Macrophages are dynamic cells that mature under the influence of signals from the local microenvironment into either classically (M1) or alternatively (M2) activated macrophages with specific functional and phenotypic properties. Although the phenotypic identification of M1 and M2 macrophages is well established in mice, this is less clear for human macrophages. In addition, the persistence and reversibility of polarized human phenotypes is not well established. Human peripheral blood monocytes were differentiated into uncommitted macrophages (M0) and then polarized to M1 and M2 phenotypes using LPS/IFN-γ and IL-4/IL-13, respectively. M1 and M2 were identified as CD64(+)CD80(+) and CD11b(+)CD209(+), respectively, by flow cytometry. Polarized M1 cells secreted IP-10, IFN-γ, IL-8, TNF-α, IL-1β, and RANTES, whereas M2 cells secreted IL-13, CCL17, and CCL18. Functionally, M2 cells were highly endocytic. In cytokine-deficient medium, the polarized macrophages reverted back to the M0 state within 12 days. If previously polarized macrophages were given the alternative polarizing stimulus after 6 days of resting in cytokine-deficient medium, a switch in polarization was seen (i.e., M1 macrophages switched to M2 and expressed CD11b(+)CD209(+) and vice versa). In summary, we report phenotypic identification of human M1 and M2 macrophages, their functional characteristics, and their ability to be reprogrammed given the appropriate stimuli.

Many human hereditary neurodegenerative diseases are caused by expanded CAG repeats, and anonymous CAG expansions have also been described in schizophrenia and bipolar disorder. We have isolated and sequenced a novel human cDNA encoding a neuronal, small conductance calcium-activated potassium channel (hSKCa3) that contains two arrays of CAG trinucleotide repeats. The second CAG repeat in hSKCa3 is highly polymorphic in control individuals, with alleles ranging in size from 12 to 28 repeats. The overall allele frequency distribution is significantly different in patients with schizophrenia compared to ethnically matched controls (Wilcoxon Rank Sum test, P = 0.024), with CAG repeats longer than the modal value being over-represented in patients (Fisher Exact test, P = 0.0035). A similar, non-significant, trend is seen for patients with bipolar disorder. These results provide evidence for a possible association between longer alleles in the hSKCa3 gene and both of these neuropsychiatric diseases, and emphasize the need for more extensive studies of this new gene. Small conductance calcium-activated K + channels play a critical role in determining the firing pattern of neurons. These polyglutamine repeats may modulate hSKCa3 channel function and neuronal excitability, and thereby increase disease risk when combined with other genetic and environmental effects.

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JAK2, a member of the Janus kinase (JAK) family of protein tyrosine kinases (PTKs), is an important intracellular mediator of cytokine signaling. Mutations of the JAK2 gene are associated with hematologic cancers, and aberrant JAK activity is also associated with a number of immune diseases, including rheumatoid arthritis. Accordingly, the development of JAK2-specific inhibitors has tremendous clinical relevance. Critical to the function of JAK2 is its PTK domain. We report the 2.0 Å crystal structure of the active conformation of the JAK2 PTK domain in complex with a high-affinity, pan-JAK inhibitor that appears to bind via an induced fit mechanism. This inhibitor, the tetracyclic pyridone 2-tert-butyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-1, was buried deep within a constricted ATP-binding site, in which extensive interactions, including residues that are unique to JAK2 and the JAK family, are made with the inhibitor. We present a structural basis of high-affinity JAK-specific inhibition that will undoubtedly provide an invaluable tool for the further design of novel, potent, and specific therapeutics against the JAK family. IntroductionThe Janus kinases (JAKs) are an important family of intracellular protein tyrosine kinases (PTKs), with 4 mammalian members, JAK1, JAK2, JAK3, and TYK2, 1-5 as well as homologs in chicken, 6 fish, 7 and Drosophila. 8 The JAKs play critical roles in several important intracellular signaling pathways, including the eponymous JAK/STAT pathway, 9 central to the mediation of cytokine signaling. 10,11 It is this pivotal role in cytokine signaling that underpins the notion that specific JAK inhibitors may be therapeutically deployed in situations where cytokine activity results in disease. Important examples of this include autoimmune diseases such as rheumatoid arthritis and psoriasis, 12,13 myeloproliferative syndromes such as polycythemia vera, 14-17 leukemias, 18-20 lymphomas, 21 and cardiovascular disease 22,23 inter alia.Members of the JAK family each share a characteristic domain structure, 2 with a C-terminal PTK domain (known as the JAK homology-1 [JH1] domain), immediately adjacent to a kinase-like domain (JH2), and 5 additional JAK homology domains (JH3-JH7). While the JH2 domain appears to possess an important regulatory role on the PTK activity of the JH1 domain, 24-29 the precise mechanism by which this control is exerted is currently poorly understood. The role of a putative SH2-like domain (JH3/JH4) 2,30 is also unknown at present, whereas the function of a well-defined band F ezrin-radixin-moesin homology (FERM) domain (JH7) 31,32 appears to be critical for interaction of the JAKs with their cognate receptors and regulatory proteins.The JAKs coordinate specifically to different receptors, for example, JAK3 appears to be associated with cytokine receptors that include the ␥c chain of the interleukin-2 (IL-2) receptor (eg, IL-4, IL-7, etc), whereas JAK2 is associated with a wide range of cytokine receptors, including those activated by growth horm...

Like most eukaryotic rDNA promoters, the promoter for rDNA in Saccharomyces cerevisiae consists of two elements: a core element, which is essential, and an upstream element, which is not essential but is required for a high level of transcription. We have demonstrated that stimulation of transcription by the upstream element is mediated by a multiprotein transcription factor, UAF (upstream activation factor) which contains three proteins encoded by RRN5, RRN9, and RRN10 genes, respectively, and probably two additional uncharacterized proteins. The three genes were originally defined by mutants that show specific reduction in the transcription of rDNA. These genes were cloned and characterized. Epitope tagging of RRN5 (or RRN9), combined with immunoaffinity purification was used to purify UAF, which complemented all three (rrn5, rrn9, and rrn10) mutant extracts. Using rrn10 mutant extracts, a large stimulation by UAF was demonstrated for template containing both the core element and the upstream element but not for a template lacking the upstream element. In the absence of UAF, the mutant extracts showed the same weak transcriptional activity regardless of the presence or absence of the upstream element. We have also demonstrated that UAF alone makes a stable complex with the rDNA template, committing that template to transcription. Conversely, no such template commitment was observed with rrn10 extracts without UAF. By using a series of deletion templates, we have found that the region necessary for the stable binding of UAF corresponds roughly to the upstream element defined previously based on its ability to stimulate rDNA transcription. Differences between the yeast UAF and the previously studied metazoan UBF are discussed.

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Somatic mutations in Janus kinase 2 (JAK2), including JAK2V617F, result in dysregulated JAK-signal transducer and activator transcription (STAT) signaling, which is implicated in myeloproliferative neoplasm (MPN) pathogenesis. CYT387 is an ATP-competitive small molecule that potently inhibits JAK1/ JAK2 kinases (IC 50 ¼ 11 and 18 nM, respectively), with significantly less activity against other kinases, including JAK3 (IC 50 ¼ 155 nM). CYT387 inhibits growth of Ba/F3-JAK2V617F and human erythroleukemia (HEL) cells (IC 50 B1500 nM) or Ba/F3-MPLW515L cells (IC 50 ¼ 200 nM), but has considerably less activity against BCR-ABL harboring K562 cells (IC ¼ 58 000 nM). Cell lines harboring mutated JAK2 alleles (CHRF-288-11 or Ba/F3-TEL-JAK2) were inhibited more potently than the corresponding pair harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3), and STAT-5 phosphorylation was inhibited in HEL cells with an IC 50 ¼ 400 nM. Furthermore, CYT387 selectively suppressed the in vitro growth of erythroid colonies harboring JAK2V617F from polycythemia vera (PV) patients, an effect that was attenuated by exogenous erythropoietin. Overall, our data indicate that the JAK1/JAK2 selective inhibitor CYT387 has potential for efficacious treatment of MPN harboring mutated JAK2 and MPL alleles.

BackgroundAirway epithelial cells (AEC) from patients with asthma, appear to have an impaired interferon (IFN)-β and -λ response to infection with rhinovirus.ObjectivesTo determine if impaired IFN responses can be identified in young children at risk of developing asthma due to atopy and/or early life wheeze, and if the site of infection or the infecting virus influence the antiviral response.MethodsNasal (N) and tracheal (T) epithelial cells (EC) were collected from children categorised with atopy and/or wheeze based on specific IgE to locally common aeroallergens and a questionnaire concerning respiratory health. Submerged primary cultures were infected with respiratory syncytial virus (RSV) or human metapneumovirus (hMPV), and IFN production, inflammatory cytokine expression and viral replication quantified.ResultsNasal epithelial cells (NEC), but not tracheal epithelial cells (TEC), from children with wheeze and/or atopy produced less IFN-β, but not IFN-λ, in response to RSV infection; this was associated with higher viral shedding. However, IFN-regulated factors IRF-7, Mx-1 and CXCL-10, and inflammatory cytokines were not differentially regulated. NECs and TECs from children with wheeze and/or atopy demonstrated no impairment of the IFN response (β or λ) to hMPV infection. Despite this, more hMPV was shed from these cells.ConclusionsAECs from children with wheeze and/or atopy do not have an intrinsic defect in the production of IFN-β or -λ, however, this response is influenced by the infecting virus. Higher viral load is associated with atopy and wheeze suggesting an impaired antiviral response to RSV and hMPV that is not influenced by production of IFNs.