V1.4 L-type calcium channels show unique biophysical properties such as slow inactivation due to the lack of calcium-dependent inactivation (CDI). These properties make CaV1.4 channels appropriate candidates for triggering persistent glutamate release at retinal photoreceptor cell synapses. Mutations in the CACNA1F gene encoding for the CaV1.4 α1 subunit are described in patients with X-linked congenital stationary night blindness type 2 (CSNB2). Impaired transmission between rod photoreceptor cells and second-order neurons manifests as night blindness and various other visual symptoms in the affected individuals.CaV1.4 mutants L849P and R1816stop compared to wild-type (WT) in transiently transfected tsA 201 cells  via whole-cell patch clamp technique using 15 mM Ba2+ and Ca2+ as charge carrier. For statistics, either Mann-Whitney (two groups) or Kruskal-Wallis test and Dunn’s Post hoc test (multiple comparisons) were used.The aim of this study was to investigate the functional properties of Ca2+), only minor, not significant (p > 0.05) changes in the voltage-dependent activation properties were observed. In presence of the dihydropyridin-activator BayK8644 (5 μM) the current density was increased ~10-fold (p < 0.001). The fold-increase in current density was comparable to WT. As expected R1816stop, which lacks an intrinsic C-terminal modulator (CTM), exhibited CDI ; R1816stop: 0.63 ± 0.02 (n = 22)) and shifted the voltage-dependence of activation to more negative voltages (V0.5act in mV: WT: 1.8 ± 0.3 (n = 74), R1816stop: −12.3 ± 0.3 (n = 23)). In presence of the CaV1.4-CTM; comprising the last 122 C-terminal residues WT conditions were fully restored, e.g. V0.5act 2.2 ± 1.0 mV (n = 14) (p < 0.0001).L849P was mainly characterized by a reduced current density (pA/pF: WT: −16.3 ± 1.6 (n = 38), L849P: −2.5 ± 0.3 (n = 12), p < 0.0001; CaV1.4 channel protein rather than a reduced open probability. Moreover, the fact that the functional phenotype of the R1816stop can be rescued bears a potential pharmacotherapeutic concept based to the C-terminal modulatory mechanism present in CaV1.4 channels.We assume that the reduced current density observed in mutant L849P derives from decreased channel expression, which might be explained by a folding defect of the Ca


Their chemical structures were elucidated by means of spectroscopic analysis. The cytotoxities of these isolated constituents were assayed.Three new compounds, including two diterpenoids, nemoralisins H and I (The online version of this article (doi:10.1007/s13659-014-0006-6) contains supplementary material, which is available to authorized users. Swietenia mahagoni, which is an economically important timber tree, has been used as a folk medicine for the treatment of hypertension, diabetes, and malaria + . The 1D NMR data  and the ketal carbon (δC 106.9)  . Fractions were monitored by TLC and spots were visualized by heating the silica gel plates sprayed with 10 % H2SO4 in EtOH. Lichroprep RP-18  and Sephadex LH-20  were also used for column chromatography.Optical rotations were obtained with a Jasco P-1020 polarimeter. UV (in MeOH) and IR  were extracted with 70 % aqueous acetone  at room temperature three times to give a dark green residue (650 g), which was then partitioned between EtOAc and water to give the EtOAc-soluble fraction (120 g). The EtOAc extract was chromatographed by silica gel column eluted with CHCl3–MeOH as a gradient  to afford four fractions. The CHCl3–MeOH (100:1) portion was evaporated to obtain a residue (20 g), which was subjected to silica gel chromatograph column with petroleum ether–EtOAc  as elution, to give fractions . Fraction B was further subjected to RP-18 chromatograph column, eluting with MeOH–H2O  to afford subfraction (E), which was then purified by semi-preparation HPLC with MeCN–H2O (50:50) to give compound 3 (6 mg), 5 (12 mg), 6 (10 mg), 7 (15 mg). Fraction C was subjected to silica gel chromatograph column with petroleum ether–EtOAc  as elution, to give fractions F, which was successively subjected to RP-18, Sephadex LH-20 and semi-preparation HPLC, compound 1 (1.5 mg), 2 (1.3 mg), and 4 (6 mg) were obtained.The air-dried powdered leaves and twigs of α]D25 − 0.48 ; UV (MeOH) λmax (log ε) 202 (3.75), 261(3.37) nm; IR (KBr) νmax 3434, 2976, 2930, 1761, 1639, 1585, 1457, 1383, 1270, 1177, 1088, and 1036 cm−1, 1H and 13C-DEPT data see Table m/z 371 [M + Na]+; HREIMS m/z 348.1932 .Colorless oil; D25 − 14.3 ; UV (MeOH) λmax (log ε) 204 (3.87), 261(3.70) nm; IR (KBr) νmax 3432, 2977, 2933, 1761, 1698, 1584, 1458, 1383, 1299, 1267, 1177, 1034 and 938 cm−1, 1H and 13C-DEPT data see Table m/z 371 [M + Na]+; HREIMS m/z 348.1927 .Colorless oil; [α]D18 + 26.3 ; UV (MeOH) λmax(log ε) 209 (3.83) nm; IR (KBr) νmax 3440, 2952, 1727, 1632, 1462, 1441, 1387, 1280, 1245, 1159, 1053, 1024, and 600 cm−1, ESIMS m/z 571 [M + Na]+; HREIMS m/z 548.2250 .White amorphous powder; [1–7 against HL-60, SMMC-7721, A549, MCF-7 and SW480 cell lines were determined by the MTT method [The cytotoxic activities of compounds Supplementary material 1 (DOC 830 kb)Below is the link to the electronic supplementary material.


V) within this nucleus. Whole-cell patch recordings were made from brain slices from P13–15 CBA mice. Slices were incubated with the inhibitor of neuronal nitric oxide synthase (nNOS) 7-nitroindazole (10 µM) and pharmacological blockers used to isolate Ca2+ current subtypes. Unpaired observations in the presence and absence of the NO-donors sodium nitroprusside  or Diethyl-ammonium-nonoate  were made to elucidate NO-dependent modulation of the expressed CaV subtypes. A differential effect of NO on the calcium channel subtypes was observed: CaV1 and CaV2.1 (L+R- and P/Q+R-type) conductances were potentiated, whereas N+R-type (CaV2.2) and R-type (CaV2.3) current amplitudes were unaffected. L+R-type currents increased from 0.36±0.04 nA to 0.64±0.11 nA and P/Q+R-type from 0.55±0.09 nA to 0.94±0.05 nA, thereby changing the balance and relative contribution of each subtype to the whole cell calcium current. In addition, N+R-type half-activation voltage was left shifted following NO exposure. NO-dependent modulation of P/Q+R and N+R-type, but not L+R-type, channels was removed by inhibition of soluble guanylyl cyclase (sGC) activity. This data demonstrates a differential effect of NO signalling on voltage-gated calcium entry, by distinct NO-dependent pathways.Nitric Oxide (NO) is a diffusible second messenger that modulates ion channels, intrinsic excitability and mediates synaptic plasticity. In light of its activity-dependent generation in the principal neurons of the medial nucleus of the trapezoid body (MNTB), we have investigated its potential modulatory effects on native voltage-gated calcium channels (Ca Nitric oxide is an important signalling molecule and volume transmitter with a functional role in both central and peripheral systems 2+-signalling is vital throughout the brain with voltage-gated Ca2+ entry being mediated by multimeric Ca2+ channels V channels are grouped into three families according to the genetic relatedness of the pore-forming α1 subunit, which confers the voltage sensitivity and conductance specificity of the channels V1), P/Q-type (CaV2.1), N-type (CaV2.2) and R-type (CaV2.3) Activity-dependent CaV1 and CaV2.1 channel amplitudes and a hyperpolarising shift of half-activation voltages of CaV2.2 by distinct pathways underlies the increase in whole-cell current amplitude.Here we show that NO-dependent potentiation of Ca2+ we found total ICa amplitudes in the range of 400 pA (not shown) consistent with previous reports 2+ improved current resolution as peak amplitudes of whole-cell currents increased over 2-fold in the presence of Ba2+ compared to Ca2+, thus the following experiments were performed using Ba2+ as the charge carrier. In order to assess nitrergic effects on CaV we applied NO-donors to slices where residual modulation of CaV by NO was removed by pre-incubation with the nNOS inhibitor 7-Nitroindazole . Raw traces from one control cell (black trace), and two from cells treated with the NO donors: Diethyl ammonium-nonoate  and sodium nitroprusside , respectively are shown in 1/2) which were calculated by fitting a Boltzmann function to the normalised conductance (G/Gmax) (1/2 was −25.5±1.5 mV (n = 6); DEA V1/2 was −30.4±2.0 mV (n = 5); SNP V1/2 was −23.5±1.5 mV (n = 3)). Overall, the data showed that treatment of 7-NI incubated slices with the NO-donors significantly potentiated whole-cell IBa without affecting channel inactivation kinetics or half-activation voltages.In physiological aCSF containing 2 mM Ca(G/Gmax)  did not V subtypes underlying the whole-cell current were affected by NO. Using pharmacological tools we isolated the four CaV subtypes contributing to the whole-cell current (We next investigated how the four Ca current  in mouse1/2) was −26.5±1.7 mV and SNP-treated V1/2 was −26.1±1.3 mV; P/Q+R-type V1/2 was −19.7±4.1 mV and SNP-treated V1/2 was −21.5±3.0 mV  and whole-cell peak current amplitudes from unpaired recordings from slices exposed to NO over time (red circles) are shown in Ba: control 7-NI treated peak IBa was 0.98±0.09 nA (n = 6) and NO treated was 1.52±0.18 nA . It also shows the potentiation of L+R- and P/Q+R-type currents by the NO donor SNP (100 µM): L+R-type control was 0.36±0.04 nA (n = 5) and was increased to 0.64±0.11 nA ; P/Q+R-type control was 0.55±0.10 nA (n = 4) and was increased to 0.94±0.05 nA . N+R- and R-type current amplitudes were unaffected in cells treated with SNP (100 µM): N+R-type control was 0.70±0.10 nA (n = 6) and N+R-type +NO was 0.69±0.08 nA (n = 8); R-type control was 0.42±0.08 nA (n = 8) and R-type +NO was 0.30±0.13 nA  and the modulated CaV channel subtypes. Additionally, the NO-induced left-shift in N-type activation was removed in the presence of ODQ (1/2: −22.2±1.3 mV), whereas half-activation voltages of P/Q+R- and L+R-type currents were not affected by ODQ in the presence of SNP  V and CaV channels or affect transmitter recycling/release remains to be elucidated. Importantly, all our recordings were performed at 37°C in unpaired experimental conditions to minimise cell dialysis thereby providing intact signalling required for the nitrergic pathways. Our results therefore represent a physiological approach where native CaV were differentially modulated by intact NO signalling.CaCa in a cGMP-dependent manner suggesting that NO regulation differs greatly between cellular systems. Different studies in rat cultured hippocampal Previous studies in native neurons, dissociated hair cells from frog and rat 2+ influx via L- and P/Q-type Ca2+ channels which could have important downstream signalling effects as various Ca2+ channel subtypes are linked to multiple signalling pathways leading to changes in gene expression through: CREB (Ca2+ response element binding protein), CaRF (the Ca2+ response factor) and NFAT (the nuclear factor of activated T-cells) 2+-induced gene expression has been linked to changes in intrinsic excitability mediated by L-type Ca2+ channels as reported in primary hippocampal cultures 2+ channel activation leads to gene induction through PKA and PKC pathways. It is important to understand how nitrergic activity might influence these Ca2+-dependent pathways. NO-mediated augmentation of P/Q- and L-type channels adds to the versatile network of nitrergic signalling between cellular systems.As illustrated in Slices were prepared from CBA/Ca mice (P13–15), which were killed by decapitation in accordance with local animal's and the UK Animals (Scientific Procedures) Act 1986. All procedures were carried out complying with the policies and regulations according to the guidelines laid down by the MRC's animal's ethics and welfare committee  approved under the Home Office Project License 80/2100. Brainstem slices containing the Superior Olivary Complex (SOC) were cut in the transverse plane using a 7550 MM Integraslice  at 200 µm at ∼0°C. Brain slices were incubated at 37°C in aCSF for an hour, before being kept at room temperature prior to experiments as described previously Cells were visualised by an upright Eclipse E600FN Microscope  using 60× objective. Whole-cell voltage clamp patch recordings were obtained from principal neurons of the MNTB at 37°C as described previously 3 (26.2); TEA-Cl (30); BaCl2 (5); MgCl2 (1.3); KCl (2.5); glucose (10); NaH2PO4 (1.25); ascorbic acid (0.5); 7-NI (0.01). TTx (0.5 µM) was used externally to block sodium channels. All chemicals were obtained from Sigma, except for Ca2+ channel blockers. All recordings were leak subtracted offline by assessing the linear leak between −110 and −70 mV for generating I/Vs. Raw traces are un-manipulated other than removal of capacitance artefacts for better visualisation.Current-voltage relationships (IVs) were generated by plotting peak current against command potential. Final whole-cell access resistance was <20 MΩ and series resistance was compensated by 70% (10 µs lag). Pipette solution contained (mM): CsCl (120); NaCl (10); TEA-Cl (10); EGTA (1); HEPES (40); Phosphocreatine (5); Mg-ATP (2); Na-GTP (0.3); ZD7288 (0.01). External aCSF solution contained (mM): NaCl (95); NaHCO2+ channel blockers as indicated (µM): Nifedipine (10), ω-Agatoxin IVA (0.2) , ω-Conotoxin GVIA (2) .All external solutions contained blockers of synaptic inputs to prevent any spontaneous events (µM): DNQX (10), strychnine (1), bicuculline (10) (non-methyl derivate) and CaL-type channels were isolated pharmacologically by blocking P/Q- and N-type channels with 2 µM ω-Conotoxin GVIA and 200 nM ω-Agatoxin IVA, respectively. P/Q-type channels were isolated pharmacologically by blocking L- and N-type channels with 10 µM Nifedipine and 2 µM ω-Conotoxin GVIA, respectively. N-type channels were isolated pharmacologically by blocking L- and P/Q-type channels with 10 µM Nifedipine and 200 nM ω-Agatoxin IVA, respectively. R-type channels were pharmacologically isolated by blocking L-, P/Q- and N-type channels with 10 µM Nifedipine, 200 nM ω-Agatoxin IVA and 2 µM ω-Conotoxin GVIA, respectively.V subtypes in control and NO-treated conditions. Our observed whole cell IBa obtained from our recordings = R+N+L+P/Q. However our estimated current size based on our recording scenarios is calculated as R+N+R+L+R+P/Q+R-3R and this = 0.767 nA. If we propagate the error (Δ) associated with each of the recording scenarios by using the formula:Ba, 0.77±0.21 nA falls in a similar range to our observed 0.98±0.09 nA. The table shows similar results for the NO-treated scenarios.t-test. *P<0.05, **P<0.01 was considered significant. All data sets were tested for normality distributions prior to comparisons. Data denote mean±SEM, n - number of neurons tested. Activation parameters were determined by a Boltzmann function: I = Imax/(1+exp(V−V1/2/k) with variables Imax, V1/2 and k (slope factor). Inactivation data was obtained by fitting a single exponential decay to the current recorded at −10 mV.Statistical analyses utilized unpaired two-tailed Student's All fits were performed using Clampfit 10.2, Sigmaplot (Systat) or Excel (Microsoft) with least squares minimization.


To potentiate this, phosphoantigen levels can be boosted using zoledronic acid (ZA). However, in vivo delivery of ZA to the tumour is inefficient owing to its poor pharmacokinetic properties. Here, we set out to develop a γδ T-cell immunotherapy for epithelial ovarian cancer (EOC) using folate-targeted liposomal ZA (FT-L-ZA) to improve drug delivery to tumor cells. Folate receptor-α is over-expressed in EOC. Peripheral blood mononuclear cells isolated from healthy donors (n=21) and EOC patients (n=13) were cultured with ZA, IL-2 and IL-15 for two weeks. γδ T-cells expanded reproducibly from healthy donors and patients, the latter having on average a 97-fold expansion. The expanded γδ T-cells expressed low levels of L-selectin, accompanied by CD45RO, CD27, CD70 and NKG2D, consistent with a combined central-memory and effector-memory phenotype, similarly to healthy controls. To evaluate whether ex-vivo cultured γδ T-cells from patients with EOC are functionally competent, we developed an autologous co-cultivation assay whereby patient-derived γδ T-cells were incubated in-vitro with primary EOC “tumorspheres”. Twenty-four hours after incubation of tumorspheres with ZA, L-ZA or FT-L-ZA, ex-vivo expanded γδ T-cells were added at a 1:100 ratio. When alone, γδ T-cells destroyed 24.5% of spheres. Addition of 1µg/ml free ZA sensitized tumorspheres to destruction by γδ T-cells (96.2%), but was relatively ineffective at 0.1µg/ml (26.8%). By contrast, FT-L-ZA was highly effective at 0.1µg/ml (92.6%) while non-targeted L-ZA was no better than γδ T-cells only. Destruction of ZA-sensitized tumorspheres by autologous γδ T-cells was accompanied by their activation and production IFN−γ (≈ 2000 pg/mL). In the absence of γδ T-cells, none of these formulations exerted any toxic effect upon tumorspheres. To model EOC in vivo, SCID Beige mice were injected intraperitoneally (IP) with luciferase-expressing SKOV-3 cells. Mice (n=5 per group) were treated IP with 5μg ZA, FT-L-ZA or PBS, followed by 10E7 ex-vivo expanded γδ T-cells from healthy controls or PBS. Tumour growth was monitored by bioluminescence. One week after treatment, mice treated with γδ T-cells or γδ T-cells and free ZA showed a tumour reduction of approximately 20%. By contrast, mice treated with FT-L-ZA followed by γδ T-cells achieved a tumour reduction of 60-70%. Drugs alone had no therapeutic effect. We show here proof of concept for a synergistic immunotherapy for EOC whereby FT-L-ZA can be used to sensitize tumors to γδ T-cells.


Leishmania mexicana develop non-healing progressively growing lesions associated with a biased Th2 response while similarly infected IL-4Rα-deficient mice fail to develop lesions and develop a robust Th1 response. In order to determine the functional target(s) for IL-4/IL-13 inducing non-healing disease, the course of L. mexicana infection was monitored in mice lacking IL-4Rα expression in specific cellular compartments. A deficiency of IL-4Rα expression on macrophages/neutrophils  had minimal effect on the outcome of L. mexicana infection compared with control (IL-4Rα−/flox) mice. In contrast, CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice infected with L. mexicana developed small lesions, which subsequently healed in female mice, but persisted in adult male mice. While a strong Th1 response was manifest in both male and female CD4+ T cell specific IL-4Rα−/− mice infected with L. mexicana, induction of IL-4 was manifest in males but not females, independently of CD4+ T cell IL-4 responsiveness. Similar results were obtained using pan-T cell specific (iLckcreIL-4Rα−/lox) IL-4Rα−/− mice. Collectively these data demonstrate that upon infection with L. mexicana, initial lesion growth in BALB/c mice is dependent on non-T cell population(s) responsive to IL-4/IL-13 while progressive infection is dependent on CD4+ T cells responsive to IL-4.Immunologically intact BALB/c mice infected with  Leishmania species are parasites, transmitted by sandflies which are of extensive public health importance in the tropical and subtropical regions of the world. A large number of distinct Leishmania species cause cutaneous disease and the vast majority of studies utilize the caustive agent of Old World cutaneous leishmaniasis, L. major. Other species, for example, L. mexicana, are associated with quite different patterns of disease following infection of mice when compared with L. major. Thus, while susceptible BALB/c mice deficient in the ability to respond to the cytokines IL-4/IL-13 are not protected against development of cutaneous leishmaniasis caused by L. major they are totally resistant to infection with L. mexicana. Here we describe the outcome of L. mexicana infection in BALB/c mice with cell-specific deletion of the receptor for IL-4/IL-13 on macrophages/neutrophils or T helper cells. Infections develop in both mutants but lesion growth is controlled only in T cell specific knockouts and female but not male mice heal. Male but not female T cell specific knockouts maintain a strong IL-4/IL-13 response. This highlights the role of IL4/IL-13 in driving a non-healing response and may in part explain why human males are more susceptible to this infection than females. Leishmania mexicana is under different genetic and immunoregulatory controls to those controlling L. major infection L. major, the majority of mouse strains are susceptible to L. mexicana infection Leishmania species, protective immunity against L. mexicana is the result of a STAT-4 dependent type-1 immune response, although this can be generated independently of IL-12 L. major infections in susceptible BALB/c mice remain somewhat controversial, IL-4 plays the major role in promoting non-healing L. mexicana infections in this mouse strain L. mexicana infection as IL-4 and IL-13 receptors share the IL-4Rα sub unit New world cutaneous leishmaniasis resulting from infection with −/−, IL-4Rα−/−, or wild type splenocytes L. mexicana infection. However, the non-healing disease phenotype was dependent on a lymphocyte source of IL-4 and, in its absence, IL-4-deficient splenocyte-reconstituted SCID mice generated a healing response −/− splenocytes demonstrated that initial lesion development was also dependent upon cells from this source responding to IL-4/IL-13 In order to better differentiate both the cellular sources and targets of IL-4/IL-13 initiating lesion growth and facilitating progressive non-healing disease, we have previously examined parasite growth in SCID mice reconstituted with IL-4in vivo, tissue specific IL-4Rα−/− mice have been produced. So far macrophage/neutrophil specific (LysMcreIL-4Rα−/lox) + T cell specific (LckcreIL-4Rα−/lox) −/− mice have been generated and the consequences for L. major infection studied. In contrast to susceptible BALB/c mice, BALB/c LysMcreIL-4Rα−/lox mice showed a significantly delayed disease progression after infection with L. major, concomitant with normal Th2 and type 2 antibody immune responses but with improved macrophage leishmanicidal activities creIL-4Rα−/lox BALB/c mice infected with L. major were significantly more resistant than global IL-4Rα−/− mice and developed a disease phenotype and clinical immunity similar to genetically resistant C57BL/6 mice + T cells in the non-healing BALB/c phenotype but paradoxically indicating a protective role for IL-4Rα signaling in a non-CD4+ T cell population.In order to better differentiate the specific role of IL-4/IL-13 responding cells from global effects L. major infection L. mexicana infection in BALB/c mice. In addition, unlike global IL-4Rα−/− mice infected with L. mexicana that display no lesion growth, infected CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice initially develop lesions indicating that early susceptibility to L. mexicana is dependent on an IL-4 responsive non-CD4+ T cell population. However, subsequent lesion growth is significantly curtailed in infected CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice compared with IL-4Rα intact mice, and a strong Th1 response generated in the presence of significant elements of Th2 activity. Despite reduced susceptibility in all CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice, a dichotomy between the sexes was identified during L. mexicana infection and while lesions in female CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice healed they persisted in male mice associated with elevated IL-4 production in this sex compared with females. Together, our results suggest that initial development of the L. mexicana lesion is dependent on an IL-4/13-responsive non-T cell population, whilst progressive infection is dependent on CD4+ T cells responsive to IL-4.In the present study we demonstrate that in contrast to creIL-4Rα−/−, LckcreIL-4Rα−/−, IL-4Rαlox/lox mice were generated and maintained as previously described creIL-4Rα−/− or LckcreIL-4Rα−/− and IL-4Rαlox/lox mice. Transgene-bearing LysMCreIL-4Rα−/lox and LckcreIL-4Rα−/lox, were identified by PCR genotyping as described LysML. mexicana (MYNC/BZ/62/M379) was maintained by serial passage of amastigotes inoculated into the shaven rumps of BALB/c mice. Amastigotes for use in infections were isolated from lesions and enumerated as described below. Two sites of infection were examined and either 5×106L. mexicana amastigotes in a final volume of 50µl were inoculated subcutaneously into the shaven base of the tail, or 2×105L. mexicana amastigotes in a final volume of 25µl were inoculated subcutaneously into the hind footpad. 6–8 week old male or female LysMCreIL-4Rα−/lox and LckcreIL-4Rα−/lox mice were used in each experiment, with age and sex matched cre negative IL-4Rα−/lox littermates used as controls. The lesion diameter was measured using a sliding gauge micrometer at weekly intervals.L. mexicana infected LysMCreIL-4Rα−/lox, LckcreIL-4Rα−/lox and IL-4Rα−/lox mice and disrupted through a metal mesh with 5mL of RPMI 1640 . The parasites were washed twice at 350g in RPMI and then enumerated using an improved Naubauer haemocytometer. Alternatively parasite numbers were quantified by limiting dilution, as previously described Lesions were excised from L. mexicana antigenic lysate, as previously described −1 purified anti-mouse IFN-γ capture antibody R4-6A2  or 500ng ml−1 IL-4 capture antibody 11B11 (BD Biosciences) in PBS (pH 9.0) overnight at 4°C. Supernatants were then added to the individual wells and either 30µL recombinant mouse IFN-γ  or recombinant mouse IL-4  added to individual wells in duplicate in a doubling dilution with a solution of pH 7.4 PBS supplemented with 10% v/v FCS , ranging form 20ng mL−1 to 0.01ng mL−1 (IFN-γ) or 2ng mL−1 to 0.977pg mL−1 (IL-4). The plates were then incubated for 2 hours at 37°C. The bound cytokines were incubated with either biotinylated rat anti-mouse IFN-γ monoclonal antibody XMG1.2 or biotinylated rat anti-mouse IL-4 antibody BVD6-24G2 (both BD Biosciences) and detected with either conjugated streptavidin-alkaline phosphatase or conjugated streptavidin-horseradish peroxidase (BD Biosciences). The appropriate substrate was then added to the wells, p-nitrophenyl-phosphate  or tetramethylbenzidine in pH 5.5 sodium acetate buffer, containing 0.0003% hydrogen peroxide . Finally the plates were read at an absorbance of 405nm for IFN-γ or at 450nm for IL-4.Splenocytes were isolated from infected mice and cultured for 72 hours in 96-well plates  in the presence or absence of L. mexicana specific-IgG1 and -IgG2a were detected in the plasma of infected mice by ELISA, as previously described −1Leishmania mexicana lysate . The substrate tetramethylbenzidine in pH 5.5 sodium acetate buffer, containing 0.0003% hydrogen peroxide, was then added to the wells and, following colour development, the reaction stopped by the addition of 10% sulphuric acid, absorbance measured at 450nm using a SOFTmax PRO  and the endpoint dilution was determined. Total IgE was detected in the plasma of infected mice by capture ELISA as previously described −1 PMA and 500 ng ml−1 Ionomycin (both Sigma-Aldrich) along with GolgiPlug (BD Biosciences). Following stimulation, cells were harvested and washed, resuspended in FACS Buffer containing Fc Block (2.4G2 hybridoma supernatant) together with the appropriate combinations of the following antibodies: CD4-APC, CD8-PerCP or B220-FITC . Intracellular cytokine staining was carried out using PE-conjugated anti-mouse IL-4 or IFN-γ with Cytofix/Cytoperm solution . Data was obtained using FACSCanto (BD Bioscience) and analysed using FlowJO .Draining lymph node cells were activated for 4 hours with 50 ng mlAntibody analysis was performed using the Mann-Whitney U test and all other analysis used an unpaired Student's t test.L. mexicana lesion growth in LysMCreIL-4Rα−/lox with IL-4Rα−/lox littermate control and global IL-4Rα−/− mice, animals were infected with 5×106 amastigotes into the shaven base of the tail. While no discernible lesions were identified in infected global IL-4Rα−/− mice, as previously demonstrated CreIL-4Rα−/lox and IL-4Rα−/lox mice  than those recorded from global IL-4Rα−/− animals  than that of global IL-4Rα−/− mice . These data suggest that the expression of IL-4Rα by a macrophage/neutrophil population is not important in determining susceptibility to L. mexicana infection.To compare the progression of lox mice . Parasit animals . In line−/− mice . Antigen−/− mice , demonst+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice with 5×106L. mexicana amastigotes into the shaven base of the tail resulted in control of the lesion growth observed in control IL-4Rα−/lox mice . Interestingly, in one experiment using male mice, lesions in LckcreIL-4Rα−/lox mice did not heal completely, while experiments utilizing female mice fully resolved  IL-4Rα−/− mice developed lesions which completely healed after 4–5 weeks (+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− developed lesions which failed to fully heal  IL-4Rα−/− mice of both sexes were able to significantly control parasite growth , male mice  IL-4Rα−/− mice resulted in an enhanced Th1 response in both male and female LckcreIL-4Rα−/lox mice compared with control IL-4Rα−/lox mice, as demonstrated by significantly enhanced antigen induced splenocyte IFN-γ production  mice and this was true whether examining female or male mice (p<0.02). Antigen stimulated splenocytes from infected female but not male LckcreIL-4Rα−/lox mice produced significantly more IFN-γ than antigen stimulated splenocytes from infected global IL-4Rα−/− animals. An expanded Th1 response was also indicated by enhanced antigen specific IgG2a production compared with control IL-4Rα−/lox mice, and in magnitude similar to that generated by global IL-4Rα−/− mice  than in infected control IL-4Rα−/lox mice as early as week 6 post-infection (creIL-4Rα−/lox mice were producing significantly more IgG2a (p<0.025) than their infected control IL-4Rα−/lox counterparts . On the other hand, antigen induced splenocyte IL-4 production was similar in all infected male mice, independent of IL-4Rα expression  but not infected male LckcreIL-4Rα−/lox mice compared with their respective control IL-4Rα−/lox counterparts , but also significantly less than similarly treated male LckcreIL-4Rα−/lox mice (p<0.02 for ConA and p<0.05 for anti-CD3 respectively). Conversely male LckcreIL-4Rα−/lox mice splenocytes produced similar quantities of IL-4 to control male IL-4Rα−/lox mice with either treatment. Examination of draining inguinal lymph node cells indicated a significantly lower (p<0.05) percentage of IL-4 and greater (p<0.05) percentage of IFN-γ secreting CD4+ T cells in infected female but not male LckcreIL-4Rα−/lox mice compared with control sex-matched IL-4Rα−/lox mice  we failed to identify any significant role for IL-4Rα signaling via macrophages/neutrophils in the normal non-healing response of BALB/c mice infected with L. mexicana. In contrast, following early lesion growth, CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice were able to inhibit disease progression. However, while lesions in female CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice healed those in male CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice persisted. Furthermore, although both female and male CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice had significantly enhanced type-1 responses compared with IL-4Rα intact (IL-4Rα−/lox) mice, male CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice maintained strong type-2 responses compared with their female counterparts.We have previously demonstrated that signaling via IL-4Rα plays the major role in the non-healing response of BALB/c mice following infection with L. mexicana as well as L. majorL. major infected BALB/c mice and macrophage/neutrophil specific (LysMcreIL-4Rα−/lox) IL-4Rα−/− mice display delayed lesion growth L. mexicana infection. The control of L. major early in infection in LysMcreIL-4Rα−/lox mice has been identified as being due to enhanced macrophage microbicidal NO mediated activity in the absence of alternative macrophage activation. What may be critical in this regard is that L. amazonensis parasites, which belong to the “mexicana” complex of parasites, have been shown to be more resistant to macrophage-mediated control than L. major requiring higher levels of NO to induce killing L. major, there is in fact enhanced replication of the amastigote stage of L. amazonensis in IFN-γ-stimulated murine macrophages L. mexicana have attenuated virulence in vitro and in vivo with the indication that the parasite arginase has a potential role in depleting host L-arginine available for iNOS activity L. mexicana and L. major as the Th2 response is blunted in animals infected with arginase null mutant L. mexicana parasites while pharmacological inhibition of arginase during L. major infection did not inhibit the Th2 immune response Although signaling via IL-4Rα plays a significant role in the outcome of infection with + T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice are more resistant than global IL-4Rα−/− mice to infection with L. major, indicating that in the absence of a polarized Th2 response, there is a role for an IL-4/IL-13 responsive non-CD4+ T cell in early resistance to infection + T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice are more susceptible than global IL-4Rα−/− mice to infection with L. mexicana, indicating a role for an IL-4/IL-13 responsive non CD4+ T cell population in early susceptibility. We have now studied the course of L. mexicana infection in newly generated iLckcreIL-4Rα−/lox female and male mice that have IL-4Rα deleted on all T cell populations + T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice (data not shown). Consequently IL-4 responsive CD8+ T cells do not play a role in early susceptibility or the non-healing response following infection with L. mexicana. Studies utilizing macrophage specific BALB/c IL-4Rα−/− mice have demonstrated that IL-4/IL-13 operates through this population to enhance L. major parasite growth via alternative macrophage activation + T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice. However, IL-4 treatment of BALB/c mice prior to T cell priming has previously been demonstrated to instruct dendritic cells to produce IL-12 and facilitate a protective Th1 response against L. majorCandidaL. major in the absence of IL-4Rα responsive CD4+ T cells in BALB/c mice. As no distinct disease phenotype could be discerned in macrophage/neutrophil specific (LysMcreIL-4Rα−/lox) IL-4Rα−/− mice compared with IL-4Rα intact animals infected with L. mexicana, no IL-4/IL13 responsive non-T cell population can easily be ruled out in promoting early infection against this parasite. While a role for B cells and antibody production in the non-healing response to L. mexicana is well established  L. donovani (humans) L. infantum (dogs) L. mexicana develop much stronger Th1 responses, as measured by IFN-γ production, delayed–type hypersensitivity and IgG2a antibody levels, than similarly infected male mice L. mexicana, females generally have increased Th1 responses as measured by DTH reactions and decreased Th2 responses as measured by IgE production than males + T cell specific IL-4Rα−/− BALB/c mice have revealed a previously undetected, underlying male susceptibility to L. mexicana involving T cells. Thus, unlike female mice, male mice were unable to resolve infection and overall had a less polarized Th1 response and more polarized Th2 than their female counterparts. This was associated with IL-4 production independently of IL-4Rα signaling in male but not female CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− BALB/c mice. The L. mexicana induced IL-4 producing Th2 phenotype in male LckcreIL-4Rα−/lox BALB/c mice was not the result of differential Cre-mediated deletion efficiency of IL-4Rα in male mice as compared with female mice, as no CD4+ T cell population expressing IL-4Rα was detected from either gender (data not shown). While IL-4 production independently of IL-4Rα signaling has been observed in a number of immunological models previously Unlike the epidemiological and experimental reports on −/− mice we demonstrate that upon infection with L. mexicana initial lesion growth is dependent on a non-CD4+ T cell population responsive to IL-4/IL-13, while progressive infection is dependent on CD4+ T cells responsive to IL-4. Furthermore whether lesions heal or not is gender determined, suggesting a subtle but significant effect of sex hormones on CD4+ T cell function whereby infected male but not female CD4+ T cell specific IL-4Rα−/− mice can drive IL-4 production independently of IL-4Rα signaling.To conclude in this study utilising tissue specific IL-4RαFigure S1−/− mice heal following L. mexicana infection. Mean lesion growth (−/lox), CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/−, and global IL-4Rα−/− mice infected sub-cutaneously with 5×106 amastigotes of L. mexicana. These are the results from 2 separate experiments carried out at different times the first using females (+ T cell specific (LckcreIL-4Rα−/lox) mice they persisted in male CD4+ T cell specific (LckcreIL-4Rα−/lox). Results are mean+/− s.e. Additional experiments utilised male and female groups infected in parallel.Female but not male T cell specific IL-4Rαn growth  in femaln growth  and malen growth  IL-4Rα i females , the sec females . While l(0.20 MB TIF)Click here for additional data file.Figure S2−/− male mice 6 weeks post-infection. Mean lesion parasite burden ± s.e. at week 6 post-infection in male IL-4Rα intact (IL-4Rα−/lox), and CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/−mice infected sub-cutaneously with 5×106 amastigotes of L. mexicana.Similar parasite burdens in wild-type and T cell specific IL-4Rα(0.11 MB TIF)Click here for additional data file.Figure S3−/− mice. L. mexicana-specific IgG1 levels (−/lox), CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/−, and global IL-4Rα−/− mice infected sub-cutaneously with 5×106 amastigotes of L. mexicana. * p<0.05, and ***p<0.001 compared with IL-4Rα intact mice. Representative of 4 separate experiments.Less IgG1 production in infected female T cell specific IL-4Rα1 levels  and tota1 levels  in femal1 levels  and male1 levels  IL-4Rα i(0.38 MB TIF)Click here for additional data file.


It exhibits a distorted octahedral coordination geometry defined by two chloride anions at trans sites and four 3-pyridyl N atoms at equatorial sites from two (6-nicotinoyl-2-pyridyl)(3-pyridyl)methanone ligands. The (6-nicotinoyl-2-pyridyl)(3-pyridyl)methanone ligand can be viewed as having two pendant 3-pyridyl rings attached to a central pyridyl skeleton via separate carbonyl bridges, acting in a κ2               N,N′-chelating mode with its 3-pyridyl N atoms bound to the CuII ion. The pendant 3-pyridyl rings make a dihedral angle of 80.76 (5)°. In the crystal, molecules are linked through intermolecular C—H⋯π and C—H⋯O interactions, forming a three-dimentional framework.In the title complex, [CuCl DOI: 10.1107/S1600536811017685/zq2102Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Their role in graft-versus-host disease and mechanisms of suppression remain elusive. In this study, we demonstrate that DN T cells can inhibit CD4+ T cell-mediated GVHD in a semi-allogeneic model of bone marrow transplantation. Furthermore, we present evidence of a novel autocrine IFNγ signaling pathway in Fas-deficient C57BL/6.lpr (B6.lpr) DN T cells. B6.lpr DN T cells lacking IFNγ or its receptor were impaired in their ability to suppress syngeneic CD4+ T cells responding to alloantigen stimulation both in vitro and in vivo. Autocrine IFNγ signaling was required for sustained B6.lpr DN T cell IFNγ secretion in vivo and for upregulation of surface Fas ligand expression during TCR stimulation. Fas ligand (FasL) expression by B6.lpr DN T cells permitted lysis of activated CD4+ T cells and was required for suppression of GVHD. Collectively, our data indicate that DN T cells can inhibit GVHD and that IFNγ plays a critical autocrine role in controlling the regulatory function of B6.lpr DN T cells.TCRαβ Human DN T cells inhibit autologous CD4+ and CD8+ T cell proliferation in vitroDN T cells express an αβ T cell receptor (TCR) but do not express either CD4 or CD8 coreceptors, nor do they express NK cell markers. This phenotype differentiates them from other unconventional T cells (e.g. NK T cells and γδ T cells). Depending on the context, DN T cells have been shown to have regulatory, innate, or pathogenic properties lpr and gld respectively) and humans (autoimmune lymphoproliferative syndrome) exhibit lymphoproliferation, autoimmunity, and DN T cell expansion. Whereas some studies implicate DN T cells in the pathogenesis of autoimmunity in these settings, lpr DN T cells can also act as Tregs in some contexts lpr mice can resist diabetes induced by adoptively transferred T cells lpr DN T cells can inhibit syngeneic CD4+ T cell responses is of interest since the latter are autoimmune effectors in lpr and gld mice Fas or FasL mutations in mice  were transplanted with T cell-depleted (TCD) BALB/c (H-2d) bone marrow cells alone (BM only) or with BALB/c CD4+ T cells, without (BM+CD4+) or with preactivated and enriched (by depleting B cells and CD8+ and CD4+ T cells) BALB/c DN T cells (BM+CD4++DN) as putative suppressor cells. GVHD clinical score + succumbed to acute GVHD F1 (CB6F1) mice  B6.lpr DN T cells pre-activated by alloantigen in vivo did not cause significant illness or mortality in lethally irradiated CB6F1 mice reconstituted with B6 (H-2b) BM . Importantly, infusion of preactivated B6.lpr DN T cells increased median survival from 12 to >80 days , and decreased illness severity compared with BM+CD4+ treated mice . Effector cell density in the intestine precluded accurate quantification, but we observed statistically significant reductions in alloreactive T cell numbers in the liver , suggesting that IFNγ might contribute to B6.lpr DN T cell-mediated suppression of GVHD.IFNγ can contribute to GVHD pathogenesis; however, early IFNγ administration can inhibit GVHD lpr DN T cell regulatory function, CFSE labelled Thy1.1+CD4+ T cells were co-cultured with B6.lpr DN T cells, with either neutralizing anti-IFNγ mAb or isotype control IgG. IFNγ neutralization slightly increased the proportion of proliferated CD4+ T cells in the absence of B6.lpr DN T cells . Importantly, IFNγ neutralization markedly hampered the function of B6.lpr DN T cells , they did not suppress this response efficiently compared with B6.lpr.IFNγ+/+ DN T cells .We next tested whether IFNγ signalling might underlie B6.lpr DN T cells lacking either IFNγ or its receptor differ in their ability to inhibit GVHD, lethally irradiated CB6F1 recipients were reconstituted with BM alone, BM+CD4+, or BM+CD4++ B6.lpr, B6.lpr.IFNγR−/− or B6.lpr.IFNγ−/− DN T cells. Consistent with our in vitro data, mice receiving either IFNγ−/− or IFNγR−/− B6.lpr DN T cells experienced higher GVHD mortality than mice receiving IFNγ+/+ and IFNγR+/+ B6.lpr DN T cells, with median survival times of 34 days  and 27 days  compared with >80 days . Consistent with a role for IFNγ signalling, supplementation of B6.lpr.IFNγ−/− DN T cell cultures with IFNγ partially restored surface FasL expression, although not to levels seen in IFNγ-sufficient B6.lpr DN T cells . Interestingly, all three types of DN T cells had similar intracellular FasL expression (data not shown), consistent with the notion that IFNγ does not influence FasL protein synthesis in B6.lpr DN T cells, but promotes its cell surface expression.Our previous work revealed that syngeneic Fas-sufficient T cells are suppressed by activated B6.lpr, B6.lpr.IFNγ−/−, and B6.lpr.IFNγR−/− DN T cells were used as effectors to kill preactivated B6 CD4+ T cells. While B6.lpr DN T cells efficiently killed the targets, B6.lpr.IFNγ−/− DN T cells exhibited reduced cytotoxicity .To investigate whether autocrine IFNγ signalling is involved in the cytotoxic function of these cells, activated B6.lpr DN T cell FasL expression in GVHD suppression, lethally irradiated CB6F1 mice were transplanted with BM+CD4+ and either B6.lpr (FasL+) or B6.gld  DN T cells. In these experiments, a dose of 2.5×106, rather than 5×106, DN T cells was given to each recipient. Despite this lower dose, B6.lpr DN T cells significantly enhanced GVHD survival compared with B6.gld DN T cells  B6.lpr.IFNγR−/− DN T cells failed to synthesize significant IFNγ in vivo . Only B6.lpr DN T cells capable of secreting and responding to IFNγ were able to express high levels of surface FasL . These data are consistent with a previous report that FasL is stored within secretory lysosomes in T cells, and subsequently externalized upon activation lpr DN T cells, TCR stimulation triggers IFNγ secretion, resulting in autocrine IFNγ signaling and thus externalization of pre-synthesized FasL.We have demonstrated that autocrine IFNγ upregulates B6.ss GVHD –Fig. S3. lpr DN T cells toward activated CD4+ T cells was only mildly impaired by IFNγ deficiency but completely abrogated by IFNγR deficiency .Deletion of IFNγ in MRL.lpr DN T cells caused only a very mild illness when infused into lethally irradiated allogeneic recipients  was approved by the University Health Network Animal Care Committee. Animal care was conducted in accordance with the policies and guidelines of the Canadian Council on Animal Care and the Province of Ontario's Animals for Research Act.b), BALB/c (H-2d) and (B6xBALB/c)F1  mice were from Jackson Laboratories  or Harlan Sprague Dawley . B6 mice carrying the Faslpr/lpr mutation (B6.lpr), the FasLgld/gld mutation (B6.gld), Thy1.1-congenic B6 mice (B6. Thy1.1), and IFNγR-targeted B6 and BALB/c mice were from Jackson Laboratories and bred in-house. IFNγ-targeted B6 mice (B6.IFNγ−/−) were a gift of Dr. D. Kelvin and bred in-house. B6. Thy1.1.IFNγR−/−, B6.lpr.IFNγ−/−, B6.lpr.IFNγR−/−, and CB6F1.IFNγR−/− mice were generated by crossing appropriate strains.C57BL/6  or BioLegend : PE-conjugated anti-IFNγ, anti-CD4, anti-CD8, anti-NK1.1, anti-CD11b, anti-CD11c, anti-CD19, anti-Gr1, Ter119, and anti-γδ TCR; allophycocyanin-conjugated anti-Thy1.1 and anti-CD4; FITC-conjugated anti-TCRβ and anti-IFNγ; biotinylated anti-CD119 (IFNγR) and anti-FasL; purified anti-CD28. Biotinylated antibodies were detected using PE-Cy7-conjugated streptavidin (eBioscience). Anti-CD3 antibody (clone 145-2C11) was prepared from a hybridoma in house. Anti-mouse IFNγ mAb (clone H22) was from R&D Systems , and IgG isotype control was from eBioscience. Flow cytometry was performed on a Cytomics FC500 , an LSR II , or an Accuri C6 . Dead cells were excluded with propidium iodide , fixable viability dye eFluor450 or 7-aminoactinomycin D .+ T cells (>90% pure) were purified from B6. Thy1.1 spleen and lymph node cells with anti-CD4 microbeads . To obtain DN T cells for BMT, BALB/c, B6.lpr, B6.lpr.IFNγ−/−, or B6.lpr.IFNγR−/− mice aged 8-12 weeks were infused with 40×106 CB6F1 splenocytes to activate and expand DN T cells lpr DN T cells were purified by removing CD4+, CD8+, NK1.1+, CD19+, CD11b+, CD11c+, γδTCR+, and Ter119+ populations with PE-conjugated mAbs and anti-PE microbeads . BALB/c DN T cells were enriched by removing B cells by adherence to IgG-coated plates followed by depletion of CD8+ and CD4+ T cells with anti-CD4 and anti-CD8 hybridoma supernatants  and Low-Tox M rabbit complement . Cell number was adjusted based DN T cell purity to ensure that a consistent number of DN T cells were used. Male CB6F1 mice aged 6–10 weeks received 13Gy γ-irradiation  in a Gammacell 40 137Cs irradiator  and were infused with 2×106 TCD BM alone or with 106 CD4+ T cells, with/without 2.5–5×106 DN T cells. Survival was monitored daily. Weights and clinical scores TCD BM (>99% T cell free) was obtained by treating erythrocyte-free BM cells with anti-Thy1.2 ascites  and Low-Tox M rabbit complement . CD4+ cells in 10 random high-powered (40× objective) fields. Photomicrographs were acquired using a Leica DM2000 microscope and Infinity 1 camera . In parallel, H&E-stained paraffin sections were examined by a pathologist (O.A.) in blinded fashion to confirm GVHD.Tissue specimens were snap-frozen and stored at −80°C. Frozen sections were stained with biotinylated anti-Thy1.1 mAb (eBioscience) and horseradish peroxidase-conjugated ultrastreptavidin . Colour was developed with NovaRed , with haematoxylin counterstaining. Sections were examined using a Nikon Eclipse E200 microscope . A blinded observer (C.J.) counted Thy1.1+ T cells (107 cells/mL) were incubated  in PBS containing 1 μM CFSE , followed by quenching with FBS . Cells were washed in α-minimum essential medium with 10% FBS, 50 mM β-mercaptoethanol, 0.1mg/mL penicillin and 0.1 mg/mL streptomycin (CM). 105 CFSE labelled cells were cultured for 5 days with 2×105 irradiated (20Gy) CB6F1 splenocytes and 50 U/mL recombinant human IL-2 . Some experiments were performed using plate bound anti-CD3 and soluble anti-CD28 (5 μg/mL) antibodies instead of irradiated CB6F1 splenocytes. Purified DN T cells were added in varying ratios. Propidium iodide-negative responder cells were identified with anti-CD4 and/or anti-Thy1.1 antibodies and CFSE and subjected to flow cytometry. Percent suppression was calculated using the formula: [(%CFSEhi (DN+CD4+) – %CFSEhi (CD4+ only))/(100-%CFSEhi (CD4+ only))] ×100%. In some experiments, anti-IFNγ mAb (2.5 μg/mL) or isotype control were added. In others, 7-AAD staining and CFSE dilution were jointly examined. Cell death in the presence of B6.lpr DN T cells was compared to cell death in their absence by calculating fold change, defined as (%7-AAD+ (DN+CD4+))/(%7-AAD+ (CD4+ only)).CD4Two million splenocytes were cultured for 4 h in 2 mL CM containing GolgiStop , with/without 50 ng/mL PMA and 500 ng/mL ionomycin (Sigma-Aldrich). Cells were stained for TCRβ, CD4, CD8, and NK1.1. IFNγ staining was performed with an intracellular staining kit (eBioscience).6 B6 CD4+ T cells were added to 10×106 irradiated CB6F1 splenocytes and 50 U/mL IL-2 in 2 mL CM. After 4 days, 10 μCi/mL 3H-thymidine  was added for 18 h. In parallel, 5×106 B6.lpr, B6.lpr.IFNγ−/−, or B6.lpr.IFNγR−/− DN T cells were activated in similar fashion without 3H-thymidine. Then, 1043H-thymidine-labelled CD4+ T cells were co-cultured in duplicate or triplicate 200 μL cultures with 5×104 fresh irradiated CB6F1 splenocytes and B6.lpr DN T cells in varying ratios. The cpm of retained DNA, measured on a TopCount NXT (Perkin-Elmer), was used as an index of cell survival. At each DN:CD4+ ratio, the percent CD4+ killing was determined using the formula: [(cpm(CD4+ only) – cpm(DN+CD4+))/cpm] ×100%.The JAM assay Serum samples were assayed in duplicate for IFNγ content using a Quantikine Mouse IFNγ ELISA kit (R&D Systems), according to the manufacturer's instructions.lpr, B6.lpr.IFNγ−/−, and B6.lpr.IFNγR−/− mice were cultured in 2 mL CM in plates coated with anti-CD3 mAb, along with 50 U/mL IL-2 and 5 μg/mL soluble anti-CD28 mAb with/without 100 ng/mL recombinant mouse IFNγ (R&D Systems). In some cases anti-IFNγ antibody (2.5 μg/mL) or isotype control were included; in others, 50 µM TAPI-1 Five million spleen and lymph node cells from B6.Flow cytometry data were analyzed and presented with FlowJo 7 . Graphical presentation and statistical analysis of data were performed with Prism 5.0 . Survival analysis was performed using the log-rank test. Variance between two groups was assessed with either Student's t test  or the Mann-Whitney test; three or more groups were analyzed by ANOVA, in some cases with Bonferroni's post-test to compare groups, or the Kruskal-Wallis test in the case of non-parametric data. A p value of less than 0.05 was considered statistically significant. Error bars in all graphs represent standard deviations.Figure S1Purification of B6.lpr DN T cells. Pooled spleen and lymph node cells from B6.lpr mice were incubated with PE-conjugated antibodies to CD4, CD8, NK1.1, CD11b, CD11c, Ter119, γδTCR, and CD19, washed, and incubated with anti-PE microbeads. PE+ cells were then removed using LD columns. Aliquots of the pre-column population (left panels) and the negative fraction (right panels) were stained with FITC-conjugated TCRβ antibody and analyzed by flow cytometry. Two examples of DN T cell purification from B6.lpr mice are shown.(TIF)Click here for additional data file.Figure S2Matrix metalloproteinase inhibition results in a slight increase in FasL expression on B6.lpr DN T cells, regardless of their ability to secrete and respond to IFNγ. Spleen and lymph node cells from B6.lpr, B6.lpr.IFNγ−/−, and B6.lpr.IFNγR−/− mice were activated as in (TIF)Click here for additional data file.Figure S3+ T cells determines their susceptibility to killing by activated DN T cells.Fas expression by proliferating CD4 CFSE-labelled B6.lpr (FasLPR/LPR) or B6. Thy1.1 (Fas+/+) CD4+ T cells were cultured with irradiated CB6F1 splenocytes and IL-2, without or with B6.lpr DN T cells for 5 days. These data are from the same experiment as in +/+ Thy1.1 cells (top row) and FasLPR/LPR cells (bottom row). Numbers inside histograms reflect the percentage of 7-AAD+ cells within the gate. The fold increase in dead CD4+ T cells, defined as the percent 7-AAD+ within the divided population divided by the percent 7-AAD+ within the undivided population, is shown in the graph at right. Data are derived from duplicate wells in one of two experiments with similar results. Two-way ANOVA p<0.0001; Bonferroni post test ***p<0.001; **p<0.01; *p<0.05.(TIF)Click here for additional data file.


Nα-terminal acetylated thymosin α1 (Tα1), a 28-amino-acid peptide, is an immune modifier that has been used in the clinic to treat hepatitis B and C virus (HBV/HCV) infections. We previously documented Nα-terminal acetylation of recombinant prothymosin α (ProTα) in E. coli. Here we present a method for production of Nα-acetylated Tα1 from recombinant ProTα. The recombinant ProTα was cleaved by human legumain expressed in Pichia pastoris to release Tα1 in vitro. The Nα-acetylated Tα1 peptide was subsequently purified by reverse phase and cation exchange chromatography. Mass spectrometry indicated that the molecular mass of recombinant Nα-acetylated Tα1 was 3108.79 in, which is identical to the mass of Nα-acetylated Tα1 produced by total chemical synthesis. This mass corresponded to the nonacetylated Tα1 mass with a 42 Da increment. The retention time of recombinant Nα-acetylated Tα1 and chemosynthetic Nα-acetylated Tα1 were both 15.4 min in RP-high performance liquid chromatography (HPLC). These data support the use of an E. coli expression system for the production of recombinant human Nα-acetylated Tα1 and also will provide the basis for the preparation of recombinant acetylated peptides in E. coli.N Acetypartners . For exapartners . Acetylated form .E. coli, examples include interferon α and γ [α and γ, are not known to be acetylated in their native forms. Thus, the acetylated forms of these proteins are of little clinical utility.Although acetylation is not as common in prokaryotes as in eukaryotes, researchers have identified acetylated forms of recombinant peptides and proteins to be expressed in  α and γ , 8, soma α and γ , interle α and γ , and che α and γ . Howeverα1 (Tα1) is an immune modifier that has been shown to trigger maturational events in lymphocytes; affect immunoregulatory T-cell function; promote IFN-α, IL-2, and IL-3 production by normal human lymphocytes; and increase lymphocyte IL-2 receptor expression [α1 is a 28-amino-acid polypeptide that was originally isolated from thymosin fraction 5. The active peptide, which contains an Nα-terminal acetylated serine residue, is cleaved from prothymosin α (ProTα) by legumain, a lysosomal asparagine endopeptidase (AEP) in mammals [α-terminal acetylated Tα1, it is currently being developed for treating several diseases, including hepatitis B and C virus (HBV/HCV) infections, nonsmall cell lung cancer (NSCLC), hepatocellular carcinoma, AIDS, and malignant melanoma [Thymosin pression . Tα1 is  mammals . Given tmelanoma –18.α-terminal acetylated Tα1 has been produced by total chemical synthesis for clinical use. However, with 28 residues, it is expensive to produce the peptide by chemical synthesis. As an alternative to synthesis, prokaryotic expression systems are widely used for producing large quantities of recombinant proteins or peptides. When prothymosin α (ProTα) and its Nα-terminal peptide, Tα1, were identified 20 years ago, no acetylation was found when they were expressed in E. coli [α1 as a fusion protein or in concatemer form in E. coli [To date, N E. coli , 20. Oth E. coli –24. α is Nα-terminal acetylated when expressed in E. coli [α-terminal acetylated Tα1 from E. coli. This method may provide a useful alternative to chemical synthesis for producing acetylated peptides for clinical applications.We have previously reported that a fraction of recombinant human ProT E. coli . In thisα expression plasmid pBV220-proTα was constructed previously [Pichia pastoris expression plasmid pPIC9, which contains the promoter of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) gene, the α-mating factor prepro secretion signal from Saccharomyces cerevisiae, and the HIS4 auxotrophic selection marker, was purchased from Invitrogen . The P. pastoris GS115 (his4) host strain was from Invitrogen. HepG2 liver carcinoma cell line was purchased from the Institute of Biochemistry and Cell biology .The human ProTeviously . The yeaα-acetylated ProTα was produced as described previously [α gene was cloned in plasmid pBV220, with expression under control of the PLPR promoter. Expression of ProTα was induced by increasing the culture temperature from 30°C to 42°C for 6 h. After induction, cells were harvested by centrifugation.Recombinant human Neviously  with somE. coli proteins. Precipitated proteins were spun down, and the supernatant containing ProTα was applied on a DEAE Sepharose Fast Flow column  equilibrated with buffer A. After washing, the bound protein was eluted with a gradient from 0 to 0.4 M NaCl. Since ProTα does not contain any aromatic residues, elution of ProTα was monitored by UV absorption at 214 nm. The level of protein and nucleic acid contamination was estimated by UV absorption at 280 nm [α in eluate fractions was analyzed by 18% SDS-PAGE.The cells were suspended in buffer A  and incubated at 85°C for 15 min. Acetic acid was added to adjust the pH to 4.0 and to precipitate t 280 nm . The purPichia pastoris expression vector pPIC9 to generate the legumain expression vector pPIC9-legumain. The sequence of legumain was verified by sequencing.Total mRNA was isolated from HepG2 cell line and converted to single-stranded cDNA using MMLV Reverse Transcriptase as described by the manufacturer . The human legumain gene without signal peptide was amplified by PCR using forward primer legu4 (5′-AGACTCGAGAAAAGAGTTCCTATAGATGATCCTGAAG-3′) and reverse primer legu5 (5′-TATGCGGCCGCTTAGTAGTGACCAAGGCACACGTG-3′); primers were designed based on the ORF of legumain reported previously . AmplifiP. pastoris following the protocol for scale-up expression in shaker flasks as described in the P. pastoris expression manual (Invitrogen Life Technologies). The pPIC9-legumain expression vector (10 μg) was digested with SalI. P. pastoris GS115 host strain was transfected with the linearized vector by electroporation at 1.5 kV, 129 Ω, and 25 μF in electroporation cuvettes . P. pastoris colonies growing on MD plates  were picked for expansion in 50 mL of yeast extract-peptone-dextrose medium (YPD) consisting of 1% yeast extract, 2% peptone, 2% dextrose. Expression of the recombinant legumain was induced by culturing P. pastoris in methanol-complex medium containing 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, pH 6.0, 1.34% yeast nitrogen base, 4 × 10−5% biotin, and 0.5% methanol. Cultures were grown for 72 h, and 0.5% methanol was added every 12 h.Recombinant prolegumain was expressed in Culture supernatant containing prolegumain was diluted with 5 volumes of buffer C  and applied to SP Fast flow column . After washing with buffer C, bound protein was eluted with a step gradient of 25%, 45%, and 100% buffer D . Eluate fractions were analyzed by 12% SDS-PAGE. Fractions containing recombinant prolegumain were dispensed into aliquots and stored at −70°C until use. μL aliquots of the recombinant prolegumain (0.2 mg/mL) were incubated in 1 mM dithiothreitol (DTT), 1 mM EDTA, and 0.1 M sodium citrate pH 4.0. Samples were incubated for 0 h, 0.25 h, 1 h, and 4 h at 37°C. Digest products were analyzed by SDS-PAGE (12%) and stained with Coomassie Blue.To determine the optimum time for autocatalytic activation, 500 α by recombinant autoactivated legumain, 250 μL aliquots of recombinant ProTα (2 mg/mL) were mixed with 50 μL of legumain in buffer containing 1 mM DTT, 1 mM EDTA, and 0.1 M sodium citrate (pH 4.0). Samples were incubated for 0, 2, 4, 6, 8, and 16 h at 37°C. Digest products were assayed by SDS-PAGE (18%) and stained with Coomassie Blue.To optimize digestion of ProTα (100 mg) was cleaved by autoactivated legumain (1 mg) for 8 h at 37°C. The pH was adjusted to 3.0 by addition of acetic acid. The sample was applied to a PoRos R50 column  equilibrated with buffer E , and washed with two bed volumes of buffer F . Protein was eluted with a step gradient of 10%, 20%, and 30% ethanol. Eluate fractions were assayed by RP-HPLC. Purified ProTα1-containing fractions were loaded onto an SP High performance column  and washed with equilibration buffer M (10 mM NaCl). Bound protein was eluted with 0.2 M NaCl, 0.3 M NaCl, and 0.5 M NaCl. Eluate fractions were assayed by reverse phase-high performance liquid chromatography (HPLC).Tμm, Ø4.6 × 250 mm, Johnson Technologies Co. Dalian, China) was equilibrated at a flow rate of 1.0 mL/min in solvent A (0.1% trifluoroacetic acid (TFA) in water). Solvent B was 0.1% TFA in acetonitrile. Samples (25 μL) were injected on the column, and peptides were eluted with a linear solvent gradient . Peptide elution was detected by UV-absorption at 214 nm. The column was reequilibrated with solvent A for 20 min between runs.RP-HPLC was performed using the HP HPLC 1090 system. A C18 column  and eluted in methanol, water, and acetic acid (50 : 50 : 0.5). The molecular mass of Nα-acetylated Tα1 was determined by nanoelectrospray ionization (nanoESI) quadrupole time-of-fight (Q-TOF MS) using a Q-TOF2 mass spectrometer . The peptide sequence was determined by tandem mass spectrometry.Purified Tα was overexpressed in E. coli [Recombinant human prothymosin  E. coli  and puriα at asparaginyl-glycine sites and release peptide products Tα1 and Tα11, in Pichia pastoris, and purified it by cation exchange chromatography. The molecular mass of recombinant prolegumain was 56 kDa. When incubated at an acidic pH, the recombinant proenzyme was autocleavaged to a 46 kDa form in a time dependent manner  in the 0.2 M NaCl elution fraction  plus an additional 42 Da increment for the acetyl group.Purified, recombinant N.386 min , which w.386 min . The mol08.79 Da ; a mass α-acetylated Tα1 was determined by tandem MS. It was found to be “Ac-SDAAVDTSSEITTKDLKEKKEVVEEAEN” , which is the same as the native and chemosynthetic Nα-acetylated Tα1. Nα-terminal acetylation was confirmed by a m/z of 130.05 for the Nα-terminal amino acid residue , which corresponds to an acetylated serine residue.The sequence of the recombinant Nα1 had been expressed as a fusion protein or in a concatemer form [α1 concatemer was successfully expressed in E. coli and cleaved by hydroxylamine to release the Tα1 monomer. However, this version of Tα1 was not acetylated on the Nα-terminal serine residue [α-acetylated Tα1 following expression of ProTα in E. coli. A number of factors make this method easy to scale up for commercial production. Firstly, E. coli can be grown to high-density and is capable of producing large quantities of heterologous protein. ZADAXIN, Nα-terminal acetylated Tα1 produced by total chemical synthesis energizes the immune response, helping patients to battle invasive cancers and secondary infections. It has been tested and proven safe, effective, and easy to tolerate in numerous patient populations, including the elderly, previous treatment failures, and patients with depressed immune systems. In this study, we provide an alternative way to produce Nα-terminal acetylated thymosin α1 by cleaving the recombinant protein with legumain, which should be cheaper for large-scale production; as E. coli is the most efficient and economic host for recombinant polypeptide production.Tmer form , 31, 32. residue . In contP. pastoris after which it can be autocleaved to yield the active enzyme. Only 2 μg of recombinant legumain is needed to process 400 μg of ProTα (data not shown). Finally, the extraction and purification of ProTα were based on thermal denaturation [In addition, recombinant human legumain is easily expressed as the proenzyme in yeast turation  and the α-acetylated Tα1 from recombinant ProTα also confirmed our previous findings that ProTα is Nα-terminally acetylated in E. coli [E. coli. However, with the exception of Nα-acetylated Tα1 described here, many of these acetylated proteins are of little clinical utility. Nα-acetylation of proteins is catalyzed by Nα-acetyltransferases (NATs). Three E. coli NATs, namely, RimL, RimJ, and RimI, have already been identified through mutant analysis and are responsible for the acetylation of ribosomal proteins L12, S5, and S18, respectively [α-acetylation of recombinant fusion proteins of Tα1 and the ribosomal protein L12 is catalyzed by RimJ in E. coli [E. coli [E. coli.The isolation of N E. coli . Acetyla E. coli . Before ectively , 35. Fan E. coli . Johnson[E. coli . These rα-acetylated Tα1 in E. coli, which is a clinical useful peptide, and was produced by chemosynthesis previously. The product consistent with the chemosynthesis of Nα-acetylated Tα1, which was confirmed as Nα-acetylated Tα1 by the elution time in HPLC assay , measured by tandem MS of Q-TOF2, which is same as the native and chemosynthesis Nα-acetylated Tα1. Nα-terminal acetylation was confirmed by m/z of 130.05 for the Nα-terminal amino acid residue, which corresponds to an acetylated serine residue (b).The sequence of recombinant NClick here for additional data file.


In the biphenyl portion of the molecule, the aromatic rings are inclined by 37.8 (2)°.Iodine adds across both triple bonds of 4,4′-bis(prop-2-ynyloxy)biphenyl, yielding the 4,4′-bisbiphenyl title compound, C DOI: 10.1107/S1600536811003874/jh2264Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


CCAAT/enhancer binding protein γ (C/EBPγ) is a member of the C/EBP family of transcription factors, which lacks known activation domains. C/EBPγ was originally described as an inhibitor of C/EBP transactivation potential. However, previous study demonstrates that C/EBPγ augments the C/EBPβ stimulatory activity in lipopolysaccharide induction of IL-6 promoter in a B lymphoblast cell line. These data indicate a complexing functional role for C/EBPγ in regulating gene expression. Furthermore, the expression and function of C/EBPγ during inflammation are still largely unknown. In this study, we demonstrate that C/EBPγ activation was induced by IL-1β treatment in lung epithelial cells. Importantly, we demonstrate for the first time that C/EBPγ plays a critical role in regulating IL-1β-induced IL-6 expression in both mouse primary alveolar type II epithelial cells and a lung epithelial cell line, MLE12. We further provide the evidence that C/EBPγ inhibits IL-6 expression by inhibiting C/EBPβ but not NF-κB stimulatory activity in MLE12 cells. These findings suggest that C/EBPγ is a key transcription factor that regulates the IL-6 expression in alveolar epithelial cells, and may play an important regulatory role in lung inflammatory responses. As one of the major cell types comprising alveolar epithelial tissue, the alveolar type II epithelial cells play an important role in maintaining alveolar integrity by forming the key alveolar barrier, repairing damaged type I cells, and being the source of alveolar surfactant 5′-T(T/G)NNGNAA(T/G)-3′C/EBPα, -β, -δ, -ε, -γ, and -ζ comprise a family of basic region-leucine zipper (bZIP) transcription factors that dimerize through a leucine zipper and bind to DNA through an adjacent basic region. All C/EBP members can form homo- and hetero-dimers with other family members. C/EBPs can activate transcription from promoters that contain a consensus binding site: Little is known about the expression and function of C/EBPγ during inflammation. We evaluated the time course of C/EBP activation in lung epithelial cells by EMSA, using nuclear extracts from MLE12, a lung epithelial cell line, obtained at various time points after IL-1β treatment. As shown in To further determine the C/EBPγ regulation of IL-6 expression, we infected MLE12 cells with adenovirus that could induce C/EBPγ expression (Adeno-C/EBPγ). As shown in To further confirm the inhibitory role of C/EBPγ in IL-6 expression observed in the MLE12 cells, we isolated primary alveolar type II epithelial cells from mouse lung. As shown in Previous studies including ours show that C/EBPβ and NF-κB synergistically activate the IL-6 expression in various immune cells. Thus, we examined the activation of C/EBPs and NF-κB in IL-1β-treated MLE12 cells. As shown in To determine if interaction of both NF-κB and C/EBPβ with the IL-6 promoter region was required for the IL-1β-induced IL-6 expression in MLE12 cells, we transfected MLE12 cells with an IL-6 promoter-luciferase construct or an L-6 promoter-luciferase construct harboring a mutant in either the NF-κB binding site or the C/EBP binding site. As shown in To further determine the role of C/EBPβ in IL-1β-induced IL-6 production, we transfected MLE12 cells with control siRNA or siRNA specific for C/EBPβ. As shown in We reason that C/EBPγ suppresses the IL-6 expression through inhibiting stimulatory C/EBP acitivity. MLE 12 cells were transfected with 2×C/EBP-Luc, a C/EBP-dependent promoter-reporter containing two copies of a C/EBP binding site, together with C/EBPγ expressing plasmid or control plasmid. As shown in Previous study shows that C/EBPγ dramatically augments the activity of C/EBPβ in LPS induction of the IL-6 and IL-8 promoters in a B lymphoblast cell line cis-acting elements of the IL-6 promoter region, such as NF-κB and C/EBPβ/δ. For example, C/EBPβ and -δ have both been shown to activate a reporter gene controlled by the IL-6 promoter in transient expression assays Although alveolar type II epithelial cells only cover about 5% of the alveolar surface area, there is increasing evidence that they play a significant role in lung inflammatory diseases. IL-1β is a critical inducer of immune and inflammatory responses by mediating activation of alveolar type II epithelial cells, leading to pro-inflammatory cytokine production such as IL-6 Our finding that C/EBPγ can regulate IL-1β-induced IL-6 production in alveolar type II epithelial cells is interesting. C/EBPγ has been considered as an inhibitor of other C/EBP family members. For example, C/EBPγ inhibits C/EBPβ-mediated HIV-1 long terminal repeat-driven transcription in human brain cells The exact molecular mechanism whereby C/EBPγ regulates gene expression is not clear. In IL-1β-stimulated alveolar epithelial cells, C/EBPγ exists as both homodimers (C/EBPγ:γ) and heterodimers (C/EBPγ:β) . It is pTaken together, we identified a previously unrecognized role for C/EBPγ in inflammation in alveolar epithelial cells. Not surprisingly, many transcription factors such as NF-κB and C/EBPβ are activated in the acute lung inflammatory reaction. However, our current study suggests that the acute inflammatory response in the lung can also be counter-regulated by other transcription factors such as C/EBPγ . Underst2. Recombinant mouse IL-1β (401-ML) was purchased from R&D Systems, Minneapolis, MN.Murine lung epithelial cells (MLE12) and HEK293 cells were obtained from American Type Culture Collection , and cultured in DMEM/F-12  supplemented with 5% fetal calf serum . Cells were maintained in a humidified incubator at 37°C with 5% COFull-length mouse C/EBPγ cDNA was amplified from total RNA of the mouse lung using reverse transcription-PCR , sequenced, and inserted into pcDNA3.1(+) . Recombinant adenovirus containing mouse C/EBPγ (Adeno-C/EBPγ) was constructed by using BD Adeno-X™ Expression System 1 . To generate the virus, Adeno-C/EBPγ was digested with PacI and transfected to HEK-293 cells according to the manufacturer's instructions. Recombinant adenoviruses were purified by BD Adeno-X virus purification kit , and stored in aliquots at −80°C. The viral stocks were tittered using Adeno-X Rapid Titer Kit . The mouse IL-6 promoter-reporter (−250 to +1), IL-6 promoter-reporter containing a mutated NF-κB binding site, as well as the C/EBPβ and NF-κB p65 expression plasmids were kindly provided by Richard C. Schwartz (Michigan State University). Mouse IL-6 promoter-reporter containing a mutated C/EBP binding site (−161 to −147) was kindly provided by Gail A. Bishop (University of Iowa). NF-κB promoter reporter was obtained from Promega. C/EBP promoter reporter, 2×C/EBP-Luc containing two copies of a C/EBP binding site, was kindly provided by Peter F. Johnson (NCI-Frederick).Cells were grown to 90% confluence, infected with Adeno-GFP and Adeno-C/EBPγ, respectively, at 20 MOI. 24 h later, proteins were harvested for the analysis of C/EBPγ expression. In some experiments, the cells were treated with 20 ng/ml IL-1β for the indicated time. The supernatants were collected for ELISA analysis.4 cells plated in 12-well plates by using 0.5 µg of total DNA and 1.5 µl of Fugene®6 Transfection Reagent  in 50 µl of Opti-MEM I medium . 24 h after transfection, the cells were either incubated with or without 20 ng/ml IL-1β for indicated time. Cell lysates were subjected to luciferase activity analysis by using the Dual-Luciferase Reporter Assay System .Transient transfections were performed with 8×10Transient siRNA transfections were performed in 6-well plates by transfecting MLE 12 cells with control siRNA or C/EBPβ/γ siRNA (40 nM)  using 5 µl Lipofectamine™ 2000 in 500 µl of Opti-MEM I medium . 12 h after siRNA transfection, the cells were treated with or without 20 ng/ml IL-1β for different time points. Supernatants were collected for ELISA.Both MLE12 cells and primary cultured alveolar type II epithelial cells were stimulated by IL-1β for the indicated time. The supernatants were centrifuged at 3000 rpm for 5 min, and the cell-free supernatants were harvested for IL-6 measurements by using a commercially available ELISA kit  according to the manufacturer's protocol.− Reverse Transcriptase . The levels of mRNA of C/EBPγ and GAPDH were determined by qPCR. PCR was performed with primers for C/EBPγ: 5′ primer, 5′-GCA AGC AAA GCA AAA AGA GC-3′ and 3′ primer, 5′-GCT TCC AAC CGT TCA TTC TC-3′; GAPDH: 5′ primer, 5′-GCC TCG TCT CAT AGA CAA GAT G-3′ and 3′ primer, 5′-CAG TAG ACT CCA CGA CAT AC-3′. Following reverse transcription, the cDNA (2 µl) was amplified and quantified using a Sequence Detection System  and a PCR universal protocol as follows: AmpliTaq Gold activation at 95°C for 15 s and, annealing/extension at 60°C for 1 min. The fluorescence of the double-stranded products accumulated was monitored in real time. The relative mRNA levels were normalized to levels of GAPDH mRNA in the same sample. The expression of IL-6 by RT-PCR was determined as previously described Total RNAs were extracted from cells with Trizol  according to the manufacturer's procedure. After isolation, total cellular RNA was incubated with RQ1 RNase-free DNase  to remove contaminating DNA. 2 µg of total RNA was submitted to reverse transcription by using the Superscript II RNase HBoth MLE12 and primary cultured AEC II were lysed in cold RIPA buffer. Samples containing 80 µg protein were electrophoresed in a 12% polyacrylamide gel and transferred to a PVDF membrane. Membranes were incubated with rabbit anti-C/EBPγ antibody (provided by Peter F. Johnson) and rabbit anti-GAPDH antibody , respectively. After 3 washes in TBST, the membranes were incubated with a 1∶5,000 dilution of horseradish peroxidase-conjugated donkey anti-rabbit IgG . The membrane was developed by enhanced chemiluminescence technique according to the manufacturer's protocol .Alveolar type II epithelial cells from C57BL6 mice were isolated and cultured using a method described previously TEM was used to characterize freshly isolated alveolar epithelial type II cells using modified Karnovsky's fixative For immunocytochemistry, the cells were fixed in 3.7% paraformaldehyde and non-specific binding was blocked with blocking buffer for 30 minutes 2, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% (v/v) Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, and complete protease inhibitors  for 10 min on ice. Nuclei were pelleted by centrifugation at 14,000× g for 20 sec at 4°C. Proteins were extracted from nuclei by incubation at 4°C with vigorous vortexing in buffer C , 0.2 mM EDTA, 25% (v/v) glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and complete protease inhibitors . Protein concentrations were determined by Bio-Rad protein assay kit . The EMSA probes were double-stranded oligonucleotides containing a murine IL-6 C/EBP binding site , or a NF-κB consensus oligonucleotide . C/EBP probes were labeled with α [32P] ATP . NF-κB probes were labeled with γ [32P] ATP . DNA binding reactions were performed at room temperature in a 25 µl reaction mixture containing 6 µl of nuclear extract (1 mg/ml in buffer C) and 5 µl of 5× binding buffer (20% (w/v) Ficoll, 50 mM HEPES pH 7.9, 5 mM EDTA, 5 mM dithiothreitol). The remainder of the reaction mixture contained KCl at a final concentration of 50 mM, Nonidet P-40 at a final concentration of 0.1%, 1 µg of poly (dI-dC), 200 pg of probe, bromphenol blue at a final concentration of 0.06% (w/v), and water to final volume of 25 µl. Samples were electrophoresed through 5.5% polyacrylamide gels in 1× TBE at 190 V for approximately 3.5 h, dried under vacuum, and exposed to X-ray film. For supershifts, nuclear extracts were preincubated with antibodies (1 to 2 µg) for 0.5 h at 4°C prior to the binding reaction. The following antibodies were purchased from Santa Cruz, CA: NF-κB p50, p52, p65, RelB, c-Rel, C/EBPα, C/EBPβ, C/EBPδ, C/EBPε, C/EBPγ, and normal rabbit immunoglobulin G.Nuclear extracts of MLE12 cells were prepared as follows. Cells were lysed in 15 mM KCl, 10 mM HEPES (pH 7.6), 2 mM MgClp<0.05. Data sets were analyzed using Student's t test or one-way ANOVA, with individual group means being compared with the Student-Newman-Keuls multiple comparison test.All values were expressed as the mean ± S. E. M. Significance was assigned where


We characterized the ability of mometasone furoate/formoterol (MF/F) combination to improve asthma control in adults/adolescents inadequately controlled on low-, medium-, and high-dose inhaled corticosteroids (ICS).Changes from baseline to endpoint  in Asthma Control Questionnaire (ACQ) scores were assessed in subjects from 3 Phase III trials . The ACQ categorizes asthma symptoms and use of rescue-medication using a 7-point scale . In two placebo controlled trials, subjects were randomized to receive MF/F (100/10μg or 200/10μg), MF (100μg or 200μg), F (10μg), or placebo ; In a non-placebo controlled trial, subjects were randomized to receive MF/F 200/10μg, MF/F 400/10μg, or MF 400μg .Baseline ACQ scores (1.23–1.38 (MF/F 100/10μg study), 1.41–1.47 (MF/F 200/10μg study), and 1.83–1.87 (MF/F 200/10 and 400/10μg study) indicated that subjects in all trials were not well controlled (<0.75) on ICS monotherapy. MF/F yielded ACQ improvements  vs MF , and deteriorations with F 10μg  and placebo . MF/F 400/10μg improved asthma control by –0.51 compared with –0.33 for MF 400μg monotherapy. Improvements for MF/F at all doses achieved minimal importance difference of ≥0.5 point increase.MF/F showed clinically important improvement in asthma control at all strengths and was better than MF, F and placebo.


In the crystal, the phenol O, amine N and pyridinium N atoms are hydrogen-bond donors to the O atoms of the nitrate counter-ions. These hydrogen bonds lead to the formation of a layer in the crystal.The planes of the aromatic rings in the cation of the title salt, C For 2- = 0.043wR(F2) = 0.116S = 1.081458 reflections184 parameters5 restraintsH atoms treated by a mixture of independent and constrained refinementmax = 0.20 e Å−3Δρmin = −0.17 e Å−3ΔρRAPID-AUTO  global, I. DOI: 10.1107/S1600536812031352/xu5582Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812031352/xu5582Isup3.cmlSupplementary material file. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


Each Ni2+ cation is coordinated by a 1,10-phenanthroline ligand, a bidentate benzene-1,2,4,5-tetracarboxylate (btec) tetra-anion and two water molecules to generate a distorted cis-NiN2O4 octahedral coordination geometry. The btec species bridges the metal ions. In the crystal, the clusters and uncoordinated water molecules are linked by O—H⋯O hydrogen bonds and π–π interactions [shortest centroid–centroid separation = 3.596 (2) Å] to form a three-dimensional network.The asymmetric unit of the title compound, [Ni DOI: 10.1107/S1600536812004618/hb6579Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


II ion in the title complex, [PtI2(C14H10N4)], exists in a distorted square-planar environment defined by the two pyridine N atoms of the chelating 2,3-di-2-pyridylpyrazine ligand and two iodide anions. The pyridine rings are inclined to the least-squares plane of the PtI2N2 unit [maximum deviation = 0.070 (3) Å] at 66.1 (2) and 65.9 (2)°; the pyrazine ring is perpendicular to this plane [dihedral angle = 89.7 (2)°]. Two intermolecular C—H⋯I hydrogen bonds, both involving the same I atom as hydrogen-bond acceptor, generate a layer structure extending parallel to (001). Molecules are stacked in columns along the a axis. Along the b axis, successive molecules stack in opposite directions.The Pt DOI: 10.1107/S1600536812023501/ng5273Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


II ion in the title complex, [PtBr2(C14H10N4)], has a slightly distorted square-planar environment defined by the two pyridyl N atoms of the chelating 2,3-di-2-pyridylpyrazine ligand and two bromide anions. In the crystal, the pyridyl rings are considerably inclined to the least-squares plane of the PtBr2N2 unit [maximum deviation = 0.064 (2) Å] with dihedral angles of 65.2 (2) and 66.0 (2)°. The nearly planar pyrazine ring [maximum deviation = 0.020 (5) Å] is almost perpendicular to the unit plane with a dihedral angle of 89.2 (2)°. Two independent weak intermolecular C—H⋯Br hydrogen bonds, both involving the same Br atom as a hydrogen-bond acceptor, give rise to chains running along the a and b axes, forming a layer structure extending parallel to (001). The complexes are stacked in columns along the a axis. When viewed down the b axis, the successive complexes stack in the opposite direction.The Pt II complexes, see: Granifo et al. ] = 0.032                           wR(F                           2) = 0.067                           S = 0.99                           3323 reflections190 parametersH-atom parameters constrainedmax = 1.66 e Å−3                        Δρmin = −0.84 e Å−3                        Δρ                                 SMART  used to solve structure: SHELXS97  global, I. DOI: 10.1107/S160053681103412X/xu5295Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The ZnII ions are connected by 5-hydroxyisophthalate (hbdc) and 2,2′-diethyl-1,1′-di-1H-imidazole (pbie) ligands, forming a threefold interpenetrating diamondoid framework. In the pbie ligand, one of the ethylimidazole groups is disordered over two positions, with a site-occupancy ratio of 0.670 (9):0.330 (9). An intermolecular O—H⋯O hydrogen bond is formed between the hydroxy and carboxylate groups of the hbdc ligands.In the title coordination polymer, [Zn(C DOI: 10.1107/S1600536811041456/hy2473Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Echinacea purpurea, a purple coneflower plant of the compositae family (Asteraceae), is native to North America and commonly used as an herbal supplement to enhance immune function. Echinacea purpurea has been shown to stimulate macrophage activity which is a known stimulator of nitric oxide (NO) production. Echinacea purpurea supplementation  in untrained (42.5 ± 1.6 mL·kg-1·min-1) males was shown to elicit a 63% increase (p < 0.05) in serum erythropoietin (EPO) following two weeks of supplementation. This is supported in part by earlier findings which indicated that four weeks of Echinacea purpurea supplementation demonstrated a non-significant increase in maximal oxygen uptake (VO2max). It is plausible that Echinacea-induced EPO production may stimulate physiological responses independent of and/or in addition to erythropoiesis. There is also evidence suggesting EPO has vasculo-protective effects including the activation of endothelial nitric oxide synthase (eNOS). Based on these findings, a proposed non-hematological response to the Echinacea-induced increase in EPO could be enhanced NO production. The purpose of this investigation was to determine whether six weeks of oral Echinacea purpurea supplementation augmented NO production as a result of an Echinacea-induced increase in EPO and/or Echinacea-induced macrophage activity.2max = 52.9 ± 0.9 mL·kg-1·min-1 were randomly grouped using a matched-pair, double-blind design and self-administered 8,000 mg·d-1  of either Echinacea purpurea (ECH) (n=12) or placebo (PLA) (n=12) for 42 consecutive days. Blood samples were collected prior to supplementation (day-0) and every two weeks during the supplementation period  and were analyzed for nitrite and total nitrite (nitrite/nitrate) concentrations. Separate 2 × 4 (Group × Time) factorial ANOVA with repeated measures on time were used to determine statistical differences with significance set at p ≤ 0.05.Twenty-four males (mean ± SE): age = 25.2 ± 1.4 yr, height = 178.1 ± 1.4 cm, mass = 78.1 ± 1.6 kg, percent body fat = 12.7 ± 0.9 %, VO-1) , nitrate (µmol·L-1) , or nitrite/nitrate (µmol·L-1)  or for any of the intermediate  measurement points.There were no significant interaction, group, or time effects observed following six weeks of supplementation for nitrite  did not significantly change nitrite, nitrate, or nitrite/nitrate. Therefore, Echinacea purpurea may not be an effective herbal supplement for enhancing NO production in apparently healthy, recreationally active, males with above average aerobic fitness (VO2max = 52.9 ± 0.9 mL·kg-1·min-1).These results suggest that six weeks of oral


The SnIV atom in the anion is N,O-chelated by the hydroxyquinolinate in a cis-SnNOCl4 octahedral geometry. In the crystal, the cation, anion and solvent molecules are linked by N—H⋯O, O—H⋯O and O—H⋯Cl hydrogen bonds, generating a three-dimensional network.In the reaction of 8-hydroxyquinoline, 2-methyl-8-hydroxyquinoline and stannic chloride, the 2-methyl-8-hydroxyquinoline is protonated, yielding the disolvated title salt, (C DOI: 10.1107/S1600536811001954/si2328Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


In the polymer, inversion-related MnII ions are bridged by the bis-chelating 2,2′-bipyrimidine (bpym) ligands, thereby forming a chain structure along the c-axis direction, and are six-coordinated in a distorted cis-N4Br2 octahedral environment by four N atoms of twofold-related bpym ligands and twofold-related bromide anions. In the crystal, the complex polymers and solvent water molecules are linked by intermolecular O—H⋯Br and C—H⋯O hydrogen bonds, forming a two-dimensional layered structure extending parallel to the ac plane.The asymmetric unit of the title compound, {[MnBr II complex polymer [MnCl2(bpym)]n.2nH2O, which is isotypic to the title compound, see: Armentano et al. ]·2H2O = 0.033                           wR(F                           2) = 0.097                           S = 1.14                           1524 reflections78 parametersH-atom parameters constrainedmax = 0.92 e Å−3                        Δρmin = −0.76 e Å−3                        Δρ                                 SMART  used to solve structure: SHELXS97  global, I. DOI: 10.1107/S1600536811049919/pk2362Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials: