Evidence for a cerebral cholinergic system and suggested pharmacological patterns of neural organization in the prostomium of the polychaete Nereis virens (SARS)

The histological visualization of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) on frozen sections of prostomia of Nereis virens indicate a concentration of cholinergic activity in the anterior brain. Components are probably sensory epithelial cells with cholinergic axons entering the brain in cephalic nerves and efferent cholinergic axons to prostomial muscle leaving the brain in the same nerves. There are also subepidermal cholinergic cells that may be second order motor neurons serving epidermal mucous cells. The smaller, second lobe of the corpora pedunculata and its associated vertical fibre tract are CAT⁴ and appear continuous, on each side of the cerebral ganglion, with a dorsal and a ventral longitudinal bundle of AChE⁺ fibers. This system tapers to nothing at the level of the posterior eyes. There is a small AChE⁺ component to each optic nerve and AChE is present in the nuchal epithelium. These observations are discussed in relation to earlier studies on aminergic and neurosecretory activity in the same ganglion.     

Bi-specific TCR-anti CD3 redirected T-cell targeting of NY-ESO-1- and LAGE-1-positive tumors

NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157–165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1_157–165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NY-ESO-1_157–165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers.     

Endogenously produced catecholamines improve the regulatory function of TLR9-activated B cells

The sympathetic nervous system (SNS) contributes to immune balance by promoting anti-inflammatory B cells. However, whether B cells possess a self-regulating mechanism by which they modulate regulatory B cell (Breg) function is not well understood. In this study, we investigated the ability of B cells to synthesize their own catecholamines upon stimulation with different B cell activators and found that expression of the enzyme tyrosine hydroxylase (TH), required to generate catecholamines, is up-regulated by Toll-like receptor (TLR)9. This TLR9-dependent expression of TH correlated with up-regulation of adrenergic receptors (ADRs), enhanced interleukin (IL)-10 production, and overexpression of the co-inhibitory ligands programmed death ligand 1 (PD-L1) and Fas ligand (FasL). Moreover, concomitant stimulation of ß1-3-ADRs together with a B cell receptor (BCR)/TLR9 stimulus clearly enhances the anti-inflammatory potential of Bregs to suppress CD4 T cells, a crucial population in the pathogenesis of autoimmune diseases, like rheumatoid arthritis (RA). Furthermore, TH up-regulation was also demonstrated in B cells during the course of collagen-induced arthritis (CIA), a mouse model for the investigation of RA. In conclusion, our data show that B cells possess an autonomous mechanism to modulate their regulatory function in an autocrine and/or paracrine manner. These findings help to better understand the function of B cells in the regulation of autoimmune diseases and the interplay of SNS.

The sympathetic nervous system produces neurotransmitters such as catecholamines which contribute to immune balance by promoting anti-inflammatory B cells. This study shows that mouse B cells can themselves synthesize, sense, and transport catecholamines, which in turn modulate regulatory B cell function in an autocrine and/or paracrine manner to suppress T cell proliferation.     

Structural characterization of eight cyclic lipopeptides produced by Bacillus subtilis HSO121

Lipopeptides are amphiphilic compounds which contain both hydrophobic fatty acid moieties and amphiphilic peptide moieties. From the cell-free broth of Bacillus subtilis HSO121, eight cyclic lipopeptides were isolated by reversed-phase high performance liquid chromatography (RP-HPLC). The peptide part of each lipopeptide was elucidated according to electrospray ionization quadruple-time-of-flight mass spectrometry (ESI Q-TOF MS) and the fatty acid part was analyzed by electroionization gas chromatography/mass spectrometry (EI GC/MS). It showed that fractions 1-8 had molecular masses of 1007, 1021, 1021, 1035, 1035, 1035, 1063, and 1049, respectively. Analysis of hydrolyzed lipopeptides revealed that they had invariant amino acid compositions. The differences in molecular weights represent changes in the number of methylene groups and different types of branched chains in fatty acids. Peptide sequences of two of the eight lipopeptides appeared to be N-Asp-Leu-Leu-Val-Glu-Leu-Leu-C, which was different from previously reported lipopeptides. The remaining six had an identical peptide sequence of N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C. The fatty acid parts were found to be mixtures of iso C(12), iso C(13), anteiso C(13), iso C(14), n C(14), iso C(15), anteiso C(15), n C(15), anteiso C(16) and anteiso C(17) beta-hydroxy fatty acids. The structure of each lipopeptide was determined to be the beta-hydroxy fatty acid bonded to the peptide chain.     

BacMam recombinant baculovirus in transporter expression: a study of BCRP and OATP1B1

Human BCRP and OATP1B1 have recently been identified as important transporters in the absorption, distribution, and elimination of clinically significant drugs. In this report, we illustrate the use of modified baculoviruses, termed BacMam viruses for the expression of functional BCRP and OATP1B1 in mammalian cells. We show a variety of host cells efficiently transduced to express BCRP including HEK 293, LLC-PK, and U-2 OS, where protein levels on the cell-surface were modulated by titrating different amounts of viral inoculum. In addition, using the BODIPY-prazosin efflux assay and the BacMam reagent we illustrate inhibition of BCRP activity with GF120918 or Fumitremorgin C. Furthermore, we present data demonstrating simultaneous expression of BCRP and OATP1B1 in BacMam transduced mammalian cells by simply adding viral inoculum of each transporter. Thus these results indicate that BacMam mediated gene delivery provides a novel and efficient research tool for the investigation of single or multiple transporters in vitro.     

A simple and efficient approach to the synthesis of 4H-furo[3,4-b]pyrans via a three-component reaction of isocyanides

A three-component reaction of an isocyanide, a dialkyl acetylenedicarboxylate, and tetronic acid in dichloromethane at room temperature afforded 4H-furo[3,4-b]pyran derivatives. These compounds are closely related with ring systems, TAN-2483B, TAN-2483A, and FD-211 which have a broad spectrum of biological activity.     

2'-O-[2-(methylthio)ethyl]-modified oligonucleotide: an analogue of 2'-O-[2-(methoxy)-ethyl]-modified oligonucleotide with improved protein binding properties and high binding affinity to target RNA

A novel 2'-modification, 2'-O-[2-(methylthio)ethyl] or 2'-O-MTE, has been incorporated into oligonucleotides and evaluated for properties relevant to antisense activity. The results were compared with the previously characterized 2'-O-[2-(methoxy)ethyl] 2'-O-MOE modification. As expected, the 2'-O-MTE modified oligonucleotides exhibited improved binding to human serum albumin compared to the 2'-O-MOE modified oligonucleotides. The 2'-O-MTE oligonucleotides maintained high binding affinity to target RNA. Nuclease digestion of 2'-O-MTE oligonucleotides showed that they have limited resistance to exonuclease degradation. We analyzed the crystal structure of a decamer DNA duplex containing the 2'-O-MTE modifcation. Analysis of the crystal structure provides insight into the improved RNA binding affinity, protein binding affinity and limited resistance of 2'-O-MTE modified oligonucleotides to exonuclease degradation.     

Usp18 Driven Enforced Viral Replication in Dendritic Cells Contributes to Break of Immunological Tolerance in Autoimmune Diabetes

Infection with viruses carrying cross-reactive antigens is associated with break of immunological tolerance and induction of autoimmune disease. Dendritic cells play an important role in this process. However, it remains unclear why autoimmune-tolerance is broken during virus infection, but usually not during exposure to non-replicating cross-reactive antigens. Here we show that antigen derived from replicating virus but not from non-replicating sources undergoes a multiplication process in dendritic cells in spleen and lymph nodes. This enforced viral replication was dependent on Usp18 and was essential for expansion of autoreactive CD8⁺ T cells. Preventing enforced virus replication by depletion of CD11c⁺ cells, genetically deleting Usp18, or pharmacologically inhibiting of viral replication blunted the expansion of autoreactive CD8⁺ T cells and prevented autoimmune diabetes. In conclusion, Usp18-driven enforced viral replication in dendritic cells can break immunological tolerance and critically influences induction of autoimmunity.