Microglial morphological/inflammatory phenotypes and endocannabinoid signaling in a preclinical model of periodontitis and depression.

BACKGROUND: Depression is a chronic psychiatric disease of multifactorial etiology, and its pathophysiology is not fully understood. Stress and other chronic inflammatory pathologies are shared risk factors for psychiatric diseases, and comorbidities are features of major depression. Epidemiological evidence suggests that periodontitis, as a source of low-grade chronic systemic inflammation, may be associated with depression, but the underlying mechanisms are not well understood. METHODS: Periodontitis (P) was induced in Wistar: Han rats through oral gavage with the pathogenic bacteria Porphyromonas gingivalis and Fusobacterium nucleatum for 12 weeks, followed by 3 weeks of chronic mild stress (CMS) to induce depressive-like behavior. The following four groups were established (n = 12 rats/group): periodontitis and CMS (P + CMS+), periodontitis without CMS, CMS without periodontitis, and control. The morphology and inflammatory phenotype of microglia in the frontal cortex (FC) were studied using immunofluorescence and bioinformatics tools. The endocannabinoid (EC) signaling and proteins related to synaptic plasticity were analyzed in FC samples using biochemical and immunohistochemical techniques. RESULTS: Ultrastructural and fractal analyses of FC revealed a significant increase in the complexity and heterogeneity of Iba1 + parenchymal microglia in the combined experimental model (P + CMS+) and increased expression of the proinflammatory marker inducible nitric oxide synthase (iNOS), while there were no changes in the expression of cannabinoid receptor 2 (CB2). In the FC protein extracts of the P + CMS + animals, there was a decrease in the levels of the EC metabolic enzymes N-acyl phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), diacylglycerol lipase (DAGL), and monoacylglycerol lipase (MAGL) compared to those in the controls, which extended to protein expression in neurons and in FC extracts of cannabinoid receptor 1 (CB1) and to the intracellular signaling molecules phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2). The protein levels of brain-derived neurotrophic factor (BDNF) and synaptophysin were also lower in P + CMS + animals than in controls. CONCLUSIONS: The combined effects on microglial morphology and inflammatory phenotype, the EC signaling, and proteins related to synaptic plasticity in P + CMS + animals may represent relevant mechanisms explaining the association between periodontitis and depression. These findings highlight potential therapeutic targets that warrant further investigation.

Características antropométricas, capacidad de salto vertical y resistencia de carrera de bomberos, en servicio activo, con relación a la edad / Anthropometric characteristics, vertical jumping capacity and running endurance, in active-duty firefighters, in relation to age

El objetivo de este estudio fue establecer relaciones entre las variables de rendimiento físico e índices antropométricos según la edad de bomberos adultos chilenos, en servicio activo, provenientes de la región de Valparaíso. Participaron 68 bomberos, hombres, sanos, en servicio activo, que fueron divididos en dos grupos según edad; 30 años (n = 32; 40,6 ± 8,5 años). Se evaluaron las variables antropométricas de masa corporal (MC), estatura, perímetro de cintura (PC), índice de masa corporal (IMC] = Peso /estatura2 [m]) e índice cintura-estatura (ICE). La capacidad de salto vertical fue evaluada con los protocolos de Squat Jump, Countermovement Jump y Abalakov Jump. La resistencia en carrera fue estimada con la prueba de Course Navette y se calculó indirectamente el consumo de oxígeno (VO2). Las variables antropométricas para el grupo > 30 años fueron mayores en comparación al grupo de menor edad en MC (p = 0.027), IMC (p = 0.015), PC (p 0.05) entre ambos grupos. Existió una correlación significativa positiva entre la edad y las variables de MC (r = 0,252), IMC (r = 0,307), ICE (r = 0,431) y PC (r= 0,401). Al comparar ambos grupos de edad hubo diferencias antropométricas, pero no en la condición física. Se sugiere reforzar programas de entrenamiento para optimizar la composición corporal y capacidad física de bomberos en servicio activo para responder a las exigentes tareas que demanda este ámbito laboral.

SUMMARY: The aim of this study was 1) to compare anthropometric characteristics, jumping ability, and running endurance according to age and 2) to determine the relationship between age and physical performance parameters in Chilean adult firefighters in active service from the Valparaíso region. Sixty-eight healthy male firefighters, in active service, were divided into two groups according to age; 30 years (n = 32; 40.6 ± 8.5 years) participated. The anthropometric variables of body mass (BM), height, waist circumference (WC), body mass index (BMI = weight/height2 [m]) and waist-to-height ratio (WHR) were evaluated. Vertical jumping ability was assessed with the Squat Jump, Countermovement Jump and Abalakov Jump protocols. Running endurance was estimated with the Course Navette test and oxygen consumption (VO2) was calculated indirectly. Anthropometric variables for the > 30 years group were higher compared to the younger age group in BM (p = 0.027), BMI (p = 0.015), WC (p 0.05). There was a significant positive correlation between age and the variables of BM (r = 0.252), BMI (r = 0.307), WHR (r = 0.431) y WC (r= 0.401). When comparing both age groups, there were anthropometric differences, but not in physical condition. It is suggested to reinforce training programs to optimize the body composition and physical capacity of firefighters in active service in order to respond to the demanding tasks demanded by this work environment.

Neuroinflammation related to the blood-brain barrier and sphingosine-1-phosphate in a pre-clinical model of periodontal diseases and depression in rats.

AIM: To explore the potential mechanisms of neuroinflammation (microglia, blood-brain barrier [BBB] permeability, and the sphingosine-1-phosphate [S1P] pathways) resulting from the association between periodontitis and depression in rats. MATERIALS AND METHODS: This pre-clinical in vivo experimental study used Wistar rats, in which experimental periodontitis (P) was induced by using oral gavages with Porphyromonas gingivalis and Fusobacterium nucleatum. Then, a chronic mild stress (CMS) model was implemented to induce a depressive-like behaviour, resulting in four groups: P with CMS (P+CMS+), P without CMS (P+CMS-), CMS without P (P-CMS+), and control (P-CMS-). After harvesting brain samples, protein/mRNA expression analyses and fluorescence immunohistochemistry were performed in the frontal cortex (FC). Results were analysed by ANOVA. RESULTS: CMS exposure increased the number of microglia (an indicator of neuroinflammation) in the FC. In the combined model (P+CMS+), there was a decrease in the expression of tight junction proteins (zonula occludens-1 [ZO-1], occludin) and an increase in intercellular and vascular cell adhesion molecules (ICAM-1, VCAM-1) and matrix metalloproteinase 9 (MMP9), suggesting a more severe disruption of the BBB. The enzymes and receptors of S1P were also differentially regulated. CONCLUSIONS: Microglia, BBB permeability, and S1P pathways could be relevant mechanisms explaining the association between periodontitis and depression.

Lipid nanoparticles for antisense oligonucleotide gene interference into brain border-associated macrophages.

A colloidal synthesis' proof-of-concept based on the Bligh-Dyer emulsion inversion method was designed for integrating into lipid nanoparticles (LNPs) cell-permeating DNA antisense oligonucleotides (ASOs), also known as GapmeRs (GRs), for mRNA interference. The GR@LNPs were formulated to target brain border-associated macrophages (BAMs) as a central nervous system (CNS) therapy platform for silencing neuroinflammation-related genes. We specifically aim at inhibiting the expression of the gene encoding for lipocalin-type prostaglandin D synthase (L-PGDS), an anti-inflammatory enzyme expressed in BAMs, whose level of expression is altered in neuropsychopathologies such as depression and schizophrenia. The GR@LNPs are expected to demonstrate a bio-orthogonal genetic activity reacting with L-PGDS gene transcripts inside the living system without interfering with other genetic or biochemical circuitries. To facilitate selective BAM phagocytosis and avoid subsidiary absorption by other cells, they were functionalized with a mannosylated lipid as a specific MAN ligand for the mannose receptor presented by the macrophage surface. The GR@LNPs showed a high GR-packing density in a compact multilamellar configuration as structurally characterized by light scattering, zeta potential, and transmission electronic microscopy. As a preliminary biological evaluation of the mannosylated GR@LNP nanovectors into specifically targeted BAMs, we detected in vivo gene interference after brain delivery by intracerebroventricular injection (ICV) in Wistar rats subjected to gene therapy protocol. The results pave the way towards novel gene therapy platforms for advanced treatment of neuroinflammation-related pathologies with ASO@LNP nanovectors.

Sphk2 deletion is involved in structural abnormalities and Th17 response but does not aggravate colon inflammation induced by sub-chronic stress.

The chronic inflammatory process that characterizes inflammatory bowel diseases (IBD) is mainly driven by T-cell response to microbial and environmental antigens. Psychological stress is a potential trigger of clinical flares of IBD, and sphingosine-1-phosphate (S1P) is involved in T-cell recruitment. Hence, stress impact and the absence of sphingosine kinase 2 (Sphk2), an enzyme of S1P metabolism, were evaluated in the colon of mice after sub-chronic stress exposure. Here, we show that sub-chronic stress increased S1P in the mouse colon, possibly due to a decrease in its degradation enzymes and Sphk2. S1P accumulation could lead to inflammation and immune dysregulation reflected by upregulation of toll-like receptor 4 (TLR4) pathway, inhibition of anti-inflammatory mechanisms, cytokine-expression profile towards a T-helper lymphocyte 17 (Th17) polarization, plasmacytosis, decrease in IgA+ lymphoid lineage cells (CD45+)/B cells/plasmablasts, and increase in IgM+ B cells. Stress also enhanced intestinal permeability. Sphk2 knockout mice presented a cytokine-expression profile towards a boosted Th17 response, lower expression of claudin 3,4,7,8, and structural abnormalities in the colon. Intestinal pathophysiology should consider stress and S1P as modulators of the immune response. S1P-based drugs, including Sphk2 potentiation, represent a promising approach to treat IBD.

Depletion of brain perivascular macrophages regulates acute restraint stress-induced neuroinflammation and oxidative/nitrosative stress in rat frontal cortex.

The central nervous system can respond to peripheral immune stimuli through the activation of the neurovascular unit. One of the cellular types implicated are perivascular macrophages (PVMs), hematopoietic-derived brain-resident cells located in the perivascular space. PVMs have been implicated in the immune surveillance and in the regulation of the accumulation/trafficking of macromolecules in brain-blood interfaces. Recent studies suggested that the role of PVMs could vary depending on the nature and duration of the immune challenge applied. Here, we investigate the role of PVMs in stress-induced neuroinflammation and oxidative/nitrosative consequences. The basal phagocytic activity of PVMs was exploited to selectively deplete them by ICV injection of liposomes encapsulating the pro-apoptotic drug clodronate. Acute restraint stress-induced neuroinflammation and oxidative/nitrosative stress in rat brain frontal cortex samples were assessed by western blot and RT-PCR analyses. The depletion of PVMs: (1) decreased tumor necrosis-α levels (2) prevented the Janus kinase/signal transducers and activators of transcription pathway and increased interleukin-6 receptor protein-expression in stress conditions; (3) prevented the stress-induced Toll-like receptor 4/Myeloid differentiation primary response 88 protein signaling pathway; (4) down-regulated the pro-inflammatory nuclear factor κB/cyclooxygenase-2 pathway; (5) prevented stress-induced lipid peroxidation and the concomitant increase of the endogenous antioxidant mediators nuclear factor (erythroid-derived 2)-like 2, glutathione reductase 1 and Parkinsonism-associated deglycase mRNA expression. Our results point to PVMs as regulators of stress-induced neuroinflammation and oxidative/nitrosative stress. Much more scientific effort is still needed to evaluate whether their selective manipulation is promising as a therapeutic strategy for the treatment of stress-related neuropsychopathologies.

Toll-like receptor 4 agonist and antagonist lipopolysaccharides modify innate immune response in rat brain circumventricular organs.

BACKGROUND: The circumventricular organs (CVOs) are blood-brain-barrier missing structures whose activation through lipopolysaccharide (LPS) is a starting point for TLR-driven (Toll-like receptors) neuroinflammation. The aim of this study was to evaluate in the CVO area postrema (AP), subfornical organ (SFO), and median eminence (ME), the inflammatory response to two TLR4 agonists: LPS from Escherichia coli (EC-LPS), the strongest endotoxin molecule described, and LPS from Porphyromonas gingivalis (PG-LPS), a pathogenic bacteria present in the periodontium related to neuroinflammation in neurodegenerative/psychiatric diseases. The response to LPS from the cyanobacteria Rhodobacter sphaeroides (RS-LPS), a TLR4 antagonist with an interesting anti-inflammatory potential, was also assessed. METHODS: LPSs were intraperitoneally administered to Wistar rats and, as indicatives of neuroinflammation in CVOs, the cellular localization of the nuclear factor NF-κB was studied by immunofluorescence, and microglia morphology was quantified by fractal and skeleton analysis. RESULTS: Data showed that EC-LPS increased NF-κB nuclear translocation in the three CVOs studied and PG-LPS only induced NF-κB nuclear translocation in the ME. RS-LPS showed no difference in NF-κB nuclear translocation compared to control. Microglia in the three CVOs showed an ameboid-shape after EC-LPS exposure, whereas PG-LPS only elicited a mild tendency to induce an ameboid shape. On the other hand, RS-LPS produced a markedly elongated morphology described as "rod" microglia in the three CVOs. CONCLUSIONS: In conclusion, at the doses tested, EC-LPS induces a stronger neuroinflammatory response than PG-LPS in CVOs, which might be related to their different potency as TLR4 agonists. The non-reduction of basal NF-κB activation and induction of rod microglia by RS-LPS, a cell morphology only present in severe brain injury and infections, suggests that this molecule must be carefully studied before being proposed as an anti-inflammatory treatment for neuroinflammation related to neurodegenerative/psychiatric diseases.

The role of ipilimumab after anti-PD-1 treatment: two case reports and a literature review.

Metastatic melanoma has been historically associated with a poor prognosis; however, the therapeutic landscape has experimented and impressive change in the last years due to rapid advances in the immunotherapy field. The first immunotherapy treatment for metastatic melanoma was ipilimumab (anti-CTLA-4), which showed a significant improvement of overall survival compared to chemotherapy. However, in 2015 anti-PD-1 pembrolizumab shown an improved overall survival, progression-free survival and response rate compared to ipilimumab with either a better toxicity profile. Moreover, other immunotherapy combinations and target therapies, such as BRAF and MEK inhibitors combinations, have shown better outcomes than ipilimumab. Thus, ipilimumab seems to have no role in frontline metastatic melanoma treatment and even their role in second line is being less frequent due to clinical efficacy of those other treatments. Actually, the role of ipilimumab in second line after anti-PD-1 progression is not clear although there is clinical evidence for its use. Here, we report two cases of treatment response with ipilimumab in second line setting after receiving anti-PD-1 combination. So that, ipilimumab may have a role after progression to an anti-PD-1 treatment.

Practical utility of on-line clearance and blood temperature monitors as noninvasive techniques to measure hemodialysis blood access flow.

BACKGROUND/AIMS: Access blood flow (Qa) measurements are recommended by the current guidelines as one of the most important components in vascular access maintenance programs. This study evaluates the efficiency of Qa measurement with on-line conductivity (OLC-Qa) and blood temperature monitoring (BTM-Qa) in comparison with the gold standard saline dilution method (SDM-Qa). SUBJECTS AND METHODS: 50 long-term hemodialysis patients (42 arteriovenous fistulas/8 arteriovenous grafts) were studied. Bland-Altman and Lin's coefficient (ρ(c)) were used to study accuracy and precision. RESULTS: Mean values were 1,021.7 ± 502.4 ml/min SDM-Qa, 832.8 ± 574.3 ml/min OLC-Qa (p = 0.007) and 1,094.9 ± 491.9 ml/min with BTM-Qa (p = NS). Biases and ρ(c) obtained were -188.8 ml/min (ρ(c) 0.58) OLC-Qa and 73.2 ml/min (ρ(c) 0.89) BTM-Qa. The limits of agreement (bias ± 1.96 SD) obtained were from -1,119 to 741.3 ml/min (OLC-Qa) and -350.6 to 497.2 ml/min (BTM-Qa). CONCLUSIONS: BTM-Qa and OLC-Qa are valid noninvasive and practical methods to estimate Qa, although BTM-Qa was more accurate and had better concordance than OLC-Qa compared with SDM-Qa.