Air travel and venous thrombosis: how much help might aspirin be?

There has been considerable attention focused recently on the risk of deep venous thrombosis (DVT) associated with air travel. Despite the lack of evidence among air travelers, a single dose of aspirin has been widely recommended as a means of preventing such thrombosis. We have calculated the potential benefit of aspirin by applying the data for aspirin in preventing DVT in hip fracture patients to the estimated rates of travel-related DVT. If the rate of travel-related DVT is 20 per 100,000 travelers, then we will have to treat 17,000 people with aspirin to prevent 1 additional DVT.     

A single nucleotide polymorphism in the<Emphasis Type="Italic"> Emp3</Emphasis> gene defines the H4 minor histocompatibility antigen

Minor histocompatibility antigens (minor H antigen) elicit strong T-cell-mediated responses during both graft rejection and graft versus leukemia (GvL) among MHC-matched individuals (where MHC is major histocompatibility complex). Employing expression-cloning methodology, we have identified a cDNA clone, MI-35, encoding the immunodominant H4^b minor H antigen within the classical mouse H4 complex. The minimal antigenic epitope derived from H4^b presented on K^b class I MHC is SGIVYIHL (SYL8) and the polymorphism is due to CT nucleotide modification in p3 resulting in the change of threonine (ACT) to isoleucine (ATT). The results presented here demonstrate that amino acid variation in the allelic epitopes results in the low abundance of H4^a peptide. The differential peptide copy number resulted in an immunodominant cytotoxic T cells (CTL) response directed against H4^b while the anti-B6 response directed against H4^a was easily dominated. These results provide a molecular mechanism for the H4 minor H antigen and suggest a novel mechanism by which alloantigenic disparity caused by conservative amino acid changes can be augmented by posttranslational antigen processing events.     

Albumin-deficient mouse models for studying metabolism of human albumin and pharmacokinetics of albumin-based drugs

Serum albumin is the major determinant of blood colloidal osmotic pressure acting as a depot and distributor of compounds including drugs. In humans, serum albumin exhibits an unusually long half-life mainly due to protection from catabolism by neonatal Fc receptor (FcRn)-mediated recycling. These properties make albumin an attractive courier of therapeutically-active compounds. However, pharmaceutical research and development of albumin-based therapeutics has been hampered by the lack of appropriate preclinical animal models. To overcome this, we developed and describe the first mouse with a genetic deficiency in albumin and its incorporation into an existing humanized FcRn mouse model, B6.Cg-Fcgrt^tm1Dcr Tg(FCGRT)32Dcr/DcrJ (Tg32). Albumin-deficient strains (Alb^-/-) were created by TALEN-mediated disruption of the albumin (Alb) gene directly in fertilized oocytes derived from Tg32 mice and its non-transgenic background control, C57BL/6J (B6). The resulting Alb^-/- strains are analbuminemic but healthy. Intravenous administration of human albumin to Tg32-Alb^-/- mFcRn^-/- hFcRn^Tg/Tg) mice results in a remarkably extended human albumin serum half-life of ∼24 days, comparable to that found in humans, and in contrast to half-lives of 2.6–5.8 d observed in B6, B6-Alb^-/- and Tg32 strains. This striking increase can be explained by the absence of competing endogenous mouse albumin and the presence of an active human FcRn. These novel albumin-deficient models provide unique tools for investigating the biology and pathobiology of serum albumin and are a more appropriate rodent surrogates for evaluating human serum albumin pharmacokinetics and albumin-based compounds.     

Quantitative analysis of anandamide and related acylethanolamides in human seminal plasma by ultra performance liquid chromatography tandem mass spectrometry

The endocannabinoids anandamide, palmitoylethanolamide and oleoylethanolamide have been detected in human seminal plasma and are bioactive lipids implicated in regulation of sperm motility, capacitation and acrosome reaction. Several methods exist for endocannabinoid quantification but none have been validated for measurement in human seminal plasma. We describe sensitive, robust, reproducible solid phase and isotope-dilution UHPLC-ESI-MS/MS methods for the extraction and quantification of anandamide, palmitoylethanolamide and oleoylethanolamide in human seminal plasma. Precision and accuracy were evaluated using pooled seminal plasma over a 4 day period. For all analytes, the inter- and intraday precision (CV%) was between 6.6-17.7% and 6.3-12.5%, respectively. Analyses were linear over the range 0.237-19nM for anandamide and oleoylethanolamide and 0.9-76nM for PEA. Limits of detection (signal-to-noise >3) were 50, 100 and 100fmol/mL and limits of quantification (signal-to-noise >10) were 100, 200 and 200fmol/mL, respectively for anandamide, palmitoylethanolamide and oleoylethanolamide. Anandamide and oleoylethanolamide were stable at -80°C for up to 4 weeks, but palmitoylethanolamide declined significantly. We assessed seminal plasma from 40 human donors with normozoospermia and found mean (inter-quartile range) concentrations of 0.21nM (0.09-0.27), 1.785nM (0.48-2.32) and 15.54nM (7.05-16.31) for anandamide, oleoylethanolamide and palmitoylethanolamide, respectively. Consequently, this UHPLC-ESI-MS/MS method represents a rapid, reliable and reproducible technique for the analysis of these endocannabinoids in fresh seminal plasma.     

The ratio of germanium to silicon in plant phytoliths: quantification of biological discrimination under controlled experimental conditions

Slight differences in the chemical behavior of germanium (Ge) and silicon (Si) during soil weathering enable Ge/Si ratios to be used as a tracer of Si pathways. Mineral weathering and biogenic silicon cycling are the primary modifiers of Ge/Si ratios, but knowledge of the biogenic cycling component is based on relatively few studies. We conducted two sets of greenhouse experiments in order to better quantify the range and variability in Ge discrimination by plants. Graminoid species commonly found in North American grassland systems, Agropyron smithii, Schizachyrium scoparium, and Andropogon gerardii were grown under controlled hydroponic environmental conditions. Silicon leaf contents were positively correlated with solution Si and ambient temperature but not with nutrient solution pH, electrical conductivity, or species. The Ge/Si ratio incorporated into phytoliths shows a distribution coefficient [(Ge/Si)_phytolith/(Ge/Si)_solution] of about 0.2 and is remarkably invariant between species, photosynthetic pathway, and solution temperature. Ge seems to be discriminated against during the uptake and translocation of Si to the opal deposition sites by about a factor of five. In the second experiment, a wider range of graminoid species (Agropyron smithii, Bouteloua gracilis, Buchloe dactyloides, Oryzopsis hymenoides, Schizachyrium scoparium and Andropogon gerardii) were grown in two different soil mediums. Plant phytoliths showed a distribution factor of about 0.4 for field grown grasses, and 0.6 for potting soil grown grasses with no clear trends among the species. Evidence of the direction and degree of biological Ge discrimination during plant uptake provides a geochemical finger print for plants and improves the utility of Ge/Si ratios in studies of terrestrial weathering and links between Si cycles in terrestrial and marine systems.     

Neonatal FcR expression in bone marrow-derived cells functions to protect serum IgG from catabolism

The neonatal FcR (FcRn) is a receptor that protects IgG from catabolism and is important in maintaining high serum Ab levels. A major site of expression of FcRn is vascular endothelial cells where FcRn functions to extend the serum persistence of IgG by recycling internalized IgG back to the surface. Because FcRn is expressed in other tissues, it is unclear whether endothelial cells are the only site of IgG protection. In this study, we used FcRn-deficient mice and specific antiserum to determine the tissue distribution of FcRn in the adult mouse. In addition to its expression in the vascular endothelium of several organs, we found FcRn to be highly expressed in bone marrow-derived cells and professional APCs in different tissues. Experiments using bone marrow chimeras showed that FcRn expression in these cells acted to significantly extend the half-life of serum IgG indicating that in addition to the vascular endothelium, bone marrow-derived phagocytic cells are a major site of IgG homeostasis.     

FcRn: the neonatal Fc receptor comes of age

The neonatal Fc receptor for IgG (FcRn) has been well characterized in the transfer of passive humoral immunity from a mother to her fetus. In addition, throughout life, FcRn protects IgG from degradation, thereby explaining the long half-life of this class of antibody in the serum. In recent years, it has become clear that FcRn is expressed in various sites in adults, where its potential function is now beginning to emerge. In addition, recent studies have examined the interaction between FcRn and the Fc portion of IgG with the aim of either improving the serum half-life of therapeutic monoclonal antibodies or reducing the half-life of pathogenic antibodies. This Review summarizes these two areas of FcRn biology.     

Systematic review and meta-analysis of randomised trials and cohort studies of mycophenolate mofetil in lupus nephritis

Mycophenolate mofetil (MMF) is an immunosuppressant drug being used for induction and maintenance of remission of lupus nephritis in systemic lupus erythematosus. Evidence about its use was sought from full publications and abstracts of randomised trials and cohort studies by using a variety of search strategies. Efficacy and adverse event outcomes were sought. Five randomised trials enrolled patients with World Health Organization (WHO) class III, IV, or V (mostly IV) lupus nephritis, predominantly comparing MMF (1 to 3 g daily) with cyclophosphamide and steroid. Complete response and complete or partial response was significantly more frequent with MMF than with cyclophosphamide, with numbers needed to treat of 8 (95% confidence interval 4.3 to 60) to induce one additional complete or partial response, with wide confidence intervals. Death was reported less frequently with MMF (0.7%, 1 death in 152 patients) than with cyclophosphamide (7.8%, 12 deaths in 154 patients), with a number needed to treat to prevent (NNTp) one death of 14 (8 to 48). Hospital admission was also lower with MMF (1.7% versus 15%; NNTp 7.4 [4.8 to 16]). Serious infections, leucopaenia, amenorrhoea, and hair loss were all significantly less frequent with MMF than with cyclophosphamide, but diarrhoea was significantly more common with MMF. Ten of 18 cohort studies enrolled only patients with lupus nephritis (author-defined or WHO class III to V). Seven of these 10 reported that complete or partial response with MMF (mostly 1 or 2 g daily) with steroid occurred in 121/151 (80%) and that treatment failure or no response occurred in 30/151 (20%). Adverse events were generally similar in cohort studies with and without only patients with lupus nephritis. In all 18 cohorts, gastrointestinal adverse events (diarrhoea, nausea, vomiting) occurred in 30%, infection in 23%, and serious infection in 4.3%. Adverse event discontinuations occurred in 14% and lack of efficacy occurred in 10%. There was a single death with MMF, a mortality rate over the course of 1 year of approximately 0.2%. The results form a basis on which to plan future studies and provide a guide for the use of MMF in lupus nephritis until results of larger studies are available. At least one such study is under way.     

Direct regulatory role of NKT cells in allogeneic graft survival is dependent on the quantitative strength of antigenicity

The role of NKT cells during immune responses is diverse, ranging from antiviral and antitumor activity to the regulation of autoimmune diseases; however, the regulatory function of CD1d-dependent NKT cells in rejection responses against allogeneic graft is uncertain. In this study, we demonstrated the direct regulatory effects of CD1d-dependent NKT cells using an allogeneic skin transplantation model. H-Y-mismatched skin graft survival was shortened in CD1d-/- recipients compared with wild-type recipients. Adoptive transfer of syngeneic NKT cells via splenocytes or hepatic mononuclear cells into CD1d-/- recipients restored graft survival times to those of wild-type recipients. alpha-Galactosylceramide, a specific activator of NKT cells, further prolonged graft survival. Although CD1d-dependent NKT cells did not extend skin graft survival in either major or complete minor histocompatibility-mismatched models, these cells affected graft survival in minor Ag mismatch models according to the magnitude of the antigenic difference. The afferent arm of NKT cell activation during transplantation required CD1d molecules expressed on host APCs and the migration of CD1d-dependent NKT cells into grafts. Moreover, the regulatory effects of CD1d-dependent NKT cells against alloantigen were primarily IL-10 dependent. Taken together, we concluded that CD1d-dependent NKT cells may directly affect the outcome of allogeneic skin graft through an IL-10-dependent regulatory mechanism.     

Osteoclast differentiation is impaired in the absence of inhibitor of kappa B kinase alpha

Signaling through the receptor activator of nuclear factor kappa B (RANK) is required for both osteoclast differentiation and mammary gland development, yet the extent to which RANK utilizes similar signaling pathways in these tissues remains unclear. Mice expressing a kinase-inactive form of the inhibitor of kappa B kinase alpha (IKK alpha) have mammary gland defects similar to those of RANK-null mice yet have apparently normal osteoclast function. Because mice that completely lack IKK alpha have severe skin and skeletal defects that are not associated with IKK alpha-kinase activity, we wished to directly examine osteoclastogenesis in IKK alpha(-/-) mice. We found that unlike RANK-null mice, which completely lack osteoclasts, IKK alpha(-/-) mice did possess normal numbers of TRAP(+) osteoclasts. However, only 32% of these cells were multinucleated compared with 57% in wild-type littermates. A more profound defect in osteoclastogenesis was observed in vitro using IKK alpha(-/-) hematopoietic cells treated with colony-stimulating factor 1 and RANK ligand (RANKL), as the cells failed to form large, multinucleated osteoclasts. Additionally, overall RANKL-induced global gene expression was significantly blunted in IKK alpha(-/-) cells, including osteoclast-specific genes such as TRAP, MMP-9, and c-Src. IKK alpha was not required for RANKL-mediated I kappa B alpha degradation or phosphorylation of mitogen-activated protein kinases but was required for RANKL-induced p100 processing. Treatment of IKK alpha(-/-) cells with tumor necrosis factor alpha (TNF alpha) in combination with RANKL led to partial rescue of osteoclastogenesis despite a lack of p100 processing. However, the ability of TNF alpha alone or in combination with transforming growth factor beta to induce osteoclast differentiation was dependent on IKK alpha, suggesting that synergy between RANKL and TNFalpha can overcome p100 processing defects in IKK alpha(-/-) cells.