Antibody B72 Research Articles

Group 2 innate lymphoid cells are a non-redundant source of interleukin-5 required for development and function of murine B1 cells

Tissue-resident immune cells, such as innate lymphoid cells, mediate protective or detrimental immune responses at barrier surfaces. Upon activation by stromal or epithelial cell-derived alarmins, group 2 innate lymphoid cells (ILC2s) are a rapid source of type 2 cytokines, such as IL-5. However, due to the overlap in effector functions, it remains unresolved whether ILC2s are an essential component of the type 2 response or whether their function can be compensated by other cells, such as T cells. Here we show a non-redundant role of ILC2s in supporting the development and function of B1 cells. We demonstrate that B1 cells fail to develop properly in the absence of ILC2s and identify the IL-33 receptor on ILC2s as an essential cell-intrinsic regulator of IL-5 production. Further, conditional deletion of Il5 in ILC2s results in defective B1 cell development and immunoglobulin production. Consequently, B1 cells with phosphatidylcholine specific B cell receptor rearrangements are diminished in ILC2-deficient mice. Thus, our data establish an essential function of ILC2s in supporting B1 cells and antibody production at barrier surfaces.

A Single-Center Pilot Study of Therapeutic Apheresis in Patients with Severe Post-COVID Syndrome.

After the COVID-19 pandemic, many patients have reported chronic fatigue and severe post-exertional malaise, with symptoms similar to those of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). The accumulation of agonistic receptor autoantibodies targeting beta-adrenergic (β1 and β2) and muscarinic (M3 and M4) neurotransmitter receptors may play a crucial role in the pathomechanism of both ME/CFS and post-COVID conditions. Therapeutic apheresis has been suggested as an effective treatment option for alleviating and mitigating symptoms in this desperate group of patients. In this single-center pilot study, we analyzed autoantibodies in a cohort of 20 post-COVID patients before and after therapeutic apheresis. Apheresis resulted in a decline of β1 or β2 adrenergic receptor antibodies in all patients. Additionally, the majority of patients experienced a concurrent reduction in symptoms such as fatigue, physical activity restrictions, myalgia, post-exertional malaise, and concentration disorders. This study clearly demonstrates an association between autoantibodies and the clinical improvement of post-COVID patients. Even if future sham-controlled trials do not show a positive outcome, extracorporeal apheresis may still be valuable for this patient group by temporarily improving microperfusion and symptoms. Success in restoring patients to work and normal life, as observed in many individuals after therapeutic apheresis, should be recognized. Therefore, we believe that extracorporeal therapeutic apheresis, as part of a multimodal treatment, should be considered an early intervention for postinfectious syndromes in selected patients.

B-357 Siemens CA 72-4: an assay for measurement of Tumor Associated Glycoprotein (TAG) 72

Abstract Background The tumor associated glycoprotein (TAG) 72, also known as CA 72-4 is a mucin protein of high molecular weight. CA 72-4 is found on the surface of many cancer cells, including stomach (gastric), ovary, breast, colon and pancreatic cells. CA 72-4 is currently one of the primary markers for the diagnosis of gastric cancer and monitoring the course and efficacy of gastric cancer. In ovarian cancer, it has a higher sensitivity for mucin- type ovarian cancer, and its combination with CA125 for detection can significantly improve the clinical sensitivity. In colorectal cancer, CA 72-4 combined with CEA detection was also found to significantly improve the sensitivity of the initial diagnosis. An assay for CA 72-4 on Siemens Atellica IM (Siemens CA 72-4) is being developed and analytical performance is being presented. Methods The Siemens CA 72-4 assay (in development) is a one-step chemiluminescent microparticle immunoassay (CMIA) for the quantitative determination of CA 72-4 in human serum and plasma. The specimen is incubated with paramagnetic microparticles coated with the monoclonal antibody (mAb) B72.3 and acridinium-labeled mAb CC49 conjugate on the Siemens Atellica IM® System. After incubation and washing, Acid and Base reagents are added. The resulting relative light units (RLUs) are directly proportional to the amount of CA 72-4 in the sample allowing for the quantitative determination of CA 72-4 in the specimen. Results The following performance were determined using 3 unique lots of reagents and calibrators with the calibration range for the assay being 0.00 to 200.00 U/mL. The limit of blank (LoB), limit of detection (LoD), and limit of quantitation at 20% CV (LoQ) were determined and met the goal of ≤ 1.00 U/mL. Linearity according to CLSI EP06 Ed2 was demonstrated for a range of 1.00 through 200.00 U/mL. A Precision study of 2 controls and 5 panels spanning the range of the assay demonstrated a total %CV ≤ 6% for samples ≥ 5.00 U/mL and ≤ 0.25 SD for samples <5.00 U/mL. In the sample tube type study, a matrix comparison of 23 matched serum and plasma samples were evaluated. Passing-Bablock slopes of < 8% and r > 0.99 were observed when evaluating samples within the measurement range for plasma (LiHep, LiHep separator, K2EDTA and K3EDTA) tubes and serum separator tubes (SST) compared to the Red Top serum samples. Five endogenous substances were evaluated for interference in the CA 72-4 assay. The average percent difference between test and control samples for all endogenous interferents was < 10%. Percent difference in the presence of 28 potentially interfering drugs was ≤ 4.0%. Conclusions The Siemens CA 72-4 assay currently in development is a sensitive and precise assay for the quantitative determination of CA 72-4 in human serum and plasma.

Conformational trajectory of the HIV-1 fusion peptide during CD4-induced envelope opening.

The hydrophobic fusion peptide (FP), a critical component of the HIV-1 entry machinery, is located at the N terminal stretch of the envelope (Env) gp41 subunit 1-3 . The receptor-binding gp120 subunit of Env forms a heterodimer with gp41 and assembles into a trimer, in which FP is accessible for antibody binding 3 . Env conformational changes or "opening" that follow receptor binding result in FP relocating to a newly formed interprotomer pocket at the gp41-gp120 interface where it is sterically inaccessible to antibody 4 . The mechanistic steps connecting the entry-related transition of antibody accessible-to-inaccessible FP configurations remain unresolved. Here, using SOSIP-stabilized Env ectodomains 5 , we visualized atomic-level details of a functional entry intermediate, where partially open Env was bound to receptor CD4, co-receptor mimetic antibody 17b, and FP-targeting antibody VRC34.01, demonstrating that FP remains antibody accessible despite substantial receptor-induced Env opening. We determined a series of structures delineating stepwise opening of Env from its closed state to a newly resolved intermediate and defining downstream re-organizations of the gp120-gp41 interface that ultimately resulted in FP burial in an antibody-inaccessible configuration. Our studies improve our understanding of HIV-1 entry and provide information on entry-related conformation reorganization of a key site of HIV vulnerability to neutralizing antibody.

Abstract AW12: Hyperinnervation and Immune-endocrine Interactions in Renal Arteriolar Hypertrophy during Inhibition of The Renin-Angiotensin System

Background: Chronic stimulation of renin cells by long-term use of renin-angiotensin system (RAS) inhibitors or renin deletion leads to concentric renal arteriolar hypertrophy frequently surrounded by immune-inflammatory cells. The disease is caused by the renin cells which increase in number and adopt an embryonic-secretory phenotype and induce the accumulation of smooth muscle cells that obstruct the vessels leading to ischemia. Normally, renin cells are innervated but it is unclear whether innervation of the renal arterial tree is affected under RAS inhibition. Hypothesis: Long-term RAS inhibition increases renal arteriolar innervation. Methods: We used two models of RAS inhibition: 1) Ren1 c-/- ; Ren1 c-Cre ; R26R mTmG mice studied at 3 months of age; 2) SMMHC CreERT2 ; R26R tdTomato ; Ren1 cYFP mice were treated with captopril for 6 months. We performed tissue clearing and 3D staining with an anti-tubulin β3 antibody. The expression of neurogenesis-related genes in renin lineage cells was examined using our previous single-cell RNA sequencing (scRNA-seq) data from mouse kidneys. We compared RNA expression of nerve growth factor (NGF) between Ren1 c−/− mice and control mice by qPCR and in situ hybridization (ISH). T cell infiltration was observed by immunostaining with anti-CD3 antibody. Results: 3-D imaging revealed that in both RAS inhibition models there was marked hyperinnervation. Renin cells expanded along and around the renal arterioles, and covered the glomeruli. Axons grew in numerous branches surrounding thickened afferent arterioles, bound tightly to renin cells and encasing the glomeruli. Our scRNA-seq data showed that Ngf , a gene important for neurogenesis and produced by immune cells, was highly expressed in renin cells. In Ren1 c−/− mice, expression of Ngf was significantly increased compared to controls (p=0.0054). ISH revealed that Ngf was highly expressed in Ren1 c−/− mice in the juxtaglomerular area and in the renal cortical stroma. Infiltration of T cells around affected arterioles was prominent in both models of RAS inhibition. Conclusion: Long-term inhibition of RAS leads to renal hyperinnervation, infiltration of immune cells and arteriolar hypertrophy. The identification of this neuro-immune-endocrine arterioles may help understand and treat vascular disease and hypertension.

Glaesserella parasuis serotype 4 exploits fibronectin via RlpA for tracheal colonization following porcine circovirus type 2 infection.

Porcine circovirus type 2 (PCV2) often causes disease through coinfection with other bacterial pathogens, including Glaesserella parasuis (G. parasuis), which causes high morbidity and mortality, but the role played by PCV2 and bacterial and host factors contributing to this process have not been defined. Bacterial attachment is assumed to occur via specific receptor-ligand interactions between adhesins on the bacterial cell and host proteins adsorbed to the implant surface. Mass spectrometry (MS) analysis of PCV2-infected swine tracheal epithelial cells (STEC) revealed that the expression of Extracellular matrix protein (ECM) Fibronectin (Fn) increased significantly on the infected cells surface. Importantly, efficient G. parasuis serotype 4 (GPS4) adherence to STECs was imparted by interactions with Fn. Furthermore, abrogation of adherence was gained by genetic knockout of Fn, Fn and Integrin β1 antibody blocking. Fn is frequently exploited as a receptor for bacterial pathogens. To explore the GPS4 adhesin that interacts with Fn, recombinant Fn N-terminal type I and type II domains were incubated with GPS4, and the interacting proteins were pulled down for MS analysis. Here, we show that rare lipoprotein A (RlpA) directly interacts with host Fibronectin mediating GPS4 adhesion. Finally, we found that PCV2-induced Fibronectin expression and adherence of GPS4 were prevented significantly by TGF-β signaling pathway inhibitor SB431542. Our data suggest the RlpA-Fn interaction to be a potentially promising novel therapeutic target to combat PCV2 and GPS4 coinfection.

Assessment of adeno-associated virus purity by capillary electrophoresis-based western

A rigorous analytical assessment of recombinant adeno-associated virus (rAAV)-based drug products is critical for their successful development as clinical candidates. It is especially important to ascertain high purity while simultaneously ensuring low levels of impurities in the final drug product. One approach to evaluate the purity of rAAV drug products is to determine the relative stoichiometry of the three viral proteins (VPs) that comprise an rAAV capsid, and the levels of impurities in the final drug product. Here we present two capillary electrophoresis-western (CE-western) assays for quantifying (1) the relative stoichiometry of VP using the anti-AAV B1 antibody, and (2) residual levels of a baculovirus protein impurity, GP64, using the anti-GP64 antibody. In each assay, various purified samples from diverse AAV serotypes were analyzed to determine their VP ratio or GP64 levels. The ratio of VP3/VP1 in rAAV samples was correlated with biological activity, and the clearance of GP64 from the manufacturing process was demonstrated. The results obtained from both assays were further supported by liquid chromatography-mass spectrometry analyses. Overall, we report that CE-western is a high-throughput platform that utilizes low sample volumes for a rapid, sensitive, and robust assessment of the identity, composition, and purity of rAAV drug products.

Highly sensitive and selective electrochemical detection of Aflatoxin B1 in water and Pistachio samples with MOF/MXene composite based sensor

Aflatoxins are poisonous secondary metabolites capable of causing toxicity in several food items and water. The routine monitoring of Aflatoxins has assumed great analytical significance. The electrochemical biosensors are attractive tools to screen the Aflatoxin-laden samples. The present study reports on a new type of electrochemical biosensor, based on an electrode prepared of multifunctional MOF (Metal-organic framework)/MXene composite material. Molybdenum-based 2-dimensional (2D) MOF and Ti3C2 MXene have been mixed to prepare the composite, which is then deposited over a screen-printed electrode (SPE). The anti-Aflatoxin B1 (AFB1) antibodies (Ab) are covalently complexed, and the prepared Ab-MOF/MXene sensor is investigated for its applicability to detect AFB1. Utilizing the electrochemical impedance spectroscopic (EIS) analysis, the Ab-MOF/MXene sensor has allowed the detection of AFB1 from 0.06 to 50 ng/mL with a very low limit of detection, i.e., 8 pg/mL. The sensor performs well with respect to all the major quality parameters, such as long-term stability, specificity in co-presence of interfering species, reproducibility, and repeatability. It is demonstrated successfully to detect AFB1 in the spiked samples of Pistachio.

Impact of immunohistochemistry staining conditions on the incidence of human epidermal growth factor receptor 2 (HER2)-low breast cancer.

We investigated frequencies of HER2-low breast cancer (BC) (immunohistochemistry [IHC] 1+ or 2+ without gene amplification) before and after IHC conditions were modified in order to understand the impact of IHC staining conditions on frequencies of HER2-low BC. Primary BC cases diagnosed at the Yeungnam University Hospital (YUH, n = 728) or Keimyung University Dongsan Hospital (KUDH, n = 290) in 2022 were reviewed, and data on HER2 status and IHC conditions were collected (cohort 1). Both institutions used the 4B5 antibody for HER2 IHC but had different staining protocols. After modifications of the IHC conditions at both institutions, primary BC cases (YUH, n = 324 and KUDH, n = 135) diagnosed from April to July 2023 (cohort 2) were reviewed to assess any changes in the frequency of HER2 status. In cohort 1, of the 728 cases diagnosed at YUH, 556 (76.4%) were HER2-zero, 76 (10.4%) were HER2-low, and 96 (13.2%) were HER2-positive, and of the 290 cases diagnosed at KUDH, 135 (46.6%) were HER2-zero, 82 (28.3%) were HER2-low, and 73 (25.2%) were HER2-positive. Modifications in HER2 IHC staining conditions dramatically increased the frequencies of HER2-low BC in cohort 2 (YUH 38.9% and KUDH 49.6%), but they did not result in significant changes in the HER2-positive rates (YUH 15.4% and KUDH 25.2%) compared to cohort 1. In conclusion, minor modifications in HER2 IHC staining conditions significantly affected the frequency of HER2-low BC but had little impact on the HER2-positivity rate. Each pathology laboratory should verify IHC conditions using control slides (including 1+) to enable the accurate identification of HER2-low BC.

Echinacoside ameliorates hepatic fibrosis and tumor invasion in rats with thioacetamide-induced hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) affects approximately 800,000 individuals globally each year. Despite advancements in HCC treatments, there is still a pressing need to identify new drugs that can combat resistance. One potential option is echinacoside, a natural caffeic acid glycoside with antioxidant, anti-inflammatory, antidepressant, and antidiabetic properties. Therefore, we aimed to investigate the ability of echinacoside to exhibit antitumor activity against HCC in rats through ameliorating hepatic fibrosis and tumor invasion. Rats were given thioacetamide to induce HCC, and some were given 30 mg/kg of echinacoside twice a week for 16 weeks. The liver impairment was assessed by measuring serum α-fetoprotein (AFP) and examining liver sections stained with Masson trichrome or anti-transforming growth factor (TGF)-β1 antibodies. The hepatic expression of mRNA and protein levels of TGF-β1, β-catenin, SMAD4, matrix metalloproteinase-9 (MMP9), phosphoinositide 3-kinases (PI3K), mammalian target of rapamycin (mTOR), connective tissue growth factor 2 (CCN2), E-Cadherin, platelets derived growth factor (PDGF)-B and fascin were also analyzed. Echinacoside improved the survival rate of rats by decreasing serum AFP and the number of hepatic nodules. Examination of micro-images indicated that echinacoside can reduce fibrosis. It also significantly decreased the expression of TGF-β1, β-catenin, SMAD4, MMP9, PI3K, mTOR, CCN2, PDGF-B, and fascin while enhancing the expression of E-Cadherin. In conclusion, echinacoside exhibits a protective effect against HCC by increasing survival rates and decreasing tumor growth. It also acts as an inhibitor of the hepatic tissue fibrosis pathway by reducing the expression of TGF-β1, β-catenin, SMAD4, PI3K, CCN2, PDGF-B and mTOR. Additionally, it prevents tumor invasion by suppressing MMP9 and fascin, and increasing the expression of E-Cadherin.

Production of Secreted Antibody in Baculovirus Expression Vector System.

Monoclonal antibodies have widespread applications in disease treatment and antigen detection. They are traditionally produced using mammalian cell expression system, which is not able to satisfy the increasing demand of these proteins at large scale. Baculovirus expression vector system (BEVS) is an attractive alternative platform for the production of biologically active monoclonal antibodies. In this chapter, we demonstrate the production of an HIV-1 broadly neutralizing antibody b12 in BEVS. The processes including transfer vector construction, recombinant baculovirus generation, and antibody production and detection are described.

Structural and functional Analysis of novel 3‐Nitrotyrosine Aβ‐specific Antibodies

AbstractBackgroundPosttranslational modified (PTM) amyloid peptides are increasingly within the spotlight of Alzheimer’s drug research. Some of these modifications, such as the formation of pyroglutamate or isoaspartate, are associated with the deposition and increased toxicity of amyloid peptides. The role of nitration at Tyr10 of Aβ and its abundance in AD brain is less well understood. How 3NY10‐Aβ is formed is controversial, as well as the influence on aggregation behaviour. Hence, we here aimed at generation and characterization of monoclonal antibodies specific for 3NY10‐Aβ as research tool and potential therapeutic molecules.MethodMonoclonal antibodies were developed by immunizing mice with 3NY10‐Aβ(6‐14) and derivation of corresponding hybridoma cell lines. Characterisation of the antibodies was achieved by a variety of biochemical and kinetic analyses: isothermal titration calorimetry (ITC), dot blot analysis and ThioflavinT aggregation studies. X‐ray crystallography was used for structure determination. We used immunohistochemical (IHC) staining of brain tissue from 5xFAD mice and enzyme linked immune sorbent assay (ELISA) to assess the formation of 3NY10‐Aβ in transgenic mice.ResultTwo highly affine 3NY10‐Aβ antibodies 4C3 and 3B3 have been generated, both binding 3NY10‐Aβ(1‐16) in the nanomolar range (KD4C3 = 11 nM, KD3B3 = 88,5 nM) as determined by ITC. The specificity of the antibodies was confirmed by dot blot analysis, using several post‐translationally modified Aβ‐peptides. Aggregation studies of 3NY10‐Aβ(1‐40/42) peptides in presence of 3B3 or 4C3 showed an influence of fibril formation, as could be verified by means of transmission electron microscopy. Co‐crystallisation of 4C3 Fab‐fragment and 3NY10‐Aβ(1‐16) at a resolution of 2.5 Å revealed a binding of 3NY10. To investigate the role of 3NY10‐Aβ in AD, IHC‐staining of brain from mice with AD‐like pathology (5xFAD mice) was performed. The data support presence of 3NY10‐Aβ within amyloid deposits, although staining appeared weak. Likewise, a Sandwich‐ELISA of homogenized murine brain tissue displayed no significant proportion of 3NY10‐modified Aβ in relation to the total Aβ(1‐40/42).ConclusionIt has been demonstrated that the unique monoclonal 3NY10‐Aβ specific antibodies 4C3 and 3B3 are a suitable tool for investigating the role of 3NY10‐Aβ in AD. Future research aims to quantify the level of 3NY10‐Aβ in human brain tissue with AD‐pathology.

Abstract 12348: Targeted Degradation of Topoisomerase 2b by Dexrazoxane for Prevention of Doxorubicin-Induced Cardiotoxicity: Dose and Time Course Study in Human

Introduction: Doxorubicin is known to induce cardiotoxicity through topoisomerase 2b (Top2b), whereas doxorubicin poisons topoisomerase 2a (Top2a) to kill cancer cells. When Top2b was genetically deleted in the cardiomyocytes of adult mice, doxorubicin-induced cardiomyopathy was prevented. Pharmacological degradation of Top2b by dexrazoxane can also prevent doxorubicin-induced cardiotoxicity in mice. However, it is not known whether dexrazoxane can induce degradation of Top2b in human. Hypothesis: Targeted degradation of Top2b by dexrazoxane can be achieved in humans. Methods: Top2b protein level in peripheral blood mononuclear cells (PBMC) were assessed serially after intravenous dexrazoxane infusion in human volunteers. The effectiveness of dexrazoxane on Top2b degradation and the time course of Top2b degradation were determined. Blood was drawn before dexrazoxane infusion, hourly after infusion up to 12 hours, at 24 hours, and at 48 hours. PBMCs were isolated using SepMate tube and fractionated into nuclear and cytosolic fractions. The nuclear fractions were used in Western blot analysis. Immunoblotting was carried out with rabbit anti-Top2b, anti-Top2a, or anti-Lamin B1 antibodies, followed by Peroxidase AffiniPure goat anti-rabbit IgG. The ratio of the intensity of the Top2b and Lamin B1 bands were normalized by setting the pre-ratio to 1. Results: One hour after dexrazoxane infusion, Top2b level in leukocytes was reduced to 14%-21% of baseline. Dexrazoxane-induced degradation of Top2b persisted for up to 12 hours and returned to baseline 24 hours after dexrazoxane infusion. Importantly, Top2a level did not vary significantly following dexrazoxane infusion. There were no serious adverse reactions in all participants. Conclusions: Human PBMC can be used as a surrogate to follow Top2b degradation. Degradation of Top2b can be achieved with dexrazoxane dose as low as 100 mg/M 2 (one-fifth of the current FDA recommended dose when used in conjunction with doxorubicin). Thus, early dexrazoxane followed by delayed-administration of doxorubicin can be studied as a new strategy to prevent doxorubicin-induced cardiotoxicity by depleting Top2b. Moreover, dexrazoxane did not affect Top2a, thus preserving doxorubicin’s efficacy.

FRI150 Individualized And Shorter Follow-up With Immunohistochemical Staining In Unilateral Primary Aldosteronism?

Abstract Disclosure: H. Gunnarsdottir: None. B.A. Agnarsson: None. S. Jonasdottir: None. J. Gudmundsson: None. G. Birgisson: None. H.Á. Sigurjónsdóttir: None. Indroduction: Clinical significance of specific antibody staining (SAS) for unilateral primary aldosteronism (PA) has been unclear. Individualized follow-up of PA patients could be in sight. Long-term outcome of patients, classified based on immunohistochemical staining, needs further investigation. Aims: We aimed to assess long-term clinical outcome (using PASO criteria) for unilateral PA, classifying patients based on SAS. Methods: The study was a nationwide observational study, with up to 11 years follow-up, executed in 2007-2016 at Landspitali National University Hospital of Iceland, tertiary referral center. Patients were diagnosed and treated in line with the current guidelines. All unilateral PA patients, ( n=26) age 28-73 years, who underwent adrenalectomy in Iceland during the study period were included. H&E stained tissue slides were stained using specific CYP11B1 and -B2 antibodies. All cases were re-evaluated and classified as adenoma (APA) or non-adenoma (adrenal hyperplasia, unilateral adrenal micronodules (UMN) or aldosterone producing cell clusters (APCC)). Data collection consisted of aldosterone, renin and cortisol values, antihypertensive drug count, potassium supplementation, blood pressure and serum potassium pre-intervention and throughout follow-up and histopathology results post-adrenalectomy. Results: Following SAS, APA was seen in 21 adrenals, APCC in three and UMN in two. Immunohistochemical staining altered histopathological diagnosis in 27% of the patients. Around 80% of each subgroup had partial clinical success. No APA patient with complete clinical success at 12 months (n=4) relapsed during follow-up. Conclusions: We found SAS mandatory for accurate histopathological diagnosis of PA. Our results indicate that a subgroup of APA patients can be discharged safely early. Presentation: Friday, June 16, 2023

Application of In vitro transcytosis models to brain targeted biologics.

The blood brain barrier (BBB) efficiently limits the penetration of biologics drugs from blood to brain. Establishment of an in vitro BBB model can facilitate screening of central nervous system (CNS) drug candidates and accelerate CNS drug development. Despite many established in vitro models, their application to biologics drug selection has been limited. Here, we report the evaluation of in vitro transcytosis of anti-human transferrin receptor (TfR) antibodies across human, cynomolgus and mouse species. We first evaluated human models including human cerebral microvascular endothelial cell line hCMEC/D3 and human colon epithelial cell line Caco-2 models. hCMEC/D3 model displayed low trans-epithelial electrical resistance (TEER), strong paracellular transport, and similar transcytosis of anti-TfR and control antibodies. In contrast, the Caco-2 model displayed high TEER value and low paracellular transport. Anti-hTfR antibodies demonstrated up to 70-fold better transcytosis compared to control IgG. Transcytosis of anti-hTfR.B1 antibody in Caco-2 model was dose-dependent and saturated at 3 μg/mL. Enhanced transcytosis of anti-hTfR.B1 was also observed in a monkey brain endothelial cell based (MBT) model. Importantly, anti-hTfR.B1 showed relatively high brain radioactivity concentration in a non-human primate positron emission tomography study indicating that the in vitro transcytosis from both Caco-2 and MBT models aligns with in vivo brain exposure. Typically, brain exposure of CNS targeted biologics is evaluated in mice. However, antibodies, such as the anti-human TfR antibodies, do not cross-react with the mouse target. Therefore, validation of a mouse in vitro transcytosis model is needed to better understand the in vitro in vivo correlation. Here, we performed transcytosis of anti-mouse TfR antibodies in mouse brain endothelial cell-based models, bEnd3 and the murine intestinal epithelial cell line mIEC. There is a good correlation between in vitro transcytosis of anti-mTfR antibodies and bispecifics in mIEC model and their mouse brain uptake. These data strengthen our confidence in the predictive power of the in vitro transcytosis models. Both mouse and human in vitro models will serve as important screening assays for brain targeted biologics selection in CNS drug development.

Immunohistochemical staining seems mandatory for individualizing and shortening follow-up in unilateral primary aldosteronism.

The clinical significance of immunohistochemistry (IHC) for unilateral primary aldosteronism (PA) has been unclear. Individualized follow-up of PA patients could be in sight. Long-term outcomes of patients, classified based on IHC, need further investigation. We aimed to assess long-term clinical outcomes for unilateral PA, classifying patients based on IHC. A nationwide observational study, with up to 16 years follow-up, executed in 2007-2016 at Landspitali University Hospital, tertiary referral center. Patients were diagnosed and treated in line with the current guidelines. Haematoxylin and eosin (H&E) tissue slides were stained using CYP11B1 and -B2 antibodies. All cases were re-evaluated and classified according to the HISTALDO consensus. Outcomes were assessed using the PASO criteria. All unilateral PA patients diagnosed in 2007-2016 in Iceland, 26 patients aged 28-73 years, who underwent adrenalectomy, were included. Aldosterone, renin, and cortisol values, use and dosage of antihypertensives, potassium supplementation, blood pressure and serum potassium pre-intervention and throughout follow-up, and histopathology results post-adrenalectomy. Following IHC, an aldosterone-producing nodule was seen in 12 adrenals, an aldosterone-producing adenoma in 10 and multiple aldosterone-producing micronodules in four. IHC altered histopathology from previous H&E diagnosis in 23% (6/26) of the patients. In total, 81% (21/26) of the patients had partial clinical success. Eight percent (2/26) of the patients needed potassium supplementation during follow-up. In the classical group, the AVS results were more determinative with significantly higher lateralization index (median 10.1 vs. 5.3, p = .04) and more contralateral suppression (median nondominant ratio 0.4 vs. 1.0, p = .03). One out of five patients with complete clinical success at 12 months post-op had severe relapse later, the other four were normotensive without antihypertensives for up to 10 years. We found IHC mandatory for accurate histopathologic diagnosis of PA. Our results support the importance of contralateral suppression when interpreting AVS results. Also, the study highlights the complicated assessment of clinical outcome and importance of aldosterone and renin measurements during follow-up.

Integrin-mediated electric axon guidance underlying optic nerve formation in the embryonic chick retina

Retinal ganglion cell (RGC) axons converge on the optic disc to form an optic nerve. However, the mechanism of RGC axon convergence remains elusive. In the embryonic retina, an electric field (EF) exists and this EF converges on the future optic disc. EFs have been demonstrated in vitro to orient axons toward the cathode. Here, I show that the EF directs RGC axons through integrin in an extracellular Ca2+-dependent manner. The cathodal growth of embryonic chick RGC axons, which express integrin α6β1, was enhanced by monoclonal anti-chicken integrin β1 antibodies. Mn2+ abolished these EF effects, as Mn2+ occupies the Ca2+-dependent negative regulatory site in the β1 subunit to eliminate Ca2+ inhibition. The present study proposes an integrin-mediated electric axon steering model, which involves directional Ca2+ movements and asymmetric microtubule stabilization. Since neuroepithelial cells generate EFs during neurogenesis, electric axon guidance may primarily be used in central nervous system development.

A novel immunosensor based on cobalt oxide nanocomposite modified single walled carbon nanohorns for the selective detection of aflatoxin B1.

We developed a novel, sensitive, and selective platform for the specific determination of aflatoxin B1 (AFB1). Single-walled carbon nanohorns decorated by a cobalt oxide composite and gold nanoparticles were created to provide facile electron transfer and improve the sensor's sensitivity. In addition, we attributed the selectivity of the proposed sensor to the specific binding property of the anti-aflatoxin B1 antibody. We clarified the specific interaction of the proposed immunosensor to AFB1 using homology modeling combined with molecular docking. In the presence of AFB1, the current signal of the modified electrode reduced; this involved specific antibody-antigen binding, including hydrophobic hydrogen bonding and pi-pi stack interactions. The new AFB1 sensor platform showed two linearity ranges of 0.01-1ngmL-1 and 1-100ngmL-1, with the limit of detection at 0.0019ngmL-1. We investigated the proposed immunosensor in real samples, including peanuts, certified reference material of a peanut sample (labeled 206μgkg-1 AFB1), corn, and chicken feed. The sensor's accuracy was 86.1-104.4% recovery, which agrees with the reference HPLC technique using paired t-test analysis. The present work shows excellent performance for AFB1 detection and could be applied for food quality control or modified to detect other mycotoxins.

A Versatile Integrated Microfluidic Chip Based on Sonic Toothbrush-Assisted Mixing for Analyses of Diverse Biomolecules.

Usually, different assays and instrumentation are required for different types of targets, e.g., nucleic acids, proteins, small molecules, etc., because of significant differences in their structures and sizes. To increase efficiency and reduce costs, a desirable solution is to develop a versatile platform suitable for diverse objectives. Here, we established a versatile detection technique: first, target separation and enrichment were carried out using magnetic beads (MBs); then, different targets were converted to same barcoded DNA strands (BDs) released from gold nanoparticles; finally, sensitive detection of three different targets (miRNA-21, digoxigenin antibody, and aflatoxin B1) was achieved through exonuclease III (Exo III) cyclic cleavage-assisted signal amplification. To simplify the operation, we integrated this technique into a microfluidic chip with multiple chambers in which the requisite reagents were prestored. Just by moving the MBs through different chambers with a magnet, multiple steps can be completed. Due to the limited space in microfluidic chips, the full mixing of MBs and solution is a key point to improve reaction efficiency. The mixing can be achieved by acoustic vibration generated by a small, portable sonic toothbrush. Based on the microfluidic chip, the detection limits of the above three targets were 0.76 pM, 0.16 ng/mL, and 0.56 nM, respectively. Furthermore, miRNA-21 and Digoxigenin antibody (Dig-Ab) in serum and AFB1 in corn powder were also used to demonstrate the performance of this chip. Our versatile platform is easy to operate and is expected to develop into an automatic "sample-to-answer" device.

Human Antibody VH Domains Targeting GPNMB and VCAM-1 as Candidate Therapeutics for Cancers

The elevated expression of GPNMB and VCAM-1 has been observed in many cancers including breast cancer, melanoma, and prostate cancers. Such overexpression of GPNMB and VCAM-1 has been associated with poor prognosis and increased cancer metastasis. Thus, GPNMB and VCAM-1 are potential targets for immunotherapies across multiple cancers. In this study, two high-affinity specific human VH domain antibody candidates, 87 (GPNMB) and 1B2 (VCAM-1), were isolated from our in-house proprietary phage-displayed human VH antibody domain libraries. The avidity was increased after conversion to VH-Fc. Domain-based bispecific T-cell engagers (DbTE) based on these two antibodies combined with the anti-CD3ε OKT3 antibody exhibited potent killing against GPNMB and VCAM-1-positive cancer cells, respectively. Hence, these two domain antibodies are promising therapeutic candidates for cancers expressing GPNMB or VCAM-1.