Indirect Immunofluorescence Assay Research Articles

Trichinella spiralis galectin binding to toll-like receptor 4 induces intestinal inflammation and mediates larval invasion of gut mucosa

Previous studies showed that Trichinella spiralis galectin (Tsgal) facilitates larval invasion of intestinal epithelium cells (IECs). However, IEC proteins binding with Tsgal were not identified, and the mechanism by which Tsgal promotes larval invasion is not clear. Toll-like receptors (TLRs) are protein receptors responsible for recognition of pathogens. The aim of this study was to investigate whether recombinant Tsgal (rTsgal) binds to TLR-4, activates inflammatory pathway in gut epithelium and mediates T. spiralis invasion. Indirect immunofluorescence (IIF), GST pull-down and co-immunoprecipitation (Co-IP) assays confirmed specific binding between rTsgal and TLR-4 in Caco-2 cells. qPCR and Western blotting showed that binding of rTsgal with TLR-4 up-regulated the TLR-4 transcription and expression in Caco-2 cells, and activated p-NF-κB p65 and p-ERK1/2. Activation of inflammatory pathway TLR-4/MAPK-NF-κB by rTsgal up-regulated pro-inflammatory cytokines (IL-1β and IL-6) and down-regulated anti-inflammatory cytokine TGF-β in Caco-2 cells, and induced intestinal inflammation. TAK-242 (TLR-4 inhibitor) and PDTC (NF-κB inhibitor) significantly inhibited the activation of TLR-4 and MAPK-NF-κB pathway. Moreover, the two inhibitors also inhibited IL-1β and IL-6 expression, and increased TGF-β expression in Caco-2 cells. In T. spiralis infected mice, the two inhibitors also inhibited the activation of TLR-4/MAPK-NF-κB pathway, ameliorated intestinal inflammation, impeded larval invasion of gut mucosa and reduced intestinal adult burdens. The results showed that rTsgal binding to TLR-4 in gut epithelium activated MAPK-NF-κB signaling pathway, induced the expression of TLR-4 and pro-inflammatory cytokines, and mediated larval invasion. Tsgal might be regarded as a candidate molecular target of vaccine against T. spiralis enteral invasive stage.

Prevalence of serum MOG antibody and AQP4 antibody in optic neuritis after SARS-CoV-2 infection.

To evaluate the prevalence of serum myelin oligodendrocyte glycoprotein antibody (MOG-Ab) and aquaporin-4 antibody (AQP4-Ab) in optic neuritis (ON) patients after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by cell-based indirect immunofluorescence assay (CBA). In this prospective case series study, 35 patients clinically diagnosed as ON and laboratory-confirmed SARS-CoV-2 infection from 8 December 2022 to 8 February 2023 were included. All patients' clinical and laboratory data were collected and analyzed. The mean age of the 35 patients (46 eyes) was 38.2 years (ranging from 6 to 69 years), and 17 cases were female patients. Thirty-three and two cases showed positive SARS-CoV-2 RNA test results before or shortly after ON onset, respectively. ON occurred unilaterally in 24 cases and bilaterally in 11 cases. Ophthalmic examination revealed swollen optic disc in 37 eyes, normal optic disc in 6 eyes, and temporally or wholly paled optic disc in 3 eyes. CBA revealed seropositive MOG-Ab in 10 cases and AQP4-Ab in 2 cases, respectively, of which 2 AQP4-Ab-seropositive cases and 1 MOG-Ab-seropositive case had a past medical history of ON. Most ON patients showed a rapid and dramatic response to pulse steroid therapy. The median of BCVA at the onset and at the last follow-up was 20/500 (ranging from light perception to 20/20) and 20/67 (ranging from counting fingers to 20/20), respectively. Serum MOG-Ab and AQP4-Ab were detected in 28.6% (10/35) and 5.7% (2/35) ON cases after SARS-CoV-2 infection. SARS-CoV-2 infection may trigger an onset or a relapse of ON, as well as the production of MOG-Ab.

Characterization of a novel dipeptidyl peptidase 1 of Trichinella spiralis and its participation in larval invasion

The research aimed to describe a new Trichinella spiralis dipeptidyl peptidase 1 (TsDPP1) and investigate its functions in the larval invasion of intestinal epithelial cells (IECs). The gene TsDPP1 was successfully replicated and produced in Escherichia coli BL21 (DE3), showing a strong immune response. TsDPP1 was detected in diverse stages of T. spiralis and showed significant expression in the intestine infective larvae (IIL) and adult worms at 6 days post infection, as confirmed by qPCR and Western blot analysis. The primary localization of TsDPP1 in this parasite was observed in cuticles, stichosomes, and embryos by using the indirect immunofluorescence assay (IIFA). rTsDPP1 exhibited the enzymatic function of natural dipeptidyl peptidase and showed specific binding to IECs, and the binding site was found to be localized on cell membrane. Following transfection with dsRNA-TsDPP1, the expression of TsDPP1 mRNA and protein in muscle larvae (ML) were decreased by approximately 63.52 % and 58.68 %, correspondingly. The activity of TsDPP1 in the ML and IIL treated with dsRNA-TsDPP1 was reduced by 42.98 % and 45.07 %, respectively. The acceleration of larval invasion of IECs was observed with rTsDPP1, while the invasion was suppressed by anti-rTsDPP1 serum. The ability of the larvae treated with dsRNA-TsDPP1 to invade IECs was hindered by 31.23 %. In mice infected with dsRNA-treated ML, the intestinal IIL, and adults experienced a significant decrease in worm burdens and a noticeable reduction in adult female length and fecundity compared to the PBS group. These findings indicated that TsDPP1 significantly impedes the invasion, growth, and reproductive capacity of T. spiralis in intestines, suggesting its potential as a target for anti-Trichinella vaccines.

Anti-SSA Ro52 and anti-Ro60 autoantibodies: association with clinical phenotypes.

Anti-SSA autoantibodies can be differentiated according to their antigenic target proteins as anti-Ro60 (60 kDa) or anti-Ro52 (52 kDa). Anti-SSA(Ro60) antibodies are clearly associated with connective tissue diseases (CTD), but the clinical significance of anti-SSA(Ro52) antibodies remains unclear. The aim of the present study was to analyse the disease phenotype of patients with anti-Ro52 and/or anti-Ro60 antibodies. A multicentre, cross-sectional study was carried out of positive anti-Ro52 and/or Ro60 antibodies patients followed at 10 Rheumatology centres from January 2018 until December 2021. Patients were categorised into 3 groups: group 1 (Ro52+/Ro60-); group 2 (Ro52-/Ro60+); group 3 (Ro52+/Ro60+). Antinuclear antibodies were evaluated by indirect immunofluorescence assay and further screened for anti-extractable nuclear antigen (ENA) antibodies. Demographicsand clinical data were compared between the 3 groups, by patients' medical chart review. Univariate analysis was performed and subsequently logistic regression was used to identify intergroup differences and calculate the odds ratio with a 95% confidence interval (95% CI). We included 776 patients [female: 83.1%; median age: 59 (46-71) years]. Groups 1, 2, and 3 comprised 31.1%, 32.6%, and 36.3% of the patients, respectively. Anti-Ro52 antibody alone was more frequently associated with non-rheumatic diseases, older age, and men (p<0.05). Among patients with CTD, the diagnosis of systemic lupus erythematosus is 3 and 2 times more prevalent in groups 2 and 3, respectively, than in group 1 [OR 2.8 (95% CI 1.60, 4.97), p<0.001; OR 2.2 (95% CI 1.28, 3.86), p<0.01]. In group 2, the diagnosis of undifferentiated CTD is more frequent than in the other groups. Group 1 was more frequently associated with inflammatory myositis than group 2 [OR 0.09 (95% CI 0.01, 0.33), p<0.001] or group 3 [OR 0.08 (95% CI 0.01, 0.29), p<0.001]. Group 1 was also more frequently associated with arthritis (p<0.01), interstitial lung disease (p<0.01), and myositis (p<0.01). Anti-Ro52+ antibody alone is frequently found in patients with non-rheumatic diseases. In addition, anti-Ro52+ antibody is also prevalent in patients with CTD and associated with clinical phenotypes that are different from anti-Ro60+ antibody.

Phenol-Soluble Modulin α3 Stimulates Autophagy in HaCaT Keratinocytes.

Phenol-soluble modulins (PSMs) are pore-forming toxins (PFTs) produced by staphylococci. PSMs exert diverse cellular effects, including lytic, pro-apoptotic, pro-inflammatory and antimicrobial actions. Since the effects of PSMs on autophagy have not yet been reported, we evaluated the autophagic activity in HaCaT keratinocytes treated with recombinant PSMα3. The autophagic flux and levels of autophagic marker proteins were determined using Western blot analysis. Subcellular localization of LC3B and Beclin-1 was investigated using an indirect immunofluorescence assay. The ultrastructural features of control and PSMα3-treated cells were evaluated via transmission electron microscopy. Cytoplasmic acidification was measured via acridine orange staining. Phosphorylation levels of protein kinases, implicated in autophagy regulation, were studied using a phospho-kinase array and Western blot analysis. PSMα3 facilitated the intracellular redistribution of LC3B, increased the average number of autophagosomes per cell, promoted the development of acidic vesicular organelles, elevated the levels of LC3B-II, stimulated autophagic flux and triggered a significant decrease in the net autophagic turnover rate. PSMα3 induced the accumulation of autophagosomes/autolysosomes, amphisomes and multilamellar bodies at the 0.5, 6 and 24 h time points, respectively. The phospho-Akt1/2/3 (T308 and S473), and phospho-mTOR (S2448) levels were decreased, whereas the phospho-Erk1/2 (T202/Y204 and T185/Y187) level was increased in PSMα3-treated cells. In HaCaT keratinocytes, PSMα3 stimulates autophagy. The increased autophagic activity elicited by sub-lytic PSM concentrations might be an integral part of the cellular defense mechanisms protecting skin homeostasis.

Prevalence and sociodemographic correlates of antinuclear antibody testing by indirect immunofluorescence or solid-phase assays in a Spanish population: the Camargo Cohort

Autoantibodies are the hallmark of autoimmunity, and specifically, antinuclear antibodies (ANA) are one of the most relevant antibodies present in systemic autoimmune diseases (AID). In the present study, we evaluate the relationship between ANA and sociodemographic and biobehavioral factors in a population with a low pre-test probability for systemic AID. ANA were determined in serum samples at baseline visit from 2997 participants from the Camargo Cohort using indirect immunofluorescence assay, and two solid phase assays (SPA), addressable laser bead immunoassay, and fluorescence enzyme immunoassay. Sociodemographic and biobehavioral features of the subjects were obtained at baseline visit using a structured questionnaire. The prevalence of ANA positive results was significantly higher when indirect immunofluorescence assay was used as screening method in comparison with SPAs, being higher in females, older subjects, and those with higher C-reactive protein levels. Considering biobehavioral features, the prevalence was higher in those individuals with a sedentary lifestyle, and in ex- and non-alcohol users. Moreover, considering the relevance of the antibody load using ANA Screen, the prevalence of the antibody load also increased with age, especially in females. In conclusion, the prevalence of ANA varies depending on sociodemographic and biobehavioral features of the subjects, which could be relevant specifically in a population with a low pre-test probability for systemic AIDs.

A bacterium-like particle vaccine displaying protective feline herpesvirus 1 antigens can induce an immune response in mice and cats

Feline herpesvirus 1 (FHV-1) is a highly transmissible virus that mainly causes ocular and upper respiratory infections in cats and seriously threatens the health of domestic cats and captive or wild cats (such as tigers, cheetahs, and lions). Vaccination is crucial to reduce the incidence rate and mortality of cats infected with FHV-1. In this study, three bacterium-like particles (BLPs) displaying the gB, gC, and gD proteins of FHV-1 were constructed based on a gram-positive enhancer matrix-protein anchor (GEM-PA) surface display system. Indirect immunofluorescence assay, western blot, and electron microscopy results showed that gB, gC or gD protein of FHV-1 was successfully displayed on the surface of GEM particles. Additionally, we designed one more BLPs, designated gB&gC&gD-GEM, which consisted of a mixture of gB-GEM, gC-GEM, and gD-GEM at a protein content ratio of 1:1:1. Mice were immunized with the four BLPs mixed with Gel02 adjuvant, and the results indicated that neutralizing antibody level in the gB&gC&gD-GEM group was superior than those in the other groups. Moreover, gB&gC&gD-GEM significantly increased the secretion of cytokines, as well as the activation and maturation of B cells. It also boosted the production of central memory T cells among CD4 + and CD8 + T cells. Moreover, gB&gC&gD-GEM mixed with Gel02 adjuvant provoked an antibody response in cats. In conclusion, the BLPs vaccine prepared from gB&gC&gD-GEM induced specific humoral and cellular immune responses to FHV-1 and be used as a potential vaccine candidate for the control of FHV-1 infection in cats.

A Novel Mechanism of hPRL-G129R, a Prolactin Antagonist, Inhibits Human Breast Cancer Cell Proliferation and Migration.

Prolactin (PRL) and its receptor, PRLR, are closely related to the occurrence and development of breast cancer. hPRL-G129R, an hPRLR antagonist, has been found to induce apoptosis in breast cancer cells via mechanisms currently unknown. Recent studies have indicated that PRLR exhibits dual functions based on its membrane/nucleus localization. In that context, we speculated whether hPRL-G129R is a dual-function antagonist. We studied the internalization of the hPRLR-G129R/PRLR complex using indirect immunofluorescence and Western blot assays. We found that hPRL-G129R not only inhibited PRLR-mediated intracellular signaling at the plasma membrane, but also blocked nuclear localization of the receptor in T-47D and MCF-7 cells in a time-dependent manner. Clone formation and transwell migration assays showed that hPRL-G129R inhibited PRL-driven proliferation and migration of tumor cells in vitro. Further, we found that increasing concentrations of hPRL-G129R inhibited the nuclear localization of PRLR and the levels of signal transducer and activator of transcription (STAT) 5 in tumor-bearing mice and hPRL-G129R also exerted an antiproliferative effect in vivo. These results indicate that hPRL-G129R is indeed a dual-function antagonist. This study lays a foundation for exploring and developing highly effective agents against the proliferation and progression of breast malignancies.

Laboratory evaluation of ELISA and indirect immunofluorescence assay in response to emergence of Japanese encephalitis virus genotype IV in Australia.

The unexpected recent emergence of Japanese encephalitis virus (JEV) genotype IV in multiple southern states of Australia necessitated an evaluation of JEV serological tests suitable for diagnosing acute infection and for seroprevalence studies. This study examined the analytical and clinical performance of two high-throughput JEV assays, Euroimmun immunofluorescence assay (IFA) and Euroimmun enzyme-linked immunosorbent assay (ELISA), across four cohorts; (1) surveillance of piggery workers in outbreak areas, (2) surveillance of residents in outbreak areas, (3) acute JEV infection and (4) post-JEV vaccination. ELISA and IFA IgM demonstrated minimal cross-reactivity (0-1.8%) with other endemic flaviviruses, with high sensitivity (100%) for acute JEV infection in this low endemicity setting. Differences in IgG serodynamics between the two assays suggest convalescent and paired testing with IgM are critical in diagnosing acute infection. High assay concordance was observed between ELISA and IFA when used in serosurveillance (97.4% agreement, Cohen' κ 0.74 [95% CI 0.614-0.860]) and vaccination cohorts (91.1% agreement, Cohen's κ 0.806 [95% CI 0.672-0.941]). In conclusion, this study highlights the clinical & epidemiological applications and limitations of these two commercial JEV assays.

Protective immunity induced by DNA vaccine containing TgGRA35, TgGRA42, and TgGRA43 against Toxoplasma gondii infection in Kunming mice.

Toxoplasma gondii can cause congenital infection and abortion in humans and warm-blooded animals. T. gondii dense granule proteins, GRA35, GRA42, and GRA43, play a critical role in the establishment of chronic infection. However, their potential to induce protective immunity against T. gondii infection remains unexplored. This study aimed to test the efficacy of a DNA vaccine encompassing GRA35, GRA42, and GRA43 in inducing protective immunity against the highly virulent T. gondii RH strain (type I) and the brain cyst-forming PRU strain (type II). The eukaryotic plasmids pVAX-GRA35, pVAX-GRA42, and pVAX-GRA43 were constructed and formulated into two- or three-gene cocktail DNA vaccines. The indirect immunofluorescence assay (IFA) was used to analyze their expression and immunogenicity. Mice were immunized with a single-gene, two-genes, or multicomponent eukaryotic plasmid, intramuscularly. We assessed antibody levels, cytotoxic T-cell (CTL) responses, cytokines, and lymphocyte surface markers by using flow cytometry. Additionally, mouse survival and cyst numbers in the brain of mice challenged 1 to 2 months postvaccination were determined. Specific humoral and cellular immune responses were elicited in mice immunized with single-, two-, or three-gene cocktail DNA vaccine, as indicated by significant increases in serum antibody concentrations of total IgG, IgG2a/IgG1 ratio, cytokine levels (IFN-γ, IL-2, IL-12, IL-4, and IL-10), lymphocyte proliferation, lymphocyte populations (CD4+ and CD8+ T lymphocytes), CTL activities, and survival, as well as decreased brain cysts, in comparison with control mice. Moreover, compared with pVAX-GRA35 + pVAX-GRA42, pVAX-GRA42 + pVAX-GRA43, or pVAX-GRA35 + pVAX-GRA43, multicomponent DNA vaccine with three genes (pVAX-GRA35 + pVAX-GRA42 + pVAX-GRA43) induced the higher humoral and cellular immune responses, including serum antibody concentrations, cytokine levels, lymphocyte proliferation, lymphocyte populations, CTL activities and survival, resulting in prolonged survival time and reduced brain cyst loads. Furthermore, mice immunized with pVAX-GRA35 + pVAX-GRA42, pVAX-GRA42 + pVAX-GRA43, or pVAX-GRA35 + pVAX-GRA43 showed greater Th1 immune responses and protective efficacy than the single-gene-vaccinated groups. These results demonstrate that TgGRA35, TgGRA42, or TgGRA43 are vaccine candidates against T. gondii infection, and the three-gene DNA vaccine cocktail conferred the strongest protection against T. gondii infection.

Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection

Eimeria necatrix is a high pathogenic pathogen, which seriously endangers the poultry industry. The surface antigens (SAGs) of Apicomplexa are a kind of membrane protein anchored on the surface of the parasites through its carboxyl terminal glycosylphosphatidylinositol (GPI) structure. However, little is known about GPI-linked surface proteins in E. necatrix. In the present work, the E. necatrix sag gene (Ensag-CAP) was amplified and cloned for expression of the recombinant protein (rEnSAG-CAP). The full length Ensag-CAP gene was 813 bp, coding 270 amino acids with a predicated molecular weight of 28.86 kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG-CAP was about 32 kDa and mainly expressed in a soluble form. Western blot analysis indicated that the rEnSAG-CAP could be recognized by anti-rEnSAG-CAP monoclonal antibody (anti-rEnSAG-CAP McAb) and the convalescent serum of chicken infected with E. necatrix. Native protein of EnSAG-CAP was detected in second-generation merozoites (MZ-2) using anti-rEnSAG-CAP polyclonal antibody (anti-rEnSAG-CAP pAb). The findings from the indirect immunofluorescence assay and enzyme digestion utilizing Bacillus cereus phosphoinositide-specific phospholipase C (PI-PLC) revealed that EnSAG-CAP predominantly localized at the surfaces of SZ and MZ-2 via a GPI anchor. It was observed that EnSAG-CAP can be cleaved from MZ-2 by PI-PLC. Real-time quantitative PCR (qPCR) analysis showed that transcript levels of Ensag-CAP in MZ-2 was significantly higher than that in SZ (P < 0.05). The anti-rEnSAG-CAP McAb in vitro could significantly inhibit the sporozoite invasion into MDBK cells (P < 0.01), which suggests that the protein might participate in sporozoite invasion into MDBK cells. rEnSAG-CAP afforded an immune protection against E. necatrix. The ACI value was 164.99 in the chickens immunized with 200 µg rEnSAG-CAP. Chickens immunized with rEnSAG-CAP had a significantly higher antigen-specific serum IgY response (P < 0.0001). The data indicates that EnSAG-CAP could serve as a potential candidate antigen for the development of a recombinant coccidiosis vaccine.

Serological evidence for potential yellow fever virus infection in non-human primates, southeastern Mexico

BackgroundArthropod-borne flaviviruses like dengue virus (DENV) and yellow fever virus (YFV) are major human pathogens. In Latin America, YFV is maintained in sylvatic cycles involving non-human primates (NHP) and forest-dwelling mosquitos. YFV supposedly does not circulate north of Panama.MethodsWe conducted a serologic study for flaviviruses and other emerging viruses in NHP from southeastern Mexico. A total of thirty sera of black-handed spider monkeys (Ateles geoffroyi, n = 25), black howler monkeys (Alouatta pigra, n = 3), and mantled howler monkeys (Al. palliata, n = 2) sampled in 2012 and 2018 were screened by an indirect immunofluorescence assay (IFA) to detected IgG antibodies against DENV, YFV, Zika virus (ZIKV), West Nile virus (WNV), Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, Middle East respiratory syndrome coronavirus, and Zaire Ebola virus, and confirmed by plaque reduction neutralization tests (PRNT90) representing all mosquito-borne flavivirus serocomplexes circulating in the Americas.ResultsA total of 16 sera (53.3%; 95% CI, 34.3–71.7) showed IFA reactivity to at least one tested flavivirus with end-point titers ranging from 1:100 to 1:1000. No serum reacted with other viruses. Monotypic and high mean PRNT90 endpoint YFV titers of 1:246 were found in 3 black-handed spider monkey sera (10.0%; 95% CI, 2.1–26.5) sampled in 2018 in Tabasco, compared to all other flaviviruses tested. Monotypic endpoint PRNT90 titers of 1:28 for Ilheus virus and 1:22 for WNV in serum of black howler monkeys sampled in 2018 in Tabasco suggested additional flavivirus exposure.ConclusionsOur findings may suggest unnoticed YFV circulation. Intensification of YFV surveillance in NHP and vectors is warranted in Mexico and potentially other areas considered free of yellow fever.

Adopting the International Consensus on ANA Patterns (ICAP) classification for reporting: the experience of Italian clinical laboratories.

The indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) is still considered the reference method to detect anti-nuclear antibodies (ANA) because of its high sensitivity and represents a relevant tool for the diagnosis of autoimmune rheumatic diseases. During the last decade, the International Consensus on ANA Patterns (ICAP) initiative promoted harmonization and understanding of HEp-2 IFA staining pattern nomenclature, as well as promoting their use in patient care by providing interpretation for HEp-2 IFA test results. In conjunction with a nationwide survey on the evolution of autoantibody diagnostics in autoimmune rheumatic diseases, we focused on the adherence of the Italian laboratories to the ICAP nomenclature analyzing its lights and shadows. The recent ICAP-oriented report, largely used today among Italian laboratories, also represents a further step in harmonizing and improving communication with the clinicians, adding value to laboratory findings and helping with critical clinical decisions.

Effect of COVID-19 on ANA positivity in the Indian population

The prevalence of Antinuclear Antibodies (ANA) positivity has increased following Covid-19 infection. This study investigates the ANA positivity rate by comparing ANA data from two distinct years, 2019 (a pre-Covid year) and 2022 (a post-Covid year). This retrospective study analyzes and compares ANA Indirect Immunofluorescence Assay (IFA) data for the years 2019 and 2022 across various parameters, including age, gender, prevalence rate, positivity rate by grade, and patterns of ANA. In the post-Covid year 2022, there was a notable increase in both the total suspected cases and the ANA-positive cases, amounting to approximately a 30% rise. Positivity rates were observed to increase with age, and a female preponderance was noted in both years. Nuclear speckled patterns remained the most common in both time periods. The post-Covid pandemic period has witnessed a significant role of immune modulation in the development of autoimmunity. This phenomenon could potentially be attributed to Molecular Mimicry, the production of Autoantibodies upon exposure to Viral epitopes through the generation of Neutrophil Extracellular Traps (NETs), or via Toll-like Receptor (TLR) pathways of immune modulation, which may activate latent autoimmunity.

Ssn6 Interacts with Polar Tube Protein 2 and Transcriptional Repressor for RNA Polymerase II: Insight into Its Involvement in the Biological Process of Microsporidium Nosema bombycis.

Nosema bombycis is a representative species of Microsporidia, and is the pathogen that causes pebrine disease in silkworms. In the process of infection, the polar tube of N. bombycis is injected into the host cells. During proliferation, N. bombycis recruits the mitochondria of host cells. The general transcriptional corepressor Ssn6 contains six tetratricopeptide repeats (TPR) and undertakes various important functions. In this study, we isolated and characterized Nbssn6 of the microsporidium N. bombycis. The Nbssn6 gene contains a complete ORF of 1182 bp in length that encodes a 393 amino acid polypeptide. Indirect immunofluorescence assay showed that the Ssn6 protein was mainly distributed in the cytoplasm and nucleus at the proliferative phase of N. bombycis. We revealed the interaction of Nbssn6 with polar tube protein 2 (Nbptp2) and the transcriptional repressor for RNA polymerase II (Nbtrrp2) by Co-IP and yeast two-hybrid assays. Results from RNA interference further confirmed that the transcriptional level of Nbptp2 and Nbtrrp2 was regulated by Nbssn6. These results suggest that Nbssn6 impacts the infection and proliferation of N. bombycis via interacting with the polar tube protein and transcriptional repressor for RNA polymerase II.

Longitudinal study of patients with discrepant results in CLIFT and a solid-phase dsDNA antibody assay: does a gold standard dsDNA assay exist?

ObjectiveAntidouble-stranded DNA (dsDNA) antibodies are essential for diagnosis and follow-up of systemic lupus erythematous (SLE). To ensure the best diagnostic approach, most healthcare laboratories opt for a combination of highly...

Diversity of Non-Influenza Respiratory Viruses Associated with Influenza-Like Illness during 2009 pre and pandemic periods in Sao Paulo, Brazil, a Historical Overview

Background and objective: During the 2009 influenza A (H1N1) pandemic, 60% of the respiratory specimens collected from patients under surveillance for influenza-like illness (ILI) in São Paulo, Brazil, tested negative for influenza A and B by Real-Time RT-PCR (RT-qPCR) assays. In this retrospective study, we identified the diversity of other respiratory viruses associated with ILI during the pandemic period and added the benefit of RT-qPCR for their detection. Methods: Five-hundred and forty-two influenza RT-qPCR negative respiratory specimens from ILI patients collected from Jan - Dec 2009, that also tested negative by a commercial Indirect Immuno Fluorescence (IIF) assay for the Respiratory Syncytial Virus (RSV), Parainfluenza (PIV) PIV1, PIV2, PIV 3 and Adenovirus (AdV) were retested by a panel of RT-qPCR assays for the other respiratory viruses, including RSV, Human Metapneumovirus (HMPV), PIV 1-4, AdV, Human Coronaviruses (HCoV) OC43, 229E, NL63 and HKU1, Rhinovirus (RV), Enterovirus (EV) and Human Bocavirus (HBoV). Results: Of the 542 negative specimens, 428 (78%) were positive by RT-qPCR for another respiratory virus: In descending order: RV (149, 27%); RSV (41, 8%); AdV (32, 6%); HMPV (29, 5%); PIV3 (19, 4%); PIV1 (3, 1%); HCoV-OC43 (9, 2%); HBoV (9, 2%); PIV4 (6, 1%); HCoV-HKU1 (6, 1%); EV (5, 1%); HCoV-229E 4 (4, 1%); HCoV-NL63 (3, 1%). Co-detections of two viruses (98, 18%) and three viruses (12, 2%) were common. Conclusion: This retrospective study revealed a high prevalence of respiratory viruses other than influenza associated with ILI during the pandemic period and highlights the superiority of RT-qPCR over IIF for ILI surveillance.

CDR2 and CDR2L line blot performance in PCA-1/anti-Yo paraneoplastic autoimmunity.

Purkinje cytoplasmic autoantibody type 1 (PCA-1)/anti-Yo autoimmunity is a common high-risk paraneoplastic neurological disorder, traditionally attributed antigenically to cerebellar degeneration-related protein 2 (CDR2), predominantly affecting women with gynecologic or breast adenocarcinoma. Single-modality CDR2 testing may produce false-positive results. We assessed the performance characteristics of the more recently purported major PCA-1/Yo antigen, CDR2-like (CDR2L), side by side with CDR2, in a line blot format. CDR2 and CDR2L were tested in six specimen groups (serum and cerebrospinal fluid (CSF)). Group 1, PCA-1/Yo mouse brain indirect immunofluorescence assay (IFA) positives; Group 2, PCA-1/Yo IFA mimics; Group 3, suspected CDR2 line blot false positives; Group 4, consecutive patient samples tested for neural antibodies over 1 year; Group 5, healthy subject serums; and Group 6, polyclonal (non-specific) immunoglobulin G (IgG)-positive serums. Group 1: Of 64 samples tested, all but two were CDR2 positive (both CSF samples) and all were CDR2L positive. In individual patients, CDR2L values were always higher than CDR2. The two "CDR2L-only" positives were CSF samples with low titer PCA-1/Yo by IFA with serum negativity but with typical clinical phenotype. Group 2: All 51 PCA-1/Yo mimics were CDR2/CDR2L negative. Group 3: Nine samples [six of 1289 (0.47%) serums and three of 700 CSF samples (0.43%) were PCA-1/Yo IFA negative/CDR2 positive; two of the six available (serums from the same patient) were also CDR2L positive; the other four CDR2L negative had low CDR2 values (17-22). Group 4: Twenty-two patients had unexpected CDR2 or CDR2L positivity; none had tissue IFA positivity. Eleven of the 2,132 serum (0.5%) and three of the 677 CSF (0.4%) samples were CDR2 positive; median value was 19 (range, 11-48). Seven of the 2,132 serum (0.3%) and three of the 677 CSF (0.4%) samples were CDR2L positive; median value was 18 (range, 11-96). Group 5: All 151 healthy serum samples were negative. Group 6: One of the 46 polyclonal serum samples was CDR2L positive. Optimum overall performance was accomplished by requiring both CDR2 and CDR2L positivity in serum (sensitivity, 100%; and specificity, 99.9%) and positivity for CDR2L in CSF (sensitivity, 100%; and specificity, 99.6%). CDR2L provides additional PCA-1/anti-Yo sensitivity in CSF, and dual positivity with CDR2 provides additional specificity assurance in serum. Combining antigen-specific and tissue-based assays optimizes PCA-1/anti-Yo testing.

B-111 Parallel Detection of Antibodies against CDR2L and CDR2 Increases the Diagnostic Specificity in Serological Diagnostics of Paraneoplastic Cerebellar Degeneration

Abstract Background Onconeuronal anti-Yo antibodies are key diagnostic markers for paraneoplastic cerebellar degeneration (PCD) and often associated with gynaecologic/breast cancer. Cerebellar degeneration-related protein 2-like (CDR2L), but not cerebellar degeneration-related protein 2 (CDR2), has recently been shown to be the major anti-Yo target (1, 2). This study investigated if inclusion of CDR2L in diagnostic tests results in increased specificity for PCD. Methods Three panels were examined for reactivity against CDR2L and CDR2 by line blot analysis (EUROLINE). Panel A comprised 14 preselected sera from women with Yo-specific reactivity by indirect immunofluorescence assay (IFA) on rat cerebellum, presenting with cerebellar ataxia (n = 13) or peripheral neuropathy (n = 1) and underlying gynaecologic/breast cancers (n = 14). Panel B included 2132 non-selected consecutive serum/CSF samples from patients with neurological symptoms. Panel C consisted of 150 samples from healthy blood donors. Results In Panel A, all samples (100%) from patients with typical clinical characteristics for Yo-associated PCD showed strong positive reactivity to both CDR2L and CDR2. In Panel B, seven (0.3%) sera were anti-CDR2 negative, but anti-CDR2L positive, all of them from patients (6M/1F) without PCD and two with cancer or pre-cancerous condition. Eleven (0.5%) patients (9M/2F) were found to be anti-CDR2 positive, but anti-CDR2L negative, including two with cerebellar ataxia and three with cancer or pre-cancerous condition. In all 18 cases, IFA on rat cerebellum was negative, suggesting non-specific line blot reactivity to CDR2L or CDR2. In Panel C, 0/150 sera were anti-CDR2L and 1/150 anti-CDR2 positive, corresponding to specificities of 100% and 99.3%, respectively. The anti-CDR2 positive blood donor was male with negative IFA result. Conclusion Using CDR2L in addition to CDR2 increases the specificity in diagnosing anti-Yo-associated PCD. Positive line blot reactivity against both parameters seems to confirm the diagnosis. In contrast, results showing isolated reactivity against either CDR2L or CDR2 have to be interpreted carefully as they may apply to non-PCD cases.

B-073 The Clinical Utility of Reporting AC-2 Pattern With Anti-DFS70 Antibody

Abstract Background The dense fine speckled (DFS) pattern (AC-2) is often observed in antinuclear antibody (ANA) indirect immunofluorescence (IIF) assay, and the related anti-DFS70 antibody is known to be negatively associated with a systemic autoimmune rheumatic disease (SARD). Identifying the AC-2 pattern is challenging, as it could be generally confused with the AC-1, AC-4, AC-5, or AC-29 patterns displaying stained metaphase plates or speckled nuclei. Since the anti-DFS70 antibody is not included in routine autoantibody tests, the clinical utility of the AC-2 pattern and anti-DFS70 antibody is not adequately applied in the laboratory and clinical practice. We evaluated the prevalence of anti-DFS70 and other autoantibodies in samples presenting AC-2 pattern in ANA IIF assay. Additionally, we analyzed the association of anti-DFS70 antibodies and clinical features in patients with AC-2 patterns. Method We retrospectively reviewed the ANA IIF images of 1839 samples made by an automated ANA analyzer classified as AC-1, AC-2, AC-4, AC-5, and AC-29 patterns. AC-1, AC-2, AC-4, AC-5, and AC-29 patterns comprised 60.5% of all ANA positives (1839/3039). Among them, the AC-2 pattern was identified in 18.5% (341/1839) by two experts, and only 197 with residual samples were available for further evaluation. Those 197 samples were tested for anti-DFS70 antibodies with enzyme-linked immunosorbent assay (ELISA) and line immunoblot assay (LIA). They were also tested for 22 other autoantibodies (dsDNA, nucleosomes, histones, SS-A/Ro-52, SS-B, RNP/Sm, Sm, Mi-2α, Mi-2β, Ku, CENP-A, CENP-B, Sp100, PML, Scl-70, PM/Scl100, PM/Scl75, RP11, RP155, gp210, PCNA) with LIA. Clinical information from 197 patients was obtained from a retrospective review of electronic medical records. Results Anti-DFS70 antibody prevalence by ELISA and LIA was 32.0% (63/197) and 19.8% (39/197), respectively. The agreement percentage between the two assays was 81.7%, with a kappa value of 0.53 (95% confidence interval: 0.40–0.66). Of the 69 DFS70+ samples (as per ELISA or LIA), the coexistence of other autoantibodies was found in 81.2% (56/69). Of the 128 DFS70- samples, 90.6% (116/128) revealed other autoantibodies. The three most frequent autoantibodies (except anti-DFS70) in 197 patients with AC-2 patterns were anti-SS-A/Ro-52 (30.5%, 60/197), anti-nucleosomes (27.4%, 54/197), and anti-histones (20.3%, 40/197). There was no significant difference in the prevalence of the other 22 autoantibodies between DFS70+ and DFS70− patients. The anti-DFS70 prevalence in the SARD group was 33.3% (50/150), and in the non-SARD was 40.4% (19/47) without significant difference. However, of the 69 DFS70+ patients, the isolated anti-DFS70 was found more frequently in the non-SARD group (36.8%, 7/19) than in the SARD group (12.0%, 6/50) (P = 0.0346). Conclusion The anti-DFS70 positive rate in AC-2 pattern samples is lower than a third, and the prevalence is affected by different detection assays. Along with the AC-2 pattern, just reporting the presence of anti-DFS70 is not valuable for SARD exclusion. Therefore, it is important to co-examine other autoantibodies, such as anti-SS-A, anti-nucleosomes, and anti-histones, to confirm the isolated anti-DFS70.