Sequence analysis of the coding region of human methionine synthase: relevance to hyperhomocysteinaemia in neural-tube defects and vascular disease

Elevated homocysteine (Hcy) levels are observed in two apparently unrelated diseases: neural-tube defects (NTD) and premature vascular disease. Defective human methionine synthase (MS) could result in elevated Hcy levels. We sequenced the coding region of MS in 8 hyperhomocysteinaemic patients (4 NTD patients and 4 patients with pregnancies complicated by spiral arterial disease, SAD). We identified only one mutation resulting in an amino acid substitution: an A-->G transition at bp 2756, converting an aspartic acid (D919) into a glycine (G). We screened genomic DNA for the presence of this mutation in 56 NTD patients, 69 mothers of children with NTD, 108 SAD patients and 364 controls. There was no increased prevalence of the GG and AG genotypes in NTD patients, their mothers or SAD patients. The D919G mutation does not seem to be a risk factor for NTD or vascular disease. We then examined the mean Hcy levels for each MS genotype. There was no correlation between GG- or AG-genotype and Hcy levels. The D919G mutation is thus a fairly prevalent, and probably benign polymorphism. This study, though limited, provides no evidence for a major involvement of MS in the aetiology of homocysteine-related diseases such as NTD or vascular disease.      

A model for estimating incremental benefits and costs of testing donated blood for human immunodeficiency virus antigen (HIV-Ag)

We propose a model for estimating the benefit of adding a test for the human immunodeficiency virus antigen (HIV-Ag) to current procedures for testing donated blood. Using this model, data on HIV infection from published studies, and certain assumptions about blood donor behavior, we estimate that the probability of detecting an additional HIV-infective blood component is approximately 1 in 4,860,000. If this estimate is correct, adding HIV-Ag testing would prevent approximately 4 cases of primary transfusion-transmitted HIV infection annually in the United States. After adjustments for the median incubation period for AIDS, and for mortality due to primary illnesses, this estimate represents prevention of approximately 1 case of AIDS per year, within the 4 years after transfusion. A primary advantage of this model is its adaptability for recalculating cost-benefit analyses if more sensitive tests for HIV infection become available. In addition, we propose that comparing the anticipated costs and benefits of HIV-Ag testing to other possible uses of these funds should be an important factor in assessing the desirability of HIV-Ag testing.     

Developmental regulation of erythropoiesis by hematopoietic growth factors: analysis on populations of BFU-E from bone marrow, peripheral blood, and fetal liver

Fetal hematopoiesis is characterized by expanding erythropoiesis to support a continuously increasing RBC mass. To explore the basis for this anabolic, nonhomeostatic erythropoiesis, the proliferative effect of recombinant hematopoietic growth factors on highly enriched hematopoietic progenitor cells from fetal and adult tissues were compared. Fetal hepatic BFU-E, unlike adult bone marrow (BM) or peripheral blood (PB) BFU-E, were capable of proliferating in response to erythropoietin in the absence of added GM colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3), and erythropoietin (Epo) directly stimulated the expansion of the fetal BFU-E pool in suspension culture. A murine monoclonal antibody (MoAb), Ep 3, was raised against enriched fetal liver progenitor cells, which detected all fetal BFU-E and which reacted with the erythropoietin-responsive, GM-CSF/IL-3-independent fraction of adult BM BFU-E and CFU-E. All adult PB BFU-E were Ep 3- but became Ep 3+ after stimulation with GM-CSF or IL-3. These data indicate that Epo plays a unique role in fetal hepatic erythropoiesis, stimulating proliferation of immature BFU-E in addition to promoting terminal differentiation of later erythroid progenitor cells. In addition, these results demonstrate a MoAb which detects all erythropoietin-responsive progenitor cells and distinguishes the BFU-E compartments in adult BM and PB.     

Human osteoblasts support human hematopoietic progenitor cells in vitro bone marrow cultures

Hematopoietic stem cell differentiation occurs in direct proximity to osteoblasts within the bone marrow cavity. Despite this striking affiliation, surprisingly little is known about the precise cellular and molecular impact of osteoblasts on the bone marrow microenvironment. Recently, we showed that human osteoblasts produce a variety of cytokine mRNAs including granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-6. We examined here the ability of osteoblasts to support the development of hematopoietic colonies from progenitors as well the ability to maintain long-term culture-initiating cells (LTC-IC) in vitro. Examination of the hematopoietic cells recovered after 2 weeks of culture showed that osteoblasts support the maintenance of immature hematopoietic phenotypes. In methylcellulose assays, osteoblasts stimulate the development of hematopoietic colonies to a level at least 10-fold over controls from progenitor cells. Using limiting dilutional bone marrow cultures, we observed an activity produced by osteoblasts resulting in an threefold to fourfold expansion of human LTC-IC and progenitor cells in vitro. Thus, the presence of hematopoietic stem cells in close proximity to endosteal surfaces in vivo may be due in part to a requirement for osteoblast-derived products.     

Famotidine for healing and maintenance in nonsteroidal anti- inflammatory drug-associated gastroduodenal ulceration

BACKGROUND & AIMS: Nonsteroidal anti-inflammatory drugs (NSAIDs) are strongly associated with gastroduodenal ulceration. How to manage patients with NSAID-associated ulcers is a common clinical dilemma. High-dose famotidine in the healing and maintenance of NSAID-associated gastroduodenal ulceration was therefore evaluated. METHODS: One hundred four patients with rheumatoid or osteoarthritis who had gastroduodenal ulceration received famotidine, 40 mg twice daily. Sixteen patients stopped and 88 continued their NSAID treatment. Ulcer healing was assessed endoscopically at 4 and 12 weeks. Seventy-eight NSAID users with healed ulcers were then randomized to receive 40 mg twice daily famotidine or placebo and underwent endoscopy at 4, 12, and 24 weeks. RESULTS: Cumulative ulcer healing rates at 12 weeks were 89.0% (95% confidence interval [CI], 82.3%-95.7%) for patients who continued NSAID treatment and 100% (95% CI, 82.9%-100.0%) for those who stopped. The subsequent estimated cumulative gastroduodenal ulcer relapse over 6 months for NSAID users who took placebo was 53.5% (95% CI, 36.6%- 70.3%). This was reduced to 26.0% (12.1%-39.9%) in patients taking famotidine (P = 0.011). CONCLUSIONS: High-dose famotidine is effective ulcer healing therapy in patients who stop or continue NSAID treatment and significantly reduced the cumulative incidence of gastroduodenal ulcer recurrence compared with placebo when given as maintenance therapy. (Gastroenterology 1997 Jun;112(6):1817-22)     

Establishment of eosinophilic sublines from human promyelocytic leukemia (HL-60) cells: demonstration of multipotentiality and single- lineage commitment of HL-60 stem cells

Recent observations indicating that the HL-60 human acute promyelocytic leukemia cell line contains a minor eosinophil population in addition to neutrophil and mononuclear phagocyte progenitors suggest the multipotentiality of HL-60 stem cells. In order to clarify multilineage differentiation and commitment to single-lineage progenitors we analyzed HL-60 colonies formed in methylcellulose. In an HL-60 parent line with a relatively high eosinophil content (5.5%), 36% of the spontaneous colonies consisted partly or wholly of eosinophilic cells. After two rounds of subcloning in methylcellulose, two eosinophilic sublines and two neutrophilic sublines were established. These lines have been in continuous liquid culture for more than four months, and they show stable single-lineage differentiation. Purified biosynthetic GM-CSF, which stimulates normal CFU-GM and CFU-EO, induced monocytic differentiation but no eosinophilic differentiation in the neutrophilic sublines and no neutrophilic or monocytic differentiation in the eosinophilic sublines. These observations indicate that HL-60 stem cells are multipotent and capable of spontaneous commitment to single- lineage progenitors. The eosinophilic HL-60 sublines should facilitate studies on the production and function of human eosinophils and the single-lineage sublines will allow further analysis of leukemic cell differentiation and stem cell commitment.     

Retroviral-mediated gene transfer in human bone marrow cells growth in continuous perfusion culture vessels

Hematopoietic stem cell gene therapy holds the promise of being able to treat a variety of inherited and acquired diseases of the hematopoietic stem cell. However, to date, genetic modification of the human hematopoietic stem cell has been relatively inefficient. Here, we report the results of using a bioreactor system to expand hematopoietic cells after a brief retrovirus infection using a high titer, replication defective virus encoding for murine CD18. The retrovirus transduced culture continued to produce genetically modified hematopoietic progenitors for up to 6 weeks, the duration of the culture period. Up to one-third of the long-term culture initiating cell (LTC-IC) are genetically modified by the culture conditions. Murine CD18 can be expressed on the cell surface of up to 20% of the mature cells generated by the culture system, suggesting that clinically significant levels of gene transfer may be occurring. These results demonstrate the feasibility of using continuous perfusion bioreactors as a method of efficiently modifying human hematopoietic stem cells.