Lymphocyte apoptosis in acute respiratory syncytial virus bronchiolitis.

Respiratory syncytial virus (RSV) infection may have an effect on the development of T cell memory responses. RSV bronchiolitis in infants is associated with a transient decline in circulating lymphocytes. We hypothesized that the mechanism underlying this lymphopenia is apoptosis. Blood was taken from 32 infants during primary RSV bronchiolitis and three months later. Using flow cytometry, we found that absolute numbers of both CD3+/CD4+ T-helper lymphocytes (P = 0.029) and CD3+/CD8+ cytotoxic lymphocytes (CTL) (P = 0.043) were significantly reduced during acute infection. Up-regulated expression both of Fas (P < 0.001) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor (P < 0.001) was found during acute illness on both CD3+/CD4+ and CD3+/CD8+ lymphocytes, when compared with convalescent samples. Expression of Fas on CD4+ lymphocytes was inversely related to CD4+ number (P = 0.03). Plasma levels of soluble Fas ligand (P = 0.028) and caspase-1 (P = 0.037), determined by enzyme-linked immunosorbent assay, were increased during bronchiolitis. Plasma interleukin-18, a product of caspase-1 activity, was not raised. Taken together, these data suggest that in acute RSV infection, CD4+ helper lymphocytes and CD8+ cytotoxic lymphocytes are primed to undergo apoptosis. This is a mechanism through which lymphopenia may occur and T cell memory may be altered.

Phosphatidylserine-dependent adhesion of T cells to endothelial cells.

Phosphatidylserine (PS) was exposed at the surface of human umbilical vein endothelial cells (HUVECs) and cultured cell lines by agonists that increase cytosolic Ca(2+), and factors governing the adhesion of T cells to the treated cells were investigated. Thrombin, ionophore A23187 and the Ca(2+)-ATPase inhibitor 2, 5-di-tert-butyl-1,4-benzohydroquinone each induced a PS-dependent adhesion of Jurkat T cells. A23187, which was the most effective agonist in releasing PS-bearing microvesicles, was the least effective in inducing the PS-dependent adhesion of Jurkat cells. Treatment of ECV304 and EA.hy926 cells with EGTA, followed by a return to normal medium, resulted in an influx of Ca(2+) and an increase in adhering Jurkat cells. Oxidised low-density lipoprotein induced a procoagulant response in cultured ECV304 cells and increased the number of adhering Jurkat cells, but adhesion was not inhibited by pretreating ECV304 cells with annexin V. PS was not significantly exposed on untreated Jurkat cells, as determined by flow cytometry with annexin V-FITC. However, after adhesion to thrombin-treated ECV304 cells for 10 min followed by detachment in 1 mM EDTA, there was a marked exposure of PS on the Jurkat cells. Binding of annexin V-FITC to the detached cells was inhibited by pretreating them with unlabelled annexin V. Contact with thrombin-treated ECV304 cells thus induced the exposure of PS on Jurkat cells and, as Jurkat cells were unable to adhere to thrombin-treated ECV304 cells in the presence of EGTA, the adhesion of the two cell types may involve a Ca(2+) bridge between PS on both cell surfaces. The number of T cells from normal, human peripheral blood that adhered to ECV304 cells was not increased by treating the latter with thrombin. However, findings made with several T cell lines were generally, but not completely, consistent with the possibility that adhesion to surface PS on endothelial cells may be a feature of T cells that express both CD4(+) and CD8(+) antigens. Possible implications for PS-dependent adhesion of T cells to endothelial cells in metastasis, and early in atherogenesis, are discussed.

Biological properties of peripheral blood progenitor cells mobilized by cyclophosphamide and granulocyte colony-stimulating factor.

Patients transplanted with mobilized blood progenitor cells (PBPC) recover their neutrophil counts more rapidly than patients transplanted with bone marrow even when they receive the same dose/kg of granulocyte-macrophage colony-forming cells (CFU-GM). Here we have sought a biological explanation for this phenomenon. Most CD34-positive PBPC are quiescent (<1% in S phase) when they are collected from the bloodstream of patients treated with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF), but we have shown that they are able to resume proliferation rapidly in vitro by measuring the kinetics of CFU-GM production by primitive plastic-adherent (Pdelta) cells. Also, Pdelta cells in PBPC harvests, unlike normal marrow Pdelta cells, were insensitive to cell-cycle restraint imposed by contact with marrow-derived stromal cells. We found that Pdelta cells in PBPC collections produce relatively more CFU-GM and relatively fewer BFU-E than Pdelta cells in bone marrow, indicating that granulopoiesis might occur at the expense of erythropoiesis, but we were unable to find any differences in the kinetics of granulocytic maturation between PBPC and bone marrow. Our interpretation of these findings is that transplanted PBPC rapidly enter the cell cycle and contact with stromal cells in the marrow does not reduce the proportion of progenitors participating in neutrophil production. Consequently. neutrophil recovery after PBPC infusion is more rapid than neutrophil recovery after marrow infusion. Granulopoiesis at the expense of erythropoiesis may also contribute to this effect.

High-dose therapy and autologous stem cell rescue for poor risk and refractory lymphoma: a single centre experience of 123 patients.

Over an 8-year period we autografted 123 patient with poor-risk lymphoma. Sixty-three patients had Hodgkin's disease (HD) and 60 non-Hodgkin's lymphoma (NHL). Of the patients with HD, 45 had responsive and 18 resistant disease prior to high-dose therapy. Fifty-three patients with NHL had responsive and seven had resistant disease at the time of transplantation. Seventy-seven patients received autologous bone marrow (BM) rescue, 39 autologous peripheral blood progenitor cell (PBPC) rescue, and seven combined BM and PBPC rescue. High-dose chemotherapy was BEM in 67, BEAM in 39, TBI and cyclophosphamide or etoposide or BCNU in 10, etoposide/mitozantrone in six and etoposide/melphalan in one. There was eight (6.5%) deaths due to treatment-related toxicity, within the first 100 days post-transplantation. Of the patients with HD 41 (65%) are alive at a median follow-up of 39 months (range 2-94). Thirty-three (52%) patients remain in CR. The median DFS of the 63 patients with HD is 34 months (95% CI 7-61). The median DFS for patients transplanted with responsive disease was significantly better than for those transplanted with refractory disease (61 vs 21 months P < 0001). Thirty-five (58%) of the patients with NHL are alive, and 20 (33%) remain in CR. The median DFS for patients transplanted with responsive and refractory disease was 11 months (95% CI 3-19) and 4 months (95% CI 0-9; P = NS) respectively. The median DFS for patients transplanted with HD was significantly better than for patients transplanted with NHL (34 vs 8 months, P < 0.002). In both groups there was no significant difference in DFS in patients receiving one, two, three or more lines of therapy prior to transplantation. In summary, in patients with poor-risk lymphoma who have responsive disease high-dose therapy may result in durable CRs. Conversely, only a small proportion of patients with HD or NHL with resistant disease achieve CR after autologous stem cell rescue.

Peripheral blood progenitor cell transplantation: a single centre experience comparing two mobilisation regimens in 67 patients.

Between June 1991 and January 1995 we performed 67 peripheral blood progenitor cell transplants (PBPCT). Ten patients (group 1) were mobilised with 7 gm/m2 of cyclophosphamide followed by daily G-CSF injections (5 micrograms/kg, subcutaneously). When the white cell count reached 1 x 10(9)/1 they were leukapheresed for 5 days. After stem cell infusion they received G-CSF (10 micrograms/kg/day) until the neutrophil count reached 1.5 x 10(9)/1. Fifty-six patients had PBPCs mobilised with 3 gm/m2 of cyclophosphamide followed by daily subcutaneous G-CSF (5 micrograms/kg) and PBPCs were harvested on 2 consecutive days, when the white cell count rose to 4 x 10(9)/1. After stem cell infusion this group did not receive G-CSF. In 47 of the 56 patients (group 2) adequate MNC (> or = 4 x 10(8)/kg) and/or CFU-GM (> or = 10 x 10(4)/kg) were obtained. Insufficient MNC and/or CFU-GM were obtained in 10 patients. They were therefore transplanted using a combination of bone marrow and peripheral blood progenitor cells (group 3). Overall 64 patients successfully engrafted. Median days to neutrophils > or = 0.5 x 10(9)/1 were 9 (range 8-13), 12 (range 8-25) and 11 (range 9-16) and to platelets > or = 50 x 10(9)/1 were 11 (range 9-23), 13 (range 9-90) and 16 (range 13-99) in groups 1, 2 and 3 respectively. Patients in group 1 had a faster neutrophil recovery than patients in group 2 (P = 0.0002). The three patients who failed to engraft all received a combination of autologous peripheral blood and bone marrow cells.

Plastic-adherent progenitor cells in mobilized peripheral blood progenitor cell collections.

The use of peripheral blood progenitor cells (PBPC) to reconstitute hematopoiesis after high-dose chemoradiotherapy is now commonplace in the treatment of malignancies. Attempts to characterize these cells have concentrated primarily on their phenotype and their content of clonogenic colony-forming cells (CFC). We have used a plastic-adherent delta (P delta) assay system to evaluate the quantity and quality of more primitive cells in addition to the conventional measurements of CFC and CD34-positive cells. The leukapheresis products from 20 patients mobilized using cyclophosphamide (Cy) and granulocyte colony-stimulating factor (G-CSF) were examined for progenitor cell content. The mean number of mononuclear cells (MNC), colony-forming units-granulocyte/macrophage (CFU-GM), and CD34-positive cells from two leukaphereses per patients were 7.9 x 10(8)/kg, 47.3 x 10(4)/kg, and 10.5 x 10(6)/kg, respectively. The mean number of P delta progenitors was 9.3 x 10(4)/kg. Limiting dilution analyses showed the frequency of P delta progenitors in PBPC to be between 1 and 5.3 per 10(5) MNC and that each P delta progenitor has the proliferative capability to generate an overall mean of 4.5 CFU-GM. Of the 20 patients, 16 underwent autografting with PBPC alone. Fifteen patients engrafted neutrophils and platelets within 16 days. One patient had delayed engraftment associated with inadequate etoposide clearance. Statistical analysis showed a strong correlation between numbers of CFU-GM and CD34 positivity. The numbers of plastic-adherent P delta progenitor cells did not correlate with CFU-GM or CD34-positive cells. We conclude that the plastic-adherent P delta progenitor cell assay is capable of measuring primitive hematopoietic cells and that it may be useful for the investigation of primitive progenitors in PBPC harvests.

Peripheral blood progenitor cell harvesting in multiple myeloma and malignant lymphoma.

Peripheral blood progenitor cells are being used increasingly as part of the treatment protocol for a variety of haematological malignancies. The most appropriate mobilisation therapy and the optimum collection procedures have yet to be fully elucidated. 28 patients with myeloma (9), NHL (11) and HD (8) underwent PBSC mobilisation and harvesting between November 1992 and October 1993. Two protocols were used; the myeloma group received high-dose cyclophosphamide, 7 g/m2 + G-CSF and were leucapheresed on 5 consecutive days during the recovery period using the Haemonetics V50 and the lymphoma group a lower dose of cyclophosphamide, 3 g/m2 + G-CSF followed by leucapheresis on 2 or 3 occasions using a Cobe Spectra. Median time to achieve a WBC of 1 x 10(9)/l during the recovery phase, was 14 days (11-16) and 10 days (9-15) respectively. Median numbers of MNC and CFU-GM collected for the myeloma group were 5.9 x 10(8)/kg (2.5-13.5) and 69.4 x 10(4)/kg (9.9-268.1) and for the lymphoma group. 5.1 x 10(8)/kg (1.2-11.1) and 35.4 x 10(4)/kg (1.2-129.7). Three patients with lymphoma had a low yield of CFU-GM, two of which did not proceed to autograft. The third patient failed to engraft and died despite receiving bone marrow backup. For the remaining 25 patients, median time to neuts > 0.5 x 10(9)/l and platelets > 50 x 10(9)/l was 9 (8-13) and 11 (9-23) days for the myeloma group and 12 (9-15) and 13 (9-180) days for the lymphomas. We found a strong correlation between CD34+ cells and CFU-GM from the last 9 patients. There is a correlation between CFU-GM infused and speed of engraftment. All patients who received > 10 x 10(4) CFU-GM/kg showed a rapid engraftment for neutrophils and platelets. In all cases, when > 4 x 10(8)/kg MNC were harvested, > 10 x 10(4) CFU-GM/kg were obtained. Sufficient cells for a rapid engraftment can be obtained from 2 leucaphereses in the majority of patients. The recovering peripheral blood WBC provides a good indicator of when to harvest. The target value of CFU-GM can be predicted by the number of cells harvested and by the number of CD34 positive cells in the leucapheresis product.

Two unusual neurological presentations of granulocytic sarcoma in Philadelphia positive chronic myeloid leukaemia.

Granulocytic sarcoma (GS) is a rare extramedullary tumour consisting of immature myeloid precursors. It occurs most commonly in association with myeloid leukaemias and myeloproliferative disorders. Rarely there may be no evidence of haematological malignancy. We describe neurological presentations of GS in two patients with Philadelphia (Ph) positive chronic myeloid leukaemia (CML). In both cases the bone marrow was in chronic phase at the time of presentation of the GS, but there was rapid subsequent transformation into the blastic phase.

BCR rearrangement in apparent essential thrombocythaemia.

The presence of the Philadelphia chromosome is a major determinant of the prognosis of patients with myeloproliferative disorders. We describe a case of apparent essential thrombocythaemia in whom cytogenetic analysis was normal. However, the presence of basophilia, the absence of abnormal megakaryocytes in a trephine biopsy and the female sex of the patient prompted Southern analysis of peripheral granulocyte DNA. This revealed a BCR rearrangement and the patient has therefore undergone allogeneic bone marrow transplantation. This case emphasizes the importance of both cytogenetic and molecular analysis of patients with apparent essential thrombocythaemia.