Aß Assemblies Promote Amyloidogenic Processing of APP and Intracellular Accumulation of Aß42 Through Go/Gßγ Signaling.

Alzheimer's disease (AD) is characterized by the deposition of aggregated species of amyloid beta (Aß) in the brain, which leads to progressive cognitive deficits and dementia. Aß is generated by the successive cleavage of the amyloid precursor protein (APP), first by ß-site APP cleaving enzyme 1 (BACE1) and subsequently by the γ-secretase complex. Those conditions which enhace or reduce its clearance predispose to Aß aggregation and the development of AD. In vitro studies have demonstrated that Aß assemblies spark a feed-forward loop heightening Aß production. However, the underlying mechanism remains unknown. Here, we show that oligomers and fibrils of Aß enhance colocalization and physical interaction of APP and BACE1 in recycling endosomes of human neurons derived from induced pluripotent stem cells and other cell types, which leads to exacerbated amyloidogenic processing of APP and intracellular accumulation of Aß42. In cells that are overexpressing the mutant forms of APP which are unable to bind Aß or to activate Go protein, we have found that treatment with aggregated Aß fails to increase colocalization of APP with BACE1 indicating that Aß-APP/Go signaling is involved in this process. Moreover, inhibition of Gßγ subunit signaling with ßARKct or gallein prevents Aß-dependent interaction of APP and BACE1 in endosomes, ß-processing of APP, and intracellular accumulation of Aß42. Collectively, our findings uncover a signaling mechanism leading to a feed-forward loop of amyloidogenesis that might contribute to Aß pathology in the early stages of AD and suggest that gallein could have therapeutic potential.

Ionic complexation improves wound healing in deep second-degree burns and reduces in-vitro ciprofloxacin cytotoxicity in fibroblasts.

The development of new treatments capable of controlling infections and pain related to burns continues to be a challenge. Antimicrobials are necessary tools, but these can be cytotoxic for regenerating cells. In this study, antibiotic-anesthetic (AA) smart systems obtained by ionic complexation of polyelectrolytes with ciprofloxacin and lidocaine were obtained as films and hydrogels. Ionic complexation with sodium alginate and hyaluronate decreased cytotoxicity of ciprofloxacin above 70% in a primary culture of isolated fibroblasts (p < 0.05). In addition, the relative levels of the proteins involved in cell migration, integrin ß1 and p-FAK, increased above 1.5 times (p < 0.05) with no significant differences in cell mobility. Evaluation of the systems in a deep second-degree burn model revealed that reepithelization rate was AA-films = AA-hydrogels > control films > no treated > reference cream (silver sulfadiazine cream). In addition, appendage conservation and complete dermis organization were achieved in AA-films and AA-hydrogels. Encouragingly, both the films and the hydrogels showed a significantly superior performance compared to the reference treatment. This work highlights the great potential of this smart system as an attractive dressing for burns, which surpasses currently available treatments.

Sustained dual release of ciprofloxacin and lidocaine from ionic exchange responding film based on alginate and hyaluronate for wound healing.

This study presents a new antibiotic-anesthetic film (AA-film) based on natural polyelectrolytes ionically complexed with lidocaine and ciprofloxacin to manage pain associated with infected wounds. The rational selection of the components resulted in the AA-films being transparent, compatible with wound skin pH and highly water vapor permeable. The drug release properties evaluated in saline solution and water revealed an ionic exchange mechanism for the release of both drugs and showed that ciprofloxacin acts as a cross-linker, as was confirmed by rheological evaluation. The in vitro antimicrobial efficacy against S. aureus and P. aeruginosa was demonstrated. Furthermore, AA-films exhibit a high fluid absorption capacity and act as a physical barrier for microorganisms. This work highlights the great potential of this smart system as an attractive dressing for skin wounds, surpassing currently available treatments.

Ciprofloxacin-lidocaine-based hydrogel: development, characterization, and in vivo evaluation in a second-degree burn model.

The purpose of this work was to develop an effective carbomer hydrogel to be used to treat second-degree burns that combined ciprofloxacin and lidocaine (CbCipLid hydrogel). Its antibiotic and anesthetic efficacy and the physical and chemical properties of the CbCipLid hydrogel (release rate and kinetics, rheology, appearance, and drug content) were evaluated both before and after a sterilization cycle and also after 6 months of storage. For the in vivo studies, second-degree burns were developed in a rat model. Animals were divided into three groups: CbCipLid hydrogel, silver sulfadiazine cream (reference), and carbomer hydrogel (as control). The treatments were applied daily for 21 days, and the healing was monitored by macroscopic observation and histologic evaluation. The anesthetic effect was evaluated through the corneal touch threshold in a rabbit eye model. The CbCipLid hydrogel obtained is transparent and allows the loading of ciprofloxacin above its solubility at a neutral pH, with a rheology which is convenient for topical administration. Its physical and chemical properties remained unchanged after sterilization and for at least six additional months. Both ciprofloxacin and lidocaine are reversibly released from the CbCipLid hydrogel with a kinetics fitting the Higuchi model. The presence of a biologic-like fluid increased the rate of drug delivery through an ionic exchange mechanism. Treatment with the CbCipLid hydrogel decreased the wound-healing period, compared with the reference, and was associated with a greater number of fibroblasts and a faster rate of epithelialization and dermis reconstruction. These differences were assigned to the moist environment provided by the hydrogel and also to the presence of a therapeutic concentration of ciprofloxacin. Moreover, CbCipLid hydrogel provides an immediate anesthetic effect, which is significantly more intense than that of the reference. Based on these results, it is believed that the CbCipLid hydrogel could be a potential candidate in the prophylaxis/treatment of second-degree burns.

Early activation of CD95 is limited and localized to the cytotoxic synapse.

The cytotoxic synapse formed between cytotoxic T lymphocytes or natural killer cells expressing CD95L and target cells with CD95 on their surface is a key pathway for apoptosis induction by the immune system. Despite similarities with the immune synapse in antigen presenting cells, little is known about the role of the spatiotemporal organization of agonistic proteins/receptor interactions for CD95 signaling. Here, we have developed an artificial cytotoxic synapse to examine how mobility and geometry of an anti-CD95 agonistic antibody affect receptor aggregation and mobility, ie the first step of receptor activation. By measuring the distribution, diffusion coefficient, and fraction of immobile CD95 receptor in living cells, we show that at short times, the initial activation of CD95 occurs locally and is limited to the contact region of the cytotoxic synapse. This anisotropic activation of apoptotic signaling supports a role for confined interactions on the efficiency of signal transduction that may have implications for biomedical applications of extrinsic apoptosis induction.

Perfil de parámetros hematológicos en el HIV pediátrico y los cambios asociados al tratamiento antirretroviral / Hematological parameters profile in pediatric HIV and changes associated with antiretroviral treatment / Perfil de parâmetros hematológicos no HIV pediátrico e as alterações associadas ao tratamento anti-retroviral

Resumen Las alteraciones hematológicas son comunes durante la infección por el virus de la inmunodeficiencia humana (HIV). El objetivo de este trabajo fue describir los perfiles hematológicos e inmunológicos de niños infectados, antes y después de 36 meses de implementado el tratamiento antirretroviral (TARV). Se revisaron historias clínicas de niños expuestos, atendidos en este hospital en el período 2008-2018, con edades entre 6 meses y 14 años. Fueron empleados un contador hematológico (ADVIA 2120), un citómetro de flujo (FACScalibur BD) y una PCR en tiempo real Nuclisens EasyQ (bioMérieux). En 486 historias clínicas se encontraron 58 pacientes sin TARV, 30 por diagnóstico reciente y 28 por adherencia incorrecta o abandono de tratamiento. En ambos grupos se observó disminución porcentual de hemoglobina (Hb) (53% y 43%), volumen corpuscular medio (VCM) (43% y 7%) y LTCD4+ (37% y 57%), respectivamente, sin alteraciones significativas en otros parámetros hematológicos. Veintidós niños con correcta adherencia al TARV incrementaron significativamente los niveles de LTCD4+ (t0:18,8±9%, t1:32,7±6%), Hb (t0:10,9±1,6 g/dL, t1:12,6±1,1g/dL) y VCM (t0:78,7±4,5 fL, t1:101,9±5,6 fL), con disminución simultánea de la carga viral (CV) (t0:4,4±0,75 log t1:<1,70 log) después del seguimiento. La disminución de Hb observada aproximadamente en el 50% de los pacientes sin TARV estaría asociada a la acción viral y al tiempo de evolución de la infección. El incremento en los niveles, asociados a macrocitosis, se relacionaría con el aumento de LTCD4+ y disminución de la CV.

Abstract Hematologic abnormalities are common during human immunodeficiency virus (HIV) infection. Our aim was to describe hematological and immunological profiles present in antiretroviral treatment (ART)-naïve infected children and the changes observed after 36 months of ART initiation. Medical records of exposed children attended at this hospital in the 2008-2018 period were reviewed. Children between 6 months and 14 years were included. An automated blood analyser ADVIA 2120, a FACScalibur BD flow cytometer, and a Nuclisens EasyQ bioMérieux real-time PCR were used to determine different parameters. In 486 medical records evaluated, 58 patients ART-naïve were found, 30 due to recent diagnosis and 28 for incorrect adherence or abandoned treatment. In both groups, a percentage decrease in hemoglobin (Hb) (53% and 43%), mean corpuscular volume (MCV) (43% and 7%) and LTCD4+ (37% and 57%) levels respectively, was observed, without significant alterations in other hematological parameters. Twenty-two children with ART correct adherence increased significantly CD4+T cells (t0:18.8±9%, t1:32.7±6%), Hb (t0:10.9±1.6 g/dL, t1:12.6±1.1 g/dL) and MCV (t0:78.7±4.5 fL, t1:101.9±5.6 fL) levels, with simultaneous decrease of viral load (VL), (t0:4.4±0.75 log, t1:<1.70 log), after 36 months of follow-up. The reduction in Hb levels observed in 50% approximately of patients without ART would be associated with viral action and time of evolution of the infection. The increase in Hb levels and an associated macrocytosis would be related to the CD4+ T cells increase and VL decrease.

Resumo Alterações hematológicas são comuns durante a infecção pelo vírus da imunodeficiência humana (HIV). Nosso objetivo foi descrever os perfis hematológicos e imunológicos em crianças infectadas, antes e após 36 meses de implementar o tratamento antirretroviral (TARV). Foram revisados os prontuários das crianças expostas atendidas neste hospital no período 2008-2018, com idade entre 6 meses e 14 anos. Um contador hematológico (ADVIA 2120), um citômetro de fluxo (FACScalibur BD) e um PCR em tempo real Nuclisens EasyQ (bioMérieux), foram usados. Em 486 prontuários foram encontrados 58 pacientes livres de TARV, 30 por diagnóstico recente e 28 por adesão incorreta ou abandono do tratamento. Em ambos os grupos, observou-se diminuição percentual de hemoglobina (Hb) (53% e 43%), volume corpuscular médio (VCM) (43% e 7%) e LTCD4+ (37% e 57%), respectivamente, sem alterações significativas nos demais parâmetros hematológicos. Vinte e duas crianças com adesão correta ao TARV aumentaram significativamente os níveis de LTCD4+ (t0:18,8±9%, t1:32,7±6%), Hb (t0:10,9±1,6 g/dL, t1:12,6±1,1 g/dL) e VCM (t0:78,7±4,5 fL, t1:101,9±5,6 fL), com diminuição simultânea da carga viral (CV) (t0:4,4±0,75 log, t1:<1,70 log), depois do seguimento. A diminuição dos níveis de Hb observada em aproximadamente 50% dos pacientes sem TARV estaria associada à ação viral e ao tempo de evolução da infecção. O aumento nos níveis, associados a macrocitose, estaria relacionado com o aumento de LTCD4+ e diminuição da CV.