Characterization of antibody-dependent cellular phagocytosis in patients infected with hepatitis C virus with different clinical outcomes.

Early neutralizing antibodies against hepatitis C virus (HCV) and CD8 + T cell effector responses can lead to viral clearance. However, these functions alone are not sufficient to protect patients against HCV infection, thus undefined additional antiviral immune mechanisms are required. In recent years, Fc-receptor-dependent antibody effector functions, particularly, antibody-dependent cellular phagocytosis (ADCP) were shown to offer immune protection against several RNA viruses. However, its development and clinical role in patients with HCV infection remain unknown. In this study, we found that patients with chronic GT1a or GT3a HCV infection had significantly higher concentrations of anti-envelope 2 (E2) antibodies, predominantly IgG1 subclass, than patients that cleared the viruses while the latter had antibodies with higher affinities. 97% of the patients with HCV had measurable ADCP of whom patients with chronic disease showed significantly higher ADCP than those who naturally cleared the virus. Epitope mapping studies showed that patients with antibodies that target antigenic domains on the HCV E2 protein that are known to associate with neutralization function are also strongly associated with ADCP, suggesting antibodies with overlapping/dual functions. Correlation studies showed that ADCP significantly correlated with plasma anti-E2 antibody levels and neutralization function regardless of clinical outcome and genotype of infecting virus, while a significant correlation between ADCP and affinity was only evident in patients that cleared the virus. These results suggest ADCP was mostly driven by antibody titer in patients with chronic disease while maintained in clearers due to the quality (affinity) of their anti-E2 antibodies despite having lower antibody titers.

What works in radiology education for medical students: a systematic review and meta-analysis.

BACKGROUND: Medical imaging related knowledge and skills are widely used in clinical practice. However, radiology teaching methods and resultant knowledge among medical students and junior doctors is variable. A systematic review and meta-analysis was performed to compare the impact of different components of radiology teaching methods (active versus passive teaching, eLearning versus traditional face-to-face teaching) on radiology knowledge / skills of medical students. METHODS: PubMed and Scopus databases were searched for articles published in English over a 15-year period ending in June 2021 quantitatively comparing the effectiveness of undergraduate medical radiology education programs regarding acquisition of knowledge and/or skills. Study quality was appraised by the Medical Education Research Study Quality Instrument (MERSQI) scoring and analyses performed to assess for risk of bias. A random effects meta-analysis was performed to pool weighted effect sizes across studies and I2 statistics quantified heterogeneity. A meta-regression analysis was performed to assess for sources of heterogeneity. RESULTS: From 3,052 articles, 40 articles involving 6,242 medical students met inclusion criteria. Median MERSQI score of the included articles was 13 out of 18 possible with moderate degree of heterogeneity (I2 = 93.42%). Thematic analysis suggests trends toward synergisms between radiology and anatomy teaching, active learning producing superior knowledge gains compared with passive learning and eLearning producing equivalent learning gains to face-to-face teaching. No significant differences were detected in the effectiveness of methods of radiology education. However, when considered with the thematic analysis, eLearning is at least equivalent to traditional face-to-face teaching and could be synergistic. CONCLUSIONS: Studies of educational interventions are inherently heterogeneous and contextual, typically tailored to specific groups of students. Thus, we could not draw definitive conclusion about effectiveness of the various radiology education interventions based on the currently available data. Better standardisation in the design and implementation of radiology educational interventions and design of radiology education research are needed to understand aspects of educational design and delivery that are optimal for learning. TRIAL REGISTRATION: Prospero registration number CRD42022298607.

A 'one-stop-shop' point-of-care hepatitis C RNA testing intervention to enhance treatment uptake in a reception prison: The PIVOT study.

BACKGROUND & AIMS: Prisons are key venues for scaling-up hepatitis C virus (HCV) testing and treatment. Complex clinical pathways and frequent movements of people in prison remain barriers to HCV care. This study evaluated the impact of a 'one-stop-shop' point-of-care HCV RNA testing intervention on treatment uptake compared with standard of care among people recently incarcerated in Australia. METHODS: PIVOT was a prospective, non-concurrent, controlled study comparing HCV treatment uptake during 'standard of care' (n = 239; November 2019-May 2020) and a 'one-stop-shop' intervention (n = 301; June 2020-April 2021) in one reception prison in Australia. The primary endpoint was uptake of direct-acting antiviral treatment at 12 weeks from enrolment. Secondary outcomes included the time taken from enrolment to each stage in the care cascade. RESULTS: A total of 540 male participants were enrolled. Median age (29 vs. 28 years) and history of injecting drug use (48% vs. 42%) were similar between standard of care and intervention phases. Among people diagnosed with current HCV infection (n = 18/63 in the standard of care phase vs. n = 30/298 in the intervention phase), the proportion initiating direct-acting antiviral treatment within 12 weeks from enrolment in the intervention phase was higher (93% [95% CI 0.78-0.99] vs. 22% [95% CI 0.64-0.48]; p <0.001), and the median time to treatment initiation was shorter (6 days [IQR 5-7] vs. 99 days [IQR 57-127]; p <0.001) compared to standard of care. CONCLUSIONS: The 'one-stop-shop' intervention enhanced treatment uptake and reduced time to treatment initiation among people recently incarcerated in Australia, thereby overcoming key barriers to treatment scale-up in the prison sector. IMPACT AND IMPLICATIONS: This study provides important insights for policymakers regarding optimal HCV testing and treatment pathways for people newly incarcerated in prisons. The findings will improve health outcomes in people in prison with chronic HCV infection by increasing testing and treatment, thereby reducing infections, liver-related morbidity/mortality, and comorbidities. The findings will change clinical practice, clinical guidelines, and international guidance, and will inform future research and national and regional strategies, in particular regarding point-of-care testing, which is being rapidly scaled-up in various settings globally. The economic impact will likely include health budget savings resulting from reduced negative health outcomes relating to HCV, and health system efficiencies resulting from the introduction of simplified models of care. CLINICAL TRIALS REGISTRATION: This study is registered at Clinicaltrials.gov (NCT04809246).

Irradiation-Induced Dysbiosis: The Compounding Effect of High-Fat Diet on Metabolic and Immune Functions in Mice.

The negative impact of irradiation or diet on the metabolic and immune profiles of cancer survivors have been previously demonstrated. The gut microbiota plays a critical role in regulating these functions and is highly sensitive to cancer therapies. The aim of this study was to investigate the effect of irradiation and diet on the gut microbiota and metabolic or immune functions. We exposed C57Bl/6J mice to a single dose of 6 Gy radiation and after 5 weeks, fed them a chow or high-fat diet (HFD) for 12 weeks. We characterised their faecal microbiota, metabolic (whole body and adipose tissue) functions, and systemic (multiplex cytokine, chemokine assay, and immune cell profiling) and adipose tissue inflammatory profiles (immune cell profiling). At the end of the study, we observed a compounding effect of irradiation and diet on the metabolic and immune profiles of adipose tissue, with exposed mice fed a HFD displaying a greater inflammatory signature and impaired metabolism. Mice fed a HFD also showed altered microbiota, irrespective of irradiation status. An altered diet may exacerbate the detrimental effects of irradiation on both the metabolic and inflammatory profiles. This could have implications for the diagnosis and prevention of metabolic complications in cancer survivors exposed to radiation.

The effect of exercise intensity on the inflammatory profile of cancer survivors: A randomised crossover study.

BACKGROUND: Systemic inflammation has been clearly linked to poorer health outcomes from cancer diagnosis through to survivorship. There is accumulating evidence that exercise can reduce inflammation. However, the optimal intensity of exercise to reduce systemic inflammation is unknown. AIMS: The aim of this randomised crossover study was to identify the difference between high- and low-intensity aerobic exercise on the inflammatory profile of cancer survivors after a single exercise session (acute) and a short training period (six sessions over 2 weeks). METHOD: Participants (n = 20) were randomised to either low- or high-intensity exercise. They underwent 2 weeks of stationary cycling at their assigned intensity and then underwent a 6-week washout period of no exercise before returning to complete 2 weeks of exercise at the remaining intensity. RESULTS: Twenty participants with a mean age of 56.4 (±9.4) years were enrolled and completed the intervention. There was no effect of exercise intensity after a single exercise session. After 2 weeks of training, there was a significant effect of intensity on chemokines CCL2 (mean difference ± SEM; 13.2 pg/mL ± 5.0, p = .04) and CXCL12 (150.3 pg/mL ± 51.8, p = .02), where CCL2 was decreased after low-intensity exercise and CXCL12 decreased after high-intensity exercise. DISCUSSION: Our data suggest that while exercise intensity may impact different cell types in the circulation, both low- and high-intensity exercise can positively modulate inflammatory markers. CONCLUSION: The potential to scale up low-intensity exercise over time is likely to be more broadly applicable and achievable for cancer survivor cohorts while still eliciting beneficial effects on systemic inflammation.

Persistence of a Frameshifting Deletion in SARS-CoV-2 ORF7a for the Duration of a Major Outbreak.

Australia experienced widespread COVID-19 outbreaks from infection with the SARS-CoV-2 Delta variant between June 2021 and February 2022. A 17-nucleotide frameshift-inducing deletion in ORF7a rapidly became represented at the consensus level (Delta-ORF7aΔ17del) in most Australian outbreak cases. Studies from early in the COVID-19 pandemic suggest that frameshift-inducing deletions in ORF7a do not persist for long in the population; therefore, Delta-ORF7aΔ17del genomes should have disappeared early in the Australian outbreak. In this study, we conducted a retrospective analysis of global Delta genomes to characterise the dynamics of Delta-ORF7aΔ17del over time, determined the frequency of all ORF7a deletions worldwide, and compared global trends with those of the Australian Delta outbreak. We downloaded all GISAID clade GK Delta genomes and scanned them for deletions in ORF7a. For each deletion we identified, we characterised its frequency, the number of countries it was found in, and how long it persisted. Of the 4,018,216 Delta genomes identified globally, 134,751 (~3.35%) possessed an ORF7a deletion, and ORF7aΔ17del was the most common. ORF7aΔ17del was the sole deletion in 28,014 genomes, of which 27,912 (~99.6%) originated from the Australian outbreak. During the outbreak, ~87% of genomes were Delta-ORF7aΔ17del, and genomes with this deletion were sampled until the outbreak's end. These data demonstrate that, contrary to suggestions early in the COVID-19 pandemic, genomes with frameshifting deletions in ORF7a can persist over long time periods. We suggest that the proliferation of Delta-ORF7aΔ17del genomes was likely a chance founder effect. Nonetheless, the frequency of ORF7a deletions in SARS-CoV-2 genomes worldwide suggests they might have some benefit for virus transmission.

The microbiome in HLA-B27-associated disease: implications for acute anterior uveitis and recommendations for future studies.

The pathogenesis of human leukocyte antigen (HLA)-B27-associated diseases such as acute anterior uveitis (AAU) and ankylosing spondylitis (AS) remains poorly understood, though Gram-negative bacteria and subclinical bowel inflammation are strongly implicated. Accumulating evidence from animal models and clinical studies supports several hypotheses, including HLA-B27-dependent dysbiosis, altered intestinal permeability, and molecular mimicry. However, the existing literature is hampered by inadequate studies designed to establish causation or uncover the role of viruses and fungi. Moreover, the unique disease model afforded by AAU to study the gut microbiota has been neglected. This review critically evaluates the current literature and prevailing hypotheses on the link between the gut microbiota and HLA-B27-associated disease. We propose a new potential role for HLA-B27-driven altered antibody responses to gut microbiota in disease pathogenesis and outline recommendations for future well-controlled human studies, focusing on AAU.

Effect of prior Zika and dengue virus exposure on the severity of a subsequent dengue infection in adults.

Given the structural similarity between Zika and dengue viruses, prior infection from one virus is hypothesized to modulate the severity of a subsequent infection from the other virus. A previous paediatric cohort study observed that a prior Zika infection may increase the risk of a subsequent symptomatic or severe dengue infection. The Colombo Dengue study is a prospective hospital-based cohort study in Sri Lanka that recruits symptomatic adult dengue patients within the first three days of fever. Anti-Dengue Envelope and anti-Zika NS1 IgG antibodies were tested by ELISA (Euroimmun, Lubeck, Germany) in all recruited patients. Associations between pre-morbid seroprevalence for either or both infections and adverse clinical outcomes of the current dengue infection were explored. A total of 507 dengue infected patients were assessed of whom 342 (68%) and 132 (26%) patients had anti-dengue IgG and anti-Zika IgG respectively. People with combined prior dengue and zika exposure as well as prior dengue exposure alone, were at increased risk of plasma leakage, compensated and uncompensated shock, and severe dengue (p < 0·05), compared to people without prior exposure to either infection. The effect of prior Zika exposure alone could not be established due to the small the number of primary dengue infections with prior Zika exposure.

Changes to the upper gastrointestinal microbiotas of children with reflux oesophagitis and oesophageal metaplasia.

Little is known of the relationships among paediatric upper gastrointestinal microbiotas, and the impact of medication use and disease on their diversity. Here, we investigated the diversity of three microbiotas in the upper gastrointestinal tract of paediatric patients in relation to each other and to host factors. Oral, oesophageal and gastric microbiotas from a prospective paediatric cohort (n=54) were profiled using the 16S rRNA gene and ITS2 amplicon sequencing. 16S rRNA gene amplicon sequencing of oesophageal biopsies from a retrospective paediatric cohort (n=96) and shotgun metagenomics data from oesophageal brushings (n=88) were employed for genomic signature validation. Bacterial diversity and composition showed substantial differences across oral, oesophageal and gastric fluid samples that were not replicated for fungi, and the presence of reflux led to increased homogeneity in the bacterial component of these three microbiotas. The oral and oesophageal microbiotas were associated with age, sex, history of oesophageal atresia and presence of oesophageal metaplasia, with the latter characterized by Prevotella enrichment. Proton pump inhibitor use was associated with increased oral bacterial richness in the gastric fluid, and this correlated with increased levels of gastric pro-inflammatory cytokines. Profiling of oesophageal biopsies from a retrospective paediatric cohort confirmed an increased Prevotella prevalence in samples with metaplasia. Analysis of metagenome-derived oesophageal Prevotella melaninogenica genomes identified strain-specific features that were significantly increased in prevalence in samples with metaplasia. Prevotella enrichment is a signature associated with paediatric oesophageal metaplasia, and proton pump inhibitor use substantially alters the paediatric gastric microenvironment.

Longitudinal Characterization of Phagocytic and Neutralization Functions of Anti-Spike Antibodies in Plasma of Patients after Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Phagocytic responses by effector cells to opsonized viruses have been recognized to play a key role in antiviral immunity. Limited data on coronavirus disease 2019 suggest that the role of Ab-dependent and -independent phagocytosis may contribute to the observed immunological and inflammatory responses; however, their development, duration, and role remain to be fully elucidated. In this study of 62 acute and convalescent patients, we found that patients with acute coronavirus disease 2019 can mount a phagocytic response to autologous plasma-opsonized Spike protein-coated microbeads as early as 10 d after symptom onset, while heat inactivation of this plasma caused 77-95% abrogation of the phagocytic response and preblocking of Fc receptors showed variable 18-60% inhibition. In convalescent patients, phagocytic response significantly correlated with anti-Spike IgG titers and older patients, while patients with severe disease had significantly higher phagocytosis and neutralization functions compared with patients with asymptomatic, mild, or moderate disease. A longitudinal subset of the convalescent patients over 12 mo showed an increase in plasma Ab affinity toward Spike Ag and preservation of phagocytic and neutralization functions, despite a decline in the anti-Spike IgG titers by >90%. Our data suggest that early phagocytosis is primarily driven by heat-liable components of the plasma, such as activated complements, while anti-Spike IgG titers account for the majority of observed phagocytosis at convalescence. Longitudinally, a significant increase in the affinity of the anti-Spike Abs was observed that correlated with the maintenance of both the phagocytic and neutralization functions, suggesting an improvement in the quality of the Abs.

Evolution of the SARS-CoV-2 omicron variants BA.1 to BA.5: Implications for immune escape and transmission.

The first dominant SARS-CoV-2 Omicron variant BA.1 harbours 35 mutations in its Spike protein from the original SARS-CoV-2 variant that emerged late 2019. Soon after its discovery, BA.1 rapidly emerged to become the dominant variant worldwide and has since evolved into several variants. Omicron is of major public health concern owing to its high infectivity and antibody evasion. This review article examines the theories that have been proposed on the evolution of Omicron including zoonotic spillage, infection in immunocompromised individuals and cryptic spread in the community without being diagnosed. Added to the complexity of Omicron's evolution are the multiple reports of recombination events occurring between co-circulating variants of Omicron with Delta and other variants such as XE. Current literature suggests that the combination of the novel mutations in Omicron has resulted in the variant having higher infectivity than the original Wuhan-Hu-1 and Delta variant. However, severity is believed to be less owing to the reduced syncytia formation and lower multiplication in the human lung tissue. Perhaps most challenging is that several studies indicate that the efficacy of the available vaccines have been reduced against Omicron variant (8-127 times reduction) as compared to the Wuhan-Hu-1 variant. The administration of booster vaccine, however, compensates with the reduction and improves the efficacy by 12-35 fold. Concerningly though, the broadly neutralising monoclonal antibodies, including those approved by FDA for therapeutic use against previous SARS-CoV-2 variants, are mostly ineffective against Omicron with the exception of Sotrovimab and recent reports suggest that the Omicron BA.2 is also resistant to Sotrovimab. Currently two new Omicron variants BA.4 and BA.5 are emerging and are reported to be more transmissible and resistant to immunity generated by previous variants including Omicron BA.1 and most monoclonal antibodies. As new variants of SARS-CoV-2 will likely continue to emerge it is important that the evolution, and biological consequences of new mutations, in existing variants be well understood.

Intranasal Delivery of Recombinant S100A8 Protein Delays Lung Cancer Growth by Remodeling the Lung Immune Microenvironment.

Lung cancer is the leading cause of cancer-related death worldwide. Increasing evidence indicates a critical role for chronic inflammation in lung carcinogenesis. S100A8 is a protein with reported pro- and anti-inflammatory functions. It is highly expressed in myeloid-derived suppressor cells (MDSC) that accumulate in the tumor microenvironment and abrogate effective anti-cancer immune responses. Mechanisms of MDSC-mediated immunosuppression include production of reactive oxygen species and nitric oxide, and depletion of L-arginine required for T cell function. Although S100A8 is expressed in MDSC, its role in the lung tumor microenvironment is largely unknown. To address this, mouse recombinant S100A8 was repeatedly administered intranasally to mice bearing orthotopic lung cancers. S100A8 treatment prolonged survival from 19 days to 28 days (p < 0.001). At midpoint of survival, whole lungs and bronchoalveolar lavage fluid (BALF) were collected and relevant genes/proteins measured. We found that S100A8 significantly lowered expression of cytokine genes and proteins that promote expansion and activation of MDSC in lungs and BALF from cancer-bearing mice. Moreover, S100A8 enhanced activities of antioxidant enzymes and suppressed production of nitrite to create a lung microenvironment conducive to cytotoxic lymphocyte expansion and function. In support of this, we found decreased MDSC numbers, and increased numbers of CD4+ T cells and natural killer T (NK-T) cells in lungs from cancer-bearing mice treated with S100A8. Ex-vivo treatment of splenocytes with S100A8 protein activated NK cells. Our results indicate that treatment with S100A8 may favourably modify the lung microenvironment to promote an effective immune response in lungs, thereby representing a new strategy that could complement current immunotherapies in lung cancer.

Maintenance of broad neutralizing antibodies and memory B cells 1 year post-infection is predicted by SARS-CoV-2-specific CD4+ T cell responses.

Understanding the long-term maintenance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity is critical for predicting protection against reinfection. In an age- and gender-matched cohort of 24 participants, the association of disease severity and early immune responses on the maintenance of humoral immunity 12 months post-infection is examined. All severely affected participants maintain a stable subset of SARS-CoV-2 receptor-binding domain (RBD)-specific memory B cells (MBCs) and good neutralizing antibody breadth against the majority of the variants of concern, including the Delta variant. Modeling these immune responses against vaccine efficacy data indicate a 45%-76% protection against symptomatic infection (variant dependent). Overall, these findings indicate durable humoral responses in most participants after infection, reasonable protection against reinfection, and implicate baseline antigen-specific CD4+ T cell responses as a predictor of maintenance of antibody neutralization breadth and RBD-specific MBC levels at 12 months post-infection.

Broadly-Neutralizing Antibodies Against Emerging SARS-CoV-2 Variants.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have become a major concern in the containment of current pandemic. The variants, including B.1.1.7 (Alpha), B.1.351 (Beta), P1 (Gamma) and B.1.617.2 (Delta) have shown reduced sensitivity to monoclonal antibodies, plasma and/or sera obtained from convalescent patients and vaccinated individuals. Development of potent therapeutic monoclonal antibodies (mAbs) with broad neutralizing breadth have become a priority for alleviating the devastating effects of this pandemic. Here, we review some of the most promising broadly neutralizing antibodies obtained from plasma of patients that recovered from early variants of SARS-CoV-2 that may be effective against emerging new variants of the virus. This review summarizes several mAbs, that have been discovered to cross-neutralize across Sarbecoviruses and SARS-CoV-2 escape mutants. Understanding the characteristics that confer this broad and cross-neutralization functions of these mAbs would inform on the development of therapeutic antibodies and guide the discovery of second-generation vaccines.

Multi-omics of the esophageal microenvironment identifies signatures associated with progression of Barrett's esophagus.

BACKGROUND: The enrichment of Gram-negative bacteria of oral origin in the esophageal microbiome has been associated with the development of metaplasia. However, to date, no study has comprehensively assessed the relationships between the esophageal microbiome and the host. METHODS: Here, we examine the esophageal microenvironment in gastro-esophageal reflux disease and metaplasia using multi-omics strategies targeting the microbiome and host transcriptome, followed by targeted culture, comparative genomics, and host-microbial interaction studies of bacterial signatures of interest. RESULTS: Profiling of the host transcriptome from esophageal mucosal biopsies revealed profound changes during metaplasia. Importantly, five biomarkers showed consistent longitudinal changes with disease progression from reflux disease to metaplasia. We showed for the first time that the esophageal microbiome is distinct from the salivary microbiome and the enrichment of Campylobacter species as a consistent signature in disease across two independent cohorts. Shape fitting and matrix correlation identified associations between the microbiome and host transcriptome profiles, with a novel co-exclusion relationship found between Campylobacter and napsin B aspartic peptidase. Targeted culture of Campylobacter species from the same cohort revealed a subset of isolates to have a higher capacity to survive within primary human macrophages. Comparative genomic analyses showed these isolates could be differentiated by specific genomic features, one of which was validated to be associated with intracellular fitness. Screening for these Campylobacter strain-specific signatures in shotgun metagenomics data from another cohort showed an increase in prevalence with disease progression. Comparative transcriptomic analyses of primary esophageal epithelial cells exposed to the Campylobacter isolates revealed expression changes within those infected with strains with high intracellular fitness that could explain the increased likelihood of disease progression. CONCLUSIONS: We provide a comprehensive assessment of the esophageal microenvironment, identifying bacterial strain-specific signatures with high relevance to progression of metaplasia.

Hepatitis C Virus Epitope Immunodominance and B Cell Repertoire Diversity.

Despite the advent of effective, curative treatments for hepatitis C virus (HCV), a preventative vaccine remains essential for the global elimination of HCV. It is now clear that the induction of broadly neutralising antibodies (bNAbs) is essential for the rational design of such a vaccine. This review details the current understanding of epitopes on the HCV envelope, characterising the potency, breadth and immunodominance of antibodies induced against these epitopes, as well as describing the interactions between B-cell receptors and HCV infection, with a particular focus on bNAb heavy and light chain variable gene usage. Additionally, we consider the importance of a public repertoire for antibodies against HCV, compiling current knowledge and suggesting that further research in this area may be critical to the rational design of an effective HCV vaccine.

Optimisation and validation of a new method for antibody dependent cellular phagocytosis in hepatitis C virus infection.

Lack of a simple, high throughput antibody-dependent cellular phagocytosis (ADCP) assay has limited our understanding of its potential role of in hepatitis C (HCV) infection. Here, we optimised a flow-cytometry based ADCP assay using HCV envelope (E2)-protein coated microbeads that were opsonised with anti-E2 monoclonal IgG antibody (αE2 mAb) and the THP-1 monocyte cell line as effector cells. We found 1.5 × 109/ml microbeads opsonised with 5 µg/ml αE2 mAb and 1.6 × 106/ml THP-1 cells were optimal conditions to distinguish between healthy controls and patients with HCV. This optimised assay was then used to investigate ADCP in plasma obtained from 72 patients with chronic HCV infection and 15 healthy controls. We found that 75% of patients with genotype 1 and 87% of patients with genotype 3 HCV infection had significantly higher levels of ADCP compared to healthy controls. In patients, there was a significant correlation between increase in ADCP and higher concentrations of anti-E2 IgG antibodies in the plasma. Taken together, we established a simple, quick and high throughput ADCP assay for HCV infection that can readily be used for screening of large cohorts of patients and investigation of the role of ADCP in the pathogenesis or protection from this disease.

Nuclear Leukocyte Immunoglobulin-like Receptor A3 Is Monomeric and Is Involved in Multiple Layers of Regulated Gene Expression and Translation.

The leukocyte immunoglobulin-like receptor A3 (LILRA3) is a soluble protein primarily expressed by peripheral blood monocytes and is abundant in sera of healthy donors. Extracellular LILRA3 is anti-inflammatory and displays neuro-regenerative functions in vitro. However, its intracellular expression, distribution, and function(s) remain unknown. Using a combination of high-resolution confocal and super-resolution microscopy, we identified intracellular expression of native LILRA3 in the nucleus of peripheral blood monocytes and in vitro-derived macrophages. This unexpected nuclear localization of LILRA3 was confirmed in LILRA3-GFP-transfected HEK293T cells. Western blot of proteins fractionated from primary macrophages and the transfected HEK293T cells confirmed nuclear localization of the native and expressed LILRA3 proteins. Interestingly, most of the LILRA3 in the nucleus was in a monomeric form like the biologically active secreted protein, while that in the other cellular compartments was in mixed monomeric, dimeric, and oligomeric forms. The predominant presence of monomeric LILRA3 in the nucleus was independently corroborated in transfected live HEK293T cells using the number and molecular brightness (N&B) analysis method. Immunoprecipitation and mass spectrometric peptide sequencing studies revealed that nuclear LILRA3 co-immunoprecipitated with several nuclear proteins involved in host protein synthesis machinery via direct interactions to a key multifunctional RNA-binding protein, the Ewing sarcoma breakpoint region 1 protein (EWS) (data are available via ProteomeXchange with identifier PXD024602). The biological significance of the nuclear expression of LILRA3 and its interaction with these key proteins remain to be elucidated.