Platelet storage pool deficiency in pigs

We report a new bleeding disease--storage pool deficiency (SPD) of platelets--in pigs from the Mayo swine colony of homozygous von Willebrand's disease (vWD) and of heterozygous carriers of vWD. Levels of factor VIII, von Willebrand factor antigen (vWF:Ag), and ristocetin cofactor (RCof) were similar in the vWD carriers and SPD pigs. The latter pigs, however, had bleeding times of 15 minutes or more and were severe bleeders, in contrast to clinically normal vWD carriers. Platelet aggregation in response to collagen was reduced in most SPD pigs. Total platelet content of ADP, ATP, and serotonin was less than that of normal pigs. While the initial uptake of 14C-labeled serotonin into platelets was similar in SPD and normal pigs, retention of serotonin was reduced in platelets of SPD pigs. Transmission electron microscopy showed a large decrease of dense bodies in the platelets of SPD pigs. These findings support a diagnosis of SPD. Genetic analyses suggest an autosomal recessive mode of inheritance. A breeding program is under way to produce pigs affected only at the SPD gene, thus allowing further characterization of SPD and SPD-carrier pigs.     

Catch-up growth up to ten years of age in children born very preterm or with very low birth weight

Background  

Improved survival due to advances in neonatal care has brought issues such as postnatal growth and development more to the focus of our attention. Most studies report stunting in children born very preterm and/or small for gestational age. In this article we study the growth pattern of these children and aim to identify factors associated with postnatal catch-up growth.     

Role of the ATP-binding cassette transporter Abcg2 in the phenotype and function of cardiac side population cells

Recently, the side population (SP) phenotype has been introduced as a reliable marker to identify subpopulations of cells with stem/progenitor cell properties in various tissues. We and others have identified SP cells from postmitotic tissues, including adult myocardium, in which they have been suggested to contribute to cellular regeneration following injury. SP cells are identified and characterized by a unique efflux of Hoechst 33342 dye. Abcg2 belongs to the ATP-binding cassette (ABC) transporter superfamily and constitutes the molecular basis for the dye efflux, hence the SP phenotype, in hematopoietic stem cells. Although Abcg2 is also expressed in cardiac SP (cSP) cells, its role in regulating the SP phenotype and function of cSP cells is unknown. Herein, we demonstrate that regulation of the SP phenotype in cSP cells occurs in a dynamic, age-dependent fashion, with Abcg2 as the molecular determinant of the cSP phenotype in the neonatal heart and another ABC transporter, Mdr1, as the main contributor to the SP phenotype in the adult heart. Using loss- and gain-of-function experiments, we find that Abcg2 tightly regulates cell fate and function. Adult cSP cells isolated from mice with genetic ablation of Abcg2 exhibit blunted proliferation capacity and augmented cell death. Conversely, overexpression of Abcg2 is sufficient to enhance cell proliferation, although with a limitation of cardiomyogenic differentiation. In summary, for the first time, we reveal a functional role for Abcg2 in modulating the proliferation, differentiation, and survival of adult cSP cells that goes beyond its distinct role in Hoechst dye efflux.     

Rare detection of hepatitis B and hepatitis C virus genomes by polymerase chain reaction in seronegative donors with elevated alanine aminotransferase

BACKGROUND: Since screening for antibody to hepatitis C virus (HCV) was introduced in 1990, posttransfusion hepatitis has been reduced to nearly background levels. This has led to reconsideration of the value of testing donated blood for elevated alanine aminotransferase (ALT). The contribution of ALT testing in detecting seronegative infection was evaluated by the performance of polymerase chain reaction (PCR) for hepatitis B virus (HBV) or HCV in plasma from ALT-elevated blood units. STUDY DESIGN AND METHODS: Testing was performed on 375 units of plasma, derived from an equivalent of 47,500 blood donations, with a highly sensitive hemi-nested PCR procedure. Using a triplet of primers directed at the conserved regions of HBV DNA and 5'-noncoding regions of HCV RNA, the hemi-nested PCR assay can reliably amplify 10 viral molecules to levels detectable in ethidium bromide-stained agarose gels. Pools of plasma from groups of four donors were screened with hemi-nested PCR. For any reactive pools, the plasma from individual donors was retested twice on different aliquots. RESULTS: Two of 375 units, both with midrange ALT elevation, were repeatedly reactive in hemi-nested PCR (one each for HBV DNA and HCV RNA). However, samples from the two suspect donors tested 9 and 5 months later revealed no seroconversion, elevated ALT, or viral genomes in hemi-nested PCR. CONCLUSION: The lack of confirmed HBV or HCV infection in this study representing an estimated 47,500 voluntary blood donations suggests that routine ALT testing for further prevention of posttransfusion hepatitis after exclusion of HBV- and/or HCV-seropositive blood may be superfluous.     

"Stem cell" origin of the hematopoietic defect in dyskeratosis congenita

We have used the long-term bone marrow culture (LTBMC) system to analyze hematopoiesis in three patients with dyskeratosis congenita (DC), two of whom had aplastic anemia, and the third had a normal blood count (apart from mild macrocytosis) and normal BM cellularity. Hematopoiesis was severely defective in all three patients, as measured by a low incidence of colony-forming cells and a low level of hematopoiesis in LTBMC. The function of the marrow stroma was normal in its ability to support the growth of hematopoietic progenitors from normal marrows seeded onto them in all three cases, but the generation of hematopoietic progenitors from patients marrow cells inoculated onto normal stromas was reduced, thus suggesting the defect to be of stem cell origin. The parents and unaffected brother of one of the families have also been studied in LTBMC and all showed normal hematopoietic and stromal cell function. From this study we speculate that there are some similarities between DC and the defect in the W/Wv mouse.     

Angiogenesis, proliferation, and apoptosis in anal high-grade squamous intraepithelial lesions

PURPOSE: Management of anal high-grade squamous intraepithelial lesions is controversial. Anal and cervical high-grade squamous intraepithelial lesions are similar in that they occur in transitional squamous epithelium, are associated with human papilloma virus infection, and have increased incidence in the immunocompromised population. Ablation of cervical high-grade squamous intraepithelial lesions is preferred, but similar ablation or excision of anal high-grade squamous intraepithelial lesions may compromise bowel control; thus, there is a need to define the malignant potential of anal high-grade squamous intraepithelial lesions. METHODS: We analyzed 50 paraffin sections of normal anoderm, anal low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and anal squamous-cell carcinoma. Microvessels were detected immunohistochemically with von Willebrand factor and counted manually along the epithelial-stromal junction. Proliferation and apoptosis were determined in the epithelial cells with MIB-1 antibody immunostaining and the terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling, respectively. RESULTS: Microvascular density was significantly greater in anal high-grade squamous intraepithelial lesions (mean, 0.50 vessels/cm)vs. normal anoderm (mean, 0.21 vessels/cm;P=0.0017, Mann-WhitneyU test). The proliferative percentages were greater in low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and squamous-cell carcinoma (mean, 20.4, 21.8, and 23.6 percent)vs. normal anoderm (mean, 14.4 percent), although not significantly (P=0.06, Kruskal-Wallis statistic). Although the mean proliferative proportions were similar in low-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions, the apoptotic proportion was lower for high-grade squamous intraepithelial lesions than low-grade squamous intraepithelial lesions (10.13vs. 19.96 percent, respectively;P=NS, Mann-WhitneyU test). CONCLUSIONS: Angiogenesis, increased proliferation, and decreased apoptosis occur in anal high-grade squamous intraepithelial lesions as they do in the cervix before the development of malignancy. These biologic markers support the importance of anal high-grade squamous intraepithelial lesions as a potential premalignant lesion warranting surgical intervention.Supported by a grant from the National Institutes of Health (DDDN, NIDDK KO8 DK 02507-03).Podium presentation at The American Society of Colon And Rectal Surgeons' 100th Anniversary and Tripartite Meeting, Washington, D.C., May 1 to 6, 1999.     

Association of granulocyte-macrophage colony-stimulating factor with the crystalloid granules of human eosinophils

We have previously shown that normal-density human peripheral blood eosinophils transcribe and translate mRNA for granulocyte-macrophage colony-stimulating factor (GM-CSF) and that the intracellular distribution was granular as assessed by light microscopy immunocytochemistry. The present study was conducted to confirm this apparent association between GM-CSF and the crystalloid granule using a subcellular fractionation method for human eosinophils and immunogold electron microscopy (EM). Highly purified (> 99%, by negative selection using anti-CD16 immunomagnetic microbeads) human peripheral blood eosinophils were obtained from four asthmatic subjects (not taking systemic medication), homogenized and density fractionated (5 x 10(7) cells/subject) on linear Nycodenz gradients. Twenty-four fractions were collected from each cell preparation and analyzed for marker enzyme activities as well as total protein. Dot blot analysis with specific monoclonal antibodies (MoAbs) was used to detect the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP). An anti-CD9 MoAb was used as an eosinophil plasma membrane marker. Lactate dehydrogenase (LDH) was used as a cytosolic marker. Immunoreactivity for GM-CSF was detected by a specific enzyme-linked immunosorbent assay using a polyclonal antihuman GM-CSF antibody and confirmed by dot blot. GM-CSF coeluted with the cellular fractions containing granule markers (MBP, ECP, eosinophil peroxidase, hexosaminidase, and arylsulphatase), but not those containing cytoplasm (LDH+) or membrane (CD9+) markers. EM examination of pooled fractions associated with the peak of GM-CSF immunoreactivity confirmed that they contained crystalloid and small granules, but not plasma membrane. In addition, quantification, using immunogold labeling with an anti/GM-CSF MoAb, indicated preferential localization of gold particles over the eosinophil granule cores of intact cells. Thus, our results indicate that GM-CSF resides as a granule-associated, stored mediator in unstimulated human eosinophils.