A new class of pluripotent stem cell cytotoxic small molecules

10.1371/journal.pone.0085039PLoS ONE93-POLN

Attenuated Bordetella pertussis Protects against Highly Pathogenic Influenza A Viruses by Dampening the Cytokine Storm▿

The threat of a pandemic spread of highly virulent influenza A viruses currently represents a top global public health problem. Mass vaccination remains the most effective way to combat influenza virus. However, current vaccination strategies face the challenge to meet the demands in a pandemic situation. In a mouse model of severe influenza virus-induced pneumonitis, we observed that prior nasal administration of an attenuated strain of Bordetella pertussis (BPZE1) provided effective and sustained protection against lethal challenge with two different influenza A virus subtypes. In contrast to most cross-protective effects reported so far, the protective window offered upon nasal treatment with BPZE1 lasted up to at least 12 weeks, suggesting a unique mechanism(s) involved in the protection. No significant differences in viral loads were observed between BPZE1-treated and control mice, indicating that the cross-protective mechanism(s) does not directly target the viral particles and/or infected cells. This was further confirmed by the absence of cross-reactive antibodies and T cells in serum transfer and in vitro restimulation experiments, respectively. Instead, compared to infected control mice, BPZE1-treated animals displayed markedly reduced lung inflammation and tissue damage, decreased neutrophil infiltration, and strong suppression of the production of major proinflammatory mediators in their bronchoalveolar fluids (BALFs). Our findings thus indicate that protection against influenza virus-induced severe pneumonitis can be achieved through attenuation of exaggerated cytokine-mediated inflammation. Furthermore, nasal treatment with live attenuated B. pertussis offers a potential alternative to conventional approaches in the fight against one of the most frightening current global public health threats

Table of structure, molecular mass, ORAC and DPPH antioxidant data for 6 JC analogues (JC005, JC007, JC010, JC011, JC017 and JC040).

<p>Table of structure, molecular mass, ORAC and DPPH antioxidant data for 6 JC analogues (JC005, JC007, JC010, JC011, JC017 and JC040).</p

JC011 treated PSCs fail to form teratomas in SCID mice and can be effectively used to enrich for specialized cells.

<p>Teratoma sections from SCID mice. Controls showing typical teratoma tissue organization representative of all 3 germ layers, A = Cartilage, B = Gut/Intestine, C, D = JC11 treated sample showing plain muscle tissue with no teratoma growth (<b>A–D</b>). BGO1V and primary neonatal cardiomyocytes were mixed and seeded in pre-determined ratios (10∶90, 20∶80, 30∶70, 60∶40 and 50∶50). The mixed cultures were treated with JC011 at 20 µM for 12 hrs followed by SSEA-4 and TRA-1-60 FACS analysis to determine enrichment ratios (<b>E</b>). Time course cell viability data for JC011 (20 µM) treated primary neonatal cardiomyocytes indicate that cardiomyocytes maintain high cell viability (>95%) even after a 5-day incubation period with JC011 (<b>F</b>).</p

Comparative microarray analysis reveals involvement of the PERK/ATF4/DDIT3 ER stress pathways.

<p>Clustering of key differentially upregulated ER stress genes in BGO1V following 6 µM JC011 (<b>A</b>). Top 10 components of the PERK/ATF4/DDIT3 ER stress pathway that were found to be rapidly upregulated in JC011 treated BGO1V cells (<b>B</b>). qRT-PCR confirmation of upregulated UPR/ER stress pathway genes following JC011 treatment (<b>C</b>).</p

siRNA knockdown of the key ER stress genes ATF-4 and DDIT3 leads to reduced sensitivity towards JC011 in NCCIT cells.

<p>ATF-4 and DDIT3 were silenced via siRNA knockdown. Sensitivity towards JC011 was attenuated in DDIT3 knockdown and ATF-4 knockdown (P<0.05) NCCIT cells thereby confirming involvement of the PERK/ATF4/DDIT3 ER stress pathway in JC011 mediated cytotoxicity (<b>A–E</b>). qRT-PCR confirmation of ATF-4 and DDIT3 transcript knockdown (<b>F</b>). Cell viability figures were normalized to untreated controls and reported as mean ± S.D. of three independent experiments (n = 3). Statistical analysis was performed with the Student's T-test.</p

Dose response cytotoxicity data.

<p>Dose response curves for 3 PSC cell lines (BGO1V, H9 and iPS-Foreskin-1) following treatment with JC011 (<b>A</b>). Dose response curves for 3 specialized somatic cell lines (MRC-5, human primary neurons and human neonatal cardiomyocytes) treated with JC011 (<b>B</b>). Time course cytotoxicity analysis for differentiating BGO1V cultures treated with JC011 at 4-day intervals following bFGF withdrawal (<b>C</b>). Dose response curves for 3 JC011 analogues (JC005, JC017 and JC040) showing that JC040 with a longer alkyl side-chain is the most potent analogue (<b>D</b>). Cell viability values were normalized to untreated controls and reported as mean ± S.D. of three independent experiments (n = 3).</p

JC011 and JC040 reduce ROS levels in PSCs.

<p>Cytotoxic JC011 and JC040 induce a small but rapid reduction of intracellular ROS levels in BGO1V cells as confirmed by DCHF-DA FACS analysis 3 hrs after treatment (<b>A</b>). JC007 (non-cytotoxic) does not alter endogenous ROS levels while JC005 (non-cytotoxic) increases ROS levels but without any corresponding cytotoxicity to BGO1V (<b>B</b>). FACS histograms are representative outcomes of 4 independent experiments. DCHF-DA stained JC011 treated BGO1V cells (<b>C, D</b>). DCHF-DA stained JC005 treated BGO1V cells (<b>E, F</b>).</p

Morphology of BGO1V-MEF cultures following treatment with 20 mM JC011 for 12 hrs.

<p>“Hollowing-out” effect in BGO1V-MEF cultures, feeders remain intact and viable (<b>A–D</b>). Trypan blue staining of untreated control BGO1V single cells (<b>E</b>). Trypan blue staining of JC011 treated BGO1V single cells showing an increase in Trypan blue uptake (<b>F</b>). Propidium Iodide DNA content analysis of control untreated BGO1V cells (<b>G</b>). Propidium Iodide DNA content analysis of JC011 treated BGO1V after 6 hrs showing a rapid increase in the sub-G1 fraction (<b>H</b>). R1 = sub-G1 fraction, R2 = G1 fraction, R3 = early/late S-phase fraction, R4 = G2/M fraction.</p