Reviews of evidence regarding interventions to reduce tobacco use and exposure to environmental tobacco smoke.

This report presents the results of systematic reviews of effectiveness, applicability, other effects, economic evaluations, and barriers to use of selected population-based interventions intended to reduce tobacco use and exposure to environmental tobacco smoke. The related systematic reviews are linked by a common conceptual approach. These reviews form the basis of recommendations by the Task Force on Community Preventive Services (TFCPS) regarding the use of these selected interventions. The TFCPS recommendations are presented on page 67 of this supplement.

A human erythropoietin receptor gene mutant causing familial erythrocytosis is associated with deregulation of the rates of Jak2 and Stat5 inactivation.

The erythropoietin receptor (EpoR) has been previously shown to contain a cytoplasmic C-terminal negative regulatory domain, experimental deletion or mutation of which leads to increased sensitivity of expressing cells to the effects erythropoietin (Epo). We have studied a naturally occurring C-terminal truncation mutant of the human EpoR by stably transfecting the growth factor-dependent hematopoietic tissue culture cell line 32D with expression plasmids containing either the wildtype or mutant human EpoR cDNA, thus rendering the cells dependent on Epo for viability and proliferation. In Epo dose-response assays, cells expressing the mutant EpoR displayed hyperresponsiveness to Epo compared with cells expressing comparable numbers of the wild-type EpoR cultured in the presence of fetal bovine serum. We investigated whether enhanced Epo sensitivity of cells expressing the truncated EpoR is associated with alteration in Epo receptor-mediated activation of Stat5, which could have a role in Epo-induced proliferation. Although maximal Stat5 activation in response to a given concentration of Epo was comparable in 32D cells expressing the wild-type or truncated EpoRs, the time course of Epo-induced Stat5 activation was very different. Gel-mobility shift studies revealed the presence of Stat5 DNA-binding activity in nuclear and cytoplasmic extracts of cells expressing the truncated EpoR for a significantly longer time than that observed in similar extracts of cells expressing the wild-type EpoR consistent with decreased rate of inactivation of Stat5 in cells expressing the mutant EpoR. Epo-dependent tyrosine phosphorylation of both Stat5 and Jak2 was also substantially prolonged in cells expressing the truncated EpoR. These results suggest a role for Stat5 in regulation of Epo-mediated cell growth and implicate altered kinetics of Epo-induced Jak2 and Stat5 activation in the pathogenesis of familial erythrocytosis associated with this naturally occurring EpoR gene mutation.

The distal cytoplasmic domain of the erythropoietin receptor induces granulocytic differentiation in 32D cells.

The role of hematopoietic growth factors in lineage commitment and differentiation is unclear. We present evidence that heterologous expression of an erythroid specific receptor allows granulocytic differentiation of a myeloid cell line. We have previously characterized a truncation mutant of the erythropoietin receptor (EpoR), which is associated with familial erythrocytosis (Blood 89:4628, 1997). This truncated EpoR lacks the distal 70 amino acids of the cytoplasmic domain. To study the functional role of this distal receptor domain, 32D cells, a murine interleukin-3 (IL-3)-dependent myeloid line, were transfected with the wild-type EpoR (32D/EpoR WT) or the truncated EpoR (32D/EpoR FE). 32D cells expressing either the full-length or truncated EpoR display equivalent proliferative rates in saturating concentrations of Epo. There is a dramatic difference in maturational phenotype between the two cell lines, however. The 32D/EpoR FE cells and mock transfected 32D cells have an immature, monoblastic morphology and do not express the primary granule protein myeloperoxidase. The 32D/EpoR WT cells, on the other hand, demonstrate granulocytic differentiation with profuse granulation, mature, clumped chromatin, and myeloperoxidase expression. There is no evidence of erythroid differentiation in 32D cells transfected with either the full-length or truncated EpoR. Treatment of the cells with the specific Jak2 inhibitor tyrphostin AG 490 inhibits myeloid differentiation driven by the distal EpoR. We conclude that: (1) the distal cytoplasmic domain of the EpoR is able to induce a specific myeloid differentiation signal distinct from mitogenic signaling, and (2) these data extend to myelopoiesis the growing body of evidence that the cellular milieu, not the specific cytokine receptor, determines the specificity of differentiation after cytokine receptor activation.

Familial erythrocytosis associated with a short deletion in the erythropoietin receptor gene.

Familial erythrocytosis (familial polycythemia) inherited as an autosomal dominant trait has recently been reported to be associated with mutations in the gene encoding the erythropoietin receptor (EpoR) in a small number of families. We studied a new kindred with dominantly inherited familial erythrocytosis associated with heterozygosity for a deletion of seven nucleotides between positions 5985 and 5991 in exon 8 of the EpoR gene, resulting in an EpoR peptide that is truncated by 59 amino acids at its C-terminus. A 7-bp direct repeat is present in the normal EpoR gene at the site of this mutation, consistent with the slipped mispairing model for the generation of short deletions during DNA replication. Hypersensitivity to Epo of erythroid progenitors from an affected individual was observed in in vitro methylcellulose cultures, as indicated by more numerous and larger colonies compared with those of a control subject. To study mutant EpoR function, the cDNA encoding the mutant EpoR was synthesized by reverse transcription-polymerase chain reaction of peripheral blood RNA from the proband and stably tranfected into murine interleukin-3-dependent 32D cells. Epo dose-response assays showed that cells expressing the mutant EpoR displayed fivefold to 10-fold increased sensitivity to Epo compared with cells expressing similar numbers of the wild-type EpoR.

Enzyme-linked immunosorbent assay detects a potential soluble form of the erythropoietin receptor in human plasma.

The erythropoietin receptor (EpoR) is a type I transmembrane protein that is a member of the family of hemopoietin receptors. Several members of this family have soluble receptor forms that are secreted by the cells rather than expressed on the cell surface. An alternatively spliced EpoR transcript has been described in human erythroid precursors that, if translated, would produce a truncated, soluble EpoR lacking the transmembrane domain. To determine if the human EpoR is expressed in a soluble form, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) for the EpoR, and we analyzed human serum and plasma. Sheep were immunized with a fusion protein (EREx) consisting of glutathione-S-transferase (GST) and the human EpoR extracellular domain. The sheep antiserum was affinity-purified on immobilized EREx, and then used in a two-stage antigen capture ELISA. The plasma from 20 normal subjects was studied with this assay. There was a wide variability in the levels of soluble EpoR in these subjects (range, <10-2,200 ng/ml). An average value of 550 +/- 735 ng/ml for soluble EpoR was obtained in these normals. Protein A adsorption of the test plasma prior to the assay had no effect on the values obtained. Assay of serum from the same normal subjects showed an average decrease of 88% in soluble EpoR levels compared to plasma. There was no correlation between hematocrit and soluble EpoR levels compared to plasma. There was no correlation between hematocrit and soluble EpoR level. This assay may have utility in the further elucidation of erythropoietin physiology.

Localization of an essential ligand binding determinant of the human erythropoietin receptor to a domain N-terminal to the WSXWS motif: implications for soluble receptor function.

The interaction of erythropoietin (Epo) with the erythropoietin receptor (EpoR) supports erythropoiesis. The EpoR is a member of the well-recognized cytokine receptor superfamily characterized by four conserved cysteines and a WSXWS domain in the extracellular portion of the molecule. To localize ligand-binding determinants of the EpoR near the WSXWS domain, we tested the ligand-binding ability of the wild-type human EpoR extracellular domain (EREx), two truncated and three chimeric constructs with the interleukin-2 receptor beta subunit (IL2R beta). Constructs were expressed in E. coli as GST fusion proteins linked to a solid-phase support and assayed for binding to 125I Epo. As previously shown, Epo bound specifically to the expressed extracellular domain, EREx. Epo did not bind to truncated receptors lacking either the entire fifth exon or the WSXWS domain. Epo also did not bind to chimeric receptors that had the amino acids encoded by the fifth exon replaced by IL2R beta or that had the amino acids subsequent to asparagine residue 209 replaced by IL2R beta. Specific binding was demonstrated for a construct in which the WSXWS was replaced by that of IL2R beta. We conclude that the amino acids encoded by this 5' portion of exon 5 of the EpoR are necessary for ligand binding and that the WSXWS domain is necessary for Epo binding but is not involved in ligand-binding specificity. We also speculate that if the putative soluble form of the EpoR is expressed (predicted to lack exon 5), it does not bind Epo and therefore may serve a physiologic purpose other than ligand binding.

Ligand binding properties of the human erythropoietin receptor extracellular domain expressed in Escherichia coli.

We developed an assay to directly measure the ligand binding properties of the cloned human erythropoietin receptor (EpoR). The cDNA encoding the extracellular domain of the human EpoR was amplified by polymerase chain reaction and ligated into the prokaryotic expression vector pGEX3X. Synthesis in Escherichia coli was induced and a soluble glutathione S-transferase fusion protein, EREx, was purified by erythropoietin affinity chromatography. Purified EREx was bound to GSH agarose beads and used in a solid phase ligand binding assay. Specific binding of 125I-erythropoietin to EREx beads was demonstrated. A single affinity class (Kd = 1.5 nM) of the binding site was evident on Scatchard analysis. The Kd of this site is quantitatively equivalent to that of the "low" affinity cellular binding site. Kinetic analysis of ligand binding to EREx revealed both the on and off rates to be rapid, with t1/2 of 60 and 40 s, respectively. EREx ligand binding exhibits no obvious metal ion dependence or cross-competition by other hemopoietins. Antibodies to EREx block the binding of erythropoietin to the cellular EpoR. We conclude that the 66-kDa EpoR protein is capable of specific ligand binding and that no covalent modifications or associated molecules are required for this interaction. We speculate that the "high" affinity cellular binding site (Kd less than 0.2 nM) results from the interaction of the EpoR with another molecule, either additional EpoR or associated subunits, that decreases the ligand off rate.

A structurally abnormal erythropoietin receptor gene in a human erythroleukemia cell line.

Restriction endonuclease mapping demonstrates a 3' end deletion of one erythropoietin receptor (EpoR) gene in TF-1 cells, a human erythroleukemia cell line that overexpresses the EpoR and proliferates in response to erythropoietin (Epo). EpoR mRNA transcripts are highly abundant and normal in size. These findings raise interesting questions about the possible role of this EpoR gene abnormality in the pathogenesis of the erythroleukemia from which this cell line was derived. This is the first report of an abnormal human erythropoietin receptor gene.

The role of the respiratory care practitioner in the evaluation of medical devices.

Health care costs continue to skyrocket, and a large portion of these costs can be attributed to technology. Some technology, such as oximetry, is relatively inexpensive compared to CAT scanning and magnetic resonance imaging. Therefore, it may elude close scrutiny and direct health care planning and, with little fanfare, drive up costs. Many of the new respiratory monitors and ventilators have not yet been subjected to the basic question: Do they affect morbidity and mortality? Technology assessment in this country is fraught with duplication of effort and complexity; the system is fragmented and in need of a strategic approach to equipment evaluation. There is meager information on cost analysis and little consensus on what needs to be done about the ethical dilemmas created by technology. Respiratory care practitioners have a significant role to play in medical device evaluation. Problem reporting of serious equipment malfunctions to the U.S. Pharmacopeia and manufacturers is essential. In-house product evaluation prior to purchase is prudent. Important feedback can be given to manufacturers through testing of prototype equipment, with the end result that our needs are met. Small departments may need to rely on information that is currently available. Laboratory and clinical research must be conducted to provide a scientific basis for the use of technology. Adequate assessment requires knowledgeable people in a position to perform the essential tasks. Respiratory care practitioners are a logical group to take an active role in the important tasks of thoroughly assessing the technologies we use.

Protein S is required for bovine platelets to support activated protein C binding and activity.

Gel-filtered platelets accelerate activated protein C inactivation of factor Va in a reaction that requires the presence of protein S. With protein S present, specific activated protein C binding to the platelet surface is observed (Kd = 11 +/- 3 nM, 203 +/- 20 sites/platelet). The concentration dependence of the activated protein C-mediated factor Va inactivation is in close agreement with the binding. The observed binding is specific since protein C does not compete with activated protein C. Platelet-bound activated protein C is approximately 8000 times more active than the solution-phase enzyme. Platelet activation with thrombin results in formation of a site capable of accelerating factor Va inactivation by activated protein C in the absence of added protein S. This cell surface site is blocked by the addition of affinity purified antibodies to protein S. We conclude that protein S is required for activated protein C binding to the platelet surface and subsequent rapid factor Va inactivation. Platelet activation leads to the expression of either protein S or an antigenically related protein which can substitute for exogenously added protein S.

Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation.

Protein C is activated rapidly when thrombin binds to a specific cell surface cofactor protein, thrombomodulin. Studies were initiated to determine the influence of thrombin-thrombomodulin complex formation on the substrate specificity of thrombin. When thrombin binds to thrombomodulin, the resultant complex retains less than 1% of the fibrinogen clotting activity of free thrombin. Permanent alteration of the thrombin molecule is not involved since full clotting activity is regenerated by incubation of the complex with excess diisopropyl phosphothrombin. Unlike the activation of protein C by the thrombin-thrombomodulin complex which is dependent on Ca2+, inhibition of fibrinogen clotting activity is not dependent on the presence of divalent metal ions. Formation of the thrombin-thrombomodulin complex also inhibits thrombin activation of factor V. Despite these changes in macromolecular substate specificity, no significant change in the hydrolysis of the synthetic substrates p-tosyl-L-arginine methyl ester and N alpha-benzoyl-L-arginine ethyl ester is detected upon formation of the thrombin-thrombomodulin complex. Formation of this complex results in a slight increase in the Km (from 9.0 +/- 0.4 to 10.2 +/- 0.6 microM) and Vmax (from 230 +/- 10 to 270 +/- 10 mol/s/mol of thrombin) for the specific thrombin substrate H-D-Phe-Pip-Arg-p-nitroanilide. These studies suggest that thrombomodulin has two distinct anticoagulant functions: 1) to inhibit the ability of thrombin to clot fibrinogen and to activate factor V; and 2) to accelerate the formation of the anticoagulant, activated protein C.