The anti-inflammatory effect of LMWF5A and N-acetyl kynurenine on macrophages: Involvement of aryl hydrocarbon receptor in mechanism of action

After a traumatic insult, macrophages can become activated leading to general inflammation at the site of injury. Activated macrophages are partially regulated by the aryl hydrocarbon receptor (AhR) which when activated suppresses inflammation by limiting the secretion of pro-inflammatory cytokines and promoting the over expression of immuno-modulatory mediators. This study aims to determine whether the low molecular weight fraction of 5% human serum albumin (LMWF5A) and N-acetyl kynurenine (NAK), an N-acetyl tryptophan (NAT) breakdown product in LMWF5A, can regulate inflammation by inhibiting macrophage activation through the AhR since kynurenine is a known AhR agonist. Using LCMS, we demonstrate that NAT is non-enzymatically degraded during accelerated aging of LMWF5A with high heat accelerating degradation. More importantly, NAK is a major degradation product found in LMWF5A. THP-1 monocytes were differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA) and pre-treated with 2-fold dilutions of LMWF5A or synthetic NAK with or without an AhR antagonist (CH223191) prior to overnight stimulation with lipopolysaccharide (LPS). Treatment with LMWF5A caused a 50–70% decrease in IL-6 release throughout the dilution series. A dose-response inhibition of IL-6 release was observed for NAK with maximal inhibition (50%) seen at the highest NAK concentration. Finally, an AhR antagonist partially blocked the anti-inflammatory effect of LMWF5A while completely blocking the effect of NAK. A similar inhibitory effect was observed for CXCL-10, but the AhR antagonist was not effective suggesting additional mechanisms for CXCL-10 release. These preliminary findings suggest that LMWF5A and NAK partially promote the suppression of activated macrophages via the AhR receptor. Therefore, LMWF5A, which contains NAK, is potentially a useful therapeutic in medical conditions where inflammation is prevalent such as trauma, sepsis, and wound healing.     

Redox Modulation of Oligomeric State in Proline Utilization A

Homooligomerization of proline utilization A (PutA) bifunctional flavoenzymes is intimately tied to catalytic function and substrate channeling. PutA from Bradyrhizobium japonicum (BjPutA) is unique among PutAs in that it forms a tetramer in solution. Curiously, a dimeric BjPutA hot spot mutant was previously shown to display wild-type catalytic activity despite lacking the tetrameric structure. These observations raised the question of what is the active oligomeric state of BjPutA. Herein, we investigate the factors that contribute to tetramerization of BjPutA in vitro. Negative-stain electron microscopy indicates that BjPutA is primarily dimeric at nanomolar concentrations, suggesting concentration-dependent tetramerization. Further, sedimentation-velocity analysis of BjPutA at high (micromolar) concentration reveals that although the binding of active-site ligands does not alter oligomeric state, reduction of the flavin adenine dinucleotide cofactor results in dimeric protein. Size-exclusion chromatography coupled with multiangle light scattering and small-angle x-ray scattering analysis also reveals that reduced BjPutA is dimeric. Taken together, these results suggest that the BjPutA oligomeric state is dependent upon both enzyme concentration and the redox state of the flavin cofactor. This is the first report, to our knowledge, of redox-linked oligomerization in the PutA family.     

Laboratory evaluation of large-scale decontamination approaches

Aims: To evaluate the effectiveness of two spray‐based decontamination methods for surface contamination reduction and to determine the potential for contamination spread by these methods. Methods and Results: Material coupons (treated plywood and concrete) were contaminated with c. 1 × 10⁷ spores of Bacillus atrophaeus by aerosol deposition. Decontaminants (pH‐adjusted bleach or Spor‐Klenz^® RTU) were applied to coupons by either backpack sprayer or gas‐powered sprayer. Contact time, reapplication frequency and rinse method were also varied. In addition to surface removal efficacy, partitioning of contamination between the rinsate and aerosol fractions was determined. Results indicated that pH‐adjusted bleach was effective (≥6 logs reduction) when two applications and a 30 min contact time were administered, regardless of the decontaminant application method or material. Spor‐Klenz^® RTU was effective on wood, but achieved ≤3 logs reduction on concrete. A shortened application procedure with pH‐adjusted bleach resulted in lower efficacy on wood, and a greater apparent potential for contamination spread. Conclusions: Consideration of material surface type is important when selecting a decontaminant. Also, achieving conditions that effectively inactivate surface biological contamination are critical to preventing the spread of contamination. Significance and Impact of the Study: Results presented here are intended to help development of remediation plans following a biological contamination incident.     

Toward routine,DNA-based detection methods for marine pests

Marine pest incursions can cause significant ongoing damage to aquaculture, biodiversity, fisheries habitat, infrastructure and social amenity. They represent a significant and ongoing economic burden. Marine pests can be introduced by several vectors including aquaculture, aquarium trading, commercial shipping, fishing, floating debris, mining activities and recreational boating. Despite the inherent risks, there is currently relatively little routine surveillance of marine pest species conducted in the majority of countries worldwide. Accurate and rapid identification of marine pest species is central to early detection and management. Traditional techniques (e.g. physical sampling and sorting), have limitations, which has motivated some progress towards the development of molecular diagnostic tools. This review provides a brief account of the techniques traditionally used for detection and describes developments in molecular-based methods for the detection and surveillance of marine pest species. Recent advances provide a platform for the development of practical, specific, sensitive and rapid diagnosis and surveillance tools for marine pests for use in effective prevention and control strategies.     

Gene-engineered T cells as a superior adjuvant therapy for metastatic cancer

The major limiting factor in the successful application of adjuvant therapy for metastatic disease is the lack of adjuvant specificity that leads to severe side effects. Reasoning that T cells of the immune system are highly specific, we generated tumor-specific T cells by genetic modification of mouse primary T cells with a chimeric receptor reactive with the human breast cancer-associated Ag erbB-2. These T cells killed breast cancer cells and secreted IFN-gamma in an Ag-specific manner in vitro. We investigated their use against metastatic breast cancer in mice in an adjuvant setting, and compared their effectiveness with the commonly applied adjuvants doxorubicin, 5-fluorouracil, and herceptin. Mice were inoculated orthotopically with the human erbB-2-expressing spontaneously metastatic mouse breast cancer 4T1.2 in mammary tissue, and the primary tumor was surgically removed 8 days later. Significant metastatic disease was demonstrated in lung and liver at the time of surgery on day 8 with increased tumor burden at later time points. T cell adjuvant treatment of day 8 metastatic disease resulted in dramatic increases in survival of mice, and this survival was significantly greater than that afforded by either doxorubicin, 5-fluorouracil, or herceptin.     

Microsatellite genotyping of red imported fire ant (Hymenoptera: Formicidae) colonies reveals that most colonies persist in plowed pastures

Our study focused on colony dynamics of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), in relation to the standard practice of planting rye grass (i.e., plowing) in the fall in Louisiana. Microsatellite molecular markers were used to determine genotypes of individuals from red imported fire ant colonies. These markers allowed us to monitor treatment effect by detecting changes in number and location of colonies in response to disking of pasture plots. Previous research on mound disturbance as a form of cultural control in pastures has produced mixed results. We found that the majority of colonies persisted on plots after plowing. Mound density and mound area, 5 mo after plowing, were not significantly different among treatments. In contrast, April measurements of mound volume were significantly smaller on plowed plots compared with control plots. A closer look at the rebuilding of mounds on plowed plots, during the 5 mo, showed that mound heights stayed below pretreatment measurements and they were significantly smaller than those of undisturbed mounds. Whether plowing has potential for use as a cultural control technique in reducing the impact of red imported fire ant mounds on agricultural practices in pastures remains to be seen. Conceivably, the best application of this technique will be in combination with other control measures in an integrated pest management approach to control red imported fire ants in pastures.     

Dimerization of the extracellular domain of the receptor for epidermal growth factor containing the membrane-spanning segment in response to treatment with epidermal growth factor

A recombinant fragment of the human receptor for epidermal growth factor containing both its extracellular domain and its membrane-spanning segment, when dissolved with Triton X-100, was observed to dimerize in response to addition of epidermal growth factor (EGF) even at the lowest concentration of this fragment that could be assayed (4 nM). Consequently, the dissociation constant for the dimer of this fragment is at least 10,000-fold smaller than that for dimers of soluble, recombinant forms of the extracellular domain lacking the membrane-spanning segment. The second-order rate constant for dimerization of the fragment containing the extracellular domain and the membrane-spanning segment was estimated to be greater than 0.3 nM(-1) min(-1), more than 10-fold that of the native enzyme under the same conditions. This result suggests that the cytoplasmic domain of the intact enzyme sterically hinders its dimerization. When EGF is removed from the dimer of the fragment, the rate constant for its dissociation is greater than 0.2 min(-1), more than 40-fold that of the native enzyme. This result suggests that interfaces between cytoplasmic domains of intact EGF receptor impart significant stabilization to the dimer of the enzyme.     

Nucleosomes bind fibroblast growth factor-2 for increased angiogenesis in vitro and in vivo

Solid tumors often display sites of necrosis near regions of angiogenesis in vivo. As tumor cell necrosis would result in the release of nucleosomes into the extracellular environment, we explored the potential role of nucleosomes in the promotion of angiogenesis. Data indicate that nucleosomes acted similar to heparin and bound to several heparin-binding, proangiogenic factors [i.e., fibroblast growth factor (FGF)-1, FGF-2, vascular endothelial growth factor, and transforming growth factor-beta1]. Nucleosomes modestly enhanced FGF-2 growth of human umbilical vein endothelial cells when grown in restricted media as well as increased human umbilical vein endothelial cell migration and primitive blood vessel tube formation in vitro. On s.c. injection in mice, nucleosomes aided FGF-2 in promoting angiogenesis. These results suggest that nucleosomes released from dying tumor cells aid in the formation of blood vessels and may provide a novel means by which tumor cells increase angiogenesis.