Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes

BACKGROUND: Vitrification of human blastocysts has been successfully applied using grids, straws and cryoloops. We assessed the survival rate of human compacted morulae and early blastocysts vitrified in pipette tips with a smaller inner diameter and solution volume than the previously described open pulled straw (OPS) method. METHODS: Excess day 5 human embryos (n = 63) were experimentally vitrified in vessels. Embryos were incubated at 37 degrees C with sperm preparation medium (SPM) for 1 min, SPM + 7.5% ethylene glycol (EG)/dimethylsulphoxide (DMSO) for 3 min, and SPM + 16.5% EG + 16.5% DMSO + 0.67 mol/l sucrose for 25 s. They were then aspirated (0.5 microl) into a plastic micropipette tip (0.36 mm inner diameter), exposed to liquid nitrogen (LN(2)) vapour for 2 min before being placed into a pre-cooled cryotube, which was then closed and plunged into LN(2). Embryos were warmed and diluted using 0.33 mol/l and 0.2 mol/l sucrose. RESULTS: The survival rate for compacted morulae was 73% (22/30) and 82% (27/33) for early blastocysts. CONCLUSIONS: The survival rates of human compacted morulae and early blastocysts after vitrification with this simple technique are similar to those reported in the literature achieved by slow cooling and other vitrification protocols.     

Connective tissue growth factor expression and Smad signaling during mouse heart development and myocardial infarction.

Connective tissue growth factor (CTGF) is reported to be a target gene of transforming growth factor beta (TGFbeta) and bone morphogenetic protein (BMP) in vitro. Its physiological role in angiogenesis and skeletogenesis during mouse development has been described recently. Here, we have mapped expression of CTGF mRNA during mouse heart development, postnatal adult life, and after experimental myocardial infarction. Furthermore, we investigated the relationship between CTGF and the BMP/TGFbeta signaling pathway in particular during heart development in mutant mice. Postnatally, CTGF expression in the heart became restricted to the atrium. Strikingly, 1 week after myocardial infarction, when myocytes have disappeared from the infarct zone, CTGF and TGFbeta expression as well as activated forms of TGFbeta but not BMP, Smad effector proteins are colocalized exclusively in the fibroblasts of the scar tissue, suggesting possible cooperation between CTGF and TGFbeta during the pathological fibrotic response.     

Association between coagulation factors VII and X with triglyceride rich lipoproteins.

The association between the concentration of different plasma lipoproteins and plasma factor VII (F VII) was analysed by isolating plasma very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) lipoproteins and assessing their in vitro interaction with F VII by immunoenzyme assay using peroxidase labelled anti-factor VII immunoglobulins to determine whether F VII coagulant activity is prognostic for cardiovascular mortality. F VII bound to triglyceride rich lipoproteins, the fixation being stronger on chylomicrons and VLDL fractions than on LDL fractions. In our experiments HDL did not bind to F VII. The fixation of coagulation factor X (FX) tested by the same method is comparable with that of F VII. The nature of this fixation seemed to arise from hydrophobic interaction as calcium was not necessary and the use of Tween 20 inhibited the interaction. The binding of factors VII and X was increased when lipids were previously treated by phospholipase C and the interaction seemed to be completely dependent on the lipid part of the lipoproteins. Hyrophobic fixation is a possible mechanism of interaction of plasma lipoproteins and F VII and X, and it may be of importance in the covariance of triglyceride concentrations and the activity of vitamin K dependent coagulation factors.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> Virulence Genotypes in Portuguese Children and Adults with Gastroduodenal Pathology

The aim of this study was to evaluate the prevalence of virulence genotypes, namely cagA, vacA and babA2, of Helicobacter pylori strains isolated from Portuguese adults and children presenting gastroduodenal pathology. One hundred thirty-six strains were studied, 82 isolated from adult patients (50 with nonulcerative gastritis and 32 with active peptic ulcer) and 58 isolated from children (54 with nonulcerative gastritis and 4 with duodenal ulcer). Genotyping of cagA, vacA and babA2 was assessed by polymerase chain reaction. Overall, Helicobacter pylori strains carrying more virulent genotypes were much more prevalent in adults than in children, particularly the type I (vacAs1- and cagA-positive) and the triple-positive (vacAs1-, cagA- and babA2-positive) strains (P<0.001). A subpopulation of adults and children with nonulcerative gastritis was also studied, and differences in the prevalence of virulent genotypes were observed, either for individual genotypes (P=0.017 for cagA, P=0.010 for vacAs1) or in combinations, i.e. the type I genotype (P=0.005) and the triple-positive strains (P=0.031). There was no difference between the two populations in the distribution of babA2 and m1/m2 genotypes. Considering the cohort effect in the epidemiology of Helicobacter pylori infection, these results suggest that different strains might circulate during different periods of time, or that, after infection in childhood, individual strains will undergo changes during the course of infection. Electronic Publication     

Helicobacter hepaticus hydrogenase mutants are deficient in hydrogen-supported amino acid uptake and in causing liver lesions in A/J mice

Helicobacter hepaticus, a causative agent of chronic hepatitis and hepatocellular carcinoma in mice, expresses a nickel-containing hydrogen-oxidizing hydrogenase enzyme. Growth of a hyaB gene-targeted mutant was unaffected by the presence of hydrogen, unlike the wild-type strain, which showed an enhanced growth rate when supplied with H(2). Hydrogenase activities in H. hepaticus were constitutive and not dependent on the inclusion of H(2) during growth. Addition of nickel during growth significantly stimulated both urease (for wild-type and hyaB) and hydrogenase (for wild-type) activities. In a 5-h period, the extent of (14)C-labeled amino acid uptake by the wild type was markedly enhanced in the presence of hydrogen and was >5-fold greater than that of the hyaB mutant strain. In the presence of H(2), the short-term whole-cell amino acid uptake V(max) of the parent strain was about 2.2-fold greater than for the mutant, but the half-saturation affinity for amino acid transport was the same for the parent and mutant strain. The liver- and cecum-colonizing abilities of the strains was estimated by real-time PCR quantitation of the H. hepaticus-specific cytolethal distending toxin gene and showed similar animal colonization for the hyaB mutant and the wild type. However, at 21 weeks postinoculation, the livers from mice inoculated with wild type exhibited moderate lobular lymphoplasmacytic hepatitis with hepatocytic coagulative necrosis, but the hydrogenase mutants exhibited no histological evidence of lobular inflammation or necrosis.     

Rat bone marrow stromal cell osteogenic differentiation and fibronectin adsorption on chitosan membranes: the effect of the degree of acetylation

Cell adhesion, migration, and proliferation of a few anchorage-dependent cells cultured on chitosan (Ch) matrices are influenced by the degree of N-acetylation (DA) of Ch. In the present work, we examined the influence of the DA on the attachment, spreading, proliferation, and osteogenic differentiation of rat bone marrow stromal cells (rBMSCs). Ch membranes were characterized in terms of surface morphology, roughness, and wettability, and in terms of adsorption of an adhesive protein, fibronectin (Fn). Chs with DAs in the range of 4 to 49% were used. Among the Ch samples, the DA of 4% led to the highest Fn surface concentration, both from single protein solution and from diluted serum. Furthermore, the levels of Fn adsorbed from serum found for this DA were threefold higher than for the tissue culture polystyrene control, indicating that in the presence of competitive proteins Ch is more specific toward Fn adsorption than tissue culture polystyrene. rBMSCs cultured on Ch carrying a DA of 4% were able to spread, proliferate, and differentiate, reaching a higher level of osteogenic differentiation than on the control, despite the lower cell attachment observed for all Ch samples. Because the Ch sample with a DA of 4% showed the highest Fn adsorption from serum, we suggest that cell adhesion, spreading, and osteogenic differentiation of rBMSCs on Ch may be mediated by the adsorbed layer of Fn.     

Basal cell adenocarcinoma of the palate with squamous metaplasia

Basal cell adenocarcinoma is a rare salivary gland tumour, especially in minor glands. The clinical, histological, and immunohistochemical features of a case involving the palate are described. Formalin fixed, paraffin embedded sections of the tumour were examined in haematoxylin and eosin (H&E) sections and also using immunostaining for cytokeratins 7, 8, 13, 14, 18, 19, vimentin, muscle specific actin (HHF35), and laminin. H&E sections showed that the tumour was composed mainly of basaloid cells and a striking feature was the presence of squamous metaplasia. Neural invasion was also conspicuous. Immunohistochemical reactions indicated that cytokeratin 14 was expressed by all tumour cells and vimentin by all cells except those in the areas of squamous metaplasia. The remaining cytokeratins and actin were present in some of the tumour cells, while laminin showed discreet positivity around cell arrangements. The foci of squamous metaplasia and the immunohistochemical findings are helpful in distinguishing basal cell adenocarcinoma from other salivary gland tumours which show basaloid cells.     

Frequent recovery of a single clonal type of multidrug-resistant Staphylococcus aureus from patients in two hospitals in Taiwan and China

One hundred thirty-two methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from patients with S. aureus infections between January 1998 and February 1999 in two hospitals, one located in Taipei, Taiwan, and another in Nanjing, People's Republic of China, were examined for antibiotic susceptibility and for clonal type by a combination of three methods: hybridization of ClaI restriction digests with mecA- and Tn554-specific DNA probes and pulsed-field gel electrophoresis of chromosomal SmaI digests. Selected isolates representing each clonal type were also analyzed by spaA typing, multilocus sequence typing, and a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome mec (SCCmec) carried by the bacteria. The overwhelming majority of isolates (126 of 132 or 95%) belonged to minor variants of a single clonal type resembling the Brazilian and Hungarian epidemic MRSA clones, which showed a common spaA type and which were either sequence type 239 (ST239) or ST241 (a single-locus variant of ST239) in association with SCCmec type III or IIIA.     

Deposition and passage of transthyretin through the blood-nerve barrier in recipients of familial amyloid polyneuropathy livers

Familial amyloid polyneuropathy (FAP) is characterized by deposition of mutated transthyretin (TTR) in the peripheral nervous system. Prior to amyloid fibrils, nonfibrillar TTR aggregates are deposited inducing oxidative stress with increased nitration (3-NT). As the major source of TTR is the liver, liver transplantation (LT) is used to halt FAP. Given the shortage of liver donors, domino LT (DLT) using FAP livers is performed. The correlation between TTR deposition in the skin and nerve was tested in biopsies from normal individuals, asymptomatic carriers (FAP 0) and FAP patients; in FAP 0, nonfibrillar TTR was observed both in the skin and nerve in the same individuals; in patients, amyloid was detected in both tissues. The occurrence of amyloidosis in recipients of FAP livers was evaluated 1-7 years after DLT: TTR deposition occurred in the skin 3 years after transplantation either as amyloid or aggregates; in one of the recipients, fibrillar TTR was present in the epineurium 6 years after DLT. Deposits were scarce and 3-NT immunostaining was irrelevant. Nerve biopsies from DLT recipients had no FAP-related neuropathy. Our findings suggest that TTR amyloid formation occurs faster than predicted and that TTR of liver origin can cross the blood-nerve barrier. Recipients of FAP livers should be under surveillance for TTR deposition and tissue damage.     

Lymphocyte traffic is modified in vivo by anti-laminin antibody.

Data emerging from recent in vivo and in vitro studies are pointing to basement membrane and other extra cellular matrix (ECM) components as likely determinants of specific lymphocyte entry and positioning in lymphoid tissues. In this report, the relevance of this notion is investigated in T-cell deficient (B) rat recipients of cardiac allografts, using an anti-laminin (LN) antibody as the probe. The 6-hr migration patterns of 111In-labelled peripheral lymph node (PLN) cells were followed in groups of engrafted B hosts treated with rabbit anti-rat LN antibody or rabbit serum. The accumulation of adoptively transferred cells in PLN and cardiac allografts of recipients pretreated with anti-LN antibody was significantly decreased compared to controls (P less than 0.05 and P less than 0.001, respectively). The transferred cells also localized in slightly lower numbers in the spleens and lungs of anti-LN conditioned rats; no differences were seen in the clearance of lymphocytes from peripheral blood, or their sequestration in liver and kidney. These data provide the first in vivo evidence that an antibody against LN selectively affects lymphocyte traffic. The results reinforce the notion that basement membrane components could be critical in 'directing' lymphocyte migration in vivo.