Current Trends in Validating Antibody Specificities for ELISpot by Western Blotting.

The enzyme-linked immunospot (ELISpot) assay is a highly useful and sensitive method to detect total immunoglobulin and antigen-specific antibody-secreting cells. In addition, this method can measure biological activity and immunological secretions from immune cells. In general, membrane-bound antigen allows binding of antibody secreted by B cells, or a membrane-bound analyte-specific antibody binds to the specific analyte (e.g., cytokines) elicited from cells added to the well containing the bound antibody. The response from added cells is then detected by using an anti-Ig antibody and a colorimetric substrate, while in the case of non-B cells, the elicited antigen is detected with appropriate antibodies and enzyme-conjugated antibodies. Specificity of antibodies binding the protein of interest is necessary to achieve correct results. Western blotting can be used for this with/without siRNA knockdown of proteins of interest or with the use of peptide inhibitors to inhibit the binding of specific antibodies to the target protein. Despite its general simplicity, western blotting is a powerful technique for immunodetection of proteins (notably low abundance proteins) as it provides simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Now, we have plethora of immunoblotting methods to validate antibodies for ELISpot.

The Potential Utility of Salivary and Tear Proteomics to Discriminate Sjögren's Disease from Non-Sjögren's Sicca.

Sjögren's Disease (SjD) is an autoimmune disorder associated with decreased saliva and/or tear secretions, resulting in patients reporting dryness in the mouth and eyes. Serum autoantibodies directed against the Ro60/SS-A and La/SS-B autoantigens are a distinctive feature of the disease. Analysis of the saliva and tear proteomes represents one promising alternative method of both classifying and monitoring the condition, and research into salivary and tear proteomics in patients with SjD, with and without sicca, has shown its efficacy and practicality in both clinical and research settings. Studies analyzing the saliva proteomics of SjD patients have generally shown an overexpression of proteins involved in T-cell activation, the immune response, ß-2 microglobulin, and the recruitment of pro-inflammatory agents. These studies also show a decrease in or downregulation of proteins involved in salivary secretion. Studies analyzing the tear proteomics of patients with SjD have generally indicated an upregulation of proteins involved with TNF-α signaling, B-cell survival, and the recruitment of pro-inflammatory agents. Studies also note the differential expression of tear protein folding as a hallmark of ocular involvement in this condition. These findings help to elucidate the biochemical relationship between the proteomes of saliva/tear fluids and the general pathophysiology of the gland involved with the pathogenesis of this condition, giving further credence to the potential role of salivary and tear proteomics in the future of diagnosis and treatment for patients with SjD.

Validating Antibody Specificities for Immunohistochemistry by Protein Blotting.

Optimized antibody reagents are important in research, and erratic antibody performance leads to variability in immunoassays. Specificity of antibodies binding the protein of interest is vital to obtain accurate results. Recommendations for validation and use of primary antibodies are unique to each type of immunoassay as the antibodies' performance is greatly affected by the assay context. Immunoblotting procedures have been used along with other important antibody-based detection methods like enzyme-linked immunosorbent assay and immunohistochemistry to confirm results in research and diagnostic testing. Specificity of antibodies employed for immunohistochemical studies is of critical importance. Therefore, the use of western blotting is imperative to address the specificity of antibodies with/without siRNA knockdown of proteins of interest or with the use of peptide inhibitors to inhibit the binding of specific antibodies to the target protein. In spite of its overall simplicity, western blotting or protein blotting is a powerful procedure for immunodetection of proteins, especially those that are of low abundance, following electrophoretic separation. The usefulness of this procedure stems from its ability to provide simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Protein blotting has evolved greatly over the last few decades, and researchers have a variety of ways and means to carry out this procedure to validate antibodies for immunohistochemistry.

47XXY and 47XXX in Scleroderma and Myositis.

OBJECTIVE: We undertook this study to examine the X chromosome complement in participants with systemic sclerosis (SSc) as well as idiopathic inflammatory myopathies. METHODS: The participants met classification criteria for the diseases. All participants underwent single-nucleotide polymorphism typing. We examined X and Y single-nucleotide polymorphism heterogeneity to determine the number of X chromosomes. For statistical comparisons, we used χ2 analyses with calculation of 95% confidence intervals. RESULTS: Three of seventy men with SSc had 47,XXY (P = 0.0001 compared with control men). Among the 435 women with SSc, none had 47,XXX. Among 709 men with polymyositis or dermatomyositis (PM/DM), seven had 47,XXY (P = 0.0016), whereas among the 1783 women with PM/DM, two had 47,XXX. Of 147 men with inclusion body myositis (IBM), six had 47,XXY, and 1 of the 114 women with IBM had 47,XXX. For each of these myositis disease groups, the excess 47,XXY and/or 47,XXX was significantly higher compared with in controls as well as the known birth rate of Klinefelter syndrome or 47,XXX. CONCLUSION: Klinefelter syndrome (47,XXY) is associated with SSc and idiopathic inflammatory myopathies, similar to other autoimmune diseases with type 1 interferon pathogenesis, namely, systemic lupus erythematosus and Sjögren syndrome.

Nanosensors in clinical development of CAR-T cell immunotherapy.

Immunotherapy using CAR-T cells is a new technological paradigm for cancer treatment. To avoid severe side effects and tumor escape variants observed for conventional CAR-T cells approach, adaptor CAR technologies are under development, where intermediate target modules redirect immune cells against cancer. In this work, silicon nanowire field-effect transistors are used to develop target modules for an optimized CAR-T cell operation. Focusing on a library of seven variants of E5B9 peptide that is used as CAR targeting epitope, we performed multiplexed binding tests using nanosensor chips. These peptides had been immobilized onto the sensor to compare the transistor signals upon titration with anti-La 5B9 antibodies. The correlation of binding affinities and sensor sensitivities enabled a selection of candidates for the interaction between CAR and target modules. An extremely low detection limit was observed for the sensor, down to femtomolar concentration, outperforming the current assay of the same purpose. Finally, the CAR T-cells redirection capability of selected peptides in target modules was proven successful in an in-vitro cytotoxicity assay. Our results open the perspective for the nanosensors to go beyond the early diagnostics in clinical cancer research towards developing and monitoring immunotherapeutic treatment, where the quantitative analysis with the standard techniques is limited.

In vitro effects of curcumin on proinflammatory cytokines and expression of their genes in minor salivary gland tissue of patients with Sjogren's syndrome.

Curcumin reduces disease severity and ameliorates lupus-like/Sjögren's Syndrome-like disease in mice model. The immunological basis of these effects is largely unknown. This study examined the effects of curcumin on pro-inflammatory cytokines secreted by minor salivary glands in patients with primary Sjögren's syndrome (pSS). Minor salivary gland (MSG) tissue samples were collected from patients undergoing biopsy for suspected pSS. The tissues were treated with phytohemagglutinin (PHA) alone as well as PHA with curcumin (30 µM) and cultured in RPMI 1640 medium for 48 h at 37 °C in CO2 incubator. After the incubation period, culture supernatant and tissues were stored in the freezer (-80 °C). IL-6 levels were measured in supernatant by ELISA kit. Gene expressions of pro-inflammatory cytokines, namely IL-6, IL-8, TNF-α, IL-1ß, IL-4, IL-10, IL-17, IL-21, and IFN-γ, were measured by qPCR. IL-6 secretion levels and gene expressions were compared statistically between groups by Student's t test. Forty-seven patients were screened. Eight patients satisfied ACR/EULAR criteria for pSS. Seven patients with absent glandular inflammation and negative serology constituted sicca controls. These 15 subjects were included in final analysis. In pSS group, but not in controls, median IL-6 levels in supernatant were less in curcumin-treated as compared to PHA-alone subset [5.5 (0.7-13.34) vs 18.3 (12-32) ng/ml; p = 0.0156]. mRNA expression levels of IL-6 were also lower in curcumin-treated samples as compared to PHA alone, when cases and controls were analyzed together as well as in cases alone (p = 0.0009 and p = 0.0078, respectively); however, mRNA expression of IL-1ß was lower in curcumin-treated samples as compared to PHA alone, only when cases and controls were analyzed together (p = 0.0215). There was no difference in other cytokine gene expression levels between the subsets under the in-vitro experimental conditions. In conclusion, curcumin reduced mRNA expression as well as secretion of IL-6 levels by salivary gland tissue of patients with pSS. Curcumin also suppressed PHA-induced mRNA expression levels of IL-6 and IL-1ß in MSG tissue of patients with pSS and controls when analyzed together as a combined group.

A Small Step, a Giant Leap: Somatic Hypermutation of a Single Amino Acid Leads to Anti-La Autoreactivity.

The anti-La mab 312B, which was established by hybridoma technology from human-La transgenic mice after adoptive transfer of anti-human La T cells, immunoprecipitates both native eukaryotic human and murine La protein. Therefore, it represents a true anti-La autoantibody. During maturation, the anti-La mab 312B acquired somatic hypermutations (SHMs) which resulted in the replacement of four aa in the complementarity determining regions (CDR) and seven aa in the framework regions. The recombinant derivative of the anti-La mab 312B in which all the SHMs were corrected to the germline sequence failed to recognize the La antigen. We therefore wanted to learn which SHM(s) is (are) responsible for anti-La autoreactivity. Humanization of the 312B ab by grafting its CDR regions to a human Ig backbone confirms that the CDR sequences are mainly responsible for anti-La autoreactivity. Finally, we identified that a single amino acid replacement (D > Y) in the germline sequence of the CDR3 region of the heavy chain of the anti-La mab 312B is sufficient for anti-La autoreactivity.

And Yet It Moves: Oxidation of the Nuclear Autoantigen La/SS-B Is the Driving Force for Nucleo-Cytoplasmic Shuttling.

Decades ago, we and many other groups showed a nucleo-cytoplasmic translocation of La protein in cultured cells. This shuttling of La protein was seen after UV irradiation, virus infections, hydrogen peroxide exposure and the Fenton reaction based on iron or copper ions. All of these conditions are somehow related to oxidative stress. Unfortunately, these harsh conditions could also cause an artificial release of La protein. Even until today, the shuttling and the cytoplasmic function of La/SS-B is controversially discussed. Moreover, the driving mechanism for the shuttling of La protein remains unclear. Recently, we showed that La protein undergoes redox-dependent conformational changes. Moreover, we developed anti-La monoclonal antibodies (anti-La mAbs), which are specific for either the reduced form of La protein or the oxidized form. Using these tools, here we show that redox-dependent conformational changes are the driving force for the shuttling of La protein. Moreover, we show that translocation of La protein to the cytoplasm can be triggered in a ligand/receptor-dependent manner under physiological conditions. We show that ligands of toll-like receptors lead to a redox-dependent shuttling of La protein. The shuttling of La protein depends on the redox status of the respective cell type. Endothelial cells are usually resistant to the shuttling of La protein, while dendritic cells are highly sensitive. However, the deprivation of intracellular reducing agents in endothelial cells makes endothelial cells sensitive to a redox-dependent shuttling of La protein.

Two Be or Not Two Be: The Nuclear Autoantigen La/SS-B Is Able to Form Dimers and Oligomers in a Redox Dependent Manner.

According to the literature, the autoantigen La is involved in Cap-independent translation. It was proposed that one prerequisite for this function is the formation of a protein dimer. However, structural analyses argue against La protein dimers. Noteworthy to mention, these structural analyses were performed under reducing conditions. Here we describe that La protein can undergo redox-dependent structural changes. The oxidized form of La protein can form dimers, oligomers and even polymers stabilized by disulfide bridges. The primary sequence of La protein contains three cysteine residues. Only after mutation of all three cysteine residues to alanine La protein becomes insensitive to oxidation, indicating that all three cysteines are involved in redox-dependent structural changes. Biophysical analyses of the secondary structure of La protein support the redox-dependent conformational changes. Moreover, we identified monoclonal anti-La antibodies (anti-La mAbs) that react with either the reduced or oxidized form of La protein. Differential reactivities to the reduced and oxidized form of La protein were also found in anti-La sera of autoimmune patients.

T Cell Mediated Conversion of a Non-Anti-La Reactive B Cell to an Autoreactive Anti-La B Cell by Somatic Hypermutation.

Since the first description of nuclear autoantigens in the late 1960s and early 1970s, researchers, including ourselves, have found it difficult to establish monoclonal antibodies (mabs) against nuclear antigens, including the La/SS-B (Sjögrens' syndrome associated antigen B) autoantigen. To date, only a few anti-La mabs have been derived by conventional hybridoma technology; however, those anti-La mabs were not bona fide autoantibodies as they recognize either human La specific, cryptic, or post-translationally modified epitopes which are not accessible on native mouse La protein. Herein, we present a series of novel murine anti-La mabs including truly autoreactive ones. These mabs were elicited from a human La transgenic animal through adoptive transfer of T cells from non-transgenic mice immunized with human La antigen. Detailed epitope and paratope analyses experimentally confirm the hypothesis that somatic hypermutations that occur during T cell dependent maturation can lead to autoreactivity to the nuclear La/SS-B autoantigen.

Sjögren Syndrome without Focal Lymphocytic Infiltration of the Salivary Glands.

OBJECTIVE: Primary Sjögren syndrome (SS) is characterized by a focal lymphocytic infiltrate in exocrine glands. We describe patients who lacked this key feature. METHODS: We evaluated patients with sicca in a comprehensive clinic at which medical, dental, and ophthalmological examinations were performed. All subjects underwent a minor salivary gland biopsy with focus score calculation. Extraglandular manifestations were also determined. We categorized subjects as high, intermediate, or low in terms of expression of interferon (IFN)-regulated genes. RESULTS: About 20% (51 of 229, 22%) of those classified as having primary SS had a focus score of zero. Compared to those with anti-Ro positivity and a focus score > 1.0, the patients with focus score of zero (who by classification criteria must be anti-Ro-positive) were statistically less likely to have anti-La (or SSB) and elevated immunoglobulin, as well as less severe corneal staining. The focus score zero patients were less likely to have elevated expression of IFN-regulated genes in peripheral blood mononuclear cells than anti-Ro-positive SS patients with a focal salivary infiltrate. CONCLUSION: There are only a few clinical differences between patients with primary SS with focus score zero and those with both anti-Ro and a focus score > 1.0. The small subset of focus score zero patients tested did not have elevated expression of IFN-regulated genes, but did have systemic disease. Thus, extraglandular manifestations are perhaps more related to the presence of anti-Ro than increased IFN. This may have relevance to pathogenesis of SS.

Characterization of cxorf21 Provides Molecular Insight Into Female-Bias Immune Response in SLE Pathogenesis.

Background: Ninety percent of systemic lupus erythematosus (SLE) patients are women. X chromosome-dosage increases susceptibility to SLE and primary Sjögren's syndrome (pSS). Chromosome X open reading frame 21 (CXorf21) escapes X-inactivation and is an SLE risk gene of previously unknown function. We undertook the present study to delineate the function of CXorf21 in the immune system as well as investigate a potential role in the sex bias of SLE and pSS. Methods: Western blot protein analysis, qPCR, BioPlex cytokine immunoassay, pHrodo™ assays, as well as in vitro CRISPR-Cas9 knockdown experiments were employed to delineate the role of CXorf21 in relevant immunocytes. Results: Expressed in monocytes and B cells, CXorf21 basal Mrna, and protein expression levels are elevated in female primary monocytes, B cells, and EBV-transformed B cells compared to male cells. We also found CXorf21 mRNA and protein expression is higher in both male and female cells from SLE patients compared to control subjects. TLR7 ligation increased CXorf21 protein expression and CXorf21 knockdown abrogated TLR7-driven increased IFNA1 mRNA expression, and reduced secretion of both TNF-alpha and IL-6 in healthy female monocytes. Similarly, we found increased pH in the lysosomes of CXorf21-deficient female monocytes. Conclusion: CXorf21 is more highly expressed in female compared to male cells and is involved in a sexually dimorphic response to TLR7 activation. In addition, CXorf21 expression regulates lysosomal pH in a sexually dimorphic manner. Thus, sexually dimorphic expression of CXorf21 skews cellular immune responses in manner consistent with expected properties of a mediator of the X chromosome dose risk in SLE and pSS.

Lysosomal pH Is Regulated in a Sex Dependent Manner in Immune Cells Expressing CXorf21.

Background:CXorf21 and SLC15a4 both contain risk alleles for systemic lupus erythematosus (SLE) and Sjögren's syndrome (pSS). The former escapes X inactivation. Our group predicts specific endolysosomal-dependent immune responses are driven by the protein products of these genes, which form a complex at the endolysosomal surface. Our previous studies have shown that knocking out CXorf21 increases lysosomal pH in female monocytes, and the present study assesses whether the lysosomal pH in 46,XX women, who overexpress CXorf21 in monocytes, B cells, and dendritic cells (DCs), differs from 46,XY men. Methods: To determine endolysosome compartment pH we used both LysoSensor™ Yellow/Blue DND-160 and pHrodo® Red AM Intracellular pH Indicator in primary monocyte, B cells, DCs, NK cells, and T cells from healthy men and women volunteers. Results: Compared to male samples, female monocytes, B cells, and DCs had lower endolysosomal pH (female/male pH value: monocytes 4.9/5.6 p < 0.0001; DCs 4.9/5.7 p = 0.044; B cells 5.0/5.6 p < 0.05). Interestingly, T cells and NK cells, which both express low levels of CXorf21, showed no differential pH levels between men and women. Conclusion: We have previously shown that subjects with two or more X-chromosomes have increased CXorf21 expression in specific primary immune cells. Moreover, knockdown of CXorf21 increases lysosomal pH in female monocytes. The present data show that female monocytes, DC, B cells, where CXorf21 is robustly expressed, have lower lysosomal pH compared to the same immune cell populations from males. The lower pH levels observed in specific female immune cells provide a function to these SLE/SS-associated genes and a mechanism for the reported inflated endolysosomal-dependent immune response observed in women compared to men (i.e., TLR7/type I Interferon activity).

Isoelectric Focusing on Non-Denaturing Flatbed Gels.

Isoelectric focusing (IEF) serves as a very useful procedure for cell protein separation and characterization. We have used this method to study antibody clonotype changes. Here we discuss the use of a sensitive native flatbed isoelectric focusing method to analyze specific antibody clonotype changes in a patient with systemic lupus erythematosus, who developed autoantibodies to the Ro 60 autoantigen under observation. Patient sera samples collected over several years were used for analysis using flatbed IEF. Following electrofocusing, the gel is analyzed by affinity immunoblotting utilizing Ro 60-coated nitrocellulose membrane to determine oligoclonality of the anti-Ro 60 containing sera.

Two-Dimensional Gel Electrophoresis by Glass Tube-Based IEF and SDS-PAGE.

The genome information combined with data derived from modern mass spectrometry enables us to determine the identity of a protein once it is isolated from a complex mixture. Two-dimensional gel electrophoresis established more than four decades ago serves as a powerful protocol to isolate many proteins at once for such protein analysis. In the first two decades, the original procedure to use a glass tube-based IEF had been commonly used. Since an IEF in glass tubes is rather difficult to maneuver, a new method to use an IEF on a thin agarose slab backed by a plastic film (IPG Dry Strip) had been invented and is now widely used. In this chapter, we describe a protocol that uses a glass tube-based IEF because the capacity of protein loading and resolving power of this type of classic two-dimensional gel is still indispensable for many applications, not only for protein identification but also for protocols that are benefited by larger amounts of materials, i.e., analysis of posttranslational modification of proteins such as phosphorylation, methylation, glycosylation, and others.

Protein Extraction from Gels: A Brief Review.

Protein gel electrophoresis is an important procedure carried out in protein studies. Elution and recovery of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are often necessary for further downstream analyses. The process involves localizing the protein of interest on the gel following SDS-PAGE, eluting the protein from the gel, removing SDS from the eluted sample, and finally renaturing the protein (e.g., enzymes) for subsequent analyses. Investigators have extracted proteins from gels by a variety of techniques. These include dissolution of the gel matrix, passive diffusion, and electrophoretic elution. Proteins eluted from gels have been used successfully in a variety of downstream applications, including protein chemistry, proteolytic cleavage, determination of amino acid composition, polypeptide identification by trypsin digestion and matrix-assisted laser desorption ionization-time of flight mass spectroscopy, as antigens for antibody production, identifying a polypeptide corresponding to an enzyme activity, and other purposes. Protein yields ranging from nanogram levels to 100 µg have been obtained. Here, we review some of the methods that have been used to elute proteins from gels.

Ultrarapid Sodium Dodecyl Sulfate Polyacrylamide Mini-Gel Electrophoresis.

We describe here an ultrafast method for electrophoresing proteins on SDS-PAGE. Previously we reported a method to complete SDS-PAGE and immunoblotting in an hour, including electrophoresing proteins at 70°C in 10 min. Here we show that we can electrophorese molecular weight standards and bovine serum albumin on a 4-20% gradient gel in well under 10 min using heated (44 °C) Laemmli running buffer and high voltage.

Artifacts and Common Errors in Protein Gel Electrophoresis.

In spite of taking precautions, some common mistakes creep into well-planned gel electrophoresis experiments. This occurs commonly in relation to calculating the cross-linking factor of a gel, polymerization temperature and time for a polyacrylamide gel, inducing aggregates in samples for electrophoresis, titrating the running buffer in electrophoresis, proper sample preparation, amount of protein to be loaded on a gel, sample buffer-to-protein ratios, incompletely removing phosphate-buffered saline from cells prior to cell lysis, and over-focusing of IPG strip in two-dimensional gel electrophoresis. In addition, subtle artifacts can have significant deleterious effects on carefully planned and executed experiments. Proteases that act at room temperature upon proteins in the sample buffer prior to heating, cleavage of the Asp-Pro bond upon prolonged heating of proteins at high temperatures, contamination of sample or sample buffer with keratin, leaching of chemicals from disposable plastic ware, and contamination of urea with ammonium cyanate are some of the common reasons for artifacts in gel electrophoresis. Taking proper heed to all these factors can greatly help generate good experimental results.

Strawberries decrease circulating levels of tumor necrosis factor and lipid peroxides in obese adults with knee osteoarthritis.

OBJECTIVE: Knee osteoarthritis (OA) is increasingly prevalent in obese people, who often have high cardio-metabolic risk factors. Among the few available non-surgical approaches, nutraceuticals have gained popularity, and dietary berries have mitigated arthritis symptoms in observational and animal studies. Clinical studies in OA are sparse, but recently we reported that strawberry supplementation can mitigate pain and reduce inflammatory markers in adults with knee OA. This study extends those observations. METHODS: We conducted a randomized cross-over double-blind placebo-controlled trial on the effects of dietary freeze-dried strawberries on obesity-related hormones, biomarkers of inflammation and lipid peroxidation. Seventeen subjects (4 men, 13 women; age 57 ± 3 year) were randomized to strawberry supplements (50 g day-1 for 12 weeks) vs. placebo (50 g day-1, matched for calories and fiber), for two 12-week intervention periods, separated by 2-week washout phase. RESULTS: Among 24 biomarkers of inflammation examined (Bioplex-Pro human inflammation panel), 12 were detectable in all samples. Among these, high-sensitivity TNF-α (hs-TNF-α) and the soluble tumor necrosis factor receptor (sTNF-R2) were significantly decreased after strawberry consumption (p < 0.05). There were no changes in other biomarkers of the TNF super family, such as APRIL and BAFF. Among serum biomarkers of oxidative stress, 4-hydroxy-2-nonenal (4-HNE) and conjugated dienes were also reduced (p < 0.05). No changes were observed in body weight, serum obesity-related hormones, or osteocalcin. CONCLUSION: Strawberries lowered TNF-α, and lipid peroxidation products in obese adults with knee OA. Since, they also mitigate pain, these findings merit further investigation in larger trials.