD neutralizes oxidative stress by induction of antioxidant enzymes activities and thus improved total antioxidant status in cellular system and hence contributes to radioprotection.

The insulin gene is transcribed in the human thymus and transcription levels correlated with allelic variation at the INS VNTR-IDDM2 susceptibility locus for type 1 diabetes.

Type 1, or insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease associated with loss of tolerance to several pancreatic islet cell molecules, including insulin, glutamic acid decarboxylase (GAD), ICA69 and the tyrosine phosphatase IA-2 (refs 1-3). Among several predisposing loci, IDDM2 maps to the insulin gene (INS) VNTR (variable number of tandem repeats) minisatellite on chromosome 11p15 (refs 4-9). Allelic variation at this VNTR locus correlates with steady-state levels of INS mRNA in pancreas and transfected rodent cell lines, but it is difficult to reconcile the association of lower INS mRNA levels in the pancreas with class III VNTRs that are dominantly protective from IDDM. We show that during fetal development and childhood, mRNAs for insulin and other islet cell autoantigens (GAD, ICA69, IA-2) are expressed at low levels in the human thymus. Critically, we also detect proinsulin and insulin protein. VNTR alleles correlate with differential INS mRNA expression in the thymus where, in contrast to the pancreas, protective class III VNTRs are associated with higher steady-state levels of INS mRNA expression. This finding provides a plausible explanation for the dominant protective effect of class III VNTRs, and suggests that diabetes susceptibility and resistance associated with IDDM2 may derive from the VNTR influence on INS transcription in the thymus. Higher levels of (pro)insulin in the thymus may promote negative selection (deletion) of insulin-specific T-lymphocytes which play a critical role in the pathogenesis of type-1 diabetes.

Resistance of CD7-deficient mice to lipopolysaccharide-induced shock syndromes.

CD7 is an immunoglobulin superfamily molecule involved in T and natural killer (NK) cell activation and cytokine production. CD7-deficient animals develop normally but have antigen-specific defects in interferon (IFN)-gamma production and CD8(+) CTL generation. To determine the in vivo role of CD7 in systems dependent on IFN-gamma, the response of CD7-deficient mice to lipopolysaccharide (LPS)-induced shock syndromes was studied. In the high-dose LPS-induced shock model, 67% of CD7-deficient mice survived LPS injection, whereas 19% of control C57BL/6 mice survived LPS challenge (P < 0.001). CD7-deficient or C57BL/6 control mice were next injected with low-dose LPS (1 microgram plus 8 mg D-galactosamine [D-gal] per mouse) and monitored for survival. All CD7-deficient mice were alive 72 h after injection of LPS compared with 20% of C57BL/6 control mice (P < 0.001). After injection of LPS and D-gal, CD7-deficient mice had decreased serum IFN-gamma and tumor necrosis factor (TNF)-alpha levels compared with control C57BL/6 mice (P < 0.001). Steady-state mRNA levels for IFN-gamma and TNF-alpha in liver tissue were also significantly decreased in CD7-deficient mice compared with controls (P < 0.05). In contrast, CD7-deficient animals had normal liver interleukin (IL)-12, IL-18, and interleukin 1 converting enzyme (ICE) mRNA levels, and CD7-deficient splenocytes had normal IFN-gamma responses when stimulated with IL-12 and IL-18 in vitro. NK1.1(+)/ CD3(+) T cells are known to be key effector cells in the pathogenesis of toxic shock. Phenotypic analysis of liver mononuclear cells revealed that CD7-deficient mice had fewer numbers of liver NK1.1(+)/CD3(+) T cells (1.5 +/- 0.3 x 10(5)) versus C57BL/6 control mice (3.7 +/- 0.8 x 10(5); P < 0.05), whereas numbers of liver NK1.1(+)/CD3(-) NK cells were not different from controls. Thus, targeted disruption of CD7 leads to a selective deficiency of liver NK1.1(+)/ CD3(+) T cells, and is associated with resistance to LPS shock. These data suggest that CD7 is a key molecule in the inflammatory response leading to LPS-induced shock.

The binding site of human adenosine deaminase for CD26/Dipeptidyl peptidase IV: the Arg142Gln mutation impairs binding to cd26 but does not cause immune deficiency.

Human, but not murine, adenosine deaminase (ADA) forms a complex with the cell membrane protein CD26/dipeptidyl peptidase IV. CD26-bound ADA has been postulated to regulate extracellular adenosine levels and to modulate the costimulatory function of CD26 on T lymphocytes. Absence of ADA-CD26 binding has been implicated in causing severe combined immunodeficiency due to ADA deficiency. Using human-mouse ADA hybrids and ADA point mutants, we have localized the amino acids critical for CD26 binding to the helical segment 126-143. Arg142 in human ADA and Gln142 in mouse ADA largely determine the capacity to bind CD26. Recombinant human ADA bearing the R142Q mutation had normal catalytic activity per molecule, but markedly impaired binding to a CD26(+) ADA-deficient human T cell line. Reduced CD26 binding was also found with ADA from red cells and T cells of a healthy individual whose only expressed ADA has the R142Q mutation. Conversely, ADA with the E217K active site mutation, the only ADA expressed by a severely immunodeficient patient, showed normal CD26 binding. These findings argue that ADA binding to CD26 is not essential for immune function in humans.

Identification and characterization of a 100-kD ligand for CD6 on human thymic epithelial cells.

CD6 is a 130-kD glycoprotein expressed on the surface of thymocytes and peripheral blood T cells that is involved in TCR-mediated T cell activation. In thymus, CD6 mediates interactions between thymocytes and thymic epithelial (TE) cells. In indirect immunofluorescence assays, a recombinant CD6-immunoglobulin fusion protein (CD6-Rg) bound to cultured human TE cells and to thymic fibroblasts. CD6-Rg binding to TF and TE cells was trypsin sensitive, and 54 +/- 4% of binding was divalent cation dependent. By screening the blind panel of 479 monoclonal antibodies (mAbs) from the 5th International Workshop on Human Leukocyte Differentiation Antigens for expression on human TE cells and for the ability to block CD6-Rg binding to TE cells, we found one mAb (J4-81) that significantly inhibited the binding of CD6-Rg to TE cells (60 +/- 7% inhibition). A second mAb to the surface antigen identified by mAb J4-81, J3-119, enhanced the binding of CD6-Rg to TE cells by 48 +/- 5%. Using covalent cross-linking and trypsin digestion, we found that mAb J4-81 and CD6-Rg both bound to the same 100-kD glycoprotein (CD6L-100) on the surface of TE cells. These data demonstrate that a 100-kD glycoprotein on TE cells detected by mAb J4-81 is a ligand for CD6.

Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow.

Leukocyte migration into sites of inflammation involves multiple molecular interactions between leukocytes and vascular endothelial cells, mediating sequential leukocyte capture, rolling, and firm adhesion. In this study, we tested the role of molecular interactions between fractalkine (FKN), a transmembrane mucin-chemokine hybrid molecule expressed on activated endothelium, and its receptor (CX3CR1) in leukocyte capture, firm adhesion, and activation under physiologic flow conditions. Immobilized FKN fusion proteins captured resting peripheral blood mononuclear cells at physiologic wall shear stresses and induced firm adhesion of resting monocytes, resting and interleukin (IL)-2-activated CD8(+) T lymphocytes and IL-2-activated NK cells. FKN also induced cell shape change in firmly adherent monocytes and IL-2-activated lymphocytes. CX3CR1-transfected K562 cells, but not control K562 cells, firmly adhered to FKN-expressing ECV-304 cells (ECV-FKN) and tumor necrosis factor alpha-activated human umbilical vein endothelial cells. This firm adhesion was not inhibited by pertussis toxin, EDTA/EGTA, or antiintegrin antibodies, indicating that the firm adhesion was integrin independent. In summary, FKN mediated the rapid capture, integrin-independent firm adhesion, and activation of circulating leukocytes under flow. Thus, FKN and CX3CR1 mediate a novel pathway for leukocyte trafficking.

Cloning, mapping, and characterization of activated leukocyte-cell adhesion molecule (ALCAM), a CD6 ligand.

Antibody-blocking studies have demonstrated the role of CD6 in thymocyte-thymic epithelial (TE) cell adhesion. Here we report that CD6 expressed by COS cells mediates adhesion to TE cells and that this interaction is specifically blocked with an anti-CD6 monoclonal antibody (mAb) or with a mAb (J4-81) that recognized a TE cell antigen. We isolated and expressed a cDNA clone encoding this antigen and show that COS cells transfected with this cDNA bind a CD6 immunoglobulin fusion protein (CD6-Rg). This antigen, which we named ALCAM (activated leukocyte-cell adhesion molecule) because of its expression on activated leukocytes, appears to be the human homologue of the chicken neural adhesion molecule BEN/SC-1/DM-GRASP. The gene was mapped to human chromosome 3q13.1-q13.2 by fluorescence in situ hybridization of cDNA probes to metaphase chromosomes. We prepared an ALCAM-Rg fusion protein and showed that it binds to COS cell transfectants expressing CD6, demonstrating that ALCAM is a CD6 ligand. The observations that ALCAM is also expressed by activated leukocytes and that both ALCAM and CD6 are expressed in the brain suggest that ALCAM-CD6 interactions may play a role in the binding of T and B cells to activated leukocytes, as well as in interactions between cells of the nervous system.

The neuropsychology of the schizo-obsessive subtype of schizophrenia: a new analysis.

BACKGROUND: Interest in the neuro-cognitive profile of patients with schizophrenia and co-morbid obsessive compulsive disorder (schizo-OCD) is rising in response to reports of high co-morbidity rates. Whereas schizophrenia has been associated with global impairment in a wide range of neuro-cognitive domains, OCD is associated with specific deficits featuring impaired performance on tasks of motor and cognitive inhibition involving frontostriatal neuro-circuitry. METHOD: We compared cognitive function using the CANTAB battery in patients with schizo-OCD (n=12) and a schizophrenia group without OCD symptoms (n=16). The groups were matched for IQ, gender, age, medication, and duration of illness. RESULTS: The schizo-OCD patients made significantly more errors on a task of attentional set-shifting (ID-ED set-shift task). By contrast, no significant differences emerged on the Stockings of Cambridge task, the Cambridge Gamble Task or the Affective Go/NoGo tasks. No correlation emerged between ID-ED performance and severity of schizophrenia, OCD or depressive symptoms, consistent with neurocognitive impairment holding trait rather than state-marker status. Schizo-obsessives also exhibited a trend toward more motor tics emphasizing a neurological contribution to the disorder.ConclusionOur findings reveal a more severe attentional set-shifting deficit and neurological abnormality that may be fundamental to the neuro-cognitive profile of schizo-OCD. The clinical implications of these impairments merit further exploration in larger studies.

The impact of chemokine receptor CX3CR1 deficiency during respiratory infections with Mycobacterium tuberculosis or Francisella tularensis.

Recruitment of immune cells to infection sites is a critical component of the host response to pathogens. This process is facilitated partly through interactions of chemokines with cognate receptors. Here, we examine the importance of fractalkine (CX3CL1) receptor, CX3CR1, which regulates function and trafficking of macrophages and dendritic cells, in the host's ability to control respiratory infections with Mycobacterium tuberculosis or Francisella tularensis. Following low-dose aerosol challenge with M. tuberculosis, CX3CR1(-/-) mice were no more susceptible to infection than wild-type C57BL/6 mice as measured by organ burden and survival time. Similarly, following inhalation of F. tularensis, CX3CR1(-/-) mice displayed similar organ burdens to wild-type mice. CX3CR1(-/-) mice had increased recruitment of monocytes and neutrophils in the lung; however, this did not result in increased abundance of infected monocytes or neutrophils. We conclude that CX3CR1-deficiency affects immune-cell recruitment; however, loss of CX3CR1 alone does not render the host more susceptible to M. tuberculosis or F. tularensis.

Serum protein signatures detect early radiographic osteoarthritis.

OBJECTIVE: To test the hypothesis that early knee and hand osteoarthritis (OA) development is characterized by detectable changes in serum proteins relevant to inflammation, cell growth, activation, and metabolism several years before OA becomes radiographically evident. METHODS: Using microarray platforms that simultaneously test 169 proteins relevant to inflammation, cell growth, activation and metabolism, we conducted a case-control study nested within the Baltimore Longitudinal Study of Aging (BLSA). Subjects included 22 incident cases of OA and 66 age-, sex- and body mass index (BMI)-matched controls. Serum samples tested were obtained at the time of radiographic classification as either case or control, and up to 10 years earlier at a time when all participants were free of radiographic OA. Proteins with mean signal intensities fourfold higher than background were compared between cases and controls using multivariate techniques. RESULTS: Sixteen proteins were different between OA cases compared to controls. Four of these proteins [matrix metalloproteinase (MMP)-7, interleukin (IL)-15, plasminogen activator inhibitor (PAI)-1 and soluble vascular adhesion protein (sVAP)-1] were already different in samples obtained 10 years before radiographic classification and remained different at the time of diagnosis. Six additional proteins were only associated with subsequent OA development and not with established OA. CONCLUSIONS: Changes in serum proteins implicated in matrix degradation, cell activation, inflammation and bone collagen degradation products accompany early OA development and can precede radiographic detection by several years.

S100A9 is not essential for disease expression in an acute (K/BxN) or chronic (CIA) model of inflammatory arthritis.

OBJECTIVE: S100A8 (calgranulin A, MRP8) and S100A9 (calgranulin B, MRP14) are calcium-binding proteins highly expressed by activated myeloid cells and thought to be involved in the pathogenesis of inflammatory diseases. Circulating levels of S100A8/S100A9 are elevated in both human and experimental models of autoimmune disease, including rheumatoid arthritis (RA). METHODS: Mice deficient in S100A9 (S100A9 - /-) and wild-type controls were immunized using standard techniques for the K/BxN serum transfer or the collagen-induced arthritis (CIA) model. RESULTS: S100A9 - /- animals, with defective expression of both S100A8 and S100A9 proteins, had similar arthritis and histopathology to that of wild-type controls in both mouse models. CONCLUSION: S100A8 and S100A9 are not essential for disease expression in either the K/BxN serum transfer or the CIA model of inflammatory arthritis.

Self-antigen-presenting cells expressing diabetes-associated autoantigens exist in both thymus and peripheral lymphoid organs.

Recent reports indicate that genes with tissue-restricted expression, including those encoding the type 1 diabetes autoantigens insulin, glutamic acid decarboxylase (GAD), and the tyrosine-phosphatase-like protein IA-2 (or ICA512), are transcribed in the thymus. The reported modulation of diabetes susceptibility by genetically determined differences in thymic insulin levels and studies in transgenic mice provide correlative and functional evidence that thymic expression of peripheral proteins is crucial for immunological self-tolerance. However, there are no specific data about the existence, tissue distribution, phenotype, and function of those cells that express insulin and other self-antigens in the human thymus. We find that the human thymus harbors specialized cells synthesizing (pro)insulin, GAD, and IA-2, mainly localized in the medulla, and we demonstrate such cells also in peripheral lymphoid organs (spleen and lymph nodes). Phenotypic analysis qualifies these cells as antigen-presenting cells (APCs), including both dendritic cells and macrophages. These cells often appear surrounded by apoptotic lymphocytes, both in thymus and spleen, and may therefore be involved in the deletion of autoreactive lymphocytes. Our findings demonstrate the existence of, and define the tissue distribution and phenotype of, a novel subset of APCs expressing self-antigens in human lymphoid organs that appear to be involved in the regulation of self-tolerance throughout life.

Analysis of the adult thymus in reconstitution of T lymphocytes in HIV-1 infection.

A key question in understanding the status of the immune system in HIV-1 infection is whether the adult thymus contributes to reconstitution of peripheral T lymphocytes. We analyzed the thymus in adult patients who died of HIV-1 infection. In addition, we studied the clinical course of HIV-1 infection in three patients thymectomized for myasthenia gravis and determined the effect of antiretroviral therapy on CD4(+) T cells. We found that five of seven patients had thymus tissue at autopsy and that all thymuses identified had inflammatory infiltrates surrounding lymphodepleted thymic epithelium. Two of seven patients also had areas of thymopoiesis; one of these patients had peripheral blood CD4(+) T-cell levels of <50/mm3 for 51 months prior to death. Of three thymectomized patients, one rapidly progressed to AIDS, one progressed to AIDS over seven years (normal progressor), whereas the third remains asymptomatic at least seven years after seroconversion. Both latter patients had rises in peripheral blood CD4(+) T cells after antiretroviral therapy. Most patients who died of complications of HIV-1 infection did not have functional thymus tissue, and when present, thymopoiesis did not prevent prolonged lymphopenia. Thymectomy before HIV-1 infection did not preclude either peripheral CD4(+) T-cell rises or clinical responses after antiretroviral therapy.

Deficiency of PPARalpha disturbs the response of lipogenic flux and of lipogenic and cholesterogenic gene expression to dietary cholesterol in mouse white adipose tissue.

PPARalpha-deficiency in mice fed a high-carbohydrate, low-cholesterol diet was associated with a decreased weight of epididymal adipose tissue and an increased concentration of adipose tissue cholesterol. Consumption of a high (2% w/w) cholesterol diet resulted in a further increase in the concentration of cholesterol and a further decrease in epididymal fat pad weight in PPARalpha-null mice, but had no effect in the wild-type. These reductions in fat pad weight were associated with an increase in hepatic triacylglycerol content, indicating that both PPARalpha-deficiency and cholesterol altered the distribution of triacylglycerol in the body. Adipose tissue de novo lipogenesis was increased in PPARalpha-null mice and was further enhanced when they were fed a cholesterol-rich diet; no such effect was observed in the wild-type mice. The increased lipogenesis in the chow-fed PPARalpha-null mice was accompanied paradoxically by lower mRNA expression of SREBP-1c and its target genes, acetyl-CoA carboxylase and fatty acid synthase. Consumption of a high-cholesterol diet increased the mRNA expression of these genes in the PPARalpha-deficient mice but not in the wild-type. De novo cholesterol synthesis was not detectable in the adipose tissue of either genotype despite a relatively high expression of the mRNA's encoding SREBP-2 and 3-hydroxy-3-methylglutaryl Coenzyme A reductase. The mRNA expression of these genes and of the LDL-receptor in adipose tissue of the PPARalpha-deficient mice was lower than that of the wild-type and was not downregulated by cholesterol feeding. The results suggest that PPARalpha plays a role in adipose tissue cholesterol and triacylglycerol homeostasis and prevents cholesterol-mediated changes in de novo lipogenesis.

Abnormal structure and co-operative binding of low-density lipoprotein receptors containing the Glu-80-->Lys mutation.

The properties of low-density lipoprotein (LDL) receptors containing a Glu to Lys substitution at position 80 have been studied in fibroblasts from a homozygous familial hypercholesterolaemic subject (MB) and in monkey COS cells transfected with the mutant cDNA. Receptors containing the Glu-80-->Lys mutation were processed more slowly than the normal protein and only approx. 50% reached the surface as the mature form. Both cell types exhibited a normal concentration binding curve for beta-very-low-density lipoproteins (beta-VLDL) but an atypical, sigmoidal curve for LDL. The mature mutant receptor protein migrated abnormally slowly on SDS-PAGE under non-reducing conditions but normally under reducing conditions or after treatment with neuraminidase. It also showed an unusual ability to form dimers that were stable in detergents. Transfected normal and mutant receptors were apparently cleaved on the surface of the cells to give a product lacking the NH2-terminal portion of the protein, which was resistant to further proteolytic digestion. The results suggest that the Glu-80-->Lys substitution produces a change in the conformation of the protein, stabilized by polysaccharide chains, which results in a strong self-association of receptor molecules that affects their ability to bind LDL.

Determinants of variable response to statin treatment in patients with refractory familial hypercholesterolemia.

Interindividual variability in low density lipoprotein (LDL) cholesterol (LDL-C) response during treatment with statins is well documented but poorly understood. To investigate potential metabolic and genetic determinants of statin responsiveness, 19 patients with refractory heterozygous familial hypercholesterolemia were sequentially treated with placebo, atorvastatin (10 mg/d), bile acid sequestrant, and the 2 combined, each for 4 weeks. Levels of LDL-C, mevalonic acid (MVA), 7-alpha-OH-4-cholesten-3-one, and leukocyte LDL receptor and hydroxymethylglutaryl coenzyme A reductase mRNA were determined after each treatment period. Atorvastatin (10 mg/d) reduced LDL-C by an overall mean of 32.5%. Above-average responders (LDL-C -39.5%) had higher basal MVA levels (34.4+/-6.1 micromol/L) than did below-average responders (LDL-C -23.6%, P<0.02; basal MVA 26.3+/-6.1 micromol/L, P<0.01). Fewer good responders compared with the poor responders had an apolipoprotein E4 allele (3 of 11 versus 6 of 8, respectively; P<0.05). There were no baseline differences between them in 7-alpha-OH-4-cholesten-3-one, hydroxymethylglutaryl coenzyme A reductase mRNA, or LDL receptor mRNA, but the latter increased in the good responders on combination therapy (P<0.05). Severe mutations were not more common in poor than in good responders. We conclude that poor responders to statins have a low basal rate of cholesterol synthesis that may be secondary to a genetically determined increase in cholesterol absorption, possibly mediated by apolipoprotein E4. If so, statin responsiveness could be enhanced by reducing dietary cholesterol intake or inhibiting absorption.

Non-saturable degradation of LDL by monocyte-derived macrophages leads to a reduction in HMG-CoA reductase activity with little synthesis of cholesteryl esters.

In monocyte-derived macrophages from both normal and familial hypercholesterolaemic (FH) subjects, degradation of low density lipoprotein (LDL) through non-saturable pathways produced the same fall in 3-hydroxy-3-methylglutaryl-CoA reductase activity as receptor-mediated degradation of acetylated LDL, yet did not lead to as great an increase in incorporation of [14C]oleate into cholesteryl esters. Studies using FH cells showed that the simultaneous addition of LDL did not reduce oleate incorporation resulting from degradation of acetylated LDL, and that there was a similar relationship for both lipoproteins between the increase in net oleate incorporation and the increase in the cholesteryl ester content of the cells. FH cells maintained in serum-free medium accumulated more free cholesterol than cells in lipoprotein-deficient serum when incubated with LDL but not when incubated with acetylated LDL. The results suggest that the cholesterol released from non-saturable degradation of LDL is more easily removed from the cells by acceptors in the medium than cholesterol released from receptor-mediated uptake of acetylated LDL, and is not readily available for esterification.

Influence of genotype at the low density lipoprotein (LDL) receptor gene locus on the clinical phenotype and response to lipid-lowering drug therapy in heterozygous familial hypercholesterolaemia. The Familial Hypercholesterolaemia Regression Study Group.

The relationship between molecular defect and clinical phenotype has been examined in 42 patients with heterozygous familial hypercholesterolaemia (FH) and premature coronary heart disease. The defined defects included mutations in the low density lipoprotein (LDL)-receptor gene (23/42) or the apolipoprotein B Arg3500Gln mutation (5/42). Mean LDL-cholesterol was higher, both before and during treatment with simvastatin and bile acid sequestrants, in patients predicted as having a 'severe' mutation than in those with a 'mild' mutation (8.72 +/- 2.02 mmol/l vs 6.63 +/- 1.8, P = 0.05 before and 4.51 +/- 0.90 mmol/l vs 3.19 +/- 0.58, P = 0.05 during treatment). Maximum inducible LDL-receptor activity in cultured lymphoblasts was inversely correlated with LDL-cholesterol before (r2 = 0.499, P = 0.002) and during (r2 = 0.478, P = 0.004) treatment in patients with a defined mutation in the LDL-receptor gene, but not in the 14 patients with no detectable molecular defect. LDL-cholesterol concentrations before and during treatment were significantly correlated in patients with a defined LDL-receptor gene mutation (r2 = 0.548, P = 0.0001), but not in those with no detectable genetic defect. All these correlations were weak, however and there were no differences in the response to treatment in terms of either relative reduction or absolute decrease in LDL-cholesterol concentration between patients with different LDL-receptor defects. We conclude that only part of the variable phenotype of heterozygous FH patients is explained by different LDL-receptor defects and that other factors determine the severity of their hypercholesterolaemia and the onset of coronary disease.

Cholesterol-lowering drug therapy in a patient with receptor-negative homozygous familial hypercholesterolaemia.

Familial hypercholesterolaemia (FH) is caused by mutations in the gene for the low density lipoprotein (LDL) receptor. It is generally believed that homozygous FH patients do not respond well to lipid-lowering drug therapy with inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase because they cannot respond to an increased demand for hepatic cholesterol by up-regulation of LDL-receptor activity. In this paper we show that serum cholesterol in a homozygous FH patient with a receptor-negative LDL-receptor phenotype was reduced by 30% after treatment with simvastatin alone and by a further 11% with simvastatin in combination with probucol and nicotinic acid. The patient was a true homozygote, with two identical alleles of the LDL receptor gene in which a previously undescribed point mutation in exon 11 introduces a premature termination codon at residue 540 in the protein; the mutant protein is predicted to be truncated in the domain with homology to the epidermal growth factor precursor. Cultured cells from the patient were unable to bind, internalise or degrade LDL by the receptor pathway and there was no immunodetectable LDL receptor protein in the cells. Thus the lipid lowering effect of simvastatin in this individual must involve mechanisms other than stimulation of LDL receptors.