Host Defense Peptide RNase 7 Is Down-regulated in the Skin of Diabetic Patients with or without Chronic Ulcers, and its Expression is Altered with Metformin.

BACKGROUND: Diabetic foot ulcers (DFUs) are one of the main complications in patients with type 2 diabetes mellitus (DM2), previous studies have reported that DM2 patients have lower production of host defense peptides (HDP). AIM OF THE STUDY: To investigate the expression of RNase 7, cathelicidin, HBD-2, and psoriasin in biopsies obtained from DM2 patients with or without DFU. METHODS: Biopsies from DFU patients grade 3 according to Wagner's classification, from diabetic patients without ulcer and from healthy donors were obtained. qPCR, immunohistochemistry and cell line cultures were performed. To assess whether L-isoleucine, calcitriol, phenyl butyrate, metformin, glyburide or insulin induced RNase 7, keratinocytes were stimulated, and RNase 7 expression was evaluated. RESULTS: Our data showed that RNase 7 levels were decreased in both diabetic groups when were compared with skin from healthy donors. Since most of the DM2 patients are treated with drugs to reduce glycemia, we investigated whether glyburide, metformin or insulin were able to induce any change regarding RNase 7 production. Results showed that metformin reduces the expression of RNase 7 in in vitro treated keratinocytes, suggesting that the chronic use of metformin should be evaluated in DFU patients, whereas calcitriol, phenyl butyrate and L-isoleucine did not increase the RNase 7 production. CONCLUSIONS: Due RNase 7 has antimicrobial activity, its downregulation can make prone to DM2 patients to develop infections and impaired wound healing.

Induction by innate defence regulator peptide 1018 of pro-angiogenic molecules and endothelial cell migration in a high glucose environment.

Synthetic innate defence regulator (IDR) peptides such as IDR-1018 modulate immunity to promote key protective functions including chemotaxis, wound healing, and anti-infective activity, while suppressing pro-inflammatory responses to non-pathological levels. Here we demonstrated that IDR-1018 induced, by up to 75-fold, pro-angiogenic VEGF-165 in keratinocytes but suppressed this isoform in endothelial cells. It also induced early angiogenin and prolonged anti-inflammatory TGFß expression on endothelial cells, while suppressing early pro-inflammatory IL-1ß expression levels. IDR-1018 also down-regulated the hypoxia induced transcription factor HIF-1α in both keratinocytes and endothelial cells. Consistent with these data, in an in vitro wound healing scratch assay, IDR-1018 induced migration of endothelial cells under conditions of hypoxia while in epithelial cells migration increased only under conditions of normoxia.

Calcitriol promotes proangiogenic molecules in keratinocytes in a diabetic foot ulcer model.

Foot ulceration is one of the most common and complex sequelae of diabetes mellitus, generally posing a therapeutic challenge due to poor healing responses and high rates of complications, including peripheral vascular disease, ischemia and infections. Calcitriol, the most active vitamin D metabolite, induces antimicrobial peptides production in keratinocytes from diabetic foot ulcers (DFU); however, little is known about its effects on angiogenic factors in this pathology. Herein we aimed at studying whether calcitriol induces angiogenic molecules in keratinocytes under normoxic and hypoxic conditions, and if these molecules are able to improve cell migration in vitro. Evaluation of DFU samples by immunohistochemistry showed increased VEGF and decreased angiogenin and HIF-1α expression compared to controls, suggesting an altered pattern of angiogenic factors in DFU. Interestingly, incubation of keratinocytes with calcitriol significantly upregulated VEGFA, HIF-1α and angiogenin gene expression, while the resulting cell culture media stimulated both endothelial cells and keratinocytes migration in an in vitro wound closure assay under a normoxic environment (p<0.05). Moreover, the culture media of calcitriol-treated keratinocytes stimulated cell migration in a similar extent as exogenous VEGF or EGF in endothelial and keratinocytes cells. These results suggest that the altered profile of angiogenic molecules in DFU might be improved by local or systemic treatment with calcitriol under normoxic conditions, which could probably be achieved with hyperbaric oxygen therapy. Given that calcitriol not only augments proangiogenic factors but also induces antimicrobial peptides expression, this hormone should be further investigated in clinical trials of DFU.

Calcitriol prevents inflammatory gene expression in macrovascular endothelial cells.

BACKGROUND: Calcitriol (vitamin D) supplementation has been proposed for therapeutical use in vascular diseases due to its immunomodulatory activity, preventing inflammation and promoting angiogenesis. In the present study, we hypothesised whether calcitriol downregulates pro-inflammatory gene expression without affecting angiogenesis and anti-inflammatory gene expression in LPS-induced endothelial cells. METHOD: In order to evaluate the effect of calcitriol in suppressing inflammatory gene expression in the endothelium, endothelial cells were exposed to the physiological concentration of calcitriol followed by stimulation with lipopolysaccharide (LPS). Gene expression of interleukin (IL)-1ß, Transforming Growth Factor (TGF)-ß, Human ß-defensin (HBD)-2, angiogenin (ANG) and cathelicidin (LL-37) were quantified by quantitative polymerase chain reaction. RESULTS: The results from six independent experiments conducted in duplicate, showed that calcitriol decreased IL-1ß (p < 0.01) and HBD-2 expression (p < 0.01) when compared to non-treated cells. However, calcitriol treatment had no effect on TGF-ß, ANG and LL-37 gene expression. CONCLUSION: Calcitriol prevents inflammatory gene expression, but does not affect expression of angiogenic genes in endothelial cells, which suggest the potential use of calcitriol to prevent endothelial activation through the downregulation of IL-1ß and HBD-2.

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

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.

Prime-boost BCG vaccination with DNA vaccines based in ß-defensin-2 and mycobacterial antigens ESAT6 or Ag85B improve protection in a tuberculosis experimental model.

The World Health Organization (WHO) has estimated that there are about 8 million new cases annually of active Tuberculosis (TB). Despite its irregular effectiveness (0-89%), the Bacillus Calmette-Guérin) BCG is the only vaccine available worldwide for prevention of TB; thus, the design is important of novel and more efficient vaccination strategies. Considering that ß-defensin-2 is an antimicrobial peptide that induces dendritic cell maturation through the TLR-4 receptor and that both ESAT-6 and Ag85B are immunodominant mycobacterial antigens and efficient activators of the protective immune response, we constructed two DNA vaccines by the fusion of the gene encoding ß-defensin-2 and antigens ESAT6 (pDE) and 85B (pDA). After confirming efficient local antigen expression that induced high and stable Interferon gamma (IFN-γ) production in intramuscular (i.m.) vaccinated Balb/c mice, groups of mice were vaccinated with DNA vaccines in a prime-boost regimen with BCG and with BCG alone, and 2 months later were challenged with the mild virulence reference strain H37Rv and the highly virulent clinical isolate LAM 5186. The level of protection was evaluated by survival, lung bacilli burdens, and extension of tissue damage (pneumonia). Vaccination with both DNA vaccines showed similar protection to that of BCG. After the challenge with the highly virulent Mycobacterium tuberculosis strain, animals that were prime-boosted with BCG and then boosted with both DNA vaccines showed significant higher survival and less tissue damage than mice vaccinated only with BCG. These results suggest that improvement of BCG vaccination, such as the prime-boost DNA vaccine, represents a more efficient vaccination scheme against TB.

Pelvic surgery and hospitalization among Chilean women after nonsurgical sterilization with quinacrine pellets between 1977 and 1989.

BACKGROUND: Concern about quinacrine lingers because of its carcinogenic effects in rats. We describe results of long-term follow-up of women who underwent quinacrine pellet sterilization in Chile between 1977 and 1989 (N=1492). METHODS: We interviewed the women or relatives in five rounds of data collection between 1991-1993 and 2006-2007, and reviewed hospital records. Median follow-up was 18.5 years; total person-time was 23,894 woman-years. This analysis focuses on pelvic and abdominal surgeries and conditions. We used survival analysis to estimate the 15-year cumulative probability of hysterectomy, other pelvic surgical procedures and relevant adverse events. RESULTS: Uterine fibroids were by far the most common gynecologic condition, reported by 11% of the cohort. Surgical procedures were recorded for 15% of the cohort; hysterectomy was the most frequent procedure (10%), followed by salpingectomy (2%). The 15-year probability of any pelvic or abdominal procedure was 14.7 per 100 women (95% confidence interval 12.4-16.9). The probability of hysterectomy was 9.3 per 100 women (95% confidence interval 7.4-11.1). Number of quinacrine insertions had little impact on the probabilities. CONCLUSION: During long-term follow-up of women who received quinacrine pellets for nonsurgical sterilization, the incidence of noncancer adverse outcomes was not unusually high, and no alarming patterns emerged.

Expression of antimicrobial peptides in diabetic foot ulcer.

BACKGROUND: Foot ulcers are one of the main diabetes complications due to its high frequency and difficulty of complete healing. There are several factors that participate in diabetic ulcers development and limited information exists about the role of antimicrobial peptides (AMP) in its pathogenesis. OBJECTIVE: The aim of this study was to analyze the expression pattern of the main AMPs: Human Neutrophil Peptide (HNP)-1, Human ß-defensin (HBD)-1, HBD-2, HBD-3, HBD-4 and cathelicidin LL-37 in biopsies from diabetic foot ulcers (DFU). METHODS: 20 biopsies from DFU grade 3 according to Wagner's classification and 20 biopsies from healthy donors were obtained. Real time PCR, immunohistochemistry and primary cell cultures were performed. RESULTS: ß-Defensins were overexpressed in DFU, whereas LL-37 has low or none expression in comparison with healthy skin. When primary cell culture from these biopsies were performed and infected with Staphylococcus aureus, epidermal cell from diabetic ulcers showed lower LL-37 expression compared with cell cultures from healthy donors skin. CONCLUSION: These results suggest that though most AMPs are expressed in DFU, this production is not appropriate to promote wound healing and contain secondary infections.

Cancer risk after sterilization with transcervical quinacrine: updated findings from a Chilean cohort.

BACKGROUND: Dating back to the 1970s, thousands of women worldwide have voluntarily been sterilized with transcervical insertion of quinacrine pellets. The safety and efficacy of the technology are still being assessed today; in particular, better estimates on the incidence of human cancers are now feasible. METHODS: We conducted a cohort study of 1492 women in Santiago and Valdivia, Chile, who received transcervical quinacrine pellets for contraceptive sterilization between 1977 and 1989. We periodically interviewed women with the last interviews in 2006-2007 and reviewed their medical records. We calculated age and site-specific incidence of invasive cancers and compared the observed cases to the number of expected cases based on data from the Cali, Colombia, cancer registry, gathered by the International Agency for Research on Cancer. RESULTS: During 23,894 person-years of follow-up, 41 invasive cancers were identified, including 16 new cases that had occurred since the previous analysis. Ten cases of cervical cancer were observed, compared with 12.1 expected. Since the initial study's confirmation of a single case of leiomyosarcoma, no other uterine cancers have been diagnosed. We would expect 2.0 uterine cancers during this number of observed women-years. One case of ovarian cancer was diagnosed, compared with 3.1 expected. CONCLUSION: Rates of cancer among women exposed to intrauterine quinacrine are similar to population-based rates.