Selective toxicity of caffeic acid in hepatocellular carcinoma cells.

Caffeic acid is a natural phytochemical structurally similar to other cinnamic acids. In this study we found caffeic acid (CA) but not ferulic, sinapic or cinnamic acids inhibited proliferation of hepatocellular carcinoma cells (HCC) and reduced cell numbers by inducing apoptosis. Only transient exposure to CA was required for these lethal effects that are associated with disruption of mitochondrial membrane potential and induction of reactive oxygen species. By comparison, primary hepatocytes resisted CA toxicity for nearly 48 h, consistent with selective sensitivity of HCC to CA. These results support use of CA as an anti-tumor agent to inhibit HCC, especially if delivered by locoregional catheterization in an embolization procedure.

The Use of the Woodchuck as an Animal Model for Evaluation of Transarterial Embolization.

There are many shortcomings of current animal models as surrogates of hepatocellular carcinoma that handicap preclinical testing of embolization agents. The present study explores the feasibility of using the woodchuck (Marmota monax) as an animal model for the testing of novel embolization agents. Four woodchucks underwent magnetic resonance imaging, angiography, and left lobar hepatic artery particle embolization. Percutaneous access, arteriography, and lobar embolization were successful in all animals, with angiographic stasis obtained in the target vessel with minimal reflux of embolic material. These results support the feasibility of the woodchuck as an animal model for preclinical testing of embolization agents.

Novel anti-CD30/CD3 bispecific antibodies activate human T cells and mediate potent anti-tumor activity.

CD30 is expressed on Hodgkin lymphomas (HL), many non-Hodgkin lymphomas (NHLs), and non-lymphoid malignancies in children and adults. Tumor expression, combined with restricted expression in healthy tissues, identifies CD30 as a promising immunotherapy target. An anti-CD30 antibody-drug conjugate (ADC) has been approved by the FDA for HL. While anti-CD30 ADCs and chimeric antigen receptors (CARs) have shown promise, their shortcomings and toxicities suggest that alternative treatments are needed. We developed novel anti-CD30 x anti-CD3 bispecific antibodies (biAbs) to coat activated patient T cells (ATCs) ex vivo prior to autologous re-infusions. Our goal is to harness the dual specificity of the biAb, the power of cellular therapy, and the safety of non-genetically modified autologous T cell infusions. We present a comprehensive characterization of the CD30 binding and tumor cell killing properties of these biAbs. Five unique murine monoclonal antibodies (mAbs) were generated against the extracellular domain of human CD30. Resultant anti-CD30 mAbs were purified and screened for binding specificity, affinity, and epitope recognition. Two lead mAb candidates with unique sequences and CD30 binding clusters that differ from the ADC in clinical use were identified. These mAbs were chemically conjugated with OKT3 (an anti-CD3 mAb). ATCs were armed and evaluated in vitro for binding, cytokine production, and cytotoxicity against tumor lines and then in vivo for tumor cell killing. Our lead mAb was subcloned to make a Master Cell Bank (MCB) and screened for binding against a library of human cell surface proteins. Only huCD30 was bound. These studies support a clinical trial in development employing ex vivo-loading of autologous T cells with this novel biAb.

Disparate Regions of the Human Chemokine CXCL10 Exhibit Broad-Spectrum Antimicrobial Activity against Biodefense and Antibiotic-Resistant Bacterial Pathogens.

CXCL10 is a pro-inflammatory chemokine produced by the host in response to microbial infection. In addition to canonical, receptor-dependent actions affecting immune-cell migration and activation, CXCL10 has also been found to directly kill a broad range of pathogenic bacteria. Prior investigations suggest that the bactericidal effects of CXCL10 occur through two distinct pathways that compromise the cell envelope. These observations raise the intriguing notion that CXCL10 features a separable pair of antimicrobial domains. Herein, we affirm this possibility through peptide-based mapping and structure/function analyses, which demonstrate that discrete peptides derived from the N- and C-terminal regions of CXCL10 mediate bacterial killing. The N-terminal derivative, peptide P1, exhibited marked antimicrobial activity against Bacillus anthracis vegetative bacilli and spores, as well as antibiotic-resistant clinical isolates of Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecium, and Staphylococcus aureus, among others. At bactericidal concentrations, peptide P1 had a minimal degree of chemotactic activity, but did not cause red blood cell hemolysis or cytotoxic effects against primary human cells. The C-terminal derivative, peptide P9, exhibited antimicrobial effects, but only against Gram-negative bacteria in low-salt medium─conditions under which the peptide can adopt an α-helical conformation. The introduction of a hydrocarbon staple induced and stabilized α-helicity; accordingly, stapled peptide P9 displayed significantly improved bactericidal effects against both Gram-positive and Gram-negative bacteria in media containing physiologic levels of salt. Together, our findings identify and characterize the antimicrobial regions of CXCL10 and functionalize these novel determinants as discrete peptides with potential therapeutic utility against difficult-to-treat pathogens.

Bispecific antibody-targeted T-cell therapy for acute myeloid leukemia.

The management of relapsed or refractory acute myeloid leukemia (AML) continues to be therapeutically challenging. Non-toxic immunotherapy approaches are needed to provide long-term anti-leukemic effects. The goal of this study was to determine whether activated T cells (ATCs) armed with bispecific antibodies (BiAbs) could target and lyse leukemic and leukemic stem cells (LSCs). Anti-CD3 × anti-CD123 BiAb (CD123Bi) and anti-CD3 × anti-CD33GO (gemtuzumab ozogamicin [GO]) BiAb (CD33GOBi) were used to arm ATCs to produce bispecific antibody armed activated T cells (designated CD123 BATs or CD33GO BATs) to target AML cell lines, peripheral blood mononuclear cells from AML patients, and in vivo treatment of AML in xenogeneic NSG mice engrafted with leukemic cells. BATs exhibited high levels of specific cytotoxicity directed at AML cell lines at low 1:1 or 1:2 effector-to-target (E:T) ratios and secrete Th1 cytokines upon target engagement. In vivo study in AML-engrafted NSG mice showed significantly prolonged survival in mice treated with CD33GO BATs (p < 0.0001) or CD123 BATs (p < 0.0089) compared to ATC-treated control mice. Patient samples containing leukemic blasts and LSCs when treated with CD33GO BATs or CD123 BATs for 18 h showed a significant reduction (50%-100%; p < 0.005) in blasts and 75%-100% reduction in LSCs (p < 0.005) in most cases compared to unarmed ATCs. This approach may provide a potent and non-toxic strategy to target AML blasts and LSCs and enhance chemo-responsiveness in older patients who are likely to develop recurrent diseases.

The Immune System Response to 15-kDa Barley Protein: A Mouse Model Study.

Barley (Hordeum vulgare L.) proteins are taxonomically homologous to wheat proteins and react with sera from patients with baker's asthma. In the current work, the crude extract of barley proteins was divided into six fractions on DEAE-Sepharose. Their immunoreactivity in reacting with sera from patients with a confirmed food allergy varied, and the 15-kDa fraction (B−FrVI) showed the strongest response. In silico analysis confirmed that 15-kDa B-FrVI protein belongs to the trypsin/amylase inhibitor family and to a group of MHC type II allergens. In the next step, the immunogenicity of the B-FrVI was examined in a mouse model. It was shown that, compared to the PBS group, administration of B-FrVI to mice induced almost 2× higher amounts of specific IgG, ~217, and IgA ~29, as early as day 28 after immunization, regardless of the route (intraperitoneal or oral) of antigen administration (p < 0.0001). An ELISpot for B-cell responses confirmed it. Stimulation of mesenteric lymphocytes with pure B-FrVI significantly increased (p < 0.001) the proliferation of lymphocytes from all groups compared to cells growing in media only and stimulated with lyophilized beer. The experiments prove the strong immunogenicity of the 15-kDa B-FrVI protein and provide a basis for future studies of the allergenic nature of this protein.

Convalescent Plasma for Preventing Critical Illness in COVID-19: a Phase 2 Trial and Immune Profile.

The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an unprecedented event requiring frequent adaptation to changing clinical circumstances. Convalescent immune plasma (CIP) is a promising treatment that can be mobilized rapidly in a pandemic setting. We tested whether administration of SARS-CoV-2 CIP at hospital admission could reduce the rate of ICU transfer or 28-day mortality or alter levels of specific antibody responses before and after CIP infusion. In a single-arm phase II study, patients >18 years-old with respiratory symptoms with confirmed COVID-19 infection who were admitted to a non-ICU bed were administered two units of CIP within 72 h of admission. Levels of SARS-CoV-2 detected by PCR in the respiratory tract and circulating anti-SARS-CoV-2 antibody titers were sequentially measured before and after CIP transfusion. Twenty-nine patients were transfused high titer CIP and 48 contemporaneous comparable controls were identified. All classes of antibodies to the three SARS-CoV-2 target proteins were significantly increased at days 7 and 14 post-transfusion compared with baseline (P < 0.01). Anti-nucleocapsid IgA levels were reduced at day 28, suggesting that the initial rise may have been due to the contribution of CIP. The groups were well-balanced, without statistically significant differences in demographics or co-morbidities or use of remdesivir or dexamethasone. In participants transfused with CIP, the rate of ICU transfer was 13.8% compared to 27.1% for controls with a hazard ratio 0.506 (95% CI 0.165-1.554), and 28-day mortality was 6.9% compared to 10.4% for controls, hazard ratio 0.640 (95% CI 0.124-3.298). IMPORTANCE Transfusion of high-titer CIP to non-critically ill patients early after admission with COVID-19 respiratory disease was associated with significantly increased anti-SARS-CoV-2 specific antibodies (compared to baseline) and a non-significant reduction in ICU transfer and death (compared to controls). This prospective phase II trial provides a suggestion that the antiviral effects of CIP from early in the COVID-19 pandemic may delay progression to critical illness and death in specific patient populations. This study informs the optimal timing and potential population of use for CIP in COVID-19, particularly in settings without access to other interventions, or in planning for future coronavirus pandemics.

Bispecific Antibody Armed Metabolically Enhanced Headless CAR T Cells.

Adoptive T cell therapies for solid tumors is challenging. We generated metabolically enhanced co-activated-T cells by transducing intracellular co-stimulatory (41BB, ICOS or ICOS-27) and CD3ζ T cell receptor signaling domains followed by arming with bispecific antibodies (BiAbs) to produce armed "Headless CAR T cells" (hCART). Various hCART armed with BiAb directed at CD3ϵ and various tumor associated antigens were tested for: 1) specific cytotoxicity against solid tumors targets; 2) repeated and dual sequential cytotoxicity; 3) survival and cytotoxicity under in vitro hypoxic condition; and 4) cytokine secretion. The 41BBζ transduced hCART (hCART41BBζ) armed with HER2 BiAb (HER2 hCART41BBζ) or armed with EGFR BiAb (EGFR hCART41BBζ) killed multiple tumor lines significantly better than control T cells and secreted Th1 cytokines/chemokines upon tumor engagement at effector to target ratio (E:T) of 2:1 or 1:1. HER2 hCART serially killed tumor targets up to 14 days. Sequential targeting of EGFR or HER2 positive tumors with HER2 hCART41BBζ followed by EGFR hCART41BBζ showed significantly increased cytotoxicity compared single antigen targeting and continue to kill under in vitro hypoxic conditions. In summary, metabolically enhanced headless CAR T cells are effective serial killers of tumor targets, secrete cytokines and chemokines, and continue to kill under in vitro hypoxic condition.

Convalescent plasma for preventing critical illness in COVID-19: A phase 2 trial and immune profile.

RATIONALE: The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an unprecedented event requiring rapid adaptation to changing clinical circumstances. Convalescent immune plasma (CIP) is a promising treatment that can be mobilized rapidly in a pandemic setting. OBJECTIVES: We tested whether administration of SARS-CoV-2 CIP at hospital admission could reduce the rate of ICU transfer or 28 day mortality. METHODS: In a single-arm phase II study, patients >18 years-old with respiratory symptoms documented with COVID-19 infection who were admitted to a non-ICU bed were administered two units of CIP within 72 hours of admission. Detection of respiratory tract SARS-CoV-2 by polymerase chain reaction and circulating anti-SARS-CoV-2 antibody titers were measured before and at time points after CIP transfusion. MEASUREMENTS AND MAIN RESULTS: Twenty-nine patients were transfused CIP and forty-eight contemporaneous controls were identified with comparable baseline characteristics. Levels of anti-SARS-CoV-2 IgG, IgM, and IgA anti-spike, anti-receptor-binding domain, and anti-nucleocapsid significantly increased from baseline to post-transfusion for all proteins tested. In patients transfused with CIP, the rate of ICU transfer was 13.8% compared to 27.1% for controls with a hazard ratio 0.506 (95% CI 0.165-1.554), and 28-day mortality was 6.9% compared to 10.4% for controls, hazard ratio 0.640 (95% CI 0.124-3.298). CONCLUSIONS: Transfusion of high-titer CIP to patients early after admission with COVID-19 respiratory disease was associated with reduced ICU transfer and 28-day mortality but was not statistically significant. Follow up randomized trials may inform the use of CIP for COVID-19 or future coronavirus pandemics.

Cinnamic Acid Derivatives Enhance the Efficacy of Transarterial Embolization in a Rat Model of Hepatocellular Carcinoma.

INTRODUCTION: We hypothesize that the combination of transarterial embolization (TAE) plus inhibition of lactate export will limit anaerobic metabolism and reduce tumor survival compared to TAE alone. The purpose of this study was to test this hypothesis in a rat model of hepatocellular carcinoma (HCC). METHODS: Rat N1-S1 hepatoma cells were assayed in vitro using the Seahorse XF analyzer to measure extracellular acidification (lactate excretion) comparing effects of the addition of caffeic acid (CA) or ferulic acid (FA) or UK-5099 with control. Monocarboxylate transporter Slc16a3 was knocked down by RNAi. N1S1 tumors were orthotopically implanted in rats and 4 groups evaluated: (1) Control, (2) TAE-only, (3) TAE plus CA, and (4) TAE plus FA. Tumor size was determined by ultrasound and analyzed by repeated measures statistics. Tumors harvested at 4 weeks were examined by microscopy. RESULTS: Seahorse assays showed that CA and FA caused a significant reduction by >90% in lactate efflux by N1S1 tumor cells (p < 0.01). Knockdown of Slc16a3 prevented inhibition by CA. In vivo tumors grew 30-fold in volume over 4 weeks in untreated controls. By comparison, TAE resulted in near cessation of growth (10% in 4-week time period). However, both TAE + CA and TAE + FA caused a significant reduction of tumor volumes (87 and 72%, respectively) compared to control and TAE (p < 0.05). Pathologic evaluation revealed residual tumor in the TAE group but no residual viable tumor cells in the TAE + CA and TAE + FA groups. CONCLUSION: Addition of CA or FA enhances the effectiveness of TAE therapy for HCC in part by blocking lactate efflux.

Poly(A) RNAs including coding proteins RNAs occur in plant Cajal bodies.

The localisation of poly(A) RNA in plant cells containing either reticular (Allium cepa) or chromocentric (Lupinus luteus, Arabidopsis thaliana) nuclei was studied through in situ hybridisation. In both types of nuclei, the amount of poly(A) RNA was much greater in the nucleus than in the cytoplasm. In the nuclei, poly(A) RNA was present in structures resembling nuclear bodies. The molecular composition as well as the characteristic ultrastructure of the bodies containing poly(A) RNA demonstrated that they were Cajal bodies. We showed that some poly(A) RNAs in Cajal bodies code for proteins. However, examination of the localisation of active RNA polymerase II and in situ run-on transcription assays both demonstrated that CBs are not sites of transcription and that BrU-containing RNA accumulates in these structures long after synthesis. In addition, it was demonstrated that accumulation of poly(A) RNA occurs in the nuclei and CBs of hypoxia-treated cells. Our findings indicated that CBs may be involved in the later stages of poly(A) RNA metabolism, playing a role storage or retention.