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Summary: Transcription factories are nuclear domains where gene transcription takes place although the molecular basis for their formation and maintenance are unknown. In this study, we explored how the properties of chromatin as a polymer may contribute to the structure of transcription factories. We found that transcriptional active chromatin contains modifications like histone H4 acetylated at Lysine 16 (H4K16ac). Single fibre analysis showed that this modification spans the entire body of the gene. Furthermore, H4K16ac genes cluster in regions up to 500 Kb alternating active and inactive chromatin. The introduction of H4K16ac in chromatin induces stiffness in the chromatin fibre. The result of this change in flexibility is that chromatin could behave like a multi-block copolymer with repetitions of stiff-flexible (active-inactive chromatin) components. Copolymers with such structure self-organize through spontaneous phase separation into microdomains. Consistent with such model H4K16ac chromatin form foci that associates with nascent transcripts. We propose that transcription factories are the result of the spontaneous concentration of H4K16ac chromatin that are in proximity, mainly in cis.

The organization of genes within the nucleus may influence transcription. We have analyzed the nuclear positioning of the coordinately regulated alpha- and beta-globin genes and show that the gene-dense chromatin surrounding the human alpha-globin genes is frequently decondensed, independent of transcription. Against this background, we show the frequent juxtaposition of active alpha- and beta-globin genes and of homologous alpha-globin loci that occurs at nuclear speckles and correlates with transcription. However, we did not see increased colocalization of signals, which would be expected with direct physical interaction. The same degree of proximity does not occur between human beta-globin genes or between murine globin genes, which are more constrained to their chromosome territories. Our findings suggest that the distribution of globin genes within erythroblast nuclei is the result of a self-organizing process, involving transcriptional status, diffusional ability of chromatin, and physical interactions with nuclear proteins, rather than a directed form of higher-order control.

To assess the relative contribution of genetic factors in the variation of F cells (FC) and other hematologic variables, we conducted a classical twin study in unselected twins. The sample included 264 identical (monozygotic [MZ]) twin pairs and 511 nonidentical (dizygotic [DZ]) same-sex twin pairs (aged 20 to 80 years) from the St. Thomas' UK Adult Twin Register. The FC values were distributed continuously and positively skewed, with values ranging from 0.6% to 22%. FC values were higher in women than in men and decreased with age, with the variables accounting for 2% of the total FC variance. The intraclass correlations of FC values were higher in MZ (rMZ = 0.89) than in DZ (rDZ = 0.49) twins. The XmnI-(G)gamma polymorphism in the beta-globin gene cluster had a significant effect on FC levels, accounting for approximately 13% of the total FC variance. Variance components analysis showed that the FC values were accounted for predominantly by additive genetic and nonshared environmental influences, with an estimate of heritability of 0.89. Hemoglobin levels and red blood cell, white blood cell, and platelet numbers were also substantially heritable, with heritability estimates of 0.37, 0.42, 0.62, and 0.57, respectively. Previously, studies of sib pairs with sickle cell disease and isolated family studies showed that high levels of Hb F and FC tend to be inherited. Here, our classical twin study demonstrated that the variance of FC levels in healthy adults is largely genetically determined. (Blood. 2000;95:342-346)

There is now good evidence that tumour necrosis factor [TNF] stimulates DNA synthesis of B-chronic lymphocytic leukaemia (B-CLL) cells. The malignant clone produces TNF, and addition of exogenous TNF up-regulates the TNF mRNA in B-CLL cells. Interleukin-6 (rIL-6) may also be important in this growth loop. We studied the interaction of TNF and IL-6 in the regulation of DNA synthesis (3H-TdR uptake), cytokine release and cell survival in CLL cells in vitro. Addition of TNF (100 U/ml over 5 days) enhanced DNA synthesis from 718 +/- 284 (mean cpm +/- SE) to 2730 +/- 545 compared to cells cultured in medium alone (n = 16, p < 0.01). TNF-alpha induced DNA synthesis was inhibited in all cases studied by the addition of anti-TNF monoclonal antibody (5 micrograms/ml) to cell cultures. Spontaneous IL-6 protein release was enhanced in the presence of TNF (100 U/ml and 250 U/ml) by CLL cells at 48 hours of culture 143.6% and 172% (p < 0.05, n = 6). At 120 hours of culture, the increase was 323% and 412.5% (4 of 7 cases) of the control respectively. IL-6 (100 U/ml or greater) increased spontaneous DNA synthesis (3H-TdR uptake) but, in the presence of high concentrations of TNF-alpha, inhibited TNF induced DNA synthesis in a dose dependent manner. Cell survival was reduced in the presence of anti-IL-6 mAb, while IL-6 was able to protect CLL cells from spontaneous apoptosis. These results suggest that IL-6 in an autocrine manner may inhibit DNA synthesis but prolongs survival in CLL cells. Increased serum IL-6 levels were detected in 27 of 50 cases of CLL, the mean level being significantly higher in Rai Stage III and IV cases compared to Rai Stage O-II cases.

Previous studies have shown that interleukin-4 (IL-4) may have both stimulatory and inhibitory effects on the growth of normal and malignant B-cells in vitro. We studied the effects of IL-4 on tumour necrosis factor (TNF) induced and spontaneous proliferation (3H-TdR incorporation) and spontaneous release of TNF and interleukin-6 (IL-6) by purified B-cell chronic lymphocytic leukaemia (CLL) cells in vitro. TNF (100 U/ml) increased 3H-TdR uptake in cells to 700 +/- 302% of control (mean +/- S.E., n = 9, p = 0.033). Recombinant IL-4 (10 ng/ml) consistently inhibited DNA synthesis in all CLL patients studied. When added at the start of 5 day cultures, IL-4 inhibited both spontaneous (41 +/- 17% inhibition, n = 3) and TNF induced (46 +/- 5% inhibition, n = 9, p = 0.01) 3H-TdR uptake. Similar results were obtained when IL-4 was added after 48 h of culture. This effect of IL-4 was dose dependent. Inhibition was not related to clinical stage. IL-4 (whether added at T0 or T48h) also inhibited spontaneous release of TNF and IL-6 measured at 48 and 120 h. TNF and IL-4 had no consistent effect on normal cord blood CD5+ B-cells. These data show that IL-4 has inhibitory effects on B-CLL DNA synthesis and also inhibits spontaneous release of IL-6 and TNF in vitro. IL-4 may have a role in vivo in reducing proliferation in these B-cell malignancies by inhibiting potential autocrine growth loops.

Cytomegalovirus (CMV) remains the most common single infective cause of death following allogeneic bone marrow transplantation (BMT) from major histocompatibility complex (MHC)-identical siblings, whereas Epstein-Barr virus (EBV)-related disease is infrequent. We show here that MHC-unrestricted cytotoxic effector cells in the peripheral blood of BMT recipients are highly effective at killing EBV-infected target cells, but are inactive against CMV-infected target cells. Differential cytotoxicity is associated with disparate target structure expression. Although both EBV- and CMV-infected target cells express viral antigens, it is only those infected with EBV that express the adhesion molecule lymphocyte function-associated antigen 1 (LFA1; CD11a/18). Thus, EBV-infected target cells are able to interact with the principal LFA1 ligand, intercellular adhesion molecule 1 (ICAM1; CD54), which is expressed on posttransplant peripheral blood mononuclear (PBM) effector cells. CMV-infected target cells cannot utilize this ligand. Posttransplant cytotoxicity against EBV-infected target cells is abolished by target and effector cell blockade with monoclonal antibodies (MoAbs) to LFA1 and ICAM1, respectively, demonstrating the functional relevance of this additional ligand interaction. These results provide an illustration both of the importance and of the limitations of MHC-unrestricted cytotoxicity in vivo and may explain the frequency of CMV disease and the relative rarity of EBV-related disease following allogeneic transplantation from MHC-matched siblings. The increased immunosuppression used following MHC-mismatched/matched unrelated-donor BMT may cause this MHC-unrestricted defense mechanism to fail and may contribute to the greatly increased incidence of EBV lymphoproliferative syndrome in these patients.

Tumour necrosis factor (TNF) can act as an autocrine growth factor for hairy cell leukaemia (HCL) cells. The TNF produced by the malignant clone may also inhibit normal haematopoiesis thereby contributing to the cytopenias observed in patients with the disease. We have studied the effects of infusing a murine monoclonal anti-TNF antibody in three patients with HCL. In two patients receiving 0.5 mg of antibody/kg on alternate days for 12 d, the drug was well tolerated. The third patient received 2 mg/kg on alternate days and developed symptoms of serum sickness by day 9. In two patients with severe B-lymphocytopenia, circulating CD19 and CD20 positive, B-cells were restored to normal, the majority of which were negative for the HCL-associated marker CD11c. B-lymphocyte recovery was associated with a rise in serum immunoreactive IL-6 and with an early rise in immunoreactive TNF. These short courses of anti-TNF MAb treatment had modest effect on the tumour burden, producing a reduction in splenomegaly in one patient. Exploration of the effects of more prolonged administration of higher dose anti-TNF antibody will only be feasible when less immunogenic MAbs are available.

Major histocompatibility complex-unrestricted lymphokine-activated killer (LAK) cells have been proposed as therapy for a variety of hematologic malignancies. Because these cells recognize and kill their targets independently of their antigen specific CD3 receptor, it is unclear how they might discriminate between normal and malignant cells. We now propose one such mechanism for the selective killing of myeloid leukemia blasts. While both CD2+ and CD2- activated killer cells may inhibit the clonogenic growth of myeloid leukemia cells, only the CD2+ subset effectively inhibits the growth of normal myeloid (granulocyte-macrophage and granulocyte) progenitors. This difference appears to reflect differential requirements for cell adhesion molecule recognition between normal and malignant progenitor cells. Inhibition of the growth of normal granulocyte-macrophage colonies by CD2+ LAK cells is blocked by antibodies to the CD2-lymphocyte function-associated antigen 3 (LFA-3) (CD58) cell adhesion system. In contrast, these antibodies have no effect on CD2+ LAK-mediated inhibition of malignant cell clonogenic growth. Instead, antibodies to the LFA-1 (CD11a/CD18)-intercellular adhesion molecule 1 (ICAM-1) (CD54) adhesion system reduce inhibition. These differences correspond to differential expression of the CD54 cell adhesion molecule by normal and malignant myeloid progenitor cells because less than 15% of normal CD34 positive cells are CD54+ while greater than 85% of CD34+ acute myeloid leukemia blasts express the CD54 antigen. LFA-3, the ligand for CD2, is strongly expressed by erythrocytes, and these cells competitively inhibit killing of normal but not malignant clonogenic cells in an analogous way to the effects of monoclonal antibody to the CD2-LFA-3 adhesion system. The operation of this effect in vivo may be a basis for selective cytotoxicity by CD2+ LAK against clonogenic myeloid blast cells, and could be exploited further with infusion of appropriate monoclonal antibodies.

Cytokine-secreting, major histocompatibility complex-unrestricted activated killer (AK) cells are toxic to a wide range of virus-infected or malignant target cells and may be generated endogenously, eg, after bone marrow transplantation, or by infusion of cytokines such as recombinant interleukin-2 (rIL-2). Although AK cells secrete cytokines such as gamma-interferon and tumor necrosis factor, which are themselves able to recruit fresh cytokine-secreting AK cells, activation in both settings is short-lived, implying the existence of homeostatic regulatory mechanisms. We now demonstrate one mechanism by which rapid homeostasis is achieved. We show that IL-4 is produced in patients with both endogenously and exogenously generated AK cells. The cytokine was detected in serum after marrow transplantation, and IL-4 transcripts appeared in circulating lymphocytes during rIL-2 infusion. Although IL-4 inhibited the induction phase of AK cell function, it had no significant inhibitory effect on the ability of AK cells from these individuals to respond to restimulation. Nonetheless, neutralization of the IL-4 induced during cell activation doubled the half-life of AK function, once activating stimuli were removed, from 18 to 44 hours and produced a 2-log increase in AK cell secretion of tumor necrosis factor and gamma-interferon. These data suggest that IL-4 induced in vivo during lymphocyte activation abbreviates AK cell responses once the triggering stimuli have been removed. Neutralization of endogenous IL-4 in vivo by appropriate monoclonal antibodies might prolong the duration of AK function.

Administration of interleukin 2 (IL2) to patients with minimal residual malignant disease following myeloablative chemo-radiotherapy may augment immune reconstitution and reduce the risk of relapse by increasing cytotoxic effector function and cytokine dependent killing directed at residual malignant cells. The ability of IL2 generated activated killer cells to inhibit haemopoietic progenitor cells and to release gamma-interferon (gamma IFN) and tumour necrosis factor (TNF) may, however, retard haemopoietic recovery, as both TNF and gamma IFN inhibit normal myelopoiesis in vitro. To determine the effect of IL2 infusion on myeloid regeneration in vivo, we have examined haemopoietic recovery in patients receiving this cytokine following autologous marrow transplantation or ablative chemotherapy. We find that IL2 infusion accelerates neutrophil recovery and that granulocyte-macrophage colony stimulating factor (GMCSF) and IL3 mRNA become detectable in circulating mononuclear cells. Induction of TNF by IL2 may also contribute to subsequent acceleration of myelopoiesis by initiation of GM-CSF mRNA synthesis in patient marrow fibroblasts. These results show that IL2 infusion may facilitate myeloid recovery when administered during the period of haemopoietic regeneration following ablative chemoradiotherapy.

Immunoglobulin and specific antibody levels are well maintained in the recipients of T-cell-depleted allogeneic bone marrow transplants (BMT), even though up to 99% of mature T cells are removed from the donor graft. For 3-8 weeks after the procedure, natural killer (NK) cells with an activated pattern of target cell killing have been shown to circulate in the recipient. This study investigates whether these recipient NK cells spontaneously secrete lymphokines that modulate B cell function in a way analogous to that of in-vitro-activated NK cells from normal individuals. Large granular lymphocytes (LGLs) (which contain a high proportion of NK cells) have been prepared from the peripheral blood of 11 recipients of T-cell-depleted major-histocompatibility-complex-matched allografts. In the first 4-6 weeks after BMT these LGLs were found spontaneously to secrete interleukin 2, interferon gamma and B cell differentiation factor. While secretion of these factors declines by 20-24 weeks after BMT, the quantities are still greater than those seen from control donors. Patient LGLs are also able to activate autologous (donor) B cells, rendering them potentially responsive to the secreted factors. It appears likely that activated NK cells (or LGL) play a significant role in maintaining B cell function in vivo after T-cell-depleted BMT.

After marrow transplantation, major histocompatibility complex (MHC)-unrestricted natural killer (NK) lymphocytes are among the first cells to appear in the circulation. After T-cell-depleted bone marrow transplantation (TD-BMT), these cells have an activated pattern of target cell killing; they also secrete lymphokines including gamma-interferon (gamma-IFN), interleukin-2 (IL-2), and tumor necrosis factor (TNF) and may have a significant role as a primary defense against viral reactivation and in the elimination of residual host malignancy. We studied 43 patients with hematologic malignancy, treated by allogeneic TD-BMT, autologous nondepleted BMT, or chemotherapy alone to investigate (a) the mechanisms underlying the generation of these activated killer cells, (b) the range of conditions under which they are produced, and (c) their surface phenotype. We showed that gamma-IFN-secreting activated killer cells with the capacity to kill MHC-nonidentical NK-resistant targets are generated 4 to 6 weeks after either allogeneic TD-BMT or autologous BMT but do not appear after treatment with chemotherapy. Production therefore is not owing to T-cell depletion per se or to host donor alloreactivity, nor is it caused by stimulation by alloantigens contained in blood product support since no significant difference exists between allograft and chemotherapy patients in the number of units of blood platelet support given in the posttreatment period. Because most patients had no evidence of stimulation from virus reactivation/infection, the phenomenon of activation therefore appears to represent posttransplant immune disregulation following repopulation of the host immune system with lymphoid subsets derived exclusively from blood and marrow. Activated killing is predominantly mediated by the CD16+ CD3- subset, but substantial activity remains in the CD16- CD3+ cell fraction. Monoclonal antibodies (MoAbs) that block interaction with class-I MHC molecules at the level of target cell (W6/32 anti-HLA class I) or effector cell (CD8) do not inhibit killing by CD16- CD3+ cells. Activated killer cells may contribute to the lower risk of relapse after marrow transplantation as compared with intensive chemotherapy.

Large granular lymphocyte clones were prepared from normal human mononuclear cells. Seven clones were studied in detail. All had high cytotoxic activity against the natural killer (NK) cell target K562 and all were T11- and DR-positive but T4- and T8-negative: none released migration inhibitory factor, a characteristic of helper T cell clones. When these NK cell clones were co-cultured with autologous B cells in a 1:1 ratio, 4 of the 7 induced both IgM and IgG synthesis. Ig production could not be further enhanced by the addition of B cell differentiation factors. The cells stimulated included antigen-specific memory B cells, as the Ig induced contained specific antibody to an antigen, tetanus toxoid, to which the B cell donors had recently been immunized. Supernatants from the helper/NK clones contained B cell differentiation factors, and were able to induce IgG synthesis from the lymphoblastoid cell line CESS and from co-cultured T and B cells. However, NK clone supernatants, unlike the clones themselves, were not effective at inducing Ig synthesis from purified B cells. Instead it appears that NK clones first activate B cells and thus render them responsive to the factors that the clones secrete.

To test the effect of transplantation of T-cell-depleted bone marrow on recipient immune function the results of pre-transplantation immunisation with tetanus toxoid and hepatitis-B vaccine were studied in 38 donor-recipient pairs. Immunisation of the donor alone resulted in transfer of an antibody response to the recipient; immunisation of both donor and recipient resulted in potentiation of the antibody response in both magnitude and duration. These findings indicate that donor T-cell-depleted marrow can transfer humoral immunity to the recipient and that appropriate pre-transplant immunisation schedules may be of benefit to the recipient.

Low-density cells (LDC) prepared from peripheral blood by fractionation over hypertonic metrizamide contain 95% of cells with veiled morphology, almost all of which are HLA-DR-positive and have characteristics of antigen-presenting cells. In normal individuals the monoclonal antibodies RFD1 and RFD2 divide these cells into three phenotypically distinct populations, D1+D2-, D1-D2+ and D1-D2-. The RFD1-positive population is nonphagocytic. We have investigated the recovery of LDC in peripheral blood after (T cell-depleted) marrow transplantation, to assess whether defects in antigen-presenting cell (APC) subpopulations could contribute to the prolonged immune-paresis of marrow graft recipients. We find that APC of donor origin and with apparently normal morphology, phenotype, and function appear within 6 weeks of BMT. By three months the donor-derived nonphagocytic RFD1-positive subset has disappeared, although phagocytic RFD2-positive cells remain. The disappearance of the RFD1-positive subset is associated with a loss of antigen presentation by patients' LDC of the soluble protein antigen tetanus toxoid, though the capacity to present alloantigen and stimulate in a mixed lymphocyte reaction is retained. Donor-derived RFD1-positive cells and soluble antigen-presenting capacity do not reappear for one year or more. This biphasic recovery of RFD1-positive cells contrasted with the continued production of RFD2-positive APC, implies that the phenotypic and functional distinction between APC subpopulations in peripheral blood also reflects a separate ontogeny. Since these marrow graft recipients retain the phagocytic (RFD2-positive) APC but lose the nonphagocytic (RFD1-positive) APC subset, there is now an opportunity to explore the role of each subset in antigen processing and presentation.

Culture of lymphocytes from allograft recipients or from normal donors with Interleukin 2 (IL2) induces high levels of gamma-interferon (gamma IFN) and tumour necrosis factor (TNF) secretion. We now show that production of these two cytokines can also be increased after autologous bone marrow transplantation (BMT) and induced following chemotherapy for haematological malignancy. These effects occur even though the regenerating IL2 responsive lymphocytes in this context have previously been extensively exposed to cytotoxic agents. IL2 induced secretion of gamma-IFN and TNF is higher in autograft recipients than in patients treated by chemotherapy alone. This effect may be related to differences in the phenotypic profile between recovering lymphocytes in the two groups and to an increased degree of prior in vivo lymphocyte activation in the autologous bone marrow transplant recipients. In both groups of patients, IL2 acts on CD3+ T cells and on CD16+ NK cells so that depletion of either subset incompletely abrogates IL2 dependent gamma-IFN secretion. As both TNF and gamma-IFN possess anti-leukaemic and anti-infective activity, enhancement of their secretion by IL2 infusion after autologous bone marrow transplant and induction after chemotherapy may be of therapeutic benefit.

Following T cell-depleted bone marrow transplantation, helper T cell numbers remain depressed for some months. Nonetheless, functional B cells can be adoptively transferred to the recipients of such grafts, where they continue to secrete antibody. We now show that immunoglobulin production by these transferred B cells is induced by activated large granular lymphocytes (LGL) which circulate in the recipients in substantial numbers during the immediate post-transplant period. The LGL are CD3 negative and therefore provide help in an antigen-unlinked manner. Helper effects for autologous (donor) B cells are augmented by the addition of anti-LFA-2 (anti-CD2) which appears to act by blocking recruitment of LGL inhibitory to developing B cells. In contrast antibody to the beta chain of LFA-1, which effectively reduces natural killer activity of LGL, does not influence their helper function. The peripheral blood LGL fraction thus contains both helper and cytotoxic activity, which can be distinguished by appropriate monoclonal antibodies.

After T-cell depleted marrow transplantation, there is a rapid recovery of cytotoxic effector cells, with activity against targets not susceptible to killing by 'resting' natural killer cells. These targets include Epstein-Barr virus transformed B cells and leukaemic cell lines. Activated killer cell function declines by 3 months after transplantation. We find that when CD3 negative effector cells are obtained from these patients and cultured in vitro with interleukin 2 there is a further enhancement of cytotoxic activity against a range of target cells in the early post-transplant period, and a restoration of high level cytotoxic activity to effector cells obtained 3 months or more after the procedure. These results may have relevance to attempts to reduce the incidence of leukaemic relapse, and EBV + ve lymphoma outgrowth after T-cell depleted BMT.

After conventional bone marrow transplantation serum IgG, IgM and IgA levels fall from pre-transplant levels and may not return to normal for 3-12 months. In contrast IgE may rise to supranormal levels, an event that may be associated with graft-versus-host disease. We have investigated the recovery of immunoglobulin isotypes in the recipients of allogeneic marrows depleted of T-cells to prevent graft-versus-host disease. We find that pre-transplant IgG, IgM and IgA levels are maintained throughout the post-transplant period but that there is a short-lived rise in IgE about 3 weeks after transplantation: this rise occurs in the absence of clinically detectable graft-versus-host disease. We conclude that specific T-cell depletion does not impair and may actually enhance the functional recovery of B cells after allogeneic BMT.

Recombinant tumour necrosis factor (TNF) promotes survival and induces proliferation in the tumour cells from two malignancies of B lymphocytes--hairy-cell leukaemia and B-chronic lymphocytic leukaemia. Culture with TNF also induces TNF mRNA and protein, so the cytokine may act as an autocrine tumour growth factor. These growth promoting effects are antagonised by alpha but not by gamma interferon.