Domain Of Antigen Research Articles

050 Eotaxin-1 and matrix metalloproteinase-9 are critical in anti-BP180 IgE-induced experimental bullous pemphigoid

Bullous pemphigoid (BP) is the most common autoimmune blistering disease characterized by chronic subepidermal blistering, autoantibodies directed against hemidesmosomal components, and a predominant eosinophil infiltrate. BP autoantibodies recognize two hemidesmosomal proteins of basal keratinocytes: BP230 (also termed BPAG1) and BP180 (also termed BPAG2 or collagen XVII) with the NC16A domain of the human BP180 antigen containing the major autoantibody-reactive epitopes. Anti-BP180 autoantibodies belong to IgG and IgE isotypes.

MO842: Maintenance of Humoral Response During a Three-Dose Immunization Program With MRNA SARS-COV-2 Vaccines in the Haemodialysis Population

BACKGROUND AND AIMSThe COVID-19 pandemic continues to be a worldwide health issue. Among haemodialysis (HD) patients, many countries opted for a three-dose immunization scheme with mRNA vaccines, as some patients did not produce a sufficient humoral response after the second one, and most developed a rapid decline in antibody levels over the months following vaccination.METHODThis observational, prospective, multi-centre study evaluated the humoral response, clinical outcomes (breakthrough infections, hospital admissions, severe COVID-19 and mortality), and their potential associated factors by determining the presence and levels of IgG antibodies to the receptor-binding domain of the S1 spike antigen of SARS-CoV-2 (anti-S1-RBD IgG) before and after each of the three doses of SARS-CoV-2 mRNA vaccines with either the mRNA-1273 (Moderna) or BNT162b2 (Pfizer) in 153 patients from three dialysis units affiliated to Hospital Clínic of Barcelona (Spain).RESULTSUnlike the general population, where most individuals seroconverted after one dose, HD patients required two doses to produce a positive anti-S1-RBD IgG response in most of them. Moreover, their antibody levels waned significantly three and 6 months after vaccination (see Figure 1). The decline in antibody levels was accompanied by seroreversion in 32% of patients. Notably, despite the precipitous drop, there was a low incidence of breakthrough infections (5.9%), with only one case of severe COVID-19 that required ICU admission and no deaths up until receiving the third vaccine dose. The administration of the third dose achieved seroconversion in 3 out of 4 non- or weak responders to two doses. Active cancer and immunosuppressive treatment were related to a worse humoral response.CONCLUSIONEvery haemodialysis patient should receive a third-dose vaccination scheme, with a booster dose three or 6 months after receiving the second one. Despite the lack of data, immunosuppressed and active cancer patients may benefit from more frequent vaccine boosters. Further monitoring of antibody levels will be needed to assess the maintenance of the humoral response in these patients.

Humoral Response after Three Doses of mRNA-1273 or BNT162b2 SARS-CoV-2 Vaccines in Hemodialysis Patients.

The COVID-19 pandemic continues to be a worldwide health issue. Among hemodialysis (HD) patients, two-dose immunization schemes with mRNA vaccines have contributed to preventing severe COVID-19 cases; however, some have not produced a sufficient humoral response, and most have developed a rapid decline in antibody levels over the months following vaccination. This observational, prospective, multi-center study evaluated the humoral response in terms of presence and levels of IgG antibodies to the receptor-binding domain of the S1 spike antigen of SARS-CoV-2 (anti-S1-RBD IgG) to the third dose of SARS-CoV-2 mRNA vaccines, either the mRNA-1273 (Moderna) or BNT162b2 (Pfizer), in 153 patients from three dialysis units affiliated to Hospital Clínic of Barcelona (Spain). Most hemodialysis patients responded intensely to this third vaccine dose, achieving the seroconversion in three out of four non- or weak responders to two doses. Moreover, 96.1% maintained the upper limit or generated higher titers than after the second. BNT162b2 vaccine, active cancer, and immunosuppressive treatment were related to a worse humoral response. Every hemodialysis patient should be administered a third vaccine dose six months after receiving the second one. Despite the lack of data, immunosuppressed patients and those with active cancer may benefit from more frequent vaccine boosters.

CD4+ T-cell activation of bone marrow causes bone fragility and insulin resistance in type 2 diabetes.

Type 2 diabetes mellitus (T2DM) causes an increased risk of bone fractures. However, the pathophysiology of diabetic bone fragility is not completely understood. It has been proposed that an inflammatory microenvironment in bone could be a major mechanism by inducing uncontrolled bone resorption, inadequate bone formation and consequently more porous bones. We propose that activated T-cells in the bone marrow cause a pro-inflammatory microenvironment in bone, and cause bone fragility in T2DM. We induced T2DM in C57BL/6 male mice through a hypercaloric diet rich in carbohydrates and low doses of streptozocin. In T2DM mice we inhibited systemic activation of T-cells with a fusion protein between the extracellular domain of Cytotoxic T-Lymphocyte Antigen 4 and the Fc domain of human immunoglobulin G (CTLA4-Ig). We analysed the effects of T2DM or CTLA4-Ig in lymphocyte cell subsets and antigen-presenting cells in peripheral blood and femoral bone marrow; and their effect on the metabolic phenotype, blood and bone cytokine concentration, femoral bone microarchitecture and biomechanical properties, and the number of osteoblast-like cells in the femoral endosteum. We performed a Pearson multiple correlation analysis between all variables in order to understand the global mechanism. Results demonstrated that CTLA4-Ig decreased the number of activated CD4+ T-cells in the femoral bone marrow and consequently decreased TNF-α and RANK-L concentration in bone, notably improved femoral bone microarchitecture and biomechanical properties, increased the number of osteoblast-like cells, and reduces osteoclastic activity compared to T2DM mice that did not receive the inhibitor. Interestingly, we observed that blood glucose levels and insulin resistance may be related to the increase in activated CD4+ T-cells in the bone marrow. We conclude that bone marrow activated CD4+ T-cells cause poor bone quality and insulin resistance in T2DM.

Functions and roles of IFIX, a member of the human HIN-200 family, in human diseases.

Pyrin and hematopoietic expression, interferon-inducible nature, and nuclear localization (HIN) domain family member 1 (PYHIN1), also known as IFIX, belongs to the family of pyrin proteins. This family includes structurally and functionally related mouse (e.g., p202, p203, and p204 proteins) and human (e.g., the interferon-inducible protein 16, absent in melanoma 2 protein, myeloid cell nuclear differentiation antigen, and pyrin and HIN domain family 1 or IFIX) proteins. The IFIX protein belongs to the HIN-200 family of interferon-inducible proteins that have a 200-amino acid signature motif at their C-termini. The increased expression of pyrin proteins in most cell types inhibits cell cycle control and modulates cell survival. Consistent with this role for pyrin proteins, IFIX is a potential antiviral DNA sensor that is essential for immune responses, the detection of viral DNA in the nucleus and cytoplasm, and the binding of foreign DNA via its HIN domain in a sequence non-specific manner. By promoting the ubiquitination and subsequent degradation of MDM2, IFIX acts as a tumor suppressor, thereby leading to p53/TP53 stabilization, HDAC1 regulation via the ubiquitin-proteasome pathway, and tumor-cell-specific silencing of the maspin gene. These data demonstrate that the potential molecular mechanism(s) underlying the action of the IFIX protein might be associated with the development of human diseases, such as viral infections, malignant tumors, and autoimmune diseases. This review summarizes the current insights into IFIX functions and how its regulation affects the outcomes of various human diseases.

Elucidating the 3D Structure of a Surface Membrane Antigen from Trypanosoma cruzi as a Serodiagnostic Biomarker of Chagas Disease.

Chagas disease (CD) is a vector-borne parasitosis, caused by the protozoan parasite Trypanosoma cruzi, that affects millions of people worldwide. Although endemic in South America, CD is emerging throughout the world due to climate change and increased immigratory flux of infected people to non-endemic regions. Containing of the diffusion of CD is challenged by the asymptomatic nature of the disease in early infection stages and by the lack of a rapid and effective diagnostic test. With the aim of designing new serodiagnostic molecules to be implemented in a microarray-based diagnostic set-up for early screening of CD, herein, we report the recombinant production of the extracellular domain of a surface membrane antigen from T. cruzi (TcSMP) and confirm its ability to detect plasma antibodies from infected patients. Moreover, we describe its high-resolution (1.62 Å) crystal structure, to which in silico epitope predictions were applied in order to locate the most immunoreactive regions of TcSMP in order to guide the design of epitopes that may be used as an alternative to the full-length antigen for CD diagnosis. Two putative, linear epitopes, belonging to the same immunogenic region, were synthesized as free peptides, and their immunological properties were tested in vitro. Although both peptides were shown to adopt a structural conformation that allowed their recognition by polyclonal antibodies raised against the recombinant protein, they were not serodiagnostic for T. cruzi infections. Nevertheless, they represent good starting points for further iterative structure-based (re)design cycles.

Abatacept downregulates Fcγ receptor I on circulating monocytes: a potential therapeutic mechanism in patients with rheumatoid arthritis

BackgroundAbatacept is a recombinant fusion protein composed of the extracellular domain of cytotoxic T-lymphocyte antigen 4 and the Fc portion of immunoglobulin (Ig) G. The mechanism of action of abatacept in rheumatoid arthritis (RA) is believed to be competitive inhibition of T cell costimulation mediated by the binding of CD28 to CD80/CD86 on antigen-presenting cells, and recent studies have shown that abatacept induces reverse signaling in macrophages and osteoclast precursors in a T cell-independent manner. This study aimed to investigate the therapeutic effects of abatacept on circulating monocytes that contribute to RA pathogenesis.MethodsPurified circulating monocytes derived from RA patients and controls were cultured in the absence or presence of abatacept or CD28-Ig for 24 h. The recovered cells were subjected to flow cytometry to evaluate the expression levels of cell surface molecules, and cytokines and chemokines in the culture supernatant were measured by multiplex bead arrays. The expression of candidate molecules was further examined by immunoblotting using total cellular extracts of the cultured monocytes. Finally, the effects of abatacept on cytokine production in monocytes stimulated with the immune complex of anti-citrullinated peptide antibodies (ACPAs) were examined.ResultsCD64/FcγRI was identified as a monocyte-derived molecule that was downregulated by abatacept but not CD28-Ig. This effect was observed in both RA patients and controls. The abatacept-induced downregulation of CD64/FcγRI was abolished by treatment with anti-CD86 antibodies but not anti-CD80 antibodies. Abatacept suppressed the production of interleukin (IL)-1β, IL-6, C-C motif chemokine ligand 2, and tumor necrosis factor-α in cultured monocytes stimulated with the ACPA immune complex.ConclusionsThe therapeutic effects of abatacept on RA are mediated, in part, by the downregulation of CD64/FcγRI on circulating monocytes via direct binding to CD86 and the suppression of immune complex-mediated inflammatory cytokine production.

Sperm quality and absence of SARS-CoV-2 RNA in semen after COVID-19 infection: a prospective, observational study and validation of the SpermCOVID test

ObjectiveTo study the contagiousness of sperm and its influence on fertility after recovery from COVID-19 infection.DesignProspective cohort study.SettingUniversity medical center.Patient(s)One hundred twenty Belgian men who had recovered from proven COVID-19 infection.Intervention(s)No intervention was performed.Main outcome measure(s)Semen quality was assessed using the World Health Organisation criteria. DNA damage to sperm cells was assessed by quantifying the DNA fragmentation index and the high density stainability. Finally antibodies against SARS-CoV2 spike-1 antigen, nuclear and S1-receptor binding domain were measured by Elisa and chemilumenscent microparticle immunoassays, respectively.Result(s)SARS-CoV-2 RNA was not detected in semen during the period shortly after infection nor at a later time. Mean progressive motility was reduced in 60% of men tested shortly (<1 month) after COVID-19 infection, 37% of men tested 1 to 2 months after COVID-19 infection, and 28% of men tested >2 months after COVID-19 infection. Mean sperm count was reduced in 37% of men tested shortly (<1 month) after COVID-19 infection, 29% of men tested 1 to 2 months after COVID-19 infection, and 6% of men tested >2 months after COVID-19 infection. The severity of COVID-19 infection and the presence of fever were not correlated with sperm characteristics, but there were strong correlations between sperm abnormalities and the titers of SARS-CoV-2 IgG antibody against spike 1 and the receptor- binding domain of spike 1, but not against nucleotide, in serum. High levels of antisperm antibodies developed in three men (2.5%).Conclusion(s)Semen is not infectious with SARS-CoV-2 at 1 week or more after COVID-19 infection (mean, 53 days). However, couples with a desire for pregnancy should be warned that sperm quality after COVID-19 infection can be suboptimal. The estimated recovery time is 3 months, but further follow-up studies are under way to confirm this and to determine if permanent damage occurred in a minority of men.

Ovarian carcinoma immunoreactive antigen domain 2 controls mitochondrial apoptosis in lung adenocarcinoma.

Ovarian carcinoma immunoreactive antigen domain 2 (OCIAD2) has been reported to show significantly higher expression in invasive lung adenocarcinoma than in lung adenocarcinoma in situ, and its abnormal expression is associated with poorer prognosis of the patients. However, the cellular function of OCIAD2 in this tumor remains poorly understood. In the present study, we first validated that OCIAD2 showed higher expression in human lung adenocarcinoma tissues or cell lines than in normal lung tissue or immortalized normal bronchial epithelial cells. OCIAD2 was localized predominantly at the mitochondrial membrane in lung adenocarcinoma cells. Interestingly, suppression of OCIAD2 led to loss of mitochondrial structure and a reduction in the number of mitochondria. Moreover, OCIAD2 suppression led to downregulation of cellular growth, proliferation, migration, and invasion, and upregulation of mitochondria‐related apoptosis. We also showed that OCIAD2 suppression induced a decrease in mitochondrial membrane potential and release of cytochrome c. Transcriptional profiling using RNA sequencing revealed a total of 137 genes whose expression was commonly altered after OCIAD2 knockdown in three lung adenocarcinoma cell lines (A549, HCC827, and PC9). Pathway enrichment analysis of those genes demonstrated significant enrichment in apoptotic signaling or endoplasmic reticulum (ER) stress pathways. Our data suggest that OCIAD2 inhibits the mitochondria‐initiated apoptosis and thus promotes the survival of lung cancer cells. Therefore, OCIAD2 may be an effective target for treatment of lung adenocarcinoma.

Cellular OCIAD2 protein is a proviral factor for hepatitis C virus replication

Hepatitis C virus (HCV) nonstructural protein NS4B is necessary for HCV replication. Our previous research found that NS4B-associated cellular proteins PREB and Surfeit 4 are involved in HCV replication. However, the molecular mechanism of HCV replication is not fully understood. Here we identified cellular ovarian cancer immunoreactive antigen domain containing 2 (OCIAD2) protein as a novel NS4B-associated HCV host cofactor by screening with small interfering RNA. Knockdown of OCIAD2 reduced significantly the HCV replication in a dose-dependent and genotype-independent manner. Further research showed that OCIAD2 was recruited into the HCV RNA replication complex by the interaction with NS4B. Interestingly, HCV replication induced OCIAD2 expression. In turn, overexpression of wild OCIAD2 also promoted virus replication whereas that of OCIAD2 mutant lacking the ability to bind NS4B exerted no effect on HCV replication. We also examined whether OCIAD2 interacted with other proteins participating in the HCV RNA replication complex including viral proteins NS5A, NS5B, and cellular proteins PREB, Surfeit 4. The results showed that OCIAD2 interacted with PREB and NS5A, but not NS5B or Surfeit 4. Our findings provide new insights into the function of OCIAD2 and HCV replication mechanism.

Absence of SARS-CoV-2 Antibodies in Natural Environment Exposure in Sheep in Close Contact with Humans.

Simple SummaryDifferent species can harbor coronavirus, including domestic and wild animals. The Coroviridae family is composed of four genera, including Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. Some domestic animals are susceptible to SARS-CoV-2 in natural and experimental infections. The infection of animals is generally a consequence of close contact with COVID-19 patients. Among domestic animals, SARS-CoV-2 replicates in respiratory ex vivo organ cultures of domestic ruminants. In this context, there is no information about the role sheep can play in the spread of the infection. This study tested the antibody response in 90 serum samples from sheep from the pre-pandemic period and 336 serum samples from sheep from the pandemic period (June 2020 to March 2021). In both cases, the animals were in close contact with a veterinary student community composed of more than 700 members. None of the serum samples analyzed were seroreactive based on an enzyme-linked immunosorbent assay (ELISA) using the receptor-binding domain (RBD) of the spike antigen. In this sense, no statistical difference was observed compared to the pre-pandemic sheep. To the authors’ knowledge, this is the first serosurvey in sheep to detect anti-SARS-CoV-2 antibodies. Future investigations should analyze the epidemiological role of sheep in SARS-CoV-2 infection, including the prevalence of infection.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the zoonotic causative agent of coronavirus disease 2019 (COVID-19) that has caused a pandemic situation with millions of infected humans worldwide. Among domestic animals, there have been limited studies regarding the transmissibility and exposure to the infection in natural conditions. Some animals are exposed and/or susceptible to SARS-CoV-2 infection, such as cats, ferrets and dogs. By contrast, there is no information about the susceptibility of ruminants to SARS-CoV-2. This study tested the antibody response in 90 ovine pre-pandemic serum samples and 336 sheep serum samples from the pandemic period (June 2020 to March 2021). In both cases, the animals were in close contact with a veterinary student community composed of more than 700 members. None of the serum samples analyzed was seroreactive based on an enzyme-linked immunosorbent assay (ELISA) using the receptor-binding domain (RBD) of the spike antigen. In this sense, no statistical difference was observed compared to the pre-pandemic sheep. Our results suggest that it seems unlikely that sheep could play a relevant role in the epidemiology of SARS-CoV-2 infection. This is the first study to report the absence of evidence of sheep exposure to SARS-CoV-2 in natural conditions.

Humoral and Cellular Responses to mRNA-1273 and BNT162b2 SARS-CoV-2 Vaccines Administered to Hemodialysis Patients

Rationale & ObjectivePatients with kidney failure who are receiving maintenance dialysis have a higher risk of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and worse clinical outcomes after coronavirus disease 2019 (COVID-19) than the general population. Therefore, immunization against SARS-CoV-2 with effective vaccines is an important component of health-maintenance strategies for these patients. This study evaluated the humoral and cellular responses to messenger RNA (mRNA) SARS-CoV-2 vaccines in this population.Study DesignObservational prospective multicenter cohort study.Setting & Participants205 patients treated at 3 dialysis units at the Hospital Clínic of Barcelona (Spain) were vaccinated from February 3 to April 4, 2021, and followed until April 23, 2021.ExposureImmunization with either the mRNA-1273 (Moderna) or BNT162b2 (Pfizer-BioNTech) SARS-CoV-2 mRNA vaccine.OutcomeSeroconversion, defined as the detection of IgG antibodies to the receptor-binding domain of the S1 spike antigen of SARS-CoV-2 (anti–S1-RBD IgG), and the identification of activated CD4+ T cells 3 weeks after completing vaccination. Anti-S1-RBD IgG levels were also analyzed as a secondary outcome.Analytical ApproachUnivariate and multivariable logistic and multiple linear regression models were used to evaluate the associations between vaccination and study outcomes.ResultsWe found that 97.7% of 175 vaccinated patients who were seronegative at baseline developed a response (humoral, cellular, or both); 95.4% of these patients seroconverted, while 62% of those tested for cellular immunity had a positive response. Greater age and immunosuppressive treatment were associated with lower antibody levels.LimitationsMandatory vaccine administration by health authorities. Anti–S1-RBD IgG levels were reported up to 150 U/mL and cellular immune responses were characterized qualitatively. Antibody assay and cellular response assessment may not be comparable with previously published laboratory approaches.ConclusionsImmunization with mRNA vaccines generated a humoral and cellular immune response in a high proportion of patients with kidney failure receiving maintenance dialysis. These findings as well as the high risk of infection and poor clinical outcomes among these patients make their vaccination a health priority.

Self-Assembling Nanoparticle Vaccines Displaying the Receptor Binding Domain of SARS-CoV-2 Elicit Robust Protective Immune Responses in Rhesus Monkeys.

SARS-CoV-2 caused the COVID-19 pandemic that lasted for more than a year. Globally, there is an urgent need to use safe and effective vaccines for immunization to achieve comprehensive protection against SARS-CoV-2 infection. Focusing on developing a rapid vaccine platform with significant immunogenicity as well as broad and high protection efficiency, we designed a SARS-CoV-2 spike protein receptor-binding domain (RBD) displayed on self-assembled ferritin nanoparticles. In a 293i cells eukaryotic expression system, this candidate vaccine was prepared and purified. After rhesus monkeys are immunized with 20 μg of RBD–ferritin nanoparticles three times, the vaccine can elicit specific humoral immunity and T cell immune response, and the neutralizing antibodies can cross-neutralize four SARS-CoV-2 strains from different sources. In the challenge protection test, after nasal infection with 2 × 105 CCID50 SARS-CoV-2 virus, compared with unimmunized control animals, virus replication in the vaccine-immunized rhesus monkeys was significantly inhibited, and respiratory pathology observations also showed only slight pathological damage. These analyses will benefit the immunization program of the RBD–ferritin nanoparticle vaccine in the clinical trial design and the platform construction to present a specific antigen domain in the self-assembling nanoparticle in a short time to harvest stable, safe, and effective vaccine candidates for new SARS-CoV-2 isolates.

Probing the naturally acquired immune response to malaria for broadly reactive antibodies targeting P. falciparum antigens linked with severe disease

Abstract Severe malaria is caused by the adhesion of Plasmodium falciparum-infected erythrocytes to host receptors on vascular endothelium. Specifically, binding of the parasite variant surface antigen domain CIDRa1 to host endothelial protein C receptor (EPCR) is strongly associated with severe disease. In malaria-endemic regions, children quickly develop immunity against severe malaria, indicative of the development of an effective immune response against CIDRa1 domains. Indeed, serum IgG from naturally immune individuals exhibited reactivity against a diverse panel of CIDRa1 variants. In an effort to analyze CIDRa1 antibody responses at a monoclonal level, we isolated CIDRa1-specific memory B cells from malaria-experienced individuals living in Uganda and expressed the corresponding monoclonal antibodies (mAbs). Our approach has yielded anti-CIDRa1 mAbs ranging in breadth from variant-specific to reactive with the full spectrum of CIDRa1 domain variants, irrespective of their extensive sequence diversity. The broadest antibodies can be separated into two categories, those that bind outside the EPCR binding site and display exceptional breadth with modest inhibition of EPCR binding and those that target the EPCR binding site and show potent EPCR-binding inhibition. Two EPCR-binding site-targeting mAbs isolated from two different donors showed signs of convergent evolution. Our results demonstrate that natural P. falciparum infection can induce a broad and inhibitory antibody response against CIDRa1. Current experiments are aimed at understanding the structural basis of antibody-mediated inhibition of CIDRa1-EPCR binding to inform the design of a vaccine that protects against severe malaria.

Natural variability in surface antigen and reverse transcriptase domain of hepatitis B virus in treatment-naïve chronic HBV-infected Egyptian patients

Hepatitis B virus (HBV) infection is a serious health problem not only in Egypt, but also worldwide. We collected 57 serum samples from treatment-naïve chronic HBV-infected Egyptians. The DNA segment encoding HBV surface antigen (HBsAg) and reverse transcriptase (RT) domain was partially sequenced. Our data revealed that all viral isolates belonged to genotype D with ayw2 as the predominant serotype (89 %).Regarding HBsAg, 45 substitutions were detected in the collected isolates. Eleven substitutions were found in the major hydrophilic region, including two novel ones (M103T and G130E) that were not correlated before with genotype D. Additionally, 11 occult samples (19 %) were detected, in which the predominant mutations of HBsAg were S143L (7 samples) followed by D144A and T125M (4 samples each). Concerning the RT domain, 26 isolates (45 %) harbored 19 natural mutations that were reported to be associated with antiviral resistance. Eleven different mutations were not correlated previously with genotype D. The most predominant mutation was Y124H (47 samples, 82 %). Interestingly, such mutation was detected in 91 % of the previous reported sequences of HBV isolates collected in Egypt (157 sequences). Furthermore, our study illustrated the presence of viral quasispecies in the HBsAg (10 samples, 17.5 %) and RT domain (9 samples, 15.7 %).In conclusion, we elucidated the presence of natural substitutions in HBsAg and RT domain of HBV isolates obtained from treatment-naïve chronic HBV-infected Egyptian patients. Additionally, we detected viral quasispecies and revealed Y124H as a characteristic substitution in the RT domain for HBV isolates in Egypt. Moreover, novel substitutions in HBsAg and RT domain were reported with genotype D.

Is it safe to start steroids at home for a COPD exacerbation after virtual assessment in the COVID-19 era?

<h3>Aims</h3> The biomarkers representing the metastatic potential of well-differentiated thyroid carcinoma remain to be established. A study was undertaken to find whether the expression status of neural cell adhesion molecule (NCAM) and/or ovarian cancer immunoreactive antigen domain containing 1 (OCIAD1) is associated with the metastatic potential of differentiated thyroid carcinoma. <h3>Methods</h3> NCAM and OCIAD1 were analysed by immunohistochemistry on tissue microarrays. <h3>Results</h3> Among 214 well-differentiated thyroid carcinomas, 68 patients had distant metastases. Immunohistochemical analyses showed that the majority of benign thyroid lesions expressed NCAM while a significant proportion of thyroid carcinomas lost or had reduced NCAM expression. Both follicular and papillary carcinomas with distant metastasis had a significantly higher frequency of preserving NCAM expression. Hierarchical clustering analysis showed that OCIAD1 had significant differential expression between benign and malignant thyroid lesions. The overall metastatic-to-localised tumour ratio was higher in NCAM-expressing clusters, but the difference between ratios of OCIAD1-positive and OCIAD1-negative subclusters was not significant. <h3>Conclusions</h3> These analyses suggest that the preservation of NCAM expression in well-differentiated thyroid carcinoma is an indicator for a higher risk of distant metastasis. OCIAD1 is a potential biomarker of thyroid carcinoma but had no significant additive effect on the risk of distant metastasis. Further elucidation of the molecular mechanisms underlying the NCAM-mediated cellular processes will be beneficial for the development of effective treatments against the metastasis of thyroid carcinoma.

Serological evidence of SARS-CoV-2 and co-infections in stray cats in Spain.

A new coronavirus known as SARS‐CoV‐2 emerged in Wuhan in 2019 and spread rapidly to the rest of the world causing the pandemic disease named coronavirus disease of 2019 (COVID‐19). Little information is known about the impact this virus can cause upon domestic and stray animals. The potential impact of SARS‐CoV‐2 has become of great interest in cats due to transmission among domestic cats and the severe phenotypes described recently in a domestic cat. In this context, there is a public health warning that needs to be investigated in relation with the epidemiological role of this virus in stray cats. Consequently, in order to know the impact of the possible transmission chain, blood samples were obtained from 114 stray cats in the city of Zaragoza (Spain) and tested for SARS‐CoV‐2 and other selected pathogens susceptible to immunosuppression including Toxoplasma gondii, Leishmania infantum, feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) from January to October 2020. Four cats (3.51%), based on enzyme‐linked immunosorbent assay (ELISA) using the receptor binding domain (RBD) of Spike antigen, were seroreactive to SARS‐CoV‐2. T. gondii, L. infantum, FeLV and FIV seroprevalence was 12.28%, 16.67%, 4.39% and 19.30%, respectively. Among seropositive cats to SARS‐CoV‐2, three cats were also seropositive to other pathogens including antibodies detected against T. gondii and FIV (n = 1); T. gondii (n = 1); and FIV and L. infantum (n = 1). The subjects giving positive for SARS‐CoV‐2 were captured in urban areas of the city in different months: January 2020 (2/4), February 2020 (1/4) and July 2020 (1/4). This study revealed, for the first time, the exposure of stray cats to SARS‐CoV‐2 in Spain and the existence of concomitant infections with other pathogens including T. gondii, L. infantum and FIV, suggesting that immunosuppressed animals might be especially susceptible to SARS‐CoV‐2 infection.

Towards Quantitative and Standardized Serological and Neutralization Assays for COVID-19.

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.

SARS-CoV-2 Seroprevalence in Household Domestic Ferrets (Mustela putorius furo).

Simple SummaryAnimal infections with SARS-CoV-2 have been reported in different countries and several animal species have been proven to be susceptible to infection with SARS-CoV-2 both naturally or by experimental infection. Moreover, infections under natural conditions in more than 20 mink farms have been reported where humans could have been the source of infection for minks. However, little information is available about the susceptibility of pet animals under natural conditions and currently there is no SARS-CoV-2 epidemiological assessment occurrence in household ferrets. In this study, the presence of SARS-CoV-2 antibodies was evaluated in serum samples obtained from 127 household ferrets (Mustela putorius furo) in the Province of Valencia (Spain). Two ferrets tested positive to SARS-CoV-2 (1.57%) by in-house enzyme-linked immunosorbent assay based on receptor binding domain (RBD) of Spike antigen. Furthermore, anti-RBD SARS-CoV-2 antibodies persisted at detectable levels in a seropositive SARS-CoV-2 domestic ferret beyond 129 days since the first-time antibodies were detected. This study reports for the first time the evidence of household pet ferrets exposure to SARS-CoV-2 in Spain to date.Animal infections with SARS-CoV-2 have been reported in different countries and several animal species have been proven to be susceptible to infection with SARS-CoV-2 both naturally and by experimental infection. Moreover, infections under natural conditions in more than 20 mink farms have been reported where humans could have been the source of infection for minks. However, little information is available about the susceptibility of pet animals under natural conditions and currently there is no SARS-CoV-2 epidemiological assessment occurrence in household ferrets. In this study, the presence of SARS-CoV-2 antibodies was evaluated in serum samples obtained from 127 household ferrets (Mustela putorius furo) in the Province of Valencia (Spain). Two ferrets tested positive to SARS-CoV-2 (1.57%) by in-house enzyme-linked immunosorbent assay based on receptor binding domain (RBD) of Spike antigen. Furthermore, anti-RBD SARS-CoV-2 antibodies persisted at detectable levels in a seropositive SARS-CoV-2 domestic ferret beyond 129 days since the first time antibodies were detected. This study reports for the first time the evidence of household pet ferrets exposure to SARS-CoV-2 in Spain to date.

P53.08 Analysis of Mitochondria-Mediated OCIAD2 Oncogenic Function in Lung Adenocarcinoma

Ovarian carcinoma immune-reactive antigen domain 2 (OCIAD2) has been reported as a novel tumor-specific protein (Cancer Sci, 2007). Overexpression of OCIAD2 is closely associated with the stepwise progression of early-stage lung adenocarcinoma and significantly associated with poorer outcomes in patients with ovarian and lung cancer (Pathol Int, 2012 and 2018). OCIAD2 is localized predominantly in the mitochondrial membrane in cancer cells. However, the oncogenic functions mediated by OCIAD2 are poorly understood. In this study, we explored the potential role of OCIAD2 in mitochondria and identified the OCIAD2 binding partner in lung adenocarcinoma cells. We performed most experiments after 72 hours of siRNA (scramble or siOCIAD2) transfection using lipofectamine RNAiMAX or after 24 hours of Flag-OCIAD2 plasmid transfection using Fugene HD. Alterations to mitochondrial morphology in the siRNA-transfected lung adenocarcinoma cells (A549 or HCC827) were observed using transmission electron microscopy (TEM). The siRNA-transfected cells were stained with MitoPT TMRM fluorescent dye and healthy cells were detected with electrical potential gradient by flow cytometry. To identify the OCIAD2-binding partners in lung adenocarcinoma cells, the membrane fraction including the mitochondria membrane was isolated from 3'-Flag-OCIAD2- or 5'-Flag-OCIAD2-transfected cells, followed by pull-down assay and LC-MS/MS analysis. We found that suppression of OCIAD2 led to marked destruction of morphologic features of mitochondria such as cristae in lung adenocarcinoma cells. The number of mitochondria per cell and the number of cristae per mitochondrion were significantly decreased, and there was a loss of mitochondrial electrical potential gradient in comparison with siCON. By LC-MS/MS analysis, we identified several OCIAD2-binding partner proteins that are significantly associated with apoptosis, crista formation, metabolic processes, membrane permeability, and transport. OCIAD2 is abnormally overexpressed in lung adenocarcinoma and may be associated with mitochondria-mediated oncogenic signaling such as blocking of apoptosis, active mitochondrial fitness, and maintenance of the mitochondrial electrical potential gradient through interaction with mitochondrial proteins. The fact that tumor cells are controlled by OCIAD2 and depend on its oncogenic function is of considerable interest.