Improvement in immune parameters and human immunodeficiency virus-1 viral response in individuals treated with 16alpha-bromoepiandrosterone (HE2000).

A randomised, double-blind, placebo-controlled study examined the safety, tolerance, immunological effect and anti-human immunodeficiency virus (HIV) activity of sub-cutaneously administered HE2000 (16alpha-bromoepiandrosterone) as monotherapy in treatment-naïve patients with HIV-1. Twenty-four patients received five sequential daily doses of 50 or 100 mg of HE2000 or placebo every 6 weeks for up to three courses, and were followed thereafter for 3 months. HE2000 was safe, with transient injection site reactions being the main side-effect. Peripheral blood samples, collected serially, were analysed for changes in immune cell phenotypes. Significant increases were observed in the numbers of circulating dendritic cells, early activated (CD69+ CD25-) CD8 T-cells and T-NK cells after administration of 50-mg doses of HE2000 (p < 0.05). Gene expression in peripheral blood mononuclear cells was analysed by real-time RT-PCR. Before treatment, HIV-1-infected patients had significantly elevated transcripts for a number of inflammatory mediators (p < 0.012). After 50 mg or 100 mg HE2000, but not after placebo, there were significant sustained decreases in IL-1beta, TNF-alpha, IL-6 and Cox-2 transcripts (p < 0.05). There were no significant differences in CD4 cell numbers, although patients receiving 50-mg doses demonstrated a significant decrease in viral load (- 0.6 log; p < 0.01). Anti-HIV-1 T-cell responses were analysed serially using GAG-peptides to stimulate cytoplasmic IFN-gamma responses. After three courses, the 50-mg dose group demonstrated a significant increase in CD8 T-cell response against two distinct GAG peptide pools (p < 0.03). These findings suggest that immune-based therapies may be able to impact viral load by decreasing inflammation and/or stimulating CD8 T-cells.

Hospice care and patients' pain: communication between patients, relatives, nurses and doctors.

This article describes a study that sought to assess how patients, relatives, doctors and nurses in a palliative care unit viewed pain and pain management, and how standards and expectations for pain relief can be raised by upholding statements of care and agreed partnership values. The results showed that research-based pain management enables the provision of pain control that is acceptable to patients, relatives, doctors and nurses. By valuing patient-centred care, where assessment tools assist communication and information sharing, a partnership of care is established in which patient and professional autonomy are recognized and respected, international recommendations for pain relief are practised and professional codes of conduct upheld. Good pain management requires accurate assessment that is best achieved by open and honest discussion in a supportive environment. Hospices provide specialist symptom control aimed at improving quality of life for patients with advanced disease. They are not only an ideal setting to provide evidence for practice, but also a learning environment for specialist understanding of symptom control and a resource base for other professionals.

Teaching palliative care principles to UK nursing home care assistants.

This paper describes the initiatives that led to a study day for Health Care Assistants in the UK, focusing on the principles and practice of palliative care for practitioners. Topics covered were 'What is palliative care?', 'How can you help patients with pain', 'Needs of the dying patient', 'I don't know what to say', 'Answering awkward questions', and 'Ways of relieving distressing symptoms'. Participants valued the study day and feedback showed that the Health Care Assistants, essentially assistants to qualified nursing staff with minimal or no training themselves, had many vocational and emotional needs that were not being met.

Selective elimination of malignant stem cells using photosensitizers followed by light treatment.

The pros and cons of purging of either bone marrow or peripheral blood stem cell preparations for autologous transplantation for cancer has been debated strongly over the past decade. Recent data implicating the role of minimal residual disease in autografted marrow in cancer relapse have renewed interest in this question. There is a considerable body of literature supporting the possibility that photosensitizer molecules in combination with light might provide a therapeutic window permitting selective elimination of malignant stem cells while sparing those of normal lineage. Molecules of this class are known to be taken up more actively by most malignant cells, and intracellular concentrations are critical in their cytotoxic effect when they are activated by light at an appropriate wavelength. The present paper reviews the observations made over the past decade on a variety of photosensitizers and their effects on hemopoietic progenitors.

Phenotypic characterization of normal and CML CD34-positive cells: only the most primitive CML progenitors include Ph-neg cells.

We studied the sequence of acquisition of CD33, CD38 and HLA-DR antigens on CD34+ cells from marrow and blood of Ph-chromosome positive CML patients and normal marrow. We examined the Ph status of the various CML cell populations. The mean proportions of normal and CML CD34+ cells expressing CD33 and CD38 were not significantly different. However, a significantly greater proportion of CML CD34+ cells expressed HLA-DR antigens compared with normal CD34+ cells and the level of HLA-DR expression per CML cell was abnormally high. When the sequence of acquisition of these antigens on normal and CML CD34+ cells was evaluated using 3-colour fluorescence analysis, the results suggested that HLA-DR was expressed earlier than CD38 or CD33 and these findings were confirmed by following the acquisition of CD38 and CD34+/DR+/CD38-subpopulation during liquid culture. We performed cytogenetic studies on CD34+ subpopulations in 6 cases. In 4 cases there were some Ph-negative metaphases detectable in the CD34+/DR-subpopulation (range 12.5 to 60%). In the CD34+/DR+ fractions, however, all 6 patients had only Ph-positive metaphases and only 1/5 patients had detectable Ph-negative metaphases in the CD34+/CD38-subpopulation. We conclude that expression of HLA-DR antigens may precede the expression of CD38 on CD34+ cells during normal stem cell differentiation. In CML DR may be expressed aberrantly and Ph-negative cells are found predominantly in the DR negative subpopulation.

Potential mechanisms of action of interferon-alpha in CML.

Treatment with interferon-alpha (IFN-alpha) adequately controls the leukemic cell mass in the majority of newly diagnosed patients with chronic myeloid leukemia (CML). However, the degree of response ranges from no 'hematologic' response to complete suppression of the leukemic clone. The mechanism(s) by which IFN-alpha elicits these responses is unknown, but in vitro studies have indicated that IFN-alpha might function by (1) selective toxicity against the leukemic clone, (2) enhancement of 'immune' regulation, and (3) modulation of bone marrow microenvironmental regulation of hematopoiesis. Using in vitro clonogenic assays we were unable to demonstrate that IFN-alpha selectively inhibited the proliferation of CML progenitor cells. We also found no difference in the expression of LFA-3 on normal or CML CD34+ cells. However, by panning and co-culturing hematopoietic cells on monolayers of bone marrow stromal cells, grown with and without IFN-alpha, we found that IFN-alpha enhanced the adhesion of CML progenitors to stromal cells, whereas adhesion by normal progenitor cells was essentially unaffected. This enhanced adhesion by CML progenitor cells was associated with a reduction in neuraminic acid levels in the extracellular matrix overlying stromal cells. Therefore, it is possible that one of the mechanisms by which IFN-alpha exerts its regulatory effect on the leukemic clone is through enhancement of hematopoietic cell-microenvironmental cell interactions.

Physical, phenotypic and cytochemical characterisation of stroma-adherent blast colony-forming cells.

Primitive cells defined as long-term culture initiating cells (LTCIC) and blast colony-forming cells (Bl-CFC) bind to cultured stromal layers, but cells at later stages of maturation [granulocyte-erythroid-macrophage-monocyte colony-forming cells (GEMM-CFC) granulocyte-macrophage (CM-CFC) and erythroid burst-forming units (BFU-E)] do not. The precise relationship between the LTCIC and Bl-CFC is not known and this study was undertaken to determine their relative positions in the haemopoietic hierarchy. We have defined the Bl-CFC population in terms of its density profile and antigenic phenotype and compared these characteristics with GM-CFC and BFU-E. The progenitor cell populations did not differ in density. The major phenotypic difference was seen using the myeloid monoclonal antibody S17-25 which reacted with fewer Bl-CFC than GM-CFC. Also, we have cytochemically analysed the cells in colonies derived from Bl-CFC. Our studies indicate that the Bl-CFC precede BFU-E and GM-CFC but not the LTCIC.

Inactivation of the retinoblastoma susceptibility gene in a human high grade non-Hodgkin's lymphoma cell line.

We studied the expression of the retinoblastoma (RB) gene product (p105) in a B-cell line established from a patient with non-Hodgkin's lymphoma (large cell type). The karyotype of this cell line, named Ri-1, showed amongst other changes an apparent deletion of one chromosome 13 on band q14. No p105 could be detected by immunoprecipitation analysis and Western blotting in Ri-1 cells. Northern blotting revealed that RB mRNA is not expressed in Ri-1. Southern blotting confirmed the loss of one RB allele but showed a normal gross structure of the remaining allele. This suggests that the inactivation of the RB gene in Ri-1 cells is due to deletion of one allele and point mutations or small deletions in the other, as is often the case in retinoblastomas. Our findings imply that inactivation of the RB gene may play a role in the pathogenesis of high grade malignant lymphomas and that studies of RB in primary lymphoma samples would be of interest.

Deficiency of a phosphatidylinositol-anchored cell adhesion molecule influences haemopoietic progenitor binding to marrow stroma in chronic myeloid leukaemia.

The interactions between haemopoietic progenitor cells and marrow stromal cells that are essential for the regulation of normal haemopoiesis are defective in chronic phase chronic myeloid leukaemia (CML). The presence of primitive progenitor cells (blast colony-forming cells, Bl-CFC) in the blood of patients with CML is reflected by their reduced capacity to bind to marrow derived stromal layers in vitro. Whereas normal bone marrow Bl-CFC bind irreversibly to cultured stromal layers (and none are found in normal blood), the Bl-CFC in CML bind transiently and then detach. The normal cell adhesion mechanism is partially sensitive to treatment with phosphatidylinositol-specific phospholipase C (Pl-PLC), indicating the participation of a phosphatidylinositol (Pl)-linked structure; however, when CML cells were treated with Pl-PLC it had no effect on progenitor binding. Two other Pl-linked structures, decay-accelerating factor (DAF) and lymphocyte function associated antigen-3 (LFA-3) were normally expressed on CD34 positive CML cells and normally susceptible to Pl-PLC treatment. The treatment of normal cells with Pl-PLC, to mimic the situation in CML, resulted in the indiscriminate and inefficient binding of Bl-CFC to stroma. Moreover, treatment of the normal cells with 5637 conditioned medium (CM), which contains haemopoietic growth factors, also reduced the binding capacity of normal Bl-CFC; 5637CM treatment did not alter the expression of DAF. It is proposed that a Pl-linked cell adhesion molecule (CAM) is deficient in CML as a consequence of the constitutive activation of ABL kinase whilst, in normal cells, CAMs attached in this manner are responsible for efficient adhesion to stroma and are regulated by growth factors.

Interferon-alpha overrides the deficient adhesion of chronic myeloid leukemia primitive progenitor cells to bone marrow stromal cells.

Primitive blast colony-forming cells (BI-CFC) from chronic myeloid leukemia (CML) patients are defective in their attachment to bone marrow-derived stromal cells compared with normal BI-CFC. We investigated the effect of recombinant interferon-alpha 2a (IFN-alpha) on this interaction between hematopoietic progenitor cells and bone marrow-derived stromal cells by culturing normal stromal cells with IFN-alpha (50 to 5,000 U/mL). At 50 U/mL we found that: (1) the capacity of stromal cells to bind two types of CML primitive progenitor cells (BI-CFC and long-term culture-initiating cells) was increased; and (2) the amount of sulfated glycosaminoglycans (GAGs) in the stromal layer was increased. However, sulfated GAGs were not directly involved in binding CML BI-CFC, unlike binding by normal BI-CFC, which is sulfated GAG-dependent. Neuraminidase-treated control stromal cells bound an increased number of CML BI-CFC, reproducing the effect of IFN-alpha, whereas the binding to IFN-alpha-treated stromal cells was unaffected by neuraminidase treatment. Thus, the enhanced attachment by primitive CML progenitor cells to INF-alpha-treated stromal cells might be due to changes in the neuraminic acid composition in the stromal cell layer. Our in vitro evidence may provide insights into the mechanism of action of IFN-alpha in vivo. Prolonged administration may alter the marrow microenvironment in some patients such that it can restrain the aberrant proliferation of Philadelphia chromosome (Ph)-positive stem cells while permitting Ph-negative stem cells to function normally.

Interferon-alpha alters the distribution of CFU-GM between the adherent and nonadherent compartments in long-term cultures of chronic myeloid leukemia marrow.

We studied the effect of recombinant interferon-alpha 2a (IFN alpha) on the interaction between stromal cells and granulocyte-macrophage colony-forming units (CFU-GM) from the marrow of normal individuals and chronic myeloid leukemia (CML) patients in chronic phase in long-term bone marrow cultures using preformed stromal layers. These stromal layers were established with marrow cells from normal allogeneic donors and grown to confluence in the presence or absence of IFN alpha at low concentration (100 U/ml). The number of CML CFU-GM localized within IFN alpha-treated stromal layers was significantly greater than the corresponding number localized within control stromal layers. Conversely, the distribution of normal CFU-GM between the adherent and nonadherent compartments was unaffected by IFN alpha treatment of the stromal layer. Preincubation of CML marrow cells with IFN alpha did not alter this distribution, so the observed effect of IFN alpha must be due to a primary action on stromal layers. Thus, in addition to its well known antiproliferative effect, IFN alpha enhances the capacity of marrow stromal cells to bind and/or retain CML progenitor cells, and the resulting restoration of normal regulatory control may be the basis for the selectivity of IFN alpha in CML.

The effects of interferon-alpha on the proliferation of CML progenitor cells in vitro are not related to the precise position of the M-BCR breakpoint.

We investigated the effects of brief (2 h) and continuous exposure to recombinant interferon-alpha (2a) (rIFN-alpha) on the proliferation of primitive (blast colony-forming cells, Bl-CFC) and committed myeloid progenitor cells (BFU-E and GM-CFC) derived from blood and bone marrow of patients with chronic myeloid leukaemia (CML) and normal subjects. In all three clonogenic assays, rIFN-alpha suppressed colony formation in a dose-dependent manner. No differences were detected in the proliferation of CML or normal Bl-CFC and GM-CFC exposed to rIFN-alpha. Erythroid colony formation by normal, but not by CML BFU-E, was inhibited by relatively low concentrations (100 U/ml) of rIFN-alpha. However, in patients whose blood or marrow contained a mixture of Philadelphia chromosome (Ph)-positive and Ph-negative BFU-E, cytogenetic analysis of individual erythroid colonies showed no differential inhibition by rIFN-alpha. We found no difference in the sensitivity to rIFN-alpha of GM-CFC from patients whose leukaemic cells expressed BCR/ABL mRNA with the b2a2 junction and that of GM-CFC from patients with the b3a2 mRNA. We conclude that (1) rIFN-alpha does not have a significant leukaemia-specific effect on the progenitor cells detected in these assays, and (2) the sensitivity of CML GM-CFC to rIFN-alpha is independent of the type of BCR/ABL message present in the cells. The clinical efficacy of rIFN-alpha could be due to selective toxicity to cells not assayed in this study, to effects on accessory cells or to alterations induced in progenitor cell/stromal cell interactions.

The role of the MDR-1/P-170 mechanism in the development of multidrug resistance in chronic myeloid leukemia.

We studied blood and bone marrow cells from 42 patients with Ph-chromosome positive chronic myeloid leukemia (CML) and 20 normal subjects for amplification of the multidrug resistance gene (MDR-1) by Southern blotting and for overexpression of P-glycoprotein (P-170) by immunocytochemistry on intact cells with the monoclonal antibody C219. No P-170 could be detected in normal bone marrow or buffy coat. Overexpression of P-170 without amplification of MDR-1 was found in four of 11 patients with chronic phase CML at diagnosis, seven of 16 patients treated with busulfan or hydroxyurea in chronic phase and four of 15 patients in blast crisis. The P-170 overexpression involved only cells of the granulocyte lineage and varied from weak to strong in individual patients. It did not correlate with duration of or response to treatment during chronic phase. In transformation P-170 expression was seen in differentiated cells of the granulocyte lineage but not in blast cells, although three patients had been treated intensively with lipophilic and other cytotoxic drugs to which they had become resistant. We conclude that resistance to busulfan and hydroxyurea in chronic phase and resistance of blast cells to other cytotoxic drugs in transformation are not mediated primarily through the MDR-1/P-170 pathway.

The position of the M-BCR breakpoint does not predict the duration of chronic phase or survival in chronic myeloid leukemia.

It has been reported that patients with chronic myeloid leukemia (CML) with 5' breakpoints within the major breakpoint cluster region (M-BCR) of the BCR gene have somewhat better prognoses than those with 3' breakpoints. We studied the position of the breakpoint in 67 patients with CML in chronic phase using conventional Southern blotting. Using restriction enzymes BglII, BamHI and HindIII and two genomic probes, a 0.6 kb (3' M-BCR) probe hybridizing to a part of the intron between exons b3 and b4 and a 2.0 kb (5' M-BCR) probe hybridizing to sequences including exon b1, we localized the breakpoint in M-BCR as occurring 5' (n = 38) or 3' (n = 28) of the HindIII restriction site located just downstream of exon b3. We failed to localize the breakpoint in one patient. The median durations of chronic phase (37 versus 44 months respectively) and of survival (50 versus 51 months respectively) for patients with 5' and 3' breakpoints were not significantly different. When we analysed only patients whose DNA was collected within 4 weeks of diagnosis (5' breakpoints, n = 30; 3' breakpoints, n = 19), there was again no significant difference in duration of chronic phase or survival. The median survivals of patients divided into good, intermediate and poor prognosis categories in accordance with the prognostic index developed by Sokal and colleagues were 54, 50 and 26 months respectively. This study confirms the value of the Sokal prognostic index but provides no support for the notion that the precise genomic position of the breakpoint in M-BCR correlates with prognosis.

Haemopoietic stem cell subpopulations in mouse and man: discrimination by differential adherence and marrow repopulating ability.

Based on the properties of differential cell adherence, we have devised two assays for early progenitor cells in human bone marrow. One progenitor cell population binds to plastic and to pre-formed bone marrow derived stromal layers (P+S+) and gives rise to non-adherent granulocyte-macrophage colony-forming cells (GM-CFC); the other binds to stromal layers but not to plastic (P-S+); both are separable from GM-CFC which are P-S-. We have evaluated the relevance of differential binding properties to marrow repopulation in a murine model. Murine stem cells (spleen colony-forming cells--CFU-S) can be separated into P+S+, P-S+ and P-S- subpopulations by differential adhesion, thus paralleling the progenitor cell subpopulations in human marrow. Post irradiation (850 cGy X-rays) studies have shown that the P+S+ cells are essential for survival and recovery of marrow, spleen and blood cell populations. Also, in a model for purging autografts, we have demonstrated that the leukaemic cells can be separated from P+S+ repopulating cells by exploiting their different binding properties.

Inhibition of P210BCR/ABL Expression in K562 Cells by Electroporation with an Antisense Oligonucleotide.

We designed experiments to study the effects on P210BCR/ABL expression of introducing antisense oligonucleotides into K562 cells. We used two antisense oligonucleotides: one (AS1) is complementary to the first coding codon of the BCR/ABL mRNA and the two 5' and three 3' codons, and the other (AS2) to BCR coding codons 5 to 11 inclusive. To facilitate entry of the oligonucleotides the K562 cells were subjected to electroporation on three occasions at 24 hr intervals (0, 24 and 48 hr). P210BCR/ABL expression was assayed by in vivo phosphorylation followed by immune precipitation with a BCR antibody. Introduction of AS1 inhibited P210BCR/ABL expression at 72 and 96 hrs, whereas AS2 and the control oligonucleotide had no effect. AS1 also killed K562 cells. We conclude that selected antisense oligonucleotides can modify leukaemia-specific protein expression in K562 cells. This approach could prove valuable for purging CML bone marrow cells in vitro.

Autografting for patients with chronic myeloid leukaemia in chronic phase: peripheral blood stem cells may have a finite capacity for maintaining haemopoiesis.

We treated 14 patients with Ph-chromosome-positive chronic myeloid leukaemia still in chronic phase by autografting with blood-derived haemopoietic stem cells. Eleven patients were autografted electively after cytoreductive treatment with busulphan (16 mg/kg) and melphalan (60 mg/m2) and three were autografted after marrow cells from HLA-identical sibling donors had failed to engraft. In 13 patients haemopoiesis recovered; one failed to engraft and died 114 d after autografting. Two other patients became pancytopenic and received further stem cell transfusions at 3 and 40 months respectively after first autografting. One patient entered lymphoid transformation and died 14 months after autografting. Twelve patients survive at a median of 41 months (range 24-53) after autografting. Nine of the survivors have required further chemotherapy after autografting and four of the nine were electively autografted on a second occasion. Three patients surviving after autografting for 28, 43 and 53 months respectively have not required further chemotherapy. In two of these patients haemopoiesis is now predominantly Ph-negative. We conclude that autografting in chronic phase might prolong survival in some cases by reducing the size of the leukaemic stem cell population. The fact that initially successful grafts failed in two patients suggests that blood-derived stem cells may have a finite potential for self-replication.

Recurrent graft failure following syngeneic bone marrow transplantation for aplastic anaemia.

We present the case of a 60-year-old woman with drug-induced aplastic anaemia with a healthy monozygotic twin. Proof of monozygosity was confirmed by studies using the hypervariable minisatellite probe to obtain identical DNA fingerprints in donor and recipient. In vitro co-culture studies performed showed no evidence of a recipient-derived cellular or humoral inhibitor of donor haemopoiesis. Despite this, there was no engraftment following simple marrow infusion without preconditioning. A second syngeneic transplant following high dose cyclophosphamide therapy produced trilineage engraftment but severe thrombocytopenia developed at 3 months, followed later by pancytopenia with generalized marrow failure. Following a third syngeneic transplant with cyclophosphamide and total lymphoid irradiation there was good initial engraftment but graft failure occurred at 14 weeks. A fourth transplant using Campath 1G as preconditioning resulted in no engraftment and the patient died of septicaemia 8 weeks following her fourth transplant. We suggest that the cause of the recurrent aplastic anaemia in this case was a defect of marrow stroma as neither an inhibitor of donor haemopoiesis nor an intrinsic defect of donor stem cell growth could be demonstrated in vitro.

The effect of cytomegalovirus on hemopoiesis: in vitro evidence for selective infection of marrow stromal cells.

We studied the effects of adding cytomegalovirus (CMV) in vitro to normal human bone marrow mononuclear cells (BM-MNCs), committed myeloid progenitor cells, primitive myeloid blast-colony forming cells, and pre-formed marrow stromal cell monolayers in order to shed light on the mechanism by which hemopoiesis is suppressed in patients who acquire systemic CMV infection after allogeneic bone marrow transplantation. Incubation of BM-MNCs or committed progenitor cells with laboratory strain AD169 or wild strain CMV had no significant effect on total colony numbers or the morphology of component cells. CMV mRNA was not identified by in situ hybridization. In contrast, incubating marrow stromal monolayers with CMV produced specific cytopathic effects in fibroblasts and adipocytes and reduced the capacity of the stromal layers to support the proliferation of primitive myeloid progenitor cells. We conclude that CMV infection may impair hemopoiesis in vivo by a direct effect on the cellular components of the marrow stroma.