Inverse Agonistic Action of 3-Iodothyronamine at the Human Trace Amine-Associated Receptor 5

ObjectiveApplication of 3-iodothyronamine (3-T₁AM) results in decreased body temperature and body weight in rodents. The trace amine-associated receptor (TAAR) 1, a family A G protein-coupled receptor, is a target of 3-T₁AM. However, 3-T₁AM effects still persist in mTaar1 knockout mice, which suggest so far unknown further receptor targets that are of physiological relevance. TAAR5 is a highly conserved TAAR subtype among mammals and we here tested TAAR5 as a potential 3-T₁AM target. First, we investigated mouse Taar5 (mTaar5) expression in several brain regions of the mouse in comparison to mTaar1. Secondly, to unravel the full spectrum of signaling capacities, we examined the distinct G_s-, G_i/o-, G_12/13-, G_q/11- and MAP kinase-mediated signaling pathways of mouse and human TAAR5 under ligand-independent conditions and after application of 3-T₁AM. We found overlapping localization of mTaar1 and mTaar5 in the amygdala and ventromedial hypothalamus of the mouse brain. Second, the murine and human TAAR5 (hTAAR5) display significant basal activity in the G_q/11 pathway but show differences in the basal activity in G_s and MAP kinase signaling. In contrast to mTaar5, 3-T₁AM application at hTAAR5 resulted in significant reduction in basal IP₃ formation and MAP kinase signaling. In conclusion, our data suggest that the human TAAR5 is a target for 3-T₁AM, exhibiting inhibitory effects on IP₃ formation and MAP kinase signaling pathways, but does not mediate G_s signaling effects as observed for TAAR1. This study also indicates differences between TAAR5 orthologs with respect to their signaling profile. In consequence, 3-T₁AM-mediated effects may differ between rodents and humans.     

Novel Mechanism of Arenavirus-Induced Liver Pathology

Viral hemorrhagic fevers (VHFs) encompass a group of diseases with cardinal symptoms of fever, hemorrhage, and shock. The liver is a critical mediator of VHF disease pathogenesis and high levels of ALT/AST transaminases in plasma correlate with poor prognosis. In fact, Lassa Fever (LF), the most prevalent VHF in Africa, was initially clinically described as hepatitis. Previous studies in non-human primate (NHP) models also correlated LF pathogenesis with a robust proliferative response in the liver. The purpose of the current study was to gain insight into the mechanism of liver injury and to determine the potential role of proliferation in LF pathogenesis. C57Bl/6J mice were infected with either the pathogenic (for NHPs) strain of lymphocytic choriomeningitis virus (LCMV, the prototypic arenavirus), LCMV-WE, or with the non-pathogenic strain, LCMV-ARM. As expected, LCMV-WE, but not ARM, caused a hepatitis-like infection. LCMV-WE also induced a robust increase in the number of actively cycling hepatocytes. Despite this increase in proliferation, there was no significant difference in liver size between LCMV-WE and LCMV-ARM, suggesting that cell cycle was incomplete. Indeed, cells appeared arrested in the G₁ phase and LCMV-WE infection increased the number of hepatocytes that were simultaneously stained for proliferation and apoptosis. LCMV-WE infection also induced expression of a non-conventional virus receptor, AXL-1, from the TAM (TYRO3/AXL/MERTK) family of receptor tyrosine kinases and this expression correlated with proliferation. Taken together, these results shed new light on the mechanism of liver involvement in VHF pathogenesis. Specifically, it is hypothesized that the induction of hepatocyte proliferation contributes to expansion of the infection to parenchymal cells. Elevated levels of plasma transaminases are likely explained, at least in part, by abortive cell cycle arrest induced by the infection. These results may lead to the development of new therapies to prevent VHF progression.     

PDADMAC flocculation of Chinese hamster ovary cells: Enabling a centrifuge-less harvest process for monoclonal antibodies

High titer (>10 g/L) monoclonal antibody (mAb) cell culture processes are typically achieved by maintaining high viable cell densities over longer culture durations. A corresponding increase in the solids and sub-micron cellular debris particle levels are also observed. This higher burden of solids (≥15%) and sub-micron particles typically exceeds the capabilities of a continuous centrifuge to effectively remove the solids without a substantial loss of product and/or the capacity of the harvest filtration train (depth filter followed by membrane filter) used to clarify the centrate. We discuss here the use of a novel and simple two-polymer flocculation method used to harvest mAb from high cell mass cell culture processes. The addition of the polycationic polymer, poly diallyldimethylammonium chloride (PDADMAC) to the cell culture broth flocculates negatively-charged cells and cellular debris via an ionic interaction mechanism. Incorporation of a non-ionic polymer such as polyethylene glycol (PEG) into the PDADMAC flocculation results in larger flocculated particles with faster settling rate compared to PDADMAC-only flocculation. PDADMAC also flocculates the negatively-charged sub-micron particles to produce a feed stream with a significantly higher harvest filter train throughput compared to a typical centrifuged harvest feed stream. Cell culture process variability such as lactate production, cellular debris and cellular densities were investigated to determine the effect on flocculation. Since PDADMAC is cytotoxic, purification process clearance and toxicity assessment were performed.     

Genomic microstructure and differential expression of the genes encoding UDP-glucose:sinapate glucosyltransferase (UGT84A9) in oilseed rape (Brassica napus)

In oilseed rape (Brassica napus), the glucosyltransferase UGT84A9 catalyzes the formation of 1-O-sinapoyl-β-glucose, which feeds as acyl donor into a broad range of accumulating sinapate esters, including the major antinutritive seed component sinapoylcholine (sinapine). Since down-regulation of UGT84A9 was highly efficient in decreasing the sinapate ester content, the genes encoding this enzyme were considered as potential targets for molecular breeding of low sinapine oilseed rape. B. napus harbors two distinguishable sequence types of the UGT84A9 gene designated as UGT84A9-1 and UGT84A9-2. UGT84A9-1 is the predominantly expressed variant, which is significantly up-regulated during the seed filling phase, when sinapate ester biosynthesis exhibits strongest activity. In the allotetraploid genome of B. napus, UGT84A9-1 is represented by two loci, one derived from the Brassica C-genome (UGT84A9a) and one from the Brassica A-genome (UGT84A9b). Likewise, for UGT84A9-2 two loci were identified in B. napus originating from both diploid ancestor genomes (UGT84A9c, Brassica C-genome; UGT84A9d, Brassica A-genome). The distinct UGT84A9 loci were genetically mapped to linkage groups N15 (UGT84A9a), N05 (UGT84A9b), N11 (UGT84A9c) and N01 (UGT84A9d). All four UGT84A9 genomic loci from B. napus display a remarkably low micro-collinearity with the homologous genomic region of Arabidopsis thaliana chromosome III, but exhibit a high density of transposon-derived sequence elements. Expression patterns indicate that the orthologous genes UGT84A9a and UGT84A9b should be considered for mutagenesis inactivation to introduce the low sinapine trait into oilseed rape.     

Ligation of B and T lymphocyte attenuator prevents the genesis of experimental cerebral malaria

B and T lymphocyte attenuator (BTLA; CD272) is a coinhibitory receptor that is predominantly expressed on T and B cells and dampens T cell activation. In this study, we analyzed the function of BTLA during infection with Plasmodium berghei ANKA. Infection of C57BL/6 mice with this strain leads to sequestration of leukocytes in brain capillaries that is associated with a pathology resembling cerebral malaria in humans. During the course of infection, we found an induction of BTLA in several organs, which was either due to up-regulation of BTLA expression on T cells in the spleen or due to infiltration of BTLA-expressing T cells into the brain. In the brain, we observed a marked induction of BTLA and its ligand herpesvirus entry mediator during cerebral malaria, which was accompanied by an accumulation of predominantly CD8+ T cells, but also CD4+ T cells. Application of an agonistic anti-BTLA mAb caused a significantly reduced incidence of cerebral malaria compared with control mice. Treatment with this Ab also led to a decreased number of T cells that were sequestered in the brain of P. berghei ANKA-infected mice. Our findings indicate that BTLA-herpesvirus entry mediator interactions are functionally involved in T cell regulation during P. berghei ANKA infection of mice and that BTLA is a potential target for therapeutic interventions in severe malaria.     

Acute exercise stimulates macrophage function: possible role of NF-kappaB pathways

Moderate physical activity when performed on a regular basis presents a number of benefits to the whole organism, especially regarding immune system function, such as augmenting resistance to infections and to cancer growth. Although glutamine production by active muscle cells as well as neuroendocrine alterations mediated by the chronic adaptation to exercise may play a role, the entire mechanism by which exercise makes the immune system aware of challenges remains mostly uncovered. This is particularly true for the effects of an acute exercise session on immune function. In this work, circulating monocytes/macrophages from sedentary rats submitted to an acute (1 h) swimming session were tested for the ability of phagocytosing zymosan particles, phorbol myristate acetate (PMA)-induced hydrogen peroxide production, nitric oxide (NO) release (assessed by nitrate and nitrite production) and the expression of NO synthases (NOS-1, NOS-2 and NOS-3). The results showed that an exercise bout induced a 2.4-fold rise in macrophage phagocytic capacity (p = 0.0041), a 9.6-fold elevation in PMA-induced hydrogen peroxide release into the incubation media (1-h, p = 0.0022) and a 95.5%-augmentation in nitrite basal production (1-h incubation; p = 0.0220), which was associated with a marked expression of NOS-2 (the inducible NOS isoform; p = 0.0319), but not in other NOS gene products. Although NOS-2 expression is nuclear factor-kappaB (NF-kappaB)-dependent, no systemic oxidative stress was found, as inferred from the data of plasma TBARS and glutathione disulphide (GSSG) to glutathione (GSH) ratio in circulating blood erythrocytes which remained constant after the acute exercise. Also, no stressful situation seemed to be faced by monocytes/macrophages, since the expression of the 70-kDa heat shock protein (HSP70) remained unchanged. We conclude that NF-kappaB-dependent induction of NOS-2 and macrophage activation must be related to local factor(s) produced in the surroundings of monocytes/macrophages.