Identification ofSchistosoma haematobium soluble egg antigens that elicit human granuloma formation in vitro

Schistosoma haematobium soluble egg antigens (SH SEAs) induce intense granulomas in human hosts that often culminate in severe disease. In an attempt to identify the SH SEA fractions that are responsible for pathology, we combined T-cell Western blotting and an in vitro model of granuloma formation. Whole SH SEAs were dotted onto nitrocellulose pieces or were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose paper. Horizontal strips bearing the separated antigens were solubilized in dimethylsulfoxide and precipitated in carbonate/bicarbonate buffer. Antigen-free and antigen-bearing particles were used to stimulate peripheral blood mononuclear cells (PBMCs) obtained fromS. haematobium-infected patients and sex- and agematched healthy controls to form granulomas in vitro. Whole SH SEA-bearing nitrocellulose particles elicited in vitro formation of granulomas by PBMCs from infected donors. The response was similar in sensitivity, specificity, and reproducibility to that evoked by SH SEA-bound polyacrylamide beads. The results obtained in samples from 30 patients and 10 controls tested with SH SEA-separated fractions revealed that SEA bands of 84 000, 63 000, 57 000, 55 000, 40 000, 30 000, and 28 000 Da elicited in vitro granuloma reactions by PBMCs of almost all infected patients. Conversely, separated soluble adult-worm antigens failed to stimulate PBMCs of infected patients to form granulomas. This study is the first to identify the SH SEA fractions that evok in vitro granuloma formation and represents an initial step toward the development of an anti-urinary schistosomiasis pathology vaccine.     

Survivorship and Patient-Reported Outcomes of an Uncemented Vitamin E–Infused Monoblock Acetabular Cup: A Multicenter Prospective Cohort Study

BackgroundAddition of vitamin E to polyethylene is theorized to reduce the potential for oxidative wear in acetabular components. This paper presents a multicenter prospective cohort study that reports on outcomes from use of a Vitamin E–infused highly cross-linked polyethylene acetabular cup.MethodsPatients were recruited across nine medical institutions. Clinical outcome measures recorded were the Harris Hip Score, visual analogue score for pain and satisfaction. Evidence of implant loosening or osteolysis was collected radiologically. Cup survival and reasons for revision in relevant cases were also recorded. Data collection was undertaken preoperatively, at 6-12 weeks, 6 months, 1 year, 2 years, and 5 years. A total of 675 patients were recruited, with 450 cases available at final review. Data regarding cup survival was available to 8 years and 9 months postoperatively.ResultsImprovements in both the Harris Hip Score and visual analogue score for pain and satisfaction were recorded at all time points, with these being maintained through the length of follow-up. In total, 89% of cups were implanted within the Lewinnek safe zone. A lucent line was identified in one case, with no evidence of acetabular osteolysis observed throughout the follow-up period. Cup survival was 98.9% at 8 years and 9 months. No revisions for aseptic loosening were observed.ConclusionsThe use of a vitamin E–infused polyethylene acetabular cup demonstrates reassuring patient-reported outcomes, radiological measures, and cup survival at medium to long-term follow-up.     

Tissue distribution and macromolecular interactions of 14[C-ring] melphalan in the rat

Summary The kinetics of uptake and elimination, covalent binding, and macromolecular interactions of ¹⁴[C-ring] melphalan was studied after a single oral dose (20 mg/kg, 0.1 mCi/kg) in normal rats. Peak radioactivity level in tissues was observed at 2–4 h after administration. Uptake of label in most tissues was rapid, with a t_1/2 of less than 1 h. Elimination was biphasic. Tissues of the gastrointestinal tract showed the most rapid rates of elimination, with t_1/2 of 13, 24, 18, and 19 h for stomach, duodenum, and small and large intestines, respectively. Bone marrow also showed a fast rate of elimination of radioactivity, with a t_1/2 of 30 h. Tissues with the slowest rates of elimination were skin, eye, spleen, pancreas, and lung, with t_1/2 of 333, 241, 149, 122, and 109 h, respectively. Covalent binding studies showed that melphalan, or its metabolites, bound irreversibly to all tissue macromolecular fractions. The percentage of covalently bound radioactivity increased with time in all tissues except kidney and eye, reaching up to 70%–80% of the total radioactivity remaining at 72 h. Elimination of covalently bound radioactivity was slower in the DNA fractions of the tissues of the gastrointestinal tract and heart compared with the elimination rate from lipid, protein, or RNA fractions. Slow elimination rates of ¹⁴[C-ring] melphalan equivalents from the protein fraction were observed in the skin, eye, and brain. Accumulation, rather than elimination, of radioactivity in this fraction was most prominent in the pancreas. In the bone marrow accumulation of radioactivity was observed in the lipid fraction.Abbreviations used in this paper are L-Pam Melphalan, l-phenylalanine mustard 4-bis (2-chloroethyl) amino-1-phenylalanine - l-DOH 4-bis (2-hydroxyethyl) amino-1-phenylalanine - l-MOH 4-2 hydroxyethyl 2 chloroethyl amino-1-phenylalanine - HPLC high-pressure liquid chromatography - TCA trichloroacetic acid - AUC area under the curve - GIT gastrointestinal tract     

Application of LC/MS to determine specific activity variation in a series of sulfonamide tracers from the [35S]methane sulfonate reagent

The specific activities for a series of S‐35 tracers were found to vary from the decay‐corrected specific activity of the labeled reagent. If not known before the stock solution preparation and binding assay, this variation would have resulted in performing the assay at approximately two to three times over the targeted concentration, thereby leading to considerable error in the calculated binding and related conclusions. Copyright © 2012 John Wiley & Sons, Ltd.     

Changes in Choroidal Thickness after Cataract Surgery

Objective: The aim of this study is to compare subfoveal choroidal thickness (SFCT) before and after uneventful phacoemulsification using enhanced depth imaging optical coherence tomography (EDI-OCT).Background: Cataract is a major cause of visual impairment in the elderly. Cataract surgery is the most common ophthalmic surgery and is performed simultaneously with glaucoma or vitreous surgery in many cases. However, according to the results in epidemiology studies, cataract surgery is associated with the onset of age-related macular degeneration (AMD) and cataract surgery increases visual acuity in these patients without an increased risk of progression to exudative AMD. Methods: A prospective study was conducted on 53 eyes of 53 patients who had phacoemulsification. Measurements of SFCT were performed preoperatively, 7 days (D7), 1 month (M1), and 3 months (M3) postoperative using the EDI-OCT technique. Central retinal thickness was also measured at the same time. Results: Twenty-seven male (50.9%) and 26 female (49.1%) with a mean age of 56.43 years ± 10.34 (SD) were analyzed. The mean choroidal thickness was 199.9 ± 60.74 µm. It significantly increased to 228.42 ± 59.77 µm at D7, then decreased to 210.78 ± 59.59 µm at M1, and then decreased to 200.63 ± 61 µm at M3. The mean retinal thickness was 260.79 ± 6.12 µm. It significantly increased to 294.09 ± 7.20 µm at D7 and then decreased to 274.70 ± 6.00 µm at M1 and 258.92 ± 5.89 µm at M3. Conclusion: Mean SFCT increased after cataract surgery. The changes in SFCT return to near the baseline after 3 months.     

Endothelin ETA receptor/lipid peroxides/COX-2/TGF-β1 signalling underlies aggravated nephrotoxicity caused by cyclosporine plus indomethacin in rats

Background and Purpose

Cyclosporine (CSA) and non-steroidal anti-inflammatory drugs (NSAIDs) are co-prescribed for some arthritic conditions. We tested the hypothesis that this combined regimen elicits exaggerated nephrotoxicity in rats via the up-regulation of endothelin (ET) receptor signalling.

Experimental Approach

The effects of a 10 day treatment with CSA (20 mg·kg⁻¹·day⁻¹), indomethacin (5 mg·kg⁻¹·day⁻¹) or their combination on renal biochemical, inflammatory, oxidative and structural profiles were assessed. The roles of ET_A receptor and COX-2 pathways in the interaction were evaluated.

Key Results

Oral treatment with CSA or indomethacin elevated serum urea and creatinine, caused renal tubular atrophy and interstitial fibrosis, increased renal TGF-β1, and reduced immunohistochemical expressions of ET_A receptors and COX-2. CSA, but not indomethacin, increased renal ET-1, the lipid peroxidation product malondialdehyde (MDA) and GSH activity. Compared with individual treatments, simultaneous CSA/indomethacin exposure caused: (i) greater elevations in serum creatinine and renal MDA; (ii) loss of the compensatory increase in GSH; (iii) renal infiltration of inflammatory cells and worsening of fibrotic and necrotic profiles; and (iv) increased renal ET-1 and decreased ET_A receptor and COX-2 expressions. Blockade of ET_A receptors by atrasentan ameliorated the biochemical, structural, inflammatory and oxidative abnormalities caused by the CSA/indomethacin regimen. Furthermore, atrasentan partly reversed the CSA/indomethacin-evoked reductions in the expression of ET_A receptor and COX-2 protein.

Conclusions and Implications

The exaggerated oxidative insult and associated dysregulation of the ET_A receptor/COX-2/TGF-β1 signalling might account for the aggravated nephrotoxicity caused by the CSA/indomethacin regimen. The potential renoprotective effect of ET_A receptor antagonism might be exploited therapeutically.Tables of Links

Application of HPLC mixed‐mode chromatography in determining radiochemical purity of [14C] labeled metformin hydrochloride

Metformin is currently prescribed worldwide to treat type 2 diabetes, and therefore, radiolabeled [¹⁴C] metformin is often prepared for clinical comparisons of new drug candidates. Prior to using the radiolabeled metformin, the purity needs to be determined to ensure the quality of the material. While typical reversed‐phase LC methods are often the first choice for purity analysis, they are not suitable for this determination because the compound is poorly retained under these conditions. Mixed‐mode chromatography has been demonstrated to overcome these retention issues, and therefore, this methodology was utilized for the purity determination of radiolabeled metformin.     

Production and role of extracellular guanosine cyclic 3', 5' monophosphate in sodium uptake in human proximal tubule cells

The present study was designed to determine the capability of human renal proximal tubule (RPT) to generate and export guanosine cyclic 3', 5' monophosphate (cGMP) in response to direct stimulation of soluble guanylyl cyclase by nitric oxide (NO) donors. In addition, we investigated whether cGMP extrusion from human RPT cells is required for inhibition of cellular sodium uptake. RPT cells were cultured from fresh human kidneys (normotensive subjects, n=4, mean age 65+/-4.7 years, 3 men, 1 woman; hypertensive patients, n=6, mean age 64+/-6.1 years, 4 men, 2 women) after unilateral nephrectomy. The fluorescence dye Sodium Green was employed to determine cytoplasmic Na+ concentration. In the presence of the Na+/K+ ATPase inhibitor ouabain, fluorescence was monitored at the appropriate wavelength (excitation 485 nm, emission 535 nm). Nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP, 10(-4) M), increased both intracellular and extracellular cGMP (from 1.26+/-0.21 to 88.7+/-12.6 pmol/mg protein and from 0.58+/-0.10 to 9.24+/-1.9 pmol/mL, respectively, P<0.01) and decreased cellular Na+ uptake by 37.4+/-6.8% (P<0.05) compared with control. The effects of SNAP on cGMP production were similar in normotensive and hypertensive subjects. The increases in intracellular and extracellular cGMP concentration because of SNAP were blocked completely by soluble guanylyl cyclase inhibitor ODQ (1-H-[1,2,4] oxadiazolo [4,2-alpha] quinoxalin-1-one). Probenecid, an organic anion transport inhibitor, augmented the SNAP (10(-6) M)-induced increase in intracellular cGMP accumulation (from 4.9+/-0.9 to 9.8+/-1.5 pmol/mg protein, P<0.05), abrogated the SNAP-induced increase in extracellular cGMP extrusion (from 1.07+/-0.4 to 0.37+/-0.1 pmol/L, P<0.05) and blocked the SNAP-induced reduction in cellular Na+ uptake. Neither intracellular nor extracellular cGMP were influenced by l-arginine, the metabolic precursor of NO, or N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase. After exogenous administration of cGMP (10(-5) M) or its membrane-permeable analogue 8-Br-cGMP (10(-5) M), only 8-Br-cGMP crossed the cell membrane to increase intracellular cGMP (from 1.36+/-0.19 to 289.7+/-29.4 pmol/mg protein, P<0.01). However, both cGMP and 8-Br-cGMP were effective in decreasing cellular Na+ uptake. In conclusion, human RPT cells contain soluble guanylyl cyclase and are able to generate and export cGMP in response to NO. Because human RPT cells do not themselves contain constitutive NO synthase, the NO-generating cGMP must be derived from sources outside the human RPT. The cGMP cellular export system is critical in the regulation of RPT cellular Na+ absorption in humans.