Evidence of Alternative Modes of B Cell Activation Involving Acquired Fab Regions of N-Glycosylation in Antibody-Secreting Cells Infiltrating the Labial Salivary Glands of Patients With Sjögren's Syndrome.

OBJECTIVE: To better understand the role of B cells, the potential mechanisms responsible for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjögren's syndrome (SS), this study explored patterns of selection pressure and sites of N-glycosylation acquired by somatic mutation (acN-glyc) in the IgG variable (V) regions of antibody-secreting cells (ASCs) isolated from the minor salivary glands of patients with SS and non-SS control patients with sicca symptoms. METHODS: A novel method to produce and characterize recombinant monoclonal antibodies (mAb) from single cell-sorted ASC infiltrates was applied to concurrently probe expressed genes (all heavy- and light-chain isotypes as well as any other gene of interest not related to immunoglobulin) in the labial salivary glands of patients with SS and non-SS controls. V regions were amplified by reverse transcription-polymerase chain reaction, sequenced, and analyzed for the incidence of N-glycosylation and selection pressure. For specificity testing, the amplified regions were expressed as either the native mAb or mutant mAb lacking the acN-glyc motif. Protein modeling was used to demonstrate how even an acN-glyc site outside of the complementarity-determining region could participate in, or inhibit, antigen binding. RESULTS: V-region sequence analyses revealed clonal expansions and evidence of secondary light-chain editing and allelic inclusion, of which neither of the latter two have previously been reported in patients with SS. Increased frequencies of acN-glyc were found in the sequences from patients with SS, and these acN-glyc regions were associated with an increased number of replacement mutations and lowered selection pressure. A clonal set of polyreactive mAb with differential framework region 1 acN-glyc motifs was also identified, and removal of the acN-glyc could nearly abolish binding to autoantigens. CONCLUSION: These findings support the notion of an alternative mechanism for the selection and proliferation of some autoreactive B cells, involving V-region N-glycosylation, in patients with SS.

Antiadrenergic autoimmunity in postural tachycardia syndrome.

AIMS: Postural tachycardia syndrome (POTS), a common and debilitating cardiovascular disorder, is characterized by an exaggerated heart rate increase during orthostasis and a wide spectrum of adrenergic-related symptoms. To determine the aetiology of POTS, we examined a possible pathophysiological role for autoantibodies against α1-adrenergic (α1AR) and ß1/2-adrenergic receptors (ß1/2AR). METHODS AND RESULTS: Immunoglobulin G (IgG) derived from 17 POTS patients, 7 with recurrent vasovagal syncope (VVS), and 11 normal controls was analysed for its ability to modulate activity and ligand responsiveness of α1AR and ß1/2AR in transfected cells and to alter contractility of isolated rat cremaster arterioles in vitro. Immunoglobulin G activation of α1AR and ß1/2AR was significantly higher in POTS compared with VVS and controls in cell-based assays. Eight, 11, and 12 of the 17 POTS patients possessed autoantibodies that activated α1AR, ß1AR and ß2AR, respectively. Pharmacological blockade suppressed IgG-induced activation of α1AR and ß1/2AR. Eight of 17 POTS IgG decreased the α1AR responsiveness to phenylephrine and 13 of 17 POTS IgG increased the ß1AR responsiveness to isoproterenol irrespective of their ability to directly activate their receptors. Postural tachycardia syndrome IgG contracted rat cremaster arterioles, which was reversed by α1AR blockade. The upright heart rate correlated with IgG-mediated ß1AR and α1AR activity but not with ß2AR activity. CONCLUSION: These data confirm a strong relationship between adrenergic autoantibodies and POTS. They support the concept that allosteric-mediated shifts in the α1AR and ß1AR responsiveness are important in the pathophysiology of postural tachycardia.

Significantly reduced lymphadenopathy, salivary gland infiltrates and proteinuria in MRL-lpr/lpr mice treated with ultrasoluble curcumin/turmeric: increased survival with curcumin treatment.

OBJECTIVES: Commercial curcumin (CU), derived from food spice turmeric (TU), has been widely studied as a potential therapeutic for a variety of oncological and inflammatory conditions. Lack of solubility/bioavailability has hindered curcumin's therapeutic efficacy in human diseases. We have solubilised curcumin in water applying heat/pressure, obtaining up to 35-fold increase in solubility (ultrasoluble curcumin (UsC)). We hypothesised that UsC or ultrasoluble turmeric (UsT) will ameliorate systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS)-like disease in MRL-lpr/lpr mice. METHODS: Eighteen female MRL-lpr/lpr (6 weeks old) and 18 female MRL-MpJ mice (6 weeks old) were used. Female MRL-lpr/lpr mice develop lupus-like disease at the 10th week and die at an average age of 17 weeks. MRL-MpJ mice develop lupus-like disease around 47 weeks and typically die at 73 weeks. Six mice of each strain received autoclaved water only (lpr-water or MpJ-water group), UsC (lpr-CU or MpJ-CU group) or UsT (lpr-TU or MpJ-TU group) in the water bottle. RESULTS: UsC or UsT ameliorates SLE in the MRL-lpr/lpr mice by significantly reducing lymphoproliferation, proteinuria, lesions (tail) and autoantibodies. lpr-CU group had a 20% survival advantage over lpr-water group. However, lpr-TU group lived an average of 16 days shorter than lpr-water group due to complications unrelated to lupus-like illness. CU/TU treatment inhibited lymphadenopathy significantly compared with lpr-water group (p=0.03 and p=0.02, respectively) by induction of apoptosis. Average lymph node weights were 2606±1147, 742±331 and 385±68 mg, respectively, for lpr-water, lpr-CU and lpr-TU mice. Transferase dUTP nick end labelling assay showed that lymphocytes in lymph nodes of lpr-CU and lpr-TU mice underwent apoptosis. Significantly reduced cellular infiltration of the salivary glands in the lpr-TU group compared with the lpr-water group, and a trend towards reduced kidney damage was observed in the lpr-CU and lpr-TU groups. CONCLUSIONS: These studies show that UsC/UsT could prove useful as a therapeutic intervention in SLE/SS.

Parafilm-M®, An Available Cost-Effective Alternative for Immuno-blot Pouches.

Commercially available standard immuno-blot pouches do play an efficient role in antibody incubation in performing an immuno-blot, but are not readily available in the laboratory and have to be specifically ordered. We have developed an equally efficient technique to make an immune-blot more cost-effective with more conservation of antibodies by using a common and readily available laboratory product Parafilm-M(®). Parafilm-M(®) which serves as a sealant for various items of laboratory equipment can be used for antibody incubation. Manually made Parafilm-M(®) pouch has a clear advantage over standard immuno-blot pouches in terms of availability, cost-effectiveness, and consumption of antibodies that ultimately reduces the cost of an immuno-blot. We have performed a series of experiments to check the efficacy of both the techniques. Samples with equal amount of protein were analyzed on separate SDS PAGE gels. The proteins were transferred electrophoretically to the nitrocellulose membrane using Trans-Blot(®) Turbo™ Mini Nitrocellulose Transfer Pack. Antibody incubation was done using standard immuno-blot pouch, standard container and Parafilm-M(®) sealed pouch. The expression of protein was determined and the results of immuno-blots were compared. We found that antibodies are binding the membrane in Parafilm-M(®) pouches as efficiently as in container method or in standard immuno-blot pouches. By restricting the membrane, the surface area of the manually made Parafilm-M(®) pouch can be reduced, less diluent is required to cover the membrane as a result less antibodies are consumed. We also calculated that each immuno-blot pouch cost around $0.1906, whereas the cost for Parafilm-M(®) pouch is 0.0695 which is almost one-third the price of an immuno-blot pouch. Thus, Parafilm-M(®) method distinctly provides a cost-effective solution for antibody incubation.

Single-cell western blotting.

Cell heterogeneity is a variation in cellular processes in functionally similar cells. Cells from the same tissue which are considered genetically identical may have difference in size, structure, and level of protein expression which can lead to major impact on the functions of cell leading to difference in physiological consequences. Single-cell proteome-wide studies are used to detect cell heterogeneity. Flow cytometry and immunocytochemistry do play an important role in evaluating cell heterogeneity. However, these methods are based on separation by antibodies with limited specificity. Cross-reactivity can occur leading to bias in result. Western blot is done to separate the proteins according to molecular weight. Therefore, off-target and on-target signals can be discriminated. Detection of protein expression from a tissue can be done with the help of western blot. However, it is unable to differentiate protein expression of individual cells. For detection of this cell-to-cell variation, a highly advanced technique termed "single-cell western blotting" is carried out. Single-cell western blot has enabled us to detect protein expression at cellular level at a fairly advanced high resolution using a western blot designed to assess cell heterogeneity.