A Point Mutation Creating a 3' Splice Site in C8A Is a Predominant Cause of C8α-γ Deficiency in African Americans.

C8α-γ deficiency was examined in four unrelated African Americans. Two individuals were compound heterozygotes for a previously reported point mutation in exon 9. mRNA from the remaining six C8A alleles contained a 10 nt insertion between nt 992 and 993 corresponding to the junction between exons 6 and 7. This suggested that C8α-γ deficiency in these individuals was caused by a splicing defect. Genomic sequencing revealed a G→A point mutation in intron 6, upstream of the exon 7 acceptor site. This mutation converts a GG to an AG, generates a consensus 3' splice site that shifts the reading frame, and creates a premature stop codon downstream. To verify that the point mutation caused a splicing defect, we tested wild-type and mutant mRNA substrates, containing 333 nt of the C8α intron 6/exon 7 boundary, in an in vitro splicing assay. This assay generated spliced RNA containing the 10 bp insertion observed in the C8α mRNA of affected patients. In addition, in mutant RNA substrates, the new 3' splice site was preferentially recognized compared with wild-type. Preferential selection of the mutant splice site likely reflects its positioning adjacent to a polypyrimidine tract that is stronger than that adjacent to the wild-type site. In summary, we have identified a G→A mutation in intron 6 of C8A as a predominant cause of C8α-γ deficiency in African Americans. This mutation creates a new and preferred 3' splice site, results in a 10 nt insertion in mRNA, shifts the reading frame, and produces a premature stop codon downstream.

Development of an in vitro colonization model to investigate Staphylococcus aureus interactions with airway epithelia.

Staphylococcus aureus is a bacterial pathogen responsible for a wide range of diseases and is also a human commensal colonizing the upper respiratory tract. Strains belonging to the clonal complex group CC30 are associated with colonization, although the colonization state itself is not clearly defined. In this work, we developed a co-culture model with S. aureus colonizing the apical surface of polarized human airway epithelial cells. The S. aureus are grown at the air-liquid interface to allow an in-depth evaluation of a simulated colonization state. Exposure to wild-type, S. aureus bacteria or conditioned media killed airway epithelial cells within 1 day, while mutant S. aureus strains lacking alpha-toxin (hla) persisted on viable cells for at least 2 days. Recent S. aureus CC30 isolates are natural hla mutants, and we observed that these strains displayed reduced toxicity toward airway epithelial cells. Quantitative real-time polymerase chain reaction of known virulence factors showed the expression profile of S. aureus grown in co-culture correlates with results from previous human colonization studies. Microarray analysis indicated significant shifts in S. aureus physiology in the co-culture model toward lipid and amino acid metabolism. The development of the in vitro colonization model will enable further study of specific S. aureus interactions with the host epithelia.

Staphylococcus aureus nuclease is an SaeRS-dependent virulence factor.

Several prominent bacterial pathogens secrete nuclease (Nuc) enzymes that have an important role in combating the host immune response. Early studies of Staphylococcus aureus Nuc attributed its regulation to the agr quorum-sensing system. However, recent microarray data have indicated that nuc is under the control of the SaeRS two-component system, which is a major regulator of S. aureus virulence determinants. Here we report that the nuc gene is directly controlled by the SaeRS two-component system through reporter fusion, immunoblotting, Nuc activity measurements, promoter mapping, and binding studies, and additionally, we were unable identify a notable regulatory link to the agr system. The observed SaeRS-dependent regulation was conserved across a wide spectrum of representative S. aureus isolates. Moreover, with community-associated methicillin-resistant S. aureus (CA MRSA) in a mouse model of peritonitis, we observed in vivo expression of Nuc activity in an SaeRS-dependent manner and determined that Nuc is a virulence factor that is important for in vivo survival, confirming the enzyme's role as a contributor to invasive disease. Finally, natural polymorphisms were identified in the SaeRS proteins, one of which was linked to Nuc regulation in a CA MRSA USA300 endocarditis isolate. Altogether, our findings demonstrate that Nuc is an important S. aureus virulence factor and part of the SaeRS regulon.

Quorum quenching and antimicrobial activity of goldenseal (Hydrastis canadensis) against methicillin-resistant Staphylococcus aureus (MRSA).

The popular herbal remedy goldenseal (Hydrastis canadensis L.) is traditionally used to treat skin infections. With this study, we show activity of H. canadensis extracts in vitro against methicillin-resistant Staphylococcus aureus (MRSA). An extract from H. canadensis leaves demonstrated more potent antimicrobial activity than the alkaloid berberine alone (MICs of 75 µg/mL and 150 µg/mL, respectively). LC-MS detected alkaloids and efflux-pump inhibitory flavonoids in the extract, and the latter may explain the enhanced efficacy of the extract compared to berberine alone. We also show evidence of anti-virulence activity as a second mechanism by which H. canadensis acts against S. aureus. The H. canadensis leaf extract (but not the isolated alkaloids berberine, hydrastine, and canadine) demonstrated quorum quenching activity against several clinically relevant MRSA isolates (USA300 strains). Our data suggest that this occurs by attenuation of signal transduction through the AgrCA two-component system. Consistent with this observation, the extract inhibited toxin production by MRSA and prevented damage by MRSA to keratinocyte cells in vitro. Collectively, our results show that H. canadensis leaf extracts possess a mixture of constituents that act against MRSA via several different mechanisms. These findings lend support for the traditional application of crude H. canadensis extracts in the prevention of infection.

agr-Dependent interactions of Staphylococcus aureus USA300 with human polymorphonuclear neutrophils.

The emergence of serious infections due to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has fueled interest in the contributions of specific staphylococcal virulence factors to clinical disease. To assess the contributions of agr-dependent factors to the fate of organisms in polymorphonuclear neutrophils (PMN), we examined the consequences for organism and host cells of feeding PMN with wild-type CA-MRSA (LAC) or CA-MRSA (LAC agr KO) at different multiplicities of infection (MOIs). Phagocytosed organisms rapidly increased the transcription of RNAIII in a time- and MOI-dependent fashion; extracellular USA300 (LAC) did not increase RNAIII expression despite having the capacity to respond to autoinducing peptide-enriched culture medium. HOCl-mediated damage and intracellular survival were the same in the wild-type and USA300 (LAC agr KO). PMN lysis by ingested USA300 (LAC) was time- and MOI-dependent and, at MOIs >1, required α-hemolysin (hla) as USA300 (LAC agr KO) and USA300 (LAC hla KO) promoted PMN lysis only at high MOIs. Taken together, these data demonstrate activation of the agr operon in human PMN with the subsequent production of α-hemolysin and PMN lysis. The extent to which these events in the phagosomes of human PMN contribute to the increased morbidity and mortality of infections with USA300 (LAC) merits further study.

Identification of qRT-PCR reference genes for analysis of opioid gene expression in a hibernator.

Previous work has suggested that central and peripheral opioid signaling are involved in regulating torpor behavior and tissue protection associated with the hibernation phenotype. We used quantitative real-time PCR (qRT-PCR) to measure mRNA levels of opioid peptide precursors and receptors in the brain and heart of summer ground squirrels (Ictidomys tridecemlineatus) and winter hibernating squirrels in the torpid or interbout arousal states. The use of appropriate reference genes for normalization of qRT-PCR gene expression data can have profound effects on the analysis and interpretation of results. This may be particularly important when experimental subjects, such as hibernating animals, undergo significant morphological and/or functional changes during the study. Therefore, an additional goal of this study was to identify stable reference genes for use in qRT-PCR studies of the 13-lined ground squirrel. Expression levels of 10 potential reference genes were measured in the small intestine, liver, brain, and heart, and the optimal combinations of the most stable reference genes were identified by the GeNorm Excel applet. Based on this analysis, we provide recommendations for reference genes to use in each tissue that would be suitable for comparative studies among different activity states. When appropriate normalization of mRNA levels was used, there were no changes in opioid-related genes in heart among the three activity states; in brain, DOR expression was highest during torpor, lowest in interbout arousal and intermediate in summer. The results support the idea that changes in DOR expression may regulate the level of neuronal activity in brain during the annual hibernation cycle and may contribute to hibernation-associated tissue protection.

Intra-arrest hypothermia: both cold liquid ventilation with perfluorocarbons and cold intravenous saline rapidly achieve hypothermia, but only cold liquid ventilation improves resumption of spontaneous circulation.

BACKGROUND: Rapid intra-arrest induction of hypothermia using total liquid ventilation (TLV) with cold perfluorocarbons improves resuscitation outcome from ventricular fibrillation (VF). Cold saline intravenous infusion during cardiopulmonary resuscitation (CPR) is a simpler method of inducing hypothermia. We compared these 2 methods of rapid hypothermia induction for cardiac resuscitation. METHODS: Three groups of swine were studied: cold preoxygenated TLV (TLV, n=8), cold intravenous saline infusion (S, n=8), and control (C, n=8). VF was electrically induced. Beginning at 8 min of VF, TLV and S animals received 3 min of cold TLV or rapid cold saline infusion. After 11 min of VF, all groups received standard air ventilation and closed chest massage. Defibrillation was attempted after 3 min of CPR (14 min of VF). The end point was resumption of spontaneous circulation (ROSC). RESULTS: Pulmonary arterial (PA) temperature decreased after 1 min of CPR from 37.2 degrees C to 32.2 degrees C in S and from 37.1 degrees C to 34.8 degrees C in TLV (S or TLV vs. C p<0.0001). Coronary perfusion pressure (CPP) was higher in TLV than S animals during the initial 3 min of CPR. Arterial pO(2) was higher in the preoxygenated TLV animals. ROSC was achieved in 7 of 8 TLV, 2 of 8 S, and 1 of 8C (TLV vs. C, p=0.03). CONCLUSIONS: Moderate hypothermia was achieved rapidly during VF and CPR using both cold saline infusion and cold TLV, but ROSC was higher than control only in cold TLV animals, probably due to better CPP and pO(2). The method by which hypothermia is achieved influences ROSC.

Liquid ventilation with perfluorocarbons facilitates resumption of spontaneous circulation in a swine cardiac arrest model.

BACKGROUND: Induced external hypothermia during ventricular fibrillation (VF) improves resuscitation outcomes. Our objectives were twofold (1) to determine if very rapid hypothermia could be achieved by intrapulmonary administration of cold perfluorocarbons (PFC), thereby using the lungs as a vehicle for targeted cardiopulmonary hypothermia, and (2) to determine if this improved resuscitation success. METHODS: Part 1: Nine female swine underwent static intrapulmonary instillation of cold perfluorocarbons (PFC) during electrically induced VF. Part 2: Thirty-three female swine in VF were immediately ventilated via total liquid ventilation (TLV) with pre-oxygenated cold PFC (-15 degrees C) or warm PFC (33 degrees C), while control swine received no ventilation during VF. All swine in both Parts 1 and 2 underwent VF arrest for 11 min, then defibrillation, ventilation and closed chest massage until resumption of spontaneous circulation (ROSC). The endpoint was continued spontaneous circulation for 1h without pharmacologic support. RESULTS: Static intrapulmonary instillation of cold PFC achieved rapid cardiopulmonary hypothermia; pulmonary artery (PA) temperature of 33.5+/-0.2 degrees C was achieved by 10 min. Nine of 9 achieved ROSC. Hypothermia was achieved faster using TLV: at 6 min VF, cold TLV temperature was 32.9+/-0.4 degrees C vs. cold static instillation temperature 34.3+/-0.2 degrees C. Nine of 11 cold TLV swine achieved ROSC for 1h vs. 3 of 11 control swine (p=0.03). Warm PFC also appeared to be beneficial, with a trend toward greater achievement of ROSC than control (ROSC; warm PFC 8 of 11 vs. control 3 of 11, p=0.09). CONCLUSION: Targeted cardiopulmonary intra-arrest moderate hypothermia was achieved rapidly by static intrapulmonary administration of cold PFC and more rapidly by total liquid ventilation with cold PFC; resumption of spontaneous circulation was facilitated. Warm PFC showed a trend toward facilitating ROSC.

Proenkephalin expression and enkephalin release are widely observed in non-neuronal tissues.

Enkephalins are opioid peptides that are found at high levels in the brain and endocrine tissues. Studies have shown that enkephalins play an important role in behavior, pain, cardiac function, cellular growth, immunity, and ischemic tolerance. Our global hypothesis is that enkephalins are released from non-neuronal tissues in response to brief ischemia or exercise, and that this release contributes to cardioprotection. To identify tissues that could serve as potential sources of enkephalins, we used real-time PCR, Western blot analysis, ELISA, immunofluorescence microscopy, and ex vivo models of enkephalin release. We found widespread expression of preproenkephalin (pPENK) mRNA and production of the enkephalin precursor protein proenkephalin (PENK) in rat and mouse tissues, as well as in tissues and cells from humans and pigs. Immunofluorescence microscopy with anti-enkephalin antisera demonstrated immunoreactivity in rat tissues, including heart and skeletal muscle myocytes, intestinal and kidney epithelium, and intestinal smooth muscle cells. Finally, isolated tissue studies showed that heart, skeletal muscle, and intestine released enkephalins ex vivo. Together our studies indicate that multiple non-neuronal tissues produce PENK and release enkephalins. These data support the hypothesis that non-neuronal tissues could play a role in both local and systemic enkephalin-mediated effects.

Exercise enhances myocardial ischemic tolerance via an opioid receptor-dependent mechanism.

Exercise increases serum opioid levels and improves cardiovascular health. Here we tested the hypothesis that opioids contribute to the acute cardioprotective effects of exercise using a rat model of exercise-induced cardioprotection. For the standard protocol, rats were randomized to 4 days of treadmill training and 1 day of vigorous exercise (day 5), or to a sham exercise control group. On day 6, animals were killed, and global myocardial ischemic tolerance was assessed on a modified Langendorff apparatus. Twenty minutes of ischemia followed by 3 h of reperfusion resulted in a mean infarct size of 42 +/- 4% in hearts from sham exercise controls and 21 +/- 3% (P < 0.001) in the exercised group. The cardioprotective effects of exercise were gone by 5 days after the final exercise period. To determine the role of opioid receptors in exercise-induced cardioprotection, rats were exercised according to the standard protocol; however, just before exercise on days 4 and 5, rats were injected subcutaneously with 10 mg/kg of the opioid receptor antagonist naltrexone. Similar injections were performed in the sham exercise control group. Naltrexone had no significant effect on baseline myocardial ischemic tolerance in controls (infarct size 43 +/- 4%). In contrast, naltrexone treatment completely blocked the cardioprotective effect of exercise (infarct size 40 +/- 5%). Exercise was also associated with an early increase in myocardial mRNA levels for several opioid system genes and with sustained changes in a number of genes that regulate inflammation and apoptosis. These findings demonstrate that the acute cardioprotective effects of exercise are mediated, at least in part, through opioid receptor-dependent mechanisms that may include changes in gene expression.

Met5-enkephalin-Arg6-Phe7 (MEAP): a cardioprotective hormonal opioid.

BACKGROUND: Acute myocardial ischemia is an important cause of morbidity and mortality worldwide. The heart and other organs can be rendered more resistant to the deleterious effects of ischemia through a variety of preconditioning strategies, including treadmill exercise and brief ischemia of skeletal muscle. Some of the beneficial effects of these preconditioning strategies appear to be mediated by as-of-yet unidentified hormonal opioids. OBJECTIVES: To test the hypothesis that endogenous opioids of the enkephalin class are capable of improving ischemic tolerance and acting in a hormonal manner. METHODS: In phase one of the investigation, the authors assessed the cardioprotective potential of all four known enkephalins. This was achieved by subjecting isolated buffer-perfused rabbit hearts to a 25-minute period of test ischemia and two hours of reperfusion (protocol 1) after receiving treatment with either saline vehicle (controls) or increasing concentrations of purified enkephalins. On the basis of results from these initial studies, the authors performed additional experiments (protocol 2) to determine whether Met5-enkephalin-Arg6-Phe7 (MEAP) could be absorbed from skeletal muscle and exert a cardioprotective effect. Specifically, MEAP or vehicle (controls) was given intramuscularly 24 hours before the hearts were harvested. A similar assessment of ischemic tolerance as described in protocol 1 was then performed. Postischemic myocardial viability (infarct size) was assessed in all cases by triphenyltetrazolium chloride (TTC) staining. Hemodynamic parameters and infarct sizes for concentration-dependence studies were compared by two-way analysis of variance, and infarct sizes from protocol 2 studies were compared by using Student's t-test (significance set at p < or = 0.05). RESULTS: Mean infarct size in control hearts (+/- SEM) was 33% (+/- 4%) and 36% (+/- 6%) for protocol 1 and 2, respectively. Of the four enkephalins tested in protocol 1, only MEAP treatment showed a tendency toward cardioprotection. Interestingly, an alternative enkephalin, methionine5-enkephalin-Arg6-Gly7-Leu8, tended to exert an injurious effect. In protocol 2, MEAP treatment 24 hours before ischemia significantly reduced infarct size (14% +/- 4%) compared with controls, suggesting that it can be released from muscle and exert a distant cardioprotective effect. CONCLUSIONS: When given either directly to the heart or absorbed from a distant tissue, MEAP induces cardioprotection, supporting the hypothesis that it can act as a hormonal modulator of ischemic tolerance.

Nuclear factor-kappaB contributes to interleukin-4- and interferon-dependent polymeric immunoglobulin receptor expression in human intestinal epithelial cells.

Polymeric immunoglobulins (pIgs) that are present at mucosal surfaces play key roles in both the innate and adaptive immune responses. These pIgs are delivered to the mucosal surface via transcytosis across the epithelium, a process mediated by the polymeric immunoglobulin receptor (pIgR). Previous studies demonstrate that expression of the pIgR is regulated by multiple immunomodulatory factors including interleukin-4 (IL-4) and interferon-gamma (IFN-gamma). In studies using human intestinal epithelial cells (HT29), multiple inhibitors of the transcription factor nuclear factor-kappaB (NF-kappaB), including a dominant negative IkappaBalpha-serine mutant, inhibited both IL-4- and IFN-dependent increases in pIgR expression. Under identical conditions, NF-kappaB inhibitors had no effect on cytokine-dependent increases in expression of the transcription factor interferon regulatory factor-1. Over-expression of the IkappaBalpha-serine mutant also inhibited reporter gene expression in response to IL-4, TNF-alpha, IL-1beta, and in some cases IFN-gamma using constructs with sequences from the pIgR promoter. Reduced levels of pIgR were observed even when inhibitors were added >/=24 hr after cytokines suggesting that prolonged activation of NF-kappaB is required. Finally, reporter gene studies with NF-kappaB enhancer elements indicated that IFN-gamma alone and IL-4 in combination with other cytokines activated NF-kappaB in HT29 cells. Together, these studies provide additional insight into the signalling pathways that contribute to expression of the pIgR, a critical player in mucosal immunity.