Host Antimicrobial Peptides: the promise of new treatment strategies against Tuberculosis

Tuberculosis (TB) continues to be a devastating infectious disease and remerges as a global health emergency due to an alarming rise of antimicrobial resistance to its treatment. Despite of the serious effort that has been applied to develop effective antitubercular chemotherapies, the potential of antimicrobial peptides (AMPs) remains underexploited. A large amount of literature is now accessible on the AMP mechanisms of action against a diversity of pathogens; nevertheless, research on their activity on mycobacteria is still scarce. In particular, there is an urgent need to integrate all available interdisciplinary strategies to eradicate extensively drug-resistant Mycobacterium tuberculosis strains. In this context, we should not underestimate our endogenous antimicrobial proteins and peptides as ancient players of the human host defense system. We are confident that novel antibiotics based on human AMPs displaying a rapid and multifaceted mechanism, with reduced toxicity, should significantly contribute to reverse the tide of antimycobacterial drug resistance. In this review, we have provided an up to date perspective of the current research on AMPs to be applied in the fight against TB. A better understanding on the mechanisms of action of human endogenous peptides should ensure the basis for the best guided design of novel antitubercular chemotherapeutics

Expresión del péptido cecropina A-1 de drosophila melanogaster en células endoteliales de bovino

Staphylococcus aureus es el principal agente causal de la mastitis bovina, enfermedad que ocasiona pérdidas económicas considerables en la industria lechera. El tratamiento convencional de la mastitis se basa en la administración de antibióticos de amplio espectro

Determination of the Potential Thermal Gradient for the Mexican Pacific Ocean

The energy potential of the oceanic thermal gradients of the Mexican Pacific Ocean was valued theoretically, using seasonal oceanographic data on surface and 1000 m depth ocean temperatures from 1955 to 2013, taken from the World Ocean Database (WOD). The study was carried out to determine possible sites for Ocean Thermal Energy Conversion (OTEC), assuming that the minimum usable gradient is 20 °C and the maximum profitable distance from the extraction site to the shore is 10 km. Geographic Information System tools were used to compute thermal gradients and distances to shore all along the Mexican coast. Then, the optimal sites were identified. The results show that the best sites for OTEC exploitation are found in the southern Pacific coast on the littoral of the states of Guerrero and Oaxaca

Peptide IDR-1002 Inhibits NF-kB Nuclear Translocation by Inhibition of IkBalpha Degradation, and Activates p38/ERK1/2-MSK1 Dependent CREB Phosphorylation in Macrophages Stimulated With Lipopolysaccharide

The inflammatory response is a critical molecular defense mechanism of the innate immune system that mediates the elimination of disease-causing bacteria. Repair of the damaged tissue, and the reestablishment of homeostasis, must be accomplished after elimination of the pathogen. The innate defense regulators (IDRs) are short cationic peptides that mimic natural host defense peptides and are effective in eliminating pathogens by enhancing the activity of the immune system while controlling the inflammatory response. Although the role of different IDRs as modulators of inflammation has been reported, there have been only limited studies of the signaling molecules regulated by this type of peptide. The present study investigated the effect of IDR-1002 on NF-kB and CREB transcription factors that are responsible for triggering and controlling inflammation, respectively, in macrophages. We found that TNF-alpha and COX-2 expression, IkBalpha phosphorylation, and NF-kB nuclear translocation were strongly inhibited in macrophages pre-incubated with IDR-1002 and then stimulated with lipopolysaccharide (LPS). IDR-1002 also increased CREB phosphorylation at Ser133 via activation of the p38/ERK1/2-MSK1/2 signaling pathways without detectable expression of the cytokines IL-4, IL-10 and IL-13 involved is suppressing inflammation or alternative activation. Transcriptional activation of NF-kB and CREB is known to require interaction with the transcriptional co-activator CREB-binding protein (CBP). To test for CBP-NF-kB and CBP-CREB complex formation, we performed co-immunoprecipitation assays. These assays showed that IDR-1002 inhibited the interaction between CBP and NF-kB in macrophages stimulated with LPS, which might explain the inhibition of TNF-alpha and COX-2 expression. Furthermore, the complex between CBP and CREB in macrophages stimulated with IDR-1002 was also inhibited, which might explain why IDR-1002 did not lead to expression of IL-4, IL-10 and IL-13, even though it induced an increase in phospho-CREB relative abundance. In conclusion, our results indicated that IDR-1002 has a dual effect. On one hand, it inhibited NF-kB nuclear translocation through a mechanism that involved inhibition of IkBalpha phosphorylation, and on the other it activated a protein kinase signaling cascade that phosphorylated CREB to selectively influence cytokine gene expression. Based on or results we think IDR-1002 could be a potential good biopharmaceutical candidate to control inflammation

The Glycogen Synthase Kinase 3α and β Isoforms Differentially Regulates Interleukin-12p40 Expression in Endothelial Cells Stimulated with Peptidoglycan from Staphylococcus aureus.

Glycogen synthase kinase 3 (GSK3) is a constitutively active regulatory enzyme that is important in cancer, diabetes, and cardiovascular, neurodegenerative, and psychiatric diseases. While GSK3α is usually important in neurodegenerative and psychiatric diseases GSK3β is fundamental in the inflammatory response caused by bacterial components. Peptidoglycan (PGN), one of the most abundant cell-wall structures of Gram-positive bacteria, is an important inducer of inflammation. To evaluate whether inhibition of GSK3α and GSK3β activity in bovine endothelial cells (BEC) regulates the expression of the pro-inflammatory cytokine IL-12p40, we treated BEC with SDS-purified PGN from Staphylococcus aureus. We found that PGN triggered a TLR2/PI3K/Akt-dependent phosphorylation of GSK3α at Ser21, GSK3β at Ser9, and NF-κB p65 subunit (p65) at Ser536, and the phosphorylation of GSK3α was consistently higher than that of GSK3β. The expression of IL-12p40 was inhibited in BEC stimulated with PGN and pre-treated with a specific neutralizing anti-TLR2 antibody that targets the extracellular domain of TLR2 or by the addition of Akt-i IV (an Akt inhibitor). Inhibition of GSK3α and GSK3β with LiCl or SB216763 induced an increase in IL-12p40 mRNA and protein. The effect of each isoform on IL-12p40 expression was evaluated by siRNA-gene expression silencing of GSK3α and GSK3β. GSK3α gene silencing resulted in a marked increase in IL-12p40 mRNA and protein while GSK3β gene silencing had the opposite effect on IL-12p40 expression. These results indicate that the TLR2/PI3K/Akt-dependent inhibition of GSK3α activity also plays an important role in the inflammatory response caused by stimulation of BEC with PGN from S. aureus

A working model of the signaling pathway involved on the IL-12p40 expression induced by PGN in BEC.

<p>PGN induces the activation of the TLR2/PI3K/Akt pathway, which in turn induces GSK3α and GSK3β phosphorylation/inhibition and this in turn up- or down-regulates IL-12p40 expression. Anti-TLR2, Wortmannin (PI3K inhibitor), LY-294002 (PI3K inhibitor) or SH-5 (Akt inhibitor) blocks the phosphorylation/inhibition of GSK3α and GSK3β. LiCl (GSK3 inhibitor), SB-216763 (GSK3 inhibitor) or siRNA GSK3α increases the expression of IL-12p40 while siRNA GSK3β decreases the expression of IL-12p40. Not represented in the diagram is the decrease in IL-12p40 expression when TLR2 is blocked with anti-TLR2 and when Akt is inhibited with Akt-i IV.</p

PGN induces PI3K/Akt-dependent phosphorylation of GSK3α and GSK3β in BEC.

<p>A-B) BEC were untreated and unstimulated (U1 and U2), stimulated with 10 μg/mL of PGN for 30 min (P1 and P2) or treated with either 10 μM of LY294002 (LY), 100 nM of Wortmannin (Wort) or 10 μM of SH-5 for 30 min, and then stimulated with 10 μg/mL of PGN for 30 min. Protein extracts were analyzed by western blot and probed with monoclonal antibodies against the phosphorylated forms of GSK3α (pGSK3α Ser21) or GSK3β (pGSK3β Ser9. To verify for equal amount of proteins, blots were stripped and reprobed with an antibody that recognizes the nonphosphorylated form of GSK3β. Blots are representative of three independent experiments. Graphs indicate the band intensity obtained by densitometric analysis. In each graph, the densitometric control values plotted were the average of U1 + U2 while the values plotted for the PGN-stimulated cells were the average of P1 + P2. Results are expressed as the mean ± S.E.M. (<i>n</i> = 3). *<i>p</i> < 0.05; **<i>p</i> < 0.01, compared with the unstimulated control.</p

Journal of research in character education

<p>A) BEC were left untransfected and unstimulated (-), stimulated with 10 μg/mL of PGN for 4 h, transfected with control siRNA (siRNA control), transfected with control siRNA and then stimulated with 10 μg/mL of PGN for 4 h, transfected with siRNA targeting GSK3β (siRNA GSK3β1) or transfected with siRNA targeting GSK3β (siRNA GSK3β1) and then stimulated with 10 μg/mL of PGN for 4 h. B) BEC were left untransfected and unstimulated (-), stimulated with 10 μg/mL of PGN for 9 h, transfected with control siRNA (siRNA control), transfected with control siRNA and then stimulated with 10 μg/mL of PGN for 9 h, transfected with siRNA targeting GSK3β (siRNA GSK3β1) or transfected with siRNA targeting GSK3β (siRNA GSK3β1) and then stimulated with 10 μg/mL of PGN for 9 h. Total RNA was extracted and relative transcript level of IL-12p40 was quantitated by qRT-PCR, using the delta-delta Ct method, and amplification of β-actin as a reference gene (A). Cell-free supernatants were analyzed by ELISA for production of IL-12p40 (B). Results are expressed as the mean ± S.E.M. (<i>n</i> = 3). *<i>p</i> <0.05. All data were compared with the untreated and untransfected controls.</p

PGN induces IL-12p40 expression through TLR2/Akt activation and GSK3 inhibition in BEC.

<p>A) BEC were left untreated and unstimulated (0) or stimulated with 10 μg/mL of PGN-prep for 2, 4 or 8 h. B) BEC were stimulated with 10 μg/mL of PGN for 4 h, treated with 5 μg/mL of neutralizing anti-TLR2 for 60 min, or treated with 5 μg/mL of anti-TLR2 for 60 min and then stimulated with 10 μg/mL of PGN for 4 h. C) BEC were stimulated with 10 μg/mL of PGN for 9 h or treated with 5 μg/mL of anti-TLR2 for 60 min and then stimulated with 10 μg/mL of PGN for 9 h. D) BEC were stimulated with 10 μg/mL of PGN for 9 h or pretreated with 1 μM of Akt inhibitor IV (Akt-i IV) for 30 min and then stimulated with 10 μg/mL for 9 h. E) BEC were treated with 10 mM NaCl for 60 min, stimulated with 10 μg/mL of PGN for 9 h, treated with 10 mM of LiCl for 60 min or treated with 10 mM of LiCl for 60 min and then stimulated with 10 μg/mL of PGN for 9 h. F) BEC were treated with 10 μM of DMSO, stimulated with 10 μg/mL of PGN for 9 h, treated with 10 μM of SB 216763 (SB) for 30 min or treated with 10 μM of SB 216763 (SB) for 30 min and then stimulated with 10 μg/mL of PGN for 9 h. As controls, in B-F BEC were left untreated and stimulated (-). Total RNA was extracted and relative transcript level of IL-12p40 was quantitated by qRT-PCR using the delta-delta Ct method, and amplification of β-actin as a reference gene (A-B). Cell-free supernatants were analyzed by ELISA for production of IL-12p40 (C-F). Results are expressed as the mean ± S.E.M. (<i>n</i> = 3). *<i>p</i> <0.05; **<i>p</i> <0.01. All data were compared with the untreated and unstimulated controls.</p