Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

Functional genomics of the yeast DNA-damage response

High-throughput approaches are beginning to have an impact on many areas of yeast biology. Two recent studies, using different experimental platforms, provide insight into new pathways involved in the response of yeast to DNA damage.     

The tomato Cf-9 disease resistance gene functions in tobacco and potato to confer responsiveness to the fungal avirulence gene product avr 9

The Cf-9 gene encodes an extracytoplasmic leucine-rich repeat protein that confers resistance in tomato to races of the fungus Cladosporium fulvum that express the corresponding avirulence gene Avr 9. We investigated whether the genomic Cf-9 gene functions in potato and tobacco. Transgenic tobacco and potato plants carrying Cf-9 exhibit a rapid hypersensitive cell death response (HR) to Avr 9 peptide injection. Cf 9 tobacco plants were reciprocally crossed to Avr 9-producing tobacco. A developmentally regulated seedling lethal phenotype occurred in F1 progeny when Cf9 was used as the male parent and Avr 9 as the female parent. However, when Cf9 was inherited in the maternal tissue and a heterozygous Avr 9 plant was used as the pollen donor, a much earlier reaction was caused, leading to no germination of any F1 seed. Detailed analysis of the Avr 9-induced responses in Cf 9 tobacco leaves revealed that (1) most mesophyll cells died within 3 hr (compared with 12 to 16 hr in tomato); (2) the macroscopic HR was visible at an Avr 9 titer five times lower than that which caused visible symptoms in tomato; (3) the HR invariably extended into noninjected panels of the tobacco leaf; (4) no HR occurred in leaves of young tobacco plants; (5) in older plants, the HR was dramatically enhanced by sequential Avr 9 challenges; and (6) coexpression of a salicylate hydroxylase transgene (nahG) from Pseudomonas putida reduced the severity of the macroscopic leaf HR and also restored germination to Cf 9 x 35S:Avr 9 F1 seedlings. Simultaneous introduction of Cf-9 homologs (Hcr 9-9 genes A and B or D) along with the native Cf-9 gene did not alter the responses that were specifically induced by Avr 9. Various ways to use the Cf-9-Avr 9 gene combination to engineer broad-spectrum disease resistance in several solanaceous species are discussed.     

Distribution of cell surface saccharides on pancreatic cells

We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a “universal” affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.     

Primary glioblastoma cultures: can profiling of stem cell markers predict radiotherapy sensitivity?

Human glioblastomas may be hierarchically organized. Within this hierarchy, glioblastoma‐initiating cells have been proposed to be more resistant to radiochemotherapy and responsible for recurrence. Here, established stem cell markers and stem cell attributed characteristics such as self‐renewal capacity and tumorigenicity have been profiled in primary glioblastoma cultures to predict radiosensitivity. Furthermore, the sensitivity to radiotherapy of different subpopulations within a single primary glioblastoma culture was analyzed by a flow cytometric approach using Nestin, SRY (sex‐determining region Y)‐box 2 (SOX2) and glial fibrillary acidic protein. The protein expression of Nestin and SOX2 as well as the mRNA levels of Musashi1, L1 cell adhesion molecule, CD133, Nestin, and pleiomorphic adenoma gene‐like 2 inversely correlated with radioresistance in regard to the clonogenic potential. Only CD44 protein expression correlated positively with radioresistance. In terms of proliferation, Nestin protein expression and Musashi1, pleiomorphic adenoma gene‐like 2, and CD133 mRNA levels are inversely correlated with radioresistance. Higher expression of stem cell markers does not correlate with resistance to radiochemotherapy in the cancer genome atlas glioblastoma collective. SOX2 expressing subpopulations exist within single primary glioblastoma cultures. These subpopulations predominantly form the proliferative pool of the primary cultures and are sensitive to irradiation. Thus, profiling of established stem cell markers revealed a surprising result. Except CD44, the tested stem cell markers showed an inverse correlation between expression and radioresistance.

     

Effects of lipid environment on the conformational changes of an ABC importer

In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.     

Autoantibodies directed to novel components of the PM/Scl complex, the human exosome

The autoantigenic polymyositis/scleroderma (PM/Scl) complex was recently shown to be the human homologue of the yeast exosome, which is an RNA-processing complex. Our aim was to assess whether, in addition to targeting the known autoantigens PM/Scl-100 and PM/Scl-75, autoantibodies also target recently identified components of the PM/Scl complex. The prevalence of autoantibodies directed to six novel human exosome components (hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp46p, hCsl4p) was determined in sera from patients with idiopathic inflammatory myopathy (n = 48), scleroderma (n = 11), or the PM/Scl overlap syndrome (n = 10). The sera were analyzed by enzyme-linked immunosorbent assays and western blotting using the affinity-purified recombinant proteins. Our results show that each human exosome component is recognized by autoantibodies. The hRrp4p and hRrp42p components were most frequently targeted. The presence of autoantibodies directed to the novel components of the human exosome was correlated with the presence of the anti-PM/Scl-100 autoantibody in the sera of patients with idiopathic inflammatory myopathy (IIM), as was previously found for the anti-PM/Scl-75 autoantibody. Other clear associations between autoantibody activities were not found. These results further support the conception that the autoimmune response may initially be directed to PM/Scl-100, whereas intermolecular epitope spreading may have caused the autoantibody response directed to the associated components.