Polymerase chain reaction on cerebrospinal fluid cells in the detection of leptomeningeal involvement by B-cell lymphoma and leukaemia: a novel strategy and its implications

In the search of B-cell lymphoma/leukaemia dissemination to cerebrospinal fluid (CSF), we used the highly sensitive semi-nested PCR (snPCR) for the analysis of IgH gene rearrangements. This method detects a rearranged IgH gene from a single B lymphocyte which may or may not represent the neoplastic B-cell population. We therefore performed multiple snPCR (three to five) experiments on the same CSF sample, postulating that the detection of a band of the same size and sequence in different PCR runs was highly indicative of a clonal population. 17 consecutive cases with a differential diagnosis of primary (P cns L) ( n =10) or secondary (S cns L) ( n =7) CNS lymphoma or leukaemia were investigated by the new strategy. The clonal nature of the B-cell population was confirmed in 3/10 of suspected P cns L, and in six other cases the PCR study was indicative of reactive lymphocytosis. One case revealed a clonal B-cell population in the clinical context of an autoimmune disorder. Evidence of clonal B-cell population was found in 4/7 of suspected S cns L. In one of these cases the detected band and its sequence proved identical to that of the primary nodal lymphoma. We believe that the evaluation of B-cell clonality in CSF requires multiple snPCR amplification on the same sample to compare the size of the products and, if necessary, the DNA sequences to ascertain the diagnosis of malignancy in equivocal cytologic and clinical findings.     

Transforming growth factor beta-1 (TGF-β1) released by an Epstein-Barr virus (EBV) positive spontaneous lymphoblastoid cell line from a patient with Kostmann's congenital neutropenia inhibits the growth of normal committed haemopoietic progenitors in vitro

Summary This study reports the characterization of a spontaneous lymphoblastoid cell line (LCL) raised from the peripheral blood of a patient with Kostmann's congenital neutropenia. The LCL was composed of EBV-infected polyclonal B cells and displayed surface markers and pattern of growth in vitro typical of normal LCLs. The supernatant of the LCL contained a colony inhibiting activity (CIA) that decreased the cloning efficiency of normal committed haemopoietic progenitors and was identified as immunoreactive transforming growth factor β1 (TGF-β1) by neutralization experiments with a specific antiserum. Control studies with a panel of LCLs spontaneously derived from the peripheral blood of patients seropositive for Epstein-Barr virus (EBV) infections showed that 5/30 LCLs produced a CIA. This CIA was not identifiable as TGF-β1 but rather was due to the combined effects of tumour necrosis factor α (TNFα). tumour necrosis factor β (TNFβ) and interferon α (IFNα), that were present in the LCL supernatants. The hypothesis that the B cells latently infected by EBV in vivo and possibly expanded as a consequence of the infection may have contributed to the inhibition of the patient granulopoiesis by releasing TGF-β1 will be discussed.     

Carboxy-terminal peptides (C1–24 and C13–24 but not C1–13) of platelet factor 4 inhibit murine megakaryocytopoiesis, an activity which is neutralized by heparin

Negative regulation of megakaryocytopoiesis is a complex process involving various cytokines. One of these cytokines is platelet factor 4 (PF4), a megakaryocyte/platelet specific protein. PF4 and a carboxy-terminal peptide related to PF4 have been reported to inhibit human and murine megakaryocytopoiesis. The growth of several megakaryoblastic cell lines: human erythroleukaemia cell line (HEL), Meg-01 and Dami, was also inhibited by PF4 and a 13–24 carboxy-terminal peptide related to PF4. We report that peptides corresponding to the 1–24 and 13–24 but not 1–13 carboxy-terminal region of PF4 inhibit murine megakaryocytopoiesis both in vivo (5 μg/inj) and in vitro (2.5 and 5 μg/ml). Moreover, such an inhibitory activity of PF4-related peptides is abrogated by heparin (5 IU/dish). These overall data indicate that carboxy-terminal PF4-related peptides retain the inhibitory effect of PF4 on both murine single MK and CFU-MK in vivo and in vitro by acting on an early stage of megakaryocytopoiesis and strongly suggest that the inhibitory activity of the multi-functional PF4 might be localized in a short carboxy-terminal region which might include, in part, the PF4 heparin binding domain.     

Carboxy-terminal peptides (C1–24 and C13–24 but not C1–13) of platelet factor 4 inhibit murine megakaryocytopoiesis,an activity which is neutralized by heparin

Negative regulation of megakaryocytopoiesis is a complex process involving various cytokines. One of these cytokines is platelet factor 4 (PF4), a megakaryocyte/platelet specific protein. PF4 and a carboxy-terminal peptide related to PF4 have been reported to inhibit human and murine megakaryocytopoiesis. The growth of several megakaryoblastic cell lines: human erythroleukaemia cell line (HEL), Meg-01 and Dami, was also inhibited by PF4 and a 13–24 carboxy-terminal peptide related to PF4. We report that peptides corresponding to the 1–24 and 13–24 but not 1–13 carboxy-terminal region of PF4 inhibit murine megakaryocytopoiesis both in vivo (5 μg/inj) and in vitro (2.5 and 5 μg/ml). Moreover, such an inhibitory activity of PF4-related peptides is abrogated by heparin (5 IU/dish). These overall data indicate that carboxy-terminal PF4-related peptides retain the inhibitory effect of PF4 on both murine single MK and CFU-MK in vivo and in vitro by acting on an early stage of megakaryocytopoiesis and strongly suggest that the inhibitory activity of the multi-functional PF4 might be localized in a short carboxy-terminal region which might include, in part, the PF4 heparin binding domain.