Dennis P. McDaniel

Rostrocaudal analysis of corpus callosum demyelination and axon damage across disease stages refines diffusion tensor imaging correlations with pathological features.

Noninvasive assessment of the progression of axon damage is important for evaluating disease progression and developing neuroprotective interventions in multiple sclerosis patients. We examined thecellular responses correlated with diffusion tensor imaging-derivedaxial (&lgr;∥) and radial (&lgr;⊥) diffusivity values throughout acute (4 weeks) and chronic (12 weeks) stages of demyelination and after 6 weeks of recovery using the cuprizone demyelination of the corpus callosum model in C57BL/6 and Thy1-YFP-16 mice. The rostrocaudal progression of pathological alterations in the corpus callosum enabled spatially and temporally defined correlations of pathological features with diffusion tensor imaging measurements. During acute demyelination, microglial/macrophage activation was most extensive and axons exhibited swellings, neurofilament dephosphorylation, and reduced diameters. Axial diffusivity values decreased in the acute phase but did not correlate with axonal atrophy during chronic demyelination. In contrast, radial diffusivity increased with the progression of demyelination but did not correlate with myelin loss orastrogliosis. Unlike other animal models with progressive neurodegeneration and axon loss, the acute axon damage did not progress to discontinuity or loss of axons even after a period of chronicdemyelination. Correlations of reversible axon pathology, demyelination, microglia/macrophage activation, and astrogliosis with regional axial and radial diffusivity measurements will facilitate the clinical application of diffusion tensor imaging in multiple sclerosis patients.

Components of myelin damage and repair in the progression of white matter pathology after mild traumatic brain injury.

Abstract White matter tracts are highly vulnerable to damage from impact-acceleration forces of traumatic brain injury (TBI). Mild TBI is characterized by a low density of traumatic axonal injury, whereas associated myelin pathology is relatively unexplored. We examined the progression of white matter pathology in mice after mild TBI with traumatic axonal injury localized in the corpus callosum. Adult mice received a closed-skull impact and were analyzed from 3 days to 6 weeks post-TBI/sham surgery. At all times post-TBI, electron microscopy revealed degenerating axons distributed among intact fibers in the corpus callosum. Intact axons exhibited significant demyelination at 3 days followed by evidence of remyelination at 1 week. Accordingly, bromodeoxyuridine pulse-chase labeling demonstrated the generation of new oligodendrocytes, identified by myelin proteolipid protein messenger RNA expression, at 3 days post-TBI. Overall oligodendrocyte populations, identified by immunohistochemical staining for CC1 and/or glutathione S-transferase pi, were similar between TBI and sham mice by 2 weeks. Excessively long myelin figures, similar to redundant myelin sheaths, were a significant feature at all post-TBI time points. At 6 weeks post-TBI, microglial activation and astrogliosis were localized to areas of axon and myelin pathology. These studies show that demyelination, remyelination, and excessive myelin are components of white matter degeneration and recovery in mild TBI with traumatic axonal injury.

Preserving Immunogenicity of Lethally Irradiated Viral and Bacterial Vaccine Epitopes Using a Radio-Protective Mn2+-Peptide Complex from Deinococcus

Although pathogen inactivation by γ-radiation is an attractive approach for whole-organism vaccine development, radiation doses required to ensure sterility also destroy immunogenic protein epitopes needed to mount protective immune responses. We demonstrate the use of a reconstituted manganous peptide complex from the radiation-resistant bacterium Deinococcus radiodurans to protect protein epitopes from radiation-induced damage and uncouple it from genome damage and organism killing. The Mn(2+) complex preserved antigenic structures in aqueous preparations of bacteriophage lambda, Venezuelan equine encephalitis virus, and Staphylococcus aureus during supralethal irradiation (25-40 kGy). An irradiated vaccine elicited both antibody and Th17 responses, and induced B and T cell-dependent protection against methicillin-resistant S. aureus (MRSA) in mice. Structural integrity of viruses and bacteria are shown to be preserved at radiation doses far above those which abolish infectivity. This approach could expedite vaccine production for emerging and established pathogens for which no protective vaccines exist.

Recombinant Bacillus anthracis spore proteins enhance protection of mice primed with suboptimal amounts of protective antigen

Inactivated Bacillus anthracis spores given with protective antigen (PA) contribute to immunity against anthrax in several animal models. Antiserum raised against whole irradiated B. anthracis spores has been shown to have anti-germination and opsonic activities in vitro. Based on these observations, we hypothesized that surface-exposed spore proteins might serve as supplemental components of a PA-based anthrax vaccine. The protective anti-spore serum was tested for reactivity with recombinant forms of 30 proteins known, or believed to be, present within the B. anthracis exosporium. Eleven of those proteins were reactive with this antiserum, and, subsequently a subset of this group was used to generate rabbit polyclonal antibodies. These sera were evaluated for recognition of the immunogens on intact spores generated from Sterne strain, as well as from an isogenic mutant lacking the spore surface protein Bacillus collagen-like antigen (BclA). The data were consistent with the notion that the antigens in question were located beneath BclA on the basal surface of the exosporium. A/J mice immunized with either the here-to-for hypothetical protein p5303 or the structural protein BxpB, each in combination with subprotective levels of PA, showed enhanced protection against subcutaneous spore challenge. While neither anti-BxpB or anti-p5303 antibodies reduced the rate of spore germination in vitro, both caused increased uptake and lead to a higher rate of destruction by phagocytic cells. We conclude that by facilitating more efficient phagocytic clearance of spores, antibodies against individual exosporium components can contribute to protection against B. anthracis infection.

Microglia activation along the corticospinal tract following traumatic brain injury in the rat: a neuroanatomical study.

Traumatic injury to the brain often manifests itself symptomatically and structurally long after the traumatic event. The cellular basis of this complex response is not completely understood. However, we hypothesized that microglia might contribute to the brain-wide process. To test this hypothesis, we employed optical and electron microscopy to study the microglia in rat brains up to 2 months after digitally controlled cortical impact (CCI) to produce traumatic brain injury (TBI). We also used antibodies against ED-1 and Iba-1, respectively, as markers for activated and resting microglia. ED-1 positive microglial cells are observed accompanying the entire corticospinal tract (CST) on the injured side, but not the control, contralateral side of the brain at 2 months. In this case, ED-1 and Iba-1 were observed to co-localize uniquely on the injured side of the brain. At earlier times following CCI, ultrastructural studies reveal that microglial cells have very irregular shapes and have many processes that intermingle with degenerating nerve axons of the CST in the hindbrain pyramids. These cells appear to be engulfing degenerating myelinated axons. The debris within the cells is converted to lipofuscin, the antigen for the ED-1 antibody, and remains in the cell cytoplasm throughout the life of the cell. We conclude, as hypothesized, that microglia are critical cellular components. Based on observed close association with myelin degeneration, interdigitating activated microglia may be contributing to damage control. Finally, based on the close neuroanatomical relationship between the lesioned corticospinal tract and the wide distribution of activated microglia, primary signals from CST neurons per se, may be directing microglial responses along the entire damaged rat neuroaxis. The role of persistent activation of microglia has not been determined.

Adaptive Redox Response of Mesenchymal Stromal Cells to Stimulation with Lipopolysaccharide Inflammagen: Mechanisms of Remodeling of Tissue Barriers in Sepsis

Acute bacterial inflammation is accompanied by excessive release of bacterial toxins and production of reactive oxygen and nitrogen species (ROS and RNS), which ultimately results in redox stress. These factors can induce damage to components of tissue barriers, including damage to ubiquitous mesenchymal stromal cells (MSCs), and thus can exacerbate the septic multiple organ dysfunctions. The mechanisms employed by MSCs in order to survive these stress conditions are still poorly understood and require clarification. In this report, we demonstrated that in vitro treatment of MSCs with lipopolysaccharide (LPS) induced inflammatory responses, which included, but not limited to, upregulation of iNOS and release of RNS and ROS. These events triggered in MSCs a cascade of responses driving adaptive remodeling and resistance to a “self-inflicted” oxidative stress. Thus, while MSCs displayed high levels of constitutively present adaptogens, for example, HSP70 and mitochondrial Sirt3, treatment with LPS induced a number of adaptive responses that included induction and nuclear translocation of redox response elements such as NFkB, TRX1, Ref1, Nrf2, FoxO3a, HO1, and activation of autophagy and mitochondrial remodeling. We propose that the above prosurvival pathways activated in MSCs in vitro could be a part of adaptive responses employed by stromal cells under septic conditions.

The Oligo-Acyl Lysyl Antimicrobial Peptide C12K-2β12 Exhibits a Dual Mechanism of Action and Demonstrates Strong In Vivo Efficacy against Helicobacter pylori

ABSTRACT Helicobacter pylori has developed antimicrobial resistance to virtually all current antibiotics. Thus, there is a pressing need to develop new anti-H. pylori therapies. We recently described a novel oligo-acyl-lysyl (OAK) antimicrobial peptidomimetic, C12K-2β12, that shows potent in vitro bactericidal activity against H. pylori. Herein, we define the mechanism of action and evaluate the in vivo efficacy of C12K-2β12 against H. pylori after experimental infection of Mongolian gerbils. We demonstrate using a 1-N-phenylnaphthylamine (fluorescent probe) uptake assay and electron microscopy that C12K-2β12 rapidly permeabilizes the bacterial membrane and creates pores that cause bacterial cell lysis. Furthermore, using nucleic acid binding assays, Western blots, and confocal microscopy, we show that C12K-2β12 can cross the bacterial membranes into the cytoplasm and tightly bind to bacterial DNA, RNA, and proteins, a property that may result in inhibition of enzymatic activities and macromolecule synthesis. To define the in vivo efficacy of C12K-2β12, H. pylori-infected gerbils were orogastrically treated with increasing doses and concentrations of C12K-2β12 1 day or 1 week postinfection. The efficacy of C12K-2β12 was strongest in animals that received the largest number of doses at the highest concentration, indicating dose-dependent activity of the peptide (P < 0.001 by analysis of variance [ANOVA]) regardless of the timing of the treatment with C12K-2β12. Overall, our results demonstrate a dual mode of action of C12K-2β12 against the H. pylori membrane and cytoplasmic components. Moreover, and consistent with the previously reported in vitro efficacy, C12K-2β12 shows significant in vivo efficacy against H. pylori when used as monotherapy. Therefore, OAK peptides may be a valuable resource for therapeutic treatment of H. pylori infection.

Autophagy-Mediated Defense Response of Mouse Mesenchymal Stromal Cells (MSCs) to Challenge with Escherichia coli

Abstract : Symbiotic microorganisms are spatially separated from their animal host, e.g., in the intestine and skin, in a manner enabling nutrient metabolism as well as evolutionary development of protective physiologic features in the host such as innate and adaptive immunity, immune tolerance, and function of tissue barriers . The major interface barrier between the microbiota and host tissue is constituted by epithelium reticuloendothelial tissue, and mucosa-associated lymphoid tissue (MALT) . Traumatic damage to skin and the internal epithelium in soft tissues can cause infections that account for 7% to 10% of hospitalizations in the United States (4). Moreover, wound infections and sepsis are an increasing cause of death in severely ill patients, especially those with immunosupression due to exposure to cytotoxic agents and chronic inflammation (4). It is well accepted that breakdown of the host-bacterial symbiotic homeostasis and associated infections are the major consequences of impairment of the first line of antimicrobial defense barriers such as the mucosal layers, MALT and reticuloendothelium. Under these impairment conditions of particular interest then is the role of sub-mucosal structures, such as connective tissue stroma, in the innate defense compensatory responses to infections. The mesenchymal connective tissue of different origins is a major source of multipotent mesenchymal stromal cells (i.e., colony-forming-unit fibroblasts). Recent discovery of immunomodulatory function of mesenchymal stromal cells (MSCs) suggests that they are essential constituents that control inflammatory responses.

Autophagy and mitochondrial remodelling in mouse mesenchymal stromal cells challenged with Staphylococcus epidermidis

The bone marrow stroma constitutes the marrow‐blood barrier, which sustains immunochemical homoeostasis and protection of the haematopoietic tissue in sequelae of systemic bacterial infections. Under these conditions, the bone marrow stromal cells affected by circulating bacterial pathogens shall elicit the adaptive stress‐response mechanisms to maintain integrity of the barrier. The objective of this communication was to demonstrate (i) that in vitro challenge of mesenchymal stromal cells, i.e. colony‐forming unit fibroblasts (CFU‐F), with Staphylococcus epidermidis can activate the autophagy pathway to execute antibacterial defence response, and (ii) that homoeostatic shift because of the bacteria‐induced stress includes the mitochondrial remodelling and sequestration of compromised organelles via mitophagy. Implication of Drp1 and PINK1–PARK2‐dependent mechanisms in the mitophagy turnover of the aberrant mitochondria in mesenchymal stromal cells is investigated and discussed.

Nelfinavir inhibits proliferation and induces DNA damage in thyroid cancer cells

The HIV protease inhibitor Nelfinavir (NFV) inhibits PI3K/AKT and MAPK/ERK signaling pathways, emerging targets in thyroid cancers. We examined the effects of NFV on cancer cells that derived from follicular (FTC), papillary (PTC) and anaplastic (ATC) thyroid cancers. NFV (1-20 µM) was tested in FTC133, BCPAP and SW1736 cell lines. The effects of NFV on cell proliferation were determined in vitro using real-time microscopy and by flow cytometry. DNA damage, apoptotic cell death and expression of molecular markers of epithelial-mesenchymal transition (EMT) were determined by Western blot and real-time PCR. Real-time imaging demonstrated that NFV (10 µM) increased the time required for the cell passage through the phases of cell cycle and induced DNA fragmentation. Growth inhibitory effects of NFV were associated with the accumulation of cells in G0/G1 phase, downregulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). NFV also induced the expression of γH2AX and p53BP1 indicating DNA damage. Treatment with NFV (20 µM) resulted in caspase-3 cleavage in all examined cells. NFV (20 µM) decreased the levels of total and p-AKT in PTEN-deficient FTC133 cells. NFV had no significant effects on total ERK and p-ERK in BRAF-positive BCPAP and SW1736 cells. NFV had no effects on the expression of EMT markers (Twist, Vimentin, E- and N-Cadherin), but inhibited the migration and decreased the abilities of thyroid cancer cells to survive in non-adherent conditions. We conclude that NFV inhibits proliferation and induces DNA damage in thyroid cancer cell lines. Our in vitro data suggest that NFV has a potential to become a new thyroid cancer therapeutic agent.