The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their liver adult bone marrow counterparts

Varying, limiting numbers of unseparated or purified cells (Ly-5.1), either from 14.5-day-old fetal liver (FL) or from adult bone marrow (BM) were coinjected with 10(5) unseparated BM cells (Ly-5.2) into lethally irradiated adult C57B1/6 recipients (Ly-5.2). The kinetics of donor cell repopulation of the lymphoid and myeloid compartments by Ly- 5.1+ donor hematopoietic stem cells (ie, competitive repopulation units [CRU]) were monitored at various time points after the transplantation by Ly-5 analysis of the peripheral white blood cells (WBC). Recipients that had received on average less than 2 adult BM or FL CRU did not show a significant difference in the level of donor-reconstitution when analyzed 4 weeks after the transplantation, However, at 8 and 16 weeks, the FL recipients showed a significantly higher percentage of donor- derived nucleated peripheral blood cells than did the recipients of adult BM cells. Analysis of individual mice showed that approximately 80% of the recipients of FL CRU showed an increase in mature WBC output between 4 and 8 weeks after transplantation, whereas this occurred in less than 40% in the recipients of adult BM cells. In addition to this effect on mature cell output, the cellularity of the reconstituted BM was significantly higher in recipients of FL CRU than in recipients of adult BM CRU, even at 7 to 9 months after transplantation, which is consistent with an increased clonal expansion of FL CRU. When marrow cells from primary recipients of FL CRU were injected into secondary recipients, a significantly higher percentage of these mice showed donor-reconstitution of their lymphoid and myeloid compartments (P < .01) and to a greater extent (P < .008) as compared with mice that had received marrow cells from primary recipients of similar numbers of adult BM CRU. Taken together, these results show that individual FL CRU exhibit a greater proliferative activity in vivo than similar cells from adult BM that is accompanied by a greater production of daughter CRU.     

Severe perinatal thrombosis in double and triple heterozygous offspring of a family segregating two independent protein S mutations and a protein C mutation

Molecular genetic and phenotypic analyses were performed in a highly unusual case of combined protein S and protein C deficiency manifesting in a family in which a child had died perinatally from renal vein thrombosis. Antenatal diagnosis in a second pregnancy was initially performed by indirect restriction fragment length polymorphism (RFLP) tracking using a neutral dimorphism within the PROS gene and served to exclude severe protein S deficiency. Am umbilical vein blood sample at 22 weeks gestation showed isolated protein C deficiency. This pregnancy proceeded to a full-term delivery without thrombotic complications. Molecular genetic analysis of the PROC and PROS gene segregating in the family then yielded one PROC gene lesion in the father and two PROS gene lesions, one in each parent. These lesions were shown to segregate with the respective deficiency states through the family pedigree. Analysis of DNA from paraffin-embedded liver tissue taken from the deceased child showed the presence of both PROS mutations, as well as the PROC mutation. Genotypic analysis of the second child showed a PROC mutation, but neither PROS mutation consistent with its possession of normal protein S levels and a low/borderline protein C level. Antenatal diagnosis was then performed in a third pregnancy by direct mutation detection. However, although the fetus carried only the paternal PROS and PROC gene lesions, the child developed renal thrombosis in utero. It may be that a further genetic lesion at a third locus still remains to be defined. Alternatively, the intrauterine development of thrombosis in this infant could have been caused, at least in part by a transplacental thrombotic stimulus arising in the protein S-deficient maternal circulation. This analysis may, therefore, serve as a warning against extrapolating too readily from genotype to phenotype in families with a complex thrombotic disorder.     

Molecular basis of the heterogeneity of expression of glycosyl phosphatidylinositol anchored proteins in paroxysmal nocturnal hemoglobinuria

The purpose of these studies was to determine the molecular basis of the phenotypic mosaicism that is a defining feature of paroxysmal nocturnal hemoglobinuria (PNH). Analysis of T cell clones from a female patient revealed four distinct phenotypes based on surface expression of glycosyl phosphatidylinositol-anchored proteins (GPI-AP). When PIG-A (the gene that is mutant in PNH) from these clones was analyzed, four discrete somatic mutations were identified. Analysis of X chromosomal inactivation among the abnormal T cell clones was consistent with polyclonality. Together, these studies demonstrate that the phenotypic mosaicism that is characteristic of PNH is a consequence of genotypic mosaicism and that, at least in this case, PNH is a polyclonal rather than a monoclonal disease. That four distinct somatic mutations were present in a single patient suggests that in conditions that predispose to PNH PIG-A may be hypermutable.     

Minactivin expression in human monocyte and macrophage populations

Adherent monolayer cultures of human blood monocytes, peritoneal macrophages, bone marrow macrophages, and colonic mucosa macrophages were examined for their ability to produce and secrete minactivin, a specific inactivator of urokinase-type plasminogen activator. All except colonic mucosa macrophages produced and secreted appreciable amounts of minactivin, but only blood monocytes were stimulated by muramyl dipeptide (adjuvant peptide) to increase production. The minactivin from each of these populations could be shown to preferentially inhibit urokinase-type plasminogen activator and not trypsin, plasmin, or "tissue"-type plasminogen activator (HPA66). A plasminogen-activating enzyme present in monocyte cultures appeared unaffected by the presence of minactivin and could be shown to be regulated independently by dexamethasone.     

The relation between plasma tyrosine concentration and fatigue in primary biliary cirrhosis and primary sclerosing cholangitis

Background  

In primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) fatigue is a major clinical problem. Abnormal amino acid (AA) patterns have been implicated in the development of fatigue in several non-hepatological conditions but for PBC and PSC no data are available. This study aimed to identify abnormalities in AA patterns and to define their relation with fatigue.     

Cyclin D1 protein analysis in the diagnosis of mantle cell lymphoma

Mantle cell lymphoma (MCL) is a clinicopathologic entity that is difficult to diagnose on histopathologic criteria. Approximately 50% to 70% of MCL contain a t(11;14)(q13;q32) translocation involving the cyclin D1 gene. Irrespective of this rearrangement, almost all MCL show overexpression of the cyclin D1 gene at the mRNA level. Other B-cell non-Hodgkin's lymphomas (NHL) do not show this rearrangement or overexpression of cyclin D1. We developed an immunohistochemical assay to detect overexpression of the cyclin D1 protein on conventional formalin-fixed, paraffin-embedded biopsies using the well-defined monoclonal antibody DCS-6. Expression in tumor cells was compared with expression of cyclin D1 in endothelial cells and fibroblasts. An exclusively nuclear staining pattern was observed. Moreover, expression was directly compared with the expression observed by immunoblot analysis with the same antibody, as well as with mRNA expression and with the occurrence of genomic rearrangements within the BCL-1 locus. Of 13 MCL that were analyzed by immunohistochemistry and immunoblot, 12 showed overexpression with both techniques, whereas no overexpression was observed in 39 other NHL. Of 13 additional MCL studied either by immunohistochemistry or immunoblot, 11 also showed overexpression. Two lymphomas morphologically indistinguishable from MCL but with an aberrant immunophenotype (CD5 negative, CD10 positive) both lacked overexpression of cyclin D1. These results underscore the significance of overexpression of the cyclin D1 protein as a specific marker for MCL. Detection of cyclin D1 overexpression on formalin-fixed, paraffin- embedded tissues using the DCS-6 monoclonal antibody can be applied for routine diagnostic purposes.     

Fibrinolytic properties of a human endothelial hybrid cell line (Ea.hy 926)

The fibrinolytic characteristics of the endothelial hybrid cell line EA.hy 926, established by fusing a human umbilical vein endothelial cell with a human carcinoma cell line, were studied. The hybrid cell line produced large amounts of tissue-type plasminogen activator (t- PA), plasminogen activator inhibitor type 1, and a small amount of urokinase. All plasminogen activator present in conditioned medium was complexed with inhibitor because the cells secreted plasminogen activator inhibitor in excess over plasminogen activator and no activator activity was detectable in conditioned media by direct activity assays. t-PA activator activity was, however, demonstrable in conditioned media after treatment with sodium dodecyl sulfate, in agreement with t-PA antigen determinations. Increased plasminogen activator inhibitor activity could be induced by incubating the cells in the presence of endotoxin or microtubule inhibitors, whereas increased t-PA activity could be induced by microtubule inhibitors. Interleukin-1 had no effect. The fibrinolytic characteristics of the hybrid cell line were stable for at least 30 passages. The perpetual human hybrid cell line EA.hy 926 therefore may be a useful tool for the study of fibrinolysis in cultured endothelial cells.     

New England Medical Center Posterior Circulation Stroke Registry: I. Methods, Data Base, Distribution of Brain Lesions, Stroke Mechanisms, and Outcomes

Among 407 New England Medical Center Posterior Circulation Registry (NEMC-PCR) patients, 59% had strokes without transient ischemic attacks (TIAs), 24% had TIAs before strokes, and 16% had only posterior circulation TIAs. Embolism was the commonest stroke mechanism accounting for 40% of cases (24% cardiac origin, 14% arterial origin, 2% had potential cardiac and arterial sources). In 32%, large artery occlusive lesions caused hemodynamic brain infarction. Stroke mechanisms in the posterior and anterior circulation are very similar. Infarcts most often included the distal posterior circulation territory (rostral brainstem, superior cerebellum and occipital and temporal lobes), while the proximal (medulla and posterior inferior cerebellum) and middle (pons and anterior inferior cerebellum) territories were equally involved. Infarcts that included the distal territory were twice as common as those that included the proximal or middle territories. Most distal territory infarcts were attributable to embolism. Thirty day mortality was low (3.6%). Embolic stroke mechanism, distal territory location, and basilar artery occlusive disease conveyed the worst prognosis.