Modified rankin scale is a reliable tool for the rapid assessment of stroke severity and predicting disability outcomes.

Background: 'Time is brain' goes the adage. Rapid and precise management of stroke is of the utmost essence. The modified National Institutes of Health Stroke Scale (mNIHSS) and the modified Rankin Scale (mRS) predict stroke severity and functional disability outcomes. However, the mRS can be administered more rapidly than the mNIHSS and therefore might be better to assess patient outcomes. Hence, the aim of this study was to assess the correlation of stroke severity on admission and functional disability outcomes on the day of discharge or on the 8th day of hospitalization. Materials and Methods: This was an observational, cross-sectional study with a sample size of 61 participants. The mNIHSS score was calculated on admission for patients with clinical features suggestive of stroke and mRS was calculated on the 8th day of hospitalization or on discharge. Evaluation of the association between continuous variables was done using Spearman's correlation analysis. Results: Correlation between mNIHSS and mRS was positive and statistically significant (rho = 0.866, 95% CI [0.751, 0.925]. For each point increase in the mNIHSS, the odds of having higher mRS scores are 153% more than the odds of having lower mRS scores (aOR = 2.534, 95% CI [1.904, 3.560]). Conclusion: Our study concluded that mRS can be reliably used to predict the functional outcomes for patients with stroke in circumstances where the mNIHSS may prove to be lengthy. Thus, where 'time is brain', the mRS can be used with a similar power to predict the outcome.

Development of 1,2,4-Oxadiazole Antimicrobial Agents to Treat Enteric Pathogens within the Gastrointestinal Tract.

Colonization of the gastrointestinal (GI) tract with pathogenic bacteria is an important risk factor for the development of certain potentially severe and life-threatening healthcare-associated infections, yet efforts to develop effective decolonization agents have been largely unsuccessful thus far. Herein, we report modification of the 1,2,4-oxadiazole class of antimicrobial compounds with poorly permeable functional groups in order to target bacterial pathogens within the GI tract. We have identified that the quaternary ammonium functionality of analogue 26a results in complete impermeability in Caco-2 cell monolayers while retaining activity against GI pathogens Clostridioides difficile and multidrug-resistant (MDR) Enterococcus faecium. Low compound recovery levels after oral administration in rats were observed, which suggests that the analogues may be susceptible to degradation or metabolism within the gut, highlighting a key area for optimization in future efforts. This study demonstrates that modified analogues of the 1,2,4-oxadiazole class may be potential leads for further development of colon-targeted antimicrobial agents.

Discovery of Potent and Fast-Acting Antimalarial Bis-1,2,4-triazines.

Novel 3,3'-disubstituted-5,5'-bi(1,2,4-triazine) compounds with potent in vitro activity against Plasmodium falciparum parasites were recently discovered. To improve the pharmacokinetic properties of the triazine derivatives, a new structure-activity relationship (SAR) investigation was initiated with a focus on enhancing the metabolic stability of lead compounds. These efforts led to the identification of second-generation highly potent antimalarial bis-triazines, exemplified by triazine 23, which exhibited significantly improved in vitro metabolic stability (8 and 42 µL/min/mg protein in human and mouse liver microsomes). The disubstituted triazine dimer 23 was also observed to suppress parasitemia in the Peters 4-day test with a mean ED50 value of 1.85 mg/kg/day and exhibited a fast-killing profile, revealing a new class of orally available antimalarial compounds of considerable interest.

Sub-national TB prevalence surveys in India, 2006-2012: Results of uniformly conducted data analysis.

SETTING: Community based tuberculosis (TB) prevalence surveys in ten sites across India during 2006-2012. OBJECTIVE: To re-analyze data of recent sub-national surveys using uniform statistical methods and obtain a pooled national level estimate of prevalence of TB. METHODS: Individuals ≥15 years old were screened by interview for symptoms suggestive of Pulmonary TB (PTB) and history of anti-TB treatment; additional screening by chest radiography was undertaken in five sites. Two sputum specimens were examined by smear and culture among Screen-positives. Prevalence in each site was estimated after imputing missing values to correct for bias introduced by incompleteness of data. In five sites, prevalence was corrected for non-screening by radiography. Pooled prevalence of bacteriologically positive PTB was estimated using Random Effects Model after excluding data from one site. Overall prevalence of TB (all ages, all types) was estimated by adjusting for extra-pulmonary TB and Pediatric TB. RESULTS: Of 769290 individuals registered, 715989 were screened by interview and 294532 also by radiography. Sputum specimen were examined from 50 852 individuals. Estimated prevalence of smear positive, culture positive and bacteriologically positive PTB varied between 108.4-428.1, 147.9-429.8 and 170.8-528.4 per 100000 populations in different sites. Pooled estimate of prevalence of bacteriologically positive PTB was 350.0 (260.7, 439.0). Overall prevalence of TB was estimated at 300.7 (223.7-377.5) in 2009, the mid-year of surveys. Prevalence was significantly higher in rural compared to urban areas. CONCLUSION: TB burden continues to be high in India suggesting further strengthening of TB control activities.

3,3'-Disubstituted 5,5'-Bi(1,2,4-triazine) Derivatives with Potent in Vitro and in Vivo Antimalarial Activity.

A series of 3,3'-disubstituted 5,5'-bi(1,2,4-triazine) derivatives was synthesized and screened against the erythrocytic stage of Plasmodium falciparum 3D7 line. The most potent dimer, 6k, with an IC50 (50% inhibitory concentration) of 0.008 µM, had high in vitro potency against P. falciparum lines resistant to chloroquine (W2, IC50 = 0.0047 ± 0.0011 µM) and artemisinin (MRA1240, IC50 = 0.0086 ± 0.0010 µM). Excellent ex vivo potency of 6k was shown against clinical field isolates of both P. falciparum (IC50 = 0.022-0.034 µM) and Plasmodium vivax (IC50 = 0.0093-0.031 µM) from the blood of outpatients with uncomplicated malaria. Despite 6k being cleared relatively rapidly in mice, it suppressed parasitemia in the Peters 4-day test, with a mean ED50 value (50% effective dose) of 1.47 mg kg-1 day-1 following oral administration. The disubstituted triazine dimer 6k represents a new class of orally available antimalarial compounds of considerable interest for further development.

1,2,4-Oxadiazole antimicrobials act synergistically with daptomycin and display rapid kill kinetics against MDR Enterococcus faecium.

Background: Enterococcus faecium is an important nosocomial pathogen. It has a high propensity for horizontal gene transfer, which has resulted in the emergence of MDR strains that are difficult to treat. The most notorious of these, vancomycin-resistant E. faecium, are usually treated with linezolid or daptomycin. Resistance has, however, been reported, meaning that new therapeutics are urgently needed. The 1,2,4-oxadiazoles are a recently discovered family of antimicrobials that are active against Gram-positive pathogens and therefore have therapeutic potential for treating E. faecium. However, only limited data are available on the activity of these antimicrobials against E. faecium. Objectives: To determine whether the 1,2,4-oxadiazole antimicrobials are active against MDR and daptomycin-non-susceptible E. faecium. Methods: The activity of the 1,2,4-oxadiazole antimicrobials against vancomycin-susceptible, vancomycin-resistant and daptomycin-non-susceptible E. faecium was determined using susceptibility testing, time-kill assays and synergy assays. Toxicity was also evaluated against human cells by XTT and haemolysis assays. Results: The 1,2,4-oxadiazoles are active against a range of MDR E. faecium, including isolates that display non-susceptibility to vancomycin and daptomycin. This class of antimicrobial displays rapid bactericidal activity and demonstrates superior killing of E. faecium compared with daptomycin. Finally, the 1,2,4-oxadiazoles act synergistically with daptomycin against E. faecium, with subinhibitory concentrations reducing the MIC of daptomycin for non-susceptible isolates to a level below the clinical breakpoint. Conclusions: The 1,2,4-oxadiazoles are active against MDR and daptomycin-non-susceptible E. faecium and hold great promise as future therapeutics for treating infections caused by these difficult-to-treat isolates.

8-Mercaptoguanine Derivatives as Inhibitors of Dihydropteroate Synthase.

Dihydropteroate synthase (DHPS) is an enzyme of the folate biosynthesis pathway, which catalyzes the formation of 7,8-dihydropteroate (DHPt) from 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPPP) and para-aminobenzoic acid (pABA). DHPS is the long-standing target of the sulfonamide class of antibiotics that compete with pABA. In the wake of sulfa drug resistance, targeting the structurally rigid (and more conserved) pterin site has been proposed as an alternate strategy to inhibit DHPS in wild-type and sulfa drug resistant strains. Following the work on developing pterin-site inhibitors of the adjacent enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), we now present derivatives of 8-mercaptoguanine, a fragment that binds weakly within both enzymes, and quantify sub-µm binding using surface plasmon resonance (SPR) to Escherichia coli DHPS (EcDHPS). Eleven ligand-bound EcDHPS crystal structures delineate the structure-activity relationship observed providing a structural framework for the rational development of novel, substrate-envelope-compliant DHPS inhibitors.

Acute and subacute microRNA dysregulation is associated with cytokine responses in the rodent model of penetrating ballistic-like brain injury.

BACKGROUND: MicroRNAs (miRNAs) are small stable RNAs that regulate translational degradation or repression of genes involved in brain trauma-mediated inflammation. More recently, miRNAs have emerged as potential novel TBI biomarkers. The aim of this study was to determine if a select set of miRNAs (miR-21, Let-7i, miR-124a, miR-146a, miR-107) that were previously associated with TBI models and clinical studies would be dysregulated and correlated to inflammatory cytokine abundance in the rat penetrating ballistic-like brain injury (PBBI) model. METHODS: Adult male Sprague-Dawley rats received a unilateral frontal 10% PBBI, which produces a temporary cavity. Sham animals received a craniotomy only. Ipsilateral brain tissue and serum were collected 4 hours to 7 days post-injury. Quantitation of miR-21, Let-7i, miR-124a, miR-146a, or miR-107 levels was conducted using Taqman PCR assays normalized to the endogenous reference, U6 snRNA. Brain tissue derived from matching cohorts was used to determine 1L-1beta and IL-6 levels by enzyme-linked immunosorbent assay. RESULTS: Brain tissue Let-7i and miR-21 increased at 4 hours and 1 day, whereas miR-124a and miR-107 were enhanced only 1 day post-injury. MiR-146a displayed a biphasic response and increased 1 day and 7 days, whereas elevation of miR-21 was sustained 1 day to 7 days after PBBI. Pathway analysis indicated that miRNAs were linked to inflammatory proteins, IL-6 and IL-1beta. Confirmation by enzyme-linked immunosorbent assay indicated that both cytokines were increased and peaked at 1 day, but fell at 3 days through 7 days after PBBI, indicating an inverse relationship with miRNA abundance. Serum Let-7i, alone, was differentially abundant 7 days after PBBI. CONCLUSION: Brain tissue-derived miRNAs linked to increased cytokine levels demonstrates a plausible therapeutic target of TBI-induced inflammation. Suppression of serum derived Let-7i may have utility as a biomarker of subacute injury progression or therapeutic responses.

A Simplified Workflow for Protein Quantitation of Rat Brain Tissues Using Label-Free Proteomics and Spectral Counting.

Mass spectrometry-based proteomics is an increasingly valuable tool for determining relative or quantitative protein abundance in brain tissues. A plethora of technical and analytical methods are available, but straightforward and practical approaches are often needed to facilitate reproducibility. This aspect is particularly important as an increasing number of studies focus on models of traumatic brain injury or brain trauma, for which brain tissue proteomes have not yet been fully described. This text provides suggested techniques for robust identification and quantitation of brain proteins by using molecular weight fractionation prior to mass spectrometry-based proteomics. Detailed sample preparation and generalized protocols for chromatography, mass spectrometry, spectral counting, and normalization are described. The rat cerebral cortex isolated from a model of blast-overpressure was used as an exemplary source of brain tissue. However, these techniques may be adapted for lysates generated from several types of cells or tissues and adapted by the end user.

Design, Synthesis, and Characterization of Cyclic Peptidomimetics of the Inducible Nitric Oxide Synthase Binding Epitope That Disrupt the Protein-Protein Interaction Involving SPRY Domain-Containing Suppressor of Cytokine Signaling Box Protein (SPSB) 2 and Inducible Nitric Oxide Synthase.

SPRY domain-containing suppressor of cytokine signaling box protein (SPSB) 2-deficient macrophages have been found to exhibit prolonged expression of inducible nitric oxide synthase (iNOS) and enhanced killing of persistent pathogens, suggesting that inhibitors of the SPSB2-iNOS interaction have potential as novel anti-infectives. In this study, we describe the design, synthesis, and characterization of cyclic peptidomimetic inhibitors of the SPSB2-iNOS interaction constrained by organic linkers to improve stability and druggability. SPR, ITC, and (19)F NMR analyses revealed that the most potent cyclic peptidomimetic bound to the iNOS binding site of SPSB2 with low nanomolar affinity (KD 29 nM), a 10-fold improvement over that of the linear peptide DINNN (KD 318 nM), and showed strong inhibition of SPSB2-iNOS interaction in macrophage cell lysates. This study exemplifies a novel approach to cyclize a Type II ß-turn linear peptide and provides a foundation for future development of this group of inhibitors as new anti-infectives.

Inactivation of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 on cantaloupes by octenidine dihydrochloride.

The efficacy of a new generation disinfectant, octenidine dihydrochloride (OH), as wash and coating treatments for reducing Listeria monocytogenes (LM), Salmonella spp. (SAL), and Escherichia coli O157:H7 (EC) on cantaloupe was investigated. Cantaloupe rind plugs inoculated separately with the three bacterial species (∼8 log CFU/cm(2)) were washed for 1, 3, 5 min at 25 °C in water, or chlorine (200 ppm), ethanol (1%), OH (0.01, 0.05, 0.1%) and surviving populations were measured after treatment. Additionally, inoculated cantaloupe rind plugs were coated with 2% chitosan or chitosan containing OH (0.01, 0.05, 0.1%) and sampled for surviving pathogens. Subsequently, the antimicrobial efficacy of OH wash and coating (0.1, 0.2%) on whole cantaloupes was determined. All OH wash reduced LM, SAL, and EC on cantaloupe rinds by > 5 log CFU/cm(2) by 2 min, and reduced populations to undetectable levels (below 2 log CFU/cm(2)) by 5 min (P < 0.05). Similarly, OH coating on cantaloupe rinds reduced the pathogens by 3-5 log /cm(2) (P < 0.05). Washing and coating whole cantaloupes with OH reduced the three pathogens by at least 5 log and 2 log CFU/cm(2), respectively (P < 0.05). Results suggest that OH could be used as antimicrobial wash and coating to reduce LM, SAL, and EC on cantaloupes.

Structural Basis for the Selective Binding of Inhibitors to 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase from Staphylococcus aureus and Escherichia coli.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a member of the folate biosynthesis pathway found in prokaryotes and lower eukaryotes that catalyzes the pyrophosphoryl transfer from the ATP cofactor to a 6-hydroxymethyl-7,8-dihydropterin substrate. We report the chemical synthesis of a series of S-functionalized 8-mercaptoguanine (8MG) analogues as substrate site inhibitors of HPPK and quantify binding against the E. coli and S. aureus enzymes (EcHPPK and SaHPPK). The results demonstrate that analogues incorporating acetophenone-based substituents have comparable affinities for both enzymes. Preferential binding of benzyl-substituted 8MG derivatives to SaHPPK was reconciled when a cryptic pocket unique to SaHPPK was revealed by X-ray crystallography. Differential chemical shift perturbation analysis confirmed this to be a common mode of binding for this series to SaHPPK. One compound (41) displayed binding affinities of 120 nM and 1.76 µM for SaHPPK and EcHPPK, respectively, and represents a lead for the development of more potent and selective inhibitors of SaHPPK.

Redox-stable cyclic peptide inhibitors of the SPSB2-iNOS interaction.

SPSB2 mediates the proteasomal degradation of iNOS. Inhibitors of SPSB2-iNOS interaction are expected to prolong iNOS lifetime and thereby enhance killing of persistent pathogens. Here, we describe the synthesis and characterization of two redox-stable cyclized peptides containing the DINNN motif required for SPSB2 binding. Both analogues bind with low nanomolar affinity to the iNOS binding site on SPSB, as determined by SPR and (19)F NMR, and efficiently displace full-length iNOS from binding to SPSB2 in macrophage cell lysates. These peptides provide a foundation for future development of redox-stable, potent ligands for SPSB proteins as a potential novel class of anti-infectives.

Serum Glial Fibrillary Acidic Protein Predicts Tissue Glial Fibrillary Acidic Protein Break-Down Products and Therapeutic Efficacy after Penetrating Ballistic-Like Brain Injury.

Acute traumatic brain injury (TBI) is associated with neurological dysfunction, changes in brain proteins, and increased serum biomarkers. However, the relationship between these brain proteins and serum biomarkers, and the ability of these serum biomarkers to indicate a neuroprotective/therapeutic response, remains elusive. Penetrating ballistic-like brain injury (PBBI) was used to systematically analyze several key TBI biomarkers, glial fibrillary acidic protein (GFAP) and its break-down products (BDPs)-ubiquitin C-terminal hydrolase-L1 (UCH-L1), α-II spectrin, and α-II spectrin BDPs (SBDPs)-in brain tissues and serum during an extended acute-subacute time-frame. In addition, neurological improvement and serum GFAP theranostic value was evaluated after neuroprotective treatment. In brain tissues, total GFAP increased more than three-fold 2 to 7 d after PBBI. However, this change was primarily due to GFAP-BDPs which increased to 2.7-4.8 arbitrary units (AU). Alpha-II spectrin was nearly ablated 3 d after PBBI, but somewhat recovered after 7 d. In conjunction with α-II spectrin loss, SBDP-145/150 increased approximately three-fold 2 to 7 d after PBBI (vs. sham, p<0.05). UCH-L1 protein levels were slightly decreased 7 d after PBBI but otherwise were unaffected. Serum GFAP was elevated by 3.2- to 8.8-fold at 2 to 4 h (vs. sham; p<0.05) and the 4 h increase was strongly correlated to 3 d GFAP-BDP abundance (r=0.66; p<0.05). Serum GFAP showed such a strong injury effect that it also was evaluated after therapeutic intervention with cyclosporin A (CsA). Administration of 2.5 mg/kg CsA significantly reduced serum GFAP elevation by 22.4-fold 2 h after PBBI (vs. PBBI+vehicle; p<0.05) and improved neurological function 1 d post-injury. Serum biomarkers, particularly GFAP, may be correlative tools of brain protein changes and feasible theranostic markers of TBI progression and recovery.

Physical covering for control of Escherichia coli O157:H7 and Salmonella spp. in static and windrow composting processes.

This study investigated the effect of a 30-cm covering of finished compost (FC) on survival of Escherichia coli O157:H7 and Salmonella spp. in active static and windrow composting systems. Feedstocks inoculated with E. coli O157:H7 (7.41 log CFU/g) and Salmonella (6.46 log CFU/g) were placed in biosentry tubes (7.5-cm diameter, 30-cm height) at three locations: (i and ii) two opposing sides at the interface between the FC cover layer (where present) and the feedstock material (each positioned approximately 10 cm below the pile's surface) and (iii) an internal location (top) (approximately 30 cm below the surface). On specific sampling days, surviving populations of inoculated E. coli O157:H7 and Salmonella, generic E. coli, and coliforms in compost samples were determined. Salmonella spp. were reduced significantly within 24 h in windrow piles and were below the detection limit after 3 and 7 days at internal locations of windrow and static piles containing FC covering, respectively. Likewise, E. coli O157:H7 was undetectable after 1 day in windrow piles covered with finished compost. Use of FC as a covering layer significantly increased the number of days that temperatures in the windrows remained ≥55°C at all locations and in static piles at internal locations. These time-temperature exposures resulted in rapid reduction of inoculated pathogens, and the rate of bacterial reduction was rapid in windrow piles. The sample location significantly influenced the survival of these pathogens at internal locations compared to that at interface locations of piles. Finished compost covering of compost piles aids in the reduction of pathogens during the composting process.

19F NMR as a probe of ligand interactions with the iNOS binding site of SPRY domain-containing SOCS box protein 2.

SPRY domain-containing SOCS box protein 2 (SPSB2) regulates inducible nitric oxide synthase (iNOS) by targeting it for proteasomal degradation. Inhibiting this interaction prolongs the intracellular lifetime of iNOS, leading in turn to enhanced killing of infectious pathogens such as bacteria and parasites. SPSB2 recognizes a linear motif (DINNN) in the disordered N-terminus of iNOS, and ligands that target the DINNN binding site on SPSB2 are potentially novel anti-infective agents. We have explored (19)F NMR as a means of probing ligand binding to SPSB2. All six Trp residues in SPSB2 were replaced with 5-fluorotryptophan (5-F-Trp) by utilizing a Trp auxotroph strain of Escherichia coli. The labeled protein was well folded and bound a DINNN-containing peptide with similar affinity to native SPSB2. Six well-resolved 5-F-Trp resonances were observed in the (19)F NMR spectrum and were assigned using site-directed mutagenesis. The (19)F resonance of W207 was significantly perturbed upon binding to DINNN-containing peptides. Other resonances were perturbed to a lesser extent although in a way that was sensitive to the composition of the peptide. Analogues of compounds identified in a fragment screen also perturbed the W207 resonance, confirming their binding to the iNOS peptide-binding site on SPSB2. (19)F NMR promises to be a valuable approach in developing inhibitors that bind to the DINNN binding site.

Temporal alterations in aquaporin and transcription factor HIF1α expression following penetrating ballistic-like brain injury (PBBI).

OBJECTIVES: Brain edema is a primary factor in the morbidity and mortality of traumatic brain injury (TBI). The various isoforms of aquaporin 4 (AQP4) and aquaporin 9 (AQP9) are important factors influencing edema following TBI. Others have reported that these AQPs are regulated by the transcription factor hypoxia inducible factor (HIF) 1α. Therefore, we examined the temporal alterations in the multiple isoforms of AQP4 and AQP9, and its possible upstream regulation by HIF1α, and evaluated whether different severities of penetrating injury influence these mechanisms. METHODS: In the penetrating ballistic-like brain injury (PBBI) model, a temporary cavity and resultant injury was formed by the rapid inflation/deflation (i.e. <40ms) of an elastic balloon attached to the end of the custom probe, injuring 10% of total rat brain volume. Tissue from the ipsilateral core and perilesional injury zones was collected. Total RNA was isolated at 4, 12, and 24h, 3 and 7days post-injury (sham and PBBI, n=6 per group). cDNA was synthesized using oligodT primers. Quantitative real time PCR was performed using Taqman expression assays for aqp4 (recognizing all isoforms), aqp9, and hif1α. Using separate animals, tissue lysate was collected at 4 and 24h, 3 and 7days post-injury and analyzed by immunoblot for protein expression of multiple isoforms of AQP4, the single known isoform of AQP9 and for expression of transcription factor HIF1α (sham, probe only control, and PBBI, n=8-10 per group). RESULTS: Global aqp4 mRNA was decreased at 24h (p<0.01) with PBBI. Three of the four known protein isoforms of AQP4 were detected, M1 (34kDa), M23 (32kDa) and isoform 3 (30kDa). AQP4 M1 decreased at 3 and 7days post-injury (p<0.001; p<0.01). AQP4 M23 levels were highly variable with no significant changes. AQP4 isoform 3 levels were decreased 3days post-PBBI (p<0.05). From 4, 12, and 24h aqp9 mRNA levels were decreased with injury (p<0.01, p<0.05, p<0.01) while AQP9 levels were decreased at 3 and 7days after PBBI (p<0.001, p<0.01). At 12 and 24h post-PBBI hif1α mRNA levels increased (p<0.05, p<0.01) but at 3 and 7days mRNA levels decreased (p<0.05, p<0.01). From 24h and 3 and 7days HIF1α protein levels were decreased (p<0.0001, p<0.0001, p<0.0001). In comparison to probe control, PBBI led to greater decreases in protein for AQP4 M1 (trend), AQP4 isoform 3 (trend), AQP9 (p<0.05) and HIF1α (p<0.05). CONCLUSION: PBBI is characterized by a loss of AQP4 M1, AQP4 isoform 3 and AQP9 at delayed time-points. The severity of the injury (PBBI versus probe control) increased these effects. Therefore, AQP9 and the AQP4 M1 isoform may be regulated by HIF1α, but not AQP4 isoform 3. This delayed loss of aquaporins may markedly reduce the ability of the brain to efflux water, contributing to the protracted edema that is a characteristic following severe penetrating TBI. Factors contributing to edema differ with different types and severities of TBI. For example, cellular based edema is more prominent in diffuse non-penetrating TBI whereas vasogenic edema is more prevalent with TBI involving hemorrhage. Molecular regulation leading to edema will likely also differ, such that treatments which have been suggested for non-hemorrhagic moderate TBI, such as the suppression of aquaporins, may be detrimental in more severe forms of TBI.

Human traumatic brain injury induces autoantibody response against glial fibrillary acidic protein and its breakdown products.

The role of systemic autoimmunity in human traumatic brain injury (TBI) and other forms of brain injuries is recognized but not well understood. In this study, a systematic investigation was performed to identify serum autoantibody responses to brain-specific proteins after TBI in humans. TBI autoantibodies showed predominant immunoreactivity against a cluster of bands from 38-50 kDa on human brain immunoblots, which were identified as GFAP and GFAP breakdown products. GFAP autoantibody levels increased by 7 days after injury, and were of the IgG subtype predominantly. Results from in vitro tests and rat TBI experiments also indicated that calpain was responsible for removing the amino and carboxyl termini of GFAP to yield a 38 kDa fragment. Additionally, TBI autoantibody staining co-localized with GFAP in injured rat brain and in primary rat astrocytes. These results suggest that GFAP breakdown products persist within degenerating astrocytes in the brain. Anti-GFAP autoantibody also can enter living astroglia cells in culture and its presence appears to compromise glial cell health. TBI patients showed an average 3.77 fold increase in anti-GFAP autoantibody levels from early (0-1 days) to late (7-10 days) times post injury. Changes in autoantibody levels were negatively correlated with outcome as measured by GOS-E score at 6 months, suggesting that TBI patients with greater anti-GFAP immune-responses had worse outcomes. Due to the long lasting nature of IgG, a test to detect anti-GFAP autoantibodies is likely to prolong the temporal window for assessment of brain damage in human patients.

Correlations between blood-brain barrier disruption and neuroinflammation in an experimental model of penetrating ballistic-like brain injury.

Abstract Blood-brain barrier (BBB) disruption is a pathological hallmark of severe traumatic brain injury (TBI) and is associated with neuroinflammatory events contributing to brain edema and cell death. The goal of this study was to elucidate the profile of BBB disruption after penetrating ballistic-like brain injury (PBBI) in conjunction with changes in neuroinflammatory markers. Brain uptake of biotin-dextran amine (BDA; 3 kDa) and horseradish peroxidase (HRP; 44 kDa) was evaluated in rats at 4 h, 24 h, 48 h, 72 h, and 7 days post-PBBI and compared with the histopathologic and molecular profiles for inflammatory markers. BDA and HRP both displayed a uniphasic profile of extravasation, greatest at 24 h post-injury and which remained evident out to 48 h for HRP and 7 days for BDA. This profile was most closely associated with markers for adhesion (mRNA for intercellular adhesion molecule-1) and infiltration of peripheral granulocytes (mRNA for matrix metalloproteinase-9 [MMP-9] and myeloperoxidase staining). Improvement of BBB dysfunction coincided with increased expression of markers implicated in tissue remodeling and repair. The results of this study reveal a uniphasic and gradient opening of the BBB after PBBI and suggest MMP-9 and resident inflammatory cell activation as candidates for future neurotherapeutic intervention after PBBI.

Mechanism of action for NNZ-2566 anti-inflammatory effects following PBBI involves upregulation of immunomodulator ATF3.

The tripeptide glycine-proline-glutamate analogue NNZ-2566 (Neuren Pharmaceuticals) demonstrates neuroprotective efficacy in models of traumatic brain injury. In penetrating ballistic-like brain injury (PBBI), it significantly decreases injury-induced upregulation of inflammatory cytokines including TNF-α, IFN-γ, and IL-6. However, the mechanism by which NNZ-2566 acts has yet to be determined. The activating transcription factor-3 (ATF3) is known to repress expression of these inflammatory cytokines and was increased at the mRNA and protein level 24-h post-PBBI. This study investigated whether 12 h of NNZ-2566 treatment following PBBI alters atf3 expression. PBBI alone significantly increased atf3 mRNA levels by 13-fold at 12 h and these levels were increased by an additional fourfold with NNZ-2566 treatment. To confirm that changes in mRNA translated to changes in protein expression, ATF3 expression levels were determined in vivo in microglia/macrophages, T cells, natural killer cells (NKCs), astrocytes, and neurons. PBBI alone significantly increased ATF3 in microglia/macrophages (820%), NKCs (58%), and astrocytes (51%), but decreased levels in T cells (48%). NNZ-2566 treatment further increased ATF3 protein expression in microglia/macrophages (102%), NKCs (308%), and astrocytes (13%), while reversing ATF3 decreases in T cells. Finally, PBBI increased ATF3 levels by 55% in neurons and NNZ-2566 treatment further increased these levels an additional 33%. Since increased ATF3 may be an innate protective mechanism to limit inflammation following injury, these results demonstrating that the anti-inflammatory and neuroprotective drug NNZ-2566 increase both mRNA and protein levels of ATF3 in multiple cell types provide a cellular mechanism for NNZ-2566 modulation of neuroinflammation following PBBI.