High Antigen Concentrations Research Articles

Recurrent herpetic erosion of the cornea: diagnosis, treatment and prevention of recurrences

A group of patients was found to have a special form of recurrent corneal erosion caused by types I and II herpes virus. This form represents an independent form of ophthalmic herpes - herpetic recurrent erosion (HRE) of the cornea. The herpetic etiology of recurrent corneal erosion was confirmed by the immunofluorescence study of scraping from the conjunctiva, which revealed a high concentration of the herpes simplex virus antigen. Treatment of patients (171 patients, 182 eyes) with HRE included 2 consecutive stages: stage I - relief of acute symptoms of the disease with the help of conservative treatment (instillations of interferon inducers, autologous serum, corneal protectors, tear substitutes, use of therapeutic soft contact lenses); in some cases, phototherapeutic keratectomy was used in the absence of the effect of conservative therapy, as well as in the localization of the focus in the optical zone. Stage II involved anti-relapse therapy based on the use of a Russian-produced herpes vaccine in the intercurrent period. After vaccination, observation for 2 years or more showed that 81.3% of patients achieved clinical recovery (complete cessation of HRE recurrences), 15.8% had a decrease in the frequency and severity of relapses, while 2.9% of patients did not respond to the treatment.

Survival outcomes of patients treated with local therapy for nonmetastatic prostate cancer with high prostate-specific antigen concentrations.

Patients with nonmetastatic prostate cancer (nmPCa) and high prostate-specific antigen (PSA) levels due to the high likelihood of metastasis pose a clinical dilemma regarding their optimal treatment and long-term outcomes after initial local therapy. We aimed to evaluate the oncologic outcomes of patients treated with radical prostatectomy (RP) or radiotherapy (RT) for nmPCa with high PSA levels. We queried the Surveillance, Epidemiology, and End Results (SEER) database to identify patients diagnosed with nmPCa who received RP or RT from 2004 through 2015. We included nmPCa patients with high PSA levels categorized as ≥50 and ≥98 ng/mL, the highest level recorded in SEER. We used the Kaplan-Meier method and Cox proportional hazards to analyze cancer-specific (CSS) and overall survival (OS). We included 6177 patients with nmPCa and PSA ≥ 50 ng/mL at diagnosis; 1698 (27%) had PSA ≥ 98 ng/mL. Of these, 1658 (26.8%) underwent RP and 4519 (73.16%) patients received primary RT. Within a median of 113 months (interquartile range 74-150 months), the 5- and 10-year CSS estimates were 92.3% and 81.5% respectively; 10-year OS was 61%. In the PSA ≥ 98 ng/mL subgroup 5- and 10-year CSS estimates were 89.2% and 76%, respectively. In multivariable analyses for CSS, ISUP grade group (p < 0.001), N stage (p < 0.001), treatment with RP (hazard ratio [HR] = 0.60, 95% confidence interval [CI] 0.43-0.83, p < 0.001), and patient's age (p < 0.05) were associated with improved CSS. In the whole cohort of patients with PSA ≥ 50 ng/mL and RP subgroup, PSA failed to retain its independent prognostic value for CSS. Patients treated with local therapy for nmPCa with very high PSA at diagnosis have relatively good long-term oncological outcomes. Therefore, among well-selected patients with nmPCa, high PSA levels alone should not preclude the use of radical local therapy. Potential selection bias limits inferences about the relative effectiveness of specific local therapies in this setting.

IgG4 Antibodies Induced by Repeated Vaccination May Generate Immune Tolerance to the SARS-CoV-2 Spike Protein.

Less than a year after the global emergence of the coronavirus SARS-CoV-2, a novel vaccine platform based on mRNA technology was introduced to the market. Globally, around 13.38 billion COVID-19 vaccine doses of diverse platforms have been administered. To date, 72.3% of the total population has been injected at least once with a COVID-19 vaccine. As the immunity provided by these vaccines rapidly wanes, their ability to prevent hospitalization and severe disease in individuals with comorbidities has recently been questioned, and increasing evidence has shown that, as with many other vaccines, they do not produce sterilizing immunity, allowing people to suffer frequent re-infections. Additionally, recent investigations have found abnormally high levels of IgG4 in people who were administered two or more injections of the mRNA vaccines. HIV, Malaria, and Pertussis vaccines have also been reported to induce higher-than-normal IgG4 synthesis. Overall, there are three critical factors determining the class switch to IgG4 antibodies: excessive antigen concentration, repeated vaccination, and the type of vaccine used. It has been suggested that an increase in IgG4 levels could have a protecting role by preventing immune over-activation, similar to that occurring during successful allergen-specific immunotherapy by inhibiting IgE-induced effects. However, emerging evidence suggests that the reported increase in IgG4 levels detected after repeated vaccination with the mRNA vaccines may not be a protective mechanism; rather, it constitutes an immune tolerance mechanism to the spike protein that could promote unopposed SARS-CoV2 infection and replication by suppressing natural antiviral responses. Increased IgG4 synthesis due to repeated mRNA vaccination with high antigen concentrations may also cause autoimmune diseases, and promote cancer growth and autoimmune myocarditis in susceptible individuals.

Factors affecting IgG4-mediated complement activation.

Of the four human immunoglobulin G (IgG) subclasses, IgG4 is considered the least inflammatory, in part because it poorly activates the complement system. Regardless, in IgG4 related disease (IgG4-RD) and in autoimmune disorders with high levels of IgG4 autoantibodies, the presence of these antibodies has been linked to consumption and deposition of complement components. This apparent paradox suggests that conditions may exist, potentially reminiscent of in vivo deposits, that allow for complement activation by IgG4. Furthermore, it is currently unclear how variable glycosylation and Fab arm exchange may influence the ability of IgG4 to activate complement. Here, we used well-defined, glyco-engineered monoclonal preparations of IgG4 and determined their ability to activate complement in a controlled system. We show that IgG4 can activate complement only at high antigen and antibody concentrations, via the classical pathway. Moreover, elevated or reduced Fc galactosylation enhanced or diminished complement activation, respectively, with no apparent contribution from the lectin pathway. Fab glycans slightly reduced complement activation. Lastly, we show that bispecific, monovalent IgG4 resulting from Fab arm exchange is a less potent activator of complement than monospecific IgG4. Taken together, these results imply that involvement of IgG4-mediated complement activation in pathology is possible but unlikely.

Chicken antibodies are highly suitable for particle enhanced turbidimetric assays.

Antibody-based assays are commonly used in clinical laboratories for analyzing plasma, serum and other samples for particular protein markers. Although such assays have been traditionally based on antibodies raised in mammals (e.g., mice, rabbits, goats), there are several advantages of using avian antibodies (IgY) raised in chickens, including production volumes, costs, and ethical/animal welfare considerations. A further disadvantage of using mammalian IgG in such assays is the potential for agglutination when exposed to rheumatoid factor (RF) in serum. However, when used in the free form the immune complexes formed with avian antibodies have been reported to have less ability than those formed with mammalian antibodies to cause the light scatter which are used for instrument measurement. In addition, when the amount of antigen exceeds the maximum precipitating point in relation to the amount of antibody, there is a rapid decline in the absorbance values of the immune complexes (antigen excess) when IgY is used. However, when avian antibodies are conjugated to a substrate and used in particle enhanced turbidimetric assays (PETIA), these problems are avoided. Here we investigated three clinical assays using chicken antibodies, one using free (unbound) IgY and two with IgY-based PETIA. The IgY PETIA demonstrated a strong scatter response, even at high antigen concentrations in contrast to the steep decline seen with free IgY antibodies. IgY PETIA reagents can provide test results with low coefficient of variation (<1% for duplicate samples). We also investigated the effect of RF on agglutination of mammalian antibodies (IgG from mouse, rabbit, sheep, and human) and chicken antibodies. Whereas agglutination was observed with all the mammalian antibodies in the presence of RF, this was not observed at all with chicken IgY. Our results support the growing body of evidence that chicken egg yolks can thus be a valuable source of antibodies for use in PETIA in clinical laboratories.

Insulin B-chain hybrid peptides are agonists for T cells reactive to insulin B:9-23 in autoimmune diabetes.

Insulin is considered to be a key antigenic target of T cells in Type 1 Diabetes (T1D) and autoimmune diabetes in the NOD mouse with particular focus on the B-chain amino acid sequence B:9-23 as the primary epitope. Our lab previously discovered that hybrid insulin peptides (HIPs), comprised of insulin C-peptide fragments fused to other β-cell granule peptides, are ligands for several pathogenic CD4 T cell clones derived from NOD mice and for autoreactive CD4 T cells from T1D patients. A subset of CD4 T cell clones from our panel react to insulin and B:9-23 but only at high concentrations of antigen. We hypothesized that HIPs might also be formed from insulin B-chain sequences covalently bound to other endogenously cleaved ß-cell proteins. We report here on the identification of a B-chain HIP, termed the 6.3HIP, containing a fragment of B:9-23 joined to an endogenously processed peptide of ProSAAS, as a strong neo-epitope for the insulin-reactive CD4 T cell clone BDC-6.3. Using an I-Ag7 tetramer loaded with the 6.3HIP, we demonstrate that T cells reactive to this B-chain HIP can be readily detected in NOD mouse islet infiltrates. This work suggests that some portion of autoreactive T cells stimulated by insulin B:9-23 may be responding to B-chain HIPs as peptide ligands.

Female ejaculation and squirting as similar but completely different phenomena: A narrative review of current research.

Women expel fluids of various quantities and compositions from the urethra during sexual arousal and orgasm. These are classified as either female ejaculation (FE) or squirting (SQ). The aim of our analysis was to present evidence that FE and SQ are similar but etiologically different phenomena. A review of studies was performed on fluids expelled from the urogenital tract during female sexual activities using the Web of Knowledge™ (Web of Science Core Collection) and MEDLINE (Ovid) databases from 1946 to 2021. Until 2011, all female orgasmic expulsions of fluids were referred to as FE. The fluid was known to be either from the paraurethral glands or as a result of coital incontinence. At present, SQ is considered as a transurethral expulsion of approximately 10 milliliters or more of transparent fluid, while FE is considered as a secretion of a few milliliters of thick fluid. The fluid in SQ is similar to urine and is expelled by the urinary bladder. The secretion in FE originates from the paraurethral glands and contains a high concentration of prostate-specific antigen. Both phenomena can occur simultaneously. The mechanisms underlying SQ and FE are entirely different. SQ is a massive transurethral orgasmic expulsion from the urinary bladder, while FE is the secretion of a very small amount of fluid from the paraurethral glands.

Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients in the DRC enrolled from 2017 to 2018

Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp2/3 deletions. Samples were assayed for HRP2, pan-Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.

Phase I safety and immunogenicity study of a Brazilian serogroup B vaccine

Meningococcal disease by serogroup B has been a public health problem in Brazil in the last decades. The Brazilian Oswaldo Cruz Foundation has been working to develop a vaccine with detergent-treated outer membrane vesicles (OMV) and detoxified endotoxin (dLOS) from Neisseria meningitidis serogroup B prevalent strains. A phase I study, enrolling 26 adults (18–44 years of age) was performed using experimental vaccines combining B components and aluminum hydroxide as adjuvant. It was a dose escalation study testing vaccines made of 25, 50, and 100 µg OMV protein/mL (sum of both strains) and dLOS in half amount of total protein concentration, with three doses given two months apart. Adverse events were mild/moderate with frequency increasing with the amount of antigens. Pain in the site of injection was the most frequent reaction in all doses, reported in more than the 85% across vaccine groups. Considering all injections, cephalea was the most common systemic adverse event, detected in 11.1%, 17.2% and 32.1%, respectively with doses of 12.5 μg, 25 μg and 50 μg. High titers of total IgG (ELISA) were observed for the vaccine components before vaccination. Protective levels of bactericidal antibodies (titer ≥1:4) for both vaccine strains were also present. Considering a 4-fold increase of IgG titers compared to pre-immune values (seroconversion), 50%-70% of those who received intermediate and highest doses of antigens presented satisfactory response for OMV of N44/89 strain. The lowest dose vaccine induced no seroconversion for strain N44/89, and 11% for strain N603/95. For the three vaccines doses, 25% of seroconversion, in total IgG against LOS, was observed. Increased antibody bactericidal activity was observed for both strains in higher antigen concentrations. For IgG against LOS, all vaccine formulations showed 25% of seroconversion. In conclusion, MenB-Bio experimental vaccines were well tolerated and immunogenic, thus allowing phase II studies.

Intranasal vaccination with protein bodies elicit strong protection against Streptococcus pneumoniae colonization

Protein bodies (PBs) are particles consisting of insoluble, aggregated proteins with potential as a vaccine formulation. PBs can contain high concentrations of antigen, are stable and relatively resistant to proteases, release antigen slowly and are cost-effective to manufacture. Yet, the capacity of PBs to provoke immune responses and protection in the upper respiratory tract, a major entry route of respiratory pathogens, is largely unknown.In this study, we vaccinated mice intranasally with PBs comprising antigens from Streptococcus pneumoniae and evaluated the level of protection against nasopharyngeal colonization. PBs composed of the α-helical domain of pneumococcal surface protein A (PspAα) provided superior protection against colonization with S. pneumoniae compared to soluble PspAα. Immunization with soluble protein or PBs induced differences in antibody binding to pneumococci as well as a highly distinct antigen-specific nasal cytokine profile upon in vivo stimulation with inactivated S. pneumoniae. Moreover, immunization with PBs composed of conserved putative pneumococcal antigens reduced colonization by S. pneumoniae in mice, both as a single- and as a multi-antigen formulation.In conclusion, PBs represent a vaccine formulation that elicits strong mucosal immune responses and protection. The versatility of this platform offers opportunities for development of next-generation vaccine formulations.

Diagnostic Methods and Risk Factors for Severe Disease and Mortality in Blastomycosis: A Retrospective Cohort Study.

Background: Blastomycosis can cause severe disease with progressive respiratory failure and dissemination even in immunocompetent individuals. We sought to evaluate risk factors for severe disease and mortality using clinical and laboratory data within a large health system in an endemic area. Methods: We performed a retrospective cohort study of patients diagnosed with blastomycosis at all Mayo Clinic sites from 1 January 2004 through 31 March 2020. Diagnosis was established by culture, histopathology/cytopathology, serology, antigen testing, or PCR. Disease was categorized as mild for patients treated in the outpatient setting, moderate for hospitalized patients who did not require intensive care, and severe for patients admitted to the intensive care unit. Logistic regression was used to evaluate risk factors for severe disease. A Cox proportional hazards model was constructed to evaluate mortality. Findings: We identified 210 patients diagnosed with blastomycosis. Mean age was 51 years (range, 6–84). Most subjects were male (71.0%). Extrapulmonary disease was confirmed in 24.8%. In this cohort, 40.5% of patients had mild disease, 37.6% had moderate disease, and 21.9% had severe disease. Independent risk factors for severe disease were neutrophilia (odds ratio (OR) 3.35 (95% CI 1.53–7.35), p = 0.002) and lymphopenia (OR 3.34 (95% CI 1.59–7.03), p = 0.001). Mortality at 90 days was 11.9%. Median time from diagnosis to death was 23 days (interquartile range 8–31 days). Independent risk factors for mortality were age (OR 1.04 (95% CI 1.01–1.08), p = 0.009), neutrophilia (OR 2.84 (95% CI 1.04–7.76), p = 0.041), and lymphopenia (OR 4.50 (95% CI 1.67–12.11), p = 0.003). Blastomyces immunodiffusion had an overall sensitivity of 39.6% (95% CI 30.1–49.8). Sensitivity was higher among those who were tested 4 weeks or longer after the onset of symptoms. Urine Blastomyces antigen had a significantly higher sensitivity of 80.8% (95% CI 68.1–89.2) compared to serology. There was a trend towards higher antigen concentration in patients with severe disease. The sensitivity of PCR from respiratory specimens was 67.6% (95% CI 50.1–85.5). Conclusion: In this cohort, we did not find an association between pharmacologic immunosuppression and disease severity. Lymphopenia at diagnosis was an independent risk factor for mortality. This simple marker may aid clinicians in determining disease prognosis.

IgM-bearing B cell affinity subpopulations possess differential antigen sensitivity in rainbow trout

The need for accurate assessments of in vitro generated antibody prompted examination of the effect of antigen on secreted antibody concentrations and affinities. It was found that the antigen concentrations commonly employed for in vitro stimulation were able to significantly compromise IgM titer and affinity estimates in rainbow trout. Specifically, IgM titers were underestimated with the high affinity antibodies being specifically blocked. To remedy this, pulsed antigen cultures were employed, and it was found to reveal more accurate IgM antibody titers and affinity estimates. Additionally, pulsed dose responses provided evidence that high antigen concentrations specifically suppressed high affinity B cell induction. Optimal expression of high affinity antibodies required exposure to lower concentrations of antigen. Each affinity subpopulation appeared to possess a graded sensitivity to each dose of antigen, revealing the complex dynamic for differential IgM-bearing B cell induction that is possible during a response. These results reveal not only the need for antigen removal prior to in vitro antibody secretion, but also the possible role of high zone immunological tolerance on IgM affinity maturation in rainbow trout.

Study of Gene Expression of Programmed Cell Death Ligand 1 (PD-L1) in Breast Cancer Patients

Introduction: Breast cancer is the most frequently occurring cancer among females and the second most common cancer overall. Programmed Cell Death Ligand 1 (PD-L1) plays an important role in blocking ‘cancer-immunity cycle’ and is considered as a major inhibitory pathway. The aim of the present study was to clarify the alterations of expression of PD-L1 in peripheral blood mononuclear cytes (PBMCs) of female breast cancer patients and analyze its association with clinico-pathological criteria as well as therapeutic response. Materials and Methods: The study was conducted on 45 female breast cancer patients and 45 female controls. Blood samples were collected followed by PBMCs isolation, total RNA extraction, reverse transcription and finally, quantitative polymerase chain reaction (qPCR) using SYBR Green DNA binding dye. Expression levels of PD-L1 were calculated and then compared with clinicopathological parameters of the patients in addition to initial therapeutic response. Results: A significant difference was detected for PD-L1 expression levels in breast cancer patients compared to controls. A significant association with age, metastatic breast cancer, estrogen receptor (ER) negative status as well as high concentrations of cancer antigen 15-3 (CA15-3) was detected. On the other hand, no significant association was recognized with tumor size, lymph nodal status, histopathological type, grade, progesterone receptor (PR) status, human epidermal growth factor receptor 2 (HER-2) status, triple negative, among de novo and recurrent metastatic patients and for the number of metastatic sites as well as the therapeutic response. Conclusions: This study paves the way of the use of PD-L1 as a noninvasive prognostic and diagnostic biomarker for poor prognosis of breast cancer.

Association between serum prostate-specific antigen concentrations and the risk of developing type2 diabetes mellitus in Chinese men: A cohort study.

Aims/IntroductionThe current literature suggests that men with diabetes have a lower prostate‐specific antigen concentration than men without diabetes, but the causal association remains unclear. We aimed to investigate the association between serum prostate‐specific antigen concentrations and the risk of type 2 diabetes mellitus in a cohort study of a Chinese population.Materials and MethodsWe designed a cohort study that comprised 16,811 initially non‐diabetic Chinese men who received annual health checkups between 2009 and 2016. The outcome of this study was type 2 diabetes mellitus, identified by medical diagnosis, self‐reportage, medication use, fasting glucose, 2‐h post oral glucose or glycated hemoglobin measurements. Cox proportional hazards models were carried out to evaluate the association.ResultsDuring a median follow‐up period of 3.8 years (interquartile range 1.91–5.73 years), 1,260 participants developed incident type 2 diabetes mellitus. The multivariable model, adjusted for various potential confounders, showed that serum prostate‐specific antigen concentrations were inversely related to type 2 diabetes mellitus risk (P for trend = 0.014). Compared with the lowest quartile of serum prostate‐specific antigen, the hazard ratio and 95% confidence intervals of type 2 diabetes mellitus risk for quartile 2–4 were 0.84 (0.66–1.07), 0.75 (0.59–0.94) and 0.77 (0.62–0.96), respectively. Subgroup analyses suggested the inverse relationship was more prominent in overweight or obese participants (P for interaction = 0.013).ConclusionsHigh serum prostate‐specific antigen concentration was associated with a low risk of type 2 diabetes mellitus in Chinese men. Future studies are required to confirm these findings and investigate underlying mechanisms.

Ultra-Sensitive Serial Profiling of SARS-CoV-2 Antigens and Antibodies in Plasma to Understand Disease Progression in COVID-19 Patients with Severe Disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 21 million people worldwide since August 16, 2020. Compared to PCR and serology tests, SARS-CoV-2 antigen assays are underdeveloped, despite their potential to identify active infection and monitor disease progression. We used Single Molecule Array (Simoa) assays to quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of patients with coronavirus disease (COVID-19). We studied plasma from 64 patients who were COVID-19 positive, 17 who were COVID-19 negative, and 34 prepandemic patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we quantified changes in 31 SARS-CoV-2 biomarkers in 272 longitudinal plasma samples obtained for 39 patients with COVID-19. Data were analyzed by hierarchical clustering and were compared to longitudinal RT-PCR test results and clinical outcomes. SARS-CoV-2 S1 and N antigens were detectable in 41 out of 64 COVID-19 positive patients. In these patients, full antigen clearance in plasma was observed a mean ± 95% CI of 5 ± 1 days after seroconversion and nasopharyngeal RT-PCR tests reported positive results for 15 ± 5 days after viral-antigen clearance. Correlation between patients with high concentrations of S1 antigen and ICU admission (77%) and time to intubation (within 1 day) was statistically significant. The reported SARS-CoV-2 Simoa antigen assay is the first to detect viral antigens in the plasma of patients who were COVID-19 positive to date. These data show that SARS-CoV-2 viral antigens in the blood are associated with disease progression, such as respiratory failure, in COVID-19 cases with severe disease.

Unraveling the Hook Effect: A Comprehensive Study of High Antigen Concentration Effects in Sandwich Lateral Flow Immunoassays.

Sandwich lateral flow immunoassays (LFIAs) are limited at high antigen concentrations by the hook effect, leading to a contradictory decrease in the test line (T) intensity and false-negative results. The hook effect is mainly associated with the loss of T, and research focuses on minimizing this effect. Nevertheless, the control line (C) intensity is also affected at higher analyte concentrations, undesirably influencing the T/C ratio in LFIA readers. The main aim of this work is to identify and understand these high antigen concentration effects in order to develop ubiquitous strategies to interpret and mitigate such effects. Four complementary experiments were performed: performance assessment of three different allergen LFIAs (two for hazelnut, one for peanut) over 0.075–3500 ppm, LFIAs with C only, surface plasmon resonance (SPR) binding experiments on the immobilized control antibody, and smartphone video recording of LFIAs during their development. As antigen concentrations increase, the C signal decreases before the T signal does, suggesting that distinct mechanisms underlie these intensity reductions. Reduced binding at the C occurred even in the absence of T, so the upfront T does not explain the loss of C. SPR confirmed that the C antibody favors binding with free labeled antibody compared with a labeled antibody–analyte complex, indicating that in antigen excess, binding is reduced at C before T. Finally, a smartphone-based video method was developed for dynamically monitoring the LFIA development in real time to distinguish between different concentration-dependent effects. Digitally analyzing the data allows clear differentiation of highly positive samples and false-negative samples and can indicate whether the LFIA is in the dynamic working range or at critically high concentrations. The aim of this work is to identify and understand such high antigen concentration effects in order to develop ubiquitous strategies to interpret and mitigate such effects.

Structurally distinct endocytic pathways for B cell receptors in B lymphocytes.

B lymphocytes play a critical role in adaptive immunity. On antigen binding, B cell receptors (BCR) cluster on the plasma membrane and are internalized by endocytosis. In this process, B cells capture diverse antigens in various contexts and concentrations. However, it is unclear whether the mechanism of BCR endocytosis changes in response to these factors. Here, we studied the mechanism of soluble antigen-induced BCR clustering and internalization in a cultured human B cell line using correlative superresolution fluorescence and platinum replica electron microscopy. First, by visualizing nanoscale BCR clusters, we provide direct evidence that BCR cluster size increases with F(ab’)2 concentration. Next, we show that the physical mechanism of internalization switches in response to BCR cluster size. At low concentrations of antigen, B cells internalize small BCR clusters by classical clathrin-mediated endocytosis. At high antigen concentrations, when cluster size increases beyond the size of a single clathrin-coated pit, B cells retrieve receptor clusters using large invaginations of the plasma membrane capped with clathrin. At these sites, we observed early and sustained recruitment of actin and an actin polymerizing protein FCHSD2. We further show that actin recruitment is required for the efficient generation of these novel endocytic carriers and for their capture into the cytosol. We propose that in B cells, the mechanism of endocytosis switches to accommodate large receptor clusters formed when cells encounter high concentrations of soluble antigen. This mechanism is regulated by the organization and dynamics of the cortical actin cytoskeleton.

Effect of Revaccination with Recombinant Hepatitis B Virus Vaccine Containing Higher Antigen Concentration in Non-responder Children

BACKGROUND: Hepatitis B is a global health problem carrying significant morbidity and mortality. Vaccination against hepatitis B virus (HBV) has been implemented with good effectiveness, but a small proportion of children do not respond well after complete routine vaccination. They are called non-responders and are at higher risk for HBV infection.&#x0D; AIM: The objective of the study was to determine the effect of revaccination with recombinant HBV vaccine containing higher antigen concentration in non-responder children.&#x0D; METHODS: A prospective study was conducted from January 2018 until December 2019. Inclusion criteria were children aged 9–15 months and had three doses of recombinant HBV at 0, 1, and 6 months old. Exclusion criteria were the presence of acute or chronic hepatitis B and conditions which alter immune function and immunosuppressant medications consumption. Demographic and clinical characteristics were gathered from each subject along with serum HBsAb titer examination. Subjects with serum anti-hepatitis B surface antigen (HBsAb) titer of &lt;10 IU/L were classified into non-responders and underwent revaccination with 3 doses of vaccine containing 20 μg HBsAg. The revaccination series was repeated until all subjects showed seroconversion. After each series, repeated serum HBsAb titer measurements were conducted.&#x0D; RESULTS: A total of 400 children were enrolled in this study with a mean age of 12.1 (SD 2.64) months. Subjects were dominated with females (54.0) and normal nutritional status (79.0). The rate of non-responsiveness was 9.0%. Of all non-responders, 88.9% experienced seroconversion after the first revaccination series. After the third revaccination series, all subjects had serum HBsAb titer at the protective level.&#x0D; CONCLUSION: Revaccination with recombinant HBV vaccine containing higher antigen concentration in non-responder children is effective.

Prevalence and Factors Associated with High Concentration of Prostate-Specific Antigen: ELSIA Study.

Prostate cancer (PC) is the second most common cancer among men, behind only non-melanoma skin cancer, and the main method of screening for PC is the prostate-specific antigen (PSA). To analyze the prevalence and the factors associated with high concentration of PSA in the elderly is essential to understand this outcome, and building strategies to decrease their rates of morbidity and mortality. We performed a cross-sectional study with 96 elderly men. A high level of PSA was defined by >4.0 ng/mL. In order to identify sociodemographic, health, functional and behavioral variables, which may be associated with high levels of PSA, we carried out a multivariate analysis using Poisson regression. The prevalence of high levels of PSA was 21.9% (n = 21). High levels of PSA was associated with years of study, race/ethnicity and family arrangement, health perception, systolic blood pressure, diastolic blood pressure, metabolic diseases, alcohol consumption and sedentary behavior. The study found a high prevalence of high PSA concentrations in the elderly and several aspects are associated, which can be a worrying factor for their health, since PSA is an important marker of prostate cancer.

225Ac-PSMA-617/177Lu-PSMA-617 tandem therapy of metastatic castration-resistant prostate cancer: pilot experience.

Up to 30% of patients with prostate-specific membrane antigen (PSMA)-positive metastatic castration-resistant prostate cancer (mCRPC) never respond or develop resistance to 177Lu-labeled PSMA-targeted radioligand monotherapy. Single-agent PSMA-targeted radioligand therapy (PRLT) with the alpha-emitter 225Ac showed promise against mCRPC but may cause severe and/or persistent xerostomia, which may substantially impair patients' quality-of-life. We hypothesized that when 177Lu-PSMA ligand alone is ineffective, tandem therapy with low-activity 225Ac-PSMA ligand plus full activity of the beta emitter may enhance efficacy while minimizing xerostomia severity. We retrospectively analyzed pilot experience with 1 course of 225Ac-PSMA-617/177Lu-PSMA-617 tandem therapy in our first 20 patients with mCRPC receiving this intervention after insufficiently responding to 177Lu-PSMA-617 monotherapy. This cohort had late-stage/end-stage disease with high baseline prostate-specific antigen (PSA) concentration (median 215 ng/mL), heavy pre-treatment (abiraterone and/or enzalutamide, and 177Lu-PRLT [median cumulative activity, 26.3 GBq] in 20/20 patients, 100%; docetaxel and/or cabazitaxel in 13/20 patients, 65%), and frequent Eastern Cooperative Oncology Group performance status of 2 (8/20 patients, 40%). Median (minimum-maximum) administered activities were 225Ac-PSMA-617, 5.3 (1.5-7.9) MBq, and 177Lu-PSMA-617, 6.9 (5.0-11.6) GBq. Significant responders to tandem therapy received 177Lu-PSMA-617 monotherapy as maintenance (median [minimum-maximum]: 1 [0-5] cycle). After a median (minimum-maximum) 22 (14-63) weeks' follow-up, 13/20 patients (65%) had as best biochemical response a PSA decline > 50%. Median (95% confidence interval) progression-free survival was 19 (12-26) weeks, and overall survival was 48 (4-92) weeks post-tandem therapy administration. Xerostomia was reported as grade 1 (very mild) in 8/20 patients (40%), grade 2 (mild) in 5/20 (25%), and grade 3/4 in 0/20. Our results suggest that a single course of tandem therapy with low-activity 225Ac-PSMA-617/full-activity 177Lu-PSMA-617 may safely enhance response to PRLT in men with late-stage/end-stage mCRPC while minimizing xerostomia severity. Formal study of this combination is warranted.