DNA Damage, n-3 Long-Chain PUFA Levels and Proteomic Profile in Brazilian Children and Adolescents.

Fatty acids play a significant role in maintaining cellular and DNA protection and we previously found an inverse relationship between blood levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and DNA damage. The aim of this study was to explore differences in proteomic profiles, for 117 pro-inflammatory proteins, in two previously defined groups of individuals with different DNA damage and EPA and DHA levels. Healthy children and adolescents (n = 140) aged 9 to 13 years old in an urban area of Brazil were divided by k-means cluster test into two clusters of DNA damage (tail intensity) using the comet assay (cluster 1 = 5.9% ± 1.2 and cluster 2 = 13.8% ± 3.1) in our previous study. The cluster with higher DNA damage and lower levels of DHA (6.2 ± 1.6 mg/dL; 5.4 ± 1.3 mg/dL, p = 0.003) and EPA (0.6 ± 0.2 mg/dL; 0.5 ± 0.1 mg/dL, p < 0.001) presented increased expression of the proteins CDK8-CCNC, PIK3CA-PIK3R1, KYNU, and PRKCB, which are involved in pro-inflammatory pathways. Our findings support the hypothesis that low levels of n-3 long-chain PUFA may have a less protective role against DNA damage through expression of pro-inflammatory proteins, such as CDK8-CCNC, PIK3CA-PIK3R1, KYNU, and PRKCB.

Anti-CV2/CRMP5 Paraneoplastic Chorea Effectively Managed with Intravenous Amantadine.

Background: Paraneoplastic chorea is typically a subacute progressive hyperkinetic movement disorder. The mainstay of treatment is managing the underlying neoplasm. However, the clinical course may be variable, and effective symptomatic management can precede the start of cancer treatment. Case report: A 63-year-old man presented with insidious onset, slowly progressive generalized chorea for 1 year, later diagnosed as anti-CV2/CRMP5 autoantibody positive paraneoplastic chorea. His chorea was markedly improved with intravenous amantadine. Discussion: In patients with anti-CV2/CRMP5 autoantibody-related chorea, sequential follow-up of brain magnetic resonance imaging reveals progression from active inflammation to atrophy. Our report highlights the efficacy of intravenous amantadine in paraneoplastic chorea.

Caloric restriction can affect one-carbon metabolism during pregnancy in the rat: A transgenerational model.

One-carbon metabolism is critical to pregnancy outcomes, because it determines the availability of nutrients involved in cell divisions and DNA methylation. The aim of this study was to analyze how 50% prenatal calorie restriction affected one-carbon metabolism in pregnant Wistar rats of the F0 to F2 generations. Mean choline (p < 0.001), betaine (p < 0.001), and S-adenosylmethionine (SAM) (p < 0.05) concentrations were respectively about 40%, 45%, and 20% lower in the F0_R (R - restricted diet) than in the F0_C (C - control diet). Homocysteine, S-adenosylhomocysteine (SAH), and trimethylamine oxide concentrations were unaffected. In the F1_R, the SAM-to-SAH ratio was 25% higher (p < 0.05) than in the F1_C. No differences between the C and R groups were observed in the F2 generation. The SAM concentrations in the F1_R were higher than in the F0_R and the F2_R (p < 0.01). The relative transcript levels of Mat1a, Bhmt, Cbs, Pemt, and Mthfr were only slightly affected by the diet, with changes of less than a factor of 2.0. Cbs activity in the F2_R was significantly higher than in the F2_C (p < 0.001). Food deprivation may affect one-carbon metabolism in pregnant rats, but it does not stimulate persistent metabolic changes that can be observed during the pregnancy of their progeny of the F1 or F2 generations.

Detection of mucopolysaccharidosis III-A (Sanfilippo Syndrome-A) in dried blood spots (DBS) by tandem mass spectrometry.

BACKGROUND: With ongoing efforts to develop improved treatments for Sanfilippo Syndrome Type A (MPS-IIIA), a disease caused by the inability to degrade heparan sulfate in lysosomes, we sought to develop an enzymatic activity assay for the relevant enzyme, sulfamidase, that uses dried blood spots (DBS). METHODS: We designed and synthesized a new sulfamidase substrate that can be used to measure sulfamidase activity in DBS using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Sulfamidase activity was readily detected in DBS using the new substrate and LC-MS/MS. Sulfamidase activity showed acceptable linearity proportional to the amount of enzyme and reaction time. Sulfamidase activity in 238 random newborns was well elevated compared to the range of activities measured in DBS from 8 patients previously confirmed to have MPS-IIIA. CONCLUSIONS: This is the first report of an assay capable of detecting sulfamidase in DBS. The new assay could be useful in diagnosis and potentially for newborn screening of MPS-IIIA.

Predictive Value of Maternal Serum Biomarkers for Preeclampsia and Birth Weight: A Case-Control Study in Chinese Pregnant Women.

BACKGROUND: In clinical practice, abnormal biochemical changes often occur in women who eventually develop preeclampsia (PE). The study aims to investigate whether maternal serum biochemical markers in the early third trimester can predict PE and neonatal birth weight. STUDY DESIGN: A retrospective case-control study was performed on 287 women who subsequently developed PE (mild = 139; severe = 148) and 143 healthy women. Fasting venous blood samples of all gravidas were drawn for routine biochemical markers screening in the early third trimester (28.49 ± 1.63 weeks). Appropriate statistical methods were selected for analysis with SPSS software. RESULTS: (1) The concentrations of plasma triglyceride (TG), low-density lipoprotein cholesterol (LDL), and uric acid (UA) in the severe and mild subgroups of the PE group were significantly higher compared with the respective levels in the normal pregnancy groups (3.90 vs. 4.03 vs. 3.14 mmol/L; 3.41 vs. 3.33 vs. 2.89 mmol/L; 365.42 vs. 318.91 vs. 284.69 µmol/L; p < 0.0001). Serum calcium levels in PE group were significantly lower than those in control group (2.10 vs. 2.18 vs. 2.22 mmol/L; p < 0.0001). (2) By using the receiver operating characteristic curve to estimate the diagnosis rate of screening for PE of each marker, the highest sensitivity appeared by the combination of TG, total cholesterol (TC), LDL, high-density lipoprotein cholesterol (HDL), LDL/HDL, UA, Ca2+, and homocysteine (HCY) (79%). The area under curve (AUC) of UA was 0.70, which was the highest among these eight markers, but the AUC of an eight-marker combination model (0.85) had a better diagnostic indication. (3) In PE, the maximum systolic/diastolic blood pressure was significantly positively correlated with serum UA (r = 0.212/0.205, p < 0.0001); and negatively correlated with serum total calcium (r = -0.193/-0.196, p = 0.001). The neonatal birth weight of PE group had a positive correlation with serum TG levels (r = 0.141, p = 0.017) and serum total calcium levels (r = 0.221, p < 0.0001), and a negative correlation with UA levels (r = -0.265, p < 0.0001). CONCLUSION: The individual marker really performs terrible in predicting PE. Joint monitoring and evaluation of these parameters may improve the screening efficiency for the prediction of PE and poor fetal growth early.

Identification of a new diagnostic antigen for glanders using immunoproteome analysis.

Glanders is a disease of horses, donkeys and mules. The causative agent Burkholderia mallei, is a biorisk group 3 pathogen and is also a biothreat agent. Simple and rapid diagnostic tool is essential for control of glanders. Using a proteomic approach and immunoblotting with equine sera, we identified 12 protein antigens that may have diagnostic potential. Various immunoreactive proteins e.g. GroEL, translation elongation factor Tu, elongation factor Ts, arginine deiminase, malate dehydrogenase, DNA directed RNA polymerase subunit alpha were identified on 2-dimentional immunoblots. One of these proteins, GroEL, was cloned and expressed in E. coli and purified using Ni-NTA affinity chromatography. The recombinant GroEL protein was evaluated in ELISA format on a panel of glanders positive (n=49) and negative (n=79) equine serum samples to determine its diagnostic potential. The developed ELISA had a sensitivity and specificity of 96 and 98.7% respectively. The results of this study highlight the potential of GroEL in serodiagnosis of glanders.

Hypersuccinylacetonaemia and normal liver function in maleylacetoacetate isomerase deficiency.

BACKGROUND: A high level of succinylacetone (SA) in blood is a sensitive, specific newborn screening marker for hepatorenal tyrosinemia type 1 (HT1, MIM 276700) caused by deficiency of fumarylacetoacetate hydrolase (FAH). Newborns with HT1 are usually clinically asymptomatic but show liver dysfunction with coagulation abnormalities (prolonged prothrombin time and/or high international normalised ratio). Early treatment with nitisinone (NTBC) plus dietary restriction of tyrosine and phenylalanine prevents the complications of severe liver disease and neurological crises. METHODS AND RESULTS: Six newborns referred for hypersuccinylacetonaemia but who had normal coagulation testing on initial evaluation had sequence variants in the GSTZ1 gene, encoding maleylacetoacetate isomerase (MAAI), the enzyme preceding FAH in tyrosine degradation. Initial plasma SA levels ranged from 233 to 1282 nmol/L, greater than normal (<24 nmol/L) but less than the initial values of patients with HT1 (16 944-74 377 nmol/L, n=15). Four individuals were homozygous for c.449C>T (p.Ala150Val). One was compound heterozygous for c.259C>T (p.Arg87Ter) and an intronic sequence variant. In one, a single heterozygous GSTZ1 sequence variant was identified, c.295G>A (p.Val99Met). Bacterial expression of p.Ala150Val and p.Val99Met revealed low MAAI activity. The six individuals with mild hypersuccinylacetonaemia (MHSA) were not treated with diet or nitisinone. Their clinical course has been normal for up to 13 years. CONCLUSIONS: MHSA can be caused by sequence variants in GSTZ1. Such individuals have thus far remained asymptomatic despite receiving no specific treatment.

Effects by periodontitis on pristane-induced arthritis in rats.

BACKGROUND: An infection-immune association of periodontal disease with rheumatoid arthritis has been suggested. This study aimed to investigate the effect of pre-existing periodontitis on the development and the immune/inflammatory response of pristane-induced arthritis. METHODS: We investigated the effect of periodontitis induced by ligature placement and Porphyromonas gingivalis (P. gingivalis) infection, in combination with Fusobacterium nucleatum to promote its colonization, on the development of pristane-induced arthritis (PIA) in rats (Dark Agouti). Disease progression and severity of periodontitis and arthritis was monitored using clinical assessment, micro-computed tomography (micro-CT)/intraoral radiographs, antibody response, the inflammatory markers such as α-1-acid glycoprotein (α-1-AGP) and c-reactive protein (CRP) as well as cytokine multiplex profiling at different time intervals after induction. RESULTS: Experimentally induced periodontitis manifested clinically (P < 0.05) prior to pristane injection and progressed steadily until the end of experiments (15 weeks), as compared to the non-ligated arthritis group. Injection of pristane 8 weeks after periodontitis-induction led to severe arthritis in all rats demonstrating that the severity of arthritis was not affected by the pre-existence of periodontitis. Endpoint analysis showed that 89% of the periodontitis-affected animals were positive for antibodies against arginine gingipain B and furthermore, the plasma antibody levels to a citrullinated P. gingivalis peptidylarginine deiminase (PPAD) peptide (denoted CPP3) were significantly (P < 0.05) higher in periodontitis rats with PIA. Additionally, there was a trend towards increased pro-inflammatory and anti-inflammatory cytokine levels, and increased α-1-AGP levels in plasma from periodontitis-challenged PIA rats. CONCLUSIONS: Pre-existence of periodontitis induced antibodies against citrullinated peptide derived from PPAD in rats with PIA. However, there were no differences in the development or severity of PIA between periodontitis challenged and periodontitis free rats.

An in-house multilocus SNP genotyping assay for evaluation of complex genetic diseases.

BACKGROUND: With an increase in the discovery of newer genetic loci/polymorphisms in complex multifactorial diseases, there is also an increased need for methods that can simultaneously genotype multiple loci in a cost-effective manner. Using coronary artery disease (CAD) as a model, the study aimed to develop an in-house multilocus assay for simultaneous detection of 17 genetic variants in 11 genes implicated in CAD. METHODS: A multiplex polymerase chain reaction (PCR)-based reverse line blot hybridization (MPCR-RLBH) approach was used, where each DNA sample was amplified using two separate MPCRs, and the alleles were genotyped using covalently immobilized, amino-linked sequence-specific oligonucleotide probes using an enhanced chemiluminescence system. The assay performance was tested on 75 healthy controls and 75 angiographically proven CAD cases. Validation was done by automated Sanger sequencing. RESULTS: The assay could successfully discriminate both the alleles at CETP (I405V), LPL (D9N), NOS3 (T-786G and E298D), LIPC (C-514T), FGB (G-455A), ITGB3 (L33P), AGT (M235T), and MTR (A2756G) loci. Certain mutations included in this assay such as ins242G, ins397G, E387K, L393K in the LDLR; N291S in the LPL; D442G in the CETP; and T833C in the CBS genes were found to be absent. The genotype results obtained using this assay showed 100% concordance with sequencing. CONCLUSION: The study demonstrated development and validation of a multiplex SNP genotyping assay that can be used to assess genetic risk factors in CAD. The assay provides a cost-effective alternative to expensive high throughput genotyping systems in common molecular research laboratories.

Cadmium-induced changes in hatchability and in the activity of aminotransaminases and selected lysosomal hydrolases in the blood plasma of Muscovy ducklings (Cairina moschata).

The aim of this study was to determine the effect of cadmium on Muscovy ducklings (Cairina moschata) based on hatching results and the activity of enzymes in the blood plasma. On day 6 of incubation, hatching eggs were injected into the egg albumen with 50 µl of saline solution containing Cd ions (CdCl2) at concentrations of 0 (control group), 1.3, 4.0, 7.5, 15.0 and 30 µg/egg, using 50 eggs per group. A gradual decrease in hatchability, from 52% in the control to 4% in the highest Cd dose group, was observed, with the LD50 calculated as 8 µg/egg. However, the impact of cadmium on the incidence of malformations of duck embryos has not been proven. Compared to the control group, N-acetyl-ß-Dglucosaminidase activity increased by 30-50% (P ≤ 0.05) in the blood serum of ducklings in the groups receiving more than 7.5 µg Cd/egg, whereas an elevated activity of arylsulphatase (by 45%) was observed for a lower dose only (4 µg Cd/egg). A gradual increase in the activity of alanine and aspartate aminotransferases was observed (P ≤ 0.05), starting from the lowest exposure of 1.3 µg Cd/egg, by 155% and 53%, respectively. In conclusion, the results prove the dosedependent toxic impact of cadmium on embryogenesis and on the studied blood plasma enzyme activities of ducklings.

Serum Immunoglobulin G Levels to Porphyromonas gingivalis Peptidylarginine Deiminase Affect Clinical Response to Biological Disease-Modifying Antirheumatic Drug in Rheumatoid Arthritis.

OBJECTIVES: To determine whether serum immunity to Porphyromonas gingivalis peptidylarginine deiminase (PPAD) affects the clinical response to biological disease-modifying antirheumatic drug (bDMARD) in patients with rheumatoid arthritis (RA). METHODS: In a retrospective study, rheumatologic and periodontal conditions of 60 patients with RA who had been treated with conventional synthetic DMARD were evaluated before (baseline) and after 3 and 6 months of bDMARD therapy. After serum levels of anti-PPAD immunoglobulin G (IgG) were determined at baseline, the patients were respectively divided into two groups for high and low anti-PPAD IgG titers according to the median measurements. Genotypes at 8 functional single nucleotide polymorphisms (SNPs) related to RA were also determined. RESULTS: After 3 and 6 months of therapy, patients with low anti-PPAD IgG titers showed a significantly greater decrease in changes in the Disease Activity Score including 28 joints using C-reactive protein (DAS28-CRP) (P = 0.04 for both) and anti-cyclic citrullinated peptide (CCP) IgG levels (P = 0.03 and P = 0.04) than patients with high anti-PPAD IgG titers, although these parameter values were comparable at baseline. The anti-PPAD IgG titers were significantly positively correlated with changes in the DAS28-CRP (P = 0.01 for both) and the anti-CCP IgG levels (P = 0.02 for both) from baseline to 3 and 6 months later. A multiple regression analysis revealed a significantly positive association between the anti-PPAD IgG titers and changes in the DAS28-CRP after 6 months of bDMARD therapy (P = 0.006), after adjusting for age, gender, smoking, periodontal condition, and RA-related SNPs. CONCLUSION: The serum IgG levels to PPAD affect the clinical response to bDMARD in patients with RA.

Prevalence of soluble peptidylarginine deiminase 4 (PAD4) and anti-PAD4 antibodies in autoimmune diseases.

The objectives of this study are to investigate the prevalence of PAD4 and anti-PAD4 antibodies (Abs) in autoimmune diseases and to clarify their association with anticitrullinated protein antibodies (ACPAs) and shared epitope (SE) in patients with rheumatoid arthritis (RA). Levels of human PAD4 and anti-PAD4 Abs in serum or plasma were measured using sandwich ELISA. Samples were obtained from patients with RA (n = 148), SLE (n = 36), or SS (n = 37) and from healthy controls (HCs; n = 40). Antibodies against cyclic citrullinated glucose-6-phosphate isomerase (GPI) (CCG)-2, CCG-7, anti-CEP-1, and anti-CCP Abs were also measured using ELISA. Patients with RA were genotyped for HLA-DRB1. The human PAD4 and anti-PAD4 Ab levels were compared with the ACPA and SE in patients with RA. The PAD4 levels were 111.9 U/ml in the RA, 30.4 U/ml in the SLE, 81.9 U/ml in the SS patients, and 46.6 U/ml in the HCs. The PAD4 levels were significantly higher in the RA than in the SLE patients or the HCs. Anti-PAD4 Abs were detected in 29.7 % of the patients with RA, but not in the patients with SLE or SS, nor in the HCs. In the RA patients, the PAD4 levels in the anti-PAD4 Ab-negative group were significantly higher than those in the anti-PAD4 Ab-positive group. Moreover, anti-CCG-2, CCG-7, CEP-1, and anti-CCP Ab levels were significantly higher in the anti-PAD4 Ab-positive group than in the anti-PAD4 Ab-negative group. In the RA patients, the PAD4 levels were not correlated with ACPAs. Neither PAD4 nor anti-PAD4 Abs were significantly correlated with the presence of SE alleles. The PAD4 levels were higher in RA than in SLE or HC. Anti-PAD4 Abs appeared specifically in patients with RA. Moreover, anti-PAD4 Abs were associated with ACPAs.

[The effect of muscle load on the post-prandial content of blood serum hydrolytic enzymes in men with different levels and specificity of daily physical activity].

The article presents data on the effect of the combined action of food and muscular load on the level of hydrolytic enzymes in blood serum of healthy young people 18-22 years old, with various levels of adaptation to the effects of physical activity. The first group (n = 8) of the examined persons were high-qualified athletes developing their speed and power qualities in anaerobic energetic regime (Greco-Roman wrestling, sambo, judo). The second group (n = 8) were athletes developing endurance in aerobic energetic regime (skiers, track and field athletes--stayers, biathletes). The control group (n = 8) consists of non-athletes. The content of hydrolytic enzymes: pepsinogen-1, pepsinogen-2, the activity of pancreatic alpha-amylase, lipase were defined by ELISA. The content and activity of ferments were defined in blood serum, taken in the morning fasting and post-prandial period in dynamics after 15, 45, 75 and 105 min after administration of the test breakfast (100 g of ground boiled beef and 200 ml of unsweetened tea) in a state of relative physiological rest and after the veloergometric exercise muscular load (at the level of 60-70% of maximal oxygen consumption) during an hour (in 7-14 days). Multidirectional changes of concentration of investigated enzymes in the postprandial period among examined were defined in the conditions of relative physiological rest and under the action of the muscular tension. For groups of athletes higher alpha-amylase and lipase blood activity were characteristic both in a state of physiological rest and under the action of muscular load. It was also determined that after the muscular tension there was an increase in activity of alpha-amylase at 75 min and lipases at 15 min relative to background indicators at non-athletes. For the athletes from the second group the increase (p < 0.01) relative to background data of activity as alpha-amylase as lipase on an empty stomach was noted. However postprandial (15-45 min) alpha-amylase (p < 0.05) and lipase (p < 0.001) activity was significantly decreased. At relatively high rates of fasting alpha-amylase activity there was a decrease of its level in the postprandial period, whereas at low rates of enzyme activity on an empty stomach its increase can be occurred in the postprandial period. Lipase activity changed in groups of athletes unidirectionally, it decreased (p < 0.01) in athletes of the first group at 45 min. and in athletes from the second group at 15-105 min (p < 0.001). For athletes of the first group, also as well for non-athletes, significantly lower blood lipase activity was noted at rest and in the conditions of muscular tension. After a physical load lipase activity in athletes from the second group was decreased throughout the postprandial period. Blood serum concentration of pepsinogen-1 and pepsinogen-2 were also significantly higher in the groups of athletes, but only in fasting conditions. After receiving the breakfast the content of these proenzymes were significantly lower in athletes comparable to the control group. Pepsinogen-2 concentration had a strong tendency to significant decrease after muscular exercise in all athletes throughout the postprandial period (at 15 and 105 min). For the athletes from the first group a decrease of pepsinogen-2 concentration to the values accepted as a norm (4-22 microg/l) was tended. The obtained data suggest that the revealed changes are associated with a sport orientation and a level of daily physical activity.

Significance of serum peptidylarginine deiminase type 4 in ANCA-associated vasculitis.

OBJECTIVE: To investigate the clinical significance of peptidylarginine deiminase type 4 (PAD4) in the pathogenesis of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). To make a primary observation on the relationship of chronic bronchitis and bronchiectasis (CB) with the pathogenesis of AAV by PAD4. METHODS: The sera from 13 patients with AAV, 13 patients with CB, 11 patients with rheumatoid arthritis (RA), 11 patients with primary chronic kidney disease (CKD) and 12 normal controls were collected. Serum PAD4 was detected using commercial ELISA kits. The serum levels of PAD4 were compared not only among the different groups but also between the activity and remission stage of the same disease. The associations between serum PAD4 and the Birmingham Vasculitis Activity Score (BVAS) of AAV were further investigated. RESULTS: (1) The serum levels of PAD4 in patients with AAV, RA and CB at activity stage were all higher than that in the normal controls (P<0.001, respectively, α'=0.007). The serum level of PAD4 in patients with CB at remission stage and that in CKD group were not found elevated compared with the normal controls (P=0.02, P=0.085, respectively, α'=0.007). (2) At activity stage, among the groups of simple AAV, AAV with a long history of CB and CB without AAV, no significant difference was detected. While at remission stage among the 3 groups, the serum level of PAD4 was at the lowest level in CB group without AAV. (3) The serum level of PAD4 in some patients with CB without AAV were found still higher at remission stage. (4) The serum level of PAD4 in AAV with renal damage at activity stage was positively correlated with BVAS (the activity score of AAV, r=0.71, P=0.02). CONCLUSION: PAD4 is involved in the pathogenesis of AAV. Whether some patients with CB might progress to AAV by the link with PAD4 still need further investigation.

Generation of monoclonal antibodies against peptidylarginine deiminase 2 (PAD2) and development of a PAD2-specific enzyme-linked immunosorbent assay.

The enzyme peptidylarginine deiminase 2 (PAD2) has been associated with inflammatory diseases, such as rheumatoid arthritis and neurodegenerative diseases including multiple sclerosis. To investigate the association of various diseases with extracellular PAD2, we raised monoclonal antibodies (mAbs) against rabbit PAD2 and evaluated their cross-reactivity with human PAD2 by indirect enzyme-linked immunosorbent assay (ELISA), western blotting and immunohistological staining of inflamed synovial tissue. Moreover, we established a sandwich ELISA detecting human PAD2, based on two different monoclonal antibodies, mAbs DN2 and DN6. The assay had a lower detection limit of 200pg/mL in serum and plasma samples, and showed dilution linearity and recovery ranging from 95 to 106%. The mAbs and the ELISA showed isotype specificity for PAD2. Circulating PAD2 was found in 8/28 (29%) serum samples from healthy donors. In conclusion, several of our mAbs proved useful in western blotting and immunohistochemistry, and the ELISA described here reliably measures PAD2 levels in blood. This allows investigation of PAD2 as a possible biomarker and further investigation of PAD2's involvement in various inflammatory diseases.

Gene transfer of mutant mouse cholinesterase provides high lifetime expression and reduced cocaine responses with no evident toxicity.

Gene transfer of a human cocaine hydrolase (hCocH) derived from butyrylcholinesterase (BChE) by 5 mutations (A199S/F227A/S287G/A328W/Y332G) has shown promise in animal studies for treatment of cocaine addiction. To predict the physiological fate and immunogenicity of this enzyme in humans, a comparable enzyme was created and tested in a conspecific host. Thus, similar mutations (A199S/S227A/S287G/A328W/Y332G) were introduced into mouse BChE to obtain a mouse CocH (mCocH). The cDNA was incorporated into viral vectors based on: a) serotype-5 helper-dependent adenovirus (hdAD) with ApoE promoter, and b) serotype-8 adeno-associated virus with CMV promoter (AAV-CMV) or multiple promoter and enhancer elements (AAV-VIP). Experiments on substrate kinetics of purified mCocH expressed in HEK293T cells showed 30-fold higher activity (U/mg) with (3)H-cocaine and 25% lower activity with butyrylthiocholine, compared with wild type BChE. In mice given modest doses of AAV-CMV-mCocH vector (0.7 or 3 × 10(11) particles) plasma hydrolase activity rose 10-fold above control for over one year with no observed immune response. Under the same conditions, transduction of the human counterpart continued less than 2 months and antibodies to hCocH were readily detected. The advanced AAV-VIP-mCocH vector generated a dose-dependent rise in plasma cocaine hydrolase activity from 20-fold (10(10) particles) to 20,000 fold (10(13) particles), while the hdAD vector (1.7 × 10(12) particles) yielded a 300,000-fold increase. Neither vector caused adverse reactions such as motor weakness, elevated liver enzymes, or disturbance in spontaneous activity. Furthermore, treatment with high dose hdAD-ApoE-mCocH vector (1.7 × 10(12) particles) prevented locomotor abnormalities, other behavioral signs, and release of hepatic alanine amino transferase after a cocaine dose fatal to most control mice (120 mg/kg). This outcome suggests that viral gene transfer can yield clinically effective cocaine hydrolase expression for lengthy periods without immune reactions or cholinergic dysfunction, while blocking toxicity from drug overdose.

Two novel sandwich ELISAs identify PAD4 levels and PAD4 autoantibodies in patients with rheumatoid arthritis.

OBJECTIVE: The peptidylarginine deiminase 4 (PAD4) gene and PAD4 autoantibodies have been associated with rheumatoid arthritis (RA) and its pathogenesis. Therefore, methods for accurately determining their levels in the peripheral blood of these patients would be a diagnostic asset. The objective of our study was to adapt the enzyme-linked immunosorbent assay (ELISA) method for evaluating PAD4 levels in human blood. METHODS: We prepared recombinant human (h)PAD1, -2, -3, and -4 proteins to develop mouse monoclonal antibodies specific to hPAD4. We then generated six monoclonal antibodies against hPAD4 and developed two new sandwich ELISA methods for evaluating hPAD4 and PAD4 autoantibodies in the peripheral blood from 32 patients with RA, ten patients with osteoarthrosis, and 20 healthy individuals. RESULTS: The distribution of hPAD4 in the patients' plasma was determined. Two populations were identified: one group with high hPAD4 levels (>0.57 ng/mL) and a second group with near-zero levels (<0.1 ng/mL). Most patients approximating zero hPAD4 levels had PAD4 autoantibodies. In contrast, most of those with higher plasma hPAD4 levels did not have detectable PAD4 autoantibodies. CONCLUSION: The combination of these sandwich ELISA methods may be a potentially beneficial clinical tool for diagnosing RA.

Species difference of esterase expression and hydrolase activity in plasma.

Differences in esterase expression among human, rhesus monkey, cynomolgus monkey, dog, minipig, rabbit, rat, and mouse plasma were identified using native polyacrylamide gel electrophoresis. Paraoxonase (PON) and butyrylcholinesterase (BChE) were ubiquitous in all species, but were highly expressed in primates and dogs, whereas carboxylesterase (CES) was only abundant in rabbits, mice, and rats. Several unknown esterases were observed in minipig and mouse plasma. These differences in plasma esterases and their expression levels result in species differences with respect to hydrolase activity. These differences were characterized using several different substrates. In contrast to the high hydrolase activity found for p-nitrophenylacetate (PNPA), a substrate of several hydrolase enzymes, irinotecan, a carbamate compound, was resistant to all plasma esterases. Oseltamivir, temocapril, and propranolol (PL) derivatives were rapidly hydrolyzed in mouse and rat plasma by their highly active CES enzyme, but rabbit plasma CES hydrolyzed only the PL derivatives. Interestingly, PL derivatives were highly hydrolyzed by monkey plasma BChE, whereas BChE from human, dog, and minipig plasma showed negligible activity. In conclusion, the esterase expression and hydrolyzing pattern of dog plasma were found to be closest to that of human plasma. These differences should be considered when selecting model animals for preclinical studies.

Methylation-dependent PAD2 upregulation in multiple sclerosis peripheral blood.

BACKGROUND: Peptidylarginine deiminase 2 (PAD2) and peptidylarginine deiminase 4 (PAD4) are two members of PAD family which are over-expressed in the multiple sclerosis (MS) brain. Through its enzymatic activity PAD2 converts myelin basic protein (MBP) arginines into citrullines - an event that may favour autoimmunity - while peptidylarginine deiminase 4 (PAD4) is involved in chromatin remodelling. OBJECTIVES: Our aim was to verify whether an altered epigenetic control of PAD2, as already shown in the MS brain, can be observed in peripheral blood mononuclear cells (PBMCs) of patients with MS since some of these cells also synthesize MBP. METHODS: The expression of most suitable reference genes and of PAD2 and PAD4 was assessed by qPCR. Analysis of DNA methylation was performed by bisulfite method. RESULTS: The comparison of PAD2 expression level in PBMCs from patients with MS vs. healthy donors showed that, as well as in the white matter of MS patients, the enzyme is significantly upregulated in affected subjects. Methylation pattern analysis of a CpG island located in the PAD2 promoter showed that over-expression is associated with promoter demethylation. CONCLUSION: Defective regulation of PAD2 in the periphery, without the immunological shelter of the blood-brain barrier, may contribute to the development of the autoimmune responses in MS.

Anti-PAD4 autoantibodies in rheumatoid arthritis: levels in serum over time and impact on PAD4 activity as measured with a small synthetic substrate.

Isoform 4 of the human peptidylarginine deiminase (hPAD4) enzyme may be responsible for the citrullination of antigens in rheumatoid arthritis (RA) and has been shown to be itself the target of disease-specific autoantibodies. Here, we have tested whether the level of serum anti-hPAD4 antibodies in RA patients is stable over a period of 10 years and whether the antibodies influence hPAD4-mediated deimination of the small substrate N-α-benzoyl-L-arginine ethyl ester. RA sera (n = 128) obtained at baseline and after 10 years were assessed for anti-hPAD4 antibodies by a specific immunoassay. For 118 RA patients, serum anti-hPAD4 IgG levels were stable over 10 years. Seven patients who were negative for anti-PAD4 IgG at baseline had become positive after 10 years. Further, total IgG from selected RA patients and controls were purified, and a fraction was depleted for anti-hPAD4 antibodies. Kinetic deimination assays were performed with total IgG and depleted fractions. The k ( cat ) and K ( m ) values of hPAD4-mediated deimination of N-α-benzoyl-L-arginine ethyl ester were not affected by the depletion of the anti-hPAD4 antibodies from the total IgG pool. In conclusion, RA patients remain positive for anti-hPAD4 antibodies over time and some patients who are initially anti-hPAD4 negative become positive later in the disease course. The anti-hPAD4 antibodies did not affect the enzymatic activity of hPAD4 when the small substrate N-α-benzoyl-L-arginine ethyl ester was used. However, this finding may not exclude an effect of these autoantibodies on citrullination of protein substrates in RA.