Calpastatin Overexpression Protects against Excitotoxic Hippocampal Injury and Traumatic Spinal Cord Injury.

Small molecule inhibitors of calcium-dependent proteases, calpains (CAPNs), protect against neurodegeneration induced by a variety of insults including excitotoxicity and spinal cord injury (SCI). Many of these compounds, however, also inhibit other proteases, which has made it difficult to evaluate the contribution of calpains to neurodegeneration. Calpastatin is a highly specific endogenous inhibitor of classical calpains, including CAPN1 and CAPN2. In the present study, we utilized transgenic mice that overexpress human calpastatin under the prion promoter (PrP-hCAST) to evaluate the hypothesis that calpastatin overexpression protects against excitotoxic hippocampal injury and contusive SCI. The PrP-hCAST organotypic hippocampal slice cultures showed reduced neuronal death and reduced calpain-dependent proteolysis (α-spectrin breakdown production, 145 kDa) at 24 h after N-methyl-D-aspartate (NMDA) injury compared with the wild-type (WT) cultures (n = 5, p < 0.05). The PrP-hCAST mice (n = 13) displayed a significant improvement in locomotor function at one and three weeks after contusive SCI compared with the WT controls (n = 9, p < 0.05). Histological assessment of lesion volume and tissue sparing, performed on the same animals used for behavioral analysis, revealed that calpastatin overexpression resulted in a 30% decrease in lesion volume (p < 0.05) and significant increases in tissue sparing, white matter sparing, and gray matter sparing at four weeks post-injury compared with WT animals. Calpastatin overexpression reduced α-spectrin breakdown by 51% at 24 h post-injury, compared with WT controls (p < 0.05, n = 3/group). These results provide support for the hypothesis that sustained calpain-dependent proteolysis contributes to pathological deficits after excitotoxic injury and traumatic SCI.

A multi-mode shock tube for investigation of blast-induced traumatic brain injury.

Blast-induced mild traumatic brain injury (bTBI) has become increasingly common in recent military conflicts. The mechanisms by which non-impact blast exposure results in bTBI are incompletely understood. Current small animal bTBI models predominantly utilize compressed air-driven membrane rupture as their blast wave source, while large animal models use chemical explosives. The pressure-time signature of each blast mode is unique, making it difficult to evaluate the contributions of the different components of the blast wave to bTBI when using a single blast source. We utilized a multi-mode shock tube, the McMillan blast device, capable of utilizing compressed air- and compressed helium-driven membrane rupture, and the explosives oxyhydrogen and cyclotrimethylenetrinitramine (RDX, the primary component of C-4 plastic explosives) as the driving source. At similar maximal blast overpressures, the positive pressure phase of compressed air-driven blasts was longer, and the positive impulse was greater, than those observed for shockwaves produced by other driving sources. Helium-driven shockwaves more closely resembled RDX blasts, but by displacing air created a hypoxic environment within the shock tube. Pressure-time traces from oxyhydrogen-driven shockwaves were very similar those produced by RDX, although they resulted in elevated carbon monoxide levels due to combustion of the polyethylene bag used to contain the gases within the shock tube prior to detonation. Rats exposed to compressed air-driven blasts had more pronounced vascular damage than those exposed to oxyhydrogen-driven blasts of the same peak overpressure, indicating that differences in blast wave characteristics other than peak overpressure may influence the extent of bTBI. Use of this multi-mode shock tube in small animal models will enable comparison of the extent of brain injury with the pressure-time signature produced using each blast mode, facilitating evaluation of the blast wave components contributing to bTBI.

Color changing photonic crystals detect blast exposure.

Blast-induced traumatic brain injury (bTBI) is the "signature wound" of the current wars in Iraq and Afghanistan. However, with no objective information of relative blast exposure, warfighters with bTBI may not receive appropriate medical care and are at risk of being returned to the battlefield. Accordingly, we have created a colorimetric blast injury dosimeter (BID) that exploits material failure of photonic crystals to detect blast exposure. Appearing like a colored sticker, the BID is fabricated in photosensitive polymers via multi-beam interference lithography. Although very stable in the presence of heat, cold or physical impact, sculpted micro- and nano-structures of the BID are physically altered in a precise manner by blast exposure, resulting in color changes that correspond with blast intensity. This approach offers a lightweight, power-free sensor that can be readily interpreted by the naked eye. Importantly, with future refinement this technology may be deployed to identify soldiers exposed to blast at levels suggested to be supra-threshold for non-impact blast-induced mild TBI.

Differential expression and glycosylation of anaplasma phagocytophilum major surface protein 2 paralogs during cultivation in sialyl Lewis x-deficient host cells.

Many microbial pathogens alter expression and/or posttranslational modifications of their surface proteins in response to dynamics within their host microenvironments to retain optimal interactions with their host cells and/or to evade the humoral immune response. Anaplasma phagocytophilum is an intragranulocytic bacterium that utilizes sialyl Lewis x (sLe(x))-modified P-selectin glycoprotein ligand 1 as a receptor for infecting myeloid cells. Bacterial populations that do not rely on this receptor can be obtained through cultivation in sLe(x)-defective cell lines. A. phagocytophilum major surface protein 2 [Msp2(P44)] is encoded by members of a paralogous gene family and is speculated to play roles in host adaptation. We assessed the complement of Msp2(P44) paralogs expressed by A. phagocytophilum during infection of sLe(x)-competent HL-60 cells and two HL-60 cell lines defective for sLe(x) expression. Multiple Msp2(P44) and N-terminally truncated 25- to 27-kDa isoforms having various isoelectric points and electrophoretic mobilities were expressed in each cell line. The complement of expressed msp2(p44) paralogs and the glycosyl residues modifying Msp2(P44) varied considerably among bacterial populations recovered from sLe(x)-competent and -deficient host cells. Thus, loss of host cell sLe(x) expression coincided with both differential expression and glycosylation of A. phagocytophilum Msp2(P44). This reinforces the hypothesis that this bacterium is able to generate a large variety of surface-exposed molecules that could provide great antigenic diversity and result in multiple binding properties.

Anaplasma phagocytophilum PSGL-1-independent infection does not require Syk and leads to less efficient AnkA delivery.

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to cause granulocytic anaplasmosis in humans and mammals. P-selectin glycoprotein ligand-1 (PSGL-1) and the tetrasaccharide sialyl Lewis x (sLe(x)), which caps the PSGL-1 N-terminus, are confirmed A. phagocytophilum receptors. A. phagocytophilum is capable of sLe(x)-modified PSGL-1-dependent and -independent infection. PSGL-1 N-terminus-mediated entry is dependent on spleen tyrosine kinase (Syk). Here, we determined that PSGL-1-independent entry does not alter bacterial replication and investigated whether it involves Syk using NCH-1A2, an enriched subpopulation of A. phagocytophilum NCH-1 obtained through cultivation in a sLe(x)-deficient HL-60 cell line, HL-60 A2. Pharmacological inhibition of Syk nearly abolishes NCH-1 infection, but does not alter NCH-1A2 invasion and only marginally reduces NCH-1A2 propagation. This phenomenon was confirmed by a competitive infection assay using PSGL-1-dependent and -independent A. phagocytophilum organisms transformed to express mCherry or green fluorescent protein respectively. We also assayed for delivery and tyrosine phosphorylation of the A. phagocytophilum effector, AnkA, following NCH-1or NCH-1A2 incubation with HL-60 or HL-60 A2 cells in the presence of PSGL-1 blocking antibody. PSGL-1 N-terminus recognition promotes optimal AnkA delivery while binding to sLe(x) or the unknown receptor is comparably less important for this process.

Anaplasma phagocytophilum infects cells of the megakaryocytic lineage through sialylated ligands but fails to alter platelet production.

Anaplasma phagocytophilum is an obligate intracellular bacterial pathogen that principally inhabits neutrophils. However, infection with A. phagocytophilum results in a moderate to marked thrombocytopenia. In host neutrophils, A. phagocytophilum uses sialylated ligands, primarily P-selectin glycoprotein ligand-1 (PSGL-1), to enter its host cell. PSGL-1 is expressed on a wide array of haematopoietic cells, including megakaryocytes. In this study, it was hypothesized that (i) cells of the megakaryocytic lineage (MEG-01 cells) would be susceptible to A. phagocytophilum infection and (ii) infection may induce alterations in platelet production contributing to infection-induced thrombocytopenia. It was found that MEG-01 cells are susceptible to infection. MEG-01 cells expressing abundant sialylated ligands were the most susceptible to infection, and the absence of sialylation, or blocking of PSGL-1, limited infection susceptibility. However, infected MEG-01 cells produced proplatelets and platelet-like particles comparable to uninfected cells. These results highlight a novel target of pathogen infection and suggest that the pathogen may utilize similar strategies to gain access to megakaryocytes. Direct pathogen modification of platelet production may not play a role in infection-induced thrombocytopenia.

Anaplasma phagocytophilum MSP2(P44)-18 predominates and is modified into multiple isoforms in human myeloid cells.

Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis. MSP2(P44), the bacterium's major surface protein, is encoded by a paralogous gene family and has been implicated in a variety of pathobiological processes, including antigenic variation, host adaptation, adhesion, porin activity, and structural integrity. The consensus among several studies performed at the DNA and RNA levels is that a heterogeneous mix of a limited number of msp2(p44) transcripts is expressed by A. phagocytophilum during in vitro cultivation. Such analyses have yet to be extended to the protein level. In this study, we used proteomic and molecular approaches to determine that MSP2(P44)-18 is the predominant if not the only paralog expressed and is modified into multiple 42- to 44-kDa isoforms by A. phagocytophilum strain HGE1 during infection of HL-60 cells. The msp2(p44) expression profile was homogeneous for msp2(p44)-18. Thus, MSP2(P44)-18 may have a fitness advantage in HL-60 cell culture in the absence of selective immune pressure. Several novel 22- to 27-kDa MSP2 isoforms lacking most of the N-terminal conserved region were also identified. A. phagocytophilum MSP2(P44) orthologs expressed by other pathogens in the family Anaplasmataceae are glycosylated. Gas chromatography revealed that recombinant MSP2(P44)-18 is modified by glucose, galactose, xylose, mannose, and trace amounts of other glycosyl residues. These data are the first to confirm differential modification of any A. phagocytophilum MSP2(P44) paralog and the first to provide evidence for expression of truncated versions of such proteins.

Sialyl-Lewis x-independent infection of human myeloid cells by Anaplasma phagocytophilum strains HZ and HGE1.

Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that infects neutrophils and neutrophil precursors. Bacterial recognition of P-selectin glycoprotein ligand-1 (PSGL-1) and the alpha2,3-sialylated- and alpha1,3-fucosylated-moiety sialyl-Lewis x (sLe(x)), which modifies the PSGL-1 N terminus, is important for adhesion to and invasion of myeloid cells. We have previously demonstrated that A. phagocytophilum organisms of the NCH-1 strain that utilize an sLe(x)-modified PSGL-1-independent means of entry can be enriched for by cultivation in undersialylated HL-60 cells that are unable to construct sLe(x). Because it was unknown whether other A. phagocytophilum isolates share this ability, we extended our studies to the geographically diverse strains HZ and HGE1. HL-60 A2 is a clonal cell line that is defective for sialylation and alpha1,3-fucosyltransferase. HL-60 A2 cell surfaces, therefore, not only lack sLe(x) but also are virtually devoid of any other sialic acid- and/or alpha1,3-fucose-modified glycan. By cultivating HZ and HGE1 in HL-60 A2 cells, we enriched for bacterial subpopulations (termed HZA2 and HGE1A2) that bind and/or infect myeloid cells in the absence of sialic acid and alpha1,3-fucose and in the presence of antibody that blocks the N terminus of PSGL-1. Thus, multiple A. phagocytophilum isolates share the ability to use sLe(x)-modified PSGL-1-dependent and -independent routes of entry into myeloid cells. HZA2 and HGE1A2 represent enriched bacterial populations that will aid dissection of the complexities of the interactions between A. phagocytophilum and host myeloid cells.

Characterization of a sialic acid- and P-selectin glycoprotein ligand-1-independent adhesin activity in the granulocytotropic bacterium Anaplasma phagocytophilum.

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that colonizes neutrophils and neutrophil precursors. The granulocytotropic bacterium uses multiple adhesins that cooperatively bind to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1) and to sialyl Lewis x (sLe(x)) expressed on myeloid cell surfaces. Recognition of sLe(x) occurs through interactions with alpha2,3-sialic acid and alpha1,3-fucose. It is unknown whether other bacteria-host cell interactions are involved. In this study, we have enriched for A. phagocytophilum organisms that do not rely on sialic acid for cellular adhesion and entry by maintaining strain NCH-1 in HL-60 cells that are severely undersialylated. The selected bacteria, termed NCH-1A, also exhibit lessened dependencies on PSGL-1 and alpha1,3-fucose. Optimal adhesion and invasion by NCH-1A require interactions with the known determinants (sialic acid, PSGL-1 and alpha1,3-fucose), but none of them is absolutely necessary. NCH-1A binding to sLe(x)-modified PSGL-1 requires recognition of the known determinants in the same manners as other A. phagocytophilum strains. These data suggest that A. phagocytophilum expresses a separate adhesin from those targeting sialic acid, alpha1,3-fucose and the N-terminal region of PSGL-1. We propose that NCH-1A upregulates expression of this adhesin.