Pulmonary function in an international sample of HIV-positive, treatment-naïve adults with CD4 counts > 500 cells/µL: a substudy of the INSIGHT Strategic Timing of AntiRetroviral Treatment (START) trial.

OBJECTIVES: The aim of the study was to describe the prevalence and correlates of chronic obstructive pulmonary disease (COPD) in a multicentre international cohort of persons living with HIV (PLWH). METHODS: We performed a cross-sectional analysis of adult PLWH, naïve to HIV treatment, with baseline CD4 cell count > 500 cells/µL enrolled in the Pulmonary Substudy of the Strategic Timing of AntiRetroviral Treatment (START) trial. We collected standardized, quality-controlled spirometry. COPD was defined as forced expiratory volume in 1 s:forced vital capacity (FEV1 :FVC) ratio less than the lower limit of normal. RESULTS: Among 1026 participants from 80 sites and 20 countries, the median age was 36 [interquartile range (IQR) 30, 44] years, 29% were female, and the median time since HIV diagnosis was 1.2 (IQR 0.4, 3.5) years. Baseline median CD4 cell count was 648 (IQR 583, 767) cells/µL, median viral load was 4.2 (IQR 3.5, 4.7) log10 HIV-1 RNA copies/mL, and 10% had a viral load ≤ 400 copies/mL despite lack of HIV treatment. Current/former/never smokers comprised 28%/11%/61% of the cohort, respectively. COPD was present in 6.8% of participants, and varied by age, smoking status and region. Forty-eight per cent of those with COPD reported lifelong nonsmoking. In multivariable regression, age and pack-years of smoking had the strongest associations with FEV1 :FVC ratio (P < 0.0001). There was a significant effect of region on FEV1 :FVC ratio (P = 0.010). CONCLUSIONS: Our data suggest that, among PLWH who were naïve to HIV treatment and had CD4 cell counts > 500 cells/µL, smoking and age were important factors related to COPD. Smoking cessation should remain a high global priority for clinical care and research in PLWH.

HCV eradication does not impact gut dysbiosis or systemic inflammation in cirrhotic patients.

BACKGROUND: Eradication of hepatitis C virus (HCV) is increasing but its residual impact on the pro-inflammatory milieu in cirrhosis, which is associated with gut dysbiosis, is unclear. AIM: To define the impact of sustained virological response (SVR) on gut dysbiosis and systemic inflammation in HCV cirrhosis patients. METHODS: Cirrhotic out-patients with HCV with/without SVR (achieved >1 year prior) and age-matched healthy controls underwent serum and stool collection. Serum was analysed for IL-6, TNF-α and endotoxin while stool microbiota analysis was performed using multitagged pyrosequencing. Microbial comparisons were made using UNIFRAC and cirrhosis dysbiosis ratio (lower score indicates dysbiosis). Comparisons were performed between cirrhotics with/without SVR and controls vs. cirrhotic patients. RESULTS: A total of 105 HCV cirrhotics and 45 age-matched healthy controls were enrolled. Twenty-one patients had achieved SVR using pegylated interferon + ribavrin a median of 15 months prior. No significant differences on demographics, cirrhosis severity, concomitant medications or diabetes were seen between cirrhotics with/without SVR. There was no significant difference in overall microbiota composition (UNIFRAC P = 0.3) overall or within specific microbial families (cirrhosis dysbiosis ratio median 1.3 vs. 1.0, P = 0.45) between groups with/without SVR. This also extended towards IL-6, TNF-α and endotoxin levels. Both cirrhosis groups, however, had significant dysbiosis compared to healthy controls [UNIFRAC P = 0.01, cirrhosis dysbiosis ratio (1.1 vs. 2.9, P < 0.001)] along with higher levels of endotoxin, IL-6 and TNF-α. CONCLUSIONS: Gut dysbiosis and a pro-inflammatory systemic milieu, are found in HCV cirrhosis regardless of SVR. This persistent dysbiosis could contribute towards varying rates of improvement after HCV eradication in cirrhosis.

Determination of silicon in breast and capsular tissue from patients with breast implants performed by inductively coupled plasma emission spectroscopy. Comparison with tissue histology.

A method for analysis of silicon in tissue was developed to determine silicon content in breast parenchymal and periprosthetic capsular tissues of patients with silicone or saline implants and to compare levels in tissues from normal (nonaugmented) breasts. It is of interest to determine whether increased silicon content in tissues can be associated with morbidity in patients who have received silicone implants. This manuscript addresses the issues involved in analysis of breast tissue samples for silicon and compares silicon levels with tissue histologic findings and patient morbidity. One hundred sixty tissue samples were obtained for silicon analysis from 72 patients during augmentation, capsulectomy with or without replacement mammoplasty, mastectomy, or biopsy procedures and were frozen in acid-washed polystyrene tubes at 220 degrees C until analysis. Samples were thawed, sectioned to approximately 0.1 g (dry weight), and digested in nitric acid before analysis by inductively coupled plasma emission spectroscopy, monitoring emission intensity at 251.6 nm. Tissue silicon levels (breast parenchymal and periprosthetic capsular tissue) in patients with silicone gel implants were much higher (mean, 9,287 micrograms/g, n = 106) than in patients with saline implants (mean, 196 micrograms/g, n = 37) or nonaugmented breasts (mean, 64 micrograms/g, n = 17). Histologic examination was performed on 54 tissue samples stained with hematoxylin-eosin. Tissue samples were rated as to degree of inflammation and calcification, and amount of giant cells, foamy histiocytes, and vacuoles containing a colorless refractory material. Vacuolization and foamy histiocyte ratings correlated significantly with tissue silicon concentration. No correlations were found between tissue silicon concentration and inflammation, calcification, or giant cell rating. Implant age (number of years an implant was in place before sampling) correlated with capsular tissue silicon concentration in patients with intact implants but not in those with ruptured implants. No difference in tissue silicon concentration was found between patients with or without signs or symptoms of morbidity. Using 0.1 g of tissue, the method was linear to 1,000 micrograms/g, and sensitivity was 3.7 micrograms/g. Precision between runs (mean, 5.1 micrograms/g; coefficient of variance, 13.7%; n = 13) was calculated from multiple analyses of a bovine liver standard (National Bureau of Standards, reference material 1577a). Significant biologic variability (21.4% to 52.5%) was seen in tissues with high silicon levels. Paraffin-embedded, formalin-fixed tissues are not amenable to silicon analysis by this method, because of leaching of silicone from the tissues during preparation. Thus only fresh frozen tissue samples were used.

Oxalate, silicon and vanadium in acquired cystic kidney disease.

We have investigated the importance of several clinical and laboratory parameters on the development of acquired cystic kidney disease (ACKD) as detected by ultrasonography in 19 patients who had received dialysis therapy for at least three years. We were particularly interested on the possible effect of the serum levels of oxalate and silicon, which can produce tubular obstruction, and that of vanadium, which can affect cell proliferation. The severity of ACKD increased with the duration of dialysis and was greater in men than in women. Positive correlations were observed between the grades of ACKD and the levels of hemoglobin, hematocrit, and parathyroid hormone, while there was a negative correlation between ACKD and serum ferritin levels. The serum levels of oxalate, silicon, and vanadium, pre- and postdialysis, were markedly and significantly higher than those in normal controls, but there was no significant correlation between these levels and the duration of dialysis therapy or severity of ACKD. The pre- and postdialysis levels of vanadium were not significantly different, while the levels of oxalate and silicon were significantly lower in the postdialysis samples. No significant correlations were detected between ACKD and age of the patients, blood pressure, protein catabolic rate, efficiency of dialysis index, or the serum levels of iron, sodium, potassium, calcium, phosphorus, aluminum, and beta 2-microglobulin.

Inorganic, organic, and total mercury in blood and urine: cold vapor analysis with automated flow injection sample delivery.

A cold vapor atomic absorption technique for blood or urine mercury analysis that uses persulfate oxidation to prepare samples for total mercury analysis and acid permanganate oxidation to prepare samples for inorganic mercury analysis is described. The linearity of the procedures ranged from 0.5 to 25 micrograms/L. Precision ranged from 20% at 1 microgram/L to 7% at 20 micrograms/L. Documentation of accuracy is based on analysis of samples prepared by an international proficiency survey program. The development of a two-step digestion procedure followed by automated flow-injection mercury analysis was a necessary precursor to the assessment of inorganic and alkylmercury exposure in a large unexposed human population. Application of this technique to 902 blood and 902 urine samples collected from a normal human population who had no extraordinary mercury exposure generated mean plus two standard deviation skewed confidence-limit ranges of results as follows: blood total mercury, 0-8.4 micrograms/L; blood inorganic mercury, 0-1.7 micrograms/L; blood organic mercury, 0-7.5 micrograms/L; urine total mercury, 0-9.9 micrograms/L; urine inorganic mercury, 0-8.6 micrograms/L; and urine organic mercury, 0-1.8 micrograms/L.

Arsenic analysis II: rapid separation and quantification of inorganic arsenic plus metabolites and arsenobetaine from urine.

We describe the rapid separation of inorganic arsenic plus metabolites from arsenobetaine or seafood arsenic in urine. Traditional, high-pressure liquid chromatography is replaced by disposable silica-based cation-exchange cartridges for this separation. Both fractions are quickly separated and collected for analysis by atomic absorption spectrophotometry. Analytical recovery of both fractions is > or = 95%, with an overall precision (CV) ranging from 1.6% to 6.4%. Using this method, we correctly identified the sources of arsenic exposure, whether of inorganic or seafood origin, in 11 urine specimens supplied by the Centre de Toxicologie du Quebec.

Blood-collection device for trace and ultra-trace metal specimens evaluated.

We evaluated the evacuated phlebotomy tube designed specifically for trace metal analysis by Sherwood Medical Co. Pools of human serum containing known concentrations of aluminum, arsenic, calcium, cadmium, copper, chromium, iron, lead, magnesium, manganese, mercury, selenium, and zinc were exposed to the tube and rubber stopper for defined periods ranging from 5 min to 24 h. Analysis for each element was performed in a randomized fashion under rigidly controlled conditions by use of standard electrothermal atomization atomic absorption spectroscopy, inductively coupled plasma atomic emission spectroscopy, and cold vapor atomic absorption spectrometry. In addition, for comparative purposes, we collected blood samples from normal volunteers by use of ultra-clean polystyrene phlebotomy syringes as well as standard evacuated phlebotomy tubes. We conclude that, except for lead, there was no significant contribution of any trace element studied from the evaluated tube and stopper to the serum. Because whole blood is the usual specimen for lead testing, the observation of a trace amount of lead in this tube designed for serum collection is trivial.

Circularization and cleavage of guinea pig cytomegalovirus genomes.

The mechanisms by which herpesvirus genome ends are fused to form circles after infection and are re-formed by cleavage from concatemeric DNA are unknown. We used the simple structure of guinea pig cytomegalovirus genomes, which have either one repeated DNA sequence at each end or one repeat at one end and no repeat at the other, to study these mechanisms. In circular DNA, two restriction fragments contained fused terminal sequences and had sizes consistent with the presence of single or double terminal repeats. This result implies a simple ligation of genomic ends and shows that circularization does not occur by annealing of single-stranded terminal repeats formed by exonuclease digestion. Cleavage to form the two genome types occurred at two sites, and homologies between these sites identified two potential cis elements that may be necessary for cleavage. One element coincided with the A-rich region of a pac2 sequence and had 9 of 11 bases identical between the two sites. The second element had six bases identical at both sites, in each case 7 bp from the termini. To confirm the presence of cis cleavage elements, a recombinant virus in which foreign sequences displaced the 6- and 11-bp elements 1 kb from the cleavage point was constructed. Cleavage at the disrupted site did not occur. In a second recombinant virus, restoration of 64 bases containing the 6- and 11-bp elements to the disrupted cleavage site restored cleavage. Therefore, cis cleavage elements exist within this 64-base region, and sequence conservation suggests that they are the 6- and 11-bp elements.

Total arsenic in urine: palladium-persulfate vs nickel as a matrix modifier for graphite furnace atomic absorption spectrophotometry.

We evaluated the effectiveness of nickel and palladium with or without added potassium persulfate as matrix modifiers for the determination of total arsenic in urine. Complete recovery of pure aqueous solutions of As(III), As(V), cacodylic acid (DMA), monomethylarsinic acid (MMA), and o-arsanilic acid was attained by using both nickel and palladium modifiers. Combined arsenobetaine and arsenocholine (so-called fish arsenic), extracted from a certified control material of dogfish muscle (DORM-1), were completely recovered with Pd-S2O8 matrix modification, but not with nickel. Excellent agreement with target values for arsenic in urines from the Centre de Toxicologie du Quebec, supplied by the Interlaboratory Comparison Program, was attained irrespective of the arsenic source when we used Pd-S2O8 as the matrix modifier.

Using 16O35Cl to correct for chloride interference improves accuracy of urine arsenic determinations by inductively coupled plasma mass spectrometry.

We have observed inaccurate urine arsenic values with the method of isobaric fractionation, which was designed to correct for the 40Ar35Cl interference with 75As quantitation by inductively coupled plasma mass spectrometry. Isobaric fractionation, which is based on ion intensities at m/z 77 and 82, consistently underestimates the 40Ar35Cl interference and overestimates urine arsenic. We present an improved method for identifying the argon-chloride interference. We observed that signal intensities for the species 16O35Cl and 40Ar35Cl are proportional (I75 = 0.0295 x I51 - 14.7, r2 = 0.998; where Ix is the normalized ion intensity at m/z X) in water and urine, over a broad range of chloride concentrations (0-800 mmol/L). The proportionality constant is remarkably stable within a run (mean and SD, 0.0295 +/- 0.0023, based on 10 replicates of five chloride calibrators, 0, 100, 200, 400, and 800 mmol/L). Increased sensitivity (50-fold) for detecting the 40Ar35Cl interference provides improved accuracy for urine arsenic quantitation as demonstrated by a split-sample comparison with graphite-furnace atomic absorption spectrophotometry.

Determination of serum nickel by graphite furnace atomic absorption spectrometry with Zeeman-effect background correction: values in a normal population and a population undergoing dialysis.

An atomic absorption spectrometric method with Zeeman-effect background correction for the determination of nickel, which requires only serum dilution with an aqueous surfactant, is described. The average nickel concentration in sera collected from 38 healthy adult volunteers was 0.14 +/- 0.09 micrograms l-1 of Ni, which is approximately four times lower than normals reported previously (0.65 +/- 0.35 or 0.46 +/- 0.26 microgram l-1 of Ni). The procedure yielded accurate results for the analysis of three different reference serum pools. A comparison of the average nickel concentrations from a patient population undergoing regular haemodialysis with our normal population showed that the average concentration of serum nickel in the dialysis patient group (n = 27 patients) was 46 times higher than normal (6.38 +/- 3.36 micrograms l-1 of Ni; n = 40 specimens).

Zinc increases the activity of vitamin D-dependent promoters in osteoblasts.

Zinc modulates the structure and binding of the DNA binding domain of the 1alpha,25-dihydroxyvitamin D(3) receptor to specific vitamin D response element DNA (Nature Biotechnology 16, 262-266, 1998). To determine whether zinc alters 1alpha,25-dihydroxyvitamin D(3)-regulated genes in cells, we permanently transfected rat osteoblasts with two vitamin D-dependent promoter-reporter systems and examined their responses to 1alpha,25-dihydroxyvitamin D(3) in the presence of increasing amounts of extracellular zinc. When extracellular zinc concentrations were increased in the presence of 1alpha,25-dihydroxyvitamin D(3), there was an increase in the activity of 1alpha,25-dihydroxyvitamin D(3)-dependent promoters with increasing concentrations of zinc. The effect was specific for zinc since metals such as copper failed to increase the activity of 1alpha,25-dihydroxyvitamin D(3)-dependent promoters. The concentration of the vitamin D receptor within the cell and the affinity of 1alpha,25-dihydroxyvitamin D(3) for its receptor remained unchanged with added zinc. Our results show that zinc increases the activity of 1alpha,25-dihydroxyvitamin D(3)-dependent promoters in osteoblasts.

The ends on herpesvirus DNA replicative concatemers contain pac2 cis cleavage/packaging elements and their formation is controlled by terminal cis sequences.

Herpesviruses have large double-stranded linear DNA genomes that are formed by site-specific cleavage from complex concatemeric intermediates. In this process, only one of the two genomic ends are formed on the concatemer. Although the mechanism underlying this asymmetry is not known, one explanation is that single genomes are cleaved off of concatemer ends in a preferred direction. This implies that cis elements control the direction of packaging. Two highly conserved cis elements named pac1 and pac2 lie near opposite ends of herpesvirus genomes and are important for cleavage and packaging. By comparison of published reports and by analysis of two additional herpesviruses, we found that pac2 elements lie near the ends formed on replicative concatemers of four herpesviruses: herpes simplex virus type 1, equine herpesvirus 1, guinea pig cytomegalovirus, and murine cytomegalovirus. Formation of pac2 ends on concatemers depended on terminal cis sequences, since ectopic cleavage sites engineered into the murine cytomegalovirus genome mediated formation of pac2 ends on concatemers regardless of the orientation of their insertion. These findings are consistent with a model in which pac2 elements at concatemer ends impart a directionality to concatemer packaging by binding proteins that initiate insertion of concatemer ends into empty capsids.

Sequences within the herpesvirus-conserved pac1 and pac2 motifs are required for cleavage and packaging of the murine cytomegalovirus genome.

The DNA sequence motifs pac1 [an A-rich region flanked by poly(C) runs] and pac2 (CGCGGCG near an A-rich region) are conserved near herpesvirus genomic termini and are believed to mediate cleavage of genomes from replicative concatemers. To determine their importance in the cleavage process, we constructed a number of recombinant murine cytomegaloviruses with a second cleavage site inserted at an ectopic location within the viral genome. Cleavage at a wild-type ectopic site occurred as frequently as at the natural cleavage site, whereas mutation of this ectopic site revealed that some of the conserved motifs of pac1 and pac2 were essential for cleavage whereas others were not. Within pac1, the left poly(C) region was very important for cleavage and packaging but the A-rich region was not. Within pac2, the A-rich region and adjacent sequences were essential for cleavage and packaging and the CGCGGCG region contributed to, but was not strictly essential for, efficient cleavage and packaging. A second A-rich region was not important at all. Furthermore, mutations that prevented cleavage also blocked duplication and deletion of the murine cytomegalovirus 30-bp terminal repeat at the ectopic site, suggesting that repeat duplication and deletion are consequences of cleavage. Given that the processes of genome cleavage and packaging appear to be highly conserved among herpesviruses, these findings should be relevant to other members of this family.

Transfusional iron overload in patients undergoing dialysis: treatment with erythropoietin and phlebotomy.

Five patients undergoing long-term hemodialysis with transfusional iron overload received treatment for 18 weeks with a regimen of recombinant human erythropoietin (150 U/kg) and regular phlebotomy to maintain the hematocrit value at 25% and reduce the total body iron burden. In the 149 phlebotomy sessions performed in these patients, a mean of 228 +/- 8 ml (mean +/- SEM) of whole blood was removed; it had a hematocrit value of 27.7% +/- 0.2%. The iron content of the erythrocytes removed (erythrocyte iron concentration, 787 +/- 11 micrograms/ml in 133 samples) accounted for more than 99% of the total iron removal by phlebotomy. Serum iron (serum iron concentration, 1.57 +/- 0.09 micrograms/ml in 65 samples) accounted for an insignificant fraction of the total iron removed. The iron removed at each phlebotomy session averaged 49.1 +/- 2.0 mg, similar to the amount of iron removed with deferoxamine administration in patients undergoing dialysis who had iron overload, but without the potential for adverse side effects reported with long-term deferoxamine therapy. Total iron removal during the 18 weeks of this study ranged from 732 to 2797 mg. Mean serum ferritin level decreased from 3189 +/- 1076 micrograms/L to 1676 +/- 342 micrograms/L (p less than 0.02, Wilcoxon signed rank test). When compared with a group of five patients without transfusional iron overload who received recombinant human erythropoietin and did not undergo therapeutic phlebotomy, the patients with iron overload had much greater iron losses and a larger decrease in serum ferritin levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Routine measurement of calcium, magnesium, copper, zinc, and iron in urine and serum by inductively coupled plasma emission spectroscopy.

We describe an inductively coupled plasma atomic emission spectrometer that has been adapted to perform routine, simultaneous, direct analyses of calcium, magnesium, copper, zinc, and iron in serum or urine without sample digestion or pretreatment. The system, constructed with inexpensive, readily available components, can analyze 1-mL or smaller samples. Results correlate nearly perfectly with those derived by standard atomic absorption techniques (r = 0.98 to 0.997). Using certified serum and urine samples from various sources, we demonstrate that the instrument yields accurate results with a precision better than certified values. The instrument is sensitive to one order of magnitude less than the lower limit of the normal range in serum or urine for all elements tested, and responds linearly to concentrations two orders of magnitude higher than the upper limit of the normal range. With the system described here, these five elements can be assayed with the same or less technical effort than needed for a single element by atomic absorption.