Tim R. Rutherford

Use of immunoglobulin gene rearrangements to show clonal lymphoproliferation in hyper-reactive malarial splenomegaly.

In Africa, hyper-reactive malarial splenomegaly (HMS), which is also known as tropical splenomegaly syndrome, can be associated with a prominent lymphocytosis in blood and bone marrow that is difficult to distinguish clinically from chronic lymphocytic leukaemia (CLL). The observation that some patients with HMS become resistant to treatment with anti-malarial drugs has led to the suggestion that HMS may evolve into a malignant lymphoproliferative disorder. To test this hypothesis, 22 Ghanaian patients with HMS and/or lymphocytosis were categorised by degree of response to proguanil according to standard clinical criteria, and DNA was extracted from peripheral blood cells and screened for rearrangements of the Jh region of the immunoglobulin gene with a DNA probe. Clonal rearrangements of the Jh region were found in all 3 patients with no response, in none of 13 patients with sustained response, and in 2 of 6 patients with moderate response or relapse on proguanil therapy. The detection of such rearrangements, and hence clonal lymphoproliferation in individuals with clinical features intermediate between HMS and CLL, supports the hypothesis that HMS may evolve into a malignant lymphoproliferative disorder.

Splenic lymphoma with villous lymphocytes in tropical West Africa

Splenic lymphoma with villous lymphocytes (SLVL) is a monoclonal B-lymphoproliferative disorder characterised by splenomegaly and distinctive villous lymphocytes in the peripheral blood. It has not previously been reported from Africa, but we describe ten Ghanaian patients with SLVL seen at one hospital during a 4-year period. The clinical presentation is similar in Africa and in temperate regions, though the lymphocyte count is higher in African patients and the disorder predominantly affects middle-aged women rather than elderly men. It is likely that SLVL has previously been classified as splenic chronic lymphocytic leukaemia or hyper-reactive malarial splenomegaly.

Circulating villous lymphocytes—a link between hyperreactive malarial splenomegaly and splenic lymphoma

Significant numbers of villous lymphocytes were noted in the blood of patients with a clinical diagnosis of hyperreactive malarial splenomegaly (HMS) in Ghana. Demographic and haematological data were recorded from 22 patients with massive splenomegaly. Additional investigations included lymphocyte immunophenotyping, protein electrophoresis and immunoglobulin gene rearrangements. Although all patients had over 30% villous lymphocytes and no leucocytosis, 7 had no evidence of a monoclonal disorder. Immunophenotyping and the presence of monoclonal lymphocytes identified 3 further patients with B-cell splenic lymphoma with villous lymphocytes (B-SLVL). HMS and SLVL co-existed in the same, predominantly female, patient population and were indistinguishable except by molecular analysis of lymphocytes. The discovery of the uncommon villous lymphocytes in both non-malignant and malignant disorders in the same geographical area suggested that HMS and SLVL are pathophysiologically related. In Caucasians with SLVL the malignant cells arise from B-cells that have undergone antigen selection. We postulate that the excessive proliferation of polyclonal B-lymphocytes, driven by frequent exposure to malaria, predisposes to the emergence of a malignant lymphoma, B-SLVL, in tropical West Africa.

Gene-specific chromatin damage in human spermatozoa can be blocked by antioxidants that target mitochondria

Incubation of gradient purified human spermatozoa, which are routinely maintained in media prior to IVF and intracytoplasmic sperm injection (ICSI), induced DNA strand breaks (up to 89 nicks x 10(-3) bp) and chromatin release. Unlike highly dispersed Alu repeat sequences, the centromeric heterochromatin was much less susceptible to endonuclease attack. In addition to chromatin release, the permeability of the sperm membrane was altered as evidenced by reduced accessibility of sperm nuclei to decondensation factors in mouse embryo extracts. Hybridization of cDNA microarrays with DNA released from spermatozoa revealed a consistent hypersensitivity of certain genes to endogenous cleavage including TP53, VHL (tumour suppressors), BRCA1 (breast cancer), NOS1 (neurotransmitter), PECAM1, FLT1 (angiogenesis) and CDKN1C (cell cycle/imprinted). N-tert-butyl hydroxylamine (NTBH), a derivative of the anti-teratogenic alpha-phenyl-N-t-butyl nitrone (PBN) and synthetic superoxide dismutase (SOD)/catalase mimetics inhibited chromatin release and sustained or dissipated relative mitochondrial membrane potential. Together, these results show a link between the hyperactivation of sperm mitochondria and chromosomal damage of specific genes in vitro, and that the potential risk of disruption of paternally contributed genes can be circumvented by antioxidants which are known to target mitochondria.

Serological similarities between hyperreactive malarial splenomegaly and splenic lymphoma in West Africa

In West Africa hyperreactive malarial splenomegaly (HMS) and splenic lymphoma with villous lymphocytes (SLVL) are demographically and clinically indistinguishable. Determination of lymphocyte clonality is needed to differentiate clearly between these 2 disorders. To obtain evidence to support our hypothesis that HMS and SLVL are aetiologically related we studied the serological profile of malaria-related antibodies in HMS and SLVL in West Africa. We found that in SLVL total immunoglobulin M and antimalarial antibody levels were markedly raised, a combination which is characteristic of HMS. These findings strongly support a developmental relationship between HMS and SLVL in tropical Africa and implicate malaria in this process.

B-lymphotropic viruses in a novel tropical splenic lymphoma

Peripheral blood from patients with a novel tropical splenic lymphoma, characterized by splenomegaly and circulating naïve CD5‐negative villous B lymphocytes, has been screened for evidence of an association with the B‐lymphotropic viruses, Epstein–Barr virus (EBV), hepatitis C virus (HCV) and human herpesvirus 8 (HHV8). No increased prevalence of EBV, HCV and HHV8 was demonstrated using serological and molecular techniques, compared with a geographical, age‐matched control group. However, lymphoma patients had markedly raised EBV antibody levels without a concomitant increase in the rate of detection of viral genomes in the peripheral blood. This phenomenon also occurred in patients with hyper‐reactive malarial splenomegaly, a condition that occurs in the same geographical area and that is clinically indistinguishable from tropical splenic lymphoma, adding further weight to the suggestion that there may be an aetiological association between these two disorders.

Fidelity and reproducibility of antisense RNA amplification for the study of gene expression in human CD34+ haemopoietic stem and progenitor cells

Summary. Microarrays provide a powerful tool for the study of haemopoietic stem and progenitor cells (HSC). Because of the low frequency of HSC, it is rarely feasible to obtain enough mRNA for microarray hybridizations, and amplification will be necessary. Antisense RNA (aRNA) amplification is reported to give high‐fidelity amplification, but most studies have used only qualitative validation. Before applying aRNA amplification to the study of HSC, we wished to determine its fidelity and reproducibility, and whether statistically significant results can be obtained. We found that aRNA amplification introduced biases into relative RNA abundance. However, these biases were extremely consistent, and valid comparisons could be made, if amplified RNA was compared with amplified RNA. By applying this method to the effect of interferon‐γ and tumour necrosis factor‐α on normal primary CD34+ HSC, biologically significant differences could be detected, including potential mechanisms for resistance of CD34+ cells to CD95‐mediated apoptosis and evidence of the differentiating effects of the cytokines. Differences of twofold or less were detected, and most of these differences attained statistical significance after triplicate experiments. These data demonstrate that aRNA amplification can be used with microarray hybridization to study the transcriptional profiles of small numbers of primary CD34+ HSC.

Absence of N-RAS point mutations in peripheral blood cells of patients with aplastic anaemia and paroxysmal nocturnal haemoglobinurea.

Summary. The myelodysplastic syndromes (MDS) have a significant frequency of evolution into acute myeloid leukaemia (AML). Approximately 30% of MDS patients show activating mutations of the N‐RAS proto‐oncogene, and these patients are at increased risk of leukaemic evolution. Long‐term survivors of aplastic anaemia (AA) and paroxysmal nocturnal haemoglobinurea (PNH) are also at significant risk of developing AML. We have screened peripheral blood DNA from 42 AA patients and 15 PNH patients for the presence of N‐RAS point mutations. No mutations were detected in these samples, indicating that the mechanisms of evolution into AML may be different from those in MDS.

Differential apoptosis and Fas expression on GPI-negative and GPI-positive stem cells: a mechanism for the evolution of paroxysmal nocturnal haemoglobinuria.

Summary. Paroxysmal nocturnal haemoglobinuria (PNH) has a dual pathogenesis. PIG‐A mutations generate clones of haemopoietic stem cells (HSC) lacking glycosylphosphatidylinositol (GPI)‐anchored proteins and, secondly, these clones expand because of a selective advantage related to bone marrow failure. The first aspect has been elucidated in detail, but the mechanisms leading to clonal expansion are not well understood. We have previously shown that apoptosis and Fas expression in HSC play a role in bone marrow failure during aplastic anaemia. We have now investigated apoptosis in PNH. Ten patients were studied. Apoptosis, measured by flow cytometry, was significantly higher among CD34+ cells from patients compared with healthy controls. Fas expression was also increased. Cells that were stained for CD34, CD59 and apoptosis showed a significantly lower apoptosis in CD34+/CD59− compared with CD34+/CD59+ cells from the same patient. In three patients, staining for CD34, CD59 and Fas revealed lower Fas expression on CD34+/CD59− cells. Differential apoptosis of CD34+/CD59− HSC may be sufficient in itself to explain the expansion of PNH clones in the context of aplastic anaemia. In addition to demonstrating a basic mechanism underlying PNH clonal expansion, these results suggest further hypotheses for the evolution of PNH, based on the direct or indirect resistance of GPI‐negative HSC to pro‐inflammatory cytokines.