Associations between clinical features and therapy with macrophage subpopulations and T cells in inflammatory lesions in the aorta from patients with Takayasu arteritis.

Takayasu arteritis (TAK) is a large-vessel granulomatous vasculitis; the inflammatory infiltration in arteries comprises macrophages, multi-nucleated giant cells, CD4+ and CD8+ T cells, γδ T cells, natural killer (NK) cells and neutrophils. However, it is unknown which subtype of macrophages predominates. This study aims to evaluate macrophages subpopulations in the aorta in TAK. Immunohistochemistry was performed in the aorta from TAK patients (n = 22), patients with atherosclerotic disease (n = 9) and heart transplant donors (n = 8) using the markers CD68, CD86, CD206, CD3, CD20 and CD56. Active disease was observed in 54·5% of patients and active histological lesions were found in 40·9%. TAK patients presented atherosclerotic lesions in 27·3% of cases. The frequency of macrophages, M1 macrophages, T, B and NK cells was higher in the aorta from TAK and atherosclerotic patients compared to heart transplant donors. In TAK, macrophages and T cells were the most abundant cells in the aorta, and the expression of CD206 was higher than CD86 (P = 0·0007). No associations were found between the expression of cell markers and active disease or with atherosclerotic lesions. In TAK patients, histological disease activity led to higher T cell counts than chronic fibrotic lesions (P = 0.030), whereas prednisone use was associated with lower T cell counts (P = 0·035). In conclusion, M1 macrophages were more frequent in TAK and atherosclerotic patients compared to heart transplant donors, while M2 macrophages dominated M1 macrophages in TAK. T cells were associated with histological disease activity and with prednisone use in TAK.

Assessing non-additive effects in GBLUP model.

Understanding non-additive effects in the expression of quantitative traits is very important in genotype selection, especially in species where the commercial products are clones or hybrids. The use of molecular markers has allowed the study of non-additive genetic effects on a genomic level, in addition to a better understanding of its importance in quantitative traits. Thus, the purpose of this study was to evaluate the behavior of the GBLUP model in different genetic models and relationship matrices and their influence on the estimates of genetic parameters. We used real data of the circumference at breast height in Eucalyptus spp and simulated data from a population of F2. Three commonly reported kinship structures in the literature were adopted. The simulation results showed that the inclusion of epistatic kinship improved prediction estimates of genomic breeding values. However, the non-additive effects were not accurately recovered. The Fisher information matrix for real dataset showed high collinearity in estimates of additive, dominant, and epistatic variance, causing no gain in the prediction of the unobserved data and convergence problems. Estimates presented differences of genetic parameters and correlations considering the different kinship structures. Our results show that the inclusion of non-additive effects can improve the predictive ability or even the prediction of additive effects. However, the high distortions observed in the variance estimates when the Hardy-Weinberg equilibrium assumption is violated due to the presence of selection or inbreeding can converge at zero gains in models that consider epistasis in genomic kinship.

Lining bunker walls with oxygen barrier film reduces nutrient losses in corn silages.

The objective of this study was to evaluate 2 systems for covering corn silage in bunker silos. The first system consisted of a sheet of 45-µm-thick oxygen barrier film (OB, polyethylene + ethylene-vinyl alcohol) placed along the length of the sidewall before filling. After filling, the excess film was pulled over the wall on top of the silage, and a sheet of polyethylene was placed on top. The second system involved using a standard sheet (ST) of 180-µm-thick polyethylene film. Eight commercial bunker silos were divided into 2 parts lengthwise so that one-half of the silo was covered with OB and the other half with a ST system. During the filling, 3 net bags with chopped corn were buried in the central part (halfway between the top and bottom of the silo) of the bunkers (CCOR) in 3 sections 10 m apart. After filling, 18 net bags (9 per covering system) were buried 40 cm below the top surface of the 3 sections. These bags were placed at 3 distances from the bunker walls (0 to 50 cm, 51 to 100 cm, and 101 to 150 cm). During unloading, the bags were removed from the silos to determine the dry matter (DM) losses, fermentation end products, and nutritive value. The Milk2006 spreadsheet was used to estimate milk per tonne of DM. The model included the fixed effect of treatment (7 different locations in the bunker) and the random effect of the silo. Two contrasts were tested to compare silages in the top laterals (shoulders) with that in the CCOR (CCOR vs. OB and CCOR vs. ST). Three contrasts compared the corresponding distances of the silage covered by the 2 systems (OB50 vs. ST50, OB100 vs. ST100 and OB150 vs. ST150). Variables were analyzed with the PROC MIXED procedure of the SAS at the 5% level. The OB method produced well-fermented silages, which were similar to CCOR, whereas the OB system showed less lactic acid and greater pH and mold counts compared with CCOR. The ST method had 116.2 kg of milk/t less than the CCOR, as the OB system and the CCOR were similar (1,258.3 and 1,294.0 kg/t, respectively). Regarding the distances from the walls, the effects were more pronounced from 0 to 101 cm. The OB50 and OB100 silages had better quality and lower mold counts and DM losses than ST50 and ST100. The OB system reduced DM and nutrient losses at the shoulders in farm bunker corn silages compared with no sidewall plastic. The OB film should lap onto the crop for at least 200 cm so that 150 cm are covered outward from the wall.

Crystal structure of oxidized trimethylamine N-oxide reductase from Shewanella massilia at 2.5 A resolution.

The periplasmic trimethylamine N-oxide (TMAO) reductase from the marine bacteria Shewanella massilia is involved in a respiratory chain, having trimethylamine N-oxide as terminal electron acceptor. This molybdoenzyme belongs to the dimethyl sulfoxide (DMSO) reductase family, but has a different substrate specificity than its homologous enzyme. While the DMSO reductases reduce a broad spectra of organic S-oxide and N-oxide compounds, TMAO reductase from Shewanella massilia reduces only TMAO as the natural compound. The crystal structure was solved by molecular replacement with the coordinates of the DMSO reductase from Rhodobacter sphaeroides. The overall fold of the protein structure is essentially the same as the DMSO reductase structures, organized into four domains. The molybdenum coordination sphere is closest to that described in the DMSO reductase of Rhodobacter capsulatus. The structural differences found in the protein environment of the active site could be related to the differences in substrate specificity of these enzymes. In close vicinity of the molybdenum ion a tyrosine residue is missing in the TMAO reductase, leaving a greater space accessible to the solvent. This tyrosine residue has contacts to the oxo groups in the DMSO reductase structures. The arrangement and number of charged residues lining the inner surface of the funnel-like entrance to the active site, is different in the TMAO reductase than in the DMSO reductases from Rhodobacter species. Furthermore a surface loop at the top of the active-site funnel, for which no density was present in the DMSO reductase structures, is well defined in the oxidized form of the TMAO reductase structure, and is located on the border of the funnel-like entrance of the active center.

Molecular analysis of the trimethylamine N-oxide (TMAO) reductase respiratory system from a Shewanella species.

Trimethylamine N-oxide (TMAO) is an abundant compound of tissues of marine fish and invertebrates. During fish spoilage, certain marine bacteria can reduce TMAO to nauseous trimethylamine (TMA). One such bacterium has been isolated and identified as a new Shewanella species, and called Shewanella massilia. The anaerobic growth of S. massilia is greatly increased when TMAO is added, indicating that TMAO reduction involves a respiratory pathway. The TorA enzyme responsible for TMAO reduction is a molybdenum cofactor-containing protein of 90 kDa located in the periplasm. Whereas TorA is induced by both TMAO and dimethylsulfoxide (DMSO), this enzyme has a high substrate specificity and appears to only efficiently reduce TMAO as a natural compound. The structural torA gene encoding the TMAO reductase (TorA) and its flanking regions were amplified using PCR techniques. The torA gene is the third gene of a TMAO-inducible operon (torECAD) encoding the TMAO respiratory components. The torC gene, located upstream from torA encodes a pentahemic c-type cytochrome, likely to be involved in electron transfer to the TorA terminal reductase. TorC was shown to be anchored to the membrane and, like TorA, is induced by TMAO. Except for the TorE protein, which is encoded by the first gene of the torECAD operon, all the tor gene products are homologous to proteins found in the TMAO/DMSO reductase systems from Escherichia coli and Rhodobacter species. In addition, the genetic organization of these systems is similar. Although these bacteria are found in different ecological niches, their respiratory systems appear to be phylogenetically related, suggesting that they come from a common ancestor.

NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli.

The formation of active membrane-bound nitrate reductase A in Escherichia coli requires the presence of three subunits, NarG, NarH and NarI, as well as a fourth protein, NarJ, that is not part of the active nitrate reductase. In narJ strains, both NarG and NarH subunits are associated in an unstable and inactive NarGH complex. A significant activation of this complex was observed in vitro after adding purified NarJ-6His polypeptide to the cell supernatant of a narJ strain. Once the apo-enzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro. NarJ protein specifically recognizes the catalytic NarG subunit. Fluorescence, electron paramagnetic resonance (EPR) spectroscopy and molybdenum quantification based on inductively coupled plasma emission spectroscopy (ICPES) clearly indicate that, in the absence of NarJ, no molybdenum cofactor is present in the NarGH complex. We propose that NarJ is a specific chaperone that binds to NarG and may thus keep it in an appropriate competent-open conformation for the molybdenum cofactor insertion to occur, resulting in a catalytically active enzyme. Upon insertion of the molybdenum cofactor into the apo-nitrate reductase, NarJ is then dissociated from the activated enzyme.

Impact of dialysis room and reuse strategies on the incidence of hepatitis C virus infection in haemodialysis units.

BACKGROUND: Despite the advent of screening of blood products for anti-hepatitis C virus (HCV), the incidence of HCV infection among haemodialysis (HD) patients is alarmingly high and suggest transmission within the HD unit. To analyse trends in the prevalence and incidence of HCV infection, and evaluate the impact of dialysis room and reuse policies on the incidence of HCV infection, a hospital survey instrument was sent out to medical directors of all 71 HD units in Portugal in August 1994. Information for the years 1991, 1992 and 1993 was requested with respect to HCV infection, defined as positive anti-HCV test. Sixty-two of 71 units (87%) treating 4232 patients in 1993 responded. Overall, data from 5774 patient-years were available for analyses. Observations over multiple intervals were pooled into a single sample, and pooled logistic regression was used to evaluate the relationship between risk factors/strategies and incidence of HCV infection. By 1993, regular anti-HCV testing of patients and staff was practised by 98% and 82% of units, respectively. There was a significant decline in the incidence of HCV infection from 9.9% in 1991 to 5.7% in 1992 and 5.1% in 1993. The incidence was directly related to the prevalence in the dialysis unit. Units with a prevalence of less than 19% had an annual incidence of 2.5% compared to a 35.3% incidence in units with a prevalence greater than 60%. There was wide variation in the incidence of HCV infection in HD units across the country, with geographical location, unit ownership and socioeconomic factors playing a significant role. The incidence was lowest among units that: (i) were located in the northern regions of the country; (ii) were private hospital-based units; and (iii) used dedicated machines or separate rooms for anti-HCV-positive patients. The incidence among units that reprocessed dialysers (6.1%) was not significantly different from that among units that did not reprocess dialysers (7.4%). However, among units that did reprocess dialysers, the incidence of HCV infection was lowest in: (i) units that used separate rooms for reprocessing dialysers from anti-HCV-positive patients or did not reprocess these dialysers; and (ii) units that used Renalin as the sterilant. These results suggest the transmission of HCV infection in HD units and that use of dedicated machines and isolation of anti-HCV-positive patients and their dialysers may reduce the incidence of HCV infection.

Report on management of renale failure in Europe, XXVI, 1995. Rare diseases in renal replacement therapy in the ERA-EDTA Registry.

The proportion of centres returning the ERA-EDTA Registry questionnaires has decreased considerably in recent years. Demographic information, based on the response rate of centres in 1994 (44%), does not allow reasonable projections for management of renal failure in Europe. To encourage the participation of non-responding centres, the timing was right to show the powerful impact of the ERA-EDTA Registry as a supra-national registry, by studying patients in renal replacement therapy (RRT) suffering from rare diseases. Four such diseases, Fabry's disease, nephropathy due to cyclosporin (CsA), nephropathy due to cisplatin and scleroderma, were studied using the records of 440665 patients on file up to 31 December 1993. There were 83 patients with Fabry's disease (0.0188%), 85 patients with CsA nephropathy (0.0193%), 120 patients with cisplatin nephropathy (0.0272%) and 625 patients with scleroderma (0.142%). Scleroderma was introduced as a primary renal disease (PRD) in the ERA-EDTA Registry in 1977. Seven patients were accepted for RRT in that year, whereas the number increased to over 50 new patients per year after 1986. More than half of the patients were aged over 55 years, and 68% of them were women. Survival rate of dialysis patients suffering from scleroderma was 22% at 5 years, compared to 51% in patients with standard primary renal diseases. The main causes of death were cardiovascular complications (41%), cachexia (15%) and infection (10%). Survival of first graft in a small number of 28 patients was 44% at 3 years, compared to 60% in standard PRD. Patient survival after first transplant, however, was higher by 32% at 3 years compared to that of dialysis patients. Cisplatin nephropathy was introduced as a PRD in the ERA-EDTA Registry in 1985, and since then six to 19 new patients have been accepted for RRT each year. The main reason for undergoing cisplatin treatment was ovarian (32%) and testicular cancer (21%), and the mean interval from treatment to RRT was 21.5 months, ranging widely from 0.1 to 131 months. Patient survival on dialysis was 22% at 5 years, compared to 51% in patients with standard PRD. Malignancy and cachexia accounted for over 60% of the total number of deaths. CsA nephropathy was introduced as a PRD in the ERA-EDTA Registry in 1985 and, despite its rarity, is of particular interest as a new iatrogenic entity resulting from CsA administration, mainly in solid organ transplantation. In 1985, two new patients commenced RRT in Europe, and the number increased to 59 in 1991-93. The main reason for undergoing CsA treatment was heart (68%) and liver transplant (22%), and the mean interval from treatment to RRT was 50.2 months, ranging from 5 to 90 months. Patient survival on dialysis was 46% at 4 years, compared to 58% in patients with standard primary nephropathies. Cardiovascular causes (48%) and infection (17%) were the main causes of death. Fabry's disease was introduced as a PRD in the ERA-EDTA Registry in 1985, and since the four to 13 new patients per year have commenced RRT in Europe. It is a sex-linked recessive disorder primarily affecting males (87%), and the mean age at start of RRT was 38 years. Proteinuria, skin lesions and painful paresthesiae were the most common presenting symptoms, and over 70% of the patients were hypertensive and had significant cardiovascular problems at RRT. Patient survival on dialysis was 41% at 5 years, compared to 68% in patients with standard primary nephropathies. Cardiovascular complications (48%) and cachexia (17%) were the main causes of death. Graft survival at 3 years in 33 patients was not inferior to that of patients with standard nephropathies (72% vs 69%), and patient survival after transplantation was comparable to that of patients under 55 years of age with standard PRD. (ABSTRACT TRUNCATED)

[Treatment of anemia in patients with chronic renal insufficiency undergoing hemodialysis with recombinant human erythropoietin: 12 months' experience]. / Correcção da anemia dos doentes insuficientes renais crónicos em hemodiálise com eritropoietina recombinante humana: experiência de 12 meses.

In this study we analyse the effects of the administration of recombinant human erythropoietin (rHuEpo) during 12 months to correct the anaemia in a group of 17 patients (9 men and 8 women; mean age 52.7 +/- 13.7; range 23 to 68 years) with end-stage renal disease (ESRD) on chronic haemodialysis (HD) for a range of 14 to 126 months (mean 43.1 +/- 29.6). In the correction period the rHuEpo was started at 50 U/Kg i.v. 3 times a week, immediately after each HD. This dose was maintained during 4 weeks and then increased in 25 U/Kg steps until haemoglobin (Hb) levels of 12 g/dl or a maximum dose of 100 U/Kg were reached. During the long-term maintenance period the individual rHuEpo dose was adjusted to keep the Hb constant at the target level of 10-12 g/dl. Baseline blood tests were done before the beginning of the treatment and every months afterwards. The levels of Hb increased significantly in week 4 and at the end of the first 3 month only 4 patients had no answer to rHuEpo. These patients had baseline serum ferritin levels below 100 ng/ml and responded well when this defficiency was corrected with oral iron. When levels of 30-35 vol% haematocrit (Hct) were reached the dose of rHuEpo could be reduced (150 to 200 U/Kg/week). The serum ferritin levels decreased 51% from a mean baseline level of 247.8 +/- 196 to 121.1 +/- 154.9 ng/ml with the onset of the maintenance phase (p less than 0.05).