Picroliv preconditioning protects the rat liver against ischemia-reperfusion injury

Cell death following ischemia-reperfusion injury is a major concern in clinical issues such as organ transplantation and trauma. The need to identify agents with a potential for preventing such damage has assumed great importance. We have evaluated the efficacy of picroliv, a potent antioxidant derived from the plant Picrorhiza kurrooa, in protecting against hepatic ischemia-reperfusion injury in vivo. Picroliv was fed to male Sprague Dawley rats in a dose of 12 mg/kg once daily by oral gavage for 7 days prior to hepatic ischemia. Ischemia was induced by occluding the hepatic pedicel with a microaneurysm clip for 30 min and reperfusion was allowed thereafter for varying period (15-120 min) by releasing the microaneurysm clip. Picroliv pretreatment resulted in better hepatocyte glycogen preservation and reduced apoptosis. Reduction in apoptosis was associated with decreased mRNA expression of caspase-3 and Fas. Oxidant induced cellular damage as measured by tissue malondialdehyde (MDA) levels was significantly less following picroliv pretreatment. Both a reduction in neutrophil infiltration and an increased level of intracellular antioxidant enzyme superoxide dismutase possibly contributed to the reduction in tissue lipid peroxidation. Tissue inflammatory cytokines level of interleukin-1alpha (IL-1alpha) and interleukin-1beta (IL-1beta) was also lower in picroliv group. Furthermore, picroliv pretreatment resulted in enhanced proliferating cell nuclear antigen (PCNA) immunoreactivity. These studies strongly suggest picroliv to be a promising agent for ameliorating injury following ischemia-reperfusion.     

Endovascular management of axillofemoral bypass graft stump syndrome

OBJECTIVE: Upper extremity embolic complications of occluded axillofemoral bypass grafts are infrequent. However, traditional management of dissection of axillary anastomosis for removal of the stump can be challenging. We report two patients with critical upper extremity ischemia secondary to stump syndrome and its successful management with endovascular techniques. METHODS: One hundred fifty-two patients underwent axillofemoral bypass grafting over 10 years from 1991-2001. Two patients from this series had acute ischemia involving the ipsilateral upper extremity of occluded axillofemoral bypass graft. Duplex ultrasound scans revealed occlusion of the axillofemoral bypass graft and acute occlusion of ipsilateral upper extremity arteries. Both patients underwent brachial artery exploration and embolectomy. Completion angiograms revealed persistent axillofemoral bypass graft stump as the source of embolus. The stump was obliterated with a 10-mm/40-mm Wallgraft introduced through the same arteriotomy made for brachial embolectomy. Transesophageal echocardiography and magnetic resonance angiography of the arch and great vessels were performed to exclude other sources of origin for the embolus. RESULTS: Both patients remained symptom-free and with patent stent grafts, as seen on duplex scans at 3, 6, and 9 months of follow-up. CONCLUSIONS: Upper extremity embolism is a rare complication after occlusion of axillofemoral bypass grafts. The endovascular approach to obliterate the stump of occluded axillofemoral bypass grafts is minimally invasive and an effective alternative treatment of this rare condition.     

A Trilocus Sequence Typing Scheme for Hospital Epidemiology and Subspecies Differentiation of an Important Nosocomial Pathogen,Enterococcus faecalis

In this study, we present a trilocus sequence typing (TLST) scheme based on intragenic regions of two antigenic genes, ace and salA (encoding a collagen/laminin adhesin and a cell wall-associated antigen, respectively), and a gene associated with antibiotic resistance, lsa (encoding a putative ABC transporter), for subspecies differentiation of Enterococcus faecalis. Each of the alleles was analyzed using 50 E. faecalis isolates representing 42 diverse multilocus sequence types (ST^M; based on seven housekeeping genes) and four groups of clonally linked (by pulsed-field gel electrophoresis [PFGE]) isolates. The allelic profiles and/or concatenated sequences of the three genes agreed with multilocus sequence typing (MLST) results for typing of 49 of the 50 isolates; in addition to the one exception, two isolates were found to have identical TLST types but were single-locus variants (differing by a single nucleotide) by MLST and were therefore also classified as clonally related by MLST. TLST was also comparable to PFGE for establishing short-term epidemiological relationships, typing all isolates classified as clonally related by PFGE with the same type. TLST was then applied to representative isolates (of each PFGE subtype and isolation year) of a collection of 48 hospital isolates and demonstrated the same relationships between isolates of an outbreak strain as those found by MLST and PFGE. In conclusion, the TLST scheme described here was shown to be successful for investigating short-term epidemiology in a hospital setting and may provide an alternative to MLST for discriminating isolates.Enterococci are commensal members of the gastrointestinal tract flora of humans and animals. Within the last 2 decades, enterococci have emerged as the second to third most frequent cause of nosocomial infections, including endocarditis and bloodstream, urinary tract, and wound infections, among others (8, 15, 19, 24, 39). These organisms are also known to have the ability to acquire and transfer antibiotic resistance genes and virulence-associated genes (37). Although there are more than 30 species of the genus Enterococcus, two species, Enterococcus faecalis and Enterococcus faecium, account for a vast majority of enterococcal clinical and nosocomial infections (15, 21, 35). In the past, several molecular typing studies have shown that specific lineages of pathogenic bacteria arise periodically, proliferate, and spread in the presence of selective pressures (34). Therefore, accurate typing of enterococcal strains is crucial for the identification of particular clones capable of causing infections and with the ability to spread in the hospital environment.A number of phenotypic and genotypic typing methods have been applied to the subspecies differentiation of enterococcal isolates. Phenotypic methods which have been used in the past include serotyping (17, 22, 26) and multilocus enzyme electrophoresis (50). Genotypic methods include, among others (3, 52, 53), ribotyping (14, 38), repetitive sequence-based PCR (25, 35), multilocus variable-number tandem-repeat analysis (49, 54), pulsed-field gel electrophoresis (PFGE) (10, 12, 49), and multilocus sequence typing (MLST) (10, 26, 31, 41). Among these methods, PFGE, based on chromosomal restriction endonuclease digestion patterns, is widely used for the study of hospital outbreaks and is considered by many to be the “gold standard” molecular typing technique (48). However, this methodology has several limitations due to the facts that it is labor-intensive and the results have poor interlaboratory transportability. This technique is also unsuitable for long-term epidemiology and population studies due to changes in restriction sites, genomic rearrangements, and/or acquisition of DNA by a clonal lineage, which may markedly change the restriction pattern (41). A more appropriate typing technique for long-term epidemiology, which is currently also widely used for subspecies differentiation, is MLST. MLST, based on the allelic variations in sequences of multiple loci, unambiguously types strains (23) and offers an advantage over other techniques used for typing, such as PFGE, since the data are objective and easily stored, compared, and shared via the Internet.Two different MLST schemes have been used successfully for differentiation of E. faecalis strains (31, 41). The first scheme, which assessed three antigenic genes and one housekeeping gene, found that the allelic profile of two antigenic genes (ace and salA) was sufficient to discriminate the 22 E. faecalis isolates included therein (31). The second MLST scheme, based on the allelic profiles of seven housekeeping genes, was used to type 110 isolates and provided insight into the population structure as well as long-term epidemiological relationships of E. faecalis strains (41). However, typing studies on other organisms, such as Salmonella enterica serovar Typhimurium and Staphylococcus aureus, have suggested that MLST based on housekeeping genes may not provide enough discriminatory power to study hospital outbreaks or to accurately determine short-term genetic relationships, which can be crucial for hospital epidemiology and infection control purposes (9, 13, 27).Our hypothesis for this work was that a sequence-based methodology applied to genes encoding antigenic or cell surface proteins (rather than housekeeping genes) may potentially be more useful to establish short-term epidemiologic relationships in E. faecalis, since these genes would be more susceptible to evolutionary selective pressures and potentially could identify and discriminate isolates from hospital outbreaks, similar to PFGE.In the present work, the trilocus sequence types (ST^T; sequence type based on three genes) of 50 isolates were compared to their multilocus sequence types (ST^M; sequence type based on seven housekeeping genes). To determine the applicability of trilocus sequence typing (TLST) for a clinical setting, the scheme was also used to type sets of predetermined (by PFGE) clones and was then applied to a collection of hospital isolates from Bogota, Colombia, recently reported by Arias et al. to belong to an ST-2 clonal lineage (1).(Part of this work was presented at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2007.)     

Wavelet decomposition of the blink reflex R2 component enables improved discrimination of multiple sclerosis.

OBJECTIVES: The blink reflex R2 component was subjected to wavelet decomposition for time feature extraction in order to classify the functional status of patients with multiple sclerosis. METHODS: The blink reflex was recorded bilaterally with unilateral stimulation of the supra-orbital nerve in 37 normal subjects and 9 patients with multiple sclerosis (MS). The late component, R2, was subjected to time-frequency decomposition using the Daubechies-4 wavelet. Using the time-frequency coefficients, the mean time of the R2 wave as well as the standard deviation of the R2 interval were calculated in each trial. The wavelet transform enables noise reduction by allowing selective use of frequency bands with high signal-to-noise ratio for time feature extraction; therefore automatic estimation of time parameters is robust. The distribution densities of the mean and the standard deviation of the R2 wave duration for the set of trials for each subject were computed. RESULTS: An appreciable difference in the densities of the two parameters extracted in the wavelet domain was seen between normals and patients. This is in contrast to the onset latency of R2 which poorly discriminates MS patients from normals. CONCLUSION: The results suggest that the mean and standard deviation of the R2-time robustly estimated using wavelet decomposition can be used to support clinical diagnosis in tracking the functional status of patients with diseases like multiple sclerosis.     

Surgery for ruptured sinus of valsalva aneurysm into right ventricular outflow tract: role of intraoperative 2D and real time 3D transesophageal echocardiography

A major limitation of the 2D echocardiography during surgery for a complex cardiac lesion is its inability to provide an accurate spatial orientation of the structure. The real time 3D transesophageal echocardiography (RT-3D-TEE) technology available in Philips IE 33 ultrasound machine is relatively new to an operation suite. We evaluated its intraoperative utility in a patient, who was operated for repair of a ruptured sinus of Valsalva aneurysm (RSOVA) and closure of a supracristal ventricular septal defect. The VSD and RSOVA were visualized through different virtual windows in a more promising way on intraoperative RT-3D-TEE than on the 2D echocardiography. The acquired images could be virtually cropped and displayed in anatomical views to the operating surgeon for a clear orientation to the anatomy of the lesion. RT-3D-TEE is a potential intraoperative monitoring tool in surgeries for complex cardiac lesions.     

The influence of the structural orientation of amide linkers on the serum compatibility and lung transfection properties of cationic amphiphiles

Understanding the structural parameters of cationic amphiphiles which can influence gene transfer efficiencies of cationic amphiphiles continues to remain important for designing efficient liposomal gene delivery reagents. Previously we demonstrated the influence of structural orientation of the ester linker (widely used in covalently tethering the polar head and the non-polar tails) in modulating in vitro gene transfer efficiencies of cationic amphiphiles. However, our previously described cationic amphiphiles with ester linkers failed to deliver genes under in vivo conditions. Herein we report on the development of a highly serum compatible cationic amphiphile with circulation stable amide linker which shows remarkable selectivity in transfecting mouse lung. We also demonstrate that reversing structural orientation of the amide linker adversely affects both serum compatibility and the lung selective gene transfer property. Dynamic laser light scattering and atomic force microscopic studies revealed smaller average hydrodynamic sizes of the liposomes of transfection efficient lipid than those for the liposomes of transfection incompetent analog (148 ± 1 nm vs 214 ± 4 nm). Average surface potential of the liposomes of transfection competent amphiphiles were found to be significantly higher than that for the liposomes of transfection incompetent analog (10.7 ± 5.4 mV vs 2.8 ± 1.3 mV, respectively). Findings in fluorescence resonance energy transfer and dye entrapment experiments support lower rigidity and higher biomembrane fusogenicity of the liposomes of the transfection efficient amphiphiles. Importantly, cationic lipoplexes of the novel amide-linker based amphiphile exhibited higher mouse lung selective gene transfer properties than DOTAP, one of the widely used commercially available liposomal lung transfection kits. In summary, the present findings demonstrate for the first time that amide linker structural orientation profoundly influences the serum compatibility and lung transfection efficiencies of cationic amphiphiles.     

Relative contributions of Ebp Pili and the collagen adhesin ace to host extracellular matrix protein adherence and experimental urinary tract infection by Enterococcus faecalis OG1RF

Previous studies have demonstrated that the ebp operon and the ace gene of Enterococcus faecalis, encoding endocarditis- and biofilm-associated pili and an adhesin to collagen of E. faecalis, respectively, are both important in experimental urinary tract infections (UTI) and endocarditis. We have also shown that growth of E. faecalis in brain heart infusion (BHI) serum enhances Ebp pilus and Ace production and increases adherence to several host extracellular matrix proteins. Here, we report that deletion of ebpABC almost eliminated serum-elicited adherence to fibrinogen (P < 0.0001), resulted in moderate reduction in adherence to collagen (P < 0.05), and had no effect on fibronectin adherence relative to that of wild-type OG1RF. An OG1RFΔaceΔebpABC double mutant showed further reduced collagen adherence versus that of the OG1RFΔace or OG1RFΔebpABC mutants (P < 0.001). These results were corroborated by complementation and/or studies with native pilus-enriched surface extracts and a collagen-secreting 3T6 fibroblast cell line, as well as antibody inhibition. In the UTI model, both the OG1RFΔace and OG1RFΔaceΔebpABC mutants were found to be significantly attenuated compared to the wild type; however, no significant differences were observed between individual ace or ebp mutants and the OG1RFΔaceΔebpABC mutant. In summary, these data implicate the Ebp pili as having some role in collagen adherence, albeit less than that of Ace, and a very major role in fibrinogen adherence, which may explain in part the importance of these pili in experimental endocarditis. The OG1RFΔaceΔebpABC mutant was attenuated in the UTI model, although not significantly more so than the Δace or ΔebpABC mutants, suggesting involvement of other E. faecalis factors in urinary tract colonization or infection.