Monoclonal antibody against the poly-γ-d-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge

Background

The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT.

Methods

To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K_d values of 0.8 μM and 2.6 μM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed.

Results

Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen.

General significance

Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.     

Synthesis,copolymerization and peptide-modification of carboxylic acid-functionalized 3,4-ethylenedioxythiophene (EDOTacid) for neural electrode interfaces

Background

Conjugated polymers have been developed as effective materials for interfacing prosthetic device electrodes with neural tissue. Recent focus has been on the development of conjugated polymers that contain biological components in order to improve the tissue response upon implantation of these electrodes.

Methods

Carboxylic acid-functionalized 3,4-ethylenedioxythiophene (EDOTacid) monomer was synthesized in order to covalently bind peptides to the surface of conjugated polymer films. EDOTacid was copolymerized with EDOT monomer to form stable, electrically conductive copolymer films referred to as PEDOT-PEDOTacid. The peptide GGGGRGDS was bound to PEDOT-PEDOTacid to create peptide functionalized PEDOT films.

Results

The PEDOT-PEDOTacid-peptide films increased the adhesion of primary rat motor neurons between 3 and 9 times higher than controls, thus demonstrating that the peptide maintained its biological activity.

Conclusions

The EDOT-acid monomer can be used to create functionalized PEDOT-PEDOTacid copolymer films that can have controlled bioactivity.

General Significance

PEDOT-PEDOTacid-peptide films have the potential to control the behavior of neurons and vastly improve the performance of implanted electrodes. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.     

Effects of oral deoxynivalenol exposure on immune-related parameters in lymphoid organs and serum of mice vaccinated with porcine parvovirus vaccine

Mice were exposed to deoxynivalenol (DON) via drinking water at a concentration of 2 mg/L for 36 days. On day 8 of treatment, inactivated porcine parvovirus vaccine (PPV) was injected intraperitoneally. The relative and absolute weight of the spleen was significantly decreased in the DON-treated group (DON). Antibody titers to parvovirus in serum were 47.9?±?2.4 in the vaccination group (Vac), but 15.2?±?6.5 in the group treated with DON and vaccine (DON?+?Vac). The IgA and IgG was not different in the DON, Vac an,d DON?+?Vac groups. IgM was significantly lower only in the DON?+?Vac group. However IgE was significantly increased in the Vac and DON?+?Vac group, but no change was observed between the Vac and DON?+?Vac groups. The concentrations of IL-2, IL-4, GM-CSF, MCP-1 and Rantes in serum, and IL-1α in mesenteric lymph node and MIP-1β in spleen were significantly increased by DON treatment compared to control. The concentrations of IL-2, IL-5, IL-6, IL-9, IL-12, IL-13 and Rantes in thymus, of IL-2 in spleen, and of IL-1α, IL-1β, IL-3, IL-5, IL-10, IL-17, G-CSF, GM-CSF and MCP-1 in mesenteric lymph nodes were significantly decreased in mice compared to those in the Vac group, while concentrations of IL-1α, IL-2, IL-9, IL-13,G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1α and TNF-α were significantly increased in serum compared to the Vac group. In conclusion, the results presented here indicate that exposure to DON at 2.0 mg/L via drinking water can disrupt the immune response in vaccinated mice by modulating cytokines and chemokines involved in their immune response to infectious disease.     

Effect of Zinc Oxide Nanoparticles on the Function of MC3T3-E1 Osteoblastic Cells

Zinc oxide nanoparticles (ZnO NPs) can be ingested directly when used in food, food packaging, drug delivery, and cosmetics. This study evaluated the cellular effects of ZnO NPs (50 and 100 nm diameter particle sizes) on the function of osteoblastic MC3T3-E1 cells. ZnO NPs showed cytotoxicity at concentrations of above 50 μg/ml, and there was no significant effect of the size on the cytotoxicity of ZnO NPs. Within the testing concentrations of 0.01～1 μg/ml, which did not cause a marked drop in cell viability, ZnO NPs (0.1 μg/ml) caused a significant elevation of alkaline phosphatase activity, collagen synthesis, mineralization, and osteocalcin content in the cells (P?<?0.05). Moreover, pretreatment with ZnO NPs (0.01～1 μg/ml) significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, and ATP loss. Measurement of reactive oxygen species (ROS) indicated decrease in ROS level upon exposure to ZnO nanoparticles (0.01 μg/ml). Hence, our study indicated that ZnO nanoparticles can have protective effects on osteoblasts at low concentrations where there are little or no observable cytotoxic effects.     

Functional Analysis of TiO2 Nanoparticle Toxicity in Three Plant Species

Titanium dioxide nanoparticles (nano-TiO₂) are manufactured and used worldwide in large quantities. However, phytotoxicity research on nano-TiO₂ has yielded confusing results, ranging from strong toxicity to positive effects. Therefore, in this research, the effects of nano-TiO₂ on the germination and root elongation of seed and seedlings were studied. Additionally, the uptake and physiological responses of mature plants were investigated. Physical chemistry data were analyzed to assess the availability of nano-TiO₂. Finally, a hydroponic system designed to overcome nano-TiO₂ precipitation was used to reproduce the environmental conditions of actual fields. Nano-TiO₂ did not have any effect on seed germination or on most of the plant species tested. Nano-TiO₂ had positive effects on root elongation in some species. No physiological differences in enzyme activities or chlorophyll content were detected, even though the plants absorbed nano-TiO₂. Physical chemistry data showed that nano-TiO₂ agglomerated rapidly and formed particles with much bigger hydrodynamic diameters, even in distilled water and especially in a hydroponic system. Furthermore, agglomerated nano-TiO₂ formed precipitates; this would be more severe in an actual field. Consequently, nano-TiO₂ would not be also readily available to plants and would not cause any significant effects on plants. Our results and other reports suggest that titanium itself is not phytotoxic, even though plants absorb titanium. In conclusion, nano-TiO₂ is not toxic to the three plant species, in vitro or in situ.     

Regional Differences in Elements of Human Peroneus LongusTendons

Many studies have been performed on the structure, molecular composition, and biochemical properties of tendons. However, comparatively little research has been conducted on the content of various trace elements within tendons. Six elements were analyzed in four regions of the peroneus longus tendon: the tensional part of the tendon immediately proximal to the lateral malleolus (region A), the compressive region of the tendon in contact with the lateral malleolus (region B), the compressive region of the tendon in contact with the deep surface of the cuboid (region C), and the tensional part of the tendon between the cuboid and first metatarsal, to which the tendon is attached (region D). Regions B and C are wraparound regions. The calcium content was higher in region C (2.10?±?0.93 mg/g) than in both regions A (1.25?±?0.51 mg/g) and D (1.43?±?0.41 mg/g) (p?<?0.05), indicating that it is likely related to regional differences in cartilage degeneration. The phosphorus content was also higher in region C, possibly because of low alkaline phosphatase activity in this region. The sulfur content was higher in the wraparound regions (region B: 0.98?±?0.09 mg/g, region C: 1.24?±?0.19 mg/g) than in both regions A (0.83?±?0.11 mg/g) and D (0.83?±?0.1 mg/g) (p?<?0.01); sulfur content is thought to be influenced by tendon–bone compression. Finally, the magnesium content in the wraparound regions was also higher, which is probably related to a higher level of fibrocartilage. No significant relationships were found with regard to zinc or iron. Overall, the findings of the present study indicate that element contents are related to function and anatomical differences in tendons, and that they may even vary within the same tendon.     

Dissecting direct and indirect genetic effects on chronic obstructive pulmonary disease (COPD) susceptibility

Cigarette smoking is the major environmental risk factor for chronic obstructive pulmonary disease (COPD). Genome-wide association studies have provided compelling associations for three loci with COPD. In this study, we aimed to estimate direct, i.e., independent from smoking, and indirect effects of those loci on COPD development using mediation analysis. We included a total of 3,424 COPD cases and 1,872 unaffected controls with data on two smoking-related phenotypes: lifetime average smoking intensity and cumulative exposure to tobacco smoke (pack years). Our analysis revealed that effects of two linked variants (rs1051730 and rs8034191) in the AGPHD1/CHRNA3 cluster on COPD development are significantly, yet not entirely, mediated by the smoking-related phenotypes. Approximately 30 % of the total effect of variants in the AGPHD1/CHRNA3 cluster on COPD development was mediated by pack years. Simultaneous analysis of modestly (r ² = 0.21) linked markers in CHRNA3 and IREB2 revealed that an even larger (~42 %) proportion of the total effect of the CHRNA3 locus on COPD was mediated by pack years after adjustment for an IREB2 single nucleotide polymorphism. This study confirms the existence of direct effects of the AGPHD1/CHRNA3, IREB2, FAM13A and HHIP loci on COPD development. While the association of the AGPHD1/CHRNA3 locus with COPD is significantly mediated by smoking-related phenotypes, IREB2 appears to affect COPD independently of smoking.     

Memory T cells are uniquely resistant to melanoma-induced suppression

We have previously observed that in vivo exposure to growing melanoma tumors fundamentally alters activated T cell homeostasis by suppressing the ability of naïve T cells to undergo antigen-driven proliferative expansion. We hypothesized that exposure of T cells in later stages of differentiation to melanoma would have similar suppressive consequences. C57BL/6 mice were inoculated with media or syngeneic B16F10 melanoma tumors 8 or 60 days after infection with lymphocytic choriomeningitis virus (LCMV), and splenic populations of LCMV-specific T cells were quantified using flow cytometry 18 days after tumor inoculation. Inoculation with melanoma on post-infection day 8 potentiated the contraction of previously activated T cells. This enhanced contraction was associated with increased apoptotic susceptibility among T cells from tumor-bearing mice. In contrast, inoculation with melanoma on post-infection day 60 did not affect the ability of previously established memory T cells to maintain themselves in stable numbers. In addition, the ability of previously established memory T cells to respond to LCMV challenge was unaffected by melanoma. Following adoptive transfer into melanoma-bearing mice, tumor-specific memory T cells were significantly more effective at controlling melanoma growth than equivalent numbers of tumor-specific effector T cells. These observations suggest that memory T cells are uniquely resistant to suppressive influences exerted by melanoma on activated T cell homeostasis; these findings may have implications for T cell–based cancer immunotherapy.     

Time-course Changes in Immunoreactivities of Glucokinase and Glucokinase Regulatory Protein in the Gerbil Hippocampus Following Transient Cerebral Ischemia

Glucose is a main energy source for normal brain functions. Glucokinase (GK) plays an important role in glucose metabolism as a glucose sensor, and GK activity is modulated by glucokinase regulatory protein (GKRP). In this study, we examined the changes of GK and GKRP immunoreactivities in the gerbil hippocampus after 5 min of transient global cerebral ischemia. In the sham-operated-group, GK and GKRP immunoreactivities were easily detected in the pyramidal neurons of the stratum pyramidale of the hippocampus. GK and GKRP immunoreactivities in the pyramidal neurons were distinctively decreased in the hippocampal CA1 region (CA), not CA2/3, 3 days after ischemia–reperfusion (I–R). Five days after I–R, GK and GKRP immunoreactivities were hardly detected in the CA1, not CA2/3, pyramidal neurons; however, at this point in time, GK and GKRP immunoreactivities were newly expressed in astrocytes, not microglia, in the ischemic CA1. In brief, GK and GKRP immunoreactivities are changed in pyramidal neurons and newly expressed in astrocytes in the ischemic CA1 after transient cerebral ischemia. These indicate that changes of GK and GKRP expression may be related to the ischemia-induced neuronal damage/death.     

Proteomic Analysis of Primary Cultured Rat Cortical Neurons in Chemical Ischemia

To elucidate the molecular events involved in early ischemic neuronal death, we performed two-dimensional proteome profiling of primary cultures of rat cortical neurons following chemical ischemia induced by the administration of sodium azide under glucose-free conditions. Using a lactic dehydrogenase assay and Western blot analysis of dephosporylation of the voltage-gated potassium channel Kv2.1, we determined duration of chemical ischemia of 2 h to be the relevant time-point for early ischemic neuronal death. Sixty-one proteins were differentially expressed, and 26 different proteins were identified by MALDI-TOF with Mascot database searching. The proteome data indicated that chemical ischemia altered the expression of 20 proteins that are involved in stress response/chaperone, brain development, cytoskeletal/structural proteins, metabolic enzymes, and calcium ion homeostasis. Western blotting and immunocytochemical studies of the 6-most functionally significant proteins showed that, in the ischemia-treated group, the expression of glucose-related protein 78, heat shock protein 90 alpha, and α-enolase was significantly increased, while the expression of inositol triphosphate receptor 1 and ATP synthase beta subunit was decreased. In addition, the expression of dihydropyrimidinase-like 3 showed a truncated pattern in the ischemia group. The changes in the expression of these proteins might be significant indicators of early ischemic neuronal death.