A Reversible Chemogenetic Switch for Chimeric Antigen Receptor T†Cells.

As a revolutionary cancer treatment, the chimeric antigen receptor (CAR) T cell therapy suffers from complications such as cytokine release syndromes and T cell exhaustion. Their mitigation desires controllable activation of CAR-T cells that is achievable through regulatory display of CARs. By embedding the hepatitis C virus NS3 protease (HCV-NS3) between the single-chain variable fragment (scFv) and the hinge domain, we showed that the display of anti-CD19 scFv on CAR-T cells was positively correlated to the presence of a clinical HCV-NS3 inhibitor asunaprevir (ASV). This novel CAR design that allows the display of anti-CD19 scFv in the presence of ASV and its removal in the absence of ASV creates a practically reversible chemical switch. We demonstrated that the intact CAR on T cells can be repeatedly turned on and off by controlling the presence of ASV in a dose dependent manner both in vitro and in vivo, which enables delicate modulation of CAR-T activation during cancer treatment.

Zebrafish prmt3 negatively regulates antiviral responses.

Arginine methylation catalyzed by protein arginine methyltransferases (PRMT) is a common post-translational modification in histone and nonhistone proteins, which regulates many cellular functions. Protein arginine methyltransferase 3 (prmt3), a type I arginine methyltransferase, has been shown to carry out the formation of stable monomethylarginine as an intermediate before the establishment of asymmetric dimethylarginine. To date, however, the role of PRMT3 in antiviral innate immunity has not been elucidated. This study showed that zebrafish prmt3 was upregulated by virus infection and that the overexpression of prmt3 suppressed cellular antiviral response. The PRMT3 inhibitor, SGC707, enhanced antiviral capability. Consistently, prmt3-null zebrafish were more resistant to Spring Viremia of Carp Virus (SVCV) and Grass Carp Reovirus (GCRV) infection. Further assays showed that the overexpression of prmt3 diminished the phosphorylation of irf3 and prmt3 interacted with rig-i. In addition, both zinc-finger domain and catalytic domain of prmt3 were required for the suppressive function of prmt3 on IFN activation. Our findings suggested that zebrafish prmt3 negatively regulated the antiviral responses, implicating the vital role of prmt3-or even arginine methylation-in antiviral innate immunity.

Optimization of the modification of carrier proteins with aminated haptens.

In this report we show that succinic groups are far more reactive to amino compounds than the carboxylic groups derived from Asp and Glu on the protein when using coupling via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) (even by an 8 fold factor). Accordingly, a new carrier-protein was designed where both natural amino and carboxylic moieties were transformed into succinic residues. To prepare this hypersuccinylated carrier, all exposed carboxylic acids were first transformed into amino groups by reaction with ethylendiamine after activation with EDCI. Secondly, all these residues together with the ones from Lys were succinylated to prepare a fully succinylated protein. This was even more relevant considering that the amount of Lysine was 2-4 fold lower than Asp and Glu in most of the proteins. These "hyper-succinylated" proteins (KLH or BSA) offer significant improvements in protein reactivity compared to the native proteins (by a factor of 8-10). The optimization of the reaction, in which the presence of dioxane was found to be influential, permitted further improvements in the modification of the protein. Finally, this new strategy was successfully used to develop antibodies against the commercial anti-tumor molecule, ET-637-NH2. Using native KLH no response was found, whereas 1/64,000 serum dilutions gave very high values in ELISA procedures when immunization was performed using the hyper-succinylated KLH.

Determination method of 1-methyl-1,2,3, 4-tetrahydroisoquinoline, an endogenous parkinsonism-preventing substance, by radioimmunoassay.

To develop a sensitive and simple assay method for 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), an endogenous parkinsonism-preventing substance, we designed two kinds of 1MeTIQ-bovine serum albumin (BSA) conjugates to recognize the methyl group at the 1 position of 1MeTIQ since this is the critical structural difference between 1MeTIQ and parkinsonism-inducing substances. These hapten antigens were synthesized from 1MeTIQ analogues and BSA. A specific antiserum against 1MeTIQ was obtained from a rabbit immunized with one of the hapten antigens. To utilize this antiserum for radioimmunoassay, detailed studies were carried out to establish optimum conditions. The antiserum recognized 1MeTIQ and showed little cross-reactivity with endogenous 1MeTIQ analogues and proteins. It was confirmed to be suitable for radioimmunoassay, and a standard curve was prepared in the range of 0.5 to 100 pmol of 1MeTIQ. This method was sensitive enough to measure endogenous 1MeTIQ in rat brain. This method should be applicable for evaluation of the progression or prognosis of Parkinson's disease (PD).

Cardiac functional and structural alterations induced by endotoxin in rats: importance of platelet-activating factor.

OBJECTIVE: In this study, we evaluated the time course of the alterations in left ventricular (LV) dimensions, LV wall thickness, and LV systolic function in rats with endotoxemia by using echocardiography as well as myocardial histopathologic assessments. Our second goal was to examine whether pretreatment with a platelet-activating factor (PAF) antagonist would ameliorate the lipopolysaccharide (LPS)-induced cardiovascular collapse during the early phase. DESIGN: A prospective, controlled, in vivo animal laboratory study. SETTING: Research laboratory at a university. SUBJECTS: Male, Wistar rats (8-9 wks old; n = 83). INTERVENTIONS: In pentobarbital-anesthetized rats, the right carotid artery was cannulated to measure the arterial blood pressure and to sample blood. The right jugular vein also was catheterized for the administration of drugs. LPS (2 mg/kg) derived from Klebsiella pneumoniae or physiologic saline was administered in the presence or absence of pretreatment with TCV-309, a specific potent PAF antagonist. Echocardiographic studies were performed with an 8- to 13-MHz transducer. MEASUREMENTS AND MAIN RESULTS: LPS administration immediately induced progressive hypotension. The maximal hypotensive response was observed at 10 mins after LPS infusion with mean arterial pressure decreasing from 119 +/- 2 to 56 +/- 3 mm Hg (p < .001). LV end-diastolic internal dimensions decreased from 6.4 +/- 0.1 to 3.1 +/- 0.1 mm (p < .001) at 30 mins after LPS and remained significantly reduced compared with control rats. LV end-systolic dimensions also decreased dramatically from 3.5 +/- 0.2 to 0.5 +/- 0.1 mm (p < .001) at 30 mins after LPS and remained significantly reduced throughout the experiment. LV fractional shortening increased from 45 +/- 1% to 84 +/- 2% (p < .001) at 30 mins after LPS and remained elevated compared with control rats. LV wall thickness increased strikingly from 15 mins until 2 hrs after LPS infusion. Pathologic studies demonstrated marked congestion of capillaries and mild edema in the LV myocardium. The hematocrit increased after the administration of LPS. LPS markedly increased sympathetic tone as demonstrated by the elevation of plasma concentrations of epinephrine and norepinephrine. There was no elevation of concentrations of nitrite and nitrate. Pretreatment with TCV-309, a specific potent PAF antagonist, reduced LPS-induced hypotension and attenuated LV functional and structural changes. TCV-309 administration reduced the LPS-induced adrenergic activation and hemoconcentration. CONCLUSIONS: The hypotension that occurred during the initial phase of LPS-induced shock was accompanied by LV functional and structural alterations. The marked increase in LV wall thickness can be ascribed to the congestion of capillaries and edema in the LV myocardium. Pretreatment with a PAF antagonist reduced LPS-induced alterations. PAF may play a pivotal role during the initial phase of LPS-induced cardiovascular responses.

Requirements for Langerhans' cell depletion following in vitro exposure of murine skin to ultraviolet-B.

Langerhans' cells found within the skin and mucous membranes are critical regulators of antimicrobial and allergic responses. Therefore, the depletion of these cells following exposure of skin to solar ultraviolet radiation (UV) has direct functional consequences on immunity within this tissue. In order to understand how Langerhans' cell depletion is regulated following exposure of skin to medium-wave UV (UVB), the role of second messengers in these responses was investigated using a novel in vitro system. This was accomplished by analysing the expression of a specific marker associated with Langerhans' cells (ATPase) among the epidermal portion of cultured sections of mouse skin following treatment with inhibitors specific for second messenger components and subsequent exposure to UVB. In this study, inhibitors of guanosine triphosphate (GTP) binding proteins, H-8, pertussis toxin and cholera toxin as well as inhibitors of RNA and protein synthesis were all capable of blocking Langerhans' cell depletion in response to UVB treatment. In contrast, an inhibitor of protein kinase C (H-7) was incapable of specifically blocking depletion following treatment with this physical agent. These results suggest that Langerhans' cell depletion mediated by UVB may involve a pertussis and cholera toxin-sensitive G protein as well as de novo protein synthesis.

Corticotropin-releasing factor-induced production of cyclic AMP by human peripheral blood immunocytes.

The immunomodulating properties of a neuropeptide hormone, corticotropin-releasing factor (CRF), led us to investigate its effect on cAMP production by human peripheral blood mononuclear cells (MNC). In response to stimulation with CRF (100 nM), a statistically significant (P = 0.019) increase occurred in the amount of cAMP produced by MNC. Purified monocytes, but not lymphocytes, also displayed a significant (P = 0.01) increase (8- to 10-fold) in intracellular cAMP after treatment with CRF (100 nM). The antagonist alpha-helical CRF9-41 (100 nM) counteracted the cAMP increase induced by CRF (100 nM). The CRF-induced cAMP production was augmented by pretreatment of MNC with a cAMP-dependent protein kinase (PKA) peptide inhibitor (PI20), but was virtually unaffected by the protein kinase C (PKC) inhibitor H7, suggesting a role for cAMP signalling. Moreover, the CRF-stimulated cAMP level was reduced to baseline by intracellular Ca2+ antagonist HA1004, indicating a role for Ca(2+)-signalling. Based on these findings, it is concluded that cAMP and/or Ca2+ play a second messenger role in the CRF signal transduction pathway.

Chimerasome-mediated gene transfer in vitro and in vivo.

Proteoliposome delivery vesicles can be prepared by the protein-cochleate method [Gould-Fogerite and Mannino, Anal. Biochem. 148 (1985) 15-25; Mannino and Gould-Fogerite, Biotechniques 6 (1988) 682-690]. Proteins which mediate the entry of enveloped viruses into cells are integrated in the lipid bilayer, and materials are encapsulated at high efficiency within the aqueous interior of these vesicles. We describe proteoliposome-mediated delivery of proteins and drugs into entire populations of cells in culture. Material can be delivered gradually by Sendai-virus-glycoprotein-containing proteoliposomes. Alternatively, synchronous delivery to a population can be achieved by exposing cell-bound influenza glycoprotein vesicles briefly to low pH buffer. When DNA is encapsulated, chimeric proteoliposome gene-transfer vesicles (chimerasomes), which mediate high-efficiency gene transfer in vitro and in vivo, are produced. Stable expression of a bovine papilloma virus-based plasmid in tissue-cultured cells, at 100,000 times greater efficiency than Ca.phosphate precipitation of DNA, with respect to the quantity of DNA used, has been achieved. Stable gene transfer and expression in mice has been obtained by subcutaneous injection of chimerasomes containing a plasmid expressing the early region of polyoma virus. In one experimental group, 50% of the mice developed tumors which were shown to express polyoma virus early proteins and contain the transferred DNA. This is the first report of stable gene transfer in animals mediated by a liposome- or proteoliposome-based system.

Cross-reactivity of metocurine, atracurium, vecuronium and fazadinium with IgE antibodies from patients unexposed to these drugs but allergic to other myoneural blocking drugs.

An inhibition assay was used to determine quantitatively the allergenic cross-reactivity of some myoneural blocking drugs not yet released for use in Australia, in the sera of patients who had experienced anaphylactic reactions to neuromuscular blocking drugs. Two of the compounds, metocurine and atracurium were highly cross-reactive with the currently used myoneural blockers; fazadinium was weakly cross-reactive and vecuronium intermediate in potency between these two extremes. From these results, we predict that anaphylactic reactions to these compounds, and particularly to metocurine and atracurium, will occur in some patients allergic to the currently used neuromuscular blocking agents.

Ex vivo antigen preparation for the serological detection of drug-dependent antibodies in immune haemolytic anaemias.

Two patients with severe, intravascular haemolysis due to drug-dependent antibodies are described. The antibodies were directed against presumptive metabolites of buthiazide (International Non-proprietary Name, butizide) and nomifensine. Their detection was possible only in the presence of ex vivo antigens (i.e. fresh serum of volunteers after ingestion of the drugs) while in vitro antigen preparations yielded inconclusive results. Both antibodies lysed normal red cells in the presence of ex vivo antigens and complement. The buthiazide-related antibody was IgG (subclass IgG1), the nomifensine-related antibody was IgM. We conclude that the use of ex vivo antigens is of great importance in the serological evaluation of cases with suspected drug-dependent immune haemolysis.

[Demonstration of drug-specific IgE and IgG antibodies using RIA: clinical importance as shown with nomifensin (Alival)]. / Nachweis von arzneimittel-spezifischen IgE- und IgG-Antikörpern mittels RIA: klinische Bedeutung am Beispiel Nomifensin (Alival).

Serum samples from 41 patients who developed adverse reactions during therapy with nomifensine were screened by RAST-based immunoassay for specific IgE and IgG antibodies against nomifensine and three of its metabolites. The results were compared with those of 10 patients without side effects and with 8 non-treated controls. Nomifensine-specific IgE antibodies were found in none of the subjects. However, all patients treated with nomifensine (with and without side effects) had specific IgG antibodies. The antibody cross-reacted in all cases with the metabolites. The titers did not discriminate clearly between the different side reactions and only partially between the presence or absence of a side reaction. The finding of specific anti-drug IgG antibodies warrants more detailed investigation of immunological mechanisms, to determine the clinical relevance of these antibodies and identify patients at risk for serious side effects.

Anti-nomifensine antibody causing immune hemolytic anemia and renal failure.

An anti-Nomifensine antibody was identified in a patient with immune hemolysis and renal failure who was under Nomifensine therapy. The anti-drug specificity of the antibody was confirmed by inhibition assays using Sepharose gel-Nomifensine conjugates. Preliminary screening tests for anti-Nomifensine antibody in a population of 104 patients treated by the drug showed no further example of such immunization.

[Radioimmuno assay of the sulfonylurea AR-DF 26(author's transl)]. / Radioimmunologische Bestimmung des Sulfonylharnstoffes AR-DF 26

A radioimmunological method was developed for 1-cyclohexyl-3-[p-(beta-4,4-dimethyl-7-methoxy-1,3-(2H,4H)-isoquinolinedion-2-yl)phenethyl]-sulfonylurea (AR-DF 26, gliquidone, Glurenorm) estimations in plasma. The antisera were obtained by immunization of rabbits with AR-DF 26-bovine serum albumin (BSA) conjugate. The estimation is based on the competitive displacement in plasma samples of known amounts of labelled AR-DF 26, bound to a specific antibody by unknown amounts of unlabelled AR-DF 26. Concentrations of as low as 1 ng of AR-DF 26 per sample can be measured by this method. The investigations into the specificity of this reaction have shown that the antiserum is extremely specific for AR-DF 26. There is good reproducibility of the results and no differences could be found by variance analysis between different measurements of the same AR-DF 26 concentration. Since more than 70% of the plasma concentration correspond to unchanged AR-DF 26 for a longer period after application, the results of this AR-DF 26-radioimmuno assay is only minimally altered by its metabolites.