Early methodist experience: some prototypical accounts

A great many 20th century studies of 18th century Methodism concern John Wesley himself, and even those which promise to tell you about early Methodist beliefs and activities often turn out to be largely based on Wesley’s alone. Few have concerned themselves with the humble folk who followed him. Yet it is impossible properly to understand even why Wesley himself believed and behaved in the way he did without taking account of the minds and desires of his disciples

Morphine-mediated induction of PDGF-B chain expression in human brain microvascular endothelial cells.

<p>(<b>A</b>) Dose-dependent induction of PDGF-BB protein by morphine (10⁻⁵, 10⁻⁶ and 10⁻⁷ M) in human brain microvascular endothelial cells (HBMECs). (<b>B</b>) Time-dependent induction of PDGF-BB protein by morphine (10⁻⁷ M) in HBMECs. (<b>C</b>) Opioid receptor antagonist naltrexone (1 µM) abrogates morphine-mediated induction of PDGF-BB. (<b>D</b>) Representative picture of PDGF-B chain staining in HBMECs treated with morphine for 12 hrs. Scale bar = 5 µm. (<b>E</b>) Induction of PDGF-B mRNA in HBMECs exposed to morphine after 6 hrs. All the data are presented as mean±SD of three individual experiments. *p<0.05, ***p<0.001 vs control group; ###p<0.001 vs morphine group.</p

Egr-1 expression is up-regulated in HBMECs exposed to morphine.

<p>(<b>A</b>) Time-dependent activation of Egr-1 protein expression by morphine in HBMECs. (<b>B</b>) Representative image of Egr-1 staining in HBMECs treated with morphine. Scale bar = 5 µm. Pretreatment of HBMECs with the inhibitors specific for Erk1/2-U0126 20 µM,JNK-SP600125 20 µM (<b>C</b>) and p38-SB203580 20 µM (<b>D</b>) but not Akt-LY294002 10 µM (<b>C</b>) pathways abrogates morphine-mediated induction of Egr-1. All the data are presented as mean±SD of three individual experiments. ***p<0.001 vs control group; #p<0.05, ###p<0.001 vs morphine group.</p

STAT-1α and NF-κB play a role in the increased induction of CXCL10 by HIV-1 Tat and the cytokines in U-87 astrocytes.

<p>A) Western Blot analysis of nuclear lysates collected from cells untreated, HIV-1 Tat treated, IFN-γ and TNF-α treated, or treated with Tat in combination with the cytokines for 60 min. The blots were probed with antibodies against phospho-NF-κB p65 and phospho-STAT-1α. Antibodies against β-actin were used to reprobe the blots for normalization. B), C) Densitometric scans illustrating the ratio of phospho-NF-κB p65 and phospho-STAT-1α to β-actin levels. E) Activation of these transcription factors was shown to be involved in the increased expression of CXCL10 through inhibition of the NF-κB by TPCK and the inhibition of the Jak/STAT pathway by a Jak I inhibitor. The data represents the mean±SD from three independent experiments (***, p<0.001).</p

Effect of morphine on BBB permeability <i>in vitro</i>.

<p>Morphine-mediated increase in BBB permeability was ameliorated in HBMECs pretreated with either naltrexone or PDGF-BB neutralizing antibody. All the data are presented as mean±SD of four individual experiments. *p<0.05 vs control group; #p<0.05 vs morphine-treated group.</p

PDGF-BB induces down regulation of ZO-1 in HBMECs.

<p>(<b>A</b>) Time-dependent reduction of ZO-1 protein expression by morphine in HBMECs. (<b>B</b>) Treatment of cells with either the opioid receptor antagonist (naltrexone) or PDGF-BB neutralizing antibody results in inhibition of ZO-1 expression. All the data are presented as mean±SD of at least three individual experiments. ***p<0.001 vs control group; ###p<0.001 vs morphine-treated group.</p

PDGF receptor is involved in PDGF-CC neuroprotection.

<p>(<b>A</b>) Expression of PDGF-αR and βR in SH-SY5Y cells by RT-PCR. (<b>B</b>) SH-SY5Y cells were exposed to PDGF-CC (50 ng/ml) for 30 min in presence or absence of tyrosine kinase inhibitor STI-571 (1 µM). Phosphorylation of PDGF-αR & βR was detected by co-immunoprecipitation. (<b>C</b>) Cell viability of SH-SY5Y cells exposed to PDGF-CC and/or Tat in presence or absence of tyrosine STI-571 was assessed by MTT assay. Figure is a representative of three independent experiments. All data in these figures are presented as mean ± SEM of three individual experiments. *p<0.05 vs control, #p<0.05 vs Tat-treated group, %p<0.05 vs PDGF-CC plus Tat-treated group.</p

Akt is involved in PDGF-CC mediated neuronal protection.

<p>(<b>A</b>) SH-SY5Y cells were treated with PDGF-CC (50 ng/ml) and/or Tat (14 nM) in the absence or presence of PI3K/Akt inhibitor PI-103 (0.2 µM) followed by detection of Akt phosphorylation by western blot. (<b>B</b>) SH-SY5Y cells were treated as described for 24 h, and subjected to MTT assay. In panels C & D, SH-SY5Y cells were infected with either the wild type (<b>C</b>) or dominant-negative form of Akt adenovirus (<b>D</b>). (E) Equal expression levels of Akt in SH-SY5Y cells infection with Akt-DN or Akt-WT adenovirus. MTT assay was used to detect the cell viability of SH-SY5Y cells exposed to PDGF-CC and/or Tat. All data in the panels B, C & D are presented as mean ± SEM of three individual experiments. *P<0.05, **P<0.01 vs control; #p<0.05, ##p<0.01 vs Tat-treated group.</p

TRPC 1 channel is critical for PDGF-CC-mediated neuroprotection.

<p>(<b>A</b>) SH-SY5Y cells were labeled with Fluo-4 prior to treatment with PDGF-CC followed by recording of the fluorescence density representing the level of intracellular Ca²⁺. A representative change of fluorescence intensity is shown in the lower panel. The arrow indicates the time of addition of PDGF-CC. Number in upper panel shows the recording time. (<b>B</b>) Change in intracellular Ca²⁺ induced by PDGF-CC (50 ng/ml) in the presence of absence of pharmacological inhibitors (STI-571, 1 µM; EGTA, 2 mM; SKF96365, 20 µM; 2APB, 100 µM; U73122, 1 µM). All the data in these figures are presented as mean ± SEM of three individual experiments. ***p<0.001 vs control; ##p<0.01 vs PDGF-CC-treated group. (<b>C</b>) Cell viability in SH-SY5Y cells pretreated with SKF96365 (20 µM) by MTT assay. All the data in these figures are presented as mean ± SEM of three individual experiments. *p<0.05 vs control, #p<0.05 vs Tat-treated group. (<b>D</b>) SH-SY5Y cells were transfected with 100 nM siRNA targeting TRPC1, 5 or 6. After 24 h cells were lysed, mRNA was isolated and subjected to RT-PCR. Arrow indicates the RT-PCR product bands. (<b>E</b>) SH-SY5Y cells seeded in 96-well plate were transfected with TRPC 1, 5 or 6 siRNAs (100 nM) for 24 h later followed by treatment of cells with PDGF-CC and/or Tat. MTT assay was conducted to detect cell viability. All the data in these figures are presented as mean ± SEM of three individual experiments. *p<0.05 vs control; ##p<0.01 vs Tat-treated group.</p

Tat in combination with IFN-γ and TNF-α increases CXCL10 protein.

<p>Increased CXCL10 protein expression in (A) primary human astrocytes or (B) U-87 and A172 astrocytes cell lines treated with HIV-1 Tat alone, the cytokines IFN-γ and TNF-α, or Tat and the cytokines together for 24 and 12 hours respectively. Both primary and cell line astrocytes showed a significant increase in CXCL10 protein levels in the cells treated with HIV-1 Tat and the cytokines, than with either treatment alone. Treatment of U-87 and A172 cells with heat inactivated (HI) Tat in conjunction with the cytokines did not lead to an increase in CXCL10 protein levels compared with cells treated with the cytokines alone. The data represents the mean±SD from three independent experiments (**, p<0.01, ***, p<0.001).</p