In-depth analysis of clotting dynamics in burn patients.

BACKGROUND: Studies associating coagulopathic changes with burn injury have relied on limited tests such as partial thromboplastin time (PTT) and international normalized ratio (INR). Understanding the clotting dynamics and associated risk factors after burn injury could influence management. This work aimed to identify real-time changes in coagulation after burn injury not indicated by PTT or INR alone. MATERIALS AND METHODS: Nine burn-injured patients at a regional burn center were enrolled for blood collection at admission and set intervals over 96 h. Patient demographics, management, and laboratory data (PTT and INR) were collected. Plasma assays determined factors II, V, VII, VIII, IX, X, XI, antithrombin, and protein C functional activity as well as PAP, D-Dimer, fibrin monomer, TFPI, IL-1b, IL-6, IL-10, IL-12p.70, and TNF-α concentrations. RESULTS: Overall, five patients died. These patients had higher mortality scores and were more acidotic. All patients had normal coagulation studies (INR < 1.5, PTT < 45 s) within 24 h of admission, and only two were abnormal after. Increased factor VIII and IX activity were identified in seven patients at admission. Decreased antithrombin and protein C activity were seen in all patients. Patients had increased PAP, D-Dimer, and fibrin monomer concentrations throughout their hospital course. At admission, increased fold changes were seen in IL-6 (2.5-117) and IL-10 (2.4-32), whereas IL-1b and TNF-α levels were depressed in all patients. CONCLUSIONS: Extensive changes not identified by PTT or INR were seen after burn injury that may explain perturbed coagulation in these patients. This approach further characterizes the impact thermal injury has on coagulation.

The evaluation of a PCR-based method for identification of Salmonella enterica serotypes from environmental samples and various food matrices.

The most commonly used method for serotyping Salmonella spp. is based on the Kaufmann-White scheme, and is composed of serological reactions using antibodies to LPS agglutinins. The multiplex PCR used in this investigation was established by Kim et al. to serotype the 30 most common clinical Salmonella serotypes, as determined by CDC. The PCR assay consists of two five-plex reactions and a single two-plex PCR reaction, based on six genetic loci from Salmonella enterica serotype Typhimurium and four loci from S. enterica serotype Typhi. In this investigation, we further evaluated the method for serotyping Salmonella spp. using a reference collection, environmental samples collected from a Mid-Atlantic region tomato farm study, four food matrices spiked with different Salmonella serotypes and a proficiency test. The PCR assay was first evaluated using DNA isolated from pure cultures of isolates obtained from various clinical and environmental samples, and then DNA isolated from broth cultures of food matrices of "Red round" and Roma tomatoes, Romaine lettuce, green onions and Serrano peppers spiked with serotypes Newport, Typhimurium, Javiana and Saintpaul, respectively. The results showed that the PCR assay correctly serotyped Salmonella spp. from the clinical, environmental, spiked food matrices, and proficiency test samples. These findings are significant because the PCR assay was successful in the identification of Salmonella in the spiked samples in a broth culture containing other non-salmonella organism. This method may be a useful resource for the food safety community.

Filifactor alocis--a new emerging periodontal pathogen.

Filifactor alocis, a previously unrecognized Gram-positive anaerobic rod, is now considered a new emerging pathogen that may play a significant role in periodontal disease. F. alocis' unique characteristics and variations at the molecular level that may be responsible for the functional changes required to mediate the pathogenic process are discussed.

Induction of indoleamine 2, 3-dioxygenase in human dendritic cells by a cholera toxin B subunit-proinsulin vaccine.

Dendritic cells (DC) interact with naïve T cells to regulate the delicate balance between immunity and tolerance required to maintain immunological homeostasis. In this study, immature human dendritic cells (iDC) were inoculated with a chimeric fusion protein vaccine containing the pancreatic ß-cell auto-antigen proinsulin linked to a mucosal adjuvant the cholera toxin B subunit (CTB-INS). Proteomic analysis of vaccine inoculated DCs revealed strong up-regulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1). Increased biosynthesis of the immunosuppressive enzyme was detected in DCs inoculated with the CTB-INS fusion protein but not in DCs inoculated with proinsulin, CTB, or an unlinked combination of the two proteins. Immunoblot and PCR analyses of vaccine treated DCs detected IDO1mRNA by 3 hours and IDO1 protein synthesis by 6 hours after vaccine inoculation. Determination of IDO1 activity in vaccinated DCs by measurement of tryptophan degradation products (kynurenines) showed increased tryptophan cleavage into N-formyl kynurenine. Vaccination did not interfere with monocytes differentiation into DC, suggesting the vaccine can function safely in the human immune system. Treatment of vaccinated DCs with pharmacological NF-κB inhibitors ACHP or DHMEQ significantly inhibited IDO1 biosynthesis, suggesting a role for NF-κB signaling in vaccine up-regulation of dendritic cell IDO1. Heat map analysis of the proteomic data revealed an overall down-regulation of vaccinated DC functions, suggesting vaccine suppression of DC maturation. Together, our experimental data indicate that CTB-INS vaccine induction of IDO1 biosynthesis in human DCs may result in the inhibition of DC maturation generating a durable state of immunological tolerance. Understanding how CTB-INS modulates IDO1 activity in human DCs will facilitate vaccine efficacy and safety, moving this immunosuppressive strategy closer to clinical applications for prevention of type 1 diabetes autoimmunity.

In vitro extinction learning in Hermissenda: involvement of conditioned inhibition molecules.

Extinction of a conditioned association is typically viewed as the establishment of new learning rather than the erasure of the original memory. However, recent research in the nudibranch, Hermissenda crassicornis (H.c.) demonstrated that extinction training (using repeated light-alone presentations) given 15 min, but not 23 h, after memory acquisition reversed both the cellular correlates of learning (enhanced Type B cell excitability) and the behavioral changes (reduced phototaxis) produced by associative conditioning (pairings of light, CS, and rotation, US). Here, we investigated the putative molecular signaling pathways that underlie this extinction in H.c. by using a novel in vitro protocol combined with pharmacological manipulations. After intact H.c. received either light-rotation pairings (Paired), random presentations of light and rotation (Random), or no stimulation (Untrained), B cells from isolated CNSs were recorded from during exposure to extinction training consisting of two series of 15 consecutive light-steps (LSs). When in vitro extinction was administered shortly (2 h, but not 24 h) after paired training, B cells from Paired animals showed progressive and robust declines in spike frequency by the 30th LS, while control cells (Random and Untrained) did not. We found that several molecules implicated in H.c. conditioned inhibitory (CI) learning, protein phosphatase 1 (PP1) and arachidonic acid (AA)/12-lipoxygenase (12-LOX) metabolites, also contributed to the spike frequency decreases produced by in vitro extinction. Protein phosphatase 2B (PP2B) also appeared to play a role. Calyculin A (PP1 inhibitor), cyclosporin A (PP2B inhibitor), and baicalein (a 12-LOX inhibitor) all blocked the spike frequency declines in Paired B cells produced by 30 LSs. Conversely, injection of catalytically-active PP1 (caPP1) or PP2B (caPP2B) into Untrained B cells partially mimicked the spike frequency declines observed in Paired cells, as did bath-applied AA, and occluded additional LS-produced reductions in spiking in Paired cells.

Behavioral and neural bases of extinction learning in Hermissenda.

Extinction of classical conditioning is thought to produce new learning that masks or interferes with the original memory. However, research in the nudibranch Hermissenda crassicornis (H.c.) has challenged this view, and instead suggested that extinction erased the original associative memory. We have re-examined extinction in H.c. to test whether extinguished associative memories can be detected on the behavioral and cellular levels, and to characterize the temporal variables involved. Associative conditioning using pairings of light (CS) and rotation (US) produced characteristic suppression of H.c. phototactic behavior. A single session of extinction training (repeated light-alone presentations) reversed suppressed behavior back to pre-training levels when administered 15 min after associative conditioning. This effect was abolished if extinction was delayed by 23 h, and yet was recovered using extended extinction training (three consecutive daily extinction sessions). Extinguished phototactic suppression did not spontaneously recover at any retention interval (RI) tested (2-, 24-, 48-, 72-h), or after additional US presentations (no observed reinstatement). Extinction training (single session, 15 min interval) also reversed the pairing-produced increases in light-evoked spike frequencies of Type B photoreceptors, an identified site of associative memory storage that is causally related to phototactic suppression. These results suggest that the behavioral effects of extinction training are not due to temporary suppression of associative memories, but instead represent a reversal of the underlying cellular changes necessary for the expression of learning. In the companion article, we further elucidate mechanisms responsible for extinction-produced reversal of memory-related neural plasticity in Type B photoreceptors.