Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.

Endogenous p53 regulation and function in early stage Friend virus-induced tumor progression differs from that following DNA damage.

Erythroleukemia induced by the anemia strain of Friend virus occurs in two stages. The first stage results in rapid expansion of pre-leukemic proerythroblasts (FVA cells) dependent on erythropoietin (Epo) for differentiation and survival in vitro. The second stage is characterized by emergence of erythroleukemic clones (MEL cells) which typically bear activation of the ets-oncogene, PU.1/spi.1, and loss of functional p53. We developed a Friend virus-sensitive, p53-deficient mouse model to investigate the biological advantage conferred by p53-loss during tumor progression. Here we report p53 was not required for cell survival or growth arrest during differentiation of FVA cells, nor was p53 required for induction of apoptosis upon Epo withdrawal. However, we detected induction of the p21Cip1 cyclin-dependent kinase inhibitor gene during differentiation, which was markedly enhanced in the presence of p53. p53-dependent expression of p21Cip1 occurred in the absence of an increase in p53 mRNA and protein levels and was specific for p21Cip1, since expression of gadd45, mdm-2, cyclin G and bax were unaffected by p53. In contrast, treatment of FVA cells with DNA damaging agents led to rapid accumulation of p53 protein resulting in transcription of multiple p53-regulated genes, leading to either apoptosis or growth arrest, depending on the agent used. These data demonstrate that p53-dependent activities during differentiation of preleukemic erythroblasts are distinct from those observed in response to genotoxic agents. We propose that enhancement of p53-dependent gene expression during differentiation may represent a tumor suppressor function which is necessary to monitor differentiation of preleukemic cells and which is selected against during tumor progression.

Modulation of hematopoietic stem/progenitor cell engraftment by transforming growth factor beta.

OBJECTIVE: To investigate if cell cycle progression plays a role in modulating the engraftment potential of mouse hematopoietic stem and progenitor cells (HSPC). MATERIALS AND METHODS: HSPC were isolated from adult mouse bone marrow, cultured in vitro under conditions promoting cell cycle arrest, and subsequently were evaluated for cell cycle status, clonogenic activity, and transplant potential. RESULTS In the presence of steel factor (STL) as a survival cytokine, transforming growth factor beta (TGF-beta) increased the G0/G1 fraction of cycling progenitor cells (Rh(high)) after a 20-hour culture. Clonogenic activity of quiescent long-term repopulating (Rh(low)) HSPC was unaffected by this culture, whereas clonogenic potential of Rh(high) cells decreased by about 30%. In competitive repopulation assays, Rh(low) cells cultured in STL + TGF-beta engrafted better than cells cultured in STL alone. However, culture in STL + TGF-beta did not overcome the failure of Rh(high) cells to engraft after transplant. We also utilized a two-stage culture system to first induce proliferation of Rh(low) HSPC by a 48-hour culture in STL + interleukin 6 + Flt-3 ligand, followed by shifting the culture to STL + TGF-beta for 24 hours to induce cycle arrest. A competitive repopulation assay demonstrated a relative decrease in repopulating potential in cultures that were cycle arrested compared to those that were not. CONCLUSION: Cell cycle progression by itself cannot account for the decrease in repopulating potential that is observed after ex vivo expansion. Other determinants of engraftment must be identified to facilitate the transplantation of cultured HSPC.

Renal transplantation. A 20-year experience in a Veterans Administration Medical Center.

Between March 1963 and December 1983, 324 renal transplants were performed in 273 veteran patients at the Veterans Administration Medical Center, Nashville, Tenn. Cadaver donors were used in 273 transplants, with an overall one-year patient survival of 72.5% and one-year functional graft survival of 50%. Twenty-four living-related transplants were performed, with an overall one-year patient survival of 89% and one-year functional graft survival of 75%. For analytical purposes the 20-year transplant experience was divided into five eras. One-year patient survival increased from 45% in era 1 to 84% in era 5, while functional graft survival increased from 45% to 70%. Death has occurred in 139 patients, with sepsis being responsible for the largest number of early deaths. Cardiovascular disease was responsible for most late deaths.

Changing prevalence of asthma in Australian children.

OBJECTIVE: To investigate whether prevalence of asthma in children increased in 10 years. DESIGN: Serial cross sectional studies of two populations of children by means of standard protocol. SETTING: Two towns in New South Wales: Belmont (coastal and humid) and Wagga Wagga (inland and dry). SUBJECTS: Children aged 8-10 years: 718 in Belmont and 769 in Wagga Wagga in 1982; 873 in Belmont and 795 in Wagga Wagga in 1992. MAIN OUTCOME MEASURES: History of respiratory illness recorded by parents in self administered questionnaire; airway hyperresponsiveness by histamine inhalation test; atopy by skin prick tests; counts of house dust mites in domestic dust. RESULTS: Prevalence of wheeze in previous 12 months increased in Belmont, from 10.4% (75/718) in 1982 to 27.6% (240/873) in 1992 (P < 0.001), and in Wagga Wagga, from 15.5% (119/769) to 23.1% (183/795) (P < 0.001). The prevalence of airway hyperresponsiveness increased twofold in Belmont to 19.8% (173/873) (P < 0.001) and 1.4-fold in Wagga Wagga to 18.1% (P < 0.05). The prevalence of airway hyperresponsiveness increased mainly in atopic children only, but the prevalence of atopy was unchanged (about 28.5% in Belmont and about 32.5% in Wagga Wagga). Numbers of house dust mites increased 5.5-fold in Belmont and 4.5-fold in Wagga Wagga. CONCLUSIONS: We suggest that exposure to higher allergen levels has increased airway abnormalities in atopic children or that mechanisms that protected airways of earlier generations of children have been altered by new environmental factors.

Modulation of Schistosoma mansoni egg-induced granuloma formation: I-J restriction of T cell-mediated suppression in a chronic parasitic infection.

Newly formed hepatic granulomas around Schistosoma mansoni eggs become progressively smaller during the chronic (greater than or equal to 15 weeks after infection) phase of the disease. This reduction in granuloma size, termed "modulation," is known to be caused in part by a T lymphocyte that can adoptively transfer modulation to 6-week-infected mice. The present study examines a possible role for the I-J locus in regulating the suppressor T lymphocyte aspects of modulation. Adoptive transfer between congeneic B10.A(3R) and B10.A(5R) mice (differing at the I-J locus) indicated that optimal suppression is dependent upon homology at the I-J locus. In vivo treatment of chronically infected mice with microliter amounts of antiserum specific for the recipient's I-J determinant blocked modulation during chronic infection and prevented adoptive transfer of suppression to 6-week-infected mice. The in vivo regimen of anti-I-J had no effect on anti-schistosomal egg antigen titers during chronic infection. These results demonstrate an I-J restriction for suppression. It appears that the suppressor T lymphocyte circuit responsible for this aspect of modulation requires an I-J positive lymphocyte.

Tyrophagus putrescentiae: an allergenically important mite.

The incidence of positive skin tests to the mite Tyrophagus putrescentiae was measured and compared with skin reactions to Dermatophagoides pteronyssinus and other allergens in Australian and New Guinean populations, and mite counts were determined in Sydney and New Guinea houses. Positive reactions to T. putrescentiae were as frequent as those for D. pteronyssinus in asthmatics in Sydney and were the commonest positive reactions in the normal New Guinea population. T. putrescentiae-specific serum IgE levels were determined and some IgG cross-reactions of D. pteronyssinus and T. putrescentiae antigens were demonstrated. It is suggested that T. putrescentiae is an important source of allergen and should be considered whenever D. pteronyssinus is thought to be a problem. A convenient method for culturing and isolating T. putrescentiae is described.

Effect of salbutamol, fenoterol, and sodium cromoglycate on the release of heparin from sensitized human lung fragments challenged with Dermatophagoides pteronyssinus allergen.

Heparin from degranulating mast cells influences a wide range of cellular and humoral reactions associated with allergic inflammation and asthma. Agents that inhibit mast cell degranulation may therefore compromise the moderating effects of heparin in the tissues and result in worsening inflammation and other associated pathology. This study measures heparin release from allergen-challenged human lung tissue and compares the effect of the mast cell stabilizing beta 2-agonists, salbutamol and fenoterol, and a non-beta 2-agonist, sodium cromoglycate, on the release of heparin. Pieces of lung tissue 2 to 3 mm3 were sensitized with high titer Dermatoaphagoides pteronyssinus-specific IgE serum and challenged with D. pteronyssinus allergen, with and without prior addition of salbutamol, fenoterol, or sodium cromoglycate. Dextran sulfate was added to the mixture to prevent the binding of heparin to tissue proteins. Heparin was released together with histamine after challenge. The mean and 95% confidence interval of prechallenge and postchallenge heparin concentrations in the lung tissue filtrates were 0.10 IU/ml (0.07, 0.12) and 0.24 IU/ml (0.17, 0.30), respectively (P < 0.001). Addition of the beta 2-agonists produced a mean inhibition of released heparin of 71% (50, 92), and 73% (55, 91), respectively. Sodium cromoglycate gave a 35% (20, 51) inhibition that was significantly less than that produced by the beta 2-agonists (P < 0.01). The beta 2-agonists salbutamol and fenoterol strongly inhibited heparin release from mast cells. The therapeutic use of mast cell stabilizing agents may therefore be potentially detrimental to the control of allergic inflammation and other associated pathologies.

Characterization of airborne mineral dusts associated with farming activities in rural Alberta, Canada.

The dry and windy climate of the Western Canadian prairie provinces, combined with large scale agricultural activities, results in aerosolization or organic and mineral dusts. The purpose of this study was to conduct an environmental and minerological analysis of these dusts in order to estimate the risk for pneumoconiosis in exposed farmer populations. Two districts in central/southern Alberta were chosen for study. One of these regions was representative of a predominantly grain growing district with minimal use of irrigation; the other region was largely devoted to forage and irrigated crop production. Air pollution statistics showed a bi-modal distribution of total suspended particulates (TSP) with peaks corresponding with maximal farm activities in the spring and early fall. Analysis of bulk dust samples obtained from tractor cab filters showed that the majority of particles from both districts were within the respirable range (less than 5 microns). Samples from the forage-crop region contained more organic material, a greater water soluble fraction and had particles that were, on average, smaller and rounder than particles from the grain district. These differences were thought to reflect differences in irrigation patterns and use of fertilizers between the two districts. Free silica (quartz) content was also very variable and ranged from 1 to 17% on a mass basis. Respirable fibrous minerals were occasionally identified, however, no asbestos fibres, fibrous tremolite, or fibrous zeolites were identified. The results indicate that there is potential risk for mineral dust pneumoconiosis in heavily exposed farmer populations and that this risk will be influenced by local and regional factors.

Changes in mite allergen Der p I in house dust following spraying with a tannic acid/acaricide solution.

The beds, carpets and furnishings in 15 houses were sprayed with a solution containing tannic acid and an acaricide in an attempt to reduce allergen concentrations. Dust was collected from these sites for 4 weeks following spraying and the mite allergen Der p I concentration was measured and compared with baseline concentrations. In a subgroup of houses, counts of live mites and estimates of aeroallergen were also made. Four untreated houses were monitored over the same period. In dust samples collected 3 days after spraying, the mean concentrations of Der p I in beds, carpets and furniture were 23%, 28% and 26% of the pretreatment levels. All these reductions were significant compared to untreated controls. Samples collected 4 weeks after treatment were not significantly different to baseline for each group. After the initial reduction, the rate of increase in allergen concentration was significantly greater in the spray-only beds than in the beds which had been both sprayed and fitted with occlusive covers. Both aeroallergen and live mites continued to be detected in houses after treatment with the spray. These studies suggest that such sprays are only temporarily effective when applied at the manufacturer's recommended volumes and additional approaches are required to control the bulk of allergens in houses.

House dust mites and skin tests in different Australian localities.

Dermatophagoides mite concentrations in household dust have been measured in three Australian towns with contrasting geographical features, climate, and degree of urbanisation. Mite allergen skin tests on school children living in these areas showed that the prevalence of positive reactions varied with mite concentrations. In Belmont, D. farinae as well as D. pteronyssinus were present in some of the dust samples. Skin test weals were larger in Busselton than in either Wagga Wagga or Belmont. The importance and practical implications of these findings are discussed.

Reduction of house dust mites and mite allergens: effects of spraying carpets and blankets with Allersearch DMS, an acaricide combined with an allergen reducing agent.

Two studies were made to determine the acaricidal and allergen reducing properties of Allersearch DMS solution. In Study 1, samples of dust and carpet containing living mites were treated, in vitro, with DMS solution. No living mites were found in dust after 3-4 min or in carpets 50 min after treatment. In Study 2, mite counts and allergen estimation made on bed blankets before and after spraying with DMS solution showed a marked reduction in mite numbers (95%) and allergenicity (100-fold). Mite numbers and allergenicity stayed at this low level for 6 weeks. At 16 weeks both mites and allergens showed a slight increase, but were still significantly (P less than 0.001) below pretreatment levels.