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.     

Persistent lymphocytosis of natural killer cells in autoimmune thrombocytopenic purpura (ATP) patients after splenectomy

We report three patients with primary autoimmune thrombocytopenic purpura (ATP) who developed an absolute lymphocytosis (lymphocyte count > 5×10⁹/I) after splenectomy and with a lymphocyte count between 5.4 and 8.9×10⁹/I. An immunophenotype study showed that the peripheral blood lymphocytosis was a persistent NK cell expansion (CD2⁺, CD56⁺, CD3^-), and was characterized by a typical large granular lymphocytes (LGL) morphology. Two of these three ATP patients were refractory to splenectomy.     

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.     

Metformin inhibits glycogen synthesis and gluconeogenesis in cultured rat hepatocytes

Aim:   Glycogen synthesis, and glucose and lactate production were examined in cultured rat hepatocytes preincubated with metformin (0–500 µ m ) for 24 h.
Methods:   Cells incubated with[1-¹³C]-glucose and [1-¹³C]-lactate allowed us to study the effect of metformin on glucose production from glycogenolysis and gluconeogenesis in a detailed manner using NMR spectroscopy. ¹H and ¹³C-filtered ¹H-NMR spectra were recorded by using flow-injection technique.
Results:   Metformin decreased glycogen synthesis in a dose-dependent manner with an IC₅₀ value of 196.5 µ m . This effect could not be reversed by the presence of the glycogen phosphorylase inhibitor DAB, suggesting that glycogenolysis was not affected. A clear correlation between glucose production and glycogen content (0.97 < R < 0.99; p < 0.001) and lactate production and glycogen content (0.97 < R < 0.99; p < 0.001) was observed. Moreover, a strong inhibition (62%, p < 0.001) of glucose produced from lactate/pyruvate (3 m m /0.3 m m ) was observed in cells treated with 350 µ m metformin.
Conclusion:   Hepatocytes preincubated for 24 h in the presence of metformin at clinically relevant concentrations showed impaired glycogenesis as well as gluconeogenesis.     

Second transplantation with CD34+ bone marrow cells selected from a two-loci HLA-mismatched sibling for a patient with chronic myeloid leukaemia

A 43-year-old man with chronic myeloid leukaemia underwent a second transplant with CD34⁺ bone marrow cells selected from his two-loci HLA-mismatched sibling after rejection of the first graft from an HLA-matched unrelated donor. By immunomagnetic positive selection, CD34⁺ marrow cells at 0.95×10⁶/kg with 97% purity and CD3⁺ T lymphocytes at 1.3×10⁴/kg were collected and transplanted. Engraftment was confirmed to be of CD34⁺ cell-donor origin. The patient developed only grade I acute graft-versus-host disease (GVHD) and no chronic GVHD to date. These observations suggest that allogeneic CD34⁺ bone marrow cells are capable of reconstituting haemopoiesis and that CD34⁺ selection could be applicable to T-cell depletion.     

Sulphated macromolecules produced by in vivo labelling in the rat gastric mucosa

Abstract The aim of this study was to investigate the nature and distribution of sulphated macromolecules of the extracellular matrix in rat gastric mucosa. This was achieved by developing an in vivo labelling system.
An intraperitoneal injection of 1 mCi [³⁵S]-sulphate was given for either 4 h (0.01% incorporation into macromolecular fraction) or 8 h (0.13% incorporation). At the end of the labelling period the stomach was removed and the mucosa and submucosa was either taken as a single combined sample or separated into four layers by blunt dissection. Each sample was papain digested and analysed by ion-exchange chromatography. This analysis revealed sulphated species of differing charge existing in differing proportions throughout the mucosa. These sulphated species eluted at NaCl concentrations of approximately 0 (A), 0.19 (B), 0.34 (C) and 0.78 mol/L (D) from a Q-Sepharose ion exchange column. Further analysis by size exclusion chromatography and chemical and enzymatic digestion showed that peaks B and C had molecular weights of 2.4 × 10⁵ and 2.8 × 10⁵, respectively and were resistant to chondroitinase ABC, heparitinase and nitrous acid digestion. Peak D was found to contain a polydisperse population of molecules with a molecular weight range of approximately 1 × 10⁴ to 6 × 10⁴. This sample was susceptible to nitrous acid and chondroitinase ABC digestion and was found predominantly in the sample isolated from deeper in the tissue.
We have thus developed an in vivo labelling technique for sulphated macromolecules that can be used in the further study of injury to the gastric mucosa.     

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.     

Ferritin and iron uptake by reticulocytes

The uptake of liver ferritin labelled with ¹²⁵I or ⁵⁹Fe by guinea-pig reticulocytes has been studied to investigate the characteristics of the uptake process, compare it with transferrin uptake and determine whether ferritin-iron is utilized by the cells in haem synthesis. The results confirmed that guinea-pig reticulocytes, but not mature erythrocytes, take up liver ferritin by a saturable, time- and temperature-dependent process. Up to 70% of the iron taken up by the cells was utilized in haem synthesis and competed directly with iron derived from transferrin. Scatchard analysis of the binding parameters indicated that 30–130 × 10³ ferritin molecules were bound per cell to high affinity specific membrane receptors ( K _a: 1·77 × 10⁷ M⁻¹). In contrast, rat took up much less ferritin than guinea-pig reticulocytes and the process was entirely non-specific. Release experiments with guinea-pig reticulocytes at 37°C showed that a maximum of about 70% of the cell-associated ¹²⁵I-ferritin was released from the cells of which up to 15% was trichloroacetic acid-soluble.
We suggest that ferritin uptake by guinea-pig reticulocytes involves receptor-mediated endocytosis. The endocytotic vesicle fuses with a lysosome, iron is removed from the protein and enters a cytosolic pool in which it competes directly with transferrin-derived iron to provide iron for mitochondrial haem synthesis. Some of the ferritin is catabolized and the rest is returned to the extracellular medium during membrane recycling.     

Quantitative Measurments Concerning A and B Antigen Sites

Using ¹²⁵I-labelled anti-A and anti-B, the number of A and B antigen sites per red cell for samples obtained from adults of different phenotypes was estimated to be: on A₁ cells, 0.81—1.17×10⁶; on A₁B cells, 0.46—0.85×10⁵; on A₂ cells, 0.24—0.29×10⁶; on A₂B cells, 0.12×10⁶. The number of sites on A₁ cells obtained from cord blood was 0.25—0.37×10⁶, and on the single example of A₂ cord cells were 0.14×10⁸ sites. The number of B sites on adult cells were as follows: on B cells, 0.61—0.83×10⁶; on A₁B cells, 0.31—0.56×10⁶ sites.
The equilibrium constants and the rates of dissociation of the reaction between anti-A and A₁ and A₂ cells were also determined. The value of the equilibrium constant was approximately three times as great and the rate of dissociation three times as slow with A₁ cells compared to A₂ cells. This is consistant with the view that theere is a small difference in the molecular structure between the A₁ and A₂ antigen.     

STEM CELL MOBILIzATION IN NORMAL DONORS FOR ALLOGENEIC TRANSPLANTATION: ANALYSIS OF SAFETY AND FACTORS AFFECTING EFFICACY

The use of peripheral blood stem cells instead of bone marrow as the source of haemopoietic cells for allogeneic transplantation is being increasingly explored. We have analysed data from 17 normal donors who underwent stem cell mobilization for allogeneic transplantation with an identical protocol using G-CSF at a dose of 10 μg/kg/d, with the first leukapheresis (LP) on the day following the fourth dose of G-CSF. Both G-CSF administration and leukapheresis were well tolerated. Donors underwent a median of two leukaphereses (range one to three) and a median of 6.80 × 10⁶ CD34⁺ cells/kg recipient weight (range 2.4–15.6 × 10⁶) were collected. The median number of CD34⁺ cells per kg donor weight was 6.05 × 10⁶, when corrected for a 12 litre leukapheresis, this gave a median total of 3.89 × 10⁶ CD34⁺ cells/kg donor weight. When analysed with respect to factors which might influence the efficacy of mobilization, male donors were associated with a superior yield. The median number of CD34⁺ cells/kg/LP harvested was 4.96 × 10⁶ in males and 2.79 × 10⁶ in females ( P <0.05). The results suggested that, given a recipient of 75 kg, in a male donor a single 12 litre leukapheresis should yield sufficient CD34⁺ cells (4 × 10⁶/kg), whereas a female donor would be likely to need two leukaphereses. Age was not found to affect donor yield. In summary, these data confirm that leukapheresis is a safe procedure in normal donors and suggest that males may be more efficient mobilizers of stem cells than females.     

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.     

Successful treatment of chronic severe neutropenia with weekly recombinant granulcyte-colony stimulating factor

Daily treatment for symptomatic chronic neutropenia with recombinant granulocyte-colony stimulating factor (rhG-CSF) filgrastim is costly and sometimes causes neutrophillia. We report the use of weekly filgrastim in a 40-year-old man with life-long symptomatic neutropenia. Baseline neutrophil counts were <1×10⁹/l 60% of the time, and fell below 0.5×10⁹/l for 7 d periods every 22 d. Following 1 year of weekly filgrastim treatment, the absolute neutrophil count was maintained >1×10⁹/l (averaging 2×10⁹/l) and the frequency and severity of symptoms were reduced by 85%. Therefore the benefits of filgrastim for the treatment of at least one form of chronic severe neutropenia can be derived from weekly rather than daily doses.     

Uptake and release of transferrin and iron by mitogen-stimulated human lymphocytes

Phytohaemagglutinin stimulation of human peripheral blood lymphocytes resulted in the expression of transferrin receptors and the uptake of iron into the cells. As assessed from the resistance of the ¹²⁵I label to pronase, transferrin was rapidly bound and internalized at 37°C, while at 4°C 85% of the ¹²⁵I label remained on the cell surface and was degraded by the pronase. Over 94% of the ¹²⁵I label associated with and subsequently released from the cells was acid precipitable, indicating that transferrin was not degraded during its uptake and release. After reincubation for 1 h in fresh medium 70% of the cell associated ¹²⁵I-transferrin was released. In contrast, less than 30% of the ⁵⁹Fe was released, showing that iron was removed from transferrin and retained by the cells.
Concentration dependent binding of ¹²⁵I-transferrin estimated at 37°C occurred with an apparent K_a of 5·7±1·1 × 10⁷l mol⁻¹ (mean ± SD, n = 4) indicating little variation between cells from different individuals, although the number of transferrin molecules associated with the cells varied greatly from 6·2 × 10⁴/cell to 1·4 × 10⁵/cell. The rate of iron uptake from ⁵⁹Fe and ¹²⁵I labelled transferrin at 37°C by the cells from different subjects was also very variable, with a range between 0·46 and 2·27 pg Fe/min/10⁶ cells ( n = 6). However, iron uptake did not correlate with the amount of transferrin bound. This suggests that transferrin uptake and the release of iron from the transferrin to the interior of the cell are controlled independently.     

Effects of 3-5 log10 pre-storage leucocyte depletion on red cell storage and metabolism

SUMMARY. A new, in-line high-efficiency 3-5 log₁₀ leucodepletion filter system (Leukotrap° RC system) was used to investigate the effect of pre-storage white cell removal on the quality of AS-3 red cell concentrates stored for 42 d at 4°. Median residual white cell content was 4 × 10⁵ when filtration was performed at 22° within 8 h of phlebotomy ( n = 20) and 3.2 × 10⁴ when filtration was performed at 4° 12-24 h after phlebotomy ( n = 24). None exceeded 1 × 10⁶ WBC per red cell product. Filtration was rapid (median 28 min), and red cell loss averaged (mean ± 1 SD) 6.4 ± 0.7%. In a paired study design, post-transfusion recoveries of 42 d stored red cells in the filtered units averaged 84 ± 6% v 82 ± 8% for unfiltered units ( P < 0.05) and post-storage haemolysis, ATP, osmotic fragility, K+ and pH were significantly ( P < 0.05) better in the filtered units. Reduced glycolytic activity was also observed in the filtered units, and there was a correlation between osmotic fragility, glucose consumption, and lactate produced in standard units that was not present in leucodepleted units. In conclusion, this study suggests that leucodepletion of AS-3 red cell concentrates prior to storage results in better maintenance of the integrity of the red cell membrane with reduced glycolytic activity. There was a modest improvement in post-infusion viability sufficient to offset the filtration-induced loss and to result in an equivalent red cell product.     

Detection of human parvovirus B19 DNA in plasma pools and blood products derived from these pools: implications for efficiency and consistency of removal of B19 DNA during manufacture

The polymerase chain reaction (PCR) assay was used to detect human parvovirus B19 DNA in 38 blood products and start plasma pools from five different manufacturers. The products examined were albumin, factor VIII, intravenous (i.v.) and intramuscular (i.m.) immunoglobulin batches. The majority of pools from all the manufacturers had detectable B19 DNA (64/75: 85%; ranging from 60% to 100% for individual manufacturers). B19 DNA was found in 3/12 albumin samples, in 7/7 factor VIII samples, 3/15 IVIG samples and 3/4 IMIG samples. The levels of B19 DNA in pools varied from 10² to 10⁹ genome equivalents/ml, whereas the levels in products varied from 10² to 10⁶ genome equivalents/ml, but there was no clear relationship between the levels of B19 DNA in start pools and final products.   The levels of B19 DNA varied between different batches of the same product from a single manufacturer, possibly due to small variations in the processing parameters. In addition, there was some indication from the study of IVIG samples that treatment at low pH may result in removal of PCR-detectable B19 DNA.     

Immunophenotype of c-kit cells in normal human bone marrow: implications for the detection of minimal residual disease in AML

Summary. In the present study, seven normal human bone marrow samples from healthy volunteers have been analysed in order to investigate the immunophenotypic characteristics of the normal CD117⁺ cells and their utility for the detection of minimal residual disease in 71 acute myeloid leukaemia patients.
Our results show that most of normal BM CD117⁺ cells coexpress the HLADR and the myeloid associated CD33 antigen. In addition, almost half of CD117⁺ cells are CD34⁺, these cells displaying a different FSC/SSC distribution when compared to the CD117⁺/CD34⁻ cells. No CD117⁺/CD15⁺ and CD117⁺/CD10⁺ cells were detected and very few CD117⁺ cells (<1 × 10⁻³) expressing the HLADR⁻/CD34⁻, CD33⁺/HLADR⁻ and CD34⁺/HLADR⁻ phenotypes were found to be present in normal BM. In contrast, from the 71 AML patients analysed, 34 had CD117⁺/CD15⁺ blast cells and eight had the CD117⁺ phenotypes detected at low frequencies (<1 × 10⁻³) in normal BM.
In summary, the present study shows that the use of the CD117 antigen in different monoclonal antibodies combinations may be of great help for the detection of minimal residual disease in a high proportion of AML cases, especially in those patients displaying the CD117⁺/CD15⁺ phenotype, because cells coexpressing both antigens in normal BM, if present, are at very low frequencies.     

Assessment of hepatitis C virus RNA and genotype from 6807 patients with chronic hepatitis C in the United States

Hepatitis C virus (HCV) RNA status and HCV genotype have become important tools in the diagnosis and monitoring of therapy in chronic HCV infection. To establish a database with respect to HCV genotype and serum HCV RNA concentrations in chronic hepatitis C patients in the United States, we analysed 6807 chronic hepatitis C patients who had HCV RNA and HCV genotype tests conducted at a central laboratory. The HCV RNA concentration cut-off for the lower 25th percentile of this population (low titre) was 0.9 × 10⁶ copies ml^–1. The median HCV RNA concentration was 3.5 × 10⁶ copies ml^–1 and the cut-off for the upper 25th percentile (high titre) was 5 × 10⁶ copies ml^–1. Male patients had a median HCV RNA concentration of 3.9 × 10⁶ copies ml^–1, which was significantly higher than the median HCV RNA level for females (2.75 × 10⁶ copies ml^–1; P  < 0.001). HCV genotype 1 was detected in 73% of patients; genotype 2 in 14%; genotype 3 in 8%; mixed genotype in 4%; and genotypes 4, 5 and 6 with a frequency of < 1%. Patients from the Northeast, Southeast and Midwest had significantly ( P  < 0.001) more infections with genotype 1 than patients from the Western and Southern regions. African–American patients were more likely to be infected with genotype 1 when compared with Caucasian, Hispanic or Asian Pacific Islanders ( P  < 0.001). Patients infected with HCV genotype 1 and mixed HCV genotypes had significantly higher serum HCV RNA concentrations when compared with HCV genotypes 2 and 3 ( P  < 0.001 for all comparisons).     

Sustained decrease of peripheral lymphocytes after allogeneic blood stem cell aphereses

48 healthy donors underwent peripheral blood stem cell (PBSC) apheresis for allogeneic transplantation beginning on day 4 of G-CSF (2 × 5 μg/kg) mobilization. In one to four (median two) large-volume mononuclear cell aphereses, a median of 55.9 × 10⁹ of lymphocytes (range 21.0–109.2 × 10⁹) were collected, an amount comparable to lymphocyte numbers removed by therapeutic lymphaphereses in autoimmune diseases. Mean peripheral lymphocyte counts decreased from premobilization values of 2.31 × 10⁹/l to 1.31 × 10⁹/l at a median of 34 d (1 month) and 1.53 × 10⁹/l at a median of 327 d (11 months). The decrease in peripheral lymphocyte counts was significantly correlated with the number of lymphocytes removed and the number of aphereses. Neutrophil and platelet counts returned to normal values after 1 month whereas monocyte counts and haemoglobin concentrations were significantly decreased at 1 month but not at 11 months.     

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.