Age-specific regulation of clotting factor IX gene expression in normal and transgenic mice

Factor IX (FIX), a circulating serine protease that serves as an essential component of the blood coagulation pathway, has been shown to increase with age in humans. We show here that murine FIX mRNA and activity levels also increase with age. Furthermore, one form of hemophilia B, hemophilia B Leyden, which is caused by mutations within the promoter region of the FIX gene, has a distinct age-dependent phenotype. To determine the source of the age-related increases in FIX gene expression, we have analyzed the regulation of the normal FIX gene promoter and FIX Leyden gene promoter with the +13 mutation during aging by generating transgenic mice that contain the -189 to +21 bp promoter segment ligated to a chloramphenicol acetyltransferase reporter gene. We have established that the normal FIX promoter and the Leyden promoter transgenes are expressed in a tissue-specific manner in vivo. The normal FIX promoter transgene does not show any differences in the pattern of expression with age or sex of the organism, whereas the Leyden promoter transgene showed age-dependent male-specific expression. This is the first demonstration of the FIX Leyden phenotype in a transgenic mouse model.     

Genetic analysis is consistent with the hypothesis that NF1 limits myeloid cell growth through p21ras

Children with neurofibromatosis, type 1 (NF-1) are at increased risk of developing malignant myeloid disorders and their bone marrows frequently show loss of the normal allele of the NF1 tumor-suppressor gene. NF1 encodes a protein called neurofibromin, which accelerates guanosine triphosphate (GTP) hydrolysis on the p21ras (Ras) family of signaling proteins. We used a genetic approach to test the hypothesis that NF1 negatively regulates myeloid cell growth through its effect on Ras. This model predicts that, if RAS mutations and loss of NF1 function deregulate myeloid growth by the same biomechanical mechanism, then activating RAS mutations will be restricted to children with malignant myeloid disorders who do not have NF-1. We studied 71 children, including 28 with bone marrow monosomy 7 syndrome (Mo7), 35with juvenile chronic myelogenous leukemia (JCML), three with other forms of preleukemia, and five with acute myelogenous leukemia (AML), for activating mutations of KRAS and NRAS. The incidence of RAS mutations was 21% (12 of 55) in patients without NF-1 and 0% (zero of 16) in children with NF-1 (P = .04). Among the 55 patients who did not have NF-1, we found RAS mutations in four of 27 with Mo 7, in five of 24 with JCML, in two of 3 with AML, and in a patient with myeloproliferative syndrome (MPS). These data from primary human cancer cells provide strong genetic evidence that NF1 limits the growth of myeloid cells by regulating Ras.     

Characterization of Thy-1 (CDw90) expression in CD34+ acute leukemia

Thy-1 (CDw90) is a phosphatidylinositol-anchored cell surface molecule which, when coexpressed with CD34 in normal human bone marrow, identifies a population of immature cells that includes putative hematopoietic stem cells. To date, the characterization of Thy-1 expression has been confined largely to normal tissues and cell lines. In this study, we evaluated the frequency and intensity of Thy-1 expression as defined by reactivity with the anti-Thy-1 antibody 5E10 in 38 cases of CD34+ acute leukemia (21 acute myelogenous leukemia [AML], 8 chronic myelogenous leukemia [CML] in blast crisis, and 9 acute lymphoblastic leukemia [ALL]). In 34 of 38 cases (89%) the CD34+ cells lacked expression of the Thy-1 antigen. High-density Thy-1 expression was found in 1 case of CML in lymphoid blast crisis, and low- density Thy-1 expression was identified on a portion of the leukemic cells in 2 cases of AML with myelodysplastic features, and 1 case of CML in myeloid blast crisis, suggesting a possible correlation between Thy-1 expression and certain instances of stem cell disorders such as CML and AML with dysplastic features. In contrast, the dissociation of Thy-1 and CD34 expression in the majority of acute leukemias studied suggests that the development of these leukemias occurs at a later stage than the hematopoietic stem cell. Characterization of Thy-1 expression in acute leukemia may eventually provide insights into the origin of the disease. In addition, separation of leukemic blasts from normal stem cells based on Thy-1 expression may prove useful in assessing residual disease, as well as in excluding leukemic blasts from stem cell preparations destined for autologous bone marrow or peripheral stem cell transplantation.     

Interaction of hemoglobin E and pyrimidine 5' nucleotidase deficiency

A Bangladeshi family is described in which the genes for both hemoglobin E (Hb E) and pyrimidine 5' nucleotidase deficiency are segregating. An individual homozygous for both these conditions has a severe hemolytic anemia, whereas family members who are homozygous for Hb E are asymptomatic and those homozygous for pyrimidine 5' nucleotidase deficiency have the mild hemolytic anemia that is characteristic of this disorder. Globin-chain synthesis experiments have shown that the mechanism underlying the interaction between these two genotypes is a marked decrease in the stability of Hb E in pyrimidine 5' nucleotidase-deficient red blood cells (RBCs). It has also been found that in the enzyme-deficient RBCs in which Hb E is highly unstable, free alpha-chains, though not beta E-chains, acoumulate on the membrane. In view of the increasing evidence that the hemolysis associated with pyrimidine 5' nucleotidase deficiency results not only from an increase in the level of erythrocyte pyrimidines, but also from inhibition of the hexose monophosphate shunt activity in young erythrocytes, it is likely that the marked instability of Hb E in the enzyme-deficient cells results from oxidant damage acting on a mildly unstable Hb variant. These observations may have important implications for the better understanding of the pathophysiology of Hb E/beta-thalassemia, globally the commonest important form of thalassemia.     

The place of high-dose BEAM therapy and autologous bone marrow transplantation in poor-risk Hodgkin's disease. A single-center eight- year study of 155 patients

Although high-dose chemotherapy and autologous bone marrow transplantation (ABMT) are increasingly being used for the treatment of relapsed and resistant Hodgkin's disease, there have been few large, single-center studies reported with adequate follow-up to allow full evaluation of this therapeutic modality. We present 155 poor-risk Hodgkin's disease patients who received high-dose BEAM (BCNU, etoposide, cytosine arabinoside, and melphalan) chemotherapy and ABMT who have been studied over a period of 8 years. All patients had either not attained a remission on mechlorethamine, vincristine, procarbazine, prednisone-type therapy and had poor prognostic features at presentation, not attained a complete remission or relapsed within 1 year of an initial alternating regimen, or not attained remission with two or more lines of treatment. At the time of ABMT the relapse status of the patients was as follows: 46 patients were primarily refractory to induction therapy, 7 were good partial responders, 52 were in first relapse, 37 in second relapse, and 13 in third relapse. Seventy-eight patients had chemoresistant disease, 33 had chemosensitive disease at the time of ABMT, and 44 were untested for chemosensitivity at latest relapse. The procedure related mortality in the first 90 days post-ABMT of 10% overall. At 3 months 43 patients (28%) were assessed as complete responders, 72 patients had a partial response (46%), and 24 patients (16%) had no response or progression of disease. However, by 6 months, 53 (24%) patients were assessed as complete responders and 51 (33%) patients had nonprogressive disease. Forty-five patients had received radiotherapy post-ABMT to residual masses (41 patients) or to previous sites of bulk disease (4 patients). The actuarial overall and progression-free survival at 5 years was 55% and 50%, respectively. On multivariate analysis patients with bulk (masses > 10 cm), heavily pretreated patients (those receiving three or more lines of treatment) and females had a significantly poorer prognosis. Relapse status was also significant for progression-free survival in that patients in second (60%) and third relapse (70%) had a better prognosis than those in first relapse (44%) or with primary refractory disease (33%). Response to prior chemotherapy did not predict for progression-free survival. These results enable comparisons to be made between high-dose chemotherapy with ABMT and conventional dose salvage therapy. Furthermore, although the results as a whole are highly encouraging, certain groups carry an unfavorable prognosis.     

A staphylococcal slide test for detection of antineutrophil antibodies

We describe a simple test for direct or indrect detection of antineutrophil antibodies. Sensitized leukocytes adherent to glass slides and fixed with paraformaldehyde can be stored in buffer for at least 3 wk. Killed Cowan I staphylococci, containing protein A, bind to sensitized but not control cells, and binding is ascertainable by light microscopy. Indirect tests were positive for 39/41 patients suspected of having immune neutropenia and found to have antineutrophil antibodies by an indirect radiochemical opsonic method. Fifty-four control sera from healthy persons, patients with bone marrow failure, or with immune complex diseases without neutropenia, gave negative indirect tests. Direct tests for cell-bound antibody could be done even during severe neutropenia by reacting fixed autologous cells with staphylococci in the absence of added serum. In some patients only the direct test was positive.     

Fibrinolysis during liver transplantation in humans: role of tissue- type plasminogen activator

Human liver transplantation is frequently associated with a coagulopathy and bleeding diathesis developing during the anhepatic phase of surgery. The hemostatic defect has been attributed in part to accelerated fibrinolysis. In this study we evaluated changes in specific blood fibrinolytic parameters occurring in eight adult patients undergoing first-time orthotopic liver transplantation. Five of the eight patients experienced moderate to severe systemic fibrinolysis as reflected by alpha 2-antiplasmin consumption and fibrinogen degradation with the concomitant appearance of fibrin(ogen) degradation products. In association with these changes, an increase in tissue-type plasminogen activator (t-PA) activity and t-PA antigen levels was also observed. Fibrinolysis was most pronounced during the anhepatic phase of surgery and decreased after revascularization of the grafted liver. Three additional patients who underwent the same procedure manifested much less evidence of systemic fibrinolytic activation and had minimal elevation of t-PA antigen levels or activity. Urokinase-type plasminogen activator levels, although elevated in three patients, were disassociated from increased t-PA levels and concomitant systemic fibrinolysis. The operative course of those patients developing t-PA-associated fibrinolysis was characterized by shock, acidosis, generalized bleeding, and a need for substantially greater blood product support during surgery. These findings suggest that the observed fibrinolytic defect is related to increased circulating plasma levels of t-PA, presumably resulting from a combination of increased intravascular release and decreased hepatic clearance of t-PA. These observations may have implications for intraoperative therapy for the transplant-related coagulopathy and its associated bleeding.     

Donor immunization with Haemophilus influenzae type b (HIB)-conjugate vaccine in allogeneic bone marrow transplantation

Bone marrow transplant patients are at increased risk for infections with polysaccharide encapsulated organisms and respond poorly to polysaccharide vaccines. We evaluated the effect of donor immunization with Haemophilus influenzae type b (HIB) polysaccharide-conjugate vaccine on recipient antibody responses following allogeneic bone marrow transplantation. Thirty-two allogeneic transplant patients and their donors were immunized before transplantation with HIB-conjugate, tetanus toxoid and 23-valent pneumococcal vaccines. Following transplantation, patients received HIB-conjugate and tetanus toxoid vaccines at 3, 6, 12, and 24 months and 23-valent pneumococcal vaccine at 12 and 24 months. Thirty-three patients with unimmunized donors were immunized following transplantation in an identical manner. Patients whose donors were immunized had significantly higher total anti-HIB antibody concentrations at 3 months (P = .0001), 6 months (P = .0001), 12 months (P = .0001), and 24 months (P = .002) after transplant compared with patients whose donors were unimmunized. Higher antitetanus toxoid antibody concentrations were also noted in patients with immunized donors, whereas donor immunization with pneumococcal vaccine had no effect on antibody concentrations following transplantation. Donor immunization with HIB-conjugate vaccine resulted in higher antibody concentrations in patients as early as 3 months after allogeneic transplantation and may be an effective strategy to prevent HIB infections.     

Does Standing Affect Acetabular Component Inclination and Version After THA?

Background  

Avoiding complications after hip arthroplasty with hard-on-hard bearings, especially metal-on-metal, correlates with the position of the acetabular component. Supine imaging with conventional radiography has traditionally been utilized to assess component inclination (abduction), as well as anteversion, after THA and surface replacement arthroplasty (SRA). However, most adverse events with hard bearings (excessive wear and squeaking) have occurred with loading. Standing imaging, therefore, should provide more appropriate measurements.