Collagen is reduced and disrupted in human aneurysms and dissections of ascending aorta

In ascending aorta aneurysms, there is an enlargement of the whole vessel, whereas aortic dissections (ADs) are characterized by the cleavage of the wall into 2 sheets at the external half. We searched if alterations in collagen could be related to these diseases. Sections of aortas from 14 case patients with acute dissections, 10 case patients with aneurysms, and 9 control subjects were stained with picrosirius. Slides were analyzed under polarized microscopy to evaluate the structure of collagen fibers. The proportion of collagen was calculated in each half of the medial layer by color detection in a computerized image analysis system. Collagen appearance under polarized light was consistent with collagenolysis. The mean collagen proportions at the inner and outer halves, respectively, were 0.50 +/- 0.13 and 0.40 +/- 0.08 in the control group, 0.20 +/- 0.10 and 0.18 +/- 0.12 in the AD group, and 0.33 +/- 0.12 and 0.19 +/- 0.12 in the aneurysm group. The AD (P < .01) and control (P = .04) groups had less collagen at the external half, no difference was found in the aneurysm group (P = .71). In both halves, there was less collagen in the case patients than in the control subjects (all P < .01), but at the internal half, the decrease was significantly greater in the case patients with aneurysms than in those with dissections (P = .03; at the external half, P = .99). Aortic dissections and aneurysms show a decrease in collagen content that could be related to a weakness of the wall underlying the diseases, but the locations of the decrease differ: in dissections, it is situated mostly at the external portion of the media (site of cleavage), whereas in aneurysms, it is more diffuse, consistent with the global enlargement. (c) 2008 Elsevier Inc. All rights reserved.

A quantitative chemiluminescent method for studying replicative and stress-induced premature senescence in cell cultures

beta-Galactosidase (beta-Gal) activity is a widely accepted biomarker to detect senescence both in situ and in vitro. A cytochemical assay based on production of a blue-dyed precipitate that results from the cleavage of the chromogenic substrate X-Gal is commonly used. Blue and nonblue cells are counted under the microscope and a semiquantitative percentage of senescent cells can be obtained. Here, we present a quantitative, fast, and easy to use chemiluminescent assay to detect senescence. The Galacton chemiluminescent method used to detect the prokaryotic beta-Gal reporter enzyme in transfection studies was adapted to assay mammalian beta-Gal. The assay showed linear production of luminescence in a time- and cell-number-dependent manner. The chemiluminescent assay showed significant correlation with the cytochemical assay in detecting replicative senescence (Pearson r = 0.8486, p < 0.005). Moreover, the chemiluminescent method (Galacton) also detected stress-induced senescence in cells treated with H2O2 similar to the cytochemical assay (X-Gal) (Galacton: control 25.207.3 +/- 6548.6. H2O, 52,487.4 +/- 16,284.9, p < 0.05; X-Gal: control 41.31 +/- 7.0%, H2O2 92.97 +/- 2.8%, p < 0.01). Thus, our method is well suited to the detection of replicative and stress-induced senescence in cell culture. (C) 2007 Elsevier Inc. All rights reserved.

Peritumoral brain edema in benign meningiomas: correlation with clinical, radiologic, and surgical factors and possible role on recurrence

Background: Approximately 60% of meningiomas are associated with peritumoral edema. Various causative factors have been discussed in the literature. The objective of this study was to investigate the correlation of PTBE with clinical, radiologic, and surgical aspects and recurrence of meningiomas. Methods: Sixty-one patients with benign meningiomas were chosen for surgical treatment by the Group of Brain Tumors and Metastasis of the Department of Neurosurgery. All patients underwent complete surgical resection (Simpson grades I and 2), and those with atypical and malignant histopathologic grades were excluded. Tumors located in the cavernous sinus, tuberculum sellae, foramen magnum, ventricles, and petroclival region were excluded. Results: Edema extension had a positive correlation with the higher recurrence rates (P=.042) and with the presence of irregular margins (P<.011) on bivariate analysis. Meningiomas with larger edema sizes also showed correlation with large meningiomas (P=.035), and the ones with smaller edema sizes correlated with the tentorial location (P=.032). Multivariate analysis showed an association between PTBE and the presence of seizures (odds ratio, 3.469), large meningiomas (odds ratio, 15.977), and for each cubic centimeter added to its size, the risk of edema increased 1.082 times (odds ratio). Conclusion: Peritumoral brain edema may be related to the invading potential of meningiomas and may play a role in the recurrence potential of the tumor. As a consequence, it is reasonable to consider the presence of edema as an additional factor to be taken into account when mapping out strategies for the treatment of meningiomas. (C) 2008 Elsevier Inc. All rights reserved.

Acute intermittent porphyria: the in vitro expression of mutant hydroxymethylbilane synthase

Acute intermittent porphyria (AIP) is an inborn error of haem biosynthesis caused by a variety of mutations in the gene coding for hydroxymethylbilane synthase (HMB-S). The entire coding sequence of this gene, from each of three South African AIP patients, was therefore screened for mutations using chemical cleavage mismatch (CCM) analysis and any changes detected characterized by DNA sequencing. Three single base changes were identified; a G(77) to A in exon 3, a C-346 to T in exon 8 and a G(518) to A in exon 10. These missense mutations, previously reported to be present in other populations, are known to be responsible for the structurally deleterious amino acid replacements R26H, R116W and R173Q, respectively. The in vitro expression of the enzymes containing these mutations and the subsequent measurement of their specific activities revealed a reduction to approximately 4% of normal activity. (C) 1997 Academic Press Limited.

Study of direct immunofluorescence, immunofluorescence mapping and light microscopy in porphyria cutanea tarda

BACKGROUND: Even though porphyria cutanea tarda is the most frequent type of porphyria, there are few studies about its cutaneous physiopathology. OBJECTIVE: To evaluate skin changes in porphyria cutanea tarda using light microscopy and direct immunofluorescence before and after treatment with chloroquine. To perform antigen immunomapping of bullae to study their level of cleavage. METHODS: Light microscopy and direct immunofluorescence of 28 patients are reported in three different phases: 23 patients with active porphyria before treatment (Phase A), 7 patients with clinical remission during treatment (Phase B), and 8 patients with biochemical remission (Phase C). Immunomapping was performed on 7 patients. RESULTS: In active porphyria, direct immunofluorescence showed homogenous and intense fluorescence on the inside and on the walls of blood vessels as well as in the dermal-epidermal junction. In clinical remission (Phase B) and biochemical remission (Phase C), the deposit of immunoglobulins was maintained, but the deposit of complement was reduced in most cases. Immunomapping revealed no standard cleavage plane. CONCLUSION: No correlation was observed between clinical response and immunoglobulin deposits. The reduction of complement favors the hypothesis that activation of the complement cascade represents an additional pathway that leads to endothelial damage.

Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification

Objective: To compare cryopreservation of mature human oocytes with slow-rate freezing and vitrification and determine which is most efficient at establishing a pregnancy. Design: Prospective randomized. Setting: Academically affiliated, private fertility center. Patient(s): Consenting patients with concerns about embryo cryopreservation and more than nine mature oocytes at retrieval were randomized to slow-rate freezing or vitrification of supernumerary (more than nine) oocytes. Intervention(s): Oocytes were frozen or vitrified, and upon request oocytes were thawed or warmed, respectively. Main Outcome Measure(s): Oocyte survival, fertilization, embryo development, and clinical pregnancy. Result(s): Patient use has resulted in 30 thaws and 48 warmings. Women`s age at time of cryopreservation was similar. Oocyte survival was significantly higher following vitrification/warming (81%) compared with freezing/thawing (67%). Fertilization was more successful in oocytes vitrified/warmed compared with frozen/thawed. Fertilized oocytes from vitrification/warming had significantly better cleavage rates (84%) compared with freezing/thawing (71%) and resulted in embryos with significantly better morphology. Although similar numbers of embryos were transferred, embryos resulting from vitrified oocytes had significantly enhanced clinical (38%) pregnancy rates compared with embryos resulting from frozen oocyte (13%). Miscarriage and/or spontaneous abortion rates were similar. Conclusion(s): Our results suggest that vitrification/warming is currently the most efficient means of oocyte cryopreservation in relation to subsequent success in establishing pregnancy. (Fertil Steril (R) 2010; 94: 2088-95. (C) 2010 by American Society for Reproductive Medicine.)

Associations of serious mental illness with earnings: results from the WHO World Mental Health surveys

Background Burden-of-illness data, which are often used in setting healthcare policy-spending priorities, are unavailable for mental disorders in most countries. Aims To examine one central aspect of illness burden, the association of serious mental illness with earnings, in the World Health Organization (WHO) World Mental Health (WMH) Surveys. Method The WMH Surveys were carried out in 10 high-income and 9 low- and middle-income countries. The associations of personal earnings with serious mental illness were estimated. Results Respondents with serious mental illness earned on average a third less than median earnings, with no significant between-country differences (chi(2)(9)=5.5-8.1, P=0.5-0.79). These losses are equivalent to 0.3-0.8% of total national earnings. Reduced earnings among those with earnings and the increased probability of not earning are both important components of these associations: Conclusions These results add to a growing body of evidence that mental disorders have high societal costs. Decisions about healthcare resource allocation should take these costs into consideration.

Cost-effectiveness analysis of routine rotavirus vaccination in Brazil

Objective. The objective of this study was to conduct a cost-effectiveness analysis of a universal rotavirus vaccination program among children : 5 years of age in Brazil. Methods. Considering a hypothetical annual cohort of approximately 3 300 000 newborns followed over 5 years, a decision-tree model was constructed to examine the possible clinical and economic effects of rotavirus infection with and without routine vaccination of children. Probabilities and unit costs were derived from published research and national administrative data. The impact of different estimates for key parameters was studied using sensitivity analysis. The analysis was conducted from both healthcare system and societal perspectives. Results. The vaccination program was estimated to prevent approximately 1735 351 (54%) of the 3 210 361 cases of rotavirus gastroenteritis and 703 (75%) of 933 rotavirus-associated deaths during the 5-year period. At a vaccine price of 18.6 Brazilian reais (R$) per dose, this program would cost R$121 673 966 and would save R$38 536 514 in direct costs to the public healthcare system and R$71 778 377 in direct and indirect costs to society. The program was estimated to cost R$1 028 and R$1 713 per life-years saved (LYS)from the societal and healthcare system perspectives, respectively. Conclusions. Universal rotavirus vaccination was a cost-effective strategy for both perspectives. However, these findings are highly sensitive to diarrhea incidence rate, proportion of severe cases, vaccine coverage, and vaccine price.

Angiotensin processing is partially carried out by carboxypeptidases in the rat mesenteric arterial bed perfusate

Here we investigated the possible association between the carboxypeptidase A (CPA)-like activity of the rat mesenteric arterial bed (MAB) perfusate and the ability of this fluid of forming angiotensin (Ang) 1-9 and Ang 1-7 upon incubation with Ang I and Ang II, respectively. Initially, we observed that anion exchange chromatography of the perfusate would consistently split the characteristic Z-Val-Phe-hydrolyzing activity of CPA-like enzymes into five distinct peaks, whose proteolytic activities were then determined using also Ang I and Ang II as substrates. The resulting proteolytic profile for each peak indicated that rat MAB perfusate contains a complex mixture of carboxypeptidases; tentatively, five carboxypeptidases were distinguished based on their substrate preferences toward Z-Val-Phe. Ang I and Ang II. The respective reactions, namely, Z-Val-Phe cleavage, Ang I to Ang 1-9 conversion and Ang II to Ang 1-7 conversion, were inhibited by 1,10-phenanthroline and nearly fully blocked by potato carboxypeptidase inhibitor. Also, all the CPA-like activity peaks prepared by anion exchange chromatography were tested negative for contaminating Ang I-converting enzyme-2, cathepsin A and prolylcarboxypeptidase. Overall, our results indicate that rat MAB perfusate contains a multiplicity of Ang I and Ang II-processing CPA-like enzymes whose proteolytic specificities suggest they might perform peculiar regulatory roles in the local resin-angiotensin system. (C) 2008 Elsevier B.V. All rights reserved.

CXCL12 rs1801157 Polymorphism in Patients with Breast Cancer, Hodgkin`s Lymphoma, and Non-Hodgkin`s Lymphoma

Chemokines and their receptors regulate the trafficking of immune cells during their development, inflammation, and tissue repair. The single-nucleotide polymorphism (SNP) rs1801157 (previously known as CXCL12-A/ stromal cell-derived factor-1 (SDF1)-3`A) in CXCL12/SDF1 gene was assessed in breast cancer, Hodgkin`s lymphoma (HL), and non-Hodgkin`s lymphoma (NHL), since the chemokine CXCL12, previously known as SDF1, and its receptor CXCR4 regulate leukocyte trafficking and many essential biological processes, including tumor growth, angiogenesis, and metastasis of different types of tumors. Genotyping was performed by PCR-RFLP (polymerase chain reaction followed by restriction fragment length polymorphism) using a restriction enzyme Hpall cleavage. No significant difference was observed in genotype distribution between breast cancer patients (GG: 57.3%; GA: 39.8%; AA: 2.9%) and healthy female controls (GG: 62.9%; GA: 33%; AA: 4.1%) nor between HL patients (GG: 61.1%; GA:27.8%; AA: 11.1%) and healthy controls (GG: 65.6%; GA: 28.9%; AA: 5.5%), whereas a significant difference was observed in genotype distribution between NHL patients (GG: 51.4%; GA: 47.1%; AA: 1.5%) and healthy controls (GG: 65.6%; GA: 28.9%; AA: 5.5%). Further studies will be necessary to elucidate the cancer chemokine network. However, this study suggests that CXCL12 rs1801157 polymorphism may have important implications in the pathogenesis of NHL. J. Clin. Lab. Anal. 23:387-393, 2009. (C) 2009 Wiley-Liss, Inc.

Analysis of Intracellular Substrates and Products of Thimet Oligopeptidase in Human Embryonic Kidney 293 Cells

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme that has been proposed to metabolize peptides within cells, thereby affecting antigen presentation and G protein-coupled receptor signal transduction. However, only a small number of intracellular substrates of EP24.15 have been reported previously. Here we have identified over 100 peptides in human embryonic kidney 293 (HEK293) cells that are derived from intracellular proteins; many but not all of these peptides are substrates or products of EP24.15. First, cellular peptides were extracted from HEK293 cells and incubated in vitro with purified EP24.15. Then the peptides were labeled with isotopic tags and analyzed by mass spectrometry to obtain quantitative data on the extent of cleavage. A related series of experiments tested the effect of overexpression of EP24.15 on the cellular levels of peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10 of the cellular peptides were incubated with purified EP24.15 in vitro, and the cleavage was monitored by high pressure liquid chromatography and mass spectrometry. Many of the EP24.15 substrates identified by these approaches are 9-11 amino acids in length, supporting the proposal that EP24.15 can function in the degradation of peptides that could be used for antigen presentation. However, EP24.15 also converts some peptides into products that are 8-10 amino acids, thus contributing to the formation of peptides for antigen presentation. In addition, the intracellular peptides described here are potential candidates to regulate protein interactions within cells.

Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules

In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24 h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured it, TCM plus FSH and 10% estrous cow serum. After fertilization. presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P < 0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless. there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis. (C) 2009 Elsevier Inc. All rights reserved.

Supplemented tissue culture medium 199 is a better medium for in vitro maturation of oocytes from women with polycystic ovary syndrome women than human tubal fluid

Objective: To compare oocyte maturation, fertilization and cleavage rates, and embryonic developmental quality after culture of human immature oocytes from polycystic ovary syndrome (PCOS) patients in human tubal fluid (HTF) or tissue culture medium (TCM) 199. Design: Prospective, randomized, controlled trial. Setting: University hospital. Patient(s): Thirteen women undergoing 23 in vitro maturation cycles, from whom 119 oocytes were retrieved. Intervention(s): Cumulus-enclosed germinal vesicle-stage oocytes matured in TCM-199-supplemented or HTF-supplemented media. Main Outcome Measure(s): Oocyte maturation and fertilization rates, embryonic developmental quality. Result(s): Significant differences were observed between TCM 199 and HTF regarding maturation rate (82% vs. 56.9%), fertilization rate (70% vs. 39.4%), and embryo quality (81.3% vs. 41.7%). Conclusion(S): Human tubal fluid medium, although widely used for embryo fertilization and maintenance in IVF techniques, is not,in appropriate medium for the maturation of oocytes obtained from PCOS patients in nonstimulated cycles. (Fertil Steril(R) 2009;91:509-13. (C)2009 by American Society for Reproductive Medicine.)

Reliability of short comparative genomic hybridization in fibroblasts and blastomeres for a comprehensive aneuploidy screening: first clinical application

BACKGROUND: Comparative genomic hybridization (CGH) is a valuable alternative to fluorescence in situ hybridization (FISH) for preimplantation genetic screening (PGS) because it allows full karyotype analysis. However, this approach requires the cryopreservation of biopsied embryos until results are available. The aim of this study is to reduce the hybridization period of CGH, in order to make this short-CGH technique suitable for PGS of Day-3 embryos, avoiding the cryopreservation step. METHODS: Thirty-two fibroblasts from six aneuploid cell lines (Coriell) and 48 blastomeres from 10 Day-4 embryos, discarded after PGS by FISH with 9 probes (9-chr-FISH), were analysed by short-CGH. A reanalysis by the standard 72 h-CGH and FISH using telomeric probes was performed when no concordant results between short-CGH and FISH diagnosis were observed. The short-CGH was subsequently applied in a clinical case of advanced maternal age. RESULTS: In 100% of the fibroblasts analysed, the characteristic aneuploidies of each cell line were detected by short-CGH. The results of the 48 blastomeres screened by short-CGH were supported by both 72 h-CGH results and FISH reanalysis. The chromosomes most frequently involved in aneuploidy were 22 and 16, but aneuploidies for the other chromosomes, excepting 1, 10 and 13, were also detected. Forty-one of the 94 aneuploid events observed (43.6%) corresponded to chromosomes which are not analysed by 9-chr-FISH. CONCLUSIONS: We have performed a preliminary validation of the short-CGH technique, including one clinical case, suggesting this approach may be applied to Day-3 aneuploidy analysis, thereby avoiding embryo cryopreservation and perhaps helping to improve implantation rate after PGS.

Lo-Fi matchmaking: A study of social pairing for backpackers

It is technically feasible for mobile social software such as pairing or ‘matchmaking’ systems to introduce people to others and assist information exchange. However, little is known about the social structure of many mobile communities or why they would want such pairing systems. While engaged in other work determining requirements for a mobile travel assistant we saw a potentially useful application for a pairing system to facilitate the exchange of travel information between backpackers. To explore this area, we designed two studies involving usage of a low-fidelity role prototype of a social pairing system for backpackers. Backpackers rated the utility of different pairing types, and provided feedback on the social implications of being paired based on travel histories. Practical usage of the social network pairing activity and the implications of broader societal usage are discussed.