FCRL1 immunoregulation in B cell development and malignancy.

Immunotherapeutic targeting of surface regulatory proteins and pharmacologic inhibition of critical signaling pathways has dramatically shifted our approach to the care of individuals with B cell malignancies. This evolution in therapy reflects the central role of the B cell receptor (BCR) signaling complex and its co-receptors in the pathogenesis of B lineage leukemias and lymphomas. Members of the Fc receptor-like gene family (FCRL1-6) encode cell surface receptors with complex tyrosine-based regulation that are preferentially expressed by B cells. Among them, FCRL1 expression peaks on naïve and memory B cells and is unique in terms of its intracellular co-activation potential. Recent studies in human and mouse models indicate that FCRL1 contributes to the formation of the BCR signalosome, modulates B cell signaling, and promotes humoral responses. Progress in understanding its regulatory properties, along with evidence for its over-expression by mature B cell leukemias and lymphomas, collectively imply important yet unmet opportunities for FCRL1 in B cell development and transformation. Here we review recent advances in FCRL1 biology and highlight its emerging significance as a promising biomarker and therapeutic target in B cell lymphoproliferative disorders.

Comprehensive mapping of SARS-CoV-2 peptide epitopes for development of a highly sensitive serological test for total and neutralizing antibodies.

Quantification of the anti-SARS-CoV-2 antibody response has proven to be a prominent diagnostic tool during the COVID-19 pandemic. Antibody measurements have aided in the determination of humoral protection following infection or vaccination and will likely be essential for predicting the prevalence of population level immunity over the next several years. Despite widespread use, current tests remain limited in part, because antibody capture is accomplished through the use of complete spike and nucleocapsid proteins that contain significant regions of overlap with common circulating coronaviruses. To address this limitation, a unique epitope display platform utilizing monovalent display and protease-driven capture of peptide epitopes was used to select high affinity peptides. A single round of selection using this strategy with COVID-19 positive patient plasma samples revealed surprising differences and specific patterns in the antigenicity of SARS-CoV-2 proteins, especially the spike protein. Putative epitopes were assayed for specificity with convalescent and control samples, and the individual binding kinetics of peptides were also determined. A subset of prioritized peptides was used to develop an antibody diagnostic assay that showed low cross reactivity while detecting 37% more positive antibody cases than a gold standard FDA EUA test. Finally, a subset of peptides were compared with serum neutralization activity to establish a 2 peptide assay that strongly correlates with neutralization. Together, these data demonstrate a novel phage display method that is capable of comprehensively and rapidly mapping patient viral antibody responses and selecting high affinity public epitopes for the diagnosis of humoral immunity.

Convalescent plasma-mediated resolution of COVID-19 in a patient with humoral immunodeficiency.

Convalescent plasma (CP) is widely used to treat COVID-19, but without formal evidence of efficacy. Here, we report the beneficial effects of CP in a severely ill COVID-19 patient with prolonged pneumonia and advanced chronic lymphocytic leukemia (CLL), who was unable to generate an antiviral antibody response of her own. On day 33 after becoming symptomatic, the patient received CP containing high-titer (ID50 > 5,000) neutralizing antibodies (NAbs), defervesced, and improved clinically within 48 h and was discharged on day 37. Hence, when present in sufficient quantities, NAbs to SARS-CoV-2 have clinical benefit even if administered relatively late in the disease course. However, analysis of additional CP units revealed widely varying NAb titers, with many recipients exhibiting endogenous NAb responses far exceeding those of the administered units. To obtain the full therapeutic benefits of CP immunotherapy, it will thus be important to determine the neutralizing activity in both CP units and transfusion candidates.

Differential binding of the HIV-1 envelope to phosphatidylserine receptors.

Prior work has shown that the HIV-1 envelope of the human immunodeficiency virus (HIV) interacts directly with T-cell immunoglobulin mucin (TIM) family proteins. Herein, we demonstrate that HIV-1 envelope glycoproteins from varying HIV-1 clades bind differentially to TIM proteins and functionally similar proteins acting as phosphatidylserine (PtdSer) receptors. Using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) technology, we show that lysate containing HIV-1 envelope and recombinant HIV-1 envelope glycoproteins bind TIM-4 and advanced glycosylation end product-specific receptor (AGER). The complex binding of HIV-1 UG21 gp140 to TIM-4 or AGER suggests a biphasic interaction with these proteins.

L-4F inhibits lipopolysaccharide-mediated activation of primary human neutrophils.

Human apolipoprotein A-I (apoA-I) mimetic L-4F inhibits acute inflammation in endotoxemic animals. Since neutrophils play a crucial role in septic inflammation, we examined the effects of L-4F, compared to apoA-I, on lipopolysaccharide (LPS)-mediated activation of human neutrophils. We performed bioassays in human blood, isolated human neutrophils (incubated in 50 % donor plasma), and isolated human leukocytes (incubated in 5 and 50 % plasma) in vitro. In whole blood, both L-4F and apoA-I inhibited LPS-mediated elevation of TNF-α and IL-6. In LPS-stimulated neutrophils, L-4F and apoA-I (40 µg/ml) also decreased myeloperoxidase and TNF-α levels; however, L-4F tended to be superior in inhibiting LPS-mediated increase in IL-6 levels, membrane lipid rafts abundance and CD11b expression. In parallel experiments, when TNF-α and IL-8, instead of LPS, was used for cell stimulation, L-4F and/or apoA-I revealed only limited efficacy. In LPS-stimulated leukocytes, L-4F was as effective as apoA-I in reducing superoxide formation in 50 % donor plasma, and more effective in 5 % donor plasma (P<0.05). Limulus ambocyte lysate (LAL) and surface plasmon resonance assays showed that L-4F neutralizes LAL endotoxin activity more effectively than apoA-I (P<0.05) likely due to avid binding to LPS. We conclude that (1) direct binding/neutralization of LPS is a major mechanism of L-4F in vitro; (2) while L-4F has similar efficacy to apoA-I in anti-endotoxin effects in whole blood, it demonstrates superior efficacy to apoA-I in aqueous solutions and fluids with limited plasma components. This study rationalizes the utility of L-4F in the treatment of inflammation that is mediated by endotoxin-activated neutrophils.

Evidence that 2-aminoethoxydiphenyl borate provokes fibrillation in perfused rat hearts via voltage-independent calcium channels.

We tested whether 2-aminoethoxydiphenyl borate (2-APB) induces arrhythmia in perfused rat hearts and whether this arrhythmia might result from the activation of voltage-independent calcium channels. Rat hearts were Langendorff perfused and beat under sinus rhythm. An isovolumic balloon inserted into the left ventricle was used to record mechanical function while bipolar electrograms were recorded from electrodes sutured to the base and the apex of hearts. Western and immunofluorescence analyses were performed on rat left ventricular protein extracts and left ventricular frozen sections, respectively. Rat ventricular myocytes express Orai 1 and Orai 3, and ventricle also contains the Orai regulator Stim1. Rat hearts (n=5) perfused with Krebs-Henseleit (KH) alone maintained sinus rhythm at 4.8 ± 0.1 Hz and stable mechanical function. By contrast, perfusing hearts (n=5) with (KH+22 µM 2-APB) provoked a period of tachycardic ectopy at rates of up to 10.8 ± 0.2 Hz. As perfusion with (KH+22 µM 2-APB) continued, the rate of spontaneous ventricular depolarization increased to 21.8 ± 1.2 Hz and became disorganized. Heart mechanical function collapsed as developed pressure decreased from 87 ± 8.8 to 3.5 ± 1.9 mm Hg. Flow rate did not change between normal (16.6 ± 0.9 ml/min) and fibrillating (17.4 ± 0.8 ml/min) hearts. The addition of 20 µM 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole (SKF-96365) to (KH+22 µM 2-APB) perfusates (n=4) restored sinus rhythm and heart mechanical output. These data indicate that activating myocardial voltage-independent calcium channels, possibly the Orais, may be a novel cause of ventricular arrhythmia.

Pharmacological evidence for Orai channel activation as a source of cardiac abnormal automaticity.

Calcium transport through plasma membrane voltage-independent calcium channels is vital for signaling events in non-excitable and excitable cells. Following up on our earlier work, we tested the hypothesis that this type of calcium transport can disrupt myocardial electromechanical stability. Our Western and immunofluorescence analyses show that left atrial and ventricular myocytes express the Orai1 and the Orai3 calcium channels. Adding the Orai activator 2-aminoethoxydiphenyl borate (2-APB) to the superfusate of rat left atria causes these non-automatic muscles to contract spontaneously and persistently at rates of up to 10 Hz, and to produce normal action potentials from normal resting potentials, all in the absence of external stimulation. 2-APB likewise induces such automatic activity in superfused rat left ventricular papillary muscles, and the EC(50)s at which 2-APB induces this activity in both muscles are similar to the concentrations which activate Orais. Importantly, the voltage-independent calcium channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole (SKF-96365) suppresses this automaticity with an IC(50) of 11 ± 0.6 µM in left atria and 6 ± 1.6 µM in papillary muscles. 1-(5-Iodonaphthalene-1-sulfonyl)-hexahydro-1,4-diazepine (ML-7), a second voltage-independent calcium channel inhibitor, and two calmodulin inhibitors also prevent 2-APB automaticity while two calmodulin-dependent protein kinase II inhibitors do not. Thus an activator of the Orai calcium channels provokes a novel type of high frequency automaticity in non-automatic heart muscle.

Mechanism by which alcohol and wine polyphenols affect coronary heart disease risk.

The reduction in coronary heart disease (CHD) from moderate alcohol intake may be mediated, in part, by increased fibrinolysis; endothelial cell (EC)-mediated fibrinolysis should decrease acute atherothrombotic consequences (eg, plaque rupture) of myocardial infarction (MI). We have shown that alcohol and individual polyphenols modulate EC fibrinolytic protein (t-PA, u-PA, PAI-1, u-PAR and Annexin-II) expression at the cellular, molecular, and gene levels to sustain increased fibrinolytic activity. Herein we describe the sequence of molecular events by which EC t-PA expression is increased through common activation of p38 MAPK signaling. Up-regulation of t-PA gene transcription, through specific alcohol and polyphenol transcription factor binding sites in the t-PA promoter, results in increased in vitro fibrinolysis and in vivo clot lytic activity (using real-time fluorescence [Fl] imaging of Cy5.5-labeled fibrin clot lysis in a mouse model). Fl-labeled fibrin clots injected into untreated C56Bl/6 wild-type control mice are lysed in approximately 2 hours and clot lytic rates significantly increased in mice treated with either alcohol, catechins, or quercetin (4-6 weeks). Fl-labeled clot lysis in ApoE knock-out mice (atherosclerosis model) showed impaired in vivo clot lysis that was "normalized" to wild-type control levels by treatment with alcohol, catechin, or quercetin for 6 to 8 weeks.

Polyphenols downregulate PAI-1 gene expression in cultured human coronary artery endothelial cells: molecular contributor to cardiovascular protection.

Epidemiologic data have indicated that the intake of polyphenols is inversely associated with mortality from cardiovascular disease. Mitogen-activated protein kinases (MAPKs) are ubiquitous signaling proteins that have been associated with gene regulation. This study determined whether polyphenols (catechin and quercetin) activated kinase-signaling cascades that suppress PAI-1 expression and whether this suppression is at the transcription level in human coronary artery endothelial cells (ECs) remains unresolved. ECs were incubated in the absence/presence of polyphenols and RNA and protein were analyzed by real-time PCR and Western blot analysis. MAPKs were analyzed using antibodies to active form of p38, JNK, and ERK1/2. ECs were transiently transfected with a 1.1-kb PAI-1 promoter (pPAI110/luc) and promoter activity were assays after treatment with polyphenols. Catechin and quercetin decreased EC PAI-1 mRNA in a time- and dose-dependent manner, reaching a maximum at 4 and 2 h, respectively. These polyphenols activated EC p38 and ERK1/2 within 2.5 and 5 min, respectively, while maximal JNK activation occurred at 10-15 min. An inhibitor of p38 MAPK had no effect on polyphenol-induced repression of PAI-1. Inhibitors of ERK or JNK prevented polyphenol repression of EC PAI-1 gene expression. Exposing ECs transiently transfected with pPAI110/luc to polyphenols decreased promoter activity 50%. Polyphenols repress EC PAI-1 expression, in part, by activating ERK and JNK signaling pathways and this repression is at transcriptional levels. Thus MAPK seem to play an important role in polyphenol-induce repression of PAI-1 expression in ECs.

Alcohol-induced up-regulation of fibrinolytic activity and plasminogen activators in human monocytes.

BACKGROUND: Moderate alcohol consumption is associated with reduced risk for coronary heart disease. This may due, in part to increased fibrinolysis. Monocytes synthesize fibrinolytic proteins, tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and their receptors. These studies were carried out to determine the effect of low alcohol on monocyte fibrinolytic activity and PA messenger RNA (mRNA) synthesis. METHODS: Peripheral blood monocytes and U937 cells were incubated in absence/presence of low alcohol (0.1%, v/v) for various times (0-1 hr), followed by incubations in the absence of alcohol (0-24 hr) before measurement of fibrinolytic activity and PA mRNA levels (reverse transcriptase polymerase chain reaction). RESULTS: Brief exposure (15 min, 4 degrees C) of U937 cells to low alcohol resulted in an approximately 2- to 3-fold increase (269.0 +/- 5.6 fmol/1 x 10 cells versus 656.0 +/- 94.0 fmol/1 x 10 cells) in fibrinolytic activity. Preincubation of U937 cells and peripheral blood monocytes in low alcohol (1 hr, 37 degrees C) followed by incubation in the absence of alcohol (24 hr) resulted in a sustained approximately 4- to 5-fold increase (414.0 +/- 174.7 vs. 965.33.0 +/- 104.8 fmol/1 x 10 cells) and an approximately 3- to 4-fold (20.5 +/- 2.14 vs. 74 +/- 2.28 fmol/2 x 10 cells, respectively) increase in fibrinolytic activity. Preincubation of monocytes with low alcohol (1 hr, 37 degrees C) followed by incubation in the absence of alcohol (6 hr) resulted in an approximately 5- to 6-fold (0.06 +/- 0.02 vs. 0.42 +/- 0.02) and an approximately 2- to 3-fold (0.89 +/- 0.04 vs. 2.07 +/- 0.29) increase in t-PA and u-PA mRNA (reverse transcriptase polymerase chain reaction; PA/glyceraldehyde-3-phosphate dehydrogenase ratio), respectively. CONCLUSIONS: These data suggest that low alcohol exerts a rapid, direct, and sustained effect on monocyte fibrinolytic activity, which may be, due in part, to increased monocyte t-PA/u-PA expression. These data provide a feasible molecular mechanism by which alcohol effects on monocyte fibrinolysis may contribute to the cardioprotective benefit associated with moderate alcohol consumption.