G-CSF alone mobilizes sufficient peripheral blood CD34+ cells for positive selection in newly diagnosed patients with myeloma and lymphoma

We have evaluated CD34⁺ cell positive selection from granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPC) in 26 patients with either multiple myeloma (MM, n  = 18) or follicular non-Hodgkin's lymphoma (NHL, n  = 8). 26 PBPC were collected with two leukaphereses: 16 contained sufficient numbers of CD34⁺ cells and were selected. The absolute number of CD34⁺ cells in the leukapheresis products was found to be significantly related to the duration of underlying disease and exposure to prior treatment. CD34⁺ cell positive selection allowed recovery of a median of 35% of CD34⁺ cells, the selected fraction containing a median number of 1.43 × 10⁶/kg CD34⁺ cells/kg (range 0.48–41.5). 10 patients were transplanted and received a median dose of 1.51 × 10⁶ CD34⁺ cells (range 0.48–4.2). The median time to granulocyte (>0.5 × 10⁹/l) and platelet (>20 × 10⁹/l) engraftment was 12 and 13 d respectively (ranges 10–13 and 0–95). Lymphoma cells were found by a sensitive polymerase chain reaction technique in four out of five CD34⁺ cell fractions tested.     

A New Method for the Simultaneous Quantitation of Platelet-bound Immunoglobuin (IgG) and Complement (C3) Employing an Enzyme-linked Immunosorbent Assay (ELISA) Procedure

An ELISA technique for the quantitation of platelet-bound IgG and C₃ is described. This is an antiglobulin consumption assay using commercial horseradish peroxidase conjugated anti-IgG and anti-C₃ antisera. The greater the amount of antiglobulin consumed by the reaction with platelets the less is available to bind to an immune adsorbent in the form of human serum-coated polystyrene balls. This test can be calibrated by adding known quantities of IgG or C₃ to the specific enzymelinked antibody and the unbound fraction of antiserum is quantitated by allowing it to react with a chromogenic substrate for the enzyme and measuring the intensity spectrophotometrically, thus establishing an inverse relationship with the amount of antigen. Platelets from normal donors and those with non-immunological throm-bocytopenia gave values of 1·3–15·5 ng IgG/10⁶ platelets and 0·22–0·96 ng C₃/10⁶ platelets which are in accordance with the normal ratio of these proteins in normal serum. Fifteen patients in whom immune destruction of platelets was suspected had excess platelet-bound IgG ranging from 30 to 450 ng IgG/10⁶ platelets. In none of these patients could excess platelet-bound C₃ be demonstrated. Compared to the antiglobulin consumption test we have found this test to be superior both technically and in terms of sensitivity and reproducibility.     

Collection of peripheral blood stem cells from normal donors 60 years of age or older

We report 14 normal peripheral blood stem cell (PBSC) donors ≥ 60 years of age who had cytokine mobilization followed by PBSC apheresis for allogeneic transplantation. Mobilization was achieved with filgrastim (6 μg/kg twice daily). Their median age was 63.5 years (range 60–77), and 43% had a positive medical history, mainly hypertension and/or cardiac problems. Their median pre-apheresis leucocyte count (×10⁹/l) was 38.6 (range 29.6–63.4). The median apheresis yield (×10⁶ CD34⁺ cells/litre blood processed, first apheresis) was 27.9 (range 1.6–54.8). The target cell dose (≥4& times; 10⁶ CD34⁺ cells/kg recipient) was reached with one procedure in eight (57%) donors. Filgrastim-related adverse events were acceptable and apheresis was well tolerated. When compared to younger donors (<60 years of age), a trend to a lower CD34⁺ apheresis yield and a requirement for more than one apheresis to achieve the collection target (≥4 ×10⁶ CD34⁺ cells/kg) was evident. Although older (≥60 years) donors seem to mobilize less effectively, these data suggest that PBSC collection from them is feasible and has an acceptable short-term safety profile.     

Cellular abnormalities of folate deficiency

S ummary . To trace the development of folate-deficient abnormalities of morphology and DNA synthesis, Friend erythroleukaemia cells were grown in media containing 10², 10³ and 10⁴ ng of [³H]PteGlu₁/ml and then transferred to folate-free media. Parameters examined were: intracellular folate levels; growth potential; morphology; dU suppression; and DNA content by flow microfluorimetry. The most sensitive indicators of folate-deficient cell growth were those related to DNA synthesis (dU and flow microfluorimetry). These became abnormal at intracellular folate levels of 0.2–0.5 ng/10⁶ cells and markedly so below 0.1 ng/10⁶ cells. Morphological criteria were less sensitive. Cells became megaloblastic at intracellular folate levels below 0.06 ng/10⁶. The capacity of the cells to replicate in folate-free media was a function of the intracellular folate (ICF): duplications = 4.01+ln(ICF)/0.67 (r = 0.993, P<0.001).
These studies demonstrate that regardless of initial intracellular folate levels, cellular stigmata of folate deficiency appear when cellular folate falls below 3 × 10⁵ molecules per cell (dU and flow microfluorimetry) and cells lose the capacity for further replication below 7–10 × 10⁵ molecules. The intracellular folate level not only predicts early defects, but also determines the replicative capacity.     

Serum oncostatin M in multiple myeloma: association with prognostic factors

We report on five children with haematological malignancies who underwent allogeneic peripheral blood progenitor cell (PBPC) transplantation. PBPC were harvested from HLA-identical sibling donors after G-CSF (10 μg/kg/d s.c.) mobilization. Aphereses were carried out on day 5 after G-CSF using a Cobe Spectra blood cell separator. All PBPC allografts were cryopreserved before transplantation. The median of CD34⁺ cells and CD3⁺ cells infused were 14.1×10⁶/kg recipient body weight (range 4.92–22.3) and 2.40×10⁸/kg recipient body weight (range 0.54–4.82), respectively. Engraftment occurred in all cases. The median time to a neutrophil count >0.5×10⁹/l and a platelet count >20×10⁹/l were 15 and 14 d, respectively. The incidence of severe acute graft-versus-host disease was 20%. These data suggest that allogeneic PBPC transplantation might be an alternative to bone marrow transplantation in children.     

Observations of the Number of Available c, D, and E Antigen Sites on Red Cells

Abstract. The number of available antigen sites within the Rh system were estimated using trace-labelled antibodies. The results were as follows, given as the number of sites per red cell: c-antigen: on cc cells, 70,000–85,000: on Cc, 37,000–53,000; D sites on cells of phenotype -D-: 110,000–202,000; E-antigen sites showed considerable heterogeneity depending on phenotype as well as source of anti-E, and estimates varied between 450 and 25,600; e-antigen: on eE cells, 13,400 and 14,500: on ee cells, 18,200 and 24,400.
The average equilibrium constants of the antibodies were: anti-E, 4 × 10⁸ 1/mol; anti-e, 2.5 × 10⁸ 1/mol; anti-c, 3.2 × 10⁷ and 5.6 × 10⁷ 1/mol.     

Granulocyte colony-stimulating factor given in addition to interferon-α to mobilize peripheral blood stem cells for autologous transplantation in chronic myeloid leukaemia

In order to potentially mobilize and harvest the Ph⁻ cells observed in most patients with chronic myeloid leukaemia (CML) during interferon-α (IF-α) therapy, G-CSF (filgrastim), 5 μg/kg/d, was administered subcutaneously together with IF-α to 30 CML patients in haematological remission but with various degrees of cytogenetic remission, after IF-α therapy. Peripheral blood stem cells (PBSC ) were harvested using standard aphereses from day 5 of G-CSF. Patients underwent one to four (median three) aphereses. Median total yields/kg were 7.6 (range 3.8–25) × 10⁸ MNC, 3.4 (0–140) × 10⁶ CD34⁺ cells, and 17 (1.1–107) × 10⁴ CFU-GM. No patient had a significant increase in the percentage of Ph⁺ cells in the bone marrow under G-CSF therapy. The percentage of Ph⁺ cells in apheresis products tended to decrease between the first and the last apheresis ( P  = 0.05). 14 patients who were not responsive to IF-α were transplanted after conditioning with busulphan 16 mg/kg and melphalan 140 mg/m². Median time to neutrophils > 0.5 × 10⁹/l was 20 d (16–114 d) and to platelets > 50 × 10⁹/l 18 d (12–149 d). Nine patients had a major cytogenetic response post graft, which correlated with the amount of Ph⁺ cells reinfused with the graft ( P  = 0.02). We conclude that this procedure is feasible, allowing the harvest of enough PBSC, some of them Ph⁻ in patients who responded to IF-α, to allow autologous transplantation.     

Immunomagnetic selection of CD34+peripheral blood stem cells for autografting in patients with breast cancer

Contamination of transplants with tumour cells may contribute to relapse after peripheral blood stem cell transplantation (PBSCT). We studied the feasibility of CD34⁺ cell selection from blood-derived autografts obtained following G-CSF-supported cytotoxic chemotherapy in a group of 25 patients with breast cancer (10 with high-risk stage II/III and 15 with stage IV without bone or bone marrow involvement).
Using immunomagnetic beads (Isolex 300 SA, Baxter) CD34⁺ cells were enriched and released by chymopapain resulting in a median purity of 95% (range 82–99%) and a median recovery of 80% (range 27–132%). The enrichment procedure did not change the proportion of CD34⁺ subsets coexpressing HLA-DR, CD38 and Thy-1, while L-selectin was removed from the cell surface following selection. Using a sensitive immunocytological technique with a cocktail of epithelial-specific antibodies (anti-cytokeratin 8, 18 and 19; HEA125; BM7 and BM8), five leukaphereses products contained epithelial cells, whereas the selected CD34⁺ cell fraction was free of tumour cells. A neutrophil count of 0.5×10⁹/l and a platelet count of 20×10⁹/l was reached after a median time of 14 and 10 d following 40 high-dose chemotherapy (HDC) cycles. Our results indicate that immunomagnetic selection of CD34⁺ cells yields highly purified autografts devoid of tumour cells whereas the engraftment ability of the progenitor and stem cells is fully retained.     

Octapeptide but not nonapeptide from HIV-1 p24gag protein upregulates cell surface HLA-C expression

Objectives   The HLA-Cw3 molecule has been reported to present peptides derived from HIV-1 p24^gag protein to a cytotoxic T lymphocyte clone. We have shown previously that the synthetic octapeptide 145–152 derived from the p24^gag sequence upregulated cell surface HLA-C expression on HLA-Cw*0303⁺ cells. Here, we examined the question of whether the nonapeptide 144–152 also exerts a similar effect.
Methods   The HLA-Cw*0303⁺ B-LCL PAJ and control HLA-Cw3-negative cells B-LCL HAJ and T-LCL 500/C9 were used. HLA expression on peptide-pulsed and non-pulsed cells was evaluated using specific antibodies and flow cytofluorimetry. Binding of dansylated peptides onto different cell lines was measured spectrofluorimetrically.
Results   The HIV-1 p24^gag octapeptide upregulated cell surface HLA-C on PAJ (Cw*0303⁺) cells, whereas the nonapeptide did not. HLA-A2 expression was not affected by these peptides. Specificity of the effect of octapeptide was confirmed by the lack of HLA-C upregulation on HLA-Cw3^– cells and by lower binding of dansylated petide to the HLA-Cw3^- cells HAJ and 500/C9.
Conclusions   The above results indicate that HLA-Cw*0303 preferentially binds the octapeptide rather than the nonapeptide derived from HIV-1 p24^gag protein .     

Progressive sensitization of circulating basophils against Ixodes ricinus L. antigens during repeated infestations of rabbits

The sensitivity of rabbit basophils to antigens from Ixodes ricinus females has been studied by a degranulation test. Observations of basophil numbers and degranulation were made on the 6th day of each of four sequential infestations. Maximal degranulation of cells was observed after challenge of cells with antigen at a concentration of 10⁶ and 10⁷ pg/ml. At these concentrations, during a 1st infestation, 21.8 and 23.6% of cells degranulated. During a 2nd infestation, these percentages increased (34.8 and 33.8%) and reached 59.8 and 63.8% by the 4th infestation. A plasma factor which partially blocks basophil degranulation, is described. This was already present during the 1st infestation, since in its presence the percentage of degranulation was reduced by 2.8 and 4.0% respectively on challenge with 10⁶ and 10⁷ pg antigen/ml. Inhibition was maximal at the 4th infestation (difference: 16.5 and 20.5%). Basophil sensitization and inhibition of the degranulation are thus both progressive phenomena. After 10–15 infestations on four other rabbits, 75. 0 and 79. 8% degranulation was obtained. The inhibition of degranulation by plasma was also greater (difference: 25. 5 and 27. 4%). IgG specific anti- I. ricinus antibodies were identified by indirect immunofluorescence. In two animals, they were detected at the 6th day of the 1st infestation. Subsequently, they were generally present for all the animals.     

Increased number of peripheral blood CD34+ cells in lithium-treated patients

Eight adult patients with bipolar disorder were prospectively examined to find whether lithium carbonate increased their peripheral blood CD34⁺ haemopoietic stem cells. Following lithium therapy for 3–4 weeks their neutrophil counts increased by a mean of 88% (from 4625 ± 1350 × 10⁹/l, mean ± SD pretreatment, to a peak of 8300 ± 3910 × 10⁹/l). Concommitantly, there was a significant increment in their CD34⁺ cells (from 0.11 ± 0.01% to a peak of 0.18 ± 0.08%). There was a significant correlation between the rise in neutrophil count and that of the CD34⁺ cells ( r  = 0.795, P  = 0.019). Lithium therapy may be used to mobilize peripheral blood CD34⁺ cells for marrow transplantation.     

Evaluation of a new, integral, whole blood filter (RS2000) system for prestorage leucodepletion of SAG-M red cells

Residual donor leucocytes are responsible for many adverse transfusion reactions. Prestorage leucodepletion may ameliorate these effects and enhance product quality. We studied a bottom and top (BAT) system incorporating an integral filter for whole blood leucodepletion. Our evaluation assessed leucodepletion efficiency as well as in vitro SAG-M red cell quality and storage characteristics.   Sixty-six units of blood were collected; test units into the Optipac^®- p L u S system and controls into the standard triple pack configuration. Test units were held for 4–6 h at room temperature (rt) or 12–18 h at 4°C. The mean leucocyte counts for the SAG-M red cells in the quality and storage trial were 0.6×10⁶ (rt hold), 0.05×10⁶ (4°C hold) and 2500×10⁶ (controls). We observed no significant differences between the groups for Na⁺, ATP, 2,3-DPG, glucose, lactate and pH during the 49 d storage. The control group, however, showed a greater increase in haemolysis and K⁺ with time. Autologous in vivo 24 h red cell recovery, after 42 d storage, was >75%. Adjustment of processing parameters in subsequent studies gave leucodepleted SAG-M red cells with minimal cell loss (9–19%) plus acceptable haemoglobin content (46–76 g/U) and haematocrit (54–62%). This system achieved >3.5 log leucodepletion with all but one unit containing <1×10⁶ leucocytes. The product quality is good and the system suitable for routine use in blood centres.     

COLLECTION AND TRANSPLANTATION OF PERIPHERAL BLOOD PROGENITOR CELLS MOBILIzED BY G-CSF ALONE IN CHILDREN WITH MALIGNANCIES

Results of collection and transplantation of peripheral blood progenitor cells (PBPC) mobilized by G-CSF in 31 children with different malignancies were analysed. A total of 43 aphereses were performed, following administration of granulocyte colony-stimulating factor (G-CSF), using a continuous flow blood cell separator (Cobe Spectra) through a central venous catheter. For patients weighing ≤25 kg the extracorporeal line was primed with red blood cells. The mean blood flow rate was 33.2ml/min (range 12–56ml/min). The mean number of mononuclear cells (MNC), granulocyte-macrophage colony-forming units (CFU-GM) and CD34⁺ cells collected were 7.22×10⁸/kg body weight (b.w.), 15.9×10⁴/kg and 5.44×10⁶/kg respectively.
The morbidity related to PBPC collection was low and the mean apheresis time was 302.7min (range 115–492). The volume of processed blood for apheresis (per kilogram body weight) ranged from 117 to 539.6 (mean 309.13ml/kg). 20 patients required only one apheresis to collect the minimum requirement of 5–7×10⁸/kg of MNC.
All patients subsequently underwent autografting with PBPC after myeloablative therapy. Days to achieve an absolute count of neutrophils (ANC) <0.5×10⁹/l and a platelet count of 20×10⁹2/l without platelet support were 9.5 and 18, respectively. The number of CD34⁺ cells infused correlated highly with engraftment kinetics. The extramedullary toxicity was low and manageable.     

Analysis of clinical AIDS-free interval after CD4+ cell counts fall below 200 × 106 L−1 in Japanese Haemophiliacs infected with HIV-1

We analysed the time from the date CD4+ cell counts fell below 200 × 10⁶ L⁻¹, defined as t _i, to the onset of clinical AIDS, according to the 1987 Centers for Disease Control and Prevention case definition, in 129 Japanese Haemophilia patients infected with HIV-1. The cumulative onset of clinical AIDS was analysed by the Kaplan–Meier method and proportional hazard model. Incorporated covariates were age of each patient at time t _i, as well as CD4+ and CD8+ cell counts, serum levels of IgG, IgA, IgM, GOT and GPT at t _i. The time of antiretroviral treatment initiation was also considered. The 50% AIDS-free interval after t _i was 3.00 years (95% confidence interval (CI), range 0.49–5.51) and 1.71 years (95% CI, range 0.66–2.76) for the patients at CDC stage II and stage III, respectively (significantly different, P = 0.0013). Among the patients at CDC stage II at t _i, higher levels of IgA were tightly associated with a shorter period from t _i to onset of clinical AIDS ( P < 0.0001), and relative hazard was 1.35 (95% CI, 1.11–1.64) with increase of IgA level by 1.0 g L⁻¹. Thus there is a broad distribution in the time to onset of clinical AIDS in Japanese Haemophiliacs even after CD4+ cell counts fall below 200 × 10⁶ L⁻¹. This should be taken into consideration in deciding upon the therapy and care of HIV-1 infected people.     

Peripheral blood stem cell mobilization using high-dose cyclophosphamide and G-CSF in pretreated patients with lymphoma

Summary. Peripheral blood stem cell (PBSC) mobilization for autologous transplantation is more difficult in treated patients and those with bone marrow involvement. In 17 pretreated lymphoma patients, cyclophosphamide (4 g/m²) and G-CSF mobilized a median circulating peak of 1959 CFU-GM/ml on day 12.5. PBSC harvesting commenced when WBC was 1×10⁹/l at day 10.5 collected a median of 21.2 × 10⁴/CFU-GM/kg. 13/17 (76%) patients exceeded the 10 × 10⁴ CFU-GM/kg threshold for engraftment. The CFU-GM yield was significantly higher in patients whose WBC recovered from 1–5 × 10⁹/1 in less than 3 days and correlated with the maximum WBC pre and post the cyclophospharnide induced nadir. This regime safely mobilized adequate PBSC in the majority of pretreated lymphoma patients.     

Hepatitis G virus infection in patients with bleeding disorders

Eighty-two patients with bleeding disorders registered with our centre were screened for infection with hepatitis G virus (HGV). 80 patients were positive for hepatitis C (HCV) antibodies, 66 of whom (83%) were HCV PCR positive. 11 patients (13%) were HGV RNA-positive, a similar prevalence rate to that of other studies of patients with bleeding disorders who received factor concentrates prior to the introduction of viral inactivation procedures. There was no significant difference in histological activity index (HAI) between the 10 HGV RNA-positive and the 31 HGV RNA-negative patients who underwent liver biopsy for assessment of HCV infection (median HAI scores 5.5, range 2–10 and four, range 0–10 respectively, P  = 0.07). One patient in each group had established cirrhosis. In patients who underwent HCV quantitation there was no significant difference in HCV viral titre between HGV RNA-positive and negative patients (median HCV titre in HGV RNA-positive patients 2.10 × 10⁵ DNA copies /ml ( n  = 8) range 4.17 × 10² to 4.17 × 10⁶, median HCV titre in HGV RNA-negative patients 3.33 × 10⁵ ( n  = 31) range 1.00 × 10³ to 6.67 × 10⁶, P  = 0.68). In this study there was no evidence that individuals co-infected with HGV and HCV have more severe liver disease than those infected with HCV alone.     

Haemopoietic progenitor cells are reduced in aplastic anaemia

Summary We investigated the frequencies of early populations of progenitors in aplastic anaemia (AA) bone marrow, from patients with a range of disease severity, compared with normal. Double-colour immunofluorescent staining for CD34 and CD33 was carried out on bone marrow mononuclear cells (BMMC) and analysed using fluorescence activated cell sorting (FACS), AA CD34⁺ cells were reduced by 68% compared to normal. In addition, AA CD33⁺ cells and the three progenitor subsets (CD34⁺/CD33^–, CD34⁺/CD33⁺ and CD34^–/CD33⁺) were reduced by 44–80%. Our data lend further support for an early stem cell deficiency in AA.     

A pilot study of low-dose recombinant human granulocyte-macrophage colony-stimulating factor in chronic neutropenia

Summary. Recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) is under investigation for the treatment of a wide range of haematological disorders. At commonly used doses of > 120 μg/m²/d, extramedullary toxicity is common. We report the effects of low-dose (LD) rhGM-CSF in patients with chronic neutropenia related to HIV infection, myelodysplastic syndrome and idiopathic neutropenia. Nine patients with a mean pre-treatment neutrophil count of 0·6 × 10⁹/1 (range 0·2–1·4 × 10⁹/1) received daily rhGM-CSF at doses of between 5 and 15 μg/m², Eight patients responded with a mean post-treatment ANC of 3·2 × 10⁹/1 (range 1·9–4·6 × 10⁹/1). There was no significant therapy-related morbidity. We conclude that in chronic neutropenia, LD rhGM-CSF is an acceptable treatment which has important cost/benefit implications.     

Immunotoxins containing saporin linked to different CD2 monoclonal antibodies: in vitro evaluation

In this study we describe immunotoxins prepared with different CD2 monoclonal antibodies (mAbs) and a ribosome-inactivating protein, saporin. The CD2 immunotoxins were tested on different models. Anti-CD2–saporin conjugates inhibited protein synthesis by a neoplastic CD2+ cell line (SKW-3) and by an interleukin 2 dependent polyclonal CD2+ lymphoid cell culture (T lymphoblasts), with IC₅₀s ranging from 10^-13 m to 10^-11 m (as saporin). Similar results were obtained with proliferation inhibition tests (³H-thymidine incorporation) on phytohaemagglutinin (PHA) driven lymphoid cultures and on mixed lymphocyte culture activated lymphocytes. Moreover a CD2–ricin A chain conjugate was less effective than an analogous immunotoxin containing the same CD2 mAb and saporin in inhibiting lymphocyte proliferation induced by PHA (IC₅₀ approximately 10^-9 m as ricin A chain versus 10^-12 m as saporin). The conjugates were not toxic on bone marrow stem cells. These results suggest that CD2–saporin immunotoxins could represent an effective tool for CD2+ lymphomas or leukaemias, and for T-dependent immune disorders, such as transplanted organ rejection and graft-versus-host disease.     

Epidemiology of Crohn's Disease in Southern Israel

Objectives: Crohn's disease in Israel was described in the past as being of low incidence, more common in Europe-America-born Jews than other Jews, and of unebaracteristically low morbidity. However, recent experience bas suggested that these premises are no longer correct. Methods: the records of all hospital and outpatient cases of Crobn's disease in soutbern Israel for the period 1968–1992 were reviewed. Private family practitioners and specialists were contacted to assure complete case ascertainment. Results: the prevalence rate of Crohn's disease among Jews on December 31, 1992, was 50.6/10⁵ (Asia-Africa-born Jews 55.0/10⁵, Europe-America-born Jews 58.7/10⁵), and the rate was 8.2/10⁵ among Bedouin Arabs. the annual incidence rate (1987–1992) was calculated as 4.2/10^%r in Jews (Asia-Africa-born 4.6/10⁵/yr, Europe-America-born 3.9/10⁵/yr). the age of presentation declined progressively over the study period, was lower in Israel-born patients than immigrants, and was lower in ileocolonic versus other sites of disease. Conclusions: The data show that Crobn's disease bas become more common in Jews in Israel, losing ethnic differences of frequency, and that it occurs at a younger age than before. In Arabs, the disease is more rare.