Increased Levels of Plasma Tumor Necrosis Factor-α Mediate Schizophrenia Symptom Dimensions and Neurocognitive Impairments and Are Inversely Associated with Natural IgM Directed to Malondialdehyde and Paraoxonase 1 Activity.

Accumulating evidence suggests that TNF-α-mediated immune-neurotoxicity contributes to cognitive impairments and the overall severity of schizophrenia (OSOS). There are no data whether peripheral IL-6 and IL-4 may affect the phenome of schizophrenia above and beyond the effects of TNF-α and whether those cytokines are regulated by lowered natural IgM to malondialdehyde (MDA) and paraoxonase 1 enzyme activity. We assessed the aforementioned biomarkers in a cross-sectional study that enrolled schizophrenia patients with (n = 40) and without (n = 40) deficit schizophrenia and 40 healthy controls. Deficit schizophrenia was best predicted by a combination of increased IL-6 and PON1 status (QQ genotype and lowered CMPAase activity) and lowered IgM to MDA. Partial least squares bootstrapping shows that 41.0% of the variance in negative symptoms, psychosis, hostility, excitation, mannerism, psychomotor retardation, and formal thought disorders was explained by increased TNF-α and PON1 status (QQ genotype and lowered CMPAase activity), which lowered IL-4 and IgM to MDA as well as male sex and lowered education. We found that 47.9% of the variance in verbal fluency, word list memory, true recall, Mini-Mental State Examination, and executive functions was predicted by increased TNF-α and lowered IL-4, IgM to MDA, and education. In addition, both TNF-α and IL-4 levels were significantly associated with lowered IgM to MDA, while TNF-α was correlated with PON1 status. These data provide evidence that the symptomatic (both the deficit subtype and OSOS) and cognitive impairments in schizophrenia are to a large extent mediated by the effects of immune-mediated neurotoxicity as well as lowered regulation by the innate immune system.

Paraoxonase 1 knockout rats have impaired T cell development at the CD4/CD8 double-negative to double-positive transition stage.

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that performs multiple physiological activities. Previous studies suggest that PON1 plays an anti-inflammatory role in the cardiovascular system, although its roles in hematopoiesis and adaptive immunity have not been clarified. To investigate the impact of PON1 on the immune system, we generated PON1-knockout (PON1-/-) rats using the CRISPR/Cas9 system. The thymus was smaller in PON1-/- rats than that in wild-type (PON1+/+) rats. Furthermore, analysis of thymocyte development revealed diminished total T cell numbers and a decrease in CD4+, CD8+ and double-positive T cells in peripheral blood and thymus from PON1-/- rats. This may be due to a block in the transition of T cells from the double-negative to the double-positive stage. We also showed that the activation of p38 MAPK phosphorylation contributed to the increased apoptosis and defective T cell development in PON-/- rats. Therefore, our results indicate that PON1 functions as a novel regulator of T cell development.

Antibodies against HDL Components in Ischaemic Stroke and Coronary Artery Disease.

Quantitative and qualitative defects of high-density lipoprotein (HDL) are important in atherogenesis. In this study, we investigated whether antibodies against HDL components had additional value to conventional cardiovascular risk factors for the diagnosis of ischaemic stroke (IS) and coronary artery disease (CAD). Cross-sectional study was conducted on 53 patients with IS, 51 with CAD and 55 healthy controls, and in vitro studies to validate findings of the clinical study. We determined serum immunoglobulin G (IgG) antibodies against HDL (aHDL), apolipoproteins (aApoA-I, aApoA-II and aApoC-I) and paraoxonase-1 (aPON1) as well as PON1 activity (PON1a), total antioxidant capacity and biomarkers of endothelial activation (serum nitric oxide metabolites, 3-nitrotyrosine, VCAM-1 and ICAM-1); in vitro assays tested the capacity of IgG aHDL purified from high titer patients to inhibit PON1a and to reverse protective effect of HDL on endothelial cells. IgG aHDL, aApoA-I and aPON1 were higher in IS and CAD than controls (p < 0.001), predicted negatively PON1a and positively VCAM-1 and ICAM-1. By adding IgG aHDL and aApoA-I to a traditional cardiovascular risk factors model for IS and by adding IgG aHDL in a similar model for CAD, we obtained better discrimination of IS and CAD from healthy controls. IgG aHDL purified from IS and CAD inhibited PON1a by 38% (p < 0.01) and abrogated the protective effect of HDL on VCAM-1 expression by 126% compared with non-specific human IgG (p < 0.001). IgG against HDL components interfere with the antioxidant and anti-inflammatory properties of HDL and may represent novel biomarkers for vascular disease that need to be investigated in prospective studies.

Serum Levels of Anti-PON1 and Anti-HDL Antibodies as Potential Biomarkers of Premature Atherosclerosis in Systemic Lupus Erythematosus.

The present study aimed to evaluate the possible role of immunoglobulin G (IgG) antibodies against high-density lipoproteins (HDL) and paraoxonase 1 (PON1) as possible biomarkers of cardiovascular disease (CVD) in systemic lupus erythematosus (SLE). To this end, levels of these autoantibodies, PON1 activity and total antioxidant capacity were quantified in serum samples from 198 SLE patients, 100 healthy controls (HC) and 42 non-autoimmune individuals with traditional cardiovascular risk factors. PON1 rs662 polymorphism was analysed in a subgroup of patients and controls. Subclinical CVD were determined by Doppler ultrasound in 118 SLE patients and 30 HC, analysing carotid intima-media thickness (IMT) and blood flow parameters in internal carotid, middle cerebral and basilar arteries. Serum levels of both anti-HDL and anti-PON1 antibodies were increased in SLE patients compared with HC (p < 0.001); however, only anti-PON1 antibodies, in addition to disease activity, were significant predictors of the impaired PON1 function in SLE (ß = -0.143, p = 0.045). Conversely, anti-HDL antibodies were associated with higher risk of CVD (odds ratio: 3.69; p = 0.012) and lower HDL levels at disease onset (ρ = -0.324, p = 0.044). Finally, anti-PON1 antibodies were associated with carotid IMT in SLE (ß = 0.201, p = 0.008) and inversely related to cranial arteries blood flow velocities in patients with clinical and subclinical CVD (all p < 0.001). In sum, these findings allowed us to propose serum levels of anti-PON1 and anti-HDL antibodies as potential early biomarkers of endothelial damage and premature atherosclerosis in SLE, thus constituting useful therapeutic targets for the prevention of future CVD in these patients.

Antibodies to paraoxonase 1 are associated with oxidant status and endothelial activation in rheumatoid arthritis.

Traditional and non-traditional cardiovascular (CV) risk factors underlie CV disease occurrence in rheumatoid arthritis (RA). Recently, a functional impairment of high-density lipoprotein (HDL) has been observed. Although the actual players are unknown, anti-HDLs were associated with altered lipid profile, decreased paraoxonase 1 (PON1) activity and CV disease in RA. Therefore, we aimed to evaluate whether the presence of antibodies against PON1 may be involved in this scenario. IgG anti-PON1 antibodies were quantified by ELISA in serum samples from 212 RA patients, 175 healthy controls (HC) and 54 subjects with traditional CV risk factors (CVR). A subgroup of 13 RA patients was prospectively followed upon tumour necrosis factor-α  (TNFα) blockade. Serum PON1 activity, nitric oxide (NO) and total antioxidant capacity (TAC) were measured. Interferon-γ (IFNγ), interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP-1), vascular endothelial growth factor (VEGF), soluble intercellular adhesion molecule (sICAM) and TNFα serum levels were assessed by immunoassays. PON1 rs662 (Q > R) status was studied by reverse transcription (RT)-PCR. IgG anti-PON1 antibodies are increased in RA patients compared with HC (P<0.0001) and CVR subjects (P<0.001), even after correcting for total IgG levels. Although no associations with lipid profile were found, a positive correlation with Health Assessment Questionnaire (HAQ) was observed (r=0.215, P=0.004). Anti-PON1 antibodies were associated with PON1 activity, NO and TAC, a rs662-mediated gene-dosage effect being found. Similarly, anti-PON1 antibodies were associated with sICAM serum levels in univariate and multivariate models. Finally, these antibodies were not affected by TNFα blockade. Anti-PON1 antibodies can be responsible for PON1 impairment in RA patients, with a potential impact on biomarkers of oxidative status and endothelial activation. A gene-environment interaction of rs662 variants is supported.

Impairment of high-density lipoprotein resistance to lipid peroxidation and adipose tissue inflammation in obesity complicated by obstructive sleep apnea.

CONTEXT: Obstructive sleep apnea (OSA) complicates morbid obesity and is associated with increased cardiovascular disease incidence. An increase in the circulating markers of chronic inflammation and dysfunctional high-density lipoprotein (HDL) occur in severe obesity. OBJECTIVE: The objective of the study was to establish whether the effects of obesity on inflammation and HDL dysfunction are more marked when complicated by OSA. DESIGN AND PATIENTS: Morbidly obese patients (n = 41) were divided into those whose apnea-hypoapnea index (AHI) was more or less than the median value and on the presence of OSA [OSA and no OSA (nOSA) groups]. We studied the antioxidant function of HDL and measured serum paraoxonase 1 (PON1) activity, TNFα, and intercellular adhesion molecule 1 (ICAM-1) levels in these patients. In a subset of 19 patients, we immunostained gluteal sc adipose tissue (SAT) for TNFα, macrophages, and measured adipocyte size. RESULTS: HDL lipid peroxide levels were higher and serum PON1 activity was lower in the high AHI group vs the low AHI group (P < .05 and P < .0001, respectively) and in the OSA group vs the nOSA group (P = .005 and P < .05, respectively). Serum TNFα and ICAM-1 levels and TNFα immunostaining in SAT increased with the severity of OSA. Serum PON1 activity was inversely correlated with AHI (r = -0.41, P < .03) in the OSA group. TNFα expression in SAT directly correlated with AHI (r = 0.53, P < .03) in the subset of 19 patients from whom a biopsy was obtained. CONCLUSION: Increased serum TNFα, ICAM-1, and TNFα expression in SAT provide a mechanistic basis for enhanced inflammation in patients with OSA. Decreased serum PON1 activity, impaired HDL antioxidant function, and increased adipose tissue inflammation in these patients could be a mechanism for HDL and endothelial dysfunction.

Heart failure is associated with impaired anti-inflammatory and antioxidant properties of high-density lipoproteins.

Oxidative stress and inflammation are hallmarks of the heart failure (HF) disease state. In the present study, we investigated the inflammatory/anti-inflammatory characteristics of high-density lipoproteins (HDL) in patients with HF. Ninety-six consecutive patients with systolic HF were followed in an advanced HF center, and 21 healthy subjects were recruited. Plasma was tested for HDL inflammatory index (HII) using a monocyte chemotactic activity assay, with HII >1.0 indicating proinflammatory HDL. We found significantly increased inflammatory properties of HDL in patients with HF (median HII 1.56 vs 0.59 in controls; p <0.0001). Serum amyloid A level was markedly elevated and the activity of paraoxonase-1, an HDL antioxidant enzyme, was significantly reduced in patients versus controls. HDL and albumin from patients with HF contained markedly elevated levels of oxidized products of arachidonic and linoleic acids. HDL function improved when plasma was treated in vitro with 4F, an apolipoprotein A-I mimetic peptide (40% reduction in HII, p <0.0001). There was no correlation found between HII level and ejection fraction or New York Heart Association functional class. In conclusion, HDL function is significantly impaired and oxidation products of arachidonic and linoleic acids are markedly elevated in patients with HF compared with non-HF controls.

Autoimmune-mediated reduction of high-density lipoprotein-cholesterol and paraoxonase 1 activity in systemic lupus erythematosus-prone gld mice.

OBJECTIVE: To characterize modifications of high-density lipoprotein (HDL) in autoimmune gld mice that may be relevant to premature atherosclerosis in systemic lupus erythematosus, and to assess their relationship to specific aspects of autoimmune disease. METHODS: HDL cholesterol (HDL-C), apolipoprotein A-I (Apo A-I), paraoxonase 1 (PON1) activity, hepatic gene expression, and HDL biogenesis were measured in aging female gld and wild-type congenic mice. Autoantibodies, lymphoid organs, and cytokines were analyzed by enzyme-linked immunosorbent assay, flow cytometry, and multiplex assay, respectively. RESULTS: Plasma HDL-C, HDL Apo A-I, and HDL-associated PON1 activity were reduced in aging gld mice in association with the development of autoimmunity, independent of changes in hepatic Apo A-I and PON1 expression or HDL biogenesis. Hepatic induction of the acute-phase reactant serum amyloid A1 resulted in its incorporation into HDL in gld mice. Deletion of the lipid-sensitive receptor G2A in gld mice (G2A-/- gld) attenuated reductions in HDL-C and PON1 activity without altering hepatic Apo A-I and PON1 expression, HDL biogenesis, or levels of acute-phase proinflammatory cytokines. Plasma anti-Apo A-I autoantibodies were elevated in aging gld mice commensurate with detectable increases in Apo A-I immune complexes. Autoantibody levels were lower in aging G2A-/- gld mice compared with gld mice, and anti-Apo A-I autoantibody levels were significantly related to HDL-C concentrations (r=-0.645, P<0.00004) and PON1 activity (r=-0.555, P<0.0007) among autoimmune gld and G2A-/- gld mice. CONCLUSION: Autoantibodies against Apo A-I contribute to reducing HDL-C and PON1 activity in autoimmune gld mice independently of hepatic HDL biogenesis, suggesting that functional impairment and premature clearance of HDL immune complexes may be principal mechanisms involved.

Improved pharmacokinetics and immunogenicity profile of organophosphorus hydrolase by chemical modification with polyethylene glycol.

A catalytic bioscavenger with broad substrate specificity for the therapeutic and prophylactic defense against recognized chemical threat agents has been a long standing objective of civilian and military research. A catalytic bioscavenger utilizing the bacterial enzyme organophosphorus hydrolase (OPH) is characterized in these studies, and has potential application for both military and civilian personnel in threat scenarios involving either nerve agents or OP pesticides. The present study examines the effects of PEGylation on the biochemical and pharmacological characteristics of OPH. The enzyme was conjugated with linear and branched methyl-PEO(n)-NHS esters of relatively small molecular mass from 333 to 2420Da. PEGylated OPH displayed a decreased maximal catalytic rate, though substantial activity was maintained against two tested substrates: up to 30% with paraoxon and up to 50-60% with demeton-S. The thermostability of the PEGylated enzymes ranged between 60 and 64 degrees C, compared to the unmodified OPH, which is approximately 67 degrees C. The enzyme conjugates revealed a significant improvement of pharmacokinetic properties in animal studies. The clearance from a guinea pig's blood stream significantly decreased relative to unmodified OPH, resulting in an increase of residence time and systemic availability. Evaluation of the humoral immune response indicated that the branched PEG-OPH conjugate significantly reduced production of anti-OPH antibodies, compared to the unmodified enzyme. The OPH-PEG conjugates with improved pharmacokinetic and immunogenicity properties, considerable catalytic activity and thermal stability provide a new opportunity for the in vivo detoxification of the neurotoxic OP compounds.

The roles of PON1 and PON2 in cardiovascular disease and innate immunity.

PURPOSE OF REVIEW: The paraoxonase (PON) gene family includes three members, PON1, PON2, and PON3. In-vitro and mouse studies have demonstrated that all three PONs are atheroprotective. Some, but not all, human epidemiologic studies have observed associations between PON gene polymorphisms and risk of cardiovascular disease (CVD). In this review, we summarize studies published within the last year, elucidating involvement of PON1 and PON2 in oxidative stress, CVD, and innate immune responses. RECENT FINDINGS: In a prospective study, the PON1 192QQ genotype and low PON1 activity were associated with increased systemic oxidative stress and increased risk for CVD. PON1 expression protected against Pseudomonas aeruginosa lethality in Drosophila, suggesting that PON1 can interfere with quorum sensing in vivo. PON2 attenuated macrophage triglyceride accumulation via inhibition of diacylglycerol acyltransferase 1. Overexpression of PON2 protected against endoplasmic reticulum stress-induced apoptosis when the stress was induced by interference with protein modification but not when endoplasmic reticulum stress was induced by Ca2+ deregulation. SUMMARY: Both mouse and human studies have demonstrated the antioxidative and atheroprotective effects of PON1. The mechanisms by which PON2 exerts its atheroprotective effects are emerging. Large-scale epidemiologic studies are needed to further examine the relationship between PON2 genetic polymorphisms and risk for CVD. Elucidation of the physiologic substrates of the PON proteins is of particular importance to further advance this field.

[Preparation and preliminary application of rabbit anti-human PON2 antibodies(paraoxonase-2)].

AIM: To preparation and characterize the rabbit polyclonal antibodies against human PON2 (paraoxonase-2). METHODS: A fragment of human PON2 gene which was of low homology with rabbits but of higher hydrophilicity and immunogenicity was selected for recombinant expression in prokaryotic expression system. The rabbits were immunized with the purified GST fusion protein 3 times. The specificity and sensitivity of the anti-human PON2 polyclonal antibodies were detected by Western blot and indirect immunofluorescence. RESULTS: The GST-PON2 fusion protein was highly expressed in Ecoli with a molecular weight of 46 kDa. Western blot analysis proved the rabbit polyclonal antibodies could specifically recognize 39 kDa native PON2 protein expressed in several cells and tissues, such as HeLa cells, U937 cells, and human liver tissue. Indirect immunofluorescence analysis confirmed that PON2 protein was located in the cytoplasm of SY5Y cells. CONCLUSION: The rabbit polyclonal antibodies against human PON2 can specifically recognize natural protein expressed in human cells and tissues, Which can be used for further study and clinical detection of human PON2.

IgG-paraoxonase-1 fusion protein for targeted drug delivery across the human blood-brain barrier.

Paraoxonase (PON)-1 is the most potent human protein with organophosphatase activity against organophosphate (OP) toxins. OP compounds readily cross the blood-brain barrier (BBB) and have lethal mechanisms of action within the brain. The production of a brain penetrating form of human PON1, which crosses the BBB, is possible with the re-engineering of the enzyme as a fusion protein with a monoclonal antibody (mAb) against the human insulin receptor (HIR). The HIRMAb crosses the BBB via the endogenous insulin receptor, and acts as a molecular Trojan horse to ferry the PON1 into brain. The human PON1 was fused to the carboxyl terminus of the heavy chain of the chimeric HIRMAb. COS cells were dual transfected with the heavy chain gene and the light chain gene, and the HIRMAb-PON1 fusion protein was affinity purified with protein A chromatography. Western blotting with antibodies to human IgG or human PON1 showed the heavy chain of the HIRMAb-PON1 fusion protein was 40 kDa larger than the heavy chain of the chimeric HIRMAb. The ED50 of binding to the HIR extracellular domain was 0.55 +/- 0.07 nM and 1.1 +/- 0.1 nM, respectively, for the chimeric HIRMAb and the HIRMAb-PON1 fusion protein. The PON1 enzyme activity of the fusion protein was approximately 25% of the enzyme activity in human plasma, based on a fluorometric enzymatic assay. In conclusion, human PON1 has been re-engineered as an IgG-organophosphatase fusion protein that penetrates the human BBB.

Trypanosoma congolense: paraoxonase 1 prolongs survival of infected mice.

In vitro studies have suggested that a fraction of human high density lipoprotein (HDL), termed trypanosome lysis factor (TLF), can protect against trypanosome infection. We examined the involvement of two proteins located in the TLF fraction, apolipoprotein A-II (apoA-II) and paraoxonase 1 (PON1), against trypanosome infection. To test whether PON1 is involved in trypanosome resistance, we infected human PON1 transgenic mice, PON1 knockout mice, and wild-type mice with Trypanosoma congolense. When challenged with the same dosage of trypanosomes, mice overexpressing PON1 lived significantly longer than wild-type mice, and mice deficient in PON1 lived significantly shorter. In contrast, mice overexpressing another HDL associated protein, apoA-II, had the same survival as wild-type mice. Together, these data suggest that PON1 provides protection against trypanosome infection. In vitro studies using T. brucei brucei indicated that HDL particles containing PON1 and those depleted of PON1 did not differ in their lysis ability, suggesting that protection by PON1 is indirect. Our data are consistent with an in vivo role of HDL protection against trypanosome infection.