Differential roles for endothelial ICAM-1, ICAM-2, and VCAM-1 in shear-resistant T cell arrest, polarization, and directed crawling on blood-brain barrier endothelium

Endothelial ICAM-1 and ICAM-2 were shown to be essential for T cell diapedesis across the blood-brain barrier (BBB) in vitro under static conditions. Crawling of T cells prior to diapedesis was only recently revealed to occur preferentially against the direction of blood flow on the endothelial surface of inflamed brain microvessels in vivo. Using live cell-imaging techniques, we prove that Th1 memory/effector T cells predominantly crawl against the direction of flow on the surface of BBB endothelium in vitro. Analysis of T cell interaction with wild-type, ICAM-1-deficient, ICAM-2-deficient, or ICAM-1 and ICAM-2 double-deficient primary mouse brain microvascular endothelial cells under physiological flow conditions allowed us to dissect the individual contributions of endothelial ICAM-1, ICAM-2, and VCAM-1 to shear-resistant T cell arrest, polarization, and crawling. Although T cell arrest was mediated by endothelial ICAM-1 and VCAM-1, T cell polarization and crawling were mediated by endothelial ICAM-1 and ICAM-2 but not by endothelial VCAM-1. Therefore, our data delineate a sequential involvement of endothelial ICAM-1 and VCAM-1 in mediating shear-resistant T cell arrest, followed by endothelial ICAM-1 and ICAM-2 in mediating T cell crawling to sites permissive for diapedesis across BBB endothelium.

Polymorphisms in the ABCB1 gene in phenobarbital responsive and resistant idiopathic epileptic Border Collies

BACKGROUND: Variation in the ABCB1 gene is believed to play a role in drug resistance in epilepsy. HYPOTHESIS/OBJECTIVES: Variation in the ABCB1 gene encoding the permeability-glycoprotein could have an influence on phenobarbital (PB) resistance, which occurs with high frequency in idiopathic epileptic Border Collies (BCs). Animals: Two hundred and thirty-six client-owned BCs from Switzerland and Germany including 25 with idiopathic epilepsy, of which 13 were resistant to PB treatment. METHODS: Prospective and retrospective case-control study. Data were collected retrospectively regarding disease status, antiepileptic drug (AED) therapy, and drug responsiveness. The frequency of a known mutation in the ABCB1 gene (4 base-pair deletion in the ABCB1 gene [c.296_299del]) was determined in all BCs. Additionally, the ABCB1 coding exons and flanking sequences were completely sequenced to search for additional variation in 41 BCs. Association analyses were performed in 2 case-control studies: idiopathic epileptic and control BCs and PB-responsive and resistant idiopathic epileptic BCs. RESULTS: One of 236 BCs (0.4%) was heterozygous for the mutation in the ABCB1 gene (c.296_299del). A total of 23 variations were identified in the ABCB1 gene: 4 in exons and 19 in introns. The G-allele of the c.-6-180T > G variation in intron 1 was significantly more frequent in epileptic BCs resistant to PB treatment than in epileptic BCs responsive to PB treatment (P(raw) = .0025). CONCLUSIONS AND CLINICAL IMPORTANCE: A variation in intron 1 of the ABCB1 gene is associated with drug responsiveness in BCs. This might indicate that regulatory mutations affecting the expression level of ABCB1 could exist, which may influence the reaction of a dog to AEDs.

Acinetobacter baumannii isolates from pets and horses in Switzerland: molecular characterization and clinical data

OBJECTIVES: We investigated whether Acinetobacter baumannii isolates of veterinary origin shared common molecular characteristics with those described in humans. METHODS: Nineteen A. baumannii isolates collected in pets and horses were analysed. Clonality was studied using repetitive extragenic palindromic PCR (rep-PCR) and multilocus sequence typing (MLST). PCR and DNA sequencing for various beta-lactamase, aminoglycoside-modifying enzyme, gyrA and parC, ISAba1 and IS1133, adeR and adeS of the AdeABC efflux pump, carO porin and class 1/2/3 integron genes were performed. RESULTS: Two main clones [A (n = 8) and B (n = 9)] were observed by rep-PCR. MLST indicated that clone A contained isolates of sequence type (ST) ST12 (international clone II) and clone B contained isolates of ST15 (international clone I). Two isolates of ST10 and ST20 were also noted. Seventeen isolates were resistant to gentamicin, 12 to ciprofloxacin and 3 to carbapenems. Isolates of ST12 carried bla(OXA-66), bla(ADC-25), bla(TEM-1), aacC2 and IS1133. Strains of ST15 possessed bla(OXA-69), bla(ADC-11), bla(TEM-1) and a class 1 integron carrying aacC1 and aadA1. ISAba1 was found upstream of bla(ADC) (one ST10 and one ST12) and/or bla(OXA-66) (seven ST12). Twelve isolates of different STs contained the substitutions Ser83Leu in GyrA and Ser80Leu or Glu84Lys in ParC. Significant disruptions of CarO porin and overexpressed efflux pumps were not observed. The majority of infections were hospital acquired and in animals with predisposing conditions for infection. CONCLUSIONS: STs and the molecular background of resistance observed in our collection have been frequently described in A. baumannii detected in human patients. Animals should be considered as a potential reservoir of multidrug-resistant A. baumannii.

Emergence of linezolid-resistant Staphylococcus aureus after prolonged treatment of cystic fibrosis patients in Cleveland, Ohio

Linezolid (LZD)-resistant Staphylococcus aureus (LRSA) isolates were monitored from 2000 to 2009 in Cleveland, OH. LRSA first emerged in 2004 only in cystic fibrosis (CF) patients, with 11 LRSA-infected CF patients being identified by 2009. LRSA was isolated from 8 of 77 CF patients with S. aureus respiratory tract infection treated with LZD from 2000 to 2006. Analysis of clinical data showed that the 8 CF patients with LRSA received more LZD courses (18.8 versus 5.9; P = 0.001) for a longer duration (546.5 versus 211.9 days; P < 0.001) and had extended periods of exposure to LZD (83.1 versus 30.1 days/year; P < 0.001) than the 69 with LZD-susceptible isolates. Five LRSA isolates included in the clinical analysis (2000 to 2006) and three collected in 2009 were available for molecular studies. Genotyping by repetitive extrapalindromic PCR and pulsed-field gel electrophoresis revealed that seven of these eight LRSA strains from unique patients were genetically similar. By multilocus sequence typing, all LRSA isolates were included in clonal complex 5 (seven of sequence type 5 [ST5] and one of ST1788, a new single-locus variant of ST5). However, seven different variants were identified by spa typing. According to the Escherichia coli numbering system, seven LRSA isolates contained a G2576T mutation (G2603T, S. aureus numbering) in one to four of the five copies of domain V of the 23S rRNA genes. One strain also contained a mutation (C2461T, E. coli numbering) not previously reported. Two strains, including one without domain V mutations, possessed single amino acid substitutions (Gly152Asp or Gly139Arg) in the ribosomal protein L3 of the peptidyltransferase center, substitutions not previously reported in clinical isolates. Emergence of LRSA is a serious concern for CF patients who undergo prolonged courses of LZD therapy.

Impaired innate interferon induction in severe therapy resistant atopic asthmatic children

Deficient type I interferon-β and type III interferon-λ induction by rhinoviruses has previously been reported in mild/moderate atopic asthmatic adults. No studies have yet investigated if this occurs in severe therapy resistant asthma (STRA). Here, we show that compared with non-allergic healthy control children, bronchial epithelial cells cultured ex vivo from severe therapy resistant atopic asthmatic children have profoundly impaired interferon-β and interferon-λ mRNA and protein in response to rhinovirus (RV) and polyIC stimulation. Severe treatment resistant asthmatics also exhibited increased virus load, which negatively correlated with interferon mRNA levels. Furthermore, uninfected cells from severe therapy resistant asthmatic children showed lower levels of Toll-like receptor-3 mRNA and reduced retinoic acid inducible gene and melanoma differentiation-associated gene 5 mRNA after RV stimulation. These data expand on the original work, suggesting that the innate anti-viral response to RVs is impaired in asthmatic tissues and demonstrate that this is a feature of STRA.

Biochemical characterization of detergent-resistant membranes: a systematic approach

Lateral segregation of cholesterol- and sphingomyelin-rich rafts and glycerophospholipid-containing non-raft microdomains has been proposed to play a role in a variety of biological processes. The most compelling evidence for membrane segregation is based on the observation that extraction with non-ionic detergents leads to solubilization of a subset of membrane components only. However, one decade later, a large body of inconsistent detergent-extraction data is threatening the very concept of membrane segregation. We have assessed the validity of the existing paradigms and we show the following. (i) The localization of a membrane component within a particular fraction of a sucrose gradient cannot be taken as a yardstick for its solubility: a variable localization of the DRMs (detergent-resistant membranes) in sucrose gradients is the result of complex associations between the membrane skeleton and the lipid bilayer. (ii) DRMs of variable composition can be generated by using a single detergent, the increasing concentration of which gradually extracts one protein/lipid after another. Therefore any extraction pattern obtained by a single concentration experiment is bound to be 'investigator-specific'. It follows that comparison of DRMs obtained by different detergents in a single concentration experiment is prone to misinterpretations. (iii) Depletion of cholesterol has a graded effect on membrane solubility. (iv) Differences in detergent solubility of the members of the annexin protein family arise from their association with chemically different membrane compartments; however, these cannot be attributed to the 'brick-like' raft-building blocks of fixed size and chemical composition. Our findings demonstrate a need for critical re-evaluation of the accumulated detergent-extraction data.

An implantable carotid sinus baroreflex activating system: surgical technique and short-term outcome from a multi-center feasibility trial for the treatment of resistant hypertension

OBJECTIVES: To assess perioperative outcomes and blood pressure (BP) responses to an implantable carotid sinus baroreflex activating system being investigated for the treatment of resistant hypertension. METHODS: We report on the first seventeen patients enrolled in a multicenter study. Bilateral perivascular carotid sinus electrodes (CSL) and a pulse generator (IPG) are permanently implanted. Optimal placement of the CSL is determined by intraoperative BP responses to test activations. Acute BP responses were tested postoperatively and during the first four months of follow-up. RESULTS: Prior to implant, BP was 189.6+/-27.5/110.7+/-15.3 mmHg despite stable therapy (5.2+/-1.8 antihypertensive drugs). The mean procedure time was 202+/-43 minutes. No perioperative strokes or deaths occurred. System tests performed 1 or up to 3 days postoperatively resulted in significant (all p < or = 0.0001) mean maximum reduction, with standard deviations and 95% confidence limits for systolic BP, diastolic BP and heart rate of 28+/-22 (17, 39) mmHg, 16+/-11 (10, 22) mmHg and 8+/-4 (6, 11) BPM, respectively. Repeated testing during 3 months of therapeutic electrical activation demonstrated a durable response. CONCLUSIONS: These preliminary data suggest an acceptable safety of the procedure with a low rate of adverse events and support further clinical development of baroreflex activation as a new concept to treat resistant hypertension.

Vancomycin acts synergistically with gentamicin against penicillin-resistant pneumococci by increasing the intracellular penetration of gentamicin

Vancomycin and gentamicin act synergistically against penicillin-resistant pneumococci in vitro and in experimental rabbit meningitis. The aim of the present study was to investigate the underlying mechanism of this synergism. The intracellular concentration of gentamicin was measured by using the following experimental setting. Bacterial cultures were incubated with either gentamicin alone or gentamicin plus vancomycin for a short period (15 min). The gentamicin concentration was determined before and after grinding of the cultures by using the COBAS INTEGRA fluorescence polarization system (Roche). The grinding efficacies ranged between 44 and 54%, as determined by viable cell counts. In the combination regimen the intracellular concentration of gentamicin increased to 186% compared to that achieved with gentamicin monotherapy. These data suggest that the synergy observed in vivo and in vitro is based on an increased intracellular penetration of the aminoglycoside, probably due to the effect of vancomycin on the permeability of the cell wall.

Skeletal muscle cells from insulin-resistant (non-diabetic) individuals are susceptible to insulin desensitization by palmitate

We recently demonstrated that in vivo insulin resistance is not retained in cultured skeletal muscle cells. In the present study, we tested the hypothesis that treating cultured skeletal muscle cells with fatty acids has an effect on insulin action which differs between insulin-sensitive and insulin-resistant subjects. Insulin effects were examined in myotubes from 8 normoglycemic non-obese insulin-resistant and 8 carefully matched insulin-sensitive subjects after preincubation with or without palmitate, linoleate, and 2-bromo-palmitate. Insulin-stimulated glycogen synthesis decreased by 27 +/- 5 % after palmitate treatment in myotubes from insulin-resistant, but not from insulin-sensitive subjects (1.50 +/- 0.08-fold over basal vs. 1.81 +/- 0.09-fold, p = 0.042). Despite this observation, we did not find any impairment in the PI 3-kinase/PKB/GSK-3 pathway. Furthermore, insulin action was not affected by linoleate and 2-bromo-palmitate. In conclusion, our data provide preliminary evidence that insulin resistance of skeletal muscle does not necessarily involve primary defects in insulin action, but could represent susceptibility to the desensitizing effect of fatty acids and possibly other environmental or adipose tissue-derived factors.

Human internal thoracic arteries from diabetic patients are resistant to endothelial dysfunction

The aim of this analysis was to compare vasoreactive properties of internal thoracic arteries (ITA) grafts from diabetic (DM) to those of non-diabetic (ND) patients. Ring segments of ITA, taken from patients undergoing coronary artery bypass grafting, were suspended in organ bath chambers filled with modified Krebs-Henseleit solution and contractile responses to potassium chloride (KCl), noradrenaline (NA), endothelin-1 (ET-l), and endothelium-dependent relaxant responses to acetylcholine (ACH) were recorded isometrically. The receptor-mediated agonists NA and ET-1 stimulated ITA from both groups within similar concentration ranges while ITA from DM patients proved to be significantly more sensitive to KCl than ITA from ND. Furthermore, maximal contractile responses indicated that KCl (3.79 +/- 0.30 g, n = 7 in DM and 2.50 +/- 0.23 g, n = 29 in ND, P < 0.05) evoked significantly higher responses in ITA from DM as compared to the ND control group while both NA and ET-l stimulated ITA from both groups with similar efficacies. Endothelium-dependent relaxant responses to ACH proved to be similar in both groups when expressed as percentages of the pre-existing tone. The present data support the contention that in comparison to ND controls arteries from DM patients are more sensitive to depolarization but endothelial dysfunction is less frequent in human ITA than expected from observations in systemic vascular beds.

Evaluation of pet contact as a risk factor for carriage of multidrug-resistant staphylococci in nursing home residents

BACKGROUND Pets, often used as companionship and for psychological support in the therapy of nursing home residents, have been implicated as reservoirs for antibiotic-resistant bacteria. We investigated the importance of pets as reservoirs of multidrug-resistant (MDR) staphylococci in nursing homes. METHODS We assessed the carriage of MDR staphylococci in pets and in 2 groups of residents, those living in nursing homes with pets and those living without pet contacts. We collected demographic, health status, and human-pet contact data by means of questionnaires. We assessed potential bacteria transmission pathways by investigating physical resident-to-pet contact. RESULTS The observed prevalence of MDR staphylococci carriage was 84/229 (37%) in residents living with pets and 99/216 (46%) in those not living with pets (adjusted odds ratio [aOR], 0.6; 95% confidence interval [CI], 0.4-0.9). Active pet contact was associated with lower carriage of MDR staphylococci (aOR, 0.5; 95% CI, 0.4-0.8). Antibiotic treatment during the previous 3 months was associated with significantly increased risk for MDR carriage in residents (aOR, 3.1; 95% CI, 1.8-5.7). CONCLUSIONS We found no evidence that the previously reported benefits of pet contact are compromised by the increased risk of carriage of MDR staphylococci in residents associated with interaction with these animals in nursing homes. Thus, contact with pets, always under good hygiene standards, should be encouraged in these settings.

Clinical and molecular features of methicillin-resistant, coagulase-negative staphylococci of pets and horses

OBJECTIVES To determine the antibiotic resistance and fingerprint profiles of methicillin-resistant coagulase-negative staphylococci (MRCoNS) from animal infections among different practices and examine the history of antibiotic treatment. METHODS Isolates were identified by mass spectrometry and tested for antimicrobial resistance by broth dilution, microarrays and sequence analysis of the topoisomerases. Diversity was assessed by PFGE, icaA PCR and staphylococcal cassette chromosome mec (SCCmec), arginine catabolic mobile element (ACME) and multilocus sequence typing. Clinical records were examined retrospectively. RESULTS MRCoNS were identified as Staphylococcus epidermidis (n=20), Staphylococcus haemolyticus (n=17), Staphylococcus hominis (n=3), Staphylococcus capitis (n=1), Staphylococcus cohnii (n=1) and Staphylococcus warneri (n=1). PFGE identified one clonal lineage in S. hominis isolates and several in S. haemolyticus and S. epidermidis. Fourteen sequence types were identified in S. epidermidis, with sequence type 2 (ST2) and ST5 being predominant. Ten isolates contained SCCmec IV, seven contained SCCmec V and the others were non-typeable. ACMEs were detected in 11 S. epidermidis isolates. One S. hominis and 10 S. epidermidis isolates were icaA positive. In addition to mecA-mediated β-lactam resistance, the most frequent resistance was to gentamicin/kanamycin [aac(6')-Ie-aph(2')-Ia, aph(3')-III] (n=34), macrolides/lincosamides [erm(C), erm(A), msr, lnu(A)] (n=31), tetracycline [tet(K)] (n=22), streptomycin [str, ant(6)-Ia] (n=20), trimethoprim [dfr(A), dfr(G)] (n=17), sulfamethoxazole (n = 34) and fluoroquinolones [amino acid substitutions in GyrA and GrlA] (n=30). Clinical data suggest selection through multiple antibiotic courses and emphasize the importance of accurate diagnosis and antibiograms. CONCLUSIONS MRCoNS from animal infection sites are genetically heterogeneous multidrug-resistant strains that represent a new challenge in the prevention and therapy of infections in veterinary clinics.

Extended-spectrum cephalosporin-resistant Escherichia coli in community, specialized outpatient clinic and hospital settings in Switzerland

OBJECTIVES Resistance to extended-spectrum cephalosporins (ESCs) in Escherichia coli can be due to the production of ESBLs, plasmid-mediated AmpCs (pAmpCs) or chromosomal AmpCs (cAmpCs). Information regarding type and prevalence of β-lactamases, clonal relations and plasmids associated with the bla genes for ESC-R E. coli (ESC-R-Ec) detected in Switzerland is lacking. Moreover, data focusing on patients referred to the specialized outpatient clinics (SOCs) are needed. METHODS We analysed 611 unique E. coli isolated during September-December 2011. ESC-R-Ec were studied with microarrays, PCR/DNA sequencing for blaESBLs, blapAmpCs, promoter region of blacAmpC, IS elements, plasmid incompatibility group, and also implementing transformation, aIEF, rep-PCR and MLST. RESULTS The highest resistance rates were observed in the SOCs, whereas those in the hospital and community were lower (e.g. quinolone resistance of 22.6%, 17.2% and 9.0%, respectively; P = 0.003 for SOCs versus community). The prevalence of ESC-R-Ec in the three settings was 5.3% (n = 11), 7.8% (n = 22) and 5.7% (n = 7), respectively. Thirty isolates produced CTX-M ESBLs (14 were CTX-M-15), 5 produced CMY-2 pAmpC and 5 hyper-expressed cAmpCs due to promoter mutations. Fourteen isolates were of sequence type 131 (ST131; 10 with CTX-M-15). blaCTX-M and blaCMY-2 were associated with an intact or truncated ISEcp1 and were mainly carried by IncF, IncFII and IncI1plasmids. CONCLUSIONS ST131 producing CTX-M-15 is the predominant clone. The prevalence of ESC-R-Ec (overall 6.5%) is low, but an unusual relatively high frequency of AmpC producers (25%) was noted. The presence of ESC-R-Ec in the SOCs and their potential ability to be exchanged between hospital and community should be taken into serious consideration.

Temporal trends of extended-spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates in in- and outpatients in Switzerland, 2004 to 2011

Increasing trends for invasive infections with extended-spectrum cephalosporin-resistant (ESC-R) Enterobacteriaceae have been described in many countries worldwide. However, data on the rates of ESC-R isolates in non-invasive infections and in the outpatient setting are scarce. We used a laboratory-based nationwide surveillance system to compare temporal trends of ESC-R rates in Escherichia coli and Klebsiella pneumoniae for in- and outpatients in Switzerland. Our data showed a significant increase in ESC-R rates from 1% to 5.8% in E. coli (p<0.001) and from 1.1% to 4.4% in K. pneumoniae (p=0.002) during an eight-year period (2004–2011). For E. coli, the increase was significantly higher in inpatients (from 1.2% to 6.6%), in patients residing in eastern Switzerland (from 1.0% to 6.2%), in patients older than 45 years (from 1.2% to 6.7%), and in male patients (from 1.2% to 8.1%). While the increase in inpatients was linear (p<0.001) for E. coli, the increase of ESC R K. pneumoniae isolates was the result of multiple outbreaks in several institutions. Notably, an increasing proportion of ESC-R E. coli was co-resistant to both trimethoprim-sulfamethoxazole and quinolones (42% in 2004 to 49.1% in 2011, p=0.009), further limiting the available oral therapeutic options.

Characterization of Neisseria gonorrhoeae isolates detected in Switzerland (1998–2012): emergence of multidrug-resistant clones less susceptible to cephalosporins

Background: The spread of Neisseria gonorrhoeae (Ng) isolates resistant to the clinically implemented antibiotics is challenging the efficacy of treatments. Unfortunately, phenotypic and molecular data regarding Ng detected in Switzerland are scarce. Methods: We compared the characteristics of Ng detected during 1998–2001 (n = 26) to those detected during 2009–2012 (n = 34). MICs were obtained with the Etest and interpreted as non-susceptible (non-S) according to EUCAST criteria. Sequence type (ST) was achieved implementing the NG-MAST. BlaTEM, ponA, penA, mtrR, penB, tet (M), gyrA, parC, mefA, ermA/B/C/F, rplD, rplV, and 23S rRNA genes were analyzed. Results: The following susceptibility results were obtained (period: % of non-S, MIC90 in mg/L): penicillin (1998–2001: 42.3%, 3; 2009–2012: 85.3%, 16), cefixime (1998–2001: 0%, ≤0.016; 2009–2012: 8.8%, 0.125), ceftriaxone (1998–2001: 0%, 0.004; 2009–2012: 0%, 0.047), ciprofloxacin (1998–2001: 7.7%, 0.006; 2009–2012: 73.5%, ≥32), azithromycin (1998–2001: 11.5%, 0.25; 2009–2012: 23.6%, 0.38), tetracycline (1998–2001: 65.4%, 12; 2009–2012: 88.2%, 24), spectinomycin (1998–2001: 0%, 12; 2009–2012: 0%, 8). The prevalence of multidrug-resistant (MDR) isolates increased from 7.7% in 1998–2001 to 70.6% in 2009–2012. International STs and genogroups (G) emerged during 2009–2012 (G1407, 29.4%; G2992, 11.7%; G225, 8.8%). These isolates possessed distinctive mechanisms of resistance (e.g., G1407: PBP1 with L421, PBP2 pattern XXXIV, GyrA with S91F and D95G, ParC with S87R, PorB with G120K and A121N, mtrR promoter with A deletion). Conclusions: The prevalence of penicillin- ciprofloxacin- and tetracycline-resistant Ng has reached dramatic levels, whereas cefixime and ceftriaxone show MICs that tend to increase during time. International MDR clones less susceptible to cephalosporins are rapidly emerging indicating that the era of untreatable gonococcal infections is close.