Flow Cytofluorimetric Analysis of Anti-LRP4 (LDL Receptor-Related Protein 4) Autoantibodies in Italian Patients with Myasthenia Gravis

Background

Myasthenia gravis (MG) is an autoimmune disease in which 90% of patients have autoantibodies against the muscle nicotinic acetylcholine receptor (AChR), while autoantibodies to muscle-specific tyrosine kinase (MuSK) have been detected in half (5%) of the remaining 10%. Recently, the low-density lipoprotein receptor-related protein 4 (LRP4), identified as the agrin receptor, has been recognized as a third autoimmune target in a significant portion of the double sero-negative (dSN) myasthenic individuals, with variable frequency depending on different methods and origin countries of the tested population. There is also convincing experimental evidence that anti-LRP4 autoantibodies may cause MG.

Methods

The aim of this study was to test the presence and diagnostic significance of anti-LRP4 autoantibodies in an Italian population of 101 myasthenic patients (55 dSN, 23 AChR positive and 23 MuSK positive), 45 healthy blood donors and 40 patients with other neurological diseases as controls. All sera were analyzed by a cell-based antigen assay employing LRP4-transfected HEK293T cells, along with a flow cytofluorimetric detection system.

Results

We found a 14.5% (8/55) frequency of positivity in the dSN-MG group and a 13% frequency of co-occurrence (3/23) in both AChR and MuSK positive patients; moreover, we report a younger female prevalence with a mild form of disease in LRP4-positive dSN-MG individuals.

Conclusion

Our data confirm LRP4 as a new autoimmune target, supporting the value of including anti-LRP4 antibodies in further studies on Myasthenia gravis.     

Age Related Macular Degeneration and Total Hip Replacement Due to Osteoarthritis or Fracture: Melbourne Collaborative Cohort Study

Osteoarthritis is the leading cause of total hip replacement, accounting for more than 80% of all total hip replacements. Emerging evidence suggests that osteoarthritis has a chronic inflammatory component to its pathogenesis similar to age-related macular degeneration. We evaluated the association between age-related macular degeneration and total hip replacement as proxy for severe osteoarthritis or fractured neck of femur in the Melbourne Collaborative Cohort Study. 20,744 participants had complete data on both age-related macular degeneration assessed from colour fundus photographs taken during 2003–2007 and total hip replacement. Total hip replacements due to hip osteoarthritis and fractured neck of femur during 2001–2011 were identified by linking the cohort records to the Australian Orthopedic Association National Joint Replacement Registry. Logistic regression was used to examine the association between age-related macular degeneration and risk of total hip replacement due to osteoarthritis and fracture separately, adjusted for confounders. There were 791 cases of total hip replacement for osteoarthritis and 102 cases of total hip replacement due to fractured neck of femur. After adjustment for age, sex, body mass index, smoking, and grouped country of birth, intermediate age-related macular degeneration was directly associated with total hip replacement for osteoarthritis (odds ratio 1.22, 95% CI 1.00–1.49). Late age-related macular degeneration was directly associated with total hip replacement due to fractured neck of femur (odds ratio 5.21, 95% CI2.25–12.02). The association between intermediate age-related macular degeneration and an increased 10-year incidence of total hip replacement due to osteoarthritis suggests the possibility of similar inflammatory processes underlying both chronic diseases. The association of late age-related macular degeneration with an increased 10-year incidence of total hip replacement due to fractured neck of femur may be due to an increased prevalence of fractures in those with poor central vision associated with the late complications of age-related macular degeneration.     

Natural history museum collections provide information on phenological change in British butterflies since the late-nineteenth century

Museum collections have the potential to provide valuable information on the phenological response of organisms to climate change. This is particularly useful for those species for which few data otherwise exist, but also to extend time series to the period before other observational data are available. To test this potential, we analysed data from 2,630 specimens of four species of British butterflies (Anthocharis cardamines, Hamearis lucina, Polyommatus bellargus and Pyrgus malvae), collected from 1876 to 1999 and stored in the Natural History Museum, London, UK (NHM). In A. cardamines, first-generation P. bellargus and P. malvae, we found that there was a strong significant negative relationship between spring temperature and 10th percentile collection dates, which approximates mean first appearance date, and median collection date, which approximates mean flight date. In all four species, there was a significant negative relationship between the 10th percentile collection date and the length of the collection period, which approximates flight period. In second-generation P. bellargus, these phenological measurements were correlated with summer temperature. We found that the rates of phenological response to temperature, based on NHM data, were similar to, or somewhat greater than, those reported for other organisms based on observational data covering the last 40 years. The lower rate of phenological response, and the significant influence of February rather than March or April temperatures, in recent decades compared with data from earlier in the twentieth century may indicate that early emerging British butterfly species are currently approaching the limits of phenological advancement in response to recent climate warming.     

β-Lactam and glycopeptide antibiotics: first and last line of defense?

Most infections are caused by bacteria, many of which are ever-evolving and resistant to nearly all available antibiotics. β-Lactams and glycopeptides are used to combat these infections by inhibiting bacterial cell-wall synthesis. This mechanism remains an interesting target in the search for new antibiotics in light of failed genomic approaches and the limited input of major pharmaceutical companies. Several strategies have enriched the pipeline of bacterial cell-wall inhibitors; examples include combining screening strategies with lesser-explored microbial diversity, or reinventing known scaffolds based on structure-function relationships. Drugs developed using novel strategies will contribute to the arsenal in fight against the continued emergence of bacterial resistance.     

Seroepidemiological Survey of Paracoccidioidomycosis Infection Among Urban and Rural Dogs from Uberaba,Minas Gerais,Brazil

There is some evidence that dogs can be naturally infected by Paracoccidioides brasiliensis in endemic areas of paracoccidioidomycosis. In order to evaluate canine infection with this fungus, a survey with 149 urban and 126 rural dogs was carried out using ELISA and intradermal tests with the gp43 antigen of P. brasiliensis in Uberaba, Minas Gerais state of Brazil. Forty-one out of 149 urban dogs were euthanatized and had their lungs, liver and spleen removed. One slice from each viscera was processed for histopathological examination and the remaining was homogenized and then cultivated on mycobiotic agar at room temperature and Fava-Netto medium at 35°C and observed for 12 weeks. Of urban dogs, 75 (50.3%) were small adult females, 56 (36%) were strays, while 93 (64%) had been donated to the municipal zoonosis control center. Nine (6.2%) had a positive intradermal test without statistical differences regarding gender, race, nutritional status or origin. No colonies with microscopic or morphology appearances resembling P. brasiliensis were isolated, nor granulomatous process or fungal structures were observed from histopathological examination. Eighty (53.6%) of the urban dogs presented seroreactivity, without statistical differences regarding gender, race, nutritional state, origin, or positive intradermal test. Of 126 rural dogs, 102 (80.5%) presented antibodies against gp43 antigen, and this was statistically significant in relation to the reactivity detected in urban dogs (P = 0.0001). Thus, dogs are commonly infected with P. brasiliensis, but they probably present natural resistance to develop paracoccidioidomycosis.     

Comparison of Humanized IgG and FvFc Anti-CD3 Monoclonal Antibodies Expressed in CHO Cells

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.     

Spectroscopic and metal-binding properties of DF3: an artificial protein able to accommodate different metal ions

The design, synthesis, and metal-binding properties of DF3, a new de novo designed di-iron protein model are described (“DF” represents due ferri, Italian for “two iron,” “di-iron”). DF3 is the latest member of the DF family of synthetic proteins. They consist of helix–loop–helix hairpins, designed to dimerize and form an antiparallel four-helix bundle that encompasses a metal-binding site similar to those of non-heme carboxylate-bridged di-iron proteins. Unlike previous DF proteins, DF3 is highly soluble in water (up to 3 mM) and forms stable complexes with several metal ions (Zn, Co, and Mn), with the desired secondary structure and the expected stoichiometry of two ions per protein. UV–vis studies of Co(II) and Fe(III) complexes confirm a metal-binding environment similar to previous di-Co(II)- and di-Fe(III)-DF proteins, including the presence of a μ-oxo-di-Fe(III) unit. Interestingly, UV–vis, EPR, and resonance Raman studies suggest the interaction of a tyrosine adjacent to the di-Fe(III) center. The design of DF3 was aimed at increasing the accessibility of small molecules to the active site of the four-helix bundle. Indeed, binding of azide to the di-Fe(III) site demonstrates a more accessible metal site compared with previous DFs. In fact, fitting of the binding curve to the Hill equation allows us to quantify a 150% accessibility enhancement, with respect to DF2. All these results represent a significant step towards the development of a functional synthetic DF metalloprotein.     

Protein expression changes induced in murine peritoneal macrophages by Group B Streptococcus

Protein expression changes induced in thioglycolate‐elicited peritoneal murine macrophages (MΦ) by infection with type III Group B Streptococcus (GBS) are described. Proteins from control MΦ and MΦ incubated 2 h with live or heat‐inactivated GBS were separated by 2‐DE. Proteins whose expression was significantly different in infected MΦ, as compared with control cells, were identified by MS/MS analysis. Changes in the expression level of proteins involved in both positive and negative modulation of phagocytic functions, stress response and cell death were induced in MΦ by GBS infection. In particular, expression of enzymes playing a key role in production of reactive oxygen species was lowered in GBS‐infected MΦ. Significant alterations in the expression of some metabolic enzymes were also observed, most of the glycolytic and of the pentose‐cycle enzymes being down‐regulated in MΦ infected with live GBS. Finally, evidence was obtained that GBS infection affects the expression of enzymes or enzyme subunits involved in ATP synthesis and in adenine nucleotides interconversion processes.     

A Genetic Engineering Solution to the ��Arginine Conversion Problem�� in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

Stable isotope labeling by amino acids in cell culture (SILAC) provides a straightforward tool for quantitation in proteomics. However, one problem associated with SILAC is the in vivo conversion of labeled arginine to other amino acids, typically proline. We found that arginine conversion in the fission yeast Schizosaccharomyces pombe occurred at extremely high levels, such that labeling cells with heavy arginine led to undesired incorporation of label into essentially all of the proline pool as well as a substantial portion of glutamate, glutamine, and lysine pools. We found that this can be prevented by deleting genes involved in arginine catabolism using methods that are highly robust yet simple to implement. Deletion of both fission yeast arginase genes or of the single ornithine transaminase gene, together with a small modification to growth medium that improves arginine uptake in mutant strains, was sufficient to abolish essentially all arginine conversion. We demonstrated the usefulness of our approach in a large scale quantitative analysis of proteins before and after cell division; both up- and down-regulated proteins, including a novel protein involved in septation, were successfully identified. This strategy for addressing the “arginine conversion problem” may be more broadly applicable to organisms amenable to genetic manipulation.Stable isotope labeling by amino acids in cell culture (SILAC)¹ (1) is one of the key methods for large scale quantitative proteomics (2, 3). In SILAC experiments, proteins are metabolically labeled by culturing cells in media containing either normal (“light”) or heavy isotope-labeled amino acids, typically lysine and arginine. Peptides derived from the light and heavy cells are thus distinguishable by mass spectrometry and can be mixed for accurate quantitation. SILAC is also possible at the whole-organism level (4).An inherent problem in SILAC is the metabolic conversion of labeled arginine to other amino acids, as this complicates quantitative analysis of peptides containing these amino acids. Arginine conversion to proline is well described in mammalian cells, although the extent of conversion varies among cell types (5). When conversion is observed, typically 10–25% of the total proline pool is found to contain label (6–11). Arginine conversion has also been reported in SILAC experiments with budding yeast Saccharomyces cerevisiae (3, 12, 13).Because more than 50% of tryptic peptides in large data sets contain proline (7), it is not practical simply to disregard proline-containing peptides during quantitation. Several methods have been proposed to either reduce arginine conversion or correct for its effects on quantitation. In some cell types, arginine conversion can be prevented by lowering the concentration of exogenous arginine (6, 14–16) or by adding exogenous proline (9). However, these methods can involve significant changes to growth media and may need to be tested for each experimental condition used. Given the importance of arginine in many metabolic pathways, careful empirical titration of exogenous arginine concentration is required to minimize negative effects on cell growth (14). In addition, low arginine medium can lead to incomplete arginine labeling, although the reasons for this are not entirely clear (7). An alternative strategy is to omit labeled arginine altogether (3, 13, 17), but this reduces the number of quantifiable peptides. Correction methods include using two different forms of labeled arginine (7) or computationally compensating for proline-containing peptides (11, 12, 18). Ultimately, none of these methods address the problem at its root, the utilization of arginine in cellular metabolism.To develop a differential proteomics work flow for the fission yeast Schizosaccharomyces pombe, we sought to adapt SILAC for use in this organism, a widely used model eukaryote with excellent classical and reverse genetics. Here we describe extremely high conversion of labeled arginine to other amino acids in fission yeast as well as a novel general solution to the problem that should be applicable to other organisms. As proof of principle, we quantitated changes in protein levels before and after cell division on a proteome-wide scale. We identified both up- and down-regulated proteins, including a novel protein involved in septation.     

Branched-chain amino acid supplementation promotes survival and supports cardiac and skeletal muscle mitochondrial biogenesis in middle-aged mice

Recent evidence points to a strong relationship between increased mitochondrial biogenesis and increased survival in eukaryotes. Branched-chain amino acids (BCAAs) have been shown to extend chronological life span in yeast. However, the role of these amino acids in mitochondrial biogenesis and longevity in mammals is unknown. Here, we show that a BCAA-enriched mixture (BCAAem) increased the average life span of mice. BCAAem supplementation increased mitochondrial biogenesis and sirtuin 1 expression in primary cardiac and skeletal myocytes and in cardiac and skeletal muscle, but not in adipose tissue and liver of middle-aged mice, and this was accompanied by enhanced physical endurance. Moreover, the reactive oxygen species (ROS) defense system genes were upregulated, and ROS production was reduced by BCAAem supplementation. All of the BCAAem-mediated effects were strongly attenuated in endothelial nitric oxide synthase null mutant mice. These data reveal an important antiaging role of BCAAs mediated by mitochondrial biogenesis in mammals.