Production, Isolation, and Detection of Poly IC in Extracellular Vesicles from U937 Cells.

Double-stranded RNA is produced by viruses during their replicative cycle. It is a potent immune modulator and indicator of viral infection within the body. Extracellular vesicles (EVs) are lipid-bound particles released from cells homeostatically. Recent studies have shown that a commercially available dsRNA, poly inosinic: poly cytidylic acid (poly IC), can be detected within EVs. This finding opens the door for studying EVs as (1) carriers for dsRNA and (2) indicators of viral infection. To study dsRNA-containing EVs, we must have reliable methods for producing, isolating, and detecting them. This chapter uses U937, a pro-monocytic, human myeloid leukemia cell line, as the EV producer following poly IC treatment, and an immunoblot using an anti-dsRNA antibody (J2) for detection. Two methods for isolating the EVs and two methods for isolating the RNA from these EVs are described. Together, these methods effectively produce, isolate, and detect long dsRNA from EVs.

Modulation and Determination of the Status of Inflammasomes in Leishmania-Infected Macrophages.

Leishmania, an intra-macrophage kinetoplastid parasite, modulates a vast array of defensive mechanisms of the host macrophages to create a comfortable environment for their survival. When the host encounters intracellular pathogens, a multimeric protein complex called NLRP3 inflammasome gets turned on, leading to caspase-1 activation-mediated maturation of IL-1ß from its pro-form. However, Leishmania often manages to neutralize inflammasome activation by manipulating negative regulatory molecules of the host itself. Exhaustion of NLRP3 and pro-IL-1ß result from decreased NF-κB activity in infection, which was attributed to increased expression of A20, a negative regulator of NF-κB signalling. Moreover, reactive oxygen species, another key requirement for inflammasome activation, are inhibited by mitochondrial uncoupling protein 2 (UCP2) which is upregulated by Leishmania. Inflammasome activation is a complex event and procedures involved in monitoring inflammasome activation need to be accurate and error-free. In this chapter, we summarize the protocol that includes various experimental procedures required for the determination of the status of inflammasomes in Leishmania-infected macrophages.

Automated Capillary Electrophoresis Immunoblot for the Detection of Alpha-Synuclein in Mouse Tissue.

Background: Alpha-synuclein (aSyn) is a key player in neurodegenerative diseases such as Parkinson's disease (PD), dementia with Lewy bodies, or multiple system atrophy. aSyn is expressed throughout the brain, and can also be detected in various peripheral tissues. In fact, initial symptoms of PD are non-motoric and include autonomic dysfunction, suggesting that the periphery might play an important role in early development of the disease. aSyn is expressed at relatively low levels in non-central tissues, which brings challenges for its detection and quantification in different tissues. Objective: Our goal was to assess the sensitivity of aSyn detection in central and peripheral mouse tissues through capillary electrophoresis (CE) immunoblot, considering the traditional SDS-PAGE immunoblot as the current standard. Methods: Tissues from central and non-central origin from wild type mice were extracted, and included midbrain, inner ear, and esophagus/stomach. aSyn detection was assessed through immunoblotting using Simple Western size-based CE and SDS-PAGE. Results: CE immunoblots show a consistent detection of aSyn in central and peripheral tissues. Through SDS-PAGE, immunoblots revealed a reliable signal corresponding to aSyn, particularly following membrane fixation. Conclusion: Our results suggest a reliable detection of aSyn in central and peripheral tissues using the CE Simple Western immunoblot system. These observations can serve as preliminary datasets when aiming to formally compare CE with SDS-PAGE, as well as for further characterization of aSyn using this technique.

Allergenicity and structural properties of new Cor a 1 isoallergens from hazel identified in different plant tissues.

The hazel allergen Cor a 1 is a PR-10 protein, closely related to the major birch pollen allergen Bet v 1. Hazel allergies are caused by cross-reactive IgE antibodies originally directed against Bet v 1. Despite the importance of PR-10 proteins in allergy development, their function and localization in the plant remain largely elusive. Therefore, the presence of Cor a 1 mRNA and proteins was investigated in different tissues, i.e., the female flower, immature and mature nuts, catkins, and pollen. Four yet unknown Cor a 1 isoallergens, i.e., Cor a 1.0501-1.0801, and one new Cor a 1.03 variant were discovered and characterized. Depending on the isoallergen, the occurrence and level of mRNA expression varied in different tissues, suggesting different functions. Interestingly, Cor a 1.04 previously thought to be only present in nuts, was also detected in catkins and pollen. The corresponding Cor a 1 genes were expressed in Escherichia coli. The purified proteins were analysed by CD and NMR spectroscopy. Immunoblots and ELISAs to determine their allergenic potential showed that the new proteins reacted positively with sera from patients allergic to birch, hazel and elder pollen and were recognized as novel isoallergens/variants by the WHO/IUIS Allergen Nomenclature Sub-Committee.

Detection and Characterization of Apoptosis-Related Proteins in Hippocampal Neurodegeneration: From mRNA Expression to Protein Quantification.

The involvement of apoptosis in neurodegeneration can be detected by quantifying the apoptotic proteins in hippocampal lysate. Apoptosis can occur due to the overproduction of apoptotic proteins under the influence of external trigger or due to the overexpression of the apoptotic genes. Thus, the imbalance in the production of apoptotic proteins can be quantified using the Western blotting technique and the overexpression of apoptotic genes in hippocampal DNA can be quantified using the real-time quantification of mRNA expression of the apoptotic proteins. Here we provide the methodology of detecting the apoptosis-related proteins like Bax and Bcl-2 and their mRNA expression in hippocampal neurodegeneration. In this chapter, we have described the methodology for quantification of mRNA expression of these apoptosis-related proteins in the hippocampal lysate using the real-time quantitative polymerase chain reaction (qPCR) technique and the methodology of detection and characterization of respective protein expression in the hippocampal lysate using the Western blotting technique.

Epidemiological, clinical, and laboratory characteristics of human granulocytic anaplasmosis in North India.

Human granulocytic anaplasmosis (HGA) is an emerging, rickettsial tick-borne disease caused by Anaplasma phagocytophilum. Sero-epidemiological data demonstrate that this pathogen has a worldwide distribution. The diagnosis of HGA requires a high index of clinical suspicion, even in endemic areas. In recent years, HGA has increasingly been reported from Asia and described in China, Japan, and Korea. We serologically and molecularly screened 467 patients with clinical suspicion of Anaplasmosis. The present study describes the epidemiology, clinical, and laboratory details of 6 confirmed and 43 probable cases of human granulocytic anaplasmosis. One of the HGA patients developed secondary invasive opportunistic Aspergillus fumigatus and Acinetobacter baumanii infection during the illness, which resulted in a fatal infection. The HGA patients without severe complications had excellent treatment responses to doxycycline. The emergence of this newly recognized tick-borne zoonotic HGA in North India is a significant concern for public health and is likely underdiagnosed, underreported, and untreated. Hence, it is also essential to establish a well-coordinated system for actively conducting tick surveillance, especially in the forested areas of the country.IMPORTANCEThe results of the present study show the clinical and laboratory evidence of autochthonous cases of Anaplasma phagocytophilum in North India. The results suggest the possibility of underdiagnosis of HGA in this geographical area. One of the HGA patients developed secondary invasive opportunistic Aspergillus fumigatus and Acinetobacter baumanii infection during the illness, which resulted in a fatal infection.

Positive predictive value of myositis antibody line blot testing in patients with suspected idiopathic inflammatory myopathy.

INTRODUCTION/AIMS: Line blot (LB) is in widespread use for myositis antibody detection. Yet, studies of its positive predictive value (PPV) in patients with suspected idiopathic inflammatory myopathy (IIM), which would be of particular relevance to neuromuscular clinicians, are lacking. We aimed to determine the PPV of myositis antibody LB testing in patients with suspected IIM, and examine whether PPV was significantly impacted by intensity of antibody positivity. METHODS: This was a retrospective study of patients who underwent myositis antibody LB testing for suspected IIM between March 2019 and August 2022. RESULTS: Of 70 patients who underwent testing for suspected IIM and had positive myositis antibody LB results, 43 (61%) were female and the median age was 61 years (range: 10-83 years). Forty-four were classified as true-positives, yielding a PPV of 63%. The PPV of patients with weak-positive myositis antibody results (14/30, 47%) was significantly lower than the PPV of patients with moderate-positive or strong-positive myositis antibody results (30/40, 75%) (p = .02). DISCUSSION: Our study found that myositis antibody LB testing in patients with suspected IIM had a modest PPV, underscoring the need for antibody interpretation in the context of all available clinical and ancillary test data to avoid misdiagnosis. The significantly lower PPV in patients with weak-positive results emphasizes the particular importance of clinical correlation in such patients. Further study into the diagnostic performance of various LBs for myositis antibody detection is needed to inform their interpretation in clinical practice.

Immunoblot Analysis from Single Cells Using Milo™ Single-Cell Western Platform.

In this new era of precision medicine, characterization of single-cell subpopulations to better understand disease etiology is paramount. It is thus an opportune time to explore techniques that allow molecular analysis of single cells and to better understand the basis of pathogenesis of diseases like cancer. Single-cell western blotting is one such method that allows analysis of single cells at the protein level. In contrast to traditional western blotting, which relies heavily on bulk analysis of lysates generated from tissues and is often indicative of the population average, this technique allows analysis of lysates from single-cell subpopulations thereby providing a glimpse into cell heterogeneity. The method entails the use of a chip containing 30 µm thick photoactivated polyacrylamide gel spotted with nearly 6400 microwells. Single cells loaded on the chip are captured in the microwells by passive gravity and are then lysed and electrophoresed using the MILO™ single-cell western platform. This method forgoes the use of transfer of proteins on a PVDF and a nitrocellulose membrane, as performed in traditional western blotting, and all other steps including probing of primary and fluorescent secondary antibodies against the protein of interest are performed directly on the chip. The proteins of interest can then be visualized by scanning a chip with the use of a microarray scanner. The entire procedure can be performed in as less as 4-6 h, and thus this method provides several advantages over traditional western blotting.

Allergen Diversity and Abundance in Different Tissues of the Redclaw Crayfish (Cherax quadricarinatus).

Shellfish allergy affects ~2.5% of the global population and is a type I immune response resulting from exposure to crustacean and/or molluscan proteins. The Australian Redclaw crayfish (Cherax quadricarinatus) is a freshwater species endemic to and farmed in northern Australia and is becoming an aquaculture species of interest globally. Despite being consumed as food, allergenic proteins from redclaw have not been identified or characterised. In addition, as different body parts are often consumed, it is conceivable that redclaw tissues vary in allergenicity depending on tissue type and function. To better understand food-derived allergenicity, this study characterised allergenic proteins in various redclaw body tissues (the tail, claw, and cephalothorax) and how the stability of allergenic proteins was affected through cooking (raw vs. cooked tissues). The potential of redclaw allergens to cross-react and cause IgE-binding in patients allergic to other shellfish (i.e., shrimp) was also investigated. Raw and cooked extracts were prepared from each body part. SDS-PAGE followed by immunoblotting was performed to determine allergen-specific antibody reactivity to sarcoplasmic calcium-binding protein and hemocyanin, as well as to identify redclaw proteins binding to IgE antibodies from individual and pooled sera of shrimp-allergic patients. Liquid chromatography-mass spectrometry (LC/MS) was utilised to identify proteins and to determine the proportion within extracts. Known crustacean allergens were found in all tissues, with a variation in tissue distribution (e.g., higher levels of hemocyanin in the claw and cephalothorax than in the tail). The proportion of some allergens as a percentage of remaining heat-stable proteins increased in cooked tissues. Previously described heat-stable allergens (i.e., hemocyanin and sarcoplasmic calcium-binding protein) were found to be partially heat-labile. Immunoblotting indicated that shrimp-allergic patients cross-react to redclaw allergens. IgE-binding bands, analysed by LC/MS, identified up to 11 known shellfish allergens. The findings of this study provide fundamental knowledge into the diagnostic and therapeutic field of shellfish allergy.

Extended autoantibody panel in Turkish patients with early-stage systemic sclerosis: Coexpressions and their influences on clinical phenotypes.

BACKGROUND/AIM: To investigate the frequency and clinical relevance of an extended autoantibody profile in patients with systemic sclerosis (SSc). MATERIALS AND METHODS: In this cross-sectional study, serum from 100 consecutive patients was subjected to indirect immunofluorescence (IIF) (HEp-20-10/primate liver mosaic) and Systemic Sclerosis Profile by EUROIMMUN to evaluate anti-nuclear antibodies (ANA) and autoantibodies against 13 different autoantibodies in patients with SSc less than 3 years. RESULTS: Ninety-three of 100 patients were positive for ANA by IIF. Fifty-three patients showed single positivity, 26 anti-topoisomerase antibodies (anti-Scl70 ab), 16 anticentromere antibodies (ACAs), six anti-RNA polymerase III antibodies (anti-RNAPIII ab), one anti-Ku antibody, one anti-PM/Scl100 antibody, two anti-PM/Scl75 antibodies, one anti-Ro52 antibody, whereas 32 patients had multiple autoantibody positivities. Among classic SSc-specific autoantibodies, anti-Scl70 and anti-RNAPIII abs showed the highest cooccurrence (n = 4). One patient was simultaneously positive for anti-RNAPIII ab and ACA, and one was positive for ACA and anti-Scl70 ab. The clinical features were not statistically different between single and multiple autoantibody-positivity for classic SSc-specific autoantibodies (ACA, anti-Scl70 ab, and anti-RNAPIII ab), except for digital ulcer in the multiantibody positive ACA group (p = .019). CONCLUSION: Based on our results, coexpression of autoantibodies is not uncommon in SSc patients. Although autoantibodies specific to SSc in early disease show generally known clinical features, it remains to be investigated how the coexpression of autoantibodies will affect clinical presentation.

Immunoblot Criteria for Diagnosis of Lyme Disease: A Comparison of CDC Criteria to Alternative Interpretive Approaches.

The current Centers for Disease Control and Prevention (CDC) interpretive criteria for serodiagnosis of Lyme disease (LD) involve a two-tiered approach, consisting of a first-tier EIA, IFA, or chemiluminescent assay, followed by confirmation of positive or equivocal results by either immunoblot or a second-tier EIA. To increase overall sensitivity, single-tier alternative immunoblot assays have been proposed, often utilizing antigens from multiple Borrelia burgdorferi strains or genospecies in a single immunoblot; including OspA and OspB in their antigen panel; requiring fewer positive bands than permitted by current CDC criteria; and reporting equivocal results. Published reports concerning alternative immunoblot assays have used relatively small numbers of LD patients and controls to evaluate novel multi-antigen assays and interpretive criteria. We compared the two most commonly used alternative immunoblot interpretive criteria (labeled A and B) to CDC criteria using data from multiple FDA-cleared IgG and IgM immunoblot test kits. These single-tier alternative interpretive criteria, applied to both IgG and IgM immunoblots, demonstrated significantly more false-positive or equivocal results in healthy controls than two-tiered CDC criteria (12.4% and 35.0% for Criteria A and B, respectively, versus 1.0% for CDC criteria). Due to limited standardization and high false-positive rates, the presently evaluated single-tier alternative immunoblot interpretive criteria appear inferior to CDC two-tiered criteria.

Contribution of serology in congenital toxoplasmosis diagnosis: results from a 10-year French retrospective study.

This study aimed to evaluate different serological strategies for the postnatal diagnosis of congenital toxoplasmosis (CT) and establish a biological algorithm for CT diagnosis. The study analyzed serological data of immunoglobulins M, A, and G (IgM, IgA, IgG) performed by immunoenzymatic and compared immunological profile (CIP) assays in 668 newborns with CT diagnosis across four testing periods: P1 (D0- D10), P2 (D11-D35), P3 (D36-D45), and P4 (>D45). Forty-nine percent of the 668 CT cases were diagnosed during P1 and 34%, 4%, and 12% during P2, P3, and P4, respectively. CIP assays detected neosynthetized IgMs/IgGs in 98% of CT cases diagnosed during P1, while IgMs and IgAs were detected in 90% and 57% of CT cases diagnosed during P2 and in 88% and 67% of diagnoses made during P3, respectively. Detection of neosynthesized IgMs/IgGs, IgMs, and IgAs by immunoassay contributed to CT diagnosis in 81%, 77%, and 60% of cases, respectively. In total, 46% of serum samples were positive for all three parameters, 27% for two, and 27% for one of the three. The study recommends using the CIP assay as standard during P1 for CT diagnosis and IgM and IgA immunoassays after P1. A clinical and biological follow-up in a specialized center with a close collaboration between biologists and clinicians is highly recommended to increase the chances of early diagnosis. Overall, this study provides useful information for the development of a biological algorithm for CT diagnosis, which can aid in early detection and appropriate treatment of this disease.

Best practices in assessing cardiac protein O-GlcNAcylation by immunoblot.

The modification of serine and threonine amino acids of proteins by O-linked N-acetylglucosamine (O-GlcNAc) regulates the activity, stability, function, and subcellular localization of proteins. Dysregulation of O-GlcNAc homeostasis is well established as a hallmark of various cardiac diseases, including cardiac hypertrophy, heart failure, complications associated with diabetes, and responses to acute injuries such as oxidative stress and ischemia-reperfusion. Given the limited availability of site-specific O-GlcNAc antibodies, studies of changes in O-GlcNAcylation in the heart frequently use pan-O-GlcNAc antibodies for semiquantitative evaluation of overall O-GlcNAc levels. However, there is a high degree of variability in many published cardiac O-GlcNAc blots. For example, many blots often have regions that lack O-GlcNAc positive staining of proteins either below 50 or above 100 kDa. In some O-GlcNAc blots, only a few protein bands are detected, while in others, intense bands around 75 kDa dominate the gel due to nonspecific IgM band staining, making it difficult to visualize less intense bands. Therefore, the goal of this study was to develop a modifiable protocol that optimizes O-GlcNAc positive banding of proteins in cardiac tissue extracts. We showed that O-GlcNAc blots using CTD110.6 antibody of proteins ranging from <30 to ∼450 kDa could be obtained while also limiting nonspecific staining. We also show that some myofilament proteins are recognized by the CTD110.6 antibody. Therefore, by protocol optimization using the widely available CTD110.6 antibody, we found that it is possible to obtain pan-O-GlcNAc blots of cardiac tissue, which minimizes common limitations associated with this technique.NEW & NOTEWORTHY The post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is recognized as mediating cardiac pathophysiology. However, there is considerable variability in the quality of O-GlcNAc immunoblots used to evaluate changes in cardiac O-GlcNAc levels. Here we show that with relatively minor changes to a commonly used protocol it is possible to minimize the intensity of nonspecific bands while also reproducibly generating O-GlcNAc immunoblots covering a range of molecular weights from <30 to ∼450 kDa.

Noncanonical NLRP3 Inflammasome Activation: Standard Protocols.

The canonical activation of multimeric inflammasomes usually occurs through caspase-1 activation, and it is characterized by the presence of extracellular IL-1ß and IL-18 or measuring danger signal proteins, such as HMGB1 using enzyme-linked immunosorbent assay (ELISA) or Western blots; these assays differentiate non-cleaved and cleaved forms of these two cytokines (the cleaved form is the mature and active form). Similar techniques can be used to assess noncanonical inflammasome activation. Real-time PCR can measure the relative mRNA expression for a specific gene, whereas Western blots or immunocytochemistry can detect the presence of proteins by binding of specific antibodies to their antigens in biological samples. Moreover, noncanonical inflammasome activation can be evaluated through the cleavage of the amino and the carboxy terminals of one important component, gasdermin D (GSDMD), whose cleavage induces its pyroptotic activity. Thus, the analysis of cleaved GSDMD is an ideal pathway to study the noncanonical inflammasome. ELISA and immunoblot can be performed on cell culture supernatants or cell extracts.

Methods to Study Inflammasome Activation in the Central Nervous System: Immunoblotting and Immunohistochemistry.

The inflammasome is a multiprotein complex that is responsible for mounting an innate immune response through the activation of caspase-1 and the cleavage of interleukin-1ß. This multiprotein complex plays an important role in a variety of central nervous system (CNS) diseases and conditions such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, stroke, and traumatic brain injury, among others. Here we describe methodological procedures to carry out immunoblotting and immunohistochemical techniques used to study inflammasome signaling in CNS tissues (brain and spinal cord).

Use of Native-PAGE for the Identification of Epichaperomes in Cell Lines.

Epichaperomes are disease-associated pathologic scaffolds, composed of tightly bound chaperones, co-chaperones, and other factors. They mediate anomalous protein-protein interactions inside cells, which aberrantly affects the function of protein networks, and in turn, cellular phenotypes. Epichaperome study necessitates the implementation of methods that retain these protein complexes in their native cellular states for analysis. Here we describe a protocol for detection and composition analysis of epichaperomes in cell homogenates through native polyacrylamide gel electrophoresis.

Characterization of trans-3-Methylglutaconyl CoA-Dependent Protein Acylation.

3-methylglutaconyl (3MGC) CoA hydratase (AUH) is the leucine catabolism pathway enzyme that catalyzes the hydration of trans-3MGC CoA to 3-hydroxy, 3-methylglutaryl (HMG) CoA. In several inborn errors of metabolism (IEM), however, metabolic dysfunction can drive this reaction in the opposite direction (the dehydration of HMG CoA). The recent discovery that trans-3MGC CoA is inherently unstable and prone to a series of non-enzymatic chemical reactions provides an explanation for 3MGC aciduria observed in these IEMs. Under physiological conditions, trans-3MGC CoA can isomerize to cis-3MGC CoA, which is structurally poised to undergo intramolecular cyclization with the loss of CoA, generating cis-3MGC anhydride. The anhydride is reactive and has two potential fates; (a) hydrolysis to yield cis-3MGC acid or (b) a reaction with lysine side-chain amino groups to 3MGCylate substrate proteins. An antibody elicited against a 3MGC hapten was employed to investigate protein acylation in incubations containing recombinant AUH, HMG CoA, and bovine serum albumin (BSA). The data obtained show that, as AUH dehydrates HMG CoA to trans-3MGC CoA, BSA is acylated. Moreover, α-3MGC IgG immunoblot signal intensity correlates with AUH concentration, HMG CoA substrate concentration, and incubation time. Thus, protein 3MGCylation may contribute to the phenotypic features associated with IEMs that manifest 3MGC aciduria.

The diagnostic and prognostic value of a line immunoblot assay in Taiwanese patients with systemic sclerosis.

BACKGROUND AND AIMS: We aimed to evaluate the diagnostic performance and prognostic value of disease-specific antibodies and anti-Ro52 using a commercial line immunoblot assay (LIA) in Taiwanese patients with systemic sclerosis (SSc). MATERIALS AND METHODS: We retrospectively enrolledall individuals at the Taichung Veterans General Hospital. We evaluated the diagnostic performance of LIA, anti-nuclear antibody (ANA) by indirect immunofluorescence (IIF) and also the association between the autoantibodies and the clinical phenotype using multivariable logistic regression. RESULTS: The LIA exhibited a sensitivity of 65.4% and a specificity of 65.4%, at the optimal cutoff values of 2 + signal intensity. By taking the result of ANA into consideration, the optimal cutoff point was redefined as 1+. We observed a higher risk of diffuse cutaneous SSc (dcSSc) in those with negative autoantibodies, positive anti-Scl-70, anti-RNA polymerase III, and anti-Ro-52. Interstitial lung disease (ILD) was associated with negative autoantibodies, as well as positive anti-Scl-70 and anti-Ro52. Anti-Ro52 positivity was also associated with pulmonary arterial hypertension (PAH) and gastrointestinal tract involvement. CONCLUSION: The presence of anti-Ro52 or the absence of SSc-specific autoantibodies may potentially indicate advanced diseases in patients with SSc. The incorporation of both IIF and LIA testing may improve the diagnostic specificity of SSc.