B. Mendelow

Abnormalities of chromosome 12p 13 and malignant proliferation of eosinophils: a nonrandom association

Summary. Four patients representing a spectrum of haematological malignancies are reported. Two patients had Philadelphia chromosome negative myeloproliferative disorders, one had acute lymphoblastic leukaemia and one had eosinophilic leukaemia. In each case eosinophilia was present and demonstrated to be part of the malignancy by the association of clonally abnormal metaphases with eosinophil granules. Abnormalities involving the short arm of chromosome 12 (12p 13) were a constant feature in all four cases and therefore a nonrandom association between this chromosome region and malignant eosinophil proliferation is proposed.

Automated malaria detection by depolarization of laser light

Anecdotal experience with full blood count (FBC) technology incorporating analysis of depolarized laser light (DLL) for the enumeration of eosinophils showed that malaria infection generated unusual distributions in the white cell channels. The objective of this study was to identify and define criteria for a diagnosis of malaria using this technology. To determine sensitivity, specificity, and positive and negative predictive values, 224 directed samples referred specifically for malaria were used; true positives were defined as those in which malaria was identified by microscopic and/or immunological methods. For the DLL method, positive was defined as one or more large mononuclear cell(s) for which the 90° depolarized signal exceeded the 90° polarized signal. To determine possible utility in a routine haematology laboratory setting, 220 random undirected FBC samples were evaluated for possible malaria infection by the DLL method. Of the 224 directed samples, 95 were malaria positive as determined by microscopic and/or immunological methods, and 129 were negative. For the DLL method, overall sensitivity was 72% (90% in the case of Black Africans), and specificity 96%. Positive and negative predictive values overall were 93% and 82% respectively. In the utility study a single positive result was identified among the 220 samples studied. This was found to be from a patient with malaria. The detection of unexpected malaria by automated screening FBC analysis could substantially lower the mortality and morbidity from unascertained infection, especially in indigenous African peoples.

CD4+ T cell enumeration in HIV infection with limited resources

The incidence of human immunodeficiency virus (HIV) infection continues to increase in South Africa. Limited resources are available for diagnosis and management of the disease and the development of affordable strategies is required. Absolute CD4 counts are used locally predominantly to monitor disease progression and institute prophylaxis against opportunistic infections. A dramatic increase in demand for CD4 counts prompted an investigation for a more cost-effective flow cytometry method than those currently recommended by the Centers for Disease Control (CDC). CD4 counts generated by two different single tube methods using CD3/CD4/CD8 [1(3)] and CD4 [1(1)] antibodies, respectively, were compared to the CDC recommended 6 tube 2 colour panel [6(2)]. Whole blood analysis using the Coulter Multi-Q-Prep system and an Epics XL Flow Cytometer (Coulter, Hialeah, FL) was performed for each of the three methods. Random samples from HIV positive adult patients were compared. A mean difference in the absolute CD4 counts of less than 10x10(6)/l was generated by both of the alternative panels when compared with the 6(2) panel. The precision of the three methods is comparable. In reagents alone, the 1(3) and 1(1) methods represent a cost saving of 76% and 93%, respectively, over the 6(2) method. The 1(3) and 1(1) panels would permit more affordable CD4 counts to be determined by the gold standard methodology of flow cytometry with no clinically significant sacrifices in accuracy or precision.

Cytogenetic findings in chronic myeloid leukemia (CML); evaluation of karyotype, blast morphology, and survival in the acute phase

Abstract Cytogenetic data on a series of 68 patients with chronic myeloid leukemia (CML) are presented, with emphasis on chromosomal findings in 19 patients who either transformed from a chronic to an acute phase (12 cases) or presented in an acute phase (7 cases). Correlation of chromosome patterns, blast morphology, and survival was attempted, following the aims and recommendations of the First International Workshop in Leukemia (Cytogenet Cell Genet (1977): 19, 321). A Philadelphia (Ph 1 ) chromosome was detected in 61 of 68 patients (90%) and identified by banding in 40 cases; 39 patients had the usual (9;22) translocation and one patient showed an unusual (17;22) Ph 1 translocation. Clonal abnormalities in addition to the Ph 1 were found in 9% of chronic patients whereas 79% of acute patients showed karyotypic clonal evolution. The three most frequently observed patterns of clonal evolution were trisomy 8, two Ph 1 chromosomes and an isochromosome 17 (i(17q)). Two Ph 1 chromosomes were observed in both chronic and acute cases, but in this series, trisomy 8 and i(17q) were detected only in acute patients. Detection of clonal evolution is generally indicative of an accelerated phase in the majority of cases and the development of an i(17q) clone is particularly ominous. After transformation, no obvious correlation between the pattern of clonal evolution, blast morphology, or survival was found.

Karyotype analysis in acute nonlymphocytic leukemia (ANLL): Comparison with ethnic group, age, morphology, and survival

The karyotype, leukemia cell morphology (FAB classification), ethnic group, age, sex, and survival were compared in 60 patients with acute nonlymphocytic leukemia (ANLL), to determine their diagnostic and prognostic significance. An ethnic age difference was observed; a significantly greater number of black patients were children. The majority of children were males. A higher frequency of chromosome abnormalities was detected in children, yet they survived longer than adults. A specific, significant association between a (8; 21) karyotype and M2-ANLL was confirmed; four of ten patients with M2-ANLL showed this translocation. The more mature morphology of M2-ANLL was associated with a longer survival irrespective of karyotype, ethnic group, and age. The specificity of t(15; 17) in M3-ANLL and nonrandom monosomy 7 in preleukemic children was confirmed. Patients, particularly adults, with normal karyotypes tended to survive longer than those with abnormal karyotypes. The patients age and the differentiative capacity of the leukemic cell appear to be as important as the karyotype in determining survival. The nonrandom association of certain chromosome aberrations in ANLL appears to be worldwide.

Acute megakaryoblastic leukaemia with 3q inversion and elevated thrombopoietin (TSF): an autocrine role for TSF?

Summary. A patient with acute megakaryoblastic leukaemia is described in whom exactly the same paracentric inversion of 3q was detected as in three previously documented cases. The patients serum thrombopoietin (TSF) was significantly raised. Based on these findings we postulate a role for a gene (? oncogene) on chromosome 3q in thrombopoietin production. Abnormalities of 3q may assist in delineating a subgroup of acute nonlymphocytic leukaemia, namely acute megakaryoblastic leukaemia.

Stabilization of HIV-1 gp120-CD4 Receptor Complex through Targeted Interchain Disulfide Exchange

HIV-1 enters cells via interaction between the trimeric envelope (Env) glycoprotein gp120/gp41 and the host cell surface receptor molecule CD4. The requirement of CD4 for viral entry has rationalized the development of recombinant CD4-based proteins as competitive viral attachment inhibitors and immunotherapeutic agents. In this study, we describe a novel recombinant CD4 protein designed to bind gp120 through a targeted disulfide-exchange mechanism. According to structural models of the gp120-CD4 receptor complex, substitution of Ser60 on the CD4 domain 1 α-helix with Cys positions a thiol in proximity of the gp120 V1/V2 loop disulfide (Cys126–Cys196), satisfying the stereochemical and geometric conditions for redox exchange between CD4 Cys60 and gp120 Cys126, and the consequent formation of an interchain disulfide bond. In this study, we provide experimental evidence for this effect by describing the expression, purification, refolding, receptor binding and antiviral activity analysis of a recombinant two-domain CD4 variant containing the S60C mutation (2dCD4-S60C). We show that 2dCD4-S60C binds HIV-1 gp120 with a significantly higher affinity than wild-type protein under conditions that facilitate disulfide exchange and that this translates into a corresponding increase in the efficacy of CD4-mediated viral entry inhibition. We propose that targeted redox exchange between conserved gp120 disulfides and nucleophilic moieties positioned strategically on CD4 (or CD4-like scaffolds) conceptualizes a new strategy in the development of high affinity HIV-1 Env ligands, with important implications for therapy and vaccine development. More generally, this chalcogen substitution approach provides a general means of stabilizing receptor-ligand complexes where the structural and biophysical conditions for disulfide exchange are satisfied.

Laboratory monitoring of HIV / AIDS in a resource-poor setting.

Currently less than about 5% of individuals requiring anti- retroviral therapy (ART) can access these medicines in a resource-poor setting. In South Africa there are at least 4.7 million HN-positive individuals each of whom has a right to therapy. Access to ART has however been limited locally to that minority of individuals fortunate enough either to be able to afford to pay independently or to have entered sponsored programmes of HIV/AIDS disease management. Sponsorship may be related to pharmaceutical drug trials grant-funded programmes or HIV/ATDS programmes offered by employers. To parallel ART initiatives laboratory monitoring of varying degrees is essential to ensure that safety is not compromised in individuals taking ART drugs. Despite recent substantial decreases in ART costs (in some instances free drugs) expensive laboratory testing may still be a limiting factor in the implementation of ART programmes. (excerpt)

Evaluation of DNA analysis for evidence of apoptosis in megaloblastic anaemia

This study involved DNA analysis of bone marrow cells of 15 patients with megaloblastic anaemia. The diagnosis was based on the morphological changes seen in the bone marrow, associated with either a low red cell folate or serum vitamin B12 level and an adequate response to appropriate therapy as confirmation of the diagnosis. Flow cytometric DNA analysis showed an increase in the S and G2 phases of the cell cycle, but conventional agarose gel electrophoretic DNA analysis did not confirm the characteristic ‘ladder pattern’ which might have been expected in classic apoptosis. In addition, cells showing morphological changes suggestive of apoptosis, such as nuclear condensation and fragmentation, did not show evidence of DNA fragmentation using the ApopTagTMin situ digoxigenin nucleotide labelled, peroxidase detection system. Further studies using annexin V flow cytometric analysis and pulsed field gel electrophoresis were also unable to detect evidence of apoptosis as a significant cause of cell death in megaloblastic anaemia.

Characterization of bone marrow stromal cells in suspension and monolayer cultures.

Summary. Aspirated marrow fragments from healthy adult baboons were cultured in a simple liquid suspension tissue culture system. During the incubation period, haemopoietic cells were discharged from the fragments and settled to the floor of the culture vessels, thus allowing a separation of marrow stromal elements, retained within the fragments. Cells associated with the marrow stroma in vitro were of four main types: adipocytes, macrophages, plasma cells and unidentified lipid‐laden cells related to fibroblasts. These last cells were capable of DNA synthesis in vitro, and were morphologically and functionally distinguishable from typical marrow macrophages. Macrophages were characterized by their phagocytic properties. Plasma cells were capable of prolonged survival and immunoglobulin output in vitro.