Annexin 5 mediates a peroxide-induced Ca(2+) influx in B cells.

Annexin 5 is a Ca(2+)-binding protein, the function of which is poorly understood. Structural and electrophysiological studies have shown that annexin 5 can mediate Ca(2+) fluxes across phospholipid membranes in vitro [1]. There is, however, no direct evidence for the existence of annexin 5 Ca(2+) channels in living cells. Here, we show that annexin 5 inserts into phospholipid vesicle membranes at neutral pH in the presence of peroxide. We then used targeted gene disruption to explore the role of annexin 5 in peroxide-induced Ca(2+) signaling in DT40 pre-B cells. DT40 clones lacking annexin 5 exhibited normal Ca(2+) responses to both thapsigargin and B-cell receptor stimulation, but lacked the sustained phase of the response to peroxide. This late phase was due to Ca(2+) influx from the extracellular space, demonstrating that annexin 5 mediates a peroxide-induced Ca(2+) influx. Thus, peroxide induces annexin 5 membrane insertion in vitro, and peroxide-induced Ca(2+) entry in vivo in DT40 cells requires annexin 5. Our results are consistent with a role for annexin 5 either as a Ca(2+) channel, or as a signaling intermediate in the peroxide-induced Ca(2+)-influx pathway.

Immunological development and cardiovascular function are normal in annexin VI null mutant mice.

Annexins are calcium-binding proteins of unknown function but which are implicated in important cellular processes, including anticoagulation, ion flux regulation, calcium homeostasis, and endocytosis. To gain insight into the function of annexin VI, we performed targeted disruption of its gene in mice. Matings between heterozygous mice produced offspring with a normal Mendelian pattern of inheritance, indicating that the loss of annexin VI did not interfere with viability in utero. Mice lacking annexin VI reached sexual maturity at the same age as their normal littermates, and both males and females were fertile. Because of interest in the role of annexin VI in cardiovascular function, we examined heart rate and blood pressure in knockout and wild-type mice and found these to be identical in the two groups. Similarly, the cardiovascular responses of both sets of mice to septic shock were indistinguishable. We also examined components of the immune system and found no differences in thymic, splenic, or bone marrow lymphocyte levels between knockout and wild-type mice. This is the first study of annexin knockout mice, and the lack of a clear phenotype has broad implications for current views of annexin function.

Calcium signaling and annexins.

The annexins, are a family of calcium ion (Ca2+)-binding proteins whose physiological functions are poorly understood. Although many diverse functions have been proposed for these proteins, such as in vesicle trafficking, this review focuses on their proposed roles as Ca2+ or other ion channels, or as intracellular ion channel regulators. Such ideas are founded mainly on in vitro and structural analyses, but there is increasing evidence that at least some members of this protein family may indeed play a part in intracellular Ca2+ signaling by acting both as atypical ion channels and as modulators of ion channel activity. This review first introduces the annexin family, then discusses intracellular localization, developmental regulation, and modes of membrane association of annexins, which suggest roles in Ca2+ homeostasis. Finally, it examines the structural and electrophysiological data that argue for key roles for annexins in the control of ion fluxes.

CD34+ cell selection in chronic phase chronic myeloid leukaemia: a comparison of laboratory grade columns.

CD34 positive (CD34+) cell selection is increasingly used for a number of important applications including gene therapy studies, ex vivo expansion and purging. However there are no data regarding the use of different technologies for CD34+ cell selection in chronic myeloid leukaemia (CML). We therefore compared the performance of three laboratory grade CD34+ selection columns (MiniMACS, Cellpro Ceprate LC and Baxter Isolex 50), using CML chronic phase peripheral blood (PB) and bone marrow (BM). With different CML samples the CD34+ purity from the three columns was equivalent, but comparing five paired samples the Ceprate purity was greater than MiniMACS, at 92.5 and 80.9%, respectively, P = 0.04. Combining results from paired and unpaired CML samples, MiniMACS (n = 7) gave a higher CD34+ yield than Ceprate LC (n = 8) or Isolex 50 (n = 4) with a mean of 51.1%, 24.3% and 13.2% respectively, (P = 0.04 and 0.01). Cell losses with all columns were similar. Attempts to improve the yield from the Ceprate LC columns by modifying the method were unsuccessful. Following MiniMACS and Ceprate LC separation the clonogenic potentials of CD34+ cells in the pre- and positive cell fractions were the same. The proportion of CD34+ 38- or CD34+ DR- cells was unchanged following column separation. These data suggest that the MiniMACS column may be the best column for CD34+ cell selection in CML but these results must be confirmed using large scale clinical columns once the MiniMACS column is licensed. It is possible that variations in CD34+ cell yields between the different columns reflect differences in antibody binding affinity to CML cells, or differences in column technologies.

Hepatitis C seroprevalence in bone marrow transplant recipients and haemophiliacs.

AIM: To determine the prevalence of antibodies to hepatitis C (anti-HCV) in patients who have undergone bone marrow transplantation in Wellington, prior to the introduction of hepatitis C screening, and to contrast these results with the prevalence of anti-HCV in the Wellington haemophiliac population. METHOD: Serum specimens were obtained from 30 patients who had undergone bone marrow transplantation for the treatment of haematological disorders, and from 29 haemophiliacs. Specimens were analysed using a second generation HCV immunoassay. RESULTS: Exposure to blood products was high in bone marrow transplant recipients with subjects receiving red cells or platelets from an average of 53 donors (range 15-100, SD 23.2) during their transplant procedure. Despite the high usage of blood products, only one of the 30 patients tested was positive for hepatitis C on the basis of second-generation antibody testing. Confirmatory testing in this patient, (anti-HCV immunoblot assay) was negative. In contrast, 26 of 29 (89%) haemophiliac patients tested were positive for anti-HCV. CONCLUSION: Although the infective risk of blood products cannot be underestimated, the risk of patients contracting hepatitis C from multiple single-unit transfusions, prior to the introduction of screening for hepatitis C was low. This contrasts with the high risk of hepatitis C seroconversion in patients exposed to pooled plasma products.

Blood cell transplantation.

Blood cell transplantation (BCT) is the procedure of choice for autologous bone marrow transplantation. In this paper we review the current status of BCT with emphasis on important recent advances. These include increasing knowledge of the biologic nature of mobilized blood cells and important evidence showing that very primitive cells are present in blood cells. An increasing understanding of mechanisms of mobilization is likely to result in the design of more rational mobilization strategies. Current mobilization methods are discussed including justification of combined chemotherapy and cytokine mobilization as the method of choice for cancer patients. Single-apheresis BCT may be possible in the future. Recent data show variable malignant contamination of blood cell harvests, but whether this contributes to relapse is unknown. Clinical applications and the efficacy of high-dose therapy are discussed. BCT may allow novel approaches to increase both total dose and dose intensity of therapy as well as application to allogeneic transplantation.

Can mandatory pretransfusion approval programmes be improved?

To improve the appropriateness of blood-component prescribing, a mandatory haematologist pretransfusion approval programme of all non-red-cell components was instituted. This was associated with a 33% decrease in the units of fresh frozen plama (FFP) transfused. Platelet transfusions increased but utilization of both platelets and FFP are now the lowest of the six comparable blood transfusion regions in New Zealand. A subsequent concurrent audit, using preset criteria, of FFP, cryoprecipitate and platelet usage over a 3-month period showed that further reductions in blood component usage could still be achieved, despite the continuing pretransfusion approval policy. This audit showed that 33% of FFP and 30% of cryoprecipitate units transfused were inappropriately given, despite prior haematologist approval. Hospital transfusion practices can be improved by mandatory blood-component pretransfusion approval but concurrent auditing of this programme is required to identify and correct continuing inappropriate blood-component prescribing. Haematologists need to agree on blood-component indications prior to instituting a pretransfusion approval programme in order to provide optimal management.

A conserved nuclear element with a role in mammalian gene regulation.

Mammalian genomes contain numerous fragments of DNA that are derived from inactivated transposable elements. The accumulation and persistence of these elements is generally attributed to transposase activity rather than through possession or acquisition of a function of value to the host genome. Here we describe such a repetitive element, named ALF (forannexin VILINE-2fragment), comprising 130 bp of DNA derived from a LINE-2 sequence, which functions as a potent T-cell-specific silencer. The expansion of the DNA database arising as a result of the human genome sequencing project enabled us to identify ALF in, or close to, several well characterized genes including those for annexin VI, interleukin-4 and protein kinase C-beta. A systematic analysis of the entire LINE-2 sequence revealed that ALF, and not other regions of the LINE-2 sequence, was especially highly represented in the human genome. Acquisition of a function by this repetitive element may explain its abundance. These data show that a conserved fragment of an interspersed nuclear element has the potential to modulate gene expression, a discovery that has broad implications for the way in which we view so-called 'junk' DNA and our understanding of eukaryotic gene regulation.

Co-ordinate regulation of the cystic fibrosis and multidrug resistance genes in cystic fibrosis knockout mice.

The cystic fibrosis (Cftr and multidrug resistance (Mdr1) genes encode structurally similar proteins which are members of the ABC transporter superfamily. These genes exhibit complementary patterns of expression in vivo, suggesting that the regulation of their expression may be co-ordinated. We have tested this hypothesis in vivo by examining Cftr and Mdr1 expression in cystic fibrosis knockout transgenic mice (Cftr(tm1CAM)). Cftr mRNA expression in Cftr(tm1CAM)/Cftr(tm1CAM) mice was 4-fold reduced in the intestine, as compared with littermate wild-type mice. All other Cftr(tm1CAM)/Cftr(tm1CAM) mouse tissues examined showed similar reductions in Cftr expression. In contrast, we observed a 4-fold increase in Mdr1 mRNA expression in the intestines of neonatal and 3- to 4-week-old Cftr(tm1CAM)/Cftr(tm1CAM) mice, as compared with age-matched +/+ mice, and an intermediate level of Mdr1 mRNA in heterozygous Cftr(tm1CAM) mice. In 10-week-old, Cftr(tm1CAM)/Cftr(tm1CAM) mice and in contrast to the younger mice, Mdr1 mRNA expression was reduced, by 3-fold. The expression of two control genes, Pgk-1 and Mdr2, was similar in all genotypes, suggesting that the changes in Mdr1 mRNA levels observed in the Cftr(tm1CAM)/Cftr(tm1CAM) mice are specific to the loss of Cftr expression and/or function. These data provide further evidence supporting the hypothesis that the regulation Cftr and Mdr1 expression is co-ordinated in vivo, and that this co-ordinate regulation is influenced by temporal factors.

Presentation of intravascular lymphomatosis as lumbosacral polyradiculopathy.

A 53-year-old man developed progressive sensory disturbance and weakness in the legs, sphincter disturbance, back pain, systemic symptoms, and pancytopenia. Electrophysiological tests indicated a widespread lumbosacral polyradiculopathy. Spinal magnetic resonance imaging and routine cerebrospinal fluid analysis showed minor nonspecific abnormalities. Bone marrow and liver biopsies showed hemophagocytosis; and polymerase chain reaction of cerebrospinal fluid, bone marrow, and serum suggested active infection with human herpesvirus-6. Autopsy revealed that his neurological symptoms resulted from intravascular lymphomatosis (angiotropic large cell lymphoma), a rare variant of lymphoma with predilection for the nervous system.