Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps.

Histone deimination regulates gene function and contributes to antimicrobial response, allowing the formation of neutrophil extracellular traps (NETs). Deiminated proteins are target of anti-citrullinated peptides antibodies (ACPA) in rheumatoid arthritis (RA). OBJECTIVE: The objective of this paper is to test the hypothesis that RA sera react with deiminated histones contained in NETs. METHODS: Neutrophils from peripheral blood were stimulated with A23187 and acid treated; NETosis was induced by phorbol myristate acetate, and NET proteins were isolated. Sera were tested by immunoblot on acid extracted proteins from neutrophils and from NETs, and by ELISA on deiminated histone H4 or H4-derived peptides. Bands reactive with RA sera were excised from gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) analysis, before and after derivatisation to detect citrullinated peptides. RESULTS: RA sera reacted with a deiminated antigen of 11 KDa from activated neutrophils, recognised also by anti-H4 and antideiminated H4 antibodies. A similar reactivity was observed with NET proteins. The antigen from neutrophils or NETs was identified as citrullinated H4 by MALDI-TOF analysis. By ELISA, RA sera bound in vitro citrullinated H4. Citrullinated H4 14-34 and 31-50 peptides detected antibodies in 67% and 63% of RA sera and in less than 5% of controls; antibody titre was correlated with anti-CCP2. CONCLUSIONS: Citrullinated H4 from activated neutrophils and NETs is a target of antibodies in RA, and synthetic citrullinated H4-derived peptides are a new substrate for ACPA detection. As NETosis can generate antigens for ACPA, these data suggest a novel connection between innate and adaptive immunity in RA

Search for genetic variants in the p66Shc longevity gene by PCR-single strand conformational polymorphism in patients with early-onset cardiovascular disease.

Background: Among the possible candidate genes for atherosclerosis experimental data point towards the longevity gene p66(Shc). The p66(Shc) gene determines an increase of intracellular reactive oxygen species (ROS), affecting the rate of oxidative damage to nucleic acids. Knock-out p66(Shc-/-) mice show reduction of systemic oxidative stress, as well as of plasma LDL oxidation, and reduced atherogenic lesions. Thus, p66(Shc) may play a pivotal role in controlling oxidative stress and vascular dysfunction in vivo. Methods: We searched for sequence variations in the p66(Shc) specific region of the Shc gene and its upstream promoter by PCR-SSCP in a selected group of early onset coronary artery disease ( CAD) subjects (n. 78, mean age 48.5 +/- 6 years) and in 93 long-living control subjects ( mean age 89 +/- 6 years). Results: The analysis revealed two variant bands. Sequencing of these variants showed two SNPs: -354T > C in the regulatory region of p66(Shc) locus and 92C > T in the p66 specific region (CH2). Both these variants have never been described before. The first substitution partially modifies the binding consensus sequence of the SpI transcription factor, and was detected only in two heterozygous carriers (1 CAD subjects and 1 control subject). The 92C > T substitution in the CH2 region consists in an amino acid substitution at codon 31 (proline to leucine, P31L), and was detected in heterozygous status only in one CAD subject. No subjects homozygous for the two newly described SNPs were found. Conclusion: Only two sequence variations in the p66(Shc) gene were observed in a total of 171 subjects, and only in heterozygotes. Our observations, in accordance to other studies, suggest that important variations in the p66(Shc) gene may be extremely rare and probably this gene is not involved in the genetic susceptibility to CAD

severe insulin resistence in disguise: a familial case of reactive hypoglycemia associated with a novel heterozygous INSR mutation

AIM: Hypoglycemia in childhood is very rare and can be caused by genetic mutations or insulin-secreting neoplasms. Postprandial hypoglycemia has previously been associated with insulin receptor (INSR) gene mutations. We aimed to identify the cause of postprandial hypoglycemia in a 10-year-old boy. SUBJECTS: We studied the symptomatic proband and his apparently asymptomatic mother and elder brother. All of them were lean. METHODS: Metabolic screening of the proband included a 5-hour oral glucose tolerance test (OGTT), angio-magnetic resonance imaging, and 18 F-dihydroxyphenylalanine positron emission tomography/computed tomography imaging of the pancreas. INSR gene sequencing and in vitro functional studies of a novel INSR mutation were also undertaken. RESULTS: Fasting hyperinsulinemia was detected during metabolic screening, and 5-hour OGTT showed hypoglycemia at 240' in the proband, his mother, and brother. Pancreatic imaging showed no evidence of neoplasia. Acanthosis nigricans with high fasting insulin levels in the proband suggested severe insulin resistance and prompted INSR gene sequencing, which revealed the novel, heterozygous p.Phe1213Leu mutation in the patient and his family members. In vitro studies showed that this mutation severely impairs insulin receptor function by abolishing tyrosine kinase activity and downstream insulin signaling. CONCLUSIONS: The identification of etiological cause of hypoglycemia in childhood may be challenging. The combination of fasting hyperinsulinemia with acanthosis nigricans in a lean subject with hypoglycemia suggests severe insulin resistance and warrants INSR gene screening

Permanent diabetes mellitus in the first year of life

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Mutations at the same residue (R50) of Kir6.2 (KCNJ11) that cause neonatal diabetes produce different functional effects

Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore-forming subunit of the ATP-sensitive K(+) channel (K(ATP) channel), are a common cause of neonatal diabetes. We identified a novel KCNJ11 mutation, R50Q, that causes permanent neonatal diabetes (PNDM) without neurological problems. We investigated the functional effects this mutation and another at the same residue (R50P) that led to PNDM in association with developmental delay. Wild-type or mutant Kir6.2/SUR1 channels were examined by heterologous expression in Xenopus oocytes. Both mutations increased resting whole-cell currents through homomeric and heterozygous K(ATP) channels by reducing channel inhibition by ATP, an effect that was larger in the presence of Mg(2+). However the magnitude of the reduction in ATP sensitivity (and the increase in the whole-cell current) was substantially larger for the R50P mutation. This is consistent with the more severe phenotype. Single-R50P channel kinetics (in the absence of ATP) did not differ from wild type, indicating that the mutation primarily affects ATP binding and/or transduction. This supports the idea that R50 lies in the ATP-binding site of Kir6.2. The sulfonylurea tolbutamide blocked heterozygous R50Q (89%) and R50P (84%) channels only slightly less than wild-type channels (98%), suggesting that sulfonylurea therapy may be of benefit for patients with either mutation