Hemopoietic origin of factor XIII A subunits in platelets, monocytes, and plasma. Evidence from bone marrow transplantation studies.

Factor XIII A subunit (FXIIIA) is found in plasma, platelets, and monocytes. The hemopoietic contributions to FXIIIA in these components were studied in patients transplanted with marrows from donors with different FXIIIA phenotypes. In three patients with successful engraftment (by DNA genotyping, red cell phenotyping, and cytogenetic studies) platelet and monocyte FXIIIA changed to donor phenotypes with hematologic recovery. Thus, FXIIIA in platelets and monocytes is synthesized de novo and/or from their progenitor cells. Plasma FXIIIA phenotype change after transplantation was more complex. Patient I changed from phenotype 1-1 (one electrophoretically fast band) to 1-2 (three bands) in 115 d; patients 2 and 3 did not change completely from phenotype 1-2 to 1-1 in up to 458 d, but did show enrichment of the fastest band. Thus, while there is a definite contribution of donor hemopoiesis to plasma FXIIIA, another source of recipient FXIIIA appears to be present to delay or prevent the phenotype change.

A hemi-nested, allele specific, whole blood PCR assay for the detection of the factor V Leiden mutation.

We describe a novel hemi-nested, allele specific whole blood PCR assay for detection of the factor V Leiden mutation associated with the plasma defect, activated protein C resistance. This assay utilizes 5 microliters of whole blood without prior DNA extraction. The hemi-nested design, employing an outer primer pair in combination with nested, allele specific primers obviates the need for restriction enzyme digestion. PCR reactions are analysed directly on agarose or polyacrylamide minigels. The assay confirmed the genotypes of 50 individuals previously categorized by PCR and Mn11 digestion, and has been subsequently utilized in the genotyping of 445 individuals referred for thrombosis studies.

Effect of the factor V Leiden mutation on the clinical expression of severe hemophilia A.

To determine whether the factor V Leiden mutation is associated with decreased bleeding in individuals with severe hemophilia A, factor concentrate utilization, maximum annual number of bleeding episodes, and the prevalence of hemophilic arthropathy between carriers and non-carriers of the factor V Leiden mutation were compared. Heterozygosity for the factor V Leiden mutation was found in 6 of 137 subjects (4.4%). Carriers of the factor V Leiden mutation utilized less factor concentrate (geometric mean: 310 vs. 1185 units/kg/year) and had fewer bleeding episodes than non-carriers (proportion with 10 or fewer bleeding episodes in their worst year: 50 vs. 11%). However, the factor V Leiden mutation was not associated with the absence of arthropathy. The intron 22 inversion mutation of the factor VIII gene was tested for in a subgroup of 80 subjects, but it was not found to be a significant variable for any of the bleeding endpoints. The results of this small study are consistent with the hypothesis that the factor V Leiden mutation imparts a protective effect; however, a larger confirmatory study in which the factor VIII molecular defects can be controlled for is needed. Furthermore, most severe hemophiliacs who used fewer than 200 units/kg/year of factor concentrate or who had experienced 10 or fewer bleeding episodes per year did not carry the factor V Leiden mutation, suggesting that the proportion of severe hemophiliacs whose mild clinical course can be attributed to the factor V Leiden mutation is small.

Speed of response characteristics of goalkeepers: a descriptive and developmental report.

Photoelectric cells and accelerometers enabled the measurement of reaction, movement and total response times of the limbs of representative goalkeepers (N = 12) from five levels of organized hockey, under simple and choice test conditions. Speed of response characteristics fundamental to success at these levels of competition and the developmental improvement, which must occur annually to permit progress through the goalkeeping hierarchy, were suggested. The point at which the beginner's "reaction-type" pattern of goaltending must be augmented with "anticipation-type" behavior was explored. The concept that the ability to react and move is specific to the direction of the response was upheld at all levels of investigation. Standard deviation of the RT/MT/TRT's of each group reflected the degree of variability of performance that is tolerable at each level. This variability generally decreased with each ascending level within both testing conditions, with the exception of the CRT measures. Data collected over a two year period, for four subjects, revealed that the most evident longitudinal changes occurred in MT and that the youngest player experienced the greatest degree of overall improvement.

The nucleated erythrocyte: a model of cell differentiation.

The process of erythropoiesis is characterized by several distinctive features which render it a very useful model of cell differentiation. Mature erythrocytes arise from stem cells in a series of intermediate stages which are fairly well defined both on morphological and on biochemical grounds. During this development, the erythrocytes genome is gradually inactivated and the cell becomes geared to the production of primarily one gene product, hemoglobin. Recently, erythropoiesis has been closely studied in avian species since it has become technically possible to fractionate the blood of anemic birds into high-yield populations of young, developing and mature red cells. Attention has focused on patterns of RNA synthesis including globin m-RNA, in relation to cytoplasmic constitutents becoming modified for reduced activity. From the point of view of gene regulation, erythrocyte development is especially interesting in non-mammals, where in contrast to mammals, even fully mature red cells retain their nuclei. These erythrocytes rank among the most extreme examples of cell specialization and gene repression known. The nuclei of avian erythrocytes and others, contain a tissue-specific histone protein in addition to the more usual complement of vertebrate histone. This histone (H5, V, F2c) has been extensively investigated with a view to linking its presence to structural and molecular changes involved in the condensation and repression of red cell nuclei. The evidence to dat suggests that H5, in conjunction with tissue-specific changes in non-histone proteins, may be responsible for keeping the genomes of nucleated erythrocytes permanently inactive.

Purification and characterization of cytoplasmic protamine messenger ribonucleoprotein particles from rainbow trout testis cells.

Poly(A)-containing protamine messenger ribonucleoprotein particles [poly(A+) pmRNP particles] have been isolated from the polysomal and free cytoplasmic subcellular fractions of trout testis cells by a two-step isolation procedure. Ethylenediaminetetraacetic acid (EDTA) treated particles from both cytoplasmic fractions were first fractionated by sucrose gradient centrifugation and the putative pmRNP particles localized by utilizing 3H-labeled protamine complementary DNA (pcDNA) probes. In addition, particles present in these fractions were characterized by their translational activity in the heterologous, rabbit reticulocyte cell-free system and the protein components of crude mRNP complexes analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoesis. The final purification step involved affinity chromatography of pooled gradient fractions on oligo(dT)-cellulose from which intact pmRNP could be eluted with distilled water at 40 degrees C. Highly purified particles from both polysomal and free cytoplasmic fractions prepared by this procedure had buoyant densities of 1.35-1.37 g/cm3 in CsCl or a protein content of approximately 82%. Particles isolated from EDTA-dissociated polysomes were actively translated in vitro, while their free cytoplasmic counterparts were not. High salt washed pmRNP particles or the RNA extracted from pmRNP preparations, however, directed the synthesis of trout protamines in this system. A model of the activation of stored pmRNP particles in vitro and in vivo is presented.

The organization, composition and matrix of hepatocyte nuclei exposed to alpha-amanitin.

Alterations in the structure and molecular composition of avian hepatocyte nuclei were compared following administration in vivo of lethal and sub-lethal doses of alpha-amanitin. This toxin interferes with extranucleolar transcription by direct inhibition of RNA polymerase II activity. the resultant effects include: extensive condensation of chromatin, displacement of nucleoplasmic contents and fragmentation of nucleoli. Changes in nuclear morphology were quantitated by stereometry and related to variations in RNA and residual, non-histone proteins (NHP). Gross alterations in nuclear structure and depletion of RNA and NHP levels were of similar magnitude with both doses of amanitin. The effects were fully reversible, however, with a minimal dose but terminal with a lethal dose. DNA and histone protein levels remained unchanged at all stages. These results imply that the process of transciption may itself keep and/or maintain chromatin in a dispersed state, and that in the absence of transcription chromatin naturally condenses. Modification of nuclear proteins may be necessary only to maintain chromatin compacted permanently or for extended periods of time. A model of nuclear organization is proposed to incorporate these considerations and to identify the probable location of the nuclear matrix in situ.

Factor VIII gene rearrangement analysis and carrier determination in hemophilia A.

Factor VIII (FVIII) gene rearrangements between the intron 22 F8A sequence in the FVIII gene and either of the two homologous F8A sequences 500 kilobases telomeric to the FVIII gene have recently been found to be responsible for the severe hemophilia A phenotype. We studied 27 patients with severe hemophilia A and 19 with moderate and mild hemophilia, and found FVIII gene rearrangement in 12 patients with severe hemophilia A and none in the patients with moderate or mild disease. Nine of the rearrangements were with the distal telomeric F8A sequence, two were with the proximal sequence, and one had variant distal rearrangement with loss of the FVIII intron 22 F8A band. Two patients with FVIII gene rearrangement had high responding inhibitors, contrary to one previous study suggesting that the presence of a FVIII gene rearrangement is correlated with the absence of inhibitor development. Carrier detection was performed in 17 female relatives, at risk of being carriers, from eight kindreds; 13 were carriers, being heterozygous for the normal and rearranged alleles. The rearrangement assay is particularly useful for carrier determination in families with sporadic cases of hemophilia not helped by linkage analysis with restriction fragment-length polymorphism or intragenic dinucleotide repeat analysis. In all five families with rearrangements and sporadic hemophilia, the mothers of all index patients were found to be carriers.

Hemophilia B carrier determination based on family-specific mutation detection by DNA single-strand conformation analysis.

Single-strand conformation (SSC) analysis can distinguish normal from variant DNA fragments containing single point mutations by conformation-induced electrophoretic mobility shifts in non-denaturing polyacrylamide gels. We studied 25 hemophilia B kindreds by using SSC analysis after polymerase chain reaction (PCR) amplification of the eight factor IX exons and their intron boundaries. Variant SSC fragments were unambiguously identified in 24 kindreds, and direct DNA sequencing of variant PCR fragments identified 20 different hemophilia B mutations. This technique was used for rapid and accurate carrier determination in female family members without the need for additional sequencing studies, because carriers have both normal and hemophilia family-specific SSC fragments. Of 25 obligate carriers from 15 kindreds, 24 were confirmed to carry variant fragments. The exception, a patient's daughter homozygous for the normal allele, was demonstrated by subsequent PCR genotyping to be the result of non-paternity. In the additional 32 at-risk females from 16 kindreds studied, 19 were identified as carriers and 13 as non-carriers. Eleven of the unique mutations affected restriction enzyme digestion sites, and carriers could then be identified by appropriate restriction enzyme digestion of amplified DNA. Our study, with hemophilia B as a model system, demonstrates the accuracy and efficiency of SSC analysis in screening and tracking unknown mutations in monogenic inherited disorders with known gene sequences.

In vitro analysis of allogeneic lymphocyte interaction. VIII. Characterization of helper components of allogeneic effect factor (AEF) that activate Lyb5+ and Lyb5- B cells to respond to thymus-dependent and thymus-independent antigens.

We analyzed the ability of an allogeneic effect factor (AEF), produced using alloactivated A.SW (H-2s) responder T cells and irradiated A/WySN (H-2a) T cell-depleted stimulator spleen cells, to enhance the responsiveness of Lyb5+ and Lyb5- B cells to thymus-dependent (TD) and thymus-independent type 1 (TI-1) and type 2 (TI-2) antigens. We found that normal adult male CBA/CaHN (Lyb5+ + Lyb5-) B cells are activated by unfractionated AEF in primary in vitro IgM antibody responses to the SRBC (TD), TNP-LPS (TI-1), and TNP-Ficoll (TI-2) antigens. Adult male xid CBA/N (Lyb5-) B cells are activated by unfractionated AEF to respond to TNP-LPS but to neither SRBC nor TNP-Ficoll. Gel filtration of AEF on ACA 54 resolves several helper components. One component, which is I-Ak restricted in its activity and may represent a secreted form of an Ia antigen T cell receptor, interacts with histocompatible and functionally competent CBA/CaHN or CBA/N antigen-presenting cells to activate a primary anti-SRBC response of CBA/CaHN B cells but not of CBA/N B cells. A second antigen-nonspecific and H-2-nonrestricted AEF component, which consists of interleukin 2 and possibly also interleukin 1, activates anti-SRBC and anti-TNP-Ficoll responses of both CBA/CaHN B cells and CBA/N B cells. Such CBA/N B cell responses were potentiated by this AEF component only after its biochemical fractionation and only in the presence of a limiting number of T cells. These data indicate that both Lyb5+ and Lyb5- B cells are responsive to TD, TI-1, and TI-2 antigens in the presence of ancillary antigen-nonspecific help. They are also consistent with the notion that T helper cell activation of Lyb5+ B cells is H-2 nonrestricted and that T helper cell activation of Lyb5- B cells is H-2 restricted.

Graft-vs-host reactions induce H-2 class II gene transcription in host kidney cells.

To study the effect of immunologic stimuli on renal expression of Ia antigens (the class II products of the major histocompatibility complex), we induced acute graft-vs-host disease (GVHD) in mice and assessed Ia expression in the host kidney. Serologic absorption analyses showed that the amount of host Ia antigen increased up to tenfold in kidney, and immunofluorescence demonstrated that this increase occurred predominantly in renal tubule epithelial cells. To determine whether these alterations reflected changes in Ia gene transcription, we hybridized DNA probes for mRNAs encoding either Ia E alpha or A beta chains to total RNA extracted from the kidneys of normal mice and mice with acute GVHD. Northern hybridization blots revealed that the level of Ia mRNA expression in GVHD kidney is enhanced about sevenfold when compared with that found in normal kidney. Using the E alpha probe, this result was shown to reflect increased expression of C3H host Ek alpha mRNA, since the Eb alpha gene is not transcribed by the C57BL/6 donor strain used. We conclude that the increase in expression of Ia in renal tubule epithelium during GVHD probably reflects increased I-region gene transcription in host kidney cells. These findings may have implications for the pathogenesis of renal allograft rejection and immunologic renal disease.

Hemophilia A carrier detection by restriction fragment length polymorphism analysis and discriminant analysis based on ELISA of factor VIII and vWf.

We performed carrier determination on female subjects from 32 hemophilia A kindreds with a combination of restriction fragment length polymorphism analysis and discriminant analysis of factor VIII antigen and von Willebrand factor antigen analyzed by enzyme-linked immunosorbent assay. Subjects included 25 obligate carriers, 30 at-risk female subjects from 19 kindreds each with two or more male subjects, with hemophilia and 28 at-risk female subjects from 13 kindreds each with a single sporadic case. Deoxyribonucleic acid (DNA) analysis with factor VIII intragenic probes clarified the carrier status of 15 female subjects, and extragenic probes classified an additional 14. Discriminant analysis, which identified 24 of 25 (96%) obligate carriers with carrier probabilities greater than or equal to 0.71 and 37 of 39 (95.0%) normal female subjects with probabilities less than or equal to 0.30, was useful for clarifying the carrier status of the female subjects who were not helped by DNA analysis and those that were classified by extragenic probes alone. Twenty-nine female subjects could not be categorized by restriction fragment length polymorphism analysis because DNA was unavailable from the subject or from key family members, key female subjects were noninformative, or carriership could not be excluded by restriction fragment length polymorphism in kindreds with sporadic cases. We studied 28 of these female subjects by discriminant analysis; 15 had carrier probabilities of greater than or equal to 0.71, 12 less than or equal to 0.30, and 1 = 0.35. All carriers by extragenic probes had carrier probability values greater than or equal to 0.71, whereas all noncarriers had values less than or equal to 0.30. In some families, particularly those in which gonadal mosaicism was a possibility, extensive family studies together with DNA and discriminant analyses were required for clarifying the source of the mutation, and hence the carrier status of the at-risk female subjects. Thus DNA and discriminant analyses complement each other, and when combined with careful pedigree analysis, the power of carrier determination is increased in some families.

Pulmonary edema during IL-2 therapy: combined effect of increased permeability and hydrostatic pressure.

Systemic administration of recombinant interleukin-2 (rIL-2) has been shown to be promising against certain metastatic cancers. However, major side effects, such as pulmonary edema, have limited its widespread use. Although this pulmonary edema has been attributed to a vascular leak syndrome, this hypothesis has not been verified in humans. The purpose of our study was to determine both the severity and mechanism of pulmonary edema in seven patients treated with rIL-2. The severity of edema was assessed by daily evaluation of chest radiographs, using a semiquantitative scale, as well as by repeated measurements of the alveolar-to-arterial oxygen gradient (A-aDO2) in each patient. To determine the mechanism of pulmonary edema, we serially measured in each patient the lung clearance of technetium 99m-diethylenetriamine pentaacetic acid (DTPA) 99mTc-DTPA), the plasma levels of Von Willebrand factor antigen, and the pulmonary capillary wedge pressure (PCWP). Our results show that there was a gradual increase in the chest radiography edema score that was paralleled by a significant increase in A-aDO2 over its baseline value. During rIL-2 treatment, 99mTc-DTPA clearance was augmented, and the plasma concentration of Von Willebrand factor antigen was elevated. PCWP climbed from 7 to 14 mm Hg and serum total protein fell from 66.1 to 42.1 gm/L. The results obtained indicate that although pulmonary edema associated with rIL-2 treatment is partially dependent on increased permeability of the lung, changes in hydrostatic and oncotic forces may be the principal determinants of edema development.