Ontogenetic transition in fetal wound transforming growth factor-beta regulation correlates with collagen organization

Fetal rat skin transitions from scarless fetal-type repair to adult-type repair with scar between day 16 (E16) and day 18 (E18) of gestation (term = 21.5 days). Deficient transforming growth factor (TGF)-beta 1 and -beta 2 injury response has been proposed as a mechanism for scarless fetal-type repair. However, previous fetal studies have inconsistently reported the degree of TGF-beta induction after injury. To minimize developmental variables in fetal versus adult TGF-beta regulation, we narrowed our study to wounded fetal animals. We hypothesize that TGF-beta ligand and receptor expression will be differentially regulated during the transition from early gestation (E16) wounds manifesting scarless fetal-type repair to late gestation (E19) wounds manifesting adult-type repair with scar. In this study, decreased and rapidly cleared TGF-beta 1 and -beta 2 expression accompanied by increased and prolonged TGF-beta 3 levels in wounded E16 animals correlated with organized collagen deposition. In contrast, increased and prolonged TGF-beta 1 and -beta 2 expression accompanied by decreased and delayed TGF-beta 3 expression in wounded E19 animals correlated with disorganized collagen architecture. Similarly, expression of TGF-beta receptors type I and II were also increased or prolonged in E19 animals. Our results implicate increased TGF-beta 1, -beta 2, and decreased TGF-beta 3 expression, as well as increased type I and II receptor expression in late gestation fetal scar formation.     

Effects of a Single Hit from the Alpha Hemolysin Produced by Escherichia coli on the Morphology of Sheep Erythrocytes

Scanning electron micrographs of sheep erythrocytes showed that attachment of the alpha hemolysin produced by Escherichia coli resulted in the formation of spherocytes, with 10 to 20 small projections spaced relatively evenly over the surface of the erythrocyte membrane. This shape change was induced within 5 min after treatment. If the hemolysin concentration was reduced to a level which would lyse only a fraction of the total erythrocytes, the affected cells were easily identified against a background of normal, unaffected cells. Unlike sodium lauryl sulfate and other amphipathic agents which enter cell membranes and increase their flexibility, low concentrations of hemolysin did not provide protection against hypotonic hemolysis. These findings indicate that the surface projections were not the result of membrane expansion caused by incorporation of hemolysin into the outer portion of the lipid bilayer. The ability of a given amount of hemolysin to release a constant amount of hemoglobin in the presence of increasing concentrations of red cells confirmed that a single hit is sufficient for lysis. These results suggest that a single hemolysin molecule can bind to a sheep erythrocyte and trigger internal reactions which result in the derangement of membrane integrity at multiple sites on the surface. Confirmation of one-hit kinetics indicates that measurement of E. coli hemolysin activity should be carried out at low ratios of hemolysin to erythrocyte to decrease the possibility of multiple hits on a single cell.     

Model for assessment of proficiency of human immunodeficiency virus type 1 sequencing-based genotypic antiretroviral assays

Use of sequencing-based genotyping as a diagnostic assay for human immunodeficiency virus (HIV) antiretroviral resistance is increasing. Periodic evaluation of the proficiency of laboratories performing this assay should be established. It is important to identify components of the assay that influence the generation of reliable sequencing data and that should and can be monitored. A model was developed to determine what parameters were reasonable and feasible for assessing the performance of genotyping assays. Ten laboratories using the genotyping platform, HIV-1 Genotyping System (HGS) v. 1 and software versions 1.1 or 2.0, participated in two rounds of testing. For each round, each group was sent a panel consisting of three clinical samples to sequence in real time. Six months later, seven laboratories using the TRUGENE HIV-1 Genotyping Kit participated in a separate round, working with both panels at the same time. Analysis of the data showed that one main indicator of genotyping proficiency was achievement of > or =98% sequence homology of a sample tested to a group consensus sequence for that sample. A second was concordant identification of codons at sites identified with resistance mutations in the sample, although scoring of these criteria is still undetermined from this study. These criteria are applicable to all sequence-based genotyping platforms and have been used as a baseline for assessing the performance of genotyping for the determination of antiretroviral resistance in our ongoing proficiency program.     

Dual function of Langerhans cells in skin TSLP-promoted TFH differentiation in mouse atopic dermatitis

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Blood endotyping distinguishes the profile of vitiligo from that of other inflammatory and autoimmune skin diseases

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p53, Ki-67, and serum alpha feto-protein as predictors of hepatocellular carcinoma recurrence in liver transplant patients.

Patients with hepatocellular carcinoma who undergo orthotopic liver transplantation (OLT) are at risk for post-transplant tumor recurrence. The aim of this study was to evaluate whether expression of p53 and Ki-67 in hepatocellular carcinoma lesions present in explanted liver tissue was associated with time to tumor recurrence after OLT. Subjects consisted of 20 consecutive patients who underwent OLT and were found to have hepatocellular carcinoma in the liver explant. Immunostaining for p53 and Ki-67 was performed by standard methods. The presence of nuclear immunostaining in >10% of the tumor tissue was considered positive. Time to recurrence of hepatocellular carcinoma after OLT was compared between patients with positive and negative immunostaining by the log rank test. Multivariate analysis was performed using a Cox regression model to control for potentially confounding clinical factors. Time to post-transplant hepatocellular carcinoma recurrence was significantly more rapid in p53+ (P=0.0007) and Ki-67+ cases (P=0.001). These associations remained significant in multivariate analysis. Furthermore, time to recurrent hepatocellular carcinoma was significantly shorter in patients with a serum alpha feto-protein (AFP) level >or=100 ng/ml at time of diagnosis, compared to those with an AFP level <100 ng/ml (P=0.003). In conclusion, expression of p53 and Ki-67 in hepatocellular carcinoma lesions, and a serum AFP level >or=100 ng/ml were associated with more rapid recurrence of hepatocellular carcinoma after OLT. Identification of patients at risk for early post-transplant recurrence could be used to guide surveillance and adjuvant treatment strategies.     

Roxithromycin, Clarithromycin, and Azithromycin Attenuate the Injurious Effects of Bioactive Phospholipids on Human Respiratory Epithelium in Vitro

The effects of the bioactive phospholipids (PL), platelet-activating factor (PAF), lyso-PAF, and lysophosphatidylcholine (LPC) on the beat frequency and structural integrity of human ciliated respiratory epithelium were studied in vitro, in the presence or absence of polymorphonuclear leukocytes (PMNL), the antimicrobial agents, roxithromycin, clarithromycin, and azithromycin and the antioxidative enzymes catalase and superoxide dismutase (SOD). All three PL caused dose-dependent slowing of ciliary beat frequency (CBF) and epithelial damage (ED) at concentrations 1 g/ml, which were unaffected by inclusion of the antimicrobial agents and antioxidative enzymes. When epithelial strips were exposed to the combination of PMNL and PL, there was significant potentiation of ciliary dysfunction and ED, which was ameliorated by pretreatment of the PMNL with the antimicrobial agents or by inclusion of catalase, but not SOD. These results demonstrate that LPC, PAF, and lyso-PAF cause epithelial damage by direct mechanisms which are oxidant-independent, as well as by indirect mechanisms involving phagocyte-derived reactive oxidants. Macrolides and azalide antimicrobial agents may have beneficial effects on airway inflammation in asthma and microbial infections by protecting ciliated epithelium against oxidative damage inflicted by PL-sensitized phagocytes.     

Estimating the relative frequency of leukodystrophies and recommendations for carrier screening in the era of next‐generation sequencing

Leukodystrophies are a heterogeneous group of heritable disorders characterized by abnormal brain white matter signal on magnetic resonance imaging (MRI) and primary involvement of the cellular components of myelin. Previous estimates suggest the incidence of leukodystrophies as a whole to be 1 in 7,000 individuals, however the frequency of specific diagnoses relative to others has not been described. Next generation sequencing approaches offer the opportunity to redefine our understanding of the relative frequency of different leukodystrophies. We assessed the relative frequency of all 30 leukodystrophies (associated with 55 genes) in more than 49,000 exomes. We identified a relatively high frequency of disorders previously thought of as very rare, including Aicardi Goutières Syndrome, TUBB4A‐related leukodystrophy, Peroxisomal biogenesis disorders, POLR3‐related Leukodystrophy, Vanishing White Matter, and Pelizaeus‐Merzbacher Disease. Despite the relative frequency of these conditions, carrier‐screening laboratories regularly test only 20 of the 55 leukodystrophy‐related genes, and do not test at all, or test only one or a few, genes for some of the higher frequency disorders. Relative frequency of leukodystrophies previously considered very rare suggests these disorders may benefit from expanded carrier screening.