Between Saying and Doing – Brandom extends and refines his views on the nature of language and philosophy by developing a position that he calls Analytic Pragmatism. Although Brandom’s project bears on an extraordinarily rich array of different philosophical issues, we focus here on the contention that certain vocabularies have a privileged status within our linguistic practices, and that when adequately understood, the practices in which these vocabularies figure can help furnish us with an account of semantic intentionality. Brandom’s claim is that such vocabularies are privileged because they are a species of what he calls universal LX vocabulary –roughly, vocabulary whose mastery is implicit in any linguistic practice whatsoever. We show that, contrary to Brandom’s claim, logical vocabulary per se fails to satisfy the conditions that must be met for something to count as universal LX vocabulary. Further, we show that exactly analogous considerations undermine his claim that modal vocabulary is universal LX. If our arguments are sound, then, contrary to what Brandom maintains, intentionality cannot be explicated as a “pragmatically mediated semantic phenomenon”, at any rate not of the sort that he proposes.In his recent John Locke Lectures – published as  Between Saying and Doing – Brandom further extends and refines his views on these matters by seeking to develop and defend a position that he calls Analytic Pragmatism. As we will see, this position combines two commitments that are exceedingly widely endorsed amongst analytic philosophers; and a careful discussion of the resulting view is of importance if for no other reason than, to date, Brandom’s position constitutes the most detailed and systematic endeavor to develop such a view.Over the past three decades or so, Robert Brandom has exerted an enormous influence on debates regarding the nature of language and philosophy. In his recent John Locke Lectures – published as universal LX vocabulary –roughly, vocabulary whose mastery is implicit in any linguistic practice whatsoever. On such a view, then, logical and modal vocabularies are privileged because they possess a kind of transcendental status. Further, according to Brandom, this transcendental status, at least in the case of modal vocabulary, is important to characterizing the sort of intentionality essential to thought and language.Although Brandom’s project bears on an extraordinarily rich array of different philosophical issues, we focus here on a single central strand that runs throughout, and without which the project as a whole fails. In brief, the strand in question concerns the contention that certain vocabularies have a privileged status within our linguistic practices, and that when adequately understood, the practices in which these vocabularies figure can help furnish us with an account of semantic intentionality. More specifically, we focus on Brandom’s discussion of two vocabularies that have long figured prominently in analytic philosophy: logical vocabulary – especially the conditional – and modal vocabulary, such as that used to express necessity and possibility.modal Kant-Sellars thesis – the thesis that the use of ordinary, empirical vocabulary presupposes those capacities required for the introduction and deployment of modal vocabulary. Finally, we argue briefly that given our criticism of the modal Kant-Sellars thesis, Brandom’s conception of intentionality, which he articulates in the final pages of his Locke Lectures, is also untenable. If our arguments are sound, then, contrary to what Brandom maintains, intentionality cannot be explicated as a “pragmatically mediated semantic phenomenon”,This paper has three main aims. First, we show that logical vocabulary per se fails to satisfy the conditions that must be met for something to count as universal LX vocabulary. We focus in particular on the case of the conditional, which Brandom takes to be a paradigmatic example of logical vocabulary. If our argument goes though, then the conditional is not universal LX, and Brandom’s efforts to defend the privileged status of logic per se fail. Second, we show that exactly analogous considerations undermine the claim that modal vocabulary is universal LX. If this is so, then another of Brandom’s central claims is undermined – what he calls the Here is how we proceed. In Section Analytic Pragmatism – can be thought of as a consequence of endorsing two commitments. The first is a methodological commitment, which he takes to be central to traditional analytic philosophy:The Analytic Project: A central goal of philosophy ought to be to establish semantic relations between vocabularies – kinds of linguistic expressions – in order to “make sense of the meanings expressed by one kind of locution in terms of the meanings expressed by another kind of locution”.Brandom’s overall project – what he calls Brandom’s second central commitment is a general thesis about the metaphysics of meaning:Semantic Pragmatism: The meaning of a linguistic expression is determined by its use in a linguistic practice.The history of analytic philosophy is, of course, littered with attempts to produce such analyses —including, the reduction of arithmetic to logic, the translation of psychological vocabulary into physical vocabulary, and the analysis of moral vocabulary in non-moral terms. By endorsing this commitment, then, Brandom locates his position as continuous with the project of traditional analytic philosophy.challenge to it. The challenge takes a variety of forms, but the rough idea is this: If our practices determine the meanings of linguistic expressions, then it is highly implausible to suppose that expressions comprising different vocabularies – and, hence, having different meaning-determining practices – will tend to bear systematic semantic relations to each other, such as translation, reduction and paraphrase. Thus by endorsing both the Analytic Project and Semantic Pragmatism, Brandom seeks to combine a pair of commitments that seem to be in tension with each other. As a consequence, a central challenge for Brandom – and one that exerts an enormous influence on the overall shape of his Analytic Pragmatism – is to reconcile this apparent tension: to show that, contrary to appearances, one can pursue the goal of philosophical analysis at the same time as cleaving to Semantic Pragmatism.This sort of meaning pragmatism has also been prominent in the history of analytic philosophy. Though we suspect that many proponents of the analytic project have also endorsed Semantic Pragmatism, far from being a natural complement to the Analytic Project, such pragmatism poses, as Brandom notes, a serious and to pursue the Analytic Project is to take the analyzing vocabulary to describe those practices that are sufficient to deploy the target vocabulary. If one could make good on this suggestion, then semantic pragmatists could have their analytic cake, and eat it too.How is this to be done? Brandom’s solution is to propose that Semantic Pragmatism requires that we rethink the fundamental semantic relations between the analyzed and analyzing vocabularies in philosophical analyses. Specifically, he proposes that if linguistic meaning is determined by use, then the relationship between distinct vocabularies should be mediated by linguistic practices.So far we have sketched the overarching shape of Brandom’s project. But why does the notion of universal LX vocabulary come to play such a central role?There are two quite different roles that the notion of universal LX vocabulary plays in Brandom’s project. The first, exemplified by its application to logic, is to justify parts of the analyzing base that everyone must presuppose in order to pursue the Analytic Project. The second, exemplified by Brandom’s discussion of modality, is to justify or vindicate the use of philosophically contested notions. In both sorts of case, however, Brandom maintains that the response should be the same. Roughly, where some vocabulary – contested or otherwise – is universal LX, we get it for free since the capacity to master that vocabulary is a necessary condition for the possibility of any language-use whatsoever. Let us consider the cases of logical vocabulary and modality in a bit more detail.any project of analysis – classical or pragmatist – logical vocabulary has a privileged status. Roughly put, no matter what the analysandum, it is taken for granted that logical vocabulary must be part of the analyzing base. If, for example, one seeks to translate psychological vocabulary into physical vocabulary, it is not merely that one uses the substantive terms of physics; one also uses – and indeed must use – such logical resources as conjunction, disjunction, quantifiers, and of course the conditional. But if this is so, then as Brandom notes, some justification or explanation is required for why logical vocabulary should have this privileged status. The notion of universal LX vocabulary is central to Brandom’s effort to address this challenge. Logical vocabulary is supposed to be privileged because it is a species of universal LX vocabulary, and universal LX vocabulary is supposed to be privileged.One way to make vivid the potential significance of the notion of universal LX vocabulary for analytic philosophy is to note, as Brandom does, that within At least since Locke, modal vocabulary has been a point of contention within philosophy, especially amongst those of an empiricist bent.any vocabulary whatsoever, andThe vocabulary allows one to describe practices that are necessary for having mastery for Once one masters the described practice, one can in principle deploy the very vocabulary used to describe it.We now need to say more about Brandom’s notion of universal LX vocabulary. Here is a first pass. Universal LX vocabulary is supposed to be a privileged class of linguistic expressions in that they satisfy the following two conditions:whatsoever, including universal LX vocabulary itself.Crudely put, Universal LX vocabulary describes the necessary conditions for mastery of any linguistic expression vocabulary. To a first approximation: A vocabulary is autonomous if and only if one could use it even though one could use no other vocabulary whatsoever. Suppose, for example, that it was possible to master the language of arithmetic and nothing else. Then the language of arithmetic would be an autonomous vocabulary in the relevant sense. The second notion of autonomy – an autonomous practice – can be defined in terms of the previous notion. To a first approximation: an autonomous practice is any practice that is sufficient for mastery of an autonomous vocabulary.Even this rough formulation is a bit of a mouthful. But to understand clearly Brandom’s notion of universal LX vocabulary we need to explain several additional notions. First, we need to explain two notions of autonomy. The first is the notion of an autonomous universal. But we are getting ahead of ourselves. Before explaining what makes an LX vocabulary universal, we first need to explain what makes a vocabulary LX, and this in turn requires that we characterize a range of what Brandom calls meaning-use relations. Given the project of Analytic Pragmatism – to specify relations between vocabularies that are mediated by practices – Brandom appeals to a number of such relations:PV-Sufficiency: Where one has mastery of any vocabulary – autonomous or otherwise – Brandom supposes that there must be some set of practices that suffices for mastery of that vocabulary. He calls this relation PV-sufficiency. Roughly: a practice is PV-sufficient for a vocabulary just in case one has mastery of the vocabulary, if one engages in that practice.PV-Necessity: Now suppose that for some vocabulary there is a practice that one must engage in to count as having mastery of the vocabulary. Suppose, for example, that the capacity to identify the successor of any natural number is required for mastery of the language of arithmetic. Then this practice – identifying successors – would be PV-necessary for mastery of arithmetic vocabulary.VP-Sufficiency: This relation between vocabularies and practices obtains when a practice can be specified in a particular vocabulary. That is, a vocabulary is VP-sufficient for a practice just in case that practice can be described using that vocabulary.PP-Sufficiency: The fourth relation that we consider here obtains between practices. If, when one engages in a particular practice, one can in principle do everything needed to engage in some other practice, then the former is PP-sufficient for the latter. For example, perhaps being able to add any three numbers between 0 and 9 is PP-sufficient for being able to find the sum of any two numbers by the usual method of addition. In characterizing PP-sufficiency, Brandom tends to express a preference for using the framework of computational theory, but there is no reason to think that this is essential to the notion.PP-Necessity: The final relation we consider is also a relation between practices. One practice is PP-necessary for another if it is impossible to engage in the second without engaging in the first. For example, the practice of asserting is PP-necessary for the practice of asserting conditionals.As we will soon see, the paired notions of an autonomous vocabulary and an autonomous practice are important because they are required in order to specify what is it for a LX vocabulary to be 1 is LX for a vocabulary V2 iff there exists practices P1 and P2, such that:2 is PV-necessary for V2,P2 is PP-sufficient for P1,P1 is PV-sufficient for V1,P1 is VP-sufficient for P2.VWith this terminology in hand, we can now provide a more explicit formulation of LX vocabulary. A vocabulary V1 specifies a practice P2 that one must be able to master in order to be a competent user of another vocabulary V2 and, moreover, the practice P2 incorporates everything that is required, in principle, in order to engage in a practice that the use of V1 depends upon –i.e., P1. This set of conditions can be captured, perhaps more intuitively, in what Brandom calls a meaning-use diagram 2 is PP-necessary for any autonomous practice – i.e. a practice that is PV-sufficient for an autonomous vocabulary.PFinally, we are in position to characterize the notion of do everything one needs to be able to do in order in principle to be able to say what logical vocabulary lets one say.”Thus if a vocabulary satisfies the conditions -(e)) for being universal LX, then not only are the practices it describes necessary for being a language user of any sort, but the practices so described also suffice for mastery of the universal LX vocabulary itself. As Brandom puts it for the case of logic, “anyone who can talk at all, hence can deploy any base vocabulary, can already 2 is any arbitrary autonomous vocabulary, and V1 is the conditional. Moreover, let us follow Brandom as characterizing P1 as the practice of using the conditional, and P2 as the practice of inferring one claim from another. In which case, again following Brandom, there are five conditions—represented by the meaning-use diagram in Fig. It is time to consider how the notion of universal LX vocabulary is supposed to apply to Brandom’s paradigmatic example of a logical expression: the conditional. In order for the conditional to be universal LX, it must be LX for any autonomous vocabulary whatsoever. So, applying the definition from Section 2.2, let us assume that VCondition 1. According to the first condition: P2, the practice of inferring one claim from another, is PV-necessary for the autonomous vocabulary, V2. In other words, one must be able to infer one claim from another in order to count as using any vocabulary whatsoever. This is a central tenet of Brandom’s inferentialism on which the capacity to draw inferences is a metaphysically necessary condition on being a concept-user – hence language-user – of any sort.inferential practices are PP-necessary components of every autonomous discursive practice, hence PV-necessary for the deployment of every autonomous vocabulary, hence PV-necessary for the deployment of every vocabulary whatsoever.Assertions are essentially, and not just accidentally, speech acts that can play the role both of premises and as conclusions of inferences … According to this way of thinking, Though there are those who might wish to take issue with this claim, in what follows we grant this assumption, at least for the sake of argument.Condition 2. The second condition concerns a relationship between practices. According to Brandom: P2, the practice of inferring, is PP-sufficient for P1, the practice of using the conditional. In other words, if a person can infer one claim from another, then she can, at least in principle, do everything that she needs to be able to do to count as having mastery of the conditional. Again, Brandom’s inferentialism, and the connection it assumes between asserting and inferring, is central here. According to this view, if we begin with an agent that can assert and infer, then:if p then q.”…The system must respond to its assertion of the conditional “if p then q” by treating the inference from p to q as a good one—for instance, by being disposed to endorse q assertionally if it is disposed to endorse p assertionally… In a clear sense, then, the capacity to distinguish good from bad inferences involving non-logical sentences is (PP-) sufficient for the ability to deploy conditionals involving those sentences.…we can teach it also to produce-assertively tokenings of the new form “Condition 3. According to the third condition that needs to be satisfied for ‘If__, then__’ to be universal LX: P1, the practice of using conditionals, is PV-sufficient for mastery of V1, conditional vocabulary. This is true given the definitions of P1 and V1.Condition 4. The fourth condition on ‘If__, then__’ being universal LX is that the vocabulary of conditionals permits us to specify adequately the practice of inferring. In other words: V1, the vocabulary of conditionals, is VP-sufficient for P2, the practice of inferring. This condition is captured by the diagonal arrow in Fig. explicit something that otherwise was implicit in the practical sorting of non-logical inferences into good and bad.”Condition 5: If a vocabulary meets conditions 1–4, then it is LX. The final condition, which renders it universal LX, is that the practice of inferring is PP-necessary for any autonomous practice. In other words, every language-user must be proficient at inferring. Again, this is controversial; but we grant it here, at least for the sake of argument.To summarize: Brandom maintains that conditionals are universal LX because they satisfy all of the above conditions. The burden of the next section is to show that he is mistaken.Our complaint against Brandom’s treatment of the conditional is threefold. First, we maintain that there are two quite different readings of VP-sufficiency and that there are serious problems with the claim that conditionals are, in the sense relevant to the Analytic Project, VP-sufficient for the practice of inferring. Second, even if the first objection is waived, we maintain that PP-sufficiency  fails. Third, even if one supplements the account so that Condition 2 holds, Condition 4 fails; and moreover it fails no matter which of the two readings of VP-sufficiency one adopts. Thus, we maintain that even if conditionals are VP-sufficient for the practice of inferring, it still follows that conditionals are not Universal LX.The first problem arises from the fact that Brandom’s discussion incorporates two rather different construals of VP-sufficiency: what we call the descriptive and expressive readings. The problem for Brandom is that, on the one hand, only the descriptive reading will do the work required by Analytic Pragmatism, whilst on other the other hand, conditionals can, at most, provide the basis for the expressive form of VP-sufficiency. Let us explain this in more detail.analyses —i.e., to specify, in one vocabulary, the practices that determine the content of another vocabulary. This is precisely the role that VP-sufficiency is supposed to play within the broader project. But talk of “specifying” appears, given the context, to involve describing a set of practices. Indeed, his paradigm cases of VP-sufficiency confirm this impression. For example, Brandom glosses what he calls the Kaplan-Stalnaker semantics for indexicals by saying the following:can serve as an adequate pragmatic metavocabulary for indexical vocabulary. That is, one can say , in wholly non-indexical terms, everything one needs to do in order to use indexical vocabulary. Non-indexical vocabulary is VP-sufficient to specify practices-or-abilities PV-sufficient to deploy indexical vocabulary.[It provides a] purely non-indexical vocabulary [that] As characterized by Brandom, the objective of Analytic Pragmatism is to provide pragmatic descriptive construal, involves the description of linguistic practices.In short: VP-sufficiency, on this expressive construal of VP-sufficiency. On this view, VP-sufficiency need not involve the description of practices. Instead it consists in one vocabulary making explicit what is only implicit in a linguistic practice. Very roughly, one makes explicit a linguistic practice or ability when one introduces an expression into the language that allows us to formulate claims about – and rationally assess – performances of the practice.Negation, as a logical connective supporting formally valid inferences, plays the same explicitating role with respect to material incompatability relations among judgeable  contents that the conditional plays with respect to the material inferential relations among such contents.Let us turn to the second, On this expressive construal of VP-sufficiency, then, no description need occur.describe the practice of inference than the negation sign can describe the practice of treating-as-incompatible. Instead, what it more plausibly does, is make some element(s) or aspect(s) of the practice explicit. And indeed, this is what Brandom ends up emphasizing when he argues for the claim that conditionals are universal LX.The problem is that a vocabulary that only makes explicit some feature of a practice need not describe that practice. Yet precisely what pragmatic analyses seem to require is a description of the practices. One way to see the point is to consider the pragmatic “analysis” that would result from merely making explicit the practice of treating two claims as incompatible. If all that were required for VP-sufficiency was that this be made explicit, then the “analysis” would consist in one word: not! By any lights, such a result would not be an analysis worthy of the name. But it should be clear that conditionals can no more Let us waive the previous objection for the sake of argument. Still, there is another – and in our view more serious – problem for the claim that conditionals are VP-sufficient for the practice of inferring. Here the problem concerns how to understand the bottom right-hand interior box in Fig. asserting and inferring are internally related practices, in the sense that each is PP-necessary for the other.”and asserting as PP-sufficient for the practice of using conditionals. But now the problem crops up elsewhere. Even if we grant that the conjoint practice of inferring-and-asserting is PP-sufficient for the practice of using conditionals and that this practice is, in turn, PV-sufficient for the mastery of the conditional, it is simply false that the conditional locution is VP-sufficient for the practice of inferring-and-asserting. Moreover, this is so irrespective of whether one adopts a descriptive or an expressive conception of the VP-sufficiency relation.Now, it should be clear what is going on here. Brandom is assuming that the practices of asserting and inferring go together. As he puts it: “the beginning of wisdom” in understanding the nature of assertion “is to see that would make asserting explicit. According to one familiar proposal, for example, the Fregean assertion stroke (├) can be used to express assertoric force. But on the face of it, the conditional, by itself, does not obviate the need for the assertion sign, any more than, say, disjunction does. When one asserts a conditional one does not thereby assert either the antecedent or the consequent. Moreover, in expressing conditionals, one does not thereby resolve the force of the utterance since conditionals can, like most other expressions, figure in assertions, suppositions, queries, commands, and so on. Thus, however we construe VP-sufficiency (whether descriptively or expressively) conditionals are not VP-sufficient for the practice of inferring-and-asserting.Consider the options. On the descriptive reading, the ‘if__then__’ locution would need to describe the practice of inferring-and-asserting, which it clearly does not: any more than ‘not’ describes the practice of treating two claims as incompatible. On the alternative expressive reading, the ‘if__then__’ locution would need to make explicit the practice of inferring-and-asserting. But even if we grant that the conditional makes the practice of inferring explicit, it surely does not make inferring-and-asserting explicit. To see this, we need only reflect upon the vocabulary that In sum: The practice of inferring, by itself, is not PP-sufficient for the practice of using conditionals. Perhaps the practice of inferring-and-asserting is PP-sufficient for the practice of using conditionals. But even if this is true, conditionals are not VP-sufficient for the practice of inferring-and-asserting. Thus, either Condition 2 fails or Condition 4 fails. Either way, conditionals turn out not to be LX, much less universal LX.Between Saying and Doing, and since the conditional is the only piece of logical vocabulary that he considers at any length, we take this to pose a serious problem for his project. We now propose to challenge another central thesis of the book – what Brandom calls the modal Kant-Sellars thesis —roughly, the claim that modal vocabulary  is universal LX. More specifically, we maintain that it is subject to exactly analogous problems to those set out earlier.In the preceding sections we argued at some length that Brandom’s primary example of logical vocabulary – the conditional – is not universal LX. Since the claim that logical vocabulary is universal LX is amongst the central claims of Between Saying and Doing is to argue for the thesis, which he calls the modal Kant-Sellars thesis, on which modal vocabulary has a privileged status akin to logical vocabulary.As noted in Section 2.1, a central goal of Brandom’s project in modal vocabulary.In using ordinary empirical vocabulary, one already knows how to do everything one needs to know how to do in order to introduce and deploy make explicit semantic, conceptual connections and commitments that are already implicit in the use of ordinary empirical vocabulary.The expressive role characteristic of alethic modal vocabulary is to Via an extensive discussion that focuses primarily on Sellars – but takes in Ryle, Kripke, and Quine along the way – Brandom fashions a characterization of the modal Kant-Sellars thesis that conforms to the pragmatic conception of meaning so central to Brandom’s overarching project. In particular, the version of the modal Kant-Sellars thesis that he endorses is formulated as the conjunction of two claims:any empirical vocabulary whatsoever. Thus, according to Brandom’s modal Kant-Sellars thesis, modal vocabulary is universal LX. This is represented diagrammatically in Fig. As Brandom observes, this formulation of the modal Kant-Sellars thesis is amenable, with minimal modification, to the sort of meaning-use analysis that we have already encountered. Claim 1 maintains that some set of practices that are PV-necessary for the use of any empirical vocabulary are PP-sufficient for practices that are PV-sufficient to deploy modal vocabulary. Claim 2 maintains that modal vocabulary makes explicit – is VP-sufficient for – those aspects of practices that are implicit in – i.e., PV-necessary for – the use of some set of practices – corresponding to the bottom-right interior box – that satisfies three conditions:It is PV-necessary for using any empirical vocabulary;It is PP-sufficient for those practices PV-sufficient for deploying modal vocabulary; andModal vocabulary is VP-sufficient for these practices.There is a crucial component of Brandom’s discussion of the modal Kant-Sellars thesis that we have yet to touch upon. From inspection of Fig. what these practices are. This is a gap that Brandom fills in the course of arguing for the modal Kant-Sellars thesis.Yet as formulated no commitment is made regarding In brief, his argumentative strategy is to justify the thesis by showing that there is a practice that satisfies the above conditions. What might it be? His answer is that it is the capacity for what he calls “counterfactually robust inference”: the capacity to associate a range of “counterfactual robustness” with each materially good inference.Although we do not deny the general thesis that modal notions are in some way implicit in ordinary descriptive, linguistic practices, we do reject the claim that modal vocabulary is universal LX, and with it Brandom’s specific formulation of the modal Kant-Sellars thesis. Moreover, our line of criticism is exactly analogous to the objection we leveled against the claim that the conditional is universal LX in Section 4.2.Modal in Fig. In order to have mastery of modal locutions, one must also be able to engage in the practice of asserting —i.e. Pand asserting as PP-sufficient for the practice of using modal locutions. But just as before, the problem now crops up elsewhere. Even if we grant that the conjoint practice of CRI-and-asserting is PP-sufficient for the practice of using conditionals, and that this practice is, in turn, PV-sufficient for the mastery of modals, it is simply false that modal locutions are VP-sufficient for the practice of CRI-and-asserting. Moreover, this is so irrespective of whether one adopts a descriptive or an expressive conception of the VP-sufficiency relation.Now, as with the case of the conditional, one might respond to this problem by maintaining that the capacity for asserting is PP-necessary for the capacity for CRI and vice versa. In which case, to capture the meaning-use relations for modals, we need to augment the lower interior right-hand box in the diagram by having the conjunction of CRI Consider the options. On the descriptive reading, modal locutions would need to describe the practice of CRI-and-asserting, which they clearly do not: any more than ‘not’ describes the practice of treating two claims as incompatible. On the alternative expressive reading, modal locutions would need to make explicit the practice of CRI-and-asserting. But even if we grant that modals make the practice of CRI explicit, they surely do not make CRI-and-asserting explicit. To see this, we need only reflect again upon the vocabulary that would make asserting explicit. According to one familiar proposal, for example, the Fregean assertion stroke can be used to express assertoric force. But on the face of it, modal vocabulary, by itself does not obviate the need for the assertion sign, any more than, say, the conditional does. Thus, whichever way we construe VP-sufficiency – whether it be descriptively or expressively – modals are not VP-sufficient for the practice of CRI-and-asserting.In sum: The practice of CRI, by itself, is not PP-sufficient for the practice of using modals. Perhaps the practice of CRI-and-asserting is PP-sufficient for the practice of using modals. But even if this is true, modals are not VP-sufficient for the practice of CRI-and-asserting. Thus, either Condition 2 fails or Condition 4 fails. Either way modals turn out not to be LX, much less universal LX.Between Saying and Doing might wonder whether any of this applies to what Brandom calls the normative Kant-Sellars thesis: the claim that in order to apply ordinary, empirical, descriptive vocabulary – and hence to deploy any autonomous vocabulary whatsoever – one must already be able to do everything needed to introduce normative vocabulary.in fact happens to characterizations of what must happen, and what could not happen. How, he wants to know, can we rationally justify the move from descriptions of matter-of-factual regularities to formulations of necessary laws? On the side of practical reasoning, Hume asks what our warrant is for moving from descriptions of how things are to prescriptions of how they ought to be. How, he wants to know, can we rationally justify the move from ‘is’ to ‘ought’? …Kant read Hume's theoretical and practical philosophies as raising variants of a single question. On the side of theoretical reasoning, Hume asks what our warrant is for moving from descriptions of what normative or practical necessity. Normative concepts make explicit commitments that are implicit in any use of concepts, whether theoretically in judgment or practically in acting intentionally.As we have seen, on the modal side, Kant's response is that Hume's predicament is not a real one. One cannot in fact fully understand the descriptive, empirical employment of ordinary determinate concepts such as cat without at least implicitly understanding also what is made explicit by the modal concepts that articulate laws. Kant mounts a corresponding line of thought on the side of We have argued that the modal Kant-Sellars thesis fails. But those familiar with In view of this apparently tight connection between the two Kant-Sellars theses one might wonder whether the normative version is subject to the same problems we level against the modal version.We suspect that the normative Kant-Sellars thesis is unscathed by the sorts of considerations raised above. In order to see this, consider the meaning-use diagram that Brandom provides for the normative Kant-Sellars thesis (‘GAR’ stands for ‘the game of giving and asking for reasons’) Fig. just is a kind of asserting. But if this is so, then we have no reason to doubt that it is PP-sufficient for the practice of using normative vocabulary. Thus no analog of our earlier objections can be run for the present case.In order for normative vocabulary to be universal LX, it must be VP-sufficient for some practice that is, in turn, PP-sufficient for the practice of using normative vocabulary. According to Brandom the practice in question is giving and asking for reasons. But this practice is – and this is the crucial point – one that incorporates the practice of assertion. Giving a reason Between Saying and Doing concerns the nature of intentionality – the world-directed character of language and thought – that we call the pragmatically mediated conception of intentionality. In our view, this proposal is of profound importance to the overarching project of Analytic Pragmatism. Yet Brandom’s discussion is underdeveloped and, at times, frustratingly obscure. With this in mind, we propose to start with a careful and charitable reconstruction of the proposal. Having done so, we show that the arguments of the foregoing sections provide reason to reject the proposal.The final central thesis of Brandom’s Semantic Pragmatism: the thesis that linguistic meaning is determined by use. Moreover, the sorts of analyses that Brandom seeks to provide consist in specifying the linguistic practices on which our mastery of various vocabularies are thought to depend. But as Brandom himself readily acknowledges, there is a worry that such analyses are, in fact, not semantic at all; and not for any reason that has anything specific to do with the particular analyses that he advocates. Rather, the worry is quite general. Such analyses fail to be semantic – and hence to do the work required of them by Analytic Pragmatism – because they conform to the general precepts of Semantic Pragmatism, and Semantic Pragmatism is unsatisfactory as an account of semantic properties. On such a view, meaning is ultimately to be understood in terms of the linguistic practices in which we engage, not the relations that terms bear to aspects of the world. But according to many, semantics, if it is anything, is concerned with precisely such word-world relations – e.g., with representation, truth, and reference.semantic analyses, it is doomed to fail. Thus whatever else the value of pragmatic analysis, it cannot be a satisfactory extension of the project initiated by Frege, Russell and Moore.One way to appreciate the central motivation for – and significance of – Brandom’s pragmatically mediated conception of intentionality is to start by focusing on a persistent criticism of the semantic assumptions of Analytic Pragmatism. More or less by definition, Analytic Pragmatism is committed to One obvious response to the above concern is simply to deny that semantics is centrally concerned with word-world relations. Yet clearly this is not Brandom’s response. Rather, he thinks that there is something importantly right about the idea that semantic properties involve word-world relations. The challenge, then, is to explain why this is so in a manner consistent with Semantic Pragmatism. This is precisely the role that his account of intentionality is supposed to fill.qua practice involves relations between subjects and the world because in general practices involve aspects of the world: they are thick.Qua practice, it too ineliminably involves subject-world relations.Brandom’s response can be viewed as having two main steps. The first is driven by what he takes to be a very general fact about the nature of practices, which he views as a central claim of the pragmatist tradition. Of course language use real problem for the semantic pragmatist is not that there are no relations between language-using subjects and the world. On the contrary, there are a great many such relations. Such relations are cheap. The real problem for semantic pragmatists is specifying the right relation(s) – those relevant for the purposes of characterizing the intentional character of language use; and this, according to Brandom, requires a more detailed, pragmatist account of intentionality.So there is a sense in which, according to Brandom, the worry with which we started is no objection at all. That is, if the concern is that Semantic Pragmatism severs the connection between word and world, then the concern is misplaced. At best it is an artifact of assuming that the relevant practices ought to be characterized “thinly”, in such a way as to avoid reference to aspects of the world. But as Brandom clearly recognizes, this does not settle the matter. The practical intentionality. Practices quite generally, you may recall, involve relations between subjects and aspects of the world. Further, as Brandom notes, practices invariably involve what he calls practical intentionality: feedback-governed relations in which the results of earlier actions are used as guides to future, goal-directed behavior. This is true, for example, of cooking and tea drinking, but also football kicking and everything else for that matter, including language use. According to Brandom, then, the semantic intentionality characteristic of language use is best thought of as a specific instance of practical intentionality.To appreciate Brandom’s account of intentionality one needs first to make three general points clear. The first, already hinted at above, is that he views intentionality of the sort manifested by the practice of language use as a specific instance of a broader phenomenon, manifested by all human practices, which he calls The second point concerns the order of explanation one ought to adopt in explaining semantic intentionality. According to Brandom, some traditionsBrandom maintains that such an approach is wrong-headed in that it gets the proper order of explanation exactly backwards, and thereby ignores precisely that which allows for intentionality in the first place: “the thick, essentially world-involving practices” in which we engage.The final general point concerns the specific practices most central to explaining intentionality. According to Brandom, the most central are those concerned with our use of normative and modal vocabularies. Indeed his strategy is, in effect, to explain intentionality by combining the two Kant-Sellars Theses discussed in Section relata of semantic intentional relations: the world and the rational subject, respectively. The practices made explicit by modal vocabulary (CRI) impose constraints on the modal structure of reality. Roughly, they reflect constraints, implicit in our thought and language, on ways that the world could be and must be. In contrast, the practices made explicit by normative vocabulary (GAR) impose constraints on rational agency. Roughly, they tell us what is it to be a rational subject – a producer and consumer of reasons. Thus for Brandom these linguistic practices, though not themselves the poles of intentional relations, are crucial to explicating the nature of intentionality, because it is by providing appropriately perspicuous characterizations of them that we are able, by abstraction, to reconstruct the nature of the relata.Recall: for Brandom, though an explanation of semantic intentionality requires an account of the relevant subject-world relations, such an account is to be provided by abstraction from the relevant linguistic practices. The primary task, then, is to supply an adequate account of the relevant practices; and for Brandom the two central practices are those concerning modal vocabulary and normative vocabulary, since they inform us about the relata. Rather, one needs to explain how each of these practices are related to each other so as to yield the larger practice on which semantic intentionality depends. In effect, Brandom must address an analog of the problem that confronts more traditional approaches to intentionality of the sort that he rejects. For as we have already noted, on some approaches to the explanation of intentionality, the challenge is to specify the relevant relation between the relata: i.e., subject and world. For Brandom, the challenge is to specify the relationship between two practices: the world-oriented practices made explicit by modal vocabulary and the subject-oriented practices made explicit by normative vocabulary.So far so good. But an account of intentionality must do more than merely explicate those practices that concern the rational rectification, which is the practice by which new commitments are integrated into one’s cognitive economy – one’s system of beliefs, desires, goals, and other intentional states. Such a practice involves the modification of one’s beliefs – often in the light of new information – so as to eliminate inconsistencies among one’s current commitments. But the capacity to do so presupposes a capacity to recognize inconsistencies, which in turn presupposes a capacity to engage in inferences that draw out the consequences of one’s current beliefs. According to Brandom, all of these cognitive capacities are aspects of the practice made explicit by use of modal vocabulary; and thus are implicit in the practice of rational rectification.How is this to be done? Brandom’s answer is that the two practices intersect in what he calls obligation to eliminate such inconsistencies – to structure one’s commitments in such a way as not to violate facts about the modal structure of the world. And according to Brandom, the practice of having one’s commitments conform to such constraints are amongst those made explicit by our use of normative vocabulary.But there is more to rational rectification than this. Rational rectification requires more of us than the mere capacity to recognize inconsistency – and whatever inferential capacities this, in turn, presupposes. Among other things, this alone would be compatible with continuing to endorse inconsistent commitments. In addition to this, rational rectification requires that one take oneself to have an It is time to bring all the pieces together. What we have said so far on the topic intentionality is quite impressionistic; and indeed most of Brandom’s own discussion is similarly gestural. Nevertheless, in accord with his practice elsewhere in the book, when Brandom seeks to spell out his views precisely, he presents a meaning-use diagram. In the case of intentionality, the diagram that he uses to distill his views is reproduced as Fig. any practice of using an autonomous vocabulary – as an object for which intentional vocabulary is VP-sufficient – i.e., either made explicit by or describable by intentional vocabulary. Second, the diagram represents any autonomous discursive practice as incorporating two essential components: a practice made explicit by modal vocabulary and a practice made explicit by normative vocabulary.Although there is a lot going on here, we restrict ourselves to the following observations. First, the diagram aims to depict a kind of practice – indeed relevant to intentionality; and with respect to this task, the primary significance of the Kant-Sellars theses is that they permit Brandom is specify central aspects of the relevant practices. Brandom is thus relying on his earlier discussion of modality and normativity in order to flesh out his account of intentionality.Finally, the diagram represents anyone who can engage in these two practices as being capable, in principle, of deploying the vocabularies that make these practices explicit – i.e., modal and normative vocabulary. In other words, the diagram represents the fact that Brandom’s account of intentionality – in conjunction with the earlier discussed claims regarding modal and normative vocabulary – entails the modal and normative Kant-Sellars theses. This is important for present purposes, because by tying his account of intentionality to his previous claims about modality and normativity, Brandom provides a partial explication of the nature of the practices on which intentionality depends. As we noted earlier, the real challenge for a pragmatic account of intentionality of the sort advocated by Brandom is to explicate the word-world relations that is obviously cannot be right.In view of our earlier discussion, one problem with Brandom’s proposed account of intentionality should be clear. As we have already seen, modal vocabulary is not universal LX. So, there might be a language user that cannot already do everything needed to be able to use modal vocabulary; and if that is right, then the account of semantic intentionality, as represented in Fig. obj-modal in Fig. How deep does this objection go? To see the significance of the objection, we need to rehearse some aspects of our earlier discussion, and introduce some features of Brandom’s theory of content. First, recall that we showed earlier that the practice of counterfactually robust inference is not PP-sufficient for the practice of using modal vocabulary. But this is just to deny that arrow 1 in Fig. obj-modal and the practices sufficient for the use of modal vocabulary by enriching Pobj-modal. Specifically, he might add the assumption that Pobj-modal is constituted, in part, by the ability to make assertions. If such a maneuver is pursued, then our earlier objection to arrow 1 fails. But now – just as before – the problem crops up elsewhere. Though arrow 2 holds more or less by definition, arrow 3 fails. That is, as argued earlier, we no longer have any reason to suppose that modal vocabulary is VP-sufficient for Pobj-modal. In which case, modal vocabulary still fails to be universal LX, and the modal Kant-Sellars Thesis is false. As a consequence, even if one pursues this escape route, Brandom’s pragmatic theory of intentionality, at least as represented by Fig. Of course, this is not the end of the matter. Following the dialectic set out in Section as such should require that modal vocabulary be VP-sufficient for Pobj-modal. Clearly, on anything like Brandom’s view, Pobj-modal should be PP-sufficient for Pmodal. Absent this connection Brandom would, by his own lights, have failed to explicate the “world pole” of the intentionality relation. Further, on anything like Brandom’s account, a relation of PV-sufficiency must hold between Pmodal and Vmodal. After all, on his view, the existence of such a connection obtains more or less by definition. In contrast, it is utterly unclear why the account of intentionality requires that Vmodal be VP-sufficient for Pobj-modal and, hence, that modal vocabulary be universal LX. To the extent that we concerned only with the account of intentionality, then, such a commitment seems entirely optional.One might, however, accept our criticism and yet insist that the objection is easily met so as to preserve the spirit, though not the letter, of Brandom’s account of intentionality. Specifically, it seems eminently reasonable to ask why the account of intentionality modal be VP-sufficient for Pobj-modal. But we think that the assumption that it should is deeply engrained in Brandom’s views about semantics and, hence, that preserving the Brandonian approach to intentionality requires that one give up a deep-seated commitment of Brandom’s philosophical worldview, sometimes called expressive equilibrium. In our experience, the issue is not easily appreciated; and so we propose to creep up on the relevant assumption by first considering another assumption – expressive completeness – also deep-seated in Brandom’s philosophy, that does not suffice to mandate the commitment to arrow 3, but that does motivate the endorsement of expressive equilibriumThere is a sense in which we think that this response is entirely correct. Brandom’s approach to intentionality, need not – and indeed should not – demand that Vitself. That is, an expressively complete semantic theory will correctly explain how the very linguistic expressions that comprise the theory acquire their meanings.Brandom is, of course, a semantic pragmatist, and so his theory of content takes the form of a theory of those discursive practices that are adequate to confer semantic content on the expressions involved. Expressive completeness is invoked as a condition of adequacy on such a theory. To a first approximation, expressive completeness requires that a theory of discursive practices should, in principle, be available to its practitioners. In other words, an adequate semantic theory should explain how linguistic expressions acquire their content in such a way that practitioners are, in principle, able to use the theory in order to explain the practices in which they participate. A corollary of this – one that is central to appreciating the significance of this constraint – is that such a theory will apply to Why endorse such a commitment? Though we will not explore the issue in detail here, the central motivation is to avoid the sorts of self-refutation worries that notoriously confront various historically influential theories of meaning, most notably early versions of verificationism, but also Wittgenstein’s Tractarian account of meaning.modal for Pobj-modal. This is because the mere commitment to expressive completeness very obviously does not demand that modal expressions make explicit the practices on which they depend. On the face of it, all that is required is that the theory not be self-defeating – that the theory possess the resources to explain how the expressions of the theory themselves acquire their meanings. What does require VP-sufficiency, however, is the strategy that Brandom adopts in order to ensure the expressive completeness of his preferred approach to semantic theorizing – what he calls expressive equilibrium.Be that as it may, the assumption of expressive completeness alone fails to explain Brandom’s commitment to the VP-sufficiency of Vbasic theory: This describes what might be called basic discursive practices – i.e. those involving no logical vocabulary. This theory will, of course, use logical vocabulary, but it will not explain how such expressions get their meanings.The extended theory: This describes the extended discursive practices in which language-users engage – i.e., those involving logical vocabulary. More specifically, it does so by showing how basic discursive practices can be extended by the introduction of logical vocabulary.The To a first approximation, the requirement for expressive equilibrium is the requirement that the broad category of logical vocabulary – which, for Brandom, includes traditional logical connectives and quantifiers, but also normative and modal notions – makes explicit not only those norms that confer content on non-logical vocabulary, but those norms that confer content on logical vocabulary as well. To see the significance of this notion for Brandom’s semantic project we need to rehearse briefly the overall structure of the approach to semantic properties that Brandom adopts. Roughly put, he advocates a two-part approach:basic practice lack the resources to state the basic theory because they posses no logical vocabulary at all. In which case, the basic theory is self-defeating. That is where the extended theory enters the picture. This theory explains how to introduce logical vocabulary – including modal and normative vocabulary – into the basic practice. So, participants in the extended practice – i.e., the practice described by the extended theory – possess the vocabulary required to state the basic theory. But now the problem recurs. In order to ensure expressive completeness of the extended theory, such practitioners must not merely have access to the basic theory, but to the extended theory as well. Otherwise, we (once more) risk regress.Now as we have seen, the overall theory – comprised of both the basic and extended theories – needs to be expressively complete. Failing that, one risks self-defeat problems of sort that stymied verificationism. Yet clearly, participants in the Between Saying and Doing, this is just the claim that logical vocabulary, broadly construed to include normative and modal vocabulary, makes explicit the very practices that one needs to master in order to possess logical vocabulary itself. In short: the notion of universal LX vocabulary is a way of making more precise the demand for expressive equilibrium: a demand that Brandom thinks is central to an account of meaning that avoids self-defeat.Brandom’s proposed solution is that logical vocabulary is in expressive equilibrium —that it makes explicit not only those norms that confer content on non-logical vocabulary, but those norms that confer content on logical vocabulary as well. And in the parlance of We are now in a position to summarize our major concerns about Brandom’s view of intentionality. First, as noted earlier, our objections to the modal Kant-Sellars thesis in Section is required if one seeks to endorse both the account of intentionality and the strategy for ensuring expressive completeness. But this is just because it is required by the theory of expressive completeness. In other words, arrow 3 comes not from the theory of intentionality but from Brandom’s strategy for achieving expressive completeness for his pragmatist theory of meaning.Third, arrow 3 irrespective of what account of intentionality Brandom endorses. That is, once one adopts expressive equilibrium as a way of attaining expressive completeness, it needs to be the case that modal vocabulary is VP-sufficient for Pobj-modal; and more generally, that logical vocabulary, broadly construed, is VP-sufficient for those practices needed to master exactly that logical vocabulary. What our arguments in the earlier sections of this paper show is that this strategy cannot succeed.Finally, once one sees the problem, it should be clear that arrow 3 is demanded by expressive equilibrium,


Valproic acid (VPA) is suggested to be therapeutically beneficial in combination with interferon-alpha (IFNα) in various cancers. Therefore, we examined IFNα and VPA alone and in combinations in selected AML models, examining immune regulators and intracellular signaling mechanisms involved in phospho-proteomics.Luc+ mouse model and the syngeneic immunocompetent BNML rat model.The anti-leukemic effects of IFNα and VPA were examined in vitro and in vivo. We mapped the in vitro phosphoprotein modulation by IFNα-2b and human IFNα-Le in MOLM-13 cells by IMAC/2D DIGE/MS analysis and phospho-flow cytometry, and in primary healthy and AML patient-derived PBMCs by CyTOF. In vivo, IFNα-Le and VPA efficacy were investigated in the immunodeficient NOD/Scid IL2γ−/− MOLM-13IFNα-2b and IFNα-Le differed in the modulation of phospho-proteins involved in protein folding, cell stress, cell death and p-STAT6 Y641, whereas VPA and IFNα-Le shared signaling pathways involving phosphorylation of Akt (T308), ERK1/2 (T202/T204), p38 (T180/Y182), and p53 (S15). Both IFNα compounds induced apoptosis synergistically with VPA in vitro. However, in vivo, VPA monotherapy increased survival, but no benefit was observed by IFNα-Le treatment. CyTOF analysis of primary human PBMCs indicated that lack of immune-cell activation could be a reason for the absence of response to IFNα in the animal models investigated.IFNα-2b and IFNα-Le showed potent and synergistic anti-leukemic effects with VPA in vitro but not in leukemic mouse and rat models in vivo. The absence of IFNα immune activation in lymphocyte subsets may potentially explain the limited in vivo anti-leukemic effect of IFNα-monotherapy in AML.The online version of this article (10.1007/s00432-019-02931-1) contains supplementary material, which is available to authorized users. Acute myeloid leukemia (AML) is a heterogeneous aggressive blood cancer characterized by a block in differentiation, elevated threshold for undergoing apoptosis and excessive proliferation of myeloid progenitor cells  has been shown beneficial in melanoma   has been reported to be synergistic in several solid cancer models  were collected after written informed consent in compliance with the Declaration of Helsinki . PBMCs were isolated by Ficoll separation  and cryopreserved in liquid nitrogen for long-term storage. The cells were thawed, centrifuged for 5 min at 300g before incubation for 15 min in StemSpan  added 9% DMEM (Sigma-Aldrich) and 1% DNase I Solution (STEMCELL Technologies). Cells were then plated at 1x106 cells/mL and added media, 2000 IU/mL IFNα-2b, 1 mM VPA or a combination of IFNα-2b and VPA for 48 h before counting, washing with Maxpar PBS , fixed with 2% paraformaldehyde (PFA) in Maxpar PBS for 10 min at 37 °C, followed by freezing at − 80 °C for storage prior to analysis.MOLM-13  and IPC-81 cells  were down-regulated. At 2000 IU/mL only cytoskeletal protein ACTG1 was down-regulated, whilst adapter protein 14-3-3 protein epsilon (YWHAE), actin regulator CAPZB, UPR-response Heat shock protein 105 kDa (HSPH1) and gene expression regulator acidic leucine-rich nuclear phosphoprotein 32 family member A (ANP32A) were up-regulated.We investigated the difference in phosphoprotein regulation between the two IFNα compounds IFNα-Le and IFNα-2b by immobilized affinity chromatography (IMAC) and 2D DIGE in the human AML cell line MOLM-13 (48 h treatment). 2D DIGE showed a total of 47 proteins with higher than 1.3 fold change and a significance level of nds Fig.  and unfonds Fig. , was fouThe expression differences induced by IFNα-2b and IFNα-Le demonstrated no overlap between proteins regulated at low and high dose Table . At 250 p ≤ 0.05) compared to untreated control cells were regarded regulated by the drug treatment. After 15 min exposure, both 250 and 2000 IU/mL IFNα induced phosphorylation of STAT1 (pY701), STAT3 , STAT5 (pY694) and STAT6 (pY641)  by IFNα-2b compared to IFNα-Le  and late (48 h) effects on phosphorylation of signaling proteins involved in cell cycle progression and cell death pathways, as well as IFNα-regulated phosphoproteins in the AML cell line MOLM-13 by phospho-flow cytometry (antibody overview in Online Resource Table 3). Only proteins with a fold change ≥ 1.3    , whereas no significant change was induced by IFNα-Le Fig.  may be cLuc+ NOD/Scid IL2 γ−/− xenograft mouse model. Tumor load evaluation by bioluminescent imaging showed that control mice and mice treated with IFNα-Le (1 × 106 IU/kg) developed tumors in femurs and lymph nodes after 21 days, whilst animals treated with VPA showed detectable tumors 7 days later . VPA-treated and VPA/IFNα-Le combination treated mice had significantly longer survival compared to mice treated with IFNα-Le as monotherapy   mono-therapy rapidly presented with hunched posture and paralysis of hind limbs due to the accumulation of leukemic blasts in the bone marrow, and showed median survival of 21 and 22 days, respectively  and compared to IFNα-Le monotherapy (p = 0.007). Combining IFNα-Le and VPA  gave a slight but non-significant prolonged survival (p = 0.07) compared to VPA alone , whereas no survival benefit was seen for IFNα-Le mono-therapy. The dose of IFNα-Le used in both the rat and mouse model was slightly higher than the dose chosen for a cutaneous melanoma study  cells; p = 0.007, NK T-cells; p = 0.007). For AML patients, the blast population had significantly increased cleaved caspase-3 by VPA . In CD4+CD7− T cells from AML patients VPA decreased (p = 0.030) and IFNα-2b increased cleaved caspase-3 levels (p = 0.017), whereas IFNα-2b gave increased caspase-3 levels in double-negative (DN) T cells . No synergistic effects of apoptosis induction were observed by combination therapy in healthy or AML samples.To assess whether the signaling and anti-apoptotic effects observed by IFNα treatment in MOLM-13 was a cell line-specific effect, we treated 12 AML patient and five healthy donor PBMC samples using the same therapy combination (48 h). Using cleaved caspase-3 (cCaspase 3) as a surrogate for apoptosis detection, we found that the two drugs affected healthy donor Fig.  and AML p = 0.049) .VPA was found to significantly induce acetylation of p53 (K382) and IFNα-2b to significantly induce phosphorylation of STAT1 (Y701) and STAT3 (Y705) both in healthy donor and AML PBMCs Figs. , validatp = 0.021), and up-regulated PD-L1 , CD45RO , CD86  and TIM3 . For AML patient PBMCs  and monocytes (p = 0.021). PD1  and PD-L1  were also increased by IFNα-2b treatment. Comparing healthy donor and AML PBMCs after IFNα-2b treatment showed that healthy donors had monocytes with stronger induction of PD-L1 (p = 0.014), CD86 (p = 0.05) and CD45RO (p = 0.049) in addition to pDCs with higher levels of CD141 (p = 0.023), whereas AML patients had CD4+CD7− T cells with increased levels of PD1 (p = 0.022). Subdividing patients according to karyotype  showed that patients with normal karyotype had higher levels of PD-L1 compared to patients with complex karyotype (pDCs); p = 0.032) after IFNα-2b treatment. B cells in NPM1 mutated patients  had higher levels of CD45RA compared to wild type patients (p = 0.032). No significant changes were found by IFNα-2b monotherapy between FLT3 internal tandem duplication (ITD) mutated and wild type patients .Investigating the immune-modulating effects of both VPA and IFNα-2b (see Online Resource Table 2 for antibody panel), we observed that IFNα-2b treatment of healthy PBMCs Fig.  up-regulThe phosphoproteome analysis identified the acetyl transferase protein NAA10 as selectively down regulated by IFNα-Le and a potential overlapping signal pathway with the histone deacetylase inhibitor VPA. Knock-down of NAA10 has been found to increase apoptosis and increase the sensitivity to daunorubicin in vitro . Particularly, lack of activation of the differentiation markers CD141, CD45RO and CD86 in AML patient monocytes and pDCs could indicate that patients with AML have an inaccessible immune system where cell subsets stay unresponsive to activating stimuli. The absence of pSTAT1 (Y701) induction in AML-derived pDCs, and not healthy pDCs, further supports the inability of these cells to respond to IFNα. The up-regulation of CD141 in response to IFNα treatment in healthy pDCs was importantly not observed in AML-derived DCs. CD141+ DCs are known to induce differentiation of IL-4- and IL-13-producing CD4+ T cells, thereby guiding the adaptive immune response Supplementary material 2 (PDF 676 kb)Supplementary material 3 (PDF 257 kb)Supplementary material 4 (PDF 428 kb)Supplementary material 5 (PDF 8535 kb)Supplementary material 6 (PDF 2143 kb)Supplementary material 7 (PDF 1588 kb)Supplementary material 8 (PDF 2171 kb)Supplementary material 9 (PDF 1955 kb)Supplementary material 10 (DOCX 73 kb)Below is the link to the electronic supplementary material.


About 99% of the cytoplasmic calcium in cardiac myocytes is bound to buffers, and their properties will therefore have a major influence on Ca2+ signaling. This article considers the fundamental properties and identities of the buffers and how to measure them. It reviews the effects of buffering on the systolic Ca2+ transient and how this may change physiologically, and in heart failure and both atrial and ventricular arrhythmias, as well. It is concluded that the consequences of this strong buffering may be more significant than currently appreciated, and a fuller understanding is needed for proper understanding of cardiac calcium cycling and contractility.Changes of intracellular Ca The systolic rise of ionized cytoplasmic calcium concentration ([Ca2+]i) activates contraction and regulates many sarcolemmal ion currents and, thereby, the electrophysiology of the cell; abnormal Ca2+ handling is implicated in the genesis of arrhythmias. Calcium is also a major factor in the control of gene expression. Work using fluorescent Ca2+ indicators has demonstrated how alterations of Ca2+ fluxes into the cytoplasm underlie the changes of contractility in health and disease. It is often overlooked, however, that only ≈1% of cytoplasmic Ca2+ is free, with the remainder being bound to cytoplasmic buffers.3 Therefore, the properties of these buffers will potentially play as large a role as Ca2+ fluxes do in determining the size and kinetics of changes of [Ca2+]i.The importance of changes in intracellular calcium concentration in cardiac function needs little introduction  has several chemical features that make it a ubiquitous second messenger.5 It forms a chemically active divalent cation in aqueous solution with an ionic radius larger than the other common divalent ion (Mg2+) resulting in higher-affinity binding. The fact that its intracellular concentration is much lower than extracellular permits large changes of concentration because of sarcolemmal fluxes. Ca2+ binding alters the tertiary structure of proteins with consequences for their catalytic activity. This is a reciprocal interaction because binding also resists changes of the free concentration of an ion by acting as a buffer.Calcium  in a claw-like or chelation arrangement  a high-affinity site that is normally bound at resting Ca2+ concentrations with a dissociation constant (Kd) of ≈100 nmol/L, (2) 2 further binding sites on the C terminal with Kd values of ≈300 nmol/L.7 The fourth binding site has the lowest affinity (Kd=10 µmol/L) and therefore binds negligible Ca2+ within the physiological range (0.1–1 µmol/L). It may have a role in controlling enzymes located near sarcoplasmic reticulum Ca2+ release sites in cardiac cells where [Ca2+]i rises to ≈100 µmol/L.8The formation of several coordinate bonds between single Cat Figure A. For ex2+ indicators , with a forward rate constant that approaches the diffusion-controlled limit .17 Ca2+ binding to the EF hand structure of the regulatory site of troponin C is slightly slower.9 With even more complex reaction schemes  before Ca2+ binding, the kinetics slows considerably. For example, Ca2+ binding to the chelator EGTA requires dissociation of protons from intermediate forms of the ligand, reducing the overall forward rate constant (Table).18 Different forms of the EF hand motif, such as the Mg2+ sites of myosin, troponin C (TnC), and parvalbumin, have high relative affinities for Mg2+ that result in significant Mg2+ bound under physiological conditions.19 The need for Mg2+ to dissociate as part of the equilibration results in a low apparent rate constant of Ca2+ binding.The speed of the chelation reaction depends on its complexity (Table). The fastest binding occurs with small molecules (molecular weight <1000) such as BAPTA or Ca20 the Table lists the major Ca2+ binding ligands, their estimated cytoplasmic concentrations, and dissociation constants (Kd) alongside estimates of the rate constants of Ca2+ binding. The ligands that bind appreciable amounts of Mg2+ under physiological conditions are grouped separately. The steady-state Ca2+ binding for several buffers as a function of [Ca2+] is shown in Figure 2+ ATPase pump (SERCA).Based on previous work,2+ binding sites  where the only available data are of a shift in the relationship between [Ca2+]i and force. It is often not certain whether this shift results from a direct effect on Ca2+ binding or a subsequent step in the contraction mechanism.25 For example, caffeine shifts the relationship to lower [Ca2+]i but this effect is not accompanied by increased Ca2+ binding.26 Finally, much less is known about the properties of the Mg2+ site on troponin than the regulatory one. One issue, which also applies to other cellular buffers, is that studies of Ca2+ binding are generally performed in vitro using artificial solutions as opposed to cytoplasm. Given that many cellular constituents may affect the properties of this important buffer, it is important to characterize Ca2+ binding to the Mg2+ sites under more physiological conditions. These sites can be mutated, and normal contraction requires only 1 of the 2 Mg2+ sites.27 It would be interesting to know the effects on cardiac function and Ca2+ cycling of the expected large decrease of Ca2+ buffering.TnC has 2 classes of Ca2+]i.28 Initial buffering by binding is followed by active sequestration. In rabbit ventricle, systole involves an increase of ≈60 µmol/L total Ca2+ resulting in a rise of free [Ca2+]i of ≈0.6 µmol/L. Because of the affinity of SERCA binding sites, ≈30 µmol/L binds immediately and, with a peak uptake rate of ≈200 µmol·L–1·s–1, only ≈2 pump cycles are required to sequester the Ca2+ associated with a Ca2+ transient. This emphasizes the importance of the initial binding/buffering by SERCA in addition to its turnover in determining the rate of decay of the cytoplasmic Ca2+ transient.29The inclusion of SERCA as a buffer emphasizes that it has 2 roles in decreasing cytoplasmic . The lag is much greater for the slower buffers (here the Mg2+ sites of myosin and TnC) and this is emphasized in the normalized data of Figure 2+. Even at 1.5 Hz, these 2 slow sites together accumulate a total of ≈10 µmol/L Ca2+ and, at higher rates, when diastolic [Ca2+]i increases, greater binding is to be expected.The importance of the different kinetics of the major buffers is illustrated in Figure 2+ binding molecules. It is often convenient to approximate this with a composite buffer value described by a single dissociation constant and ligand concentration. The simplest method is by titration. Solaro et al31 studied isolated cardiac myofilaments and calculated that about 22 µmol of Ca2+ per kg heart is required to produce 50% maximum contraction. This was accompanied by a rise of free Ca2+ of ≈1.4 µmol/L, indicating that the myofilaments alone can bind >90% of the total Ca2+. A subsequent approach, using cardiac homogenates, found that to raise free [Ca2+] to 1 µmol/L required 72 µmol/kg total Ca2+.32 Hove-Madsen and Bers33 performed similar experiments using permeabilized cells. This removed complications of extracellular components and allowed study of mitochondrial and sarcoplasmic reticulum (SR) buffering separately from cytoplasmic. They found that cytoplasmic buffering could be described by a Kd of 0.42 µmol/L, plus a much lower-affinity component (Kd=79 µmol/L).As discussed above, buffering depends on the summed effects of a variety of Ca2+ current34 under conditions in which Ca2+ removal mechanisms were inhibited  with [Ca2+]i as estimated indirectly from changes of cell length.37 A limitation of the method of Berlin et al is that it requires the irreversible SERCA inhibitor thapsigargin, precluding repeated measurements before and after other interventions. An alternative approach uses rapid application of caffeine to release calcium from the SR resulting in an abrupt increase of [Ca2+]i which then decays as NCX removes Ca2+ from the cell. Integrating the NCX current gives a measure of the change of total Ca2+ concentration that is compared continuously with the change of free [Ca2+]i to characterize buffers36  contribute to Ca-dependent arrhythmias. A more complicated question is what is the effect of increased buffering on SR Ca2+ content in the steady state? Experimental studies have found that adding exogenous cytoplasmic buffers decreases SR Ca2+.48 One explanation is that SERCA activity depends in a cooperative manner on [Ca2+]i,49 whereas NCX has a linear dependence.50 The decreased amplitude of the systolic Ca2+ transient may therefore decrease SERCA activity more than NCX, leading to a net loss of SR Ca2+. Further studies are required to see if the decrease of SR content with increased buffering is a general phenomenon.It should be noted that, in the steady state, averaged over the cardiac cycle, CaT]) and free ([Ca2+]) are related by:For a simple buffer, total  is defined as the change of total Ca2+]i ≈100 nmol/L, the Mg2+ sites on TnC make the largest contribution, whereas, at >200 nmol/L, these are tending to saturation, and the regulatory site and SERCA contribute most. The lower graph of Figure Bmax/Kd) at low [Ca2+] and decreases as [Ca2+] increases. When [Ca2+]=Kd the buffer power is 30% of the value at 0.1 Kd and, at 2 Kd, it is only 13% of this level. Consequently, the greater the diastolic level of [Ca2+]i, the larger will be the increase of [Ca2+]i produced by a given release of total Ca from the SR.52 Some appreciation of the importance of this effect is provided by the buffer curve of Figure 2+]i, an increase of 60 µmol/L total [Ca2+] produces a larger increase of free [Ca2+] when applied from a higher diastolic [Ca2+]i than from a lower. This is clear in the simulated transients of the upper graph of Figure 2+]i of only 100 nmol/L (from 50 to 150) increases systolic [Ca2+]i by 500 nmol/L. Therefore, an increase of diastolic [Ca2+]i alone can lead to an increase of systolic, which is predicted . Figure Kd with the maximum being reached when Kd=[Ca2+]i. Therefore, increasing buffer affinity will increase buffering at diastolic levels of [Ca2+]i but decrease it at peak systolic ones.Equation 2 Figure A shows t2+ buffering is the same in all cells in the ventricle. Given the regional differences of expression of other proteins including pumps59 and channels,60 heterogeneity of buffering would not be surprising. Likewise, possible variations of Ca2+ buffering between individuals, because of mutations and polymorphisms, do not appear to have been considered.One issue that has received no attention is whether Ca2+ buffers, TnC and SERCA, are regulated by the phosphorylation of troponin I and phospholamban, respectively, resulting in increased affinity of Ca2+ for SERCA61 and decreased affinity of Ca2+ for TnC.62 One might therefore expect that β-adrenergic stimulation would alter the buffer power. Experimental measurements, however, found no such effect,40 possibly because of 2 opposing factors: phosphorylation increases the affinity of Ca2+ binding to SERCA, but lowers it for troponin. If the Kd values are above the range of [Ca2+]i considered, these effects will respectively increase and decrease buffer power  and the systolic Ca2+ transient begins at the periphery of the cell and then propagates toward the center.69 Increasing Ca2+ buffering by incorporation of EGTA can prevent this propagation.70 A modeling study also predicted this inhibitory effect of high buffer concentrations but pointed out that lower concentrations of mobile buffers such as ATP facilitate propagation.30 Greiser et al71 investigated the effects of rapid atrial pacing in rabbits to mimic the effects of atrial fibrillation. This resulted in a 2- to 3-fold increase of buffering power, at least in part, because of decreased phosphorylation of troponin I which was accompanied by  of the total troponin85 and will not therefore significantly affect cellular buffering.Ca86 We speculate that the consequent decrease of Ca2+ buffering would compensate in those situations where an increase of myofilament buffering is expected and worsen where there is a decrease. Again, it will be important to repeat these studies of heart failure while measuring buffering directly.Finally, it should be noted that many studies of heart failure find a decrease of SERCA expression.2+]i and, thence, of buffering power on the amplitude of the calcium transient. A major problem here is the paucity of measurements of diastolic [Ca2+]i. It is essential that studies on heart failure ask the simple question: how accurately has diastolic [Ca2+]i been measured and can it be excluded that changes  account for the observed changes of systolic [Ca2+]i?It is also important to reemphasize the potential effects Figure C of chan2+ buffering may not simply be involved in the development and consequences of heart failure, but may also be used to treat it. They suggested that impaired relaxation in heart failure could be ameliorated by adding intracellular buffers. They noted that fast Ca2+ buffers slow both the rise and fall of [Ca2+]i and decrease the amplitude of the Ca2+ transient, and, instead, they advocated the use of parvalbumin, a skeletal muscle Ca2+ buffer. This has the important property that it binds Ca2+ slowly because Mg2+ has to dissociate first and, therefore, there is little attenuation of the peak Ca2+ transient. It will bind Ca2+ during diastole thereby improving diastolic performance. Incorporation of α-parvalbumin was shown to accelerate the decay of [Ca2+]i with no effect on peak [Ca2+]i and  and presumably therefore Ca2+ buffering. They proposed that alterations of buffering might lead to pathological changes of the Ca2+ transient. Troponin mutations were subsequently investigated in a mouse model of the related condition of restrictive cardiomyopathy and the predicted decreased amplitude and slowed decay of the Ca2+ transient observed.90 In addition, myofilament Ca2+ sensitization with EMD 57033 mimicked the effects of troponin T mutations on Ca2+ buffering and the Ca2+ transient.23 A recent study used adenovirus to infect isolated myocytes with troponin or tropomyosin mutations and, again, found an increase of diastolic [Ca2+]i.24 Although the above results would be expected from an increase of buffering power, it has been reported that there is a decrease of SERCA expression that may also contribute.91 This study also found that the late Na+ current inhibitor ranolazine abolished the slowing of decay of the Ca2+ current. Although no data are available, it seems unlikely that ranolazine would affect Ca2+ buffering. It may therefore be that some of the effects of thin filament mutations are directly attributable to Ca2+ buffering, and others are a secondary consequence of the resulting heart failure, possibly attributable to decreased SERCA.Several studies have examined the molecular basis of familial hypertrophic cardiomyopathy (FHC). Much of this work involves the effects of mutations in thin filament proteins such as troponin and tropomyosin, which are among the causes of FHC. Robinson et al2+ sites on troponin are important contributors to buffering at low [Ca2+]i. It is therefore interesting that one of the mutations associated with FHC (D145E) greatly decreases the affinity of Ca2+ binding to these sites.92 At first sight, this might appear to contrast with the association between FHC and the increased affinity reviewed above. These observations may be reconciled by noting that a decrease in affinity of the very-high-affinity Mg2+ TnC sites will actually increase Ca2+ buffering power in the systolic range of [Ca2+]i.As mentioned in an earlier section, the Mg93 The Knollmann group has investigated the underlying mechanisms in transgenic mice. Incorporation of mutations in troponin T or tropomyosin led to ventricular tachycardia. These mutations also sensitized the contractile machinery to activation by Ca2+ . Subsequent work using myocytes derived from human-induced pluripotent stem cells reproduced these effects of increased Ca2+ buffering by myofilaments on action potential shape and suggested that the shortened, triangulated action potential could be attributable to increased buffering decreasing the amplitude of the systolic Ca2+ transient and thereby the inward (depolarizing) NCX current.94 Although this is an attractive explanation, it is also worth noting that (see above), as well as decreasing the amplitude of the Ca2+ transient, increased buffering slows decay, making it harder to predict the net effect of increased buffering on NCX current. Another article showed that, when regular pacing was terminated by a pause, the next Ca2+ transient was larger than control and this effect was more prominent in troponin T mutations that sensitize to activation by Ca2+.23 This effect was attributed to a higher cell Ca2+ content in the mutant during stimulation, with the excess Ca2+ being taken up by the SR such that release after a pause results in a prolonged action potential, increasing the probability of an arrhythmogenic early afterdepolarization. Any increase of diastolic [Ca2+]i and consequent decrease of buffer power may also increase the rise of [Ca2+]i caused by release from the SR. In the work reviewed above, the increase of buffering was a consequence of genetic changes. Similar increases of myofilament Ca2+ sensitivity have been reported following myocardial infarction81 where manoeuvers that decrease Ca2+ sensitivity were found to abolish ventricular tachycardia following pauses of stimulation. As reviewed above, an increase of Ca2+ buffering is proarrhythmogenic. It has also been shown, however, that addition of the buffer EGTA can prevent the propagation of arrhythmogenic Ca2+ waves,95 and therefore the net effect may be more complicated.Ventricular arrhythmias constitute a major cause of death in FHC.+ Figure C with th2+ buffering attributable to thin filament mutations73 may be a consequence of the slowed decay of the Ca2+ transient, resulting in incomplete recovery at the time of the next stimulus at increased rates. This would be analogous to the idea that the increased propensity of endocardium in comparison with epicardium to alternans is associated with a more slowly decaying Ca2+ transient because of lower SERCA expression.59 In contrast, a modeling study has predicted that increasing cytoplasmic Ca2+ buffering should decrease the probability of alternans occurring by decreasing the probability that Ca2+ released from the SR induces further Ca2+ release from neighboring release sites.96 This is consistent with the experimental demonstration, in whole mouse hearts, that addition of the buffer EGTA decreased the occurrence of alternans48 and may be related to the experimental observation that increasing cytoplasmic Ca2+ buffering decreases the frequency of propagating Ca2+ waves97 and makes Ca2+ sparks terminate earlier.98 Further work is clearly required in understanding the relationship between Ca2+ buffering and alternans.The induction of alternans of the action potential duration by increased Ca2+ buffering is not unique to cardiac myocytes. For example, ≈99% of the Ca2+ entering chromaffin cells binds to cytoplasmic buffers.99 The presence of Ca2+ buffers means that much larger movements of total Ca2+ are required to produce a given change of [Ca2+]i. Given that calcium movements account for up to 30% of the total energy consumption of the heart,100 one might wonder why evolution has resulted in such strong buffering. There are several explanations. (1) It may be an inescapable consequence of the fact that using Ca2+ as a second messenger requires high concentrations of Ca2+ binding proteins, for example, to activate contraction. (2) A high Ca2+ buffering may stabilize Ca2+ signaling by stopping an abnormal increase of [Ca2+]i in one part of a cell propagating throughout the cell. In this context, it is worth noting that  The need for buffering may relate to the low intracellular concentration of calcium. A diastolic concentration of 100 nmol/L equates to 6×1016 ions per liter corresponding to a mean distance between ions of 0.25 µm. Soeller and Cannell8 have modeled Ca2+ fluxes into the space between the transverse tubule and SR (dyad). They calculated that, at a concentration of 100 nmol/L, each dyad would contain between 0.007 and 0.028 Ca2+ ions. At 10 µmol/L, there will be between 0.7 and 2.8 ions. This would make it impossible to control [Ca2+]i in a stable manner because a single Ca2+ transported into or out of the space would result in an enormous fractional change of [Ca2+]i. As pointed out previously by Bers,35 at such low concentrations, chance will determine whether a transporter interacts with an ion. In contrast, if total Ca2+ is 100 times the free then there will be between 70 and 280 ions per cleft. (4) If troponin was the only buffer, then virtually all the total Ca2+ would be bound to troponin irrespective of its Kd. This would make it impossible to change force by altering Kd because this requires other buffers to take up a fraction of the total Ca.A high level of Ca2+]i, contribute to alterations of calcium handling and contractility. The limited human data reviewed above and extrapolated from animal models argue that changes of Ca2+ buffering are important in determining both inotropy and proarrhythmic status in conditions such as cardiomyopathies (dilated cardiomyopathy and hypertrophic cardiomyopathy) and ischemic heart failure in both health and disease. Clarification will require more work on human tissue.The concentration of buffered calcium in cytoplasm is 2 orders of magnitude greater than that of the free concentration, and, therefore, the buffers have an enormous effect on calcium signaling. There is a need for more work investigating whether changes of buffer properties, either directly or secondary to changes of diastolic [CaWe are indebted to F. Burton and S. Wray for comments on an earlier version of the manuscript and to Q. Lachaud for the design of Figure This work from is supported by grants from the British Heart Foundation (PG/17/12/32847 to Dr Smith and CH/2000004/12801 to Dr Eisner).None.