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1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model.

Gonzalez-Curiel I, Trujillo V, Montoya-Rosales A, Rincon K, Rivas-Calderon B, deHaro-Acosta J, Marin-Luevano P, Lozano-Lopez D, Enciso-Moreno JA, Rivas-Santiago B - PLoS ONE (2014)

Bottom Line: Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant.These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration.In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Unit Zacatecas, Mexican Institute of Social Security-IMSS, Zacatecas, Mexico; Department of Immunology, Faculty of Medicine, Autonomous University of San Luis Potosi, San Luis Potosi, Mexico.

ABSTRACT
Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

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Production of HBD-2 and LL-37 in primary cell cultures from healthy donors and DFU patient stimulated with 1,25(OH)2D3 and L-isoleucine.HBD-2 (A) and LL-37 (B) production was measured by ELISA assay from supernant concentrates. Data is expressed as median ± interquartile range. Statistics were calculated by Kruskal-Wallis test. In each experimental group, n = 15.
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pone-0111355-g003: Production of HBD-2 and LL-37 in primary cell cultures from healthy donors and DFU patient stimulated with 1,25(OH)2D3 and L-isoleucine.HBD-2 (A) and LL-37 (B) production was measured by ELISA assay from supernant concentrates. Data is expressed as median ± interquartile range. Statistics were calculated by Kruskal-Wallis test. In each experimental group, n = 15.

Mentions: Given that not all transcribed mRNA is traduced into AMPs, we determined the production LL-37 and HBD-2 in KCM from primary cells cultures by Enzyme-linked Immunosorbent Assay (ELISA). The results showed that 1,25(OH)2D3 induced HBD-2 secretion in 5 out of 15 cell cultures from DFUs reaching levels up to 8000 ng/ml even above of the levels seen for the cell culture stimulated from healthy donors. Whereas, L-isoleucine treatment induced a mild HBD-2 production only in 3 out of 15 cell cultures from DFU (Figure 3a).


1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model.

Gonzalez-Curiel I, Trujillo V, Montoya-Rosales A, Rincon K, Rivas-Calderon B, deHaro-Acosta J, Marin-Luevano P, Lozano-Lopez D, Enciso-Moreno JA, Rivas-Santiago B - PLoS ONE (2014)

Production of HBD-2 and LL-37 in primary cell cultures from healthy donors and DFU patient stimulated with 1,25(OH)2D3 and L-isoleucine.HBD-2 (A) and LL-37 (B) production was measured by ELISA assay from supernant concentrates. Data is expressed as median ± interquartile range. Statistics were calculated by Kruskal-Wallis test. In each experimental group, n = 15.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206472&req=5

pone-0111355-g003: Production of HBD-2 and LL-37 in primary cell cultures from healthy donors and DFU patient stimulated with 1,25(OH)2D3 and L-isoleucine.HBD-2 (A) and LL-37 (B) production was measured by ELISA assay from supernant concentrates. Data is expressed as median ± interquartile range. Statistics were calculated by Kruskal-Wallis test. In each experimental group, n = 15.
Mentions: Given that not all transcribed mRNA is traduced into AMPs, we determined the production LL-37 and HBD-2 in KCM from primary cells cultures by Enzyme-linked Immunosorbent Assay (ELISA). The results showed that 1,25(OH)2D3 induced HBD-2 secretion in 5 out of 15 cell cultures from DFUs reaching levels up to 8000 ng/ml even above of the levels seen for the cell culture stimulated from healthy donors. Whereas, L-isoleucine treatment induced a mild HBD-2 production only in 3 out of 15 cell cultures from DFU (Figure 3a).

Bottom Line: Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant.These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration.In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Unit Zacatecas, Mexican Institute of Social Security-IMSS, Zacatecas, Mexico; Department of Immunology, Faculty of Medicine, Autonomous University of San Luis Potosi, San Luis Potosi, Mexico.

ABSTRACT
Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

Show MeSH
Related in: MedlinePlus