The Combination of Synoeca-MP Antimicrobial Peptide with IDR-1018 Stimulates Proliferation, Migration, and the Expression of Pro-Regenerative Genes in Both Human Skin Cell Cultures and 3D Skin Equivalents.

In skin lesions, the development of microbial infection affects the healing process, increasing morbidity and mortality rates in patients with severe burns, diabetic foot, and other types of skin injuries. Synoeca-MP is an antimicrobial peptide (AMP) that exhibits activity against several bacteria of clinical importance, but its cytotoxicity can represent a problem for its positioning as an effective antimicrobial compound. In contrast, the immunomodulatory peptide IDR-1018 presents low toxicity and a wide regenerative potential due to its ability to reduce apoptotic mRNA expression and promote skin cell proliferation. In the present study, we used human skin cells and a 3D skin equivalent models to analyze the potential of the IDR-1018 peptide to attenuate the cytotoxicity of synoeca-MP, as well as the influence of synoeca-MP/IDR-1018 combination on cell proliferation, regenerative processes, and wound repair. We found that the addition of IDR-1018 significantly improved the biological properties of synoeca-MP on skin cells without modifying its antibacterial activity against S. aureus. Likewise, in both melanocytes and keratinocytes, the treatment with synoeca-MP/IDR-1018 combination induces cell proliferation and migration, while in a 3D human skin equivalent model, it can accelerate wound reepithelization. Furthermore, treatment with this peptide combination generates an up-regulation in the expression of pro-regenerative genes in both monolayer cell cultures and in 3D skin equivalents. This data suggests that the synoeca-MP/IDR-1018 combination possesses a good profile of antimicrobial and pro-regenerative activity, opening the door to the development of new strategies for the treatment of skin lesions.

Antibiofilm and immunomodulatory resorbable nanofibrous filing for dental pulp regenerative procedures.

Multifunctional scaffolds with host defense peptides designed for regenerative endodontics are desirable nanobiotechnological tools for dentistry. Here, different scaffolds were tested for use during the pulp revascularization process, including poly(vinyl alcohol)-PVA hydrogels or resins, collagen hydrogels and poly(vinyl alcohol) PVA/Chitosan (PVA/CS) nanofibers. Based on time to degradation (21 days), nanofibers were chosen to be incorporated with ciprofloxacin and IDR-1002 (each at 50 mg/g). Nanofibers containing ciprofloxacin and IDR-1002 had anti-biofilm activity against Enterococcus faecalis, Staphylococcus aureus and a multispecies oral biofilm, besides anti-inflammatory activities. The in vivo subcutaneous tissue response to tooth fragments filled with nanofibers demonstrated a pulp-like tissue formation, when compared to empty teeth fragments. Thus, we designed a strong antimicrobial, immunomodulatory and regenerative candidate for pulp revascularization and regeneration procedures.

Comparative efficiency of oestrus synchronization in sheep with progesterone/eCG and progesterone/GnRH during breeding and non-breeding season.

The present study compared the occurrence of oestrus behaviour and ovulation in response to the insertion of CIDR devices plus a classical treatment with equine chorionic gonadotrophin (eCG; single dose at CIDR removal) or alternative treatments with gonadotrophin-releasing hormone (GnRH, either in a single dose at 56 hr after CIDR removal, or in one dose at CIDR insertion and another dose at 56 hr after CIDR removal). The appearance of oestrus behaviour during reproductive season ranged between 84% and 95% and all females showing oestrus signs had subsequent ovulations. The response, during seasonal anoestrus, was similar in the group treated with eCG, but less than half of the females in the groups treated with GnRH showed oestrus signs in response to the treatment, although more than 80% of them showed resumption of ovulatory activity after the treatment. In conclusion, protocols based on GnRH administration offer similar yields to eCG-based protocols during the reproductive season but occurrence of oestrus in response to GnRH-based treatments is highly compromised during seasonal anoestrus.

IDR-1018 induces cell proliferation, migration, and reparative gene expression in 2D culture and 3D human skin equivalents.

Skin lesions are associated with functional/cosmetic problems for those afflicted. Scarless regeneration is a challenge, not limited to the skin, and focus of active investigation. Recently, the host defense peptide innate defense regulatory peptide 1018 (IDR-1018) has shown exciting regenerative properties. Nevertheless, literature regarding IDR-1018 regenerative potential is scarce and limited to animal models. Here, we evaluated the regenerative potential of IDR-1018 using human 2D and 3D human skin equivalents. First, we investigated IDR-1018 using human cells found in skin-primary fibroblasts, primary keratinocytes, and the MeWo melanocytes cell line. IDR-1018 promoted cell proliferation and expression of marker of proliferation Ki-67, matrix metalloproteinase 1, and hyaluronan synthase 2 by fibroblasts. In keratinocytes, a drastic increase in expression was observed for Ki-67, matrix metalloproteinase 1, C-X-C motif chemokine receptor type 4, C-X-C motif chemokine receptor type 7, fibroblast growth factor 2, hyaluronan synthase 2, vascular endothelial growth factor, and elastin, reflecting an intense stimulation of these cells. In melanocytes, increased migration and proliferation were observed following IDR-1018 treatment. The capacity of IDR-1018 to promote dermal contraction was verified using a dermal model. Finally, using a 3D human skin equivalent lesion model, we revealed that the regenerative potential of IDR1018, previously tested in mice and pigs, is valid for human skin tissue. Lesions closed faster in IDR-1018-treated samples, and the gene expression signature observed in 2D was reproduced in the 3D human skin equivalents. Overall, the present data show the regenerative potential of IDR-1018 in an experimental system comprising human cells, underscoring the potential application for clinical investigation.

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.

An Immunomodulatory Peptide Confers Protection in an Experimental Candidemia Murine Model.

Fungal Candida species are commensals present in the mammalian skin and mucous membranes. Candida spp. are capable of breaching the epithelial barrier of immunocompromised patients with neutrophil and cell-mediated immune dysfunctions and can also disseminate to multiple organs through the bloodstream. Here we examined the action of innate defense regulator 1018 (IDR-1018), a 12-amino-acid-residue peptide derived from bovine bactenecin (Bac2A): IDR-1018 showed weak antifungal and antibiofilm activity against a Candida albicans laboratory strain (ATCC 10231) and a clinical isolate (CI) (MICs of 32 and 64 µg · ml-1, respectively), while 8-fold lower concentrations led to dissolution of the fungal cells from preformed biofilms. IDR-1018 at 128 µg · ml-1 was not hemolytic when tested against murine red blood cells and also has not shown a cytotoxic effect on murine monocyte RAW 264.7 and primary murine macrophage cells at the tested concentrations. IDR-1018 modulated the cytokine profile during challenge of murine bone marrow-derived macrophages with heat-killed C. albicans (HKCA) antigens by increasing monocyte chemoattractant protein 1 (MCP-1) and interleukin-10 (IL-10) levels, while suppressing tumor necrosis factor alpha (TNF-α), IL-1ß, IL-6, and IL-12 levels. Mice treated with IDR-1018 at 10 mg · kg-1 of body weight had an increased survival rate in the candidemia model compared with phosphate-buffered saline (PBS)-treated mice, together with a diminished kidney fungal burden. Thus, IDR-1018 was able to protect against murine experimental candidemia and has the potential as an adjunctive therapy.

Incidence of sports-related concussion among youth football players aged 8-12 years.

OBJECTIVE: To determine the risk of concussion among youth football players (ages 8-12 years). STUDY DESIGN: Participants included 468 male youth football players in western Pennsylvania during the 2011 youth football season. Incidence rates (IRs) and incidence density ratios (IDRs) of concussion were calculated for games and practices and for age groups. RESULTS: There was a total of 11,338 (8415 practice and 2923 game) athletic exposures (AEs) in the study period, during which 20 medically diagnosed concussions occurred. A majority of concussions were the result of head-to-head (45%) contact. The combined concussion IR for practices and games was 1.76 per 1000 AEs (95% CI 0.99-2.54). The concussion IR was 0.24 per 1000 AEs (95% CI 0.04-0.79) in practices and 6.16 per 1000 AEs (95% CI 3.76-9.54) in games. The IDR for concussions in games to practices was 25.91 (95% CI 6.01-111.70). The IDR of concussions for youth aged 11-12 years compared with youth aged 8-10 years was 2.72 (95% CI 0.66-4.78). CONCLUSIONS: The overall IR for concussion in youth football players aged 8-12 years was comparable with that reported previously for high school and collegiate samples. However, participation in games was associated with an increase in risk of concussion compared with practices, which was higher than rates previously reported for high school and collegiate athletes. Younger players were slightly less likely to incur a concussion than were older players.

Indução e sincronização do estro e da ovulação em cabras leiteiras Saanen com uso de dispositivos vaginais associados ou não à eCG ou efeito macho / Estrous and ovulation induction in Saanen dairy goats using different intravaginals devices with or without eCG or male effect

O objetivo deste estudo foi comparar a eficiência de três métodos de sincronização do estro em cabras leiteiras. Foram utilizadas 59 cabras Saanen distribuídas em três grupos: G-1 (n=20) usou-se esponjas impregnadas com 60mg de acetato de medroxiprogesterona (MAP), G-2 (n=19) e G-3 (n=20) com CIDR® contendo progesterona natural (P4). Os dispositivos do G-1 permaneceram nos animais por 11 dias, no nono dia aplicou-se 250UI de eCG e 50µg de PGF2a. No G-2 e G-3 os CIDRⓇ permaneceram nas fêmeas por 5 dias; no terceiro dia aplicou-se 50µg de PGF2a e, somente no G-2, 250UI de eCG, na retirada do dispositivo. No G-3, 48 h antes da retirada do dispositivo iniciou-se o efeito macho. As montas naturais foram realizadas 20 h após a detecção do estro. A resposta estral para o G-1 e G-2 foi de 100% e 90% no G-3. O intervalo médio entre o fim do tratamento e o início do estro foi de 31,53+2,81 h (MAP), 24,39±1,21 h (CIDRⓇ+ eCG) e 43,56±2,19 h (CIDRⓇ+ efeito macho). As taxas de gestação, parição e prolificidade foram de 95%, 80% e 1,83 no G-1, de 52,6%, 47% e 1,77 para o G-2 e 61,1%, 50% e 1,70 no G-3, respectivamente. Quando comparado com a esponja e CIDRⓇ+ efeito macho, o CIDRⓇ+ eCG antecipa o início do estro e o sincroniza melhor, por outro lado, a baixa taxa de gestação e parição do CIDRⓇ em relação à esponja, nos revela, que nessas condições experimentais, a esponja é melhor que o CIDR®.

The objective of this study was to compare the efficiency of three methods of estrus synchronization in dairy goats. Fifty nine Saanen goats were used and separated in three groups: G-1 (n=20), sponge impregnated with 60mg of medroxiprogesterone acetate (MAP), G-2 (n=19) and G-3 (n=20), CIDRⓇ containing natural progesterone (P4). The devices of G-1 remained in animals for 11 days; on the ninth day it was applied 250IU eCG and 50µg of PGF2a. In G-2 and G-3 the CIDRⓇ remained in females for 5 days; on the third day it was applied 50µg of PGF2a and only the G-2, 250IU of eCG, in the withdrawal of the device. In G-3, 48h before the removal of the device, the male effect began. The natural mountings were performed 20 h after the detection of estrus. The estrus response to the G-1 and G-2 was 100% and 90% in G-3. The average interval between the end of the treatment and estrus onset was 31.53+2.81 h (MAP), 24.39±1.21 h (CIDRⓇ+ eCG) and 43.56+2.19 h (CIDRⓇ+ male effect). The pregnancy and kidding rates and prolificacy was 95%, 80% and 1.83 in G-1, 52.6%, 47% and 1.77 for the G-2 and 61.1%, 50% and 1.70 in G-3, respectively. When compared with the sponge and CIDRⓇ+ male effect, the CIDRⓇ+ eCG anticipates the onset of estrus and synchronize better, on the other hand, the low pregnancy and kidding rates in CIDRⓇ in relation to the sponge, reveals that under these experimental conditions, the sponge is better than the CIDR®.

Detección de infección por Coccidioides immitis en zonas del estado de Coahuila, México / Detection of Coccidioides immitis infection in Coahuila, Mexico

La coccidioidomicosis es una micosis inicialmente pulmonar causada por Coccidioides immitis; puede diseminarse principalmente a sistema nervioso central, huesos y piel. En México se desconoce la frecuencia exacta de esta enfermedad. Nuestro objetivo fue determinar, por intradermorreacción y por serología, los casos de infección por C. immitis en 12 comunidades (10 rurales y dos urbanas) atendidas en el Hospital Rural Nº 79 del Instituto Mexicano del Seguro Social (IMSS) del estado de Coahuila, México. Se estudiaron 668 individuos adultos de ambos sexos; se les aplicó 0,1 ml de coccidioidina por vía intradérmica; después de 72 hs. se midió el diámetro de induración. Fueron seleccionados 180 individuos y a partir del suero se determinaron los niveles de inmunoglobulinas anti-C. immitis por ELISA. Fueron positivos a la coccidioidina 621 sujetos (93%), frecuencia mucho mayor a la reportada previamente en Coahuila. De los 180 sueros estudiados los promedios de densidad óptica (DO) fueron: IgG1, 1,55; IgG2, 0,94; IgG total, 0,33; IgG3, 0,29; IgG4, 0,27; IgM, 0,08. Los valores de IgG1, IgG2 e IgM comparados con las otras inmunoglobulinas fueron estadísticamente significativos. Los valores de IgG1 e IgG2 sugieren contacto frecuente con los antígenos e incluso probables casos de enfermedad no diagnosticada.

Coccidioidomycosis is a mycosis firstly pulmonar caused by Coccidioides immitis; it can be disseminated to central nervous system, bones and skin, principaly. In Mexico, the real frequency of the disease is unknown. The aim of this work was to determine, by skin test and by serology, the infection cases by C. immitis in twelve communities (10 rural and two urban), attended in the Hospital Rural Nº 79 at the Instituto Mexicano del Seguro Social (IMSS) from the Coahuila State, Mexico. Six hundred and sixty eight adult individuals of both sexes were studied, to whom 0.1 ml of coccidioidin by intradermal route was applied; 72 h after, the induration diameter was measured. One hundred eighty individuals were selected and seric anti-C. immitis immunoglobulins levels were determined by ELISA. Six hundred twenty one individuals (93%) were positive to coccidioidin, the frequency was much higher than that previously reported in Coahuila. From 180 sera studied, the means of optical density (OD) were: IgG1, 1.55; IgG2, 0.94; total IgG, 0.33; IgG3, 0.29; IgG4, 0.27; IgM, 0.08. The values of IgG1, IgG2 and IgM compared with the other immunoglobulins were statistically significant. The high values of IgG1 and IgG2 suggest frequent contact with the antigen, and probable cases of undiagnosed disease.

Bases para estimar las necesidades de vitamina C en la gestación / Bases to determine the dose of vitamin C in pregnancy

Objetivo: Establecer la dosis de vitamina C para mantener una concentración leucocitaria >18 µg/10(8) células en mujeres embarazadas entre las semanas 28 y 32 de la gestación, con la finalidad de establecerla como base para la estimación de la ingestión diaria recomendada (IDR). Metodología: Etapa 1: estudio agudo de suplementación. Se suplementó a 10 gestantes al inicio del segundo trimestre con dosis que fueron de 0 a 200 mg/día de vitamina C (sin contar con el aporte dietético), cada dosis se administró durante una semana. Etapa 2: estudio doble ciego aleatorizado (100 mg/día de vitamina C vs. Placebo) entre la semana 20 y el término de la gestación con 52 mujeres. Resultados: Etapa 1. Con 100 mg/día de vitamina C se alcanzó la saturación leucocitaria sin incrementar significativamente la excreción urinaria. Etapa 2. Las mujeres que recibieron placebo disminuyeron significativamente su concentración leucocitaria de vitamina C a lo largo de la gestación mientras que las gestantes que recibieron el suplemento la incrementaron en forma significativa manteniéndola por arriba de >18 µg/10(8) células. Conclusión: Una dosis diaria de 100 mg de vitamina C durante la segunda mitad del embarazo ocasiona una reserva leucocitaria adecuada y puede ser considerada como referencia para establecer la IDR.

Objective: Determine the dose of Vitamin C able to maintain a leukocyte Vitamin C concentration of >18 µg/10(8)cells, in pregnant women with 28 to 32 weeks of gestation, in order to compile a database to estimate the daily recommended intake (DRI) during pregnancy. Methodology: Stage 1: acute supplementation study. A group of 10 healthy women in late first and early second trimester pregnancy were supplemented with 0 to 200 mg vitamin C/day during one week each. Stage 2: a randomized double blind study (placebo vs. vitamin C [100 mg/d]) was carried out with 52 women studied from week 20 to week 32 of pregnancy. Their plasma and leukocyte vitamin C concentrations were measured every 4 weeks to evaluate the previously established supplementation dose. Results: Stage 1: with the 100 mg/day dose, leukocyte vitamin C saturation was reached without increasing urinary excretion. Stage 2: leukocyte concentration of vitamin C decreased throughout pregnancy in women receiving placebo, while supplemented women maintained their concentrations >18 µg/10(8) cells. Conclusion: A 100 mg/day dose of vitamin C during the second half of pregnancy keeps leucocyte storage and could be useful to establish the DRI of Vitamin C during pregnancy.