Fatal embryonic bleeding events in mice lacking tissue factor, the cell-associated initiator of blood coagulation.

Tissue factor (TF) is the cellular receptor for coagulation factor VI/VIIa and is the membrane-bound glycoprotein that is generally viewed as the primary physiological initiator of blood coagulation. To define in greater detail the physiological role of TF in development and hemostasis, the TF gene was disrupted in mice. Mice heterozygous for the inactivated TF allele expressed approximately half the TF activity of wild-type mice but were phenotypically normal. However, homozygous TF-/- pups were never born in crosses between heterozygous mice. Analysis of mid-gestation embryos showed that TF-/- embryos die in utero between days 8.5 and 10.5. TF-/- embryos were morphologically distinct from their TF+/+ and TF+/- littermates after day 9.5 in that they were pale, edematous, and growth retarded. Histological studies showed that early organogenesis was normal. The initial failure in TF-/- embryos appeared to be hemorrhaging, leading to the leakage of embryonic red cells from both extraembryonic and embryonic vessels. These studies indicate that TF plays an indispensable role in establishing and/or maintaining vascular integrity in the developing embryo at a time when embryonic and extraembryonic vasculatures are fusing and blood circulation begins.     

Demonstration of active tolerance in maintenance of established islet of Langerhans allografts.

Streptozotocin-induced diabetes in mice can be reversed by transplantation of islets of Langerhans from histoincompatible mice if the islets are treated with anti-Ia-serum and complement before transplantation. Here we show that anti-Ia-treated islets most likely induce tolerance in the recipient animals. Daily injections of recipient-specific anti-I-J-serum (beginning 80 or more days after transplantation) and small numbers of donor splenocytes caused the prompt rejection of the islets in half of the animals; neither anti-I-J-serum nor donor splenocytes alone were effective. It is likely that rejection of established allografts after this treatment is the result of abolition of the activity of allograft-specific suppressor cells.     

Role of fibrinogen alpha and gamma chain sites in platelet aggregation.

Fibrinogen (Fbg) mediates platelet aggregation by its interaction with the platelet glycoprotein IIb-IIIa (integrin alpha IIb beta 3). Peptides containing the amino acid sequence RGD derived from the alpha chain (residues alpha 95-97 and residues alpha 572-574) and the sequence HHLGGAKQAGDV derived from the carboxyl terminus of the gamma chain of Fbg (residues gamma 400-411) inhibit these interactions. To determine the role of these sequences in intact Fbg, recombinant human Fbg (rFbg), mutant rFbgs with an RGD-->RGE substitution at either position alpha 97 or alpha 574, and a rFbg gamma'-containing variant that has a carboxyl-terminal interruption in the HHLGGAKQAGDV sequence have been expressed in transfected BHK cells. Purified rFbg and the two RGE mutant Fbgs were similar to plasma Fbg in platelet aggregation assays. In contrast, the gamma' variant Fbg was markedly defective in platelet aggregation. These data support the proposals that the carboxyl-terminal region of the gamma chain of Fbg is essential for optimal platelet aggregation and that the alpha-chain RGD sequences are neither necessary nor sufficient for platelet aggregation.     

Cryopreservation of human granulocytes in liquid nitrogen.

Human granulocytes (PMNL) were successfully cryopreserved for up to 14 months. The PMNL (1-2 X 10(7)/ml) were stored in 2-ml ampoules in the gas phase of liquid nitrogen at a temperature between -160 degrees C and -196 degrees C using dimethylsulphoxide (DMSO 10%) as cryoprotectant. Morphology and phagocytic and bactericidal capacity were best preserved by adding fetal calf serum to the freezing mixture, by using an interrupted cooling process, by washing the thawed PMNL in fresh freeze-dried plasma, and centrifuging at 600 g for no more than two minutes. Careful post-thaw handling of the cells was an important factor in preserving function. These preliminary studies indicate that useful numbers of PMNL can be recovered in a functional state after storage for long periods in liquid nitrogen.     

B-cell subsets: functional and structural characteristics

The review will examine B-cell differentiation with an emphasis on B-cell subpopulations. We will begin with an analysis of current evidence concerning B-cell ontogeny. The development of the B-cell repertoire will be traced from stem cell to effector cells. The CBA/N mouse, which expresses an X-linked immunodeficiency, will serve as a basis for discussing the delineation of B cells into subpopulations. The CBA/N mouse provides evidence for distinct populations of B cells which can be differentiated by cell surface antigens as well as function. We intend to focus on the functional diversity of B-cell subpopulations and how they develop. The CBA/N mouse is not the only evidence for distinct B cell subpopulations and we will attempt to organize these data into a coherent story of B-cell subsets.     

Isoforms of bone alkaline phosphatase: characterization and origin in human trabecular and cortical bone.

Alkaline phosphatase (ALP) is a glycoprotein and functions as an ectoenzyme attached to the cell membrane by a hydrophobic glycosyl-phosphatidylinositol (GPI) anchor. Three bone ALP (BALP) isoforms in human serum were separated and quantitated by high-performance liquid chromatography. B/I, a minor fraction, is composed on average of bone (70%) and intestinal (30%) ALP, and two major isoforms, B1 and B2. Treatment with GPI-specific phospholipase C (GPI-PLC) did not influence the activities or retention times for B1 and B2, indicating that the biochemical differences between B1 and B2 are likely to be due to different glycosylation patterns. The B/I fraction in serum, on average 4% of total ALP, was found to be composed of B1 and B2 isoforms, each with an intact hydrophobic GPI cell membrane anchor. We investigated the origin of these three BALP isoforms and osteocalcin in human femora from five healthy individuals (four males), mean age 51 years, obtained from a tissue bank. Bone was sampled from three sites: cortical bone, trabecular bone from the diaphysis, and trabecular bone from the greater trochanter. Trabecular bone, from both sites, had higher BALP activities compared with cortical bone. Conversely, the osteocalcin content of cortical bone was more than 3-fold greater than that of trabecular bone. Cortical bone had approximately 2-fold higher activity of B1 compared with B2, whereas trabecular bone had approximately 2-fold higher activity of B2 compared with B1. We observed a previously undescribed BALP isoform (B1x) in all bone samples. B1x was also observed in sera from some patients (60%) with severe renal insufficiency and on chronic dialysis therapy (n = 20). The isoforms of BALP may provide information relating to bone metabolism within specific bone compartments.     

Case Report and Cohort Analysis of Drug-Induced Liver Injury Associated with Daptomycin

A patient receiving daptomycin developed asymptomatic transaminitis and hyperbilirubinemia without concurrent multiorgan dysfunction or elevation of his creatinine kinase level. After ruling out other etiologies, the liver injury was attributed to daptomycin and was subsequently resolved. A single-center retrospective cohort analysis of baseline and follow-up liver function panels (n = 614) from all admissions from 2008 to 2013 during which daptomycin was administered did not reveal any other cases of probable or definite drug-induced liver injury associated with daptomycin.     

Posterior corneal curvature change and recovery after 6 months of overnight orthokeratology treatment

Purpose: To investigate the changes in, and recovery of, posterior corneal curvature after 6 months of overnight orthokeratology (ortho‐k). Methods: Twenty‐eight healthy young adults were recruited for a 6‐month period of ortho‐k treatment and data from their right eyes were analyzed. The mean ± standard deviation spherical equivalent refraction (SER) at baseline was ?2.95 ± 0.88 D. Posterior simulated keratometry (Sim K) readings were measured with a corneal topographer based on rotating Scheimpflug imaging. The three phases in the study were the 6‐month treatment period (Phase I); diurnal changes over a period of 8 h immediately after lens removal at the completion of the treatment period (Phase II); and a 2‐month recovery period after cessation of treatment (Phase III). Measurements were taken after lens wear overnight, and after 1 week, and 1, 2, 3 and 6 months of lens wear in Phase I. In Phase II, measurements were taken immediately, and then 2, 4 and 8 h after lens removal. In Phase III, corneal parameters were monitored 1 week, 2 weeks, 1 month and 2 months after cessation of ortho‐k treatment. Results: In Phase I, the posterior Sim K readings were significantly steepened after the first overnight lens wear. These significant changes were not found at other visits. In Phase II, the posterior Sim K readings were the steepest immediately after lens removal and significantly flattened 2 h after lens removal. In Phase III, all the posterior Sim K readings were similar to the baseline results. Conclusions: Steepening of the posterior cornea was only observed immediately after lens removal. It returns to its original shape within 2 h after cessation of lens wear. These changes appear to be in line with recent reports of the diurnal variation in the posterior corneal shape in non‐contact lens wearers. The reduction in myopia from ortho‐k treatment is therefore mainly due to a flattening of the anterior cornea.     

Effect of vitamin C on glycosylation of proteins.

Twelve nondiabetic subjects consumed 1 g/day vitamin C for 3 mo. A fasting blood sample was taken at the start of the study and at the end of each month for the measurement of plasma and intraerythrocyte glucose, vitamin C, glycosylated hemoglobin (affinity chromatography and electrophoresis), and glycosylated albumin (affinity chromatography). Although there were no significant changes in fasting glycemia, glycosylated hemoglobin (affinity chromatography) decreased 18%, from 6.18 +/- 0.48% (mean +/- SD) at the start to 5.05 +/- 0.50% (P less than 0.0001) after 3 mo, whereas, HbA1 measured by electrophoresis increased 16%, from 6.17 +/- 0.61 to 7.16 +/- 0.59% (P less than 0.0001) in this period. Glycosylated albumin decreased 33%, from 1.56 +/- 0.24 to 1.04 +/- 1.01% (P less than 0.0001) after 3 mo. This discrepancy between glycosylated hemoglobin measured by electrophoresis and affinity chromatography was due to methodological differences between the two techniques, with affinity chromatography measuring "true" glycosylated hemoglobin. The greater decrease found with glycosylated albumin was probably due to the different distribution of vitamin C between plasma and within the erythrocyte, levels after 1 mo of supplementation being 109 +/- 19 and 59 +/- 9 microM, respectively (P less than 0.001). This indicates that administration of oral vitamin C may inhibit the glycosylation of proteins in vivo by a competitive mechanism.