Unique features of recombinant heme oxygenase of Drosophila melanogaster compared with those of other heme oxygenases studied.

We cloned a cDNA for a Drosophila melanogaster homologue of mammalian heme oxygenase (HO) and constructed a bacterial expression system of a truncated, soluble form of D. melanogaster HO (DmDeltaHO). The purified DmDeltaHO degraded hemin to biliverdin, CO and iron in the presence of reducing systems such as NADPH/cytochrome P450 reductase and sodium ascorbate, although the reaction rate was slower than that of mammalian HOs. Some properties of DmHO, however, are quite different from other known HOs. Thus DmDeltaHO bound hemin stoichiometrically to form a hemin-enzyme complex like other HOs, but this complex did not show an absorption spectrum of hexa-coordinated heme protein. The absorption spectrum of the ferric complex was not influenced by changing the pH of the solution. Interestingly, an EPR study revealed that the iron of heme was not involved in binding heme to the enzyme. Hydrogen peroxide failed to convert it into verdoheme. A spectrum of the ferrous-CO form of verdoheme was not detected during the reaction from hemin under oxygen and CO. Degradation of hemin catalyzed by DmDeltaHO yielded three isomers of biliverdin, of which biliverdin IXalpha and two other isomers (IXbeta and IXdelta) accounted for 75% and 25%, respectively. Taken together, we conclude that, although DmHO acts as a real HO in D. melanogaster, its active-site structure is quite different from those of other known HOs.     

Studies on Tobacco Lignin

A study was made of such characteristics as infrared and ultraviolet absorption spectra, alkaline nitrobenzene oxidation, elementary composition and constituent groups of tobacco milled wood lignin (Japanese flue-cured and Matsukawa M. W. L.).

Methoxyl contents of mid-rib and stalk M. W. L. were 12.3~13.8% and 16.6~17.0% respectively. Infrared absorption spectra showed that the relation of the absorption intensities between at 1270 cm^?1 and at 1220 cm^?1 of stalk M. W. L. was 1270 cm^?1 < 1220 cm^?1 but, on the contrary, in the case of mid-rib M. W. L., the relation of the intensities was 1270 cm^?1 = 1220 cm^?1. Alkaline nitrobenzene oxidation indicated that the nuclear structures of tobacco M. W. L. were mainly composed of p-hydroxyphenyI and guaiacyl for mid-rib, and the former two and syringyl for stalk.     

Reference concentrations of cholecalciferol in animals: a basis for establishing non-target exposure

Abstract

Cholecalciferol (vitamin D₃) is widely used as a vertebrate pesticide in New Zealand. However, cholecalciferol also occurs naturally in animals. Therefore, when trying to determine whether a non-target animal has been exposed to cholecalciferol baits, knowledge of the baseline cholecalciferol concentrations in the animal's plasma and tissue is required. We analysed cattle, sheep, pig, deer, dog and cat plasma and liver samples for the vitamin D₃ metabolite 25-hydroxycholecalciferol (25-OHD), a sensitive biomarker for cholecalciferol. Based on these data and a literature search we present 25-OHD reference ranges. We also examined the literature for 25-OHD concentrations in poisoned animals and compared these to the reference ranges. Where plasma and liver samples have 25-OHD concentrations at least four times higher than our reference ranges it is likely that the animal has been exposed to cholecalciferol baits. 25-OHD concentrations 10 times higher than the reference range indicate ingestion of abnormally high amounts of cholecalciferol.     

Association of Increased Frequencies of HLA-DPB1*05∶01 with the Presence of Anti-Ro/SS-A and Anti-La/SS-B Antibodies in Japanese Rheumatoid Arthritis and Systemic Lupus Erythematosus Patients

Introduction

Autoantibodies to ribonucleoprotein are associated with a variety of autoimmune diseases, including rheumatoid arthritis (RA). Many studies on associations between human leukocyte antigen (HLA) alleles and RA have been reported, but few have been validated in RA subpopulations with anti-La/SS-B or anti-Ro/SS-A antibodies. Here, we investigated associations of HLA class II alleles with the presence of anti-Ro/SS-A or anti-La/SS-B antibodies in RA.

Methods

An association study was conducted for HLA-DRB1, DQB1, and DPB1 in Japanese RA and systemic lupus erythematosus (SLE) patients that were positive or negative for anti-Ro/SS-A and/or anti-La/SS-B antibodies.

Results

An increased prevalence of certain class II alleles was associated with the presence of anti-Ro/SS-A antibodies as follows: DRB1*08∶03 (Pc = 3.79×10⁻⁵, odds ratio [OR] 3.06, 95% confidence interval [CI] 1.98–4.73), DQB1*06∶01 (Pc = 0.0106, OR 1.70, 95%CI 1.26–2.31), and DPB1*05∶01 (Pc = 0.0040, OR 1.55, 95%CI 1.23–1.96). On the other hand, DRB1*15∶01 (Pc = 0.0470, OR 3.14, 95%CI 1.63–6.05), DQB1*06∶02 (Pc = 0.0252, OR 3.14, 95%CI 1.63–6.05), and DPB1*05∶01 (Pc = 0.0069, OR 2.27, 95% CI 1.44–3.57) were associated with anti-La/SS-B antibodies. The DPB1*05∶01 allele was associated with anti-Ro/SS-A (Pc = 0.0408, OR 1.69, 95% CI 1.19–2.41) and anti-La/SS-B antibodies (Pc = 2.48×10⁻⁵, OR 3.31, 95%CI 2.02–5.43) in SLE patients.

Conclusion

HLA-DPB1*05∶01 was the only allele associated with the presence of both anti-Ro/SS-A and anti-La/SS-B antibodies in Japanese RA and SLE patients.     

End-of-life care issues in advanced dementia

Appropriate management of advanced dementia requires it to be recognised as a terminal condition that needs palliative care. Interventions during this stage should be carefully chosen to ensure the improvement or maintenance of the quality of life of the person with dementia. Advanced care planning is an important aspect of dementia care. Carers and relatives should be educated and encouraged to actively participate in discussions related to artificial nutrition, cardiopulmonary resuscitation (CPR) and other medical interventions.     

Defect of suppression of inflammasome-independent interleukin-8 secretion from SW982 synovial sarcoma cells by familial Mediterranean fever-derived pyrin mutations

Familial Mediterranean fever (FMF) is a recessive inherited autoinflammatory syndrome. Patients with FMF have symptoms such as recurrent fever and abdominal pain, sometimes accompanied by arthralgia. Biopsy specimens have revealed substantial neutrophil infiltration into synovia. FMF patients have a mutation in the Mediterranean fever gene, encoding pyrin, which is known to regulate the inflammasome, a platform for processing interleukin (IL)-1β. FMF patients heterozygous for E148Q mutation, heterozygous for M694I mutation, or combined heterozygous for E148Q and M694I mutations, which were found to be major mutations in an FMF study group in Japan, suffer from arthritis, the severity of which is likely to be lower than in FMF patients with M694V mutations. Expression plasmids of wild-type (WT) pyrin and mutated pyrin, such as E148Q, M694I, M694V, and E148Q+M694I, were constructed, and SW982 synovial sarcoma cells were transfected with these expression plasmids. IL-8 and IL-6 were spontaneously secreted from the culture supernatant of SW982 cells without any stimulation, whereas IL-1β and TNF-α could not be detected even when stimulated with lipopolysaccharide. Notably, two inflammasome components, ASC and caspase-1, could not be detected in SW982 cells by Western blotting. IL-8 but not IL-6 secretion from SW982 cells was largely suppressed by WT pyrin, but less suppressed by mutated pyrin, which appeared to become weaker in the order of E148Q, M694I, E148Q+M694I, and M694V mutations. As for IL-8 and IL-6, similar results were obtained using stable THP-1 cells expressing the WT pyrin or mutated pyrins, such as M694V or E148Q, when stimulated by LPS. In addition, IL-8 secretion from mononuclear cells of FMF patients was significantly higher than that of healthy volunteers when incubated on a culture plate. Thus, our results suggest that IL-8 secretion from SW982 synovial sarcoma cells suppressed by pyrin independently of inflammasome is affected by pyrin mutations, which may reflect the activity in FMF arthritis.     

Head-to-head comparison of the safety of tocilizumab and tumor necrosis factor inhibitors in rheumatoid arthritis patients (RA) in clinical practice: results from the registry of Japanese RA patients on biologics for long-term safety (REAL) registry

IntroductionThe objective of this study was to directly compare the safety of tocilizumab (TCZ) and TNF inhibitors (TNFIs) in rheumatoid arthritis (RA) patients in clinical practice.MethodsThis prospective cohort study included RA patients starting TCZ [TCZ group, n = 302, 224.68 patient-years (PY)] or TNFIs [TNFI group, n = 304, 231.01 PY] from 2008 to 2011 in the registry of Japanese RA patients on biologics for long-term safety registry. We assessed types and incidence rates (IRs) of serious adverse events (SAEs) and serious infections (SIs) during the first year of treatment. Risks of the biologics for SAEs or SIs were calculated using the Cox regression hazard analysis.ResultsPatients in the TCZ group had longer disease duration (P <0.001), higher disease activity (P = 0.019) and more frequently used concomitant corticosteroids (P <0.001) than those in the TNFI group. The crude IR (/100 PY) of SIs [TCZ 10.68 vs. TNFI 3.03; IR ratio (95% confidence interval [CI]), 3.53 (1.52 to 8.18)], but not SAEs [21.36 vs. 14.72; 1.45 (0.94 to 2.25)], was significantly higher in the TCZ group compared with the TNFI group. However, after adjusting for covariates using the Cox regression hazard analysis, treatment with TCZ was not associated with higher risk for SAEs [hazard ratio (HR) 1.28, 95% CI 0.75 to 2.19] or SIs (HR 2.23, 95% CI 0.93 to 5.37).ConclusionsThe adjusted risks for SAEs and SIs were not significantly different between TCZ and TNFIs, indicating an influence of clinical characteristics of the patients on the safety profile of the biologics in clinical practice.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0583-8) contains supplementary material, which is available to authorized users.     

Pneumococcal polysaccharide vaccination in rheumatoid arthritis patients receiving tacrolimus

IntroductionIn rheumatoid arthritis (RA) patients receiving immunosuppressive treatments, vaccination against Streptococcus pneumoniae is recommended. The objective of the study was to evaluate the effects of tacrolimus (TAC) on immune response following administration of a 23-valent pneumococcal polysaccharide vaccine (PPSV23) in patients with established RA.MethodsPatients with RA (n = 133) were vaccinated with PPSV23. Patients were classified into TAC (n = 29), methotrexate (MTX) (n = 55), control (n = 35), and TAC/MTX (n = 14) treatment groups. We measured the concentrations of pneumococcal serotypes 6B and 23F by using an enzyme-linked immunosorbent assay and determined antibody functionality by using a multiplexed opsonophagocytic killing assay, reported as the opsonization index (OI), before and 4 to 6 weeks after vaccination. A positive antibody response was defined as at least a twofold increase in the IgG concentration or as at least a 10-fold increase in the OI.ResultsIgG concentrations and OIs were significantly increased in all treatment groups after PPSV23 vaccination. The TAC treatment group appears to respond in a manner similar to that of the RA control group in terms of 6B and 23F serotype concentration and function. In contrast, the MTX group had the lowest immune response. Patients who received a combination of TAC and MTX (TAC/MTX) also had a diminished immune response compared with those who received TAC alone.ConclusionsTAC monotherapy does not appear to impair PPSV23 immunogenicity in patients with RA, whereas antibody production and function may be reduced when TAC is used with MTX. Thus, PPSV23 administration during ongoing TAC treatment should be encouraged for infection-prone TAC-treated patients with rheumatic diseases.

Trial registration

University Hospital Medical Information Network Clinical Trials Registry: UMIN000009566. Registered 12 December 2012.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0662-x) contains supplementary material, which is available to authorized users.