Stereospecific high-performance liquid chromatographic analysis of eriodictyol in urine

A stereospecific method of analysis of eriodictyol [5,7,3',4'-tetrahydroxyflavanone] in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism, and tissue distribution in fruits and humans. A simple high-performance liquid chromatographic method was developed for the stereospecific determination of eriodictyol in rat and human urine. Separation was achieved on a Chiralpak OJ-RH column with UV detection at 288 nm. The stereospecific calibration curves were linear ranging from 0.5 to 100 microg/ml. The mean extraction efficiency was >98.8%. Precision of the assay was <15% (CV), and was within 12% at the limit of quantitation (0.5 microg/ml). Bias of the assay was lower than 8%, and was within 6% at the limit of quantitation. The assay was applied successfully to the urinary excretion of eriodictyol in rats and humans, and to the stereospecific quantification of eriodictyol in raw lemon juice, conventional and organic lemonade.     

Reduction in dietary omega-6 polyunsaturated fatty acids: eicosapentaenoic acid plus docosahexaenoic acid ratio minimizes atherosclerotic lesion formation and inflammatory response in the LDL receptor null mouse

Dietary very long chain omega (omega)-3 polyunsaturated fatty acids (PUFA) have been associated with reduced CVD risk, the mechanisms of which have yet to be fully elucidated. LDL receptor null mice (LDLr-/-) were used to assess the effect of different ratios of dietary omega-6 PUFA to eicosapentaenoic acid plus docosahexaenoic acid (omega-6:EPA+DHA) on atherogenesis and inflammatory response. Mice were fed high saturated fat diets without EPA and DHA (HSF omega-6), or with omega-6:EPA+DHA at ratios of 20:1 (HSF R=20:1), 4:1 (HSF R=4:1), and 1:1 (HSF R=1:1) for 32 weeks. Mice fed the lowest omega-6:EPA+DHA ratio diet had lower circulating concentrations of non-HDL cholesterol (25%, P<0.05) and interleukin-6 (IL-6) (44%, P<0.05) compared to mice fed the HSF omega-6 diet. Aortic and elicited peritoneal macrophage (Mphi) total cholesterol were 24% (P=0.07) and 25% (P<0.05) lower, respectively, in HSF R=1:1 compared to HSF omega-6 fed mice. MCP-1 mRNA levels and secretion were 37% (P<0.05) and 38% (P<0.05) lower, respectively, in elicited peritoneal Mphi isolated from HSF R=1:1 compared to HSF omega-6 fed mice. mRNA and protein levels of ATP-binding cassette A1, and mRNA levels of TNFalpha were significantly lower in elicited peritoneal Mphi isolated from HSF R=1:1 fed mice, whereas there was no significant effect of diets with different omega-6:EPA+DHA ratios on CD36, Mphi scavenger receptor 1, scavenger receptor B1 and IL-6 mRNA or protein levels. These data suggest that lower omega-6:EPA+DHA ratio diets lowered some measures of inflammation and Mphi cholesterol accumulation, which was associated with less aortic lesion formation in LDLr-/- mice.     

The value of microsurgery in liver research

The use of an operating microscope in rat liver surgery makes it possible to obtain new experimental models and improve the already existing macrosurgical models. Thus, microsurgery could be a very valuable technique to improve experimental models of hepatic insufficiency. In the current review, we present the microsurgical techniques most frequently used in the rat, such as the portacaval shunt, the extrahepatic biliary tract resection, partial and total hepatectomies and heterotopic and orthotopic liver transplantation. Hence, reducing surgical complications allows for perfecting the resulting experimental models. Thus, liver atrophy related to portacaval shunt, prehepatic portal hypertension secondary to partial portal vein ligation, cholestasis by resection of the extrahepatic biliary tract, hepatic regeneration after partial hepatectomies, acute liver failure associated with subtotal or total hepatectomy and finally complications derived from preservation or rejection in orthotopic and heterotopic liver transplantation can be studied in more standardized experimental models. The results obtained are therefore more reliable and facilitates the flow of knowledge from the bench to the bedside. Some of these microsurgical techniques, because of their simplicity, can be performed by researchers without any prior surgical training. Other more complex microsurgical techniques require in‐depth surgical training. These techniques are ideal for achieving a complete surgical training and more select microsurgical models for hepatology research.     

Panoramic View of The Fifth International Symposium on Stem Cell Therapy and Applied Cardiovascular Biotechnology, April 2008, Madrid (Spain)

The Fifth International Symposium on Stem Cell Therapy and Applied Cardiovascular Biotechnology was held on April 24th–25th, 2008, at the Auditorium of the High Council of Scientific Research of Spain (CSIC) in Madrid, as a continuation of a series of yearly meetings, organized in an attempt to encourage translational research in this field and facilitate a positive interaction among experts from several countries, along with industry representatives and journalists. In addition, members of the Task Force of the European Society concerning the clinical investigation of the use of autologous adult stem cells for repair of the heart gathered and discussed an update of the previous consensus, still pending of publication. In this article, we summarize some of the main topics of discussion, the state-of-the-art and latest advances in this field, and new challenges brought up for the near future.     

<Emphasis Type="Italic">In Vitro</Emphasis> Comparison of the Effect of Stent Configuration on Wall Shear Stress Using Time-resolved Particle Image Velocimetry

Time resolved particle image velocimetry was used to measure wall shear stress (WSS) and oscillatory shear index (OSI) within a 3.0 mm diameter compliant vessel model implanted with an Abbott Vascular XIENCE V^® stent in five configurations: baseline, over-expanded, increased vessel diameter, two overlapped stents, and increased stent length. Flow through unstented vessels was also tested for comparison. Flow conditions featured a realistic coronary pressure-flow offset and reversal at average flow rates corresponding to resting (Re = 160, f = 70 bpm) and exercise conditions (Re = 300, f = 120 bpm). Comparisons revealed that the WSS was similar for all cases behind the first strut and downstream of the device, indicating that changes in configuration have little effect downstream. However, there were notable differences within each stent revealing reduced WSS values for all cases due to the stent-imposed expansion of the vessel wall (0.20–9.29 dynes/cm² for Re = 160 and d = 3.0 mm). Over-expanding the stent with a second balloon affected the alignment of the stent geometry, and led to higher WSS at the inlet and lower values at mid-stent. The overlapped stents showed disturbed flow and a WSS deficit region downstream of the overlapped region. Analysis of the longer stent showed that the WSS within the vessel recovers with distance. An overall correlation was noted between decreased WSS values and elevated OSI. Results of this study are important because decreased WSS has been implicated in endothelial cell changes and increased restenosis, and clinical research has shown that a link exists between deployment configurations and negative patient outcomes.     

Genomic Signatures of the Haarlem Lineage of Mycobacterium tuberculosis: Implications of Strain Genetic Variation in Drug and Vaccine Development

Tuberculosis is the world''s leading cause of death due to a single infectious agent, and efforts aimed at its control require a better understanding of host, environmental, and bacterial factors that govern disease outcome. Growing evidence indicates that certain Mycobacterium tuberculosis strains of distinct phylogeographic lineages elicit unique immunopathological events. However, identifying the genetic basis of these phenotypic peculiarities has proven difficult. Here we report the presence of six large sequence polymorphisms which, together with two single-nucleotide changes previously described by our group, consistently differentiate Haarlem strains from the remaining M. tuberculosis lineages. The six newly found Haarlem-specific genetic events are four deletions, which altogether involve more than 13 kb, and two intragenic insertions of the element IS6110. The absence of the genes involved in these polymorphisms could have an important physiological impact on Haarlem strains, i.e., by affecting key genes, such as Rv1354c and cyp121, which have been recently proposed as plausible drug targets. These lineage-specific polymorphisms can serve as genetic markers for the rapid PCR identification of Haarlem strains, providing a useful tool for strain surveillance and molecular epidemiology studies. Strain variability such as that described here underscores the need for the definition of a core set of essential genes in M. tuberculosis that are ubiquitously present in all circulating lineages, as a requirement in the development of effective antituberculosis drugs and vaccines.Mycobacterium tuberculosis is the causative agent of tuberculosis, the leading cause of death by a single bacterial agent in the world (36). Infection with M. tuberculosis has historically shown to result in a variety of clinical outcomes that are usually associated with host inherited susceptibility and environmental risk factors (2, 31, 32). Moreover, increasing evidence suggests that genetic variation in the tubercle bacilli also plays an important role in the outcome of the disease (4, 19, 33). Due to the absence of exchange of genetic material with a global microbial gene pool, M. tuberculosis had long been considered to have a clonal population structure. However, a significant strain-to strain genetic variation within M. tuberculosis has recently been unveiled (11, 19).Changes in neutral regions of the chromosome, such as the direct repeat (DR) locus, and in the mycobacterial interspersed repetitive units (MIRUs) are useful in epidemiological and phylogenetic analyses and in describing the most conspicuous M. tuberculosis lineages (3, 21). In addition to the variation in neutral regions, genetic polymorphisms involving coding regions have been described to occur through single-nucleotide changes and through deletion and insertion events, the latter mediated mainly by the IS6110 element (23, 30). Although these genomic alterations are thought to be among the principal sources of phenotypic variation in M. tuberculosis, the specific genomic changes that define each lineage have not yet been fully defined.There are currently six phylogeographic lineages that make up the M. tuberculosis global population (10). One is the Euro-American group, which includes all the spoligotype families predominating in the Western world, such as Haarlem, LAM, and the ill-defined T group (3). In particular, the Haarlem genotype is ubiquitous worldwide (15) and represents about 25% of the isolates in Europe, Central America, and the Caribbean, suggesting a link with the post-Columbus European colonization (8). Haarlem strains are actively transmitted in urban settings in Colombia, causing major public health problems (N. E. Correa, E. Zapata, V. Gómez, G. E. Mejia, A. Restrepo, J. Robledo, and CCITB, presented at the 107th General Meeting of the American Society for Microbiology, Toronto, Canada, 2007) and have also been responsible for a prolonged outbreak of multidrug-resistant tuberculosis in Argentina (26, 29).An intriguing question is whether M. tuberculosis strains differ in terms of pathogenic characteristics as a consequence of long-standing interactions of particular lineages with specific human populations. Animal models that take advantage of an identical genetic background, and therefore a uniform host immune response, have given insight regarding the contribution of strain genetic diversity to the outcome of the infectious process (7, 20). It is currently accepted that genetically different M. tuberculosis strains produce markedly different immunopathological events in isogenic mice (4, 18). Thus, understanding genotypic differences and mechanisms underlying infection variability and identifying specific changes or genes associated with both virulence and immunopathogenicity of the different M. tuberculosis lineages have important implications for the future effective control of tuberculosis (7, 33).In a recent bioinformatic study using multiple genome alignments of six fully sequenced M. tuberculosis strains belonging to different lineages, we showed a trend toward accumulation of a limited number of genome-specific polymorphisms preferentially associated with circulating strains and underrepresented in laboratory strains. This suggests that such polymorphisms arise as active mechanisms of adaptation to the human host (5). We speculated that some of these genome-specific polymorphisms might be common to strains of a particular lineage rather than being an exclusive property of the isolate examined. To test this, in the present study we examined whether genome-specific polymorphisms previously identified in fully sequenced strains were present in a broader group of strains and could thus represent a lineage-wide condition. In particular, we explored whether polymorphisms identified as specific to the sequenced M. tuberculosis Haarlem strain (5) were prevalent in additional members of the Haarlem lineage and absent from other lineages. In the present paper, we report the presence of eight genomic signatures highly exclusive to the M. tuberculosis Haarlem lineage that can prove important for the rapid identification of these strains and also contribute to our understanding of the genetic variations underlying phenotypic differences among the various lineages of the tubercle bacilli.     

EBNA1 sequences in Argentinean pediatric acute and latent Epstein–Barr virus infection reflect circulation of novel South American variants

Epstein–Barr virus (EBV) is related to the development of lymphomas and is also the etiological agent for infectious mononucleosis (IM). Sequence variation of the EBNA1 gene, consistently expressed in all EBV‐positive cells, has been widely studied. Based on the amino acid at codon 487 five major EBNA1 variants have been described, two closely related prototypic variants (P‐ala and P‐thr) and three variant sequences (V‐leu, V‐val, and V‐pro). Sub‐variants were then further classified based on mutations other than the originally described. While several studies proposed associations with tumors and/or anatomical compartments, others argued in favor of a geographical distribution of these variants. In the present study, EBNA1 variants in 11 pediatric patients with IM and 19 pediatric EBV lymphomas from Argentina were compared as representatives of benign and malignant infection in children, respectively. A 3‐month follow‐up study of EBNA1 variants in peripheral blood cells and in oral secretions of patients with IM was performed. A new V‐ala variant which includes five V‐ala sub‐variants and three new V‐leu sub‐variants was described. These data favor the geographical association hypothesis since no evidence for a preferential compartment distribution of EBNA1 variants and sub‐variants was found. This is the first study to characterize EBNA1 variants in pediatric patients with infection mononucleosis worldwide. J. Med. Virol. 82:1730–1738, 2010. © 2010 Wiley‐Liss, Inc.     

Human caliciviruses detected in Mexican children admitted to hospital during 1998–2000, with severe acute gastroenteritis not due to other enteropathogens

Few studies exist regarding the frequency of human caliciviruses as single etiologic agents in sporadic cases, or in outbreaks occurring in children hospitalized for acute gastroenteritis. In this study, a total of 1,129 children of <5 years of age and hospitalized due to acute diarrhea were enrolled from three main hospitals in Mexico City during a period of 3 years (March 1998 to December 2000). After analyzing all fecal samples for several enteropathogens, 396 stools that remained negative were further screened for human caliciviruses by RT‐PCR using a primer set specific to norovirus and sapovirus. Human caliciviruses were detected in 5.6% (22/396) of the children. The minimum incidence rate for 1999 were 5.3% (7/132) for 1999 and 7.8% (13/167) for 2000, since only fecal specimens that tested negative to other enteric pathogens were examined. Positive samples were further characterized using specific GI and GII primers and sequencing. Norovirus GII was detected in 19/22 samples, most of them were GII/4, while sapovirus GI/2 was detected in one sample. Associations between the presence of human calicivirus and clinical and epidemiological data revealed that diarrhea occurred with a seasonal pattern, and that children hospitalized due to human calicivirus disease scored an average of 13 ± 3.2 (SD) points on the Vesikari scale, which corresponded to severe episodes. These results highlight that human caliciviruses, by themselves, are enteropathogens of acute severe diarrhea among young Mexican children requiring hospitalization and that their detection is important in order to reduce the diagnosis gap. J. Med. Virol. 82:632–637, 2010. © 2010 Wiley‐Liss, Inc.     

Use of titanium mesh for staged localized alveolar ridge augmentation: clinical and histologic-histomorphometric evaluation

The use of titanium mesh for localized alveolar ridge augmentation was evaluated by clinical, radiographic, laboratory, and histologic-histomorphometric evaluation. Seventeen patients participated in this study. All patients required localized alveolar ridge augmentation before placement of dental implants. An equal mixture of autogenous bone graft and inorganic bovine mineral (Bio-Oss) was used as a bone graft material. Autogenous bone graft was harvested intraorally. Titanium mesh was submerged for 8.47 months (SD 2.83). Impressions were taken intraorally before bone grafting, 6 months after bone grafting, and 6 months after implant placement. Impressions were used to measure the volume of alveolar ridge augmentation and provide linear laboratory measurements regarding the results of bone augmentation. Bone quality (type II-IV) was recorded during implant surgery. Standardized linear tomographs were taken before bone grafting and before implant placement. A biopsy was harvested with a trephine bur from the grafted area during implant surgery for histologic-histomorphometric evaluation. In all cases the grafted area had adequate bone volume and consistency for placement of dental implants. Early mesh exposure (2 weeks) was observed in 2 patients, and late exposure (>3 months) was observed in 4 patients. Volumetric laboratory measurements indicated 0.86 cc (SD 0.69) alveolar augmentation 1 month after bone grafting, 0.73 cc (SD 0.60) 6 months after bone grafting, and 0.71 cc (SD 0.57) 6 months after implant placement. This indicated 15.11% resorption 6 months after bone grafting, and no further resorption occurred after implant placement. Linear laboratory measurements indicated vertical augmentation of 2.94 mm (SD 0.86) 1 month after bone grafting, 2.59 mm (SD 0.91) 6 months after bone grafting, and 2.65 mm (SD 1.14) 6 months after implant placement. The corresponding measurements for labial-buccal augmentation were 4.47 mm (SD 1.55), 3.88 mm (SD 1.43), and 3.82 mm (SD 1.47). Radiographic evaluation indicated 2.56 mm (SD 1.32) vertical augmentation and 3.75 mm (SD 1.33) labial-buccal augmentation. Histomorphometric evaluation indicated 36.47% (SD 10.05) new bone formation, 49.18% (SD 6.92) connective tissue, and 14.35% (SD 5.85) residual Bio-Oss particles; 44.65% (SD 22.58) of the Bio-Oss surface was in tight contact with newly formed bone. The use of titanium mesh for localized alveolar ridge augmentation with a mixture of autogenous intraorally harvested bone graft and Bio-Oss offered adequate bone volume for placement of dental implants. Intraorally harvested autogenous bone graft mixed with Bio-Oss under a titanium mesh offered 36.47% new bone formation, and 15.11% resorption occurred 6 months after bone grafting.     

Chronic graft versus host disease of oral mucosa: review of available therapies

The use of hematopoetic stem cell transplantation (HSCT) has greatly expanded in the recent years for many neoplastic and hematological disorders. Chronic graft versus host disease (cGVHD) is a major complication of allogeneic HSCT and a major cause of morbidity and mortality. Oral mucosal involvement is frequent in cGVHD and contributes significantly to the overall burden of the condition. Oral medicine professionals should be familiar with various treatment options for oral cGVHD. This review discusses treatment modalities available for the management of oral mucosal manifestations of cGVHD. Available evidence for efficacy and safety of various systemic and topical agents, including corticosteroids, calcineurin antagonists, mycophenolate mofetil, and extracorporeal photopheresis, is reviewed.