Conditional reprogramming of pediatric airway epithelial cells: A new human model to investigate early-life respiratory disorders.

BACKGROUND: Airway epithelial cells (AEC) are quite difficult to access in newborns and infants. It is critically important to develop robust life-extended models to conduct translational studies in this age group. We propose the use of a recently described cell culture technology (conditionally reprogrammed cells-CRC) to generate continuous primary cell cultures from nasal and bronchial AEC of young children. METHODS: We collected nasal and/or bronchial AEC from a total of 23 subjects of different ages including newborns/infants/toddlers (0-2 years; N = 9), school-age children (4-11 years; N = 6), and a group of adolescent/adult donors (N = 8). For CRC generation, we used conditioned medium from mitotically inactivated 3T3 fibroblasts and Rho-associated kinase (ROCK) inhibitor (Y-27632). Antiviral immune responses were studied using 25 key antiviral genes and protein production of type III epithelial interferon (IFN λ1) after double-stranded (ds) RNA exposure. RESULTS: CRC derived from primary AEC of neonates/infants and young children exhibited: (i) augmented proliferative capacity and life extension, (ii) preserved airway epithelial phenotype after multiple passages, (iii) robust immune responses characterized by the expression of innate antiviral genes and parallel nasal/bronchial production of IFN λ1 after exposure to dsRNA, and (iv) induction of airway epithelial inflammatory and remodeling responses to dsRNA (eg, CXCL8 and MMP9). CONCLUSION: Conditional reprogramming of AEC from young children is a feasible and powerful translational approach to investigate early-life airway epithelial immune responses in humans.

Shaping magnetic fields to direct therapy to ears and eyes.

Magnetic fields have the potential to noninvasively direct and focus therapy to disease targets. External magnets can apply forces on drug-coated magnetic nanoparticles, or on living cells that contain particles, and can be used to manipulate them in vivo. Significant progress has been made in developing and testing safe and therapeutic magnetic constructs that can be manipulated by magnetic fields. However, we do not yet have the magnet systems that can then direct those constructs to the right places, in vivo, over human patient distances. We do not yet know where to put the external magnets, how to shape them, or when to turn them on and off to direct particles or magnetized cells-in blood, through tissue, and across barriers-to disease locations. In this article, we consider ear and eye disease targets. Ear and eye targets are too deep and complex to be targeted by a single external magnet, but they are shallow enough that a combination of magnets may be able to direct therapy to them. We focus on how magnetic fields should be shaped (in space and time) to direct magnetic constructs to ear and eye targets.

Peripheral neurotrophin levels during controlled crack/cocaine abstinence: a systematic review and meta-analysis.

Cocaine/crack abstinence periods have higher risk of relapse. Abstinence as initial part of the recovery process is affected by learning and memory changes that could preserve the addictive cycle. To further understand how the interruption of cocaine/crack consumption affects neurotrophin level we performed the present systematic review and meta-analysis following the PRISMA statement (number CRD42019121643). The search formula was conducted in PubMed, Web of Science, Embase, ScienceDirect, and Google Scholar databases. The inclusion criterion was cocaine use disorder in 18 to 60-year-old people, measuring at least one neurotrophin in blood before and after a controlled abstinence period. Studies without pre-post design were excluded. Five investigations had nine different reports, four of them were subjected to a meta-analysis (n = 146). GRADE risk of bias method was followed. Individual studies reported increased peripheral brain derived neurotrophic factor (BDNF) after abstinence, evidence pooled by Hedge's g showed no significant change in BDNF after abstinence. Relevant heterogeneity in the length of the abstinence period (12-32 days), last cocaine/crack consumption monitoring and blood processing were detected that could help to explain non-significant results. Further improved methods are suggested, and a potential BDNF augmentation hypothesis is proposed that, if true, would help to understand initial abstinence as a re-adaptation period influenced by neurotrophins such as the BDNF.

Effect of chronic unpredictable stress in female Wistar-Kyoto rats subjected to progesterone withdrawal: Relevance for Premenstrual Dysphoric Disorder neurobiology.

Premenstrual Dysphoric Disorder (PMDD) is related to an abrupt drop in progesterone and impairments in the HPA axis that cause anxiety. Suffering persons report higher daily-life stress and anxiety proneness that may contribute to developing PMDD, considered a chronic stress-related disorder. Here, we explored the effect of chronic unpredictable stress (CUS) in rats subjected to progesterone withdrawal (PW) and evaluated gene expression of HPA axis activation in the stress-vulnerable Wistar-Kyoto (WKY) rat strain that is prone to anxiety. Ovariectomized WKY rats were randomly assigned to CUS or Standard-housed conditions (SHC) for 30 days. To induce PW, animals received 2 mg/kg of progesterone on day 25th for 5 days; 24 h later, they were tested using the anxiety-like burying behavior test (BBT). After behavioral completion, rats were euthanized, and brains were extracted to measure Crh (PVN) and Nr3c1 (hippocampus) mRNA. Blood corticosterone and vasopressin levels were determined. Results showed that PW exacerbated anxiety-like behaviors through passive coping in CUS-WKY. PW decreased Crh-PVN mRNA and the Nr3c1-hippocampal mRNA expression in SHC. CUS decreased Crh-PVN mRNA compared to SHC, and no further changes were observed by PW or BBT exposure. CUS reduced Nr3c1-hippocampal gene expression compared to SHC animals, and lower Nr3c1 mRNA was detected due to BBT. The PW increased corticosterone in SHC and CUS rats; however, CUS blunted corticosterone when combined with PW+BBT and similarly occurred in vasopressin concentrations. Chronic stress blunts the response of components of the HPA axis regulation when PW and BBT (systemic and psychogenic stressors, respectively) are presented. This response may facilitate less adaptive behaviors through passive coping in stress-vulnerable subjects in a preclinical model of premenstrual anxiety.

Combinatorial gene therapy induces regression of hepatic encephalopathy.

Capillarization of the sinusoid impedes the clearance of neurotoxic substances in liver fibrosis. These events may result in hepatic encephalopathy. Neurological and hepatic features of rats after bile duct ligation (BDL) supplemented with Manganese (BDL+Mn(2+)) were examined. The 4-week-old BDL rats had elevated levels of ammonia and were concomitantly fed with 1 mg ml(-1) of MnCl(2) in drinking water (BDL/Mn(+2)). Five out of fifteen rats were killed and the serum, liver and brain tissue (striatum and substantia nigra) were recovered. Of the remaining BDL/Mn(+2)-cirrhotic animals (n=10), five were injected with a combination of Adenovirus-human plasminogen activator (Ad-huPA) and Adenovirus-matrix metalloproteinase-8 (Ad-MMP-8) (3 × 10(11)+1.5 × 10(11) vector particles per kg), and five with 4.5 × 10(11) vector particles per kg of Adenovirus-ß-galactosidase (Ad-ß-Gal). This treatment was carried on for 10 days. The BDL/Mn(+2) rats displayed tremor, rigidity and gait abnormalities, which improved notably with combinatorial gene therapy, as well as motor coordination. Liver fibrosis was evidently less after treatment with Ad-huPA+Ad-MMP-8 (25%). In the brain (striatum), Ad-huPA+Ad-MMP-8 treatment rendered higher concentrations of dopamine compared with Ad-ß-Gal-treated encephalopathic rats (210 and 162 ng g(-1) of tissue, respectively). The BDL/Mn(+2) animals and controls treated with Ad-ß-Gal showed abnormal morphology in astrocytes (gliosis) in striatum and substantia nigra, in which expressions of green fibrillar acidic protein and tyrosine hydroxylase were altered. These abnormalities decreased with Ad-huPA+Ad-MMP-8 treatment. Importantly, the latter animals showed an increment in sprouting of nervous fibers in substantia nigra. Combinatorial gene therapy improves neuroanatomical and neurochemical characteristics similar to human hepatic encephalopathy.

Panel 7 - Pathogenesis of otitis media - a review of the literature between 2015 and 2019.

OBJECTIVE: To perform a comprehensive review of the literature from July 2015 to June 2019 on the pathogenesis of otitis media. Bacteria, viruses and the role of the microbiome as well as the host response are discussed. Directions for future research are also suggested. DATA SOURCES: PubMed database of the National Library of Medicine. REVIEW METHODS: PubMed was searched for any papers pertaining to OM pathogenesis between July 2015 and June 2019. If in English, abstracts were assessed individually for their relevance and included in the report. Members of the panel drafted the report based on these searches and on new data presented at the 20th International Symposium on Recent Advances in Otitis Media. CONCLUSIONS: The main themes that arose in OM pathogenesis were around the need for symptomatic viral infections to develop disease. Different populations potentially having different mechanisms of pathogenesis. Novel bacterial otopathogens are emerging and need to be monitored. Animal models need to continue to be developed and used to understand disease pathogenesis. IMPLICATIONS FOR PRACTICE: The findings in the pathogenesis panel have several implications for both research and clinical practice. The most urgent areas appear to be to continue monitoring the emergence of novel otopathogens, and the need to develop prevention and preventative therapies that do not rely on antibiotics and protect against the development of the initial OM episode.

Aqueous extract of pomegranate enriched in ellagitannins prevents anxiety-like behavior and metabolic changes induced by cafeteria diet in an animal model of menopause.

Women around menopause are vulnerable to present psychiatric and metabolic disorders; thus, therapies that contribute to treat both pathologies are required. Previous reports showed that an aqueous extract of pomegranate (Punica granatum), enriched in ellagitannins, exerts an antidepressant-like effect in ovariectomized rats. We analyze whether this aqueous extract of P. granatum (AE-PG) prevents the anxiety-like behavior induced by a cafeteria diet (CAF) in middle-aged ovariectomized rats at the same time that it prevents an increase in body weight, glucose, lipids, and the changes on mRNA expression of the peroxisome proliferator-activated receptor-gamma (PPAR-γ) in the liver. Also, the effects of AE-PG on the protein levels of PPAR-γphospho-PPAR-γ, extracellular signal-regulated protein kinase (ERK1/2) and phospho-ERK1/2 were measured in the hippocampus and amygdala. CAF induced anxiety-like behavior, augmented lipids and glucose blood levels, body weight, visceral fat, insulin resistance, and decreased mRNA expression of PPAR-γ in the liver. In rats fed with the CAF, AE-PG prevented the anxiety-like behavior, reduced body weight, lowered lipid levels, reduced insulin resistance, and increased PPAR-γ mRNA expression in the liver. In the hippocampus, ERK1/2 but not PPAR-γ protein levels were decreased by CAF, while AE-PG prevented these effects. In the amygdala, CAF increased the phosphorylation of PPARγ, and AE-PG prevented it. In contrast, AE-PG rescued the decreased ERK1/2 protein level in the hippocampus caused by CAF. In conclusion, AE-PG treatment prevented anxiogenic and metabolic effects induced by CAF, and its effects appear to be mediated by ERK1/2 and PPARγ depending on the brain area studied.

Environmental enrichment prevents anxiety-like behavior induced by progesterone withdrawal in two strains of rats.

Stress vulnerability could influence the treatment response to anxiety associated with abrupt hormonal suppression. The present study explored the effects of different treatments on experimental anxiety induced by progesterone withdrawal (PW) in a stress-sensitive rat strain, Wistar Kyoto (WKY), in the burying behavior test (BBT). The following experimental series was conducted using independent groups of Wistar (control strain) and WKY ovariectomized rats: Experiment 1: Rats were treated for 5days with oil, a constant dose of progesterone (0.5mg/rat, s.c) or a combination of progesterone (0.5mg/rat, s.c) plus fluoxetine (10 mg/kg, i.p); on day 6, all rats were subjected to BBT. Experiment 2: Rats received corn oil or decreasing doses of progesterone (0.84, 0.67, 0.5, 0.33 and 0.17mg/rat; one dose daily); on day 6, the rats were subjected to BBT. Experiment 3: Rats were divided into two groups that were subjected to 30days of standard conditions or environmental enrichment (EE); from days 25 to 30, all rats received a fixed dose of progesterone (0.5mg/rat, s.c.) or vehicle. On day 31, the rats were tested with BBT. Results showed that PW increased anxiety in both strains, and fluoxetine prevented anxiety in WKY rats. In contrast, a gradual reduction of progesterone prevents the anxiety in Wistar but not in WKY. EE was preventive against the anxiety induced by PW in both strains of rats. Thus, the results suggest that anxiety induced by PW is prevented by EE while the anxiolytic effect of pharmacological treatments depends on stress vulnerability.

A review of type 3 tympanoplasty.

With the introduction of newer technologies, the advent of antibiotics, and improved surgical methods; the past 25 years have seen a great revolution, refinement, and improvement of tympanoplastic procedures. Furthermore, the methods of classification have been modified. The introduction of ossicular reconstruction with biocompatible implants greatly enhanced the success of type 3 tympanoplasty. This article reviews the classification, indications, surgical techniques, biomechanical properties, and expected hearing results of type 3 tympanoplasty.

Screening of 47 organic microcontaminants in agricultural irrigation waters and their soil loading.

Reclaimed water usage for crop irrigation is viewed both as an excellent sustainable water source and as a potential entrance for emerging organics into the food chain. This concern is backed by the already documented pollutant crop uptake potential. In the present study, irrigation waters used in agricultural fields (Torroella de Montgri, NE Spain) were screened for 47 analytes in a two year study (2007-2008). A total of 26 contaminants belonging to different chemical classes namely, pesticides, pharmaceuticals, personal care products, phenolic estrogens, antioxidants and disinfection by-products, were detected. Marked differences in concentration trends for the different chemical classes were evidenced from 2007 to 2008, and attributed to a persistent drought endured by the region in 2008. Also, loading mass rates of chemical classes were estimated based on crop irrigation regimes and they ranged from 0.8 to 121.3 g ha(-1) per crop cycle. These values were contrasted with those obtained for other water sources from countries where crop irrigation is commonly practiced. Finally, crops grown under these irrigation regimes, namely alfalfa and apple, were analyzed and 5 anthropogenic compounds were identified and quantitated, whose concentrations ranged from 13.9 to 532 ng g(-1) (fresh weight).

The role of inhibitor of DNA-binding (Id1) in hyperproliferation of keratinocytes: the pathological basis for middle ear cholesteatoma from chronic otitis media.

OBJECTIVES: A hallmark of cholesteatoma is hyperproliferation of keratinocytes with abundant production of keratins in the middle ear under chronic inflammatory conditions. However, little is known about the driving force of cellular proliferation and keratin production of cholesteatomal matrix. The purpose of this study was to investigate the cellular proliferation and keratin production of keratinocytes under the influence of Id1, a candidate transcription factor to cell proliferation. MATERIALS AND METHODS: Keratinocytes were transfected with Id1 and the responses of keratinocytes to Id1 were studied by using cellular and molecular biologic methods. RESULTS: Id1 positively contributed to the cell cycle progression and negatively to the p16(Ink4a) downregulation via the nuclear factor-kappa B (NF-κB)/cyclin D1 pathway. Id1 significantly increased the promoter activity of NF-κB which, in turn, up-regulated the expression of cyclin D1 and keratin 10 in keratinocytes. Specific NF-κB inhibitors (pyrrolidine dithiocarbamate, PDTC), or dominant-negative inhibitor (I kappa B alpha mutant, IκBαM) abrogated the Id1-induced cell proliferation and keratin 10 production whereas p65, a subunit of the NF-κB heterodimer and an enhancer of the NF-κB activity, strengthened the Id1-induced cell proliferation and keratin 10 production. CONCLUSIONS: Id1 contributed to hyperproliferation of keratinocytes via enhancement of cell cycle progression, removal of cell cycle inhibition, and simultaneously increased keratin production.

Uso de esteroides sistémicos en quemaduras de segundo grado en modelo animal / Systemic steroid use in second degree burns in an animal model

El uso de esteroide está reconocido en el tratamiento crítico del paciente quemado y es útil en el choque séptico que no responde a vasopresores. Este grupo de medicamentos ayuda a regular la respuesta hemodinámica mejorando el aporte de sangre a la piel, aunque sabemos que tienen un efecto nocivo en el proceso de cicatrización. Evaluamos el efecto histopatológico del empleo de esteroides en quemaduras usando un modelo animal. Empleamos 2 grupos de 10 ratas (Wistar) en las que colocamos un cilindro de metal en el dorso durante 15 segundos a 95oC, produciendo una quemadura. En ese momento, uno de los grupos recibió esteroide a dosis de estrés (hidrocortisona 5 mg/kg) y el otro no recibió ningún medicamento. Al quinto día resecamos la escara y cubrimos el defecto con cultivo de queratinocitos. Los animales fueron sacrificadas a los 14 días. Realizamos análisis histopatológico. Macroscópicamente evaluamos la (..) (AU)

The use of steroids is well recognized in critical care specially in septic shock. There are some reports of their utility in severe burns. It helps to regulate the hemodynamic response in order to improve the blood supply to the skin, although it is well known their negative effect in wound healing. Our objective is to know the hystopathologic effect of steroids in burn healing. We used 2 groups of 10 rats (Wistar). Both groups were exposed in their backs to a metallic cylinder at 95 oC for 15 seconds. At the moment of the burn, one group was given steroid (hydrocortisone at stress dose 5 mg/kg) and the other group didn't received any medication. The scar was removed at the 5th day and the burn injury was covered with queratinocyte culture. The rats were sacrificed at 14th day. We evaluated the presence of clinical signs of infection and the percentage of new epithelium. In the microscope we evaluated the following parameters: fibrosis, inflammatory process, presence of fibroblast and vascular proliferation. We compared both groups using Chi2 test (SPSS program version 10). A p =/<. 05 was considered as statistical significant. We found no difference between each group in fibrosis (p .47), inflammatory process (p .27), or fibroblast presence (p.16). But there was a difference in vascular proliferation (p .05) against the first group (steroid group). There were no signs of infection and all of them were epithelised at the 14th day. In conclusion, the use of steroids in burns in an animal model could have a final effect in wound healing. In humans it is important to say that they can be helpful in those cases with clear evidence of benefit, as for example failure to vassopresor response in septic shock. We are not sure about the final effect in wound healing in the steroid group as for example wound contracture in long term (AU)