Background Extracellular nucleotides (ATP, ADP, UTP and UDP) exert a wide range of biological effects in blood cells mediated by multiple ionotropic P2X receptors and G protein-coupled P2Y receptors. Although pharmacological experiments have suggested the presence of several P2 receptor subtypes on monocytes and lymphocytes, some results are contradictory. Few physiological functions have been firmly established to a specific receptor subtype, partly because of a lack of truly selective agonists and antagonists. This stimulated us to investigate the expression of P2X and P2Y receptors in human lymphocytes and monocytes with a newly established quantitative mRNA assay for P2 receptors. In addition, we describe for the first time the expression of P2 receptors in CD34 +  stem and progenitor cells implicating a potential role of P2 receptors in hematopoietic lineage and progenitor/stem cell function.
Background
Extracellular nucleotides (ATP, ADP, UTP and UDP) exert a wide range of biological effects in blood cells mediated by multiple ionotropic P2X receptors and G protein-coupled P2Y receptors. Although pharmacological experiments have suggested the presence of several P2 receptor subtypes on monocytes and lymphocytes, some results are contradictory. Few physiological functions have been firmly established to a specific receptor subtype, partly because of a lack of truly selective agonists and antagonists. This stimulated us to investigate the expression of P2X and P2Y receptors in human lymphocytes and monocytes with a newly established quantitative mRNA assay for P2 receptors. In addition, we describe for the first time the expression of P2 receptors in CD34 +  stem and progenitor cells implicating a potential role of P2 receptors in hematopoietic lineage and progenitor/stem cell function.Results Using a quantitative mRNA assay, we assessed the hypothesis that there are specific P2 receptor profiles in inflammatory cells. The P2X 4  receptor had the highest expression in lymphocytes and monocytes. Among the P2Y receptors, P2Y 12  and P2Y 2  had highest expression in lymphocytes, while the P2Y 2  and P2Y 13  had highest expression in monocytes. Several P2 receptors were expressed (P2Y 2 , P2Y 1 , P2Y 12 , P2Y 13 , P2Y 11 , P2X 1 , P2X 4 ) in CD34+ stem and progenitor cells.
Results
Using a quantitative mRNA assay, we assessed the hypothesis that there are specific P2 receptor profiles in inflammatory cells. The P2X 4  receptor had the highest expression in lymphocytes and monocytes. Among the P2Y receptors, P2Y 12  and P2Y 2  had highest expression in lymphocytes, while the P2Y 2  and P2Y 13  had highest expression in monocytes. Several P2 receptors were expressed (P2Y 2 , P2Y 1 , P2Y 12 , P2Y 13 , P2Y 11 , P2X 1 , P2X 4 ) in CD34+ stem and progenitor cells.Conclusions The most interesting findings were the high mRNA expression of P2Y 12  receptors in lymphocytes potentially explaining the anti-inflammatory effects of clopidogrel, P2Y 13  receptors in monocytes and a previously unrecognised expression of P2X 4  in lymphocytes and monocytes. In addition, for the first time P2 receptor mRNA expression patterns was studied in CD34 +  stem and progenitor cells. Several P2 receptors were expressed (P2Y 2 , P2Y 1 , P2Y 12 , P2Y 13 , P2Y 11 , P2X 1 , P2X 4 ), indicating a role in differentiation and proliferation. Thus, it is possible that specific antibodies to P2 receptors could be used to identify progenitors for monocytes, lymphocytes and megakaryocytes.
Conclusions
The most interesting findings were the high mRNA expression of P2Y 12  receptors in lymphocytes potentially explaining the anti-inflammatory effects of clopidogrel, P2Y 13  receptors in monocytes and a previously unrecognised expression of P2X 4  in lymphocytes and monocytes. In addition, for the first time P2 receptor mRNA expression patterns was studied in CD34 +  stem and progenitor cells. Several P2 receptors were expressed (P2Y 2 , P2Y 1 , P2Y 12 , P2Y 13 , P2Y 11 , P2X 1 , P2X 4 ), indicating a role in differentiation and proliferation. Thus, it is possible that specific antibodies to P2 receptors could be used to identify progenitors for monocytes, lymphocytes and megakaryocytes.

Background Extracellular nucleotides (ATP, ADP, UTP and UDP) exert a wide range of biological effects in blood cells mediated by multiple ionotropic P2X receptors and G protein-coupled P2Y receptors [ 1 - 3 ]. So far, the P2Y family is composed of eight cloned and functionally distinct subtypes (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , P2Y 14 ) [ 4 , 5 ]; the P2X family is composed of seven cloned subtypes (P2X 1 -P2X 7 ) [ 6 , 7 ]. We have previously quantified P2 receptor mRNA expression in platelets (representing megakaryocyte expression), and demonstrated a selective expression of the ADP receptors P2Y 12  and P2Y 1 , together with the ATP receptor P2X 1  [ 8 ]. This is consistent with the clinical effect of the P2Y 12  antagonist clopidogrel for the prevention of myocardial infarctions in patients with acute coronary syndromes [ 9 , 10 ]. However, virtually every hematopoietic cell is responsive to nucleotides [ 2 ]. Because effects as different as proliferation, differentiation, chemotaxis and release of cytokines are regulated by nucleotides, they could play a role in the atherosclerotic inflammatory process. Human lymphocytes, monocytes and macrophages constitute an important line of defence upon infection and exposure to inflammatory stimuli [ 11 ]. Circulating blood monocytes become activated, migrate to tissues, and undergo differentiation into macrophages during inflammation [ 12 ]. Monocytes have been shown to express several P2Y receptors and up-regulation of P2X 7  receptor mRNA in monocytes has been observed upon cell differentiation to macrophages [ 13 , 14 ]. Although pharmacological experiments have suggested the presence of several P2 receptor subtypes on monocytes and lymphocytes, some results are contradictory [ 1 , 2 ]. Few physiological functions have been firmly established to a specific receptor subtype, partly because of a lack truly selective agonists and antagonists. This stimulated us to investigate the expression of P2X and P2Y receptors in human lymphocytes and monocytes with a newly established quantitative mRNA assay for P2 receptors [ 8 , 15 ]. In addition, we describe for the first time the mRNA expression of P2 receptors in CD34 +  stem and progenitor cells implicating a potential role of P2 receptors in hematopoietic lineage and progenitor/stem cell function.
Background
Extracellular nucleotides (ATP, ADP, UTP and UDP) exert a wide range of biological effects in blood cells mediated by multiple ionotropic P2X receptors and G protein-coupled P2Y receptors [ 1 - 3 ]. So far, the P2Y family is composed of eight cloned and functionally distinct subtypes (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , P2Y 14 ) [ 4 , 5 ]; the P2X family is composed of seven cloned subtypes (P2X 1 -P2X 7 ) [ 6 , 7 ].We have previously quantified P2 receptor mRNA expression in platelets (representing megakaryocyte expression), and demonstrated a selective expression of the ADP receptors P2Y 12  and P2Y 1 , together with the ATP receptor P2X 1  [ 8 ]. This is consistent with the clinical effect of the P2Y 12  antagonist clopidogrel for the prevention of myocardial infarctions in patients with acute coronary syndromes [ 9 , 10 ]. However, virtually every hematopoietic cell is responsive to nucleotides [ 2 ]. Because effects as different as proliferation, differentiation, chemotaxis and release of cytokines are regulated by nucleotides, they could play a role in the atherosclerotic inflammatory process. Human lymphocytes, monocytes and macrophages constitute an important line of defence upon infection and exposure to inflammatory stimuli [ 11 ]. Circulating blood monocytes become activated, migrate to tissues, and undergo differentiation into macrophages during inflammation [ 12 ]. Monocytes have been shown to express several P2Y receptors and up-regulation of P2X 7  receptor mRNA in monocytes has been observed upon cell differentiation to macrophages [ 13 , 14 ].Although pharmacological experiments have suggested the presence of several P2 receptor subtypes on monocytes and lymphocytes, some results are contradictory [ 1 , 2 ]. Few physiological functions have been firmly established to a specific receptor subtype, partly because of a lack truly selective agonists and antagonists. This stimulated us to investigate the expression of P2X and P2Y receptors in human lymphocytes and monocytes with a newly established quantitative mRNA assay for P2 receptors [ 8 , 15 ]. In addition, we describe for the first time the mRNA expression of P2 receptors in CD34 +  stem and progenitor cells implicating a potential role of P2 receptors in hematopoietic lineage and progenitor/stem cell function.Results and Discussion Our previous studies of P2 receptor mRNA expression in man with real-time PCR has shown a good resemblance with pharmacological and physiological experiments in vascular smooth muscle cells, endothelial cells and platelets [ 8 , 15 ]. It is therefore likely that our present mRNA findings in inflammatory, progenitor and stem cells are physiologically relevant. The lack of selective agonists and antagonists for most of the receptor subtypes combined with the absence of studies focused on several of the more recently cloned receptors makes the findings important. Furthermore, no pharmacological studies have been made on CD34 +  stem and progenitor cells. Expression of P2Y receptors in lymphocytes In lymphocytes, all the target genes P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , and P2Y 13  could be detected (n = 6). To illustrate expression of the P2 receptors relative to each other the P2Y 1  receptor was used as calibrator for the others, i. e. the other receptors were expressed as a ratio of the P2Y 1 . Among the P2Y receptor subtypes the P2Y 12  and P2Y 2  had highest expression (Figure  1A ). The lowest expressed P2Y receptor was P2Y 4 . Figure 1 Relative P2 gene expression in lymphocytes.  A, Bar graph shows relative P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12  and P2Y 13  receptor gene expression normalized to GAPDH. B, Bar graph shows relative P2X 1 , P2X 4  and P2X 7  receptor gene expression normalized to GAPDH. P2Y 1  was chosen to be calibrator. Extracellular nucleotides and their P2 receptors are involved in the regulation, proliferation but also apoptosis and cell death in lymphocytes and monocytes [ 3 , 16 ]. Previous studies have shown that ATP, ADP, UTP and UDP stimulate phospholipase C and Ca 2+  release from intracellular stores, that fits well with the highly expressed P2Y 2  receptor, together with the lesser expressed P2Y 1  and P2Y 6  receptors. ATP and ADP, but not UTP, can also increase cAMP [ 17 ]. This is in agreement with the P2Y 11  receptor that had the third highest mRNA expression. The most interesting finding was that P2Y 12  had the highest expression among the P2Y receptors in lymphocytes. It is not likely that this is the result of platelet contamination, because platelets contain very low amounts of RNA. To the best of our knowledge, there are no studies that have examined the effects of P2Y 12  on lymphocytes, even though selective antagonists exist. It is expected to inhibit cAMP generation and may activate lymphocytes. This could explain the antiinflammatory effect of clopidogrel. Clopidogrel is a P2Y 12  antagonist used in the clinic as a platelet aggregation inhibitor that reduces thrombotic cardiovascular events such as myocardial infarctions. However, it has also been shown to reduce CRP, even though aspirin in antiplatelet doses lacks this effect [ 18 ]. This effect may be mediated via P2Y 12  receptors in lymphocytes. Expression of P2X receptors in lymphocytes The most abundant P2X receptor in lymphocytes was the P2X 4  receptor. As showed in Figure  1B , the expression of P2X 4  was 3.2 times higher than P2Y 1 . The expression of P2X 4  was significantly higher than the expression of the other P2X receptors; P2X 1  (P < 0.001) and P2X 7 (P < 0.01). Selective pharmacological tools to discriminate between P2X receptors are scarce. Nevertheless, several studies have suggested the importance of P2X 7  in lymphocyte regulation. However, B lymphocytes stimulated with ATP do not undergo the typical increase in permeability up to 900 Da that is typical for the P2X 7  receptor. On the other hand, P2X 7  mediated effects on Ba 2+  and ethidium influx, phospholipase D activity and shedding of L-selectin have been blocked by the P2X 7  selective antagonist KN-62 in human lymphocytes [ 19 ]. Thus it is a surprising finding that the P2X 4  receptor was the highest expressed subtype in lymphocytes at the mRNA level. Even though we have demonstrated that more than 90% of the preparation consists of lymphocytes (see methods), it is possible that a small contamination of monocytes may have influenced the results, at least regarding P2X 4  receptor mRNA expression, because of its high expression levels in monocytes. P2X 4  receptors have indeed been demonstrated at the protein level in human B lymphocytes by confocal immunohistochemistry, in which P2X 1 , P2X 4  and P2X 7  were detected at the protein level [ 20 ]. However, the P2X 4  receptor staining was the most variable of the P2X receptors with weak to moderate levels of staining in a large proportion of cells in three patients and weak levels in only a minority of the cells from the other three patients examined [ 20 ]. Expression of P2Y receptors in monocytes Again, the P2Y 1  expression was used as calibrator for the others, i. e. the other receptors were expressed as a ratio of the P2Y 1 . Among the P2Y receptors, the P2Y 2 , P2Y 13  and P2Y 11  had highest expression (Figure  2A , n = 6). The presence of P2Y receptor mRNA in monocytes and lymphocytes is in agreement with previous studies using regular RT-PCR [ 21 ]. Figure 2 Relative P2 gene expression in monocytes.  A, Bar graph shows relative P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12  and P2Y 13  receptor gene expression normalized to GAPDH. B, Bar graph shows relative P2X 1 , P2X 4  and P2X 7  receptor gene expression normalized to GAPDH. P2Y 1  was chosen to be calibrator. Extracellular nucleotides stimulate interleukin secretion, iNOS-generation in monocytes, are involved in differentiation, cytotoxicity and killing of pathogens. All monocyte/macrophage cell lines express P2Y receptors coupled to IP 3  generation and intracellular Ca 2+  release, but the individual subtypes have not been investigated in detail in monocytes [ 2 , 3 ]. However, both ATP and UTP are active agonists, which is in agreement with the highest mRNA expression of the ATP/UTP receptor P2Y 2  (Fig  2 ). ATP mediated increase in cAMP has suggested the presence of P2Y 11 , with a suggested role in maturation of human monocyte-dendritic cells [ 22 ]. A relatively high expression of P2Y 11  was confirmed in our experiments. Interestingly, the P2Y 13  had even higher mRNA levels. To our knowledge, no experiments have addressed the presence of this cAMP inhibitory ADP receptor in monocytes. In fact, it has been an unresolved issue in what tissue this receptor is expressed. High levels in the spleen could be in agreement with monocyte expression [ 23 ]. Thus, the presence of P2Y 2  and P2Y 11  receptors are confirmed as expected, with the interesting addition of P2Y 13  receptors. Future experiments addressing the physiological role of P2Y 13  receptors in monocytes are needed. Expression of P2X receptors in monocytes Early studies demonstrated that ATP activates a receptor on macrophages that increase cell permeability eventually leading to cell death [ 2 , 3 ]. P2X 7  receptor transfection confers susceptibility to ATP-dependent permeabilization and ATP-resistant clones lack the P2X 7  receptor, demonstrating that it is present on macrophages and necessary for permabilization. However, it is not known whether P2X 7  is the only constitutive subunit or if it assembles with other subunits. As showed in Figure  2B , P2X 4  was by far the highest expressed P2 receptor in monocytes and the P2X 1  (P < 0.01) and P2X 7  (P < 0.01) had lower levels. Thus, unexpectedly the P2X 7  receptor was not the highest expressed P2X receptor in monocytes. This is in agreement with patch-clamp experiments suggesting that other P2X receptors are involved [ 24 ]. Interrelation of these experiments has suggested the contribution of P2X 4  receptors, which is supported by our findings [ 25 ]. It should be noted that all the three P2X receptors were expressed at very high levels compared to other cell types (30-fold more than the calibrator gene for P2X 4  and 6–7-fold more for P2X 1  and P2X 7 ). A physiological role for all three subtypes can therefore be expected. Expression of P2 receptors in CD34 +  stem and progenitor cells CD34 +  stem and progenitor cells are receiving an increasing attention because of their extensive self-renewal and multilineage differentiation ability making them attractive for cellular therapy [ 26 ]. Knowledge of their P2 receptor expression could be used for directing differentiation or for further subtype selection of early progenitors types. There are no previous pharmacological or expression studies of P2 receptors on human CD34 +  stem and progenitor cells. We found expression of several P2Y receptors, especially P2Y 1  and P2Y 2  (Figure  3A , n = 3). This indicates that both ATP and UTP are agonists for CD34 +  stem and progenitor cells and may stimulate IP 3  and intracellular Ca 2+  release. Figure 3 Relative P2 gene expression in CD34 +  stem and progenitor cells.  A, Bar graph shows relative P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12  and P2Y 13  receptor gene expression normalized to GAPDH. B, Bar graph shows relative P2X 1 , P2X 4  and P2X 7  receptor gene expression normalized to GAPDH. P2Y 1  was chosen to be calibrator. Among the P2X receptors the P2X 1  receptor had the highest expression followed by P2X 4  (P2X 1  had significantly higher expression than P2X 7 , P < 0.05) (Figure  3B , n = 3), suggesting a potential role of these receptors in regulation of stem and progenitor cells. P2Y 1 , P2Y 2  and P2X 1  receptors have all been shown to stimulate proliferation, but also to be able to mediate apoptosis [ 26 ]. Such roles could be of major importance in the highly proliferative CD34 +  stem and progenitor cells. Antagonists or agonists of these receptors could be used to control their differentiation or proliferation.
Results and Discussion
Our previous studies of P2 receptor mRNA expression in man with real-time PCR has shown a good resemblance with pharmacological and physiological experiments in vascular smooth muscle cells, endothelial cells and platelets [ 8 , 15 ]. It is therefore likely that our present mRNA findings in inflammatory, progenitor and stem cells are physiologically relevant. The lack of selective agonists and antagonists for most of the receptor subtypes combined with the absence of studies focused on several of the more recently cloned receptors makes the findings important. Furthermore, no pharmacological studies have been made on CD34 +  stem and progenitor cells.Expression of P2Y receptors in lymphocytes In lymphocytes, all the target genes P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , and P2Y 13  could be detected (n = 6). To illustrate expression of the P2 receptors relative to each other the P2Y 1  receptor was used as calibrator for the others, i. e. the other receptors were expressed as a ratio of the P2Y 1 . Among the P2Y receptor subtypes the P2Y 12  and P2Y 2  had highest expression (Figure  1A ). The lowest expressed P2Y receptor was P2Y 4 . Figure 1 Relative P2 gene expression in lymphocytes.  A, Bar graph shows relative P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12  and P2Y 13  receptor gene expression normalized to GAPDH. B, Bar graph shows relative P2X 1 , P2X 4  and P2X 7  receptor gene expression normalized to GAPDH. P2Y 1  was chosen to be calibrator. Extracellular nucleotides and their P2 receptors are involved in the regulation, proliferation but also apoptosis and cell death in lymphocytes and monocytes [ 3 , 16 ]. Previous studies have shown that ATP, ADP, UTP and UDP stimulate phospholipase C and Ca 2+  release from intracellular stores, that fits well with the highly expressed P2Y 2  receptor, together with the lesser expressed P2Y 1  and P2Y 6  receptors. ATP and ADP, but not UTP, can also increase cAMP [ 17 ]. This is in agreement with the P2Y 11  receptor that had the third highest mRNA expression. The most interesting finding was that P2Y 12  had the highest expression among the P2Y receptors in lymphocytes. It is not likely that this is the result of platelet contamination, because platelets contain very low amounts of RNA. To the best of our knowledge, there are no studies that have examined the effects of P2Y 12  on lymphocytes, even though selective antagonists exist. It is expected to inhibit cAMP generation and may activate lymphocytes. This could explain the antiinflammatory effect of clopidogrel. Clopidogrel is a P2Y 12  antagonist used in the clinic as a platelet aggregation inhibitor that reduces thrombotic cardiovascular events such as myocardial infarctions. However, it has also been shown to reduce CRP, even though aspirin in antiplatelet doses lacks this effect [ 18 ]. This effect may be mediated via P2Y 12  receptors in lymphocytes.
Expression of P2Y receptors in lymphocytes
In lymphocytes, all the target genes P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , and P2Y 13  could be detected (n = 6). To illustrate expression of the P2 receptors relative to each other the P2Y 1  receptor was used as calibrator for the others, i. e. the other receptors were expressed as a ratio of the P2Y 1 . Among the P2Y receptor subtypes the P2Y 12  and P2Y 2  had highest expression (Figure  1A ). The lowest expressed P2Y receptor was P2Y 4 .Extracellular nucleotides and their P2 receptors are involved in the regulation, proliferation but also apoptosis and cell death in lymphocytes and monocytes [ 3 , 16 ]. Previous studies have shown that ATP, ADP, UTP and UDP stimulate phospholipase C and Ca 2+  release from intracellular stores, that fits well with the highly expressed P2Y 2  receptor, together with the lesser expressed P2Y 1  and P2Y 6  receptors. ATP and ADP, but not UTP, can also increase cAMP [ 17 ]. This is in agreement with the P2Y 11  receptor that had the third highest mRNA expression.The most interesting finding was that P2Y 12  had the highest expression among the P2Y receptors in lymphocytes. It is not likely that this is the result of platelet contamination, because platelets contain very low amounts of RNA. To the best of our knowledge, there are no studies that have examined the effects of P2Y 12  on lymphocytes, even though selective antagonists exist. It is expected to inhibit cAMP generation and may activate lymphocytes. This could explain the antiinflammatory effect of clopidogrel. Clopidogrel is a P2Y 12  antagonist used in the clinic as a platelet aggregation inhibitor that reduces thrombotic cardiovascular events such as myocardial infarctions. However, it has also been shown to reduce CRP, even though aspirin in antiplatelet doses lacks this effect [ 18 ]. This effect may be mediated via P2Y 12  receptors in lymphocytes.Expression of P2X receptors in lymphocytes The most abundant P2X receptor in lymphocytes was the P2X 4  receptor. As showed in Figure  1B , the expression of P2X 4  was 3.2 times higher than P2Y 1 . The expression of P2X 4  was significantly higher than the expression of the other P2X receptors; P2X 1  (P < 0.001) and P2X 7 (P < 0.01). Selective pharmacological tools to discriminate between P2X receptors are scarce. Nevertheless, several studies have suggested the importance of P2X 7  in lymphocyte regulation. However, B lymphocytes stimulated with ATP do not undergo the typical increase in permeability up to 900 Da that is typical for the P2X 7  receptor. On the other hand, P2X 7  mediated effects on Ba 2+  and ethidium influx, phospholipase D activity and shedding of L-selectin have been blocked by the P2X 7  selective antagonist KN-62 in human lymphocytes [ 19 ]. Thus it is a surprising finding that the P2X 4  receptor was the highest expressed subtype in lymphocytes at the mRNA level. Even though we have demonstrated that more than 90% of the preparation consists of lymphocytes (see methods), it is possible that a small contamination of monocytes may have influenced the results, at least regarding P2X 4  receptor mRNA expression, because of its high expression levels in monocytes. P2X 4  receptors have indeed been demonstrated at the protein level in human B lymphocytes by confocal immunohistochemistry, in which P2X 1 , P2X 4  and P2X 7  were detected at the protein level [ 20 ]. However, the P2X 4  receptor staining was the most variable of the P2X receptors with weak to moderate levels of staining in a large proportion of cells in three patients and weak levels in only a minority of the cells from the other three patients examined [ 20 ].
Expression of P2X receptors in lymphocytes
The most abundant P2X receptor in lymphocytes was the P2X 4  receptor. As showed in Figure  1B , the expression of P2X 4  was 3.2 times higher than P2Y 1 . The expression of P2X 4  was significantly higher than the expression of the other P2X receptors; P2X 1  (P < 0.001) and P2X 7 (P < 0.01).Selective pharmacological tools to discriminate between P2X receptors are scarce. Nevertheless, several studies have suggested the importance of P2X 7  in lymphocyte regulation. However, B lymphocytes stimulated with ATP do not undergo the typical increase in permeability up to 900 Da that is typical for the P2X 7  receptor. On the other hand, P2X 7  mediated effects on Ba 2+  and ethidium influx, phospholipase D activity and shedding of L-selectin have been blocked by the P2X 7  selective antagonist KN-62 in human lymphocytes [ 19 ]. Thus it is a surprising finding that the P2X 4  receptor was the highest expressed subtype in lymphocytes at the mRNA level. Even though we have demonstrated that more than 90% of the preparation consists of lymphocytes (see methods), it is possible that a small contamination of monocytes may have influenced the results, at least regarding P2X 4  receptor mRNA expression, because of its high expression levels in monocytes. P2X 4  receptors have indeed been demonstrated at the protein level in human B lymphocytes by confocal immunohistochemistry, in which P2X 1 , P2X 4  and P2X 7  were detected at the protein level [ 20 ]. However, the P2X 4  receptor staining was the most variable of the P2X receptors with weak to moderate levels of staining in a large proportion of cells in three patients and weak levels in only a minority of the cells from the other three patients examined [ 20 ].Expression of P2Y receptors in monocytes Again, the P2Y 1  expression was used as calibrator for the others, i. e. the other receptors were expressed as a ratio of the P2Y 1 . Among the P2Y receptors, the P2Y 2 , P2Y 13  and P2Y 11  had highest expression (Figure  2A , n = 6). The presence of P2Y receptor mRNA in monocytes and lymphocytes is in agreement with previous studies using regular RT-PCR [ 21 ]. Figure 2 Relative P2 gene expression in monocytes.  A, Bar graph shows relative P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12  and P2Y 13  receptor gene expression normalized to GAPDH. B, Bar graph shows relative P2X 1 , P2X 4  and P2X 7  receptor gene expression normalized to GAPDH. P2Y 1  was chosen to be calibrator. Extracellular nucleotides stimulate interleukin secretion, iNOS-generation in monocytes, are involved in differentiation, cytotoxicity and killing of pathogens. All monocyte/macrophage cell lines express P2Y receptors coupled to IP 3  generation and intracellular Ca 2+  release, but the individual subtypes have not been investigated in detail in monocytes [ 2 , 3 ]. However, both ATP and UTP are active agonists, which is in agreement with the highest mRNA expression of the ATP/UTP receptor P2Y 2  (Fig  2 ). ATP mediated increase in cAMP has suggested the presence of P2Y 11 , with a suggested role in maturation of human monocyte-dendritic cells [ 22 ]. A relatively high expression of P2Y 11  was confirmed in our experiments. Interestingly, the P2Y 13  had even higher mRNA levels. To our knowledge, no experiments have addressed the presence of this cAMP inhibitory ADP receptor in monocytes. In fact, it has been an unresolved issue in what tissue this receptor is expressed. High levels in the spleen could be in agreement with monocyte expression [ 23 ]. Thus, the presence of P2Y 2  and P2Y 11  receptors are confirmed as expected, with the interesting addition of P2Y 13  receptors. Future experiments addressing the physiological role of P2Y 13  receptors in monocytes are needed.
Expression of P2Y receptors in monocytes
Again, the P2Y 1  expression was used as calibrator for the others, i. e. the other receptors were expressed as a ratio of the P2Y 1 . Among the P2Y receptors, the P2Y 2 , P2Y 13  and P2Y 11  had highest expression (Figure  2A , n = 6). The presence of P2Y receptor mRNA in monocytes and lymphocytes is in agreement with previous studies using regular RT-PCR [ 21 ].Extracellular nucleotides stimulate interleukin secretion, iNOS-generation in monocytes, are involved in differentiation, cytotoxicity and killing of pathogens. All monocyte/macrophage cell lines express P2Y receptors coupled to IP 3  generation and intracellular Ca 2+  release, but the individual subtypes have not been investigated in detail in monocytes [ 2 , 3 ]. However, both ATP and UTP are active agonists, which is in agreement with the highest mRNA expression of the ATP/UTP receptor P2Y 2  (Fig  2 ). ATP mediated increase in cAMP has suggested the presence of P2Y 11 , with a suggested role in maturation of human monocyte-dendritic cells [ 22 ]. A relatively high expression of P2Y 11  was confirmed in our experiments. Interestingly, the P2Y 13  had even higher mRNA levels. To our knowledge, no experiments have addressed the presence of this cAMP inhibitory ADP receptor in monocytes. In fact, it has been an unresolved issue in what tissue this receptor is expressed. High levels in the spleen could be in agreement with monocyte expression [ 23 ]. Thus, the presence of P2Y 2  and P2Y 11  receptors are confirmed as expected, with the interesting addition of P2Y 13  receptors. Future experiments addressing the physiological role of P2Y 13  receptors in monocytes are needed.Expression of P2X receptors in monocytes Early studies demonstrated that ATP activates a receptor on macrophages that increase cell permeability eventually leading to cell death [ 2 , 3 ]. P2X 7  receptor transfection confers susceptibility to ATP-dependent permeabilization and ATP-resistant clones lack the P2X 7  receptor, demonstrating that it is present on macrophages and necessary for permabilization. However, it is not known whether P2X 7  is the only constitutive subunit or if it assembles with other subunits. As showed in Figure  2B , P2X 4  was by far the highest expressed P2 receptor in monocytes and the P2X 1  (P < 0.01) and P2X 7  (P < 0.01) had lower levels. Thus, unexpectedly the P2X 7  receptor was not the highest expressed P2X receptor in monocytes. This is in agreement with patch-clamp experiments suggesting that other P2X receptors are involved [ 24 ]. Interrelation of these experiments has suggested the contribution of P2X 4  receptors, which is supported by our findings [ 25 ]. It should be noted that all the three P2X receptors were expressed at very high levels compared to other cell types (30-fold more than the calibrator gene for P2X 4  and 6–7-fold more for P2X 1  and P2X 7 ). A physiological role for all three subtypes can therefore be expected.
Expression of P2X receptors in monocytes
Early studies demonstrated that ATP activates a receptor on macrophages that increase cell permeability eventually leading to cell death [ 2 , 3 ]. P2X 7  receptor transfection confers susceptibility to ATP-dependent permeabilization and ATP-resistant clones lack the P2X 7  receptor, demonstrating that it is present on macrophages and necessary for permabilization. However, it is not known whether P2X 7  is the only constitutive subunit or if it assembles with other subunits.As showed in Figure  2B , P2X 4  was by far the highest expressed P2 receptor in monocytes and the P2X 1  (P < 0.01) and P2X 7  (P < 0.01) had lower levels. Thus, unexpectedly the P2X 7  receptor was not the highest expressed P2X receptor in monocytes. This is in agreement with patch-clamp experiments suggesting that other P2X receptors are involved [ 24 ]. Interrelation of these experiments has suggested the contribution of P2X 4  receptors, which is supported by our findings [ 25 ]. It should be noted that all the three P2X receptors were expressed at very high levels compared to other cell types (30-fold more than the calibrator gene for P2X 4  and 6–7-fold more for P2X 1  and P2X 7 ). A physiological role for all three subtypes can therefore be expected.Expression of P2 receptors in CD34 +  stem and progenitor cells CD34 +  stem and progenitor cells are receiving an increasing attention because of their extensive self-renewal and multilineage differentiation ability making them attractive for cellular therapy [ 26 ]. Knowledge of their P2 receptor expression could be used for directing differentiation or for further subtype selection of early progenitors types. There are no previous pharmacological or expression studies of P2 receptors on human CD34 +  stem and progenitor cells. We found expression of several P2Y receptors, especially P2Y 1  and P2Y 2  (Figure  3A , n = 3). This indicates that both ATP and UTP are agonists for CD34 +  stem and progenitor cells and may stimulate IP 3  and intracellular Ca 2+  release. Figure 3 Relative P2 gene expression in CD34 +  stem and progenitor cells.  A, Bar graph shows relative P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12  and P2Y 13  receptor gene expression normalized to GAPDH. B, Bar graph shows relative P2X 1 , P2X 4  and P2X 7  receptor gene expression normalized to GAPDH. P2Y 1  was chosen to be calibrator. Among the P2X receptors the P2X 1  receptor had the highest expression followed by P2X 4  (P2X 1  had significantly higher expression than P2X 7 , P < 0.05) (Figure  3B , n = 3), suggesting a potential role of these receptors in regulation of stem and progenitor cells. P2Y 1 , P2Y 2  and P2X 1  receptors have all been shown to stimulate proliferation, but also to be able to mediate apoptosis [ 26 ]. Such roles could be of major importance in the highly proliferative CD34 +  stem and progenitor cells. Antagonists or agonists of these receptors could be used to control their differentiation or proliferation.
Expression of P2 receptors in CD34 +  stem and progenitor cells
CD34 +  stem and progenitor cells are receiving an increasing attention because of their extensive self-renewal and multilineage differentiation ability making them attractive for cellular therapy [ 26 ]. Knowledge of their P2 receptor expression could be used for directing differentiation or for further subtype selection of early progenitors types. There are no previous pharmacological or expression studies of P2 receptors on human CD34 +  stem and progenitor cells. We found expression of several P2Y receptors, especially P2Y 1  and P2Y 2  (Figure  3A , n = 3). This indicates that both ATP and UTP are agonists for CD34 +  stem and progenitor cells and may stimulate IP 3  and intracellular Ca 2+  release.Among the P2X receptors the P2X 1  receptor had the highest expression followed by P2X 4  (P2X 1  had significantly higher expression than P2X 7 , P < 0.05) (Figure  3B , n = 3), suggesting a potential role of these receptors in regulation of stem and progenitor cells. P2Y 1 , P2Y 2  and P2X 1  receptors have all been shown to stimulate proliferation, but also to be able to mediate apoptosis [ 26 ]. Such roles could be of major importance in the highly proliferative CD34 +  stem and progenitor cells. Antagonists or agonists of these receptors could be used to control their differentiation or proliferation.Conclusions The P2X 4  receptor had the highest mRNA expression in lymphocytes and monocytes. Among the P2Y receptors, P2Y 12  and P2Y 2  had highest expression in lymphocytes, while the P2Y 2  and P2Y 13  had highest expression in monocytes. The most interesting findings were the high mRNA expression of P2Y 12  receptors in lymphocytes potentially explaining the anti-inflammatory effects of clopidogrel, P2Y 13  receptors in monocytes and a previously unrecognised expression of P2X 4  in lymphocytes and monocytes. In addition, for the first time P2 receptor mRNA expression patterns have been studied in CD34 +  stem and progenitor cells. Several P2 receptors were expressed (P2Y 2 , P2Y 1 , P2Y 12 , P2Y 13 , P2Y 11 , P2X 1 , P2X 4 ), indicating a role in differentiation and proliferation. Thus, it is possible that specific antibodies to P2 receptors could be used to identify progenitors for monocytes, lymphocytes and megakaryocytes.
Conclusions
The P2X 4  receptor had the highest mRNA expression in lymphocytes and monocytes. Among the P2Y receptors, P2Y 12  and P2Y 2  had highest expression in lymphocytes, while the P2Y 2  and P2Y 13  had highest expression in monocytes. The most interesting findings were the high mRNA expression of P2Y 12  receptors in lymphocytes potentially explaining the anti-inflammatory effects of clopidogrel, P2Y 13  receptors in monocytes and a previously unrecognised expression of P2X 4  in lymphocytes and monocytes. In addition, for the first time P2 receptor mRNA expression patterns have been studied in CD34 +  stem and progenitor cells. Several P2 receptors were expressed (P2Y 2 , P2Y 1 , P2Y 12 , P2Y 13 , P2Y 11 , P2X 1 , P2X 4 ), indicating a role in differentiation and proliferation. Thus, it is possible that specific antibodies to P2 receptors could be used to identify progenitors for monocytes, lymphocytes and megakaryocytes.Methods The studies were approved by the local Ethics Committee of the Lund University and were conducted according to the principles of the Declaration of Helsinki. Preparation of monocytes and lymphocytes Peripheral blood was drawn from each of 6 healthy volunteers (after informed consent) into heparin vials. The mononuclear cells were isolated by density gradient centrifugation on Lymphoprep™ (Axis Shield Poc AS, Oslo, Norway) at 605 g for 30 minutes. The lymphocytes and monocytes thus obtained were washed three times in RPMI 1640 medium with L-glutamine (Gibco/BRL, Life Technologies Ltd, Paisleys, Scotland) and 0.1% human serum albumin (Sigma, St Louise, MO, USA), (medium), and centrifuged each time at 605 g for 5 minutes. The fraction of lymphocytes and monocytes obtained according to this procedure was resuspended in medium with 15% normal human serum (NHS) added to a concentration of 4 × 10 6  cells/ml. Flow cytometry (Epics XL-MCL Beckman-Coulter, Florida, USA) analysis on these cells by detection of cell surface CD14 and CD45 showed that approximately 10% of the cells were monocytes. 800 μl of this cell-suspension was plated on a chamber slide 4 well glass slide (Nalge Nunc International, IL, USA) at 37°C in an atmosphere containing 5% CO 2  and 96% humidity for 1 h in order for the monocytes to adhere. Nonadherent cells were removed by washing three times with medium. Flow cytometry analysis of these nonadherent cells showed that at least 90% were lymphocytes, and were therefore used as source of lymphocytes. The cells attached to the glass slides (<90% monocytes as assessed by flow cytometry) were detached by adding first PBS and then 0.5 mM EDTA-PBS for 3 min in room temperature. Preparation of CD34 +  stem and progenitor cells Bone marrow samples were obtained from healthy volunteers (n = 3), after informed consent, using guidelines approved by the Ethical Committee, Lund University. Mononuclear cells were isolated by density gradient centrifugation (Ficoll-Paque; Pharmacia, Uppsala, Sweden). CD34 +  cells were isolated by 2passages through magnetic columns (MidiMacs;Miltenyi Biotec, Bergish Gladbach, Germany) by using a hapten-conjugated CD34 antibody (Qbend/10) and an antihapten antibody conjugated to magnetic beads (CD34 +  isolation kit; Miltenyi Biotec). CD34 expression was analyzed by immunostaining with a FACSCalibur flow cytometer (Becton Dickinson) by using the CellQuest program (Becton Dickinson) and the purity of isolated populations was reproducibly > 95% [ 27 ]. RNA extraction Total cellular RNAs were extracted using TRIzol reagent (Gibco BRL, Life Technology) according to the supplier's instructions, dissolved in diethyl-pyrocarbonate (DEPC) treated water and stored at -70°C until used. Quantitative analysis of P2 receptors by real-time reverse transcription polymerase chain reaction TaqMan Reverse Transcription Reagents Kit was used to transcribe mRNA into cDNA. Real-time PCR were performed by means of a PRISM 7700Sequence Detector as described previously [ 8 , 15 , 28 , 29 ]. Oligonucleotide primers and TaqMan probes were designed using the Primer Express software, based on sequences from the GenBank database [ 8 , 15 ]. Constitutively expressed GAPDH were selected as endogenous control to correct for potential variation in RNA loading or efficiency of the amplification reaction. Previous analysis showed that amplification efficiencies were almost identical for GADPH and the following receptor mRNAs: P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , P2X 1 , P2X 4 , and P2X 7  normalized to GAPDH [ 8 , 15 ]. To confirm equal amplification efficiencies, we used the criterion of a regression slope of less than 0.1 for each gene normalized to GAPDH. This confirms that we could use the comparative C T  method for the relative quantification of target without running standard curves on the same plate (Perkin-Elmer Applied Biosystems Inc; User Bulletin No. 2, December 1997). The amount of target and endogenous reference was determined from the comparative C T  method. The target gene normalized to GAPDH was expressed as ΔC T  (C T  of target gene minus C T  of GAPDH). P2Y 1  was arbitrarily chosen to be the calibrator in the comparative analysis and is expressed as ΔC TP2Y1  (C T  of target minus C T  of GAPDH for P2Y 1 ). The normalized calibrated value is given by the equation 2 -ΔΔCt , where ΔΔC T  is ΔC T  -ΔC TP2Y1 . To further verify the specificity of PCR assays, the PCR was performed with non-reverse-transcribed total cellular RNA and samples lacking the DNA template. No significant amplifications were obtained in any of these samples (data not shown). Drugs Unless otherwise stated, all reagents and drugs were purchased from Sigma Chemical Corp, St. Louis, MI, USA. PCR consumables were obtained from Perkin-Elmer Applied Biosystems Inc, Foster City, CA, USA. Statistical methods Data are expressed as mean and standard error of the mean (SEM) unless otherwise stated. n indicates the number of subjects that were tested. Statistical analysis of the normalized C T  values (ΔC T ) was performed with a one-way ANOVA, followed by a multiple comparison post test (Tukey's test) using GraphPad InStat version 3.00 (GraphPad Software Inc., USA). Significant differences were considered at P < 0.05 (two-tailed test).
Methods
The studies were approved by the local Ethics Committee of the Lund University and were conducted according to the principles of the Declaration of Helsinki.Preparation of monocytes and lymphocytes Peripheral blood was drawn from each of 6 healthy volunteers (after informed consent) into heparin vials. The mononuclear cells were isolated by density gradient centrifugation on Lymphoprep™ (Axis Shield Poc AS, Oslo, Norway) at 605 g for 30 minutes. The lymphocytes and monocytes thus obtained were washed three times in RPMI 1640 medium with L-glutamine (Gibco/BRL, Life Technologies Ltd, Paisleys, Scotland) and 0.1% human serum albumin (Sigma, St Louise, MO, USA), (medium), and centrifuged each time at 605 g for 5 minutes. The fraction of lymphocytes and monocytes obtained according to this procedure was resuspended in medium with 15% normal human serum (NHS) added to a concentration of 4 × 10 6  cells/ml. Flow cytometry (Epics XL-MCL Beckman-Coulter, Florida, USA) analysis on these cells by detection of cell surface CD14 and CD45 showed that approximately 10% of the cells were monocytes. 800 μl of this cell-suspension was plated on a chamber slide 4 well glass slide (Nalge Nunc International, IL, USA) at 37°C in an atmosphere containing 5% CO 2  and 96% humidity for 1 h in order for the monocytes to adhere. Nonadherent cells were removed by washing three times with medium. Flow cytometry analysis of these nonadherent cells showed that at least 90% were lymphocytes, and were therefore used as source of lymphocytes. The cells attached to the glass slides (<90% monocytes as assessed by flow cytometry) were detached by adding first PBS and then 0.5 mM EDTA-PBS for 3 min in room temperature.
Preparation of monocytes and lymphocytes
Peripheral blood was drawn from each of 6 healthy volunteers (after informed consent) into heparin vials. The mononuclear cells were isolated by density gradient centrifugation on Lymphoprep™ (Axis Shield Poc AS, Oslo, Norway) at 605 g for 30 minutes. The lymphocytes and monocytes thus obtained were washed three times in RPMI 1640 medium with L-glutamine (Gibco/BRL, Life Technologies Ltd, Paisleys, Scotland) and 0.1% human serum albumin (Sigma, St Louise, MO, USA), (medium), and centrifuged each time at 605 g for 5 minutes.The fraction of lymphocytes and monocytes obtained according to this procedure was resuspended in medium with 15% normal human serum (NHS) added to a concentration of 4 × 10 6  cells/ml. Flow cytometry (Epics XL-MCL Beckman-Coulter, Florida, USA) analysis on these cells by detection of cell surface CD14 and CD45 showed that approximately 10% of the cells were monocytes. 800 μl of this cell-suspension was plated on a chamber slide 4 well glass slide (Nalge Nunc International, IL, USA) at 37°C in an atmosphere containing 5% CO 2  and 96% humidity for 1 h in order for the monocytes to adhere. Nonadherent cells were removed by washing three times with medium. Flow cytometry analysis of these nonadherent cells showed that at least 90% were lymphocytes, and were therefore used as source of lymphocytes. The cells attached to the glass slides (<90% monocytes as assessed by flow cytometry) were detached by adding first PBS and then 0.5 mM EDTA-PBS for 3 min in room temperature.Preparation of CD34 +  stem and progenitor cells Bone marrow samples were obtained from healthy volunteers (n = 3), after informed consent, using guidelines approved by the Ethical Committee, Lund University. Mononuclear cells were isolated by density gradient centrifugation (Ficoll-Paque; Pharmacia, Uppsala, Sweden). CD34 +  cells were isolated by 2passages through magnetic columns (MidiMacs;Miltenyi Biotec, Bergish Gladbach, Germany) by using a hapten-conjugated CD34 antibody (Qbend/10) and an antihapten antibody conjugated to magnetic beads (CD34 +  isolation kit; Miltenyi Biotec). CD34 expression was analyzed by immunostaining with a FACSCalibur flow cytometer (Becton Dickinson) by using the CellQuest program (Becton Dickinson) and the purity of isolated populations was reproducibly > 95% [ 27 ].
Preparation of CD34 +  stem and progenitor cells
Bone marrow samples were obtained from healthy volunteers (n = 3), after informed consent, using guidelines approved by the Ethical Committee, Lund University. Mononuclear cells were isolated by density gradient centrifugation (Ficoll-Paque; Pharmacia, Uppsala, Sweden). CD34 +  cells were isolated by 2passages through magnetic columns (MidiMacs;Miltenyi Biotec, Bergish Gladbach, Germany) by using a hapten-conjugated CD34 antibody (Qbend/10) and an antihapten antibody conjugated to magnetic beads (CD34 +  isolation kit; Miltenyi Biotec). CD34 expression was analyzed by immunostaining with a FACSCalibur flow cytometer (Becton Dickinson) by using the CellQuest program (Becton Dickinson) and the purity of isolated populations was reproducibly > 95% [ 27 ].RNA extraction Total cellular RNAs were extracted using TRIzol reagent (Gibco BRL, Life Technology) according to the supplier's instructions, dissolved in diethyl-pyrocarbonate (DEPC) treated water and stored at -70°C until used.
RNA extraction
Total cellular RNAs were extracted using TRIzol reagent (Gibco BRL, Life Technology) according to the supplier's instructions, dissolved in diethyl-pyrocarbonate (DEPC) treated water and stored at -70°C until used.Quantitative analysis of P2 receptors by real-time reverse transcription polymerase chain reaction TaqMan Reverse Transcription Reagents Kit was used to transcribe mRNA into cDNA. Real-time PCR were performed by means of a PRISM 7700Sequence Detector as described previously [ 8 , 15 , 28 , 29 ]. Oligonucleotide primers and TaqMan probes were designed using the Primer Express software, based on sequences from the GenBank database [ 8 , 15 ]. Constitutively expressed GAPDH were selected as endogenous control to correct for potential variation in RNA loading or efficiency of the amplification reaction. Previous analysis showed that amplification efficiencies were almost identical for GADPH and the following receptor mRNAs: P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , P2X 1 , P2X 4 , and P2X 7  normalized to GAPDH [ 8 , 15 ]. To confirm equal amplification efficiencies, we used the criterion of a regression slope of less than 0.1 for each gene normalized to GAPDH. This confirms that we could use the comparative C T  method for the relative quantification of target without running standard curves on the same plate (Perkin-Elmer Applied Biosystems Inc; User Bulletin No. 2, December 1997). The amount of target and endogenous reference was determined from the comparative C T  method. The target gene normalized to GAPDH was expressed as ΔC T  (C T  of target gene minus C T  of GAPDH). P2Y 1  was arbitrarily chosen to be the calibrator in the comparative analysis and is expressed as ΔC TP2Y1  (C T  of target minus C T  of GAPDH for P2Y 1 ). The normalized calibrated value is given by the equation 2 -ΔΔCt , where ΔΔC T  is ΔC T  -ΔC TP2Y1 . To further verify the specificity of PCR assays, the PCR was performed with non-reverse-transcribed total cellular RNA and samples lacking the DNA template. No significant amplifications were obtained in any of these samples (data not shown).
Quantitative analysis of P2 receptors by real-time reverse transcription polymerase chain reaction
TaqMan Reverse Transcription Reagents Kit was used to transcribe mRNA into cDNA. Real-time PCR were performed by means of a PRISM 7700Sequence Detector as described previously [ 8 , 15 , 28 , 29 ]. Oligonucleotide primers and TaqMan probes were designed using the Primer Express software, based on sequences from the GenBank database [ 8 , 15 ]. Constitutively expressed GAPDH were selected as endogenous control to correct for potential variation in RNA loading or efficiency of the amplification reaction.Previous analysis showed that amplification efficiencies were almost identical for GADPH and the following receptor mRNAs: P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , P2X 1 , P2X 4 , and P2X 7  normalized to GAPDH [ 8 , 15 ]. To confirm equal amplification efficiencies, we used the criterion of a regression slope of less than 0.1 for each gene normalized to GAPDH. This confirms that we could use the comparative C T  method for the relative quantification of target without running standard curves on the same plate (Perkin-Elmer Applied Biosystems Inc; User Bulletin No. 2, December 1997). The amount of target and endogenous reference was determined from the comparative C T  method. The target gene normalized to GAPDH was expressed as ΔC T  (C T  of target gene minus C T  of GAPDH). P2Y 1  was arbitrarily chosen to be the calibrator in the comparative analysis and is expressed as ΔC TP2Y1  (C T  of target minus C T  of GAPDH for P2Y 1 ). The normalized calibrated value is given by the equation 2 -ΔΔCt , where ΔΔC T  is ΔC T  -ΔC TP2Y1 . To further verify the specificity of PCR assays, the PCR was performed with non-reverse-transcribed total cellular RNA and samples lacking the DNA template. No significant amplifications were obtained in any of these samples (data not shown).Drugs Unless otherwise stated, all reagents and drugs were purchased from Sigma Chemical Corp, St. Louis, MI, USA. PCR consumables were obtained from Perkin-Elmer Applied Biosystems Inc, Foster City, CA, USA.
Drugs
Unless otherwise stated, all reagents and drugs were purchased from Sigma Chemical Corp, St. Louis, MI, USA. PCR consumables were obtained from Perkin-Elmer Applied Biosystems Inc, Foster City, CA, USA.Statistical methods Data are expressed as mean and standard error of the mean (SEM) unless otherwise stated. n indicates the number of subjects that were tested. Statistical analysis of the normalized C T  values (ΔC T ) was performed with a one-way ANOVA, followed by a multiple comparison post test (Tukey's test) using GraphPad InStat version 3.00 (GraphPad Software Inc., USA). Significant differences were considered at P < 0.05 (two-tailed test).
Statistical methods
Data are expressed as mean and standard error of the mean (SEM) unless otherwise stated. n indicates the number of subjects that were tested. Statistical analysis of the normalized C T  values (ΔC T ) was performed with a one-way ANOVA, followed by a multiple comparison post test (Tukey's test) using GraphPad InStat version 3.00 (GraphPad Software Inc., USA). Significant differences were considered at P < 0.05 (two-tailed test).Authors' contributions LW designed the study, carried out the RNA isolation and real-time PCR, and wrote the manuscript. SEWJ supervised the isolation of CD34 +  stem and progenitor cells, and participated in writing the manuscript. AB supervised the isolation of monocytes and lymphocytes, and participated in writing the manuscript. DE conceived the study, guided throughout the study, and wrote the manuscript. All authors read and approved the final manuscript.
Authors' contributions
LW designed the study, carried out the RNA isolation and real-time PCR, and wrote the manuscript.SEWJ supervised the isolation of CD34 +  stem and progenitor cells, and participated in writing the manuscript.AB supervised the isolation of monocytes and lymphocytes, and participated in writing the manuscript.DE conceived the study, guided throughout the study, and wrote the manuscript.All authors read and approved the final manuscript.


RNA interference (RNAi) is used in the lab to silence virtually any gene. Previously, antisense and ribozymes were successful in the lab, but have been disappointing in the clinic. Will RNAi succeed where these two have not?

RNA interference (RNAi) has been called “one of the most has exciting discoveries in biology in the last couple decades,” and since it was first recognized by Andrew Fire et al. in 1998, it has quickly become one of the most powerful and indispensable tools in the molecular biologist's toolkit. Using short double-stranded RNA (dsRNA) molecules, RNAi can selectively silence essentially any gene in the genome. It is an ancient mechanism of gene regulation, found in eukaryotes as diverse as yeast and mammals, and probably plays a central role in controlling gene expression in all eukaryotes. In the lab, RNAi is routinely used to reveal the genetic secrets of development, intracellular signaling, cancer, infection, and a full range of other phenomena. But can the phenomenon hailed by the journal  Science  as the “Breakthrough of the Year” in 2002 break out of the lab and lead to novel therapies as well? Pharmaceutical giants are hoping so, and several biotech companies have bet their futures on it, but not everyone is sanguine about the future of RNAi therapy.ScienceAt the heart of its promise as a powerful therapeutic drug lies the exquisite selectivity of RNAi: like the fabled “magic bullet,” an RNAi sequence seeks out and destroys its target without affecting other genes. The clinical applications appear endless: any gene whose expression contributes to disease is a potential target, from viral genes to oncogenes to genes responsible for heart disease, Alzheimer's disease, diabetes, and more.But for all its promise, RNAi therapy is a long way from entering the clinic. While it is a proven wunderkind in the lab, to date no tests have been done in humans, and only the most modest and circumscribed successes have been demonstrated in animals. The road to clinical success is littered with great ideas that have come a cropper along the way, including two other RNA-based therapies, antisense and ribozymes, both of which showed promise at the bench but have largely stumbled before reaching the bedside. Is RNAi also likely to fall short? Or is it different enough to make this third try the charm?Clinical Naïveté, Mysterious Mechanisms To be a successful drug, a molecule must overcome a long set of hurdles. First, it must be able to be manufactured at reasonable cost and administered safely and conveniently. Then, and even more importantly, it must be stable enough to reach its target cells before it is degraded or excreted; it must get into those cells, link up with its intracellular target, and exert its effect; and it must exert enough of an effect to improve the health of the person taking it. And, finally, it must do all this without causing significant toxic effects in either target or nontarget tissues. No matter how good a compound looks in the lab, if it fails to clear any one of these hurdles, it is useless as a drug. For RNA-based therapies, manufacture has been seen as a soluble problem, while delivery, stability, and potency have been the most significant obstacles. “There was a lot of clinical naïveté” in the early days of antisense and ribozymes, according to Nassim Usman, Vice President for Research and Development at Sirna Therapeutics in Boulder, Colorado. “Compounds were pushed into the clinic prematurely.” Sirna began as the biotech startup Ribozyme Pharmaceuticals, which tried to develop ribozymes to treat several conditions, including hepatitis C. A ribozyme is an RNA molecule whose sequence and structure allow it to cleave specific target RNA molecules (see  Figure 1 ). “The initial results with hepatitis C were not that inspiring,” says Usman, because the molecule they used had low potency and a short half-life once in the body. Despite “enormous doses,” the viral load was not significantly affected. “It just didn't have the characteristics to be a drug,” he says. No ribozyme has yet been approved for use by the United States Food and Drug Administration (FDA). Figure 1 Ribozymes A ribozyme binds to a specific mRNA, cleaves it, and thus prevents it from functioning. Similarly, despite much initial enthusiasm, attempts to develop antisense drugs have been largely disappointing. Antisense is a single strand of RNA or DNA, complementary to a target messenger RNA (mRNA) sequence; by pairing up with it, the antisense strand prevents translation of the mRNA (see  Figure 2 ). At least that was the theory, and early clinical results seemed to support the theory: antisense drugs effectively reduced tumor sizes in anticancer trials and viral loads in antiviral trials. But closer inspection revealed these results were largely due to an increase in production of interferons by the immune system in response to high doses of the foreign RNA, rather than to specific silencing of any target genes. (The relatively high proportion of C–G sequences in antisense mimics bacterial and viral genes, thus triggering the immune response.) When the antisense dose was lowered to prevent the interferon response, the clinical benefit largely disappeared as well. Thus, rather than being a highly specific therapy, antisense seemed to be a general immune system booster. Figure 2 Antisense Antisense DNA or RNA binds to a specific mRNA and prevents it from being translated into protein. But as long as patients were getting better, does it matter what the mechanism was? “It doesn't matter if you are a patient, but it does matter if you are trying to develop the next drug,” says Cy Stein, Associate Professor of Medicine and Pharmacology at Columbia University College of Physicians and Surgeons in New York City. Stein has researched antisense for more than a decade. “If you get the mechanism wrong, you're not going to be able to do it.” To date, only one antisense drug has received FDA approval. Vitravene, from Isis Pharmaceuticals in Carlsbad, California, is used to treat cytomegalovirus infections in the eye for patients with HIV. Vitravene is actually a DNA antisense drug, which binds to viral DNA, though, says Usman, “it's unclear whether it actually works by an antisense mechanism.” Stein expresses a similar skepticism about the mechanism of a second antisense drug, Genasense. Genasense is a DNA-based treatment that targets Bcl-2, a protein expressed in high levels in cancer cells, which is thought to protect them from standard chemotherapy. The FDA is currently reviewing an application for Genasense, based on promising results in the treatment of malignant melanoma.
Clinical Naïveté, Mysterious Mechanisms
To be a successful drug, a molecule must overcome a long set of hurdles. First, it must be able to be manufactured at reasonable cost and administered safely and conveniently. Then, and even more importantly, it must be stable enough to reach its target cells before it is degraded or excreted; it must get into those cells, link up with its intracellular target, and exert its effect; and it must exert enough of an effect to improve the health of the person taking it. And, finally, it must do all this without causing significant toxic effects in either target or nontarget tissues. No matter how good a compound looks in the lab, if it fails to clear any one of these hurdles, it is useless as a drug.For RNA-based therapies, manufacture has been seen as a soluble problem, while delivery, stability, and potency have been the most significant obstacles. “There was a lot of clinical naïveté” in the early days of antisense and ribozymes, according to Nassim Usman, Vice President for Research and Development at Sirna Therapeutics in Boulder, Colorado. “Compounds were pushed into the clinic prematurely.” Sirna began as the biotech startup Ribozyme Pharmaceuticals, which tried to develop ribozymes to treat several conditions, including hepatitis C. A ribozyme is an RNA molecule whose sequence and structure allow it to cleave specific target RNA molecules (see  Figure 1 ). “The initial results with hepatitis C were not that inspiring,” says Usman, because the molecule they used had low potency and a short half-life once in the body. Despite “enormous doses,” the viral load was not significantly affected. “It just didn't have the characteristics to be a drug,” he says. No ribozyme has yet been approved for use by the United States Food and Drug Administration (FDA).Similarly, despite much initial enthusiasm, attempts to develop antisense drugs have been largely disappointing. Antisense is a single strand of RNA or DNA, complementary to a target messenger RNA (mRNA) sequence; by pairing up with it, the antisense strand prevents translation of the mRNA (see  Figure 2 ). At least that was the theory, and early clinical results seemed to support the theory: antisense drugs effectively reduced tumor sizes in anticancer trials and viral loads in antiviral trials. But closer inspection revealed these results were largely due to an increase in production of interferons by the immune system in response to high doses of the foreign RNA, rather than to specific silencing of any target genes. (The relatively high proportion of C–G sequences in antisense mimics bacterial and viral genes, thus triggering the immune response.) When the antisense dose was lowered to prevent the interferon response, the clinical benefit largely disappeared as well. Thus, rather than being a highly specific therapy, antisense seemed to be a general immune system booster.But as long as patients were getting better, does it matter what the mechanism was? “It doesn't matter if you are a patient, but it does matter if you are trying to develop the next drug,” says Cy Stein, Associate Professor of Medicine and Pharmacology at Columbia University College of Physicians and Surgeons in New York City. Stein has researched antisense for more than a decade. “If you get the mechanism wrong, you're not going to be able to do it.”To date, only one antisense drug has received FDA approval. Vitravene, from Isis Pharmaceuticals in Carlsbad, California, is used to treat cytomegalovirus infections in the eye for patients with HIV. Vitravene is actually a DNA antisense drug, which binds to viral DNA, though, says Usman, “it's unclear whether it actually works by an antisense mechanism.” Stein expresses a similar skepticism about the mechanism of a second antisense drug, Genasense. Genasense is a DNA-based treatment that targets Bcl-2, a protein expressed in high levels in cancer cells, which is thought to protect them from standard chemotherapy. The FDA is currently reviewing an application for Genasense, based on promising results in the treatment of malignant melanoma.RNAi: A Natural Alternative Growing disillusionment with antisense and ribozymes coincided with the discovery of RNAi and the realization that it was a far more potent way to silence gene expression. RNAi uses short dsRNA molecules whose sequence matches that of the gene of interest. Once in a cell, a dsRNA molecule is cleaved into segments approximately 22 nucleotides long, called short interfering RNAs (siRNAs) (see  Figure 3 ). siRNAs become bound to the RNA-induced silencing complex (RISC), which then also binds any matching mRNA sequence. Once this occurs, the mRNA is degraded, effectively silencing the gene from which it came. (Details of the structure and function of the RISC are still largely unknown, but it is thought to act as a true enzyme complex, requiring only one or several siRNA molecules to degrade many times that number of matching mRNAs.) Figure 3 RNAi RNAi is a recently described naturally occurring process in which small RNA molecules activate a cellular process that results in the destruction of a specific mRNA. The extraordinary selectivity of RNAi, combined with its potency—in theory, only a few dsRNAs are needed per cell—quickly made it the tool of choice for functional genomics (determining what a gene product does and with what other products it interacts) and for drug target discovery and validation. By “knocking down” a gene with RNAi and determining how a cell responds, a researcher can, in the course of only a few days, develop significant insight into the function of the gene and determine whether reducing its expression is likely to be therapeutically useful. But does RNAi have a better chance to succeed as a drug than antisense or ribozymes? “The fundamental difference favoring RNAi is that we're harnessing an endogenous, natural pathway,” says Nagesh Mahanthappa, Director of Corporate Development at Alnylam Pharmaceuticals in Cambridge, Massachusetts, the second of two major biotech company developing RNAi-based therapy. The exploitation of a pre-existing mechanism, he says, is the main reason RNAi is orders of magnitude more potent than either of the other two types of RNA strategies.
RNAi: A Natural Alternative
Growing disillusionment with antisense and ribozymes coincided with the discovery of RNAi and the realization that it was a far more potent way to silence gene expression. RNAi uses short dsRNA molecules whose sequence matches that of the gene of interest. Once in a cell, a dsRNA molecule is cleaved into segments approximately 22 nucleotides long, called short interfering RNAs (siRNAs) (see  Figure 3 ). siRNAs become bound to the RNA-induced silencing complex (RISC), which then also binds any matching mRNA sequence. Once this occurs, the mRNA is degraded, effectively silencing the gene from which it came. (Details of the structure and function of the RISC are still largely unknown, but it is thought to act as a true enzyme complex, requiring only one or several siRNA molecules to degrade many times that number of matching mRNAs.)The extraordinary selectivity of RNAi, combined with its potency—in theory, only a few dsRNAs are needed per cell—quickly made it the tool of choice for functional genomics (determining what a gene product does and with what other products it interacts) and for drug target discovery and validation. By “knocking down” a gene with RNAi and determining how a cell responds, a researcher can, in the course of only a few days, develop significant insight into the function of the gene and determine whether reducing its expression is likely to be therapeutically useful. But does RNAi have a better chance to succeed as a drug than antisense or ribozymes?“The fundamental difference favoring RNAi is that we're harnessing an endogenous, natural pathway,” says Nagesh Mahanthappa, Director of Corporate Development at Alnylam Pharmaceuticals in Cambridge, Massachusetts, the second of two major biotech company developing RNAi-based therapy. The exploitation of a pre-existing mechanism, he says, is the main reason RNAi is orders of magnitude more potent than either of the other two types of RNA strategies.Delivery, Delivery, Delivery More potent in the test tube, at least. But stability and delivery are also the major obstacles to successful RNAi therapy, obstacles that are intrinsic to the biochemical nature of RNA itself, as well as the body's defenses against infection with foreign nucleotides. “For the strongest reasons, you can't get away from this,” says Stein. “The problem is that a charged oligonucleotide will not pass through a lipid layer,” which it must do in order to enter a cell. John Rossi, Director of the Department of Molecular Biology at City of Hope Hospital in Duarte, California, who has worked on RNA-based therapies for 15 years, concurs. “The cell doesn't want to take up RNA,” he says, which makes evolutionary sense, since extracellular RNA usually signifies a viral infection. Injected into the bloodstream, unmodified RNA is rapidly excreted by the kidneys or degraded by enzymes. To solve the problem of cell penetration, most researchers have either complexed the RNA with a lipid or modified the RNA's phosphate backbone to minimize its charge. Mahanthappa thinks the complexing approach is unlikely to be a simple solution, since drug approval would require independent testing of the lipid delivery system as well. Instead, Alnylam is pursuing backbone modification. “Some minimal modification is going to be necessary” to increase cell uptake and to improve stability in the blood stream, Mahanthappa says. “What we have learned from the antisense field is that even without other delivery strategies, when you administer RNA at sufficient doses, if it's stable, it gets taken up by cells.” “Anything that can be done to increase half-life in circulation would improve delivery,” says Judy Lieberman, a Senior Investigator at the Center for Blood Research and Associate Professor of Pediatrics at Harvard Medical School in Cambridge, Massachusetts. But that may not be the only problem, she cautions. Lieberman's lab recently demonstrated the ability of RNAi to silence expression of the Fas gene in mice, protecting them from fulminant hepatitis. Fas triggers apoptosis, or programmed cell death, in response to a variety of cell insults. In her experiment, Lieberman delivered the RNA by high-pressure injection into the tail. The RNA got to the liver, silenced Fas, and protected the mice from hepatitis. However, a significant fraction of animals died of heart failure, brought on because the injection volume was about 20% of the mouse blood volume. Such a delivery scheme simply will not work in humans. “Delivery to the cell is still an obstacle,” Lieberman explains. “Unless you really focus on how to solve that problem, you're not going to get very far.”
Delivery, Delivery, Delivery
More potent in the test tube, at least. But stability and delivery are also the major obstacles to successful RNAi therapy, obstacles that are intrinsic to the biochemical nature of RNA itself, as well as the body's defenses against infection with foreign nucleotides. “For the strongest reasons, you can't get away from this,” says Stein. “The problem is that a charged oligonucleotide will not pass through a lipid layer,” which it must do in order to enter a cell. John Rossi, Director of the Department of Molecular Biology at City of Hope Hospital in Duarte, California, who has worked on RNA-based therapies for 15 years, concurs. “The cell doesn't want to take up RNA,” he says, which makes evolutionary sense, since extracellular RNA usually signifies a viral infection. Injected into the bloodstream, unmodified RNA is rapidly excreted by the kidneys or degraded by enzymes.To solve the problem of cell penetration, most researchers have either complexed the RNA with a lipid or modified the RNA's phosphate backbone to minimize its charge. Mahanthappa thinks the complexing approach is unlikely to be a simple solution, since drug approval would require independent testing of the lipid delivery system as well. Instead, Alnylam is pursuing backbone modification. “Some minimal modification is going to be necessary” to increase cell uptake and to improve stability in the blood stream, Mahanthappa says. “What we have learned from the antisense field is that even without other delivery strategies, when you administer RNA at sufficient doses, if it's stable, it gets taken up by cells.”“Anything that can be done to increase half-life in circulation would improve delivery,” says Judy Lieberman, a Senior Investigator at the Center for Blood Research and Associate Professor of Pediatrics at Harvard Medical School in Cambridge, Massachusetts. But that may not be the only problem, she cautions. Lieberman's lab recently demonstrated the ability of RNAi to silence expression of the Fas gene in mice, protecting them from fulminant hepatitis. Fas triggers apoptosis, or programmed cell death, in response to a variety of cell insults. In her experiment, Lieberman delivered the RNA by high-pressure injection into the tail. The RNA got to the liver, silenced Fas, and protected the mice from hepatitis. However, a significant fraction of animals died of heart failure, brought on because the injection volume was about 20% of the mouse blood volume. Such a delivery scheme simply will not work in humans. “Delivery to the cell is still an obstacle,” Lieberman explains. “Unless you really focus on how to solve that problem, you're not going to get very far.”Unanswered Questions Even assuming delivery problems can be solved, other questions remain, including that of whether therapeutic levels of RNAi may be toxic. Mahanthappa says, “The conservative answer is we just don't know. The more aggressive answer is there's no reason to think so.” Rossi isn't so sure. “The target of interest may be in normal cells as well as cancer cells,” he says. “That's where you get toxicity.” But if small RNAs can be delivered to target cells efficiently and without significant toxicity, will they be effective medicines? Usman of Sirna is confident they will be. “If you can get it there, and if it's in one piece, there no doubt in our minds that it will work,” he says. To date, numerous experiments in animal models suggest RNAi can downregulate a variety of target genes effectively. However, there are still two unanswered questions about whether that will translate into effective therapy. The first is whether RNAi's exquisite specificity is really an advantage beyond the bench. “It's unclear whether highly specific drugs give you a big therapeutic effect,” says Cy Stein. For instance, he says, “most active antitumor medicines have multiple mechanisms of action. The more specific you make it, the less robust the therapeutic activity is likely to be.” Rossi agrees: “Overspecificity has never worked,” he says. The second question is what effect an excess of RNA from outside the cell will have on the normal function of the RISC, the complex at the heart of the RNAi mechanism. The number of RISCs in the cell is unknown, and one concern is that the amount of RNA needed to have a therapeutic effect may occupy all the available complexes. “We are usurping a natural cell system that is there for some other purpose, for knocking out endogenous gene function,” says Rossi. With the introduction of foreign RNA, will the system continue to perform its normal function as well, or will it become saturated? “That's the big black box in the field,” he says.
Unanswered Questions
Even assuming delivery problems can be solved, other questions remain, including that of whether therapeutic levels of RNAi may be toxic. Mahanthappa says, “The conservative answer is we just don't know. The more aggressive answer is there's no reason to think so.” Rossi isn't so sure. “The target of interest may be in normal cells as well as cancer cells,” he says. “That's where you get toxicity.”But if small RNAs can be delivered to target cells efficiently and without significant toxicity, will they be effective medicines? Usman of Sirna is confident they will be. “If you can get it there, and if it's in one piece, there no doubt in our minds that it will work,” he says. To date, numerous experiments in animal models suggest RNAi can downregulate a variety of target genes effectively. However, there are still two unanswered questions about whether that will translate into effective therapy.The first is whether RNAi's exquisite specificity is really an advantage beyond the bench. “It's unclear whether highly specific drugs give you a big therapeutic effect,” says Cy Stein. For instance, he says, “most active antitumor medicines have multiple mechanisms of action. The more specific you make it, the less robust the therapeutic activity is likely to be.” Rossi agrees: “Overspecificity has never worked,” he says.The second question is what effect an excess of RNA from outside the cell will have on the normal function of the RISC, the complex at the heart of the RNAi mechanism. The number of RISCs in the cell is unknown, and one concern is that the amount of RNA needed to have a therapeutic effect may occupy all the available complexes. “We are usurping a natural cell system that is there for some other purpose, for knocking out endogenous gene function,” says Rossi. With the introduction of foreign RNA, will the system continue to perform its normal function as well, or will it become saturated? “That's the big black box in the field,” he says.Looking Ahead to the Clinic Despite the questions and unsolved problems, Sirna, Alnylam, and several other companies are moving ahead to develop RNAi therapy; indeed, some outstanding questions are probably only likely to be answered in the process of therapeutic development. The first applications are likely to be in cancer (targeting out-of-control oncogenes) or viral infection (targeting viral genes). To avoid some of the problems of delivery, initial trials may deliver the RNA by direct injection into the target tissue (for a tumor, for instance) or ex vivo, treating white blood cells infected with HIV, for example. Having spent a decade trying to develop ribozymes, says Usman, Sirna is prepared for the rough road it faces. “We haven't solved all the problems, but we know how to proceed to work through them. It's no surprise to us—we've seen this movie before.” Usman expects Sirna to file an Investigational New Drug Application with the FDA by the end of 2004 and to have a human clinical trial in progress in 2005. “Without a doubt, there will be an RNAi-based drug within ten years,” Usman predicts. Stein isn't so sure and thinks that too much is still to be learned about RNAi and the body's reaction to it to be confident that RNA-based therapies will ultimately be successful. “The whole field was founded on the belief it was rational, but there are huge gaps in our knowledge, and so you need a bit of luck to be successful,” he says. “I think people are surprised at how complicated it is, but why should it be any other way? It's an intellectual conceit to think that nature is simple.”
Looking Ahead to the Clinic
Despite the questions and unsolved problems, Sirna, Alnylam, and several other companies are moving ahead to develop RNAi therapy; indeed, some outstanding questions are probably only likely to be answered in the process of therapeutic development. The first applications are likely to be in cancer (targeting out-of-control oncogenes) or viral infection (targeting viral genes). To avoid some of the problems of delivery, initial trials may deliver the RNA by direct injection into the target tissue (for a tumor, for instance) or ex vivo, treating white blood cells infected with HIV, for example.Having spent a decade trying to develop ribozymes, says Usman, Sirna is prepared for the rough road it faces. “We haven't solved all the problems, but we know how to proceed to work through them. It's no surprise to us—we've seen this movie before.” Usman expects Sirna to file an Investigational New Drug Application with the FDA by the end of 2004 and to have a human clinical trial in progress in 2005. “Without a doubt, there will be an RNAi-based drug within ten years,” Usman predicts.Stein isn't so sure and thinks that too much is still to be learned about RNAi and the body's reaction to it to be confident that RNA-based therapies will ultimately be successful. “The whole field was founded on the belief it was rational, but there are huge gaps in our knowledge, and so you need a bit of luck to be successful,” he says. “I think people are surprised at how complicated it is, but why should it be any other way? It's an intellectual conceit to think that nature is simple.”