Automated direct screening for resistance of Gram-negative blood cultures using the BD Kiestra WorkCell.

Early detection of resistance in sepsis due to Gram-negative organisms may lead to improved outcomes by reducing the time to effective antibiotic therapy. Traditional methods of resistance detection require incubation times of 18 to 48 h to detect resistance. We have utilised automated specimen processing, digital imaging and zone size measurements in conjunction with direct disc susceptibility testing to develop a method for the rapid screening of Gram-negative blood culture isolates for resistance. Positive clinical blood cultures with Gram-negative organisms were prospectively identified and additional resistant mock specimens were prepared. Broth was plated and antibiotic-impregnated discs (ampicillin, ceftriaxone, piperacillin-tazobactam, meropenem, ciprofloxacin, gentamicin) were added. Plates were incubated, digitally imaged and zone sizes were measured using the BD Kiestra WorkCell laboratory automation system. Minimum, clinically useful, incubation times and optimised zone size cut-offs for resistance detection were determined. We included 187 blood cultures in the study. At 5 h of incubation, > 90% of plates yielded interpretable results. Using optimised zone size cut-offs, the sensitivity for resistance detection ranged from 87 to 100%, while the specificity ranged from 84.7 to 100%. The sensitivity and specificity for piperacillin-tazobactam resistance detection was consistently worse than for the other agents. Automated direct disc susceptibility screening is a rapid and sensitive tool for resistance detection in Gram-negative isolates from blood cultures for most of the agents tested.

Establishment of an AUC0-24 Threshold for Nephrotoxicity Is a Step towards Individualized Vancomycin Dosing for Methicillin-Resistant Staphylococcus aureus Bacteremia.

Unlike vancomycin trough concentrations, data on the utility of vancomycin pharmacokinetic (PK) parameters, namely, the area under the concentration-time curve from 0 to 24 h (AUC0-24), in predicting acute kidney injury (AKI) are limited. Our aim was to investigate this relationship in patients receiving vancomycin therapy for methicillin-resistant Staphylococcus aureus bacteremia (MRSA-B). A single-center retrospective observational cohort study involving 127 consecutive MRSA-B patients was conducted to examine the incidence of AKI (defined as serum creatinine of ≥0.5 mg/liter and a 50% increase from baseline) and vancomycin exposure parameters associated with nephrotoxicity. Bayesian estimation was used to predict individual vancomycin AUC0-24 All patients received vancomycin monotherapy for a minimum of 14 days following the diagnosis of MRSA-B. AKI was observed in 15.7% of patients (20/127). Clinical characteristics were similar between patients with and without AKI. At steady state, higher vancomycin trough concentrations were associated with AKI (17.2 mg/liter versus 13.1 mg/liter; P = 0.003). A vancomycin AUC0-24 threshold for AKI of >563 mg · h/liter was detected by classification and regression tree (CART) analysis; patients with exposures above this threshold were significantly more likely to experience AKI than patients with lower vancomycin exposures (40% [8/20] versus 11.2% [12/107]; P = 0.002). This parameter remained an independent predictor of AKI on multivariate logistic regression (odds ratio [OR], 5.07; 95% confidence interval [CI], 1.57 to 16.29; P = 0.006) and was a better predictor of nephrotoxicity than vancomycin trough concentrations. Overall, AKI is associated with higher vancomycin exposure as measured by AUC0-24 These results suggest that individualized patient dosing may be possible with dose modifications directed toward established pharmacodynamic targets while balancing AKI risks.

Exposure to viral and bacterial pathogens among Soay sheep (Ovis aries) of the St Kilda archipelago.

We assessed evidence of exposure to viruses and bacteria in an unmanaged and long-isolated population of Soay sheep (Ovis aries) inhabiting Hirta, in the St Kilda archipelago, 65 km west of Benbecula in the Outer Hebrides of Scotland. The sheep harbour many metazoan and protozoan parasites but their exposure to viral and bacterial pathogens is unknown. We tested for herpes viral DNA in leucocytes and found that 21 of 42 tested sheep were infected with ovine herpesvirus 2 (OHV-2). We also tested 750 plasma samples collected between 1997 and 2010 for evidence of exposure to seven other viral and bacterial agents common in domestic Scottish sheep. We found evidence of exposure to Leptospira spp., with overall seroprevalence of 6·5%. However, serological evidence indicated that the population had not been exposed to border disease, parainfluenza, maedi-visna, or orf viruses, nor to Chlamydia abortus. Some sheep tested positive for antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) but, in the absence of retrospective faecal samples, the presence of this infection could not be confirmed. The roles of importation, the pathogen-host interaction, nematode co-infection and local transmission warrant future investigation, to elucidate the transmission ecology and fitness effects of the few viral and bacterial pathogens on Hirta.

Short duration of lamivudine for the prevention of hepatitis B virus transmission in pregnancy: lack of potency and selection of resistance mutations.

This study sought to assess the antiviral efficacy of lamivudine (LMV) administered during third trimester to reduce maternal viraemia and to identify the emergence of LMV resistance. A prospective observational analysis was performed on 26 mothers with high viral load (>107 IU/mL). Twenty-one women received LMV (treated group) for an average of 53 days (range 22-88 days), and the remaining five formed the untreated control group. Serum samples from two time points were used to measure HBV DNA levels and antiviral drug resistance. The LMV-treated women achieved a median HBV DNA reduction of 2.6-log10 IU/mL. Although end-of-treatment (EOT) HBV DNA in four (18%) LMV-treated women remained at >10(7) IU/mL (± 0.5 log IU/mL), no mother-to-baby transmission was observed. In contrast, a baby from the untreated mother was HBsAg positive at 9 months postpartum. Four technologies were used for drug resistance testing. Only ultra-deep pyrosequencing (UDPS) was sufficiently sensitive to detect minor viral variants down to <1%. UDPS showed that LMV therapy resulted in increased viral quasispecies diversity and positive selection of HBV variants with reverse transcriptase amino acid substitutions at sites associated with primary LMV resistance (rtM204I/V and rtA181T) in four (19%) women. These viral variants were detected mostly at low frequencies (0.63-5.92%) at EOT, but one LMV-treated mother had an rtA181T variant that increased from 2.2% pretherapy to 25.59% at EOT. This mother was also infected with the vaccine escape variant (sG145R), which was inhibited by LMV treatment. LMV therapy during late pregnancy only reduced maternal viraemia moderately, and drug-resistant viral variants emerged.

Validation of a multiplex-tandem RT-PCR for the detection of bovine respiratory disease complex using Scottish bovine lung samples.

The welfare and economic impact of bovine respiratory disease complex (BRDC), and its associated antibiotic usage, are major challenges to cattle rearing and beef cattle finishing industries. Accurate pathogen diagnosis is important to undertake appropriate treatment and long-term management strategies, such as vaccine selection. Conventional diagnostic approaches have several limitations including high costs, long turnaround times and difficulty in test interpretation, which could delay treatment decisions and lead to unnecessary animal losses. We describe the validation of a multiplex-tandem (MT) reverse transcription-polymerase chain reaction (RT-PCR) for the detection of seven common pathogens associated with BRDC. This test has the potential to advance pathogen identification and to overcome many of the limitations of current testing methods. It requires a single sample and results are obtained quickly and not influenced by prior antimicrobial therapy or overgrowth of contaminating organisms. We demonstrated a test specificity of 100% and sensitivity ranging from 93.5% to 100% for these seven common pathogens. This test will be a useful addition to advance BRDC investigation and diagnosis.

Molecular typing of enteroviruses: comparing 5'UTR, VP1 and whole genome sequencing methods.

Enteroviruses (EV) commonly cause hand, foot and mouth disease (HFMD), and can also cause potentially fatal neurological and systemic complications. In our laboratory, sequencing 5' untranslated region (UTR) of the viral genome has been the routine method of genotyping EVs. During a recent localised outbreak of aseptic meningitis, sequencing the 5'UTR identified the causative virus as EV-A71, which did not fit with the clinical syndrome or illness severity. When genotyped using a different target gene, VP1, the result was different. This led us to evaluate the accuracy of the two different target genome regions and compare them against whole genome sequencing (WGS). We aimed to optimise the algorithm for detection and characterisation of EVs in the diagnostic laboratory. We hypothesised that VP1 and WGS genotyping would provide different results than 5'UTR in a subset of samples. Clinical samples from around New South Wales which were positive for EV by commercial polymerase chain reaction (PCR) assays were genotyped by targeting three different viral genome regions: the 5'UTR, VP1 and WGS. Sequencing was performed by Sanger and next generation sequencing. The subtyping results were compared. Of the 74/118 (63%) samples that were successfully typed using both the 5'UTR and the VP1 method, the EV typing result was identical for 46/74 (62%) samples compared to WGS as the gold standard. The same EV group but different EV types were found in 22/74 (30%) samples, and 6/74 (8%) samples belonged to different EV groups depending on typing method used. Genotyping with WGS and VP1 is more accurate than 5'UTR. Genotyping by the 5'UTR method is very sensitive, but less specific.

Multi-centre evaluation of pre-transfusional routine tests using 8-column format gel cards (DG Gel®).

BACKGROUND: Advances in immunohaematology laboratory practice to improve performance, cost-effectiveness and patient safety are desirable. OBJECTIVES: To perform a multi-centre evaluation of the 8-column Grifols DG Gel(®) cards and reagent system to assess its performance, suitability and adaptability to the daily blood transfusion laboratory routine in the United Kingdom. METHODS/MATERIALS: A total of 4281 immunohematological analyses {1825 ABO/D grouping, 1921 antibody screening, 75 Rh phenotyping and K antigen determination, 361 antibody identification and 99 neonates [ABO/D and DAT (direct anti-globulin test)]} were performed on 2255 specimens. All cases were run in parallel with the reference method of each laboratory (DiaMed-ID(®) cards or conventional tube technique in some cases). RESULTS: Concordant results between Grifols DG Gel(®) system and the reference method were obtained in 97·7% of tests. For ABO grouping by the Grifols DG Gel(®) system, sensitivity was 99·95%, specificity was 99·96%, predictive positive value (PPV) was 99·89% and predictive negative value (PNV) was 99·98%. For D grouping, sensitivity was 99·78%, specificity was 100%, PPV was 100% and PNV was 99·78%. For antibody screening, sensitivity was 90·63%, specificity was 99·94%, PPV was 99·32% and PNV was 99·15%. Of the Rh subgroups and K types, results were 100% concordant. For antibody specificity detection, accuracy was 96·95% for Grifols DG Gel(®) system and 95·29% for DiaMed-ID(®) system. For the newborn tests, concordant results were obtained in 100% of ABO/D grouping and in 89·9% of DAT. CONCLUSION: The Grifols DG Gel(®) 8-column system is reliable and safe for routine tests performed in the immunohaematology laboratory.

Adjuvant immunosuppression for paradoxical deterioration in tuberculous meningitis including one case responsive to cyclosporine. A tertiary referral hospital experience.

BACKGROUND: Tuberculous meningitis (TBM) accounts for 1-4% of all tuberculosis (TB) presentations. Paradoxical deterioration in non-HIV patients is a common manifestation of anti-tuberculosis therapy, characterised by clinico-radiological deterioration. We report a case series of TBM admissions to our institution including one case with paradoxical deterioration refractory to corticosteroids who responded to adjuvant cyclosporine. METHODS: Retrospective review of 12 HIV-negative patients admitted to Liverpool Hospital, Sydney (2005-2016) with laboratory and/or radiologically confirmed TBM. RESULTS: Median patient age was 40 (range 22-81 years), M:F = 7:5. Eleven patients (92%) were of Asia-Pacific origin. Eleven initially presented with central nervous system manifestations and one had preceding miliary TB. Nine patients had extra-cranial TB involvement including eight with past or current pulmonary disease. Cerebrospinal fluid (CSF) TB PCR/culture was positive in 10 patients. Paradoxical deterioration developed in three patients despite concomitant corticosteroids in two. One patient with paradoxical deterioration was refractory to corticosteroids: A 22-year-old Vietnamese male with TBM developed worsening headaches and altered mentation after seven weeks concomitant anti-TB and corticosteroid treatment. Interval MRI brain demonstrated increased size and number of tuberculomas as well as hydrocephalus. Cyclosporine was added with gradual improvement and ultimately good outcome. CONCLUSION: Our case series highlights the seriousness of paradoxical deterioration in TBM and the potential role of adjuvant cyclosporine in patients refractory to corticosteroids.

Utility of 16S rRNA PCR performed on clinical specimens in patient management.

BACKGROUND: Broad-range 16S rRNA PCR can be used for the detection and identification of bacteria from clinical specimens in patients for whom there is a high suspicion of infection and cultures are negative. The aims of this study were (1) to compare 16S rRNA PCR results with microbiological culture results, (2) to assess the utility of 16S rRNA PCR with regard to antimicrobial therapy, and (3) to compare the yield of 16S rRNA PCR for different types of clinical specimen and to perform a cost analysis of the test. METHODS: A retrospective study was performed on different clinical specimens which had 16S performed over 3 years (2012-2015). Standard microbiological cultures were performed on appropriate media, as per the laboratory protocol. Patient clinical and microbiological data were obtained from the electronic medical records and laboratory information system, respectively. 16S rRNA PCR was performed in a reference laboratory using a validated method for amplification and sequencing. The outcomes assessed were the performance of 16S rRNA PCR, change of antimicrobials (rationalization, cessation, or addition), and duration of therapy. Concordance of 16S rRNA PCR with bacterial cultures was also determined for tissue specimens. RESULTS: Thirty-two patients were included in the study, for whom an equal number of specimens (n=32) were sent for 16S rRNA PCR. 16S rRNA PCR could identify an organism in 10 of 32 cases (31.2%), of which seven were culture-positive and three were culture-negative. The sensitivity was 58% (confidence interval (CI) 28.59-83.5%) and specificity was 85% (CI 61.13-96%), with a positive predictive value of 70% (CI 35.3-91.9%) and negative predictive value of 77.2% (CI 54.17-91.3%). Antimicrobial therapy was rationalized after 16S rRNA PCR results in five patients (15.6%) and was ceased in four based on negative results (12.5%). Overall the 16S rRNA PCR result had an impact on antimicrobial therapy in 28% of patients (9/32). The highest concordance of 16S rRNA PCR with bacterial culture was found for heart valve tissue (80%), followed by joint fluid/tissue (50%). CONCLUSIONS: Despite the low diagnostic yield, results of 16S rRNA PCR can still have a significant impact on patient management due to rationalization or cessation of the antimicrobial therapy. The yield of 16S rRNA PCR was highest for heart valves.

Development of a real time RT-PCR to detect and type ovine pestiviruses.

A real time one-step RT-PCR was designed to detect and type border disease virus (BDV), bovine viral diarrhea virus (BVDV) type 1 and BVDV type 2 in ovine samples. The real time RT-PCR was shown to behave in a linear manner and had limits of detection of 100-1000 copies of viral RNA as judged by in vitro transcribed RNA. The real time RT-PCR was validated on 50 clinical samples from UK flocks and was more sensitive than a virus isolation and a classical nested RT-PCR (nRT-PCR). The results of real time RT-PCR virus typing agreed completely with sequencing. The majority of ovine isolates were BDV; a small proportion were BVDV type 1. BVDV type 2 was not detected in any sample. This test appears reliable and can be used for the typing of ovine pestiviruses in the UK.

Sequential study of gamma-glutamyltransferase during complete and two-stage mouse skin carcinogenesis.

The histochemical pattern of gamma-glutamyltransferase (GGT) was studied in benign and malignant tumors produced by two different experimental protocols, two-stage carcinogenesis and complete carcinogenesis. Six percent of all papillomas produced by two-stage carcinogenesis were GGT positive, whereas 14% of benign tumors produced by complete carcinogenesis exhibited GGT-positive areas. The incidence of GGT-positive papillomas in the two-step carcinogenesis protocol increased up to wk 28 of treatment. After 32 wk, the incidence decreased abruptly, coinciding with an abrupt increase in the incidence of squamous cell carcinomas. On the other hand, the incidence of GGT-positive benign tumors produced during the course of complete carcinogenesis increased gradually up to wk 32 of treatment, coinciding with the increased incidence of squamous cell carcinomas. The incidence of GGT-positive keratoacanthomas and GGT-positive papillomas produced with the complete carcinogenesis protocol exhibited different patterns, suggesting different histogenesis and biological behavior of these two types of tumors. In addition, the labeling index of GGT-positive areas was lower (17 +/- 3%) than that of the GGT-negative areas (41 +/- 0.18%) of the same papillomas, indicating that the presence of GGT may be related to abnormal keratocyte differentiation rather than to proliferative changes.

Biochemical characterization of a genetically altered calmodulin in Paramecium.

Recent evidence proposes that the calcium-binding protein, calmodulin, plays a crucial role in the regulation or modulation of the calcium-dependent potassium conductance in Paramecium tetraurelia (Hinrichsen, R.D., Burgess-Cassler, A., Soltvedt, B.C., Hennessey, T. and Kung, C. (1986) Science 323, 503-506). We purified the calmodulins from both the wild type and pantophobiac A (a mutant lacking the above-mentioned conductance and whose phenotypic defect is traceable to its calmodulin) by hydrophobic interaction and immunoaffinity chromatographies, and examined them biochemically. In this paper we address the preliminary characterization of the two calmodulins and discuss the consequences of the genetic alteration. The differences described here are in their electrophoretic mobilities in polyacrylamide gel electrophoresis and in their binding characteristics to monoclonal antibodies raised against calmodulin from wild-type paramecia. Also, we present data which indicate a difference in the stimulation of the calmodulin-dependent enzyme bovine brain phosphodiesterase under certain conditions.

Development and clinical assessment of an artificial cornea.

Keratoprosthesis research has been a gradual, rather fragmentary process with advances being made by isolated groups of researchers. This has arisen partly because of poor funding in the area; research groups which have achieved commercial support have often had constraints upon the full disclosure of their findings. Despite these difficulties there has been real progress over the last decade by several independent groups. This article concentrates upon our own development of a hydrogel core-and-skirt keratoprosthesis, the Chirila KPro, in order to illustrate the scientific and clinical problems common to keratoprosthesis research. Pilot data from a clinical trial is presented and the priorities for future research are discussed.

The incidence of red cell alloantibodies underlying panreactive warm autoantibodies.

A recognized hazard of administering blood transfusions to patients with panreactive warm autoantibodies is that alloantibodies may be masked. Studies have shown the incidence of underlying alloantibodies to be 30 to 40 percent. Adsorption procedures can be used to remove autoantibodies and allow detection and identification of underlying alloantibodies. This study contains data from 126 patients referred to the Red Cell Immunohaematology laboratory at the National Blood Service, Newcastle upon Tyne, United Kingdom. These patients were from the northeast of England, a population for which data have not previously been reported. Samples identified as containing panreactive warm autoantibodies were subjected to adsorption procedures (95 by alloadsorption and 31 by autoadsorption). Absorbed sera were then tested to identify underlying alloantibodies. Of 126 samples, 39 (31%) contained a total of 61 RBC alloantibodies; 15 (12%) contained 2 or more antibody specificities; and 14 (11%) contained alloantibodies not found within the Rh or Kell blood group systems. Antibodies identified included the following specificities: E (19), D (9), c (7), C (6), S (5), Fy(a) (3), Jk(a) (2), Jkb (2), K (2),Kp(a) (2), Fy(b),C(w),N, and f (ce). This study reinforces the value of adsorption studies, whether using autologous or allogeneic RBCs, when panreactive warm autoantibodies are present. In addition, this study confirms that it is not appropriate in these cases simply to issue blood which is "least incompatible" or Rh phenotype- and K antigen-matched.

In vivo Paramecium mutants show that calmodulin orchestrates membrane responses to stimuli.

Paramecium generates a Ca2+ action potential and can be considered a one-cell animal. Rises in internal [Ca2+] open membrane channels that specifically pass K+, or Na+. Mutational and patch-clamp studies showed that these channels, like enzymes, are activated by Ca(2+)-calmodulin. Viable CaM mutants of Paramecium have altered transmembrane currents and easily recognizable eccentricities in their swimming behavior, i.e. in their responses to ionic, chemical, heat, or touch stimuli. Their CaMs have amino-acid substitutions in either C- or N-terminal lobes but not the central helix. Surprisingly, these mutations naturally fall into two classes: C-lobe mutants (S101F, I136T, M145V) have little or no Ca(2+)-dependent K+ currents and thus over-react to stimuli. N-lobe mutants (E54K, G40E+D50N, V35I+D50N) have little or no Ca(2+)-dependent Na+ current and thus under-react to certain stimuli. Each mutation also has pleiotropic effects on other ion currents. These results suggest a bipartite separation of CaM functions, a separation consistent with the recent studies of Ca(2+)-ATPase by Kosk-Kosicka et al. [41, 55]. It appears that a major function of Ca(2+)-calmodulin in vivo is to orchestrate enzymes and channels, at or near the plasma membrane. The orchestrated actions of these effectors are not for vegetative growth at steady state but for transient responses to stimuli epitomized by those of electrically excitable cells.

Alterations in arginine metabolic enzymes in trauma.

Arginine is the sole substrate for nitric oxide (NO) synthesis by NO synthases (NOS) and promotes the proliferation and maturation of human T-cells. Arginine is also metabolized by the enzyme arginase, producing urea and ornithine, the precursor for polyamine production. We sought to determine the molecular mechanisms regulating arginase and NOS in splenic immune cells after trauma. C3H/HeN mice underwent laparotomy as simulated moderate trauma or anesthesia alone (n = 24 per group). Six, 12, 24, or 48 h later, 6 animals from each group were sacrificed, and splenectomy was performed and plasma collected. Six separate animals had neither surgery nor anesthesia and were sacrificed to provide resting values (t = 0 h). Spleen arginase I and II and iNOS mRNA abundance, arginase I protein expression, and arginase activity were determined. Plasma NO metabolites (nitrite + nitrate) were also measured. Trauma increased spleen arginase I protein expression and activity (P = 0.01) within 12 and for at least 48 h after injury and coincided with up-regulated arginase I mRNA abundance at 24 h. Neither arginase II nor iNOS mRNA abundance in the spleen was significantly increased by trauma at 24 h. Plasma nitrite + nitrate was decreased in animals 48 h post-injury compared to anesthesia controls (P < 0.05). Trauma induces up-regulation of arginase I gene expression in splenic immune cells within 24 h of injury. Arginase II is not significantly up-regulated at that time point. Arginase I, rather than iNOS appears to be the dominant route for arginine metabolism in splenic immune cells 24 h after trauma.

Beneficial effects of S-adenosyl-L-methionine on blood-brain barrier breakdown and neuronal survival after transient cerebral ischemia in gerbils.

We have studied the beneficial effects of S-adenosyl-L-methionine (SAM) tosylate on blood-brain barrier (BBB) breakdown and neuronal survival after transient cerebral ischemia in gerbils. BBB breakdown experiments were performed in pentobarbital anesthetized gerbils subjected to 10 min of bilateral carotid artery occlusion and 6 h of reperfusion. For BBB breakdown measurements, SAM (120 mg/kg, i.p.) was administered to gerbils just after occlusion and thereafter every hour up to 5 h. Fluorometric measurements quantified the blood-brain permeability tracer, Evans blue (EB). SAM treatment significantly reduced the BBB breakdown as indicated by reduced levels of EB fluorescence. Neuronal count experiments were conducted in gerbils subjected to transient ischemia and 7 days of reperfusion. For neuronal count experiments SAM (15-120 mg/kg) was administered at 6 and 12 h after reperfusion, and twice each day thereafter for 7 days. SAM dose dependently protected the hippocampal CA1 neurons assessed by histopathological methods. SAM has a beneficial effect on the outcome of ischemic injury by reducing the BBB breakdown and neuronal death.

The modulation of corneal keratocyte and epithelial cell responses to poly(2-hydroxyethyl methacrylate) hydrogel surfaces: phosphorylation decreases collagenase production in vitro.

We examined the regulation of collagenase production by rabbit keratocyte, epithelial and mixed keratocyte/epithelial cell cultures which were exposed to poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel surfaces with different chemistries and morphologies (sponge and homogeneous gels). Tissue culture modified polystyrene (TCP), used as a control surface, induced the maximum collagenase response with all cell culture types. Copolymer homogeneous gels containing 2-ethoxyethyl methacrylate (EEMA) or methyl methacrylate (MMA) induced a high response in keratocyte cultures, whilst PHEMA hydrogels induced a moderate response and the phosphorylated PHEMA (phos-PHEMA) hydrogel induced no response. Epithelial cells cultured on PHEMA, copolymer and phos-PHEMA hydrogels produced less collagenase activity than the keratocyte cells. The profile of collagenases produced by epithelial cells in response to phos-PHEMA was different to that for the other hydrogels. Co-cultured cells produced higher levels of collagenase (relative to the TCP) in response to hydrogels than did either the keratocytes or epithelial cells alone, but the response of phos-PHEMA was still the lowest. The overall enzyme response to the sponge hydrogels was lower than that to the homogeneous hydrogels, although this effect was less prominent in the keratocyte cultures. The markedly reduced and alternative collagenase responses to phosphorylated surfaces was not a consequence of cell death, and may be a phenomenon related to changes in cell surface charge and morphology.

Beta adrenoceptor regulation of macrophage arginase activity.

BACKGROUND: Arginase, which metabolizes L-arginine within the urea cycle, is essential for production of polyamines and affects production of nitric oxide by depletion of L-arginine, the common substrate for both arginase and nitric oxide synthase. Having shown that trauma increases splenic macrophage arginase activity, we seek to define the mechanisms for this. RAW macrophage arginase activity and expression are increased by 8-bromo-cAMP in vitro. We hypothesize that since catecholamines increase cAMP, trauma-induced splenic arginase activity may be mediated by post-injury catecholamine release. METHODS: RAW 264.7 macrophage arginase activity was measured in vitro in response to 4 catecholamines with or without propranolol or lipopolysaccharide (LPS). C57BL/6 mice underwent laparotomy as a model of moderate trauma after propranolol treatment, with and without intraperitoneal Escherichia coli LPS administration as a simulated pro-inflammatory stimulus. RESULTS: Macrophage arginase activity increased in vitro in response to catecholamines or LPS (P < .05). Propranolol pretreatment blocked macrophage arginase activity induced by epinephrine (10 mumol/L) in vitro (P < .05). Trauma or LPS alone increased splenic arginase activity in vivo (P < .05). Propranolol did not alter LPS-induced splenic arginase activity but did significantly reduce trauma-induced splenic arginase activity (P < .05). CONCLUSIONS: Catecholamines alone increase macrophage arginase activity through beta-adrenoceptor activation. Increased splenic arginase activity induced by moderate trauma is decreased by beta-adrenoceptor blockade, suggesting that trauma-induced arginase activity is partly mediated by endogenous catecholamines.