Characteristics and Predictors of Intensive Care Unit Admission in Pediatric Blunt Abdominal Trauma

BACKGROUND: Pediatric trauma patients sustaining blunt abdominal trauma (BAT) with intra-abdominal injury (IAI) are frequently admitted to the intensive care unit (ICU). This study was performed to identify predictors for ICU admission following BAT. METHODS: Prospective study of children (\u3c 16 years) who presented to 14 Level-One Pediatric Trauma Centers following BAT over a 1-year period. Patients were categorized as ICU or non-ICU patients. Data collected included vitals, physical exam findings, laboratory results, imaging, and traumatic injuries. A multivariable hierarchical logistic regression model was used to identify predictors of ICU admission. Predictive ability of the model was assessed via tenfold cross-validated area under the receiver operating characteristic curves (cvAUC). RESULTS: Included were 2,182 children with 21% (n = 463) admitted to the ICU. On univariate analysis, ICU patients were associated with abnormal age-adjusted shock index, increased injury severity scores (ISS), lower Glasgow coma scores (GCS), traumatic brain injury (TBI), and severe solid organ injury (SOI). With multivariable logistic regression, factors associated with ICU admission were severe trauma (ISS \u3e 15), anemia (hematocrit \u3c 30), severe TBI (GCS \u3c 8), cervical spine injury, skull fracture, and severe solid organ injury. The cvAUC for the multivariable model was 0.91 (95% CI 0.88-0.92). CONCLUSION: Severe solid organ injury and traumatic brain injury, in association with multisystem trauma, appear to drive ICU admission in pediatric patients with BAT. These results may inform the design of a trauma bay prediction rule to assist in optimizing ICU resource utilization after BAT. STUDY DESIGN: Prognosis study

Comparative genomic analysis of toxin-negative strains of Clostridium difficile from humans and animals with symptoms of gastrointestinal disease

Background: Clostridium difficile infections (CDI) are a significant health problem to humans and food animals. Clostridial toxins ToxA and ToxB encoded by genes tcdA and tcdB are located on a pathogenicity locus known as the PaLoc and are the major virulence factors of C. difficile. While toxin-negative strains of C. difficile are often isolated from faeces of animals and patients suffering from CDI, they are not considered to play a role in disease. Toxin-negative strains of C. difficile have been used successfully to treat recurring CDI but their propensity to acquire the PaLoc via lateral gene transfer and express clinically relevant levels of toxins has reinforced the need to characterise them genetically. In addition, further studies that examine the pathogenic potential of toxin-negative strains of C. difficile and the frequency by which toxin-negative strains may acquire the PaLoc are needed. Results: We undertook a comparative genomic analysis of five Australian toxin-negative isolates of C. difficile that lack tcdA, tcdB and both binary toxin genes cdtA and cdtB that were recovered from humans and farm animals with symptoms of gastrointestinal disease. Our analyses show that the five C. difficile isolates cluster closely with virulent toxigenic strains of C. difficile belonging to the same sequence type (ST) and have virulence gene profiles akin to those in toxigenic strains. Furthermore, phage acquisition appears to have played a key role in the evolution of C. difficile. Conclusions: Our results are consistent with the C. difficile global population structure comprising six clades each containing both toxin-positive and toxin-negative strains. Our data also suggests that toxin-negative strains of C. difficile encode a repertoire of putative virulence factors that are similar to those found in toxigenic strains of C. difficile, raising the possibility that acquisition of PaLoc by toxin-negative strains poses a threat to human health. Studies in appropriate animal models are needed to examine the pathogenic potential of toxin-negative strains of C. difficile and to determine the frequency by which toxin-negative strains may acquire the PaLoc

The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation

Ordered Processing of the Human Immunodeficiency Virus Type 1 GagPol Precursor Is Influenced by the Context of the Embedded Viral Protease

Ordered and accurate processing of the human immunodeficiency virus type 1 (HIV-1) GagPol polyprotein precursor by a virally encoded protease is an indispensable step in the appropriate assembly of infectious viral particles. The HIV-1 protease (PR) is a 99-amino-acid enzyme that is translated as part of the GagPol precursor. Previously, we have demonstrated that the initial events in precursor processing are accomplished by the PR domain within GagPol in cis, before it is released from the polyprotein. Despite the critical role that ordered processing of the precursor plays in viral replication, the forces that define the order of cleavage remain poorly understood. Using an in vitro assay in which the full-length HIV-1 GagPol is processed by the embedded PR, we examined the effect of PR context (embedded within GagPol versus the mature 99-amino-acid enzyme) on precursor processing. Our data demonstrate that the PR domain within GagPol is constrained in its ability to cleave some of the processing sites in the precursor. Further, we find that this constraint is dependent upon the presence of a proline as the initial amino acid in the embedded PR; substitution of an alanine at this position produces enhanced cleavage at additional sites when the precursor is processed by the embedded, but not the mature, PR. Overall, our data support a model in which the selection of processing sites and the order of precursor processing are defined, at least in part, by the structure of GagPol itself

Initial Cleavage of the Human Immunodeficiency Virus Type 1 GagPol Precursor by Its Activated Protease Occurs by an Intramolecular Mechanism

Processing of the GagPol polyprotein precursor of human immunodeficiency virus type 1 (HIV-1) is a critical step in viral assembly and replication. The HIV-1 protease (PR) is translated as part of GagPol and is both necessary and sufficient for precursor processing. The PR is active only as a dimer; enzyme activation is initiated when the PR domains in two GagPol precursors dimerize. The precise mechanism by which the PR becomes activated and the subsequent initial steps in precursor processing are not well understood. However, it is clear that processing is initiated by the PR domain that is embedded within the precursor itself. We have examined the earliest events in precursor processing using an in vitro assay in which full-length GagPol is cleaved by its embedded PR. We demonstrate that the embedded, immature PR is as much as 10,000-fold less sensitive to inhibition by an active-site PR inhibitor than is the mature, free enzyme. Further, we find that different concentrations of the active-site inhibitor are required to inhibit the processing of different cleavage sites within GagPol. Finally, our results indicate that the first cleavages carried out by the activated PR within GagPol are intramolecular. Overall, our data support a model of virus assembly in which the first cleavages occur in GagPol upstream of the PR. These intramolecular cleavages produce an extended form of PR that completes the final processing steps accompanying the final stages of particle assembly by an intermolecular mechanism

Auristatin PYE, a novel synthetic derivative of dolastatin 10, is highly effective in human colon tumour models.

NoDespite promising early data, the natural product dolastatin 10 has not been successful as a single agent in phase II clinical trials. Herein the mechanism of action and efficacy of a synthetic analogue, auristatin PYE, was investigated in 2 human colon adenocarcinoma models, DLD-1 and COLO 205. In vivo efficacy was assessed in subcutaneous xenografts following intravenous administration. Mechanistic studies investigated effects of auristatin PYE on microtubule disruption using immunocytochemistry, whilst cell cycle effects were studied using flow cytometry. Possible effects on tumour functional blood vasculature were assessed in tumour-bearing mice. Auristatin PYE was less potent in vitro than dolastatin 10, but was significantly more effective (p<0.01) in vivo against both tumours. Significant effects on tumour blood vasculature were seen, with optimal shutdown at 6-h post-treatment. Extensive necrosis became more evident over time after treatment. Auristatin PYE caused severe disruption of normal microtubule structure at concentrations and times comparable with the IC50 data, and also instigated a G2/M cell cycle block. Auristatin PYE was more effective in the DLD-1 and COLO 205 models than dolastatin 10, with anti-tumour effects mediated through vascular shutdown. These data suggest that auristatin PYE has good potential as an anti-cancer agent

Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (<it>Oryza sativa</it> L.)

Abstract Background Transferrin (TF) plays a critical physiological role in cellular iron delivery via the transferrin receptor (TFR)-mediated endocytosis pathway in nearly all eukaryotic organisms. Human serum TF (hTF) is extensively used as an iron-delivery vehicle in various mammalian cell cultures for production of therapeutic proteins, and is also being explored for use as a drug carrier to treat a number of diseases by employing its unique TFR-mediated endocytosis pathway. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasma-derived TF, recombinant hTF is preferred to use for these applications. Here, we carry out comparative studies of the TFR binding, TFR-mediated endocytosis and cellular iron delivery of recombinant hTF from rice (rhTF), and evaluate its suitability for biopharmaceutical applications. Result Through a TFR competition binding affinity assay with HeLa human cervic carcinoma cells (CCL-2) and Caco-2 human colon carcinoma cells (HTB-37), we show that rhTF competes similarly as hTF to bind TFR, and both the TFR binding capacity and dissociation constant of rhTF are comparable to that of hTF. The endocytosis assay confirms that rhTF behaves similarly as hTF in the slow accumulation in enterocyte-like Caco-2 cells and the rapid recycling pathway in HeLa cells. The pulse-chase assay of rhTF in Caco-2 and HeLa cells further illustrates that rice-derived rhTF possesses the similar endocytosis and intracellular processing compared to hTF. The cell culture assays show that rhTF is functionally similar to hTF in the delivery of iron to two diverse mammalian cell lines, HL-60 human promyelocytic leukemia cells (CCL-240) and murine hybridoma cells derived from a Sp2/0-Ag14 myeloma fusion partner (HB-72), for supporting their proliferation, differentiation, and physiological function of antibody production. Conclusion The functional similarity between rice derived rhTF and native hTF in their cellular iron delivery, TFR binding, and TFR-mediated endocytosis and intracellular processing support that rice-derived rhTF can be used as a safe and animal-free alternative to serum hTF for bioprocessing and biopharmaceutical applications.</p