Amino Acid Derivatives as Palmitoylethanolamide Prodrugs: Synthesis,In Vitro Metabolism and In Vivo Plasma Profile in Rats

Palmitoylethanolamide (PEA) has antinflammatory and antinociceptive properties widely exploited in veterinary and human medicine, despite its poor pharmacokinetics. Looking for prodrugs that could progressively release PEA to maintain effective plasma concentrations, we prepared carbonates, esters and carbamates at the hydroxyl group of PEA. Chemical stability (pH 7.4) and stability in rat plasma and liver homogenate were evaluated by in vitro assays. Carbonates and carbamates resulted too labile and too resistant in plasma, respectively. Ester derivatives, prepared by conjugating PEA with various amino acids, allowed to modulate the kinetics of PEA release in plasma and stability in liver homogenate. L-Val-PEA, with suitable PEA release in plasma, and D-Val-PEA, with high resistance to hepatic degradation, were orally administered to rats and plasma levels of prodrugs and PEA were measured at different time points. Both prodrugs showed significant release of PEA, but provided lower plasma concentrations than those obtained with equimolar doses of PEA. Amino-acid esters of PEA are a promising class to develop prodrugs, even if they need further chemical optimization.     

Impact of Childhood Maltreatment on the Recognition of Facial Expressions of Emotions

The development of the explicit recognition of facial expressions of emotions can be affected by childhood maltreatment experiences. A previous study demonstrated the existence of an explicit recognition bias for angry facial expressions among a population of adolescent Sierra Leonean street-boys exposed to high levels of maltreatment. In the present study, the recognition bias for angry facial expressions was investigated in a younger population of street-children and age-matched controls. Participants performed a forced-choice facial expressions recognition task. Recognition bias was measured as participants’ tendency to over-attribute anger label to other negative facial expressions. Participants’ heart rate was assessed and related to their behavioral performance, as index of their stress-related physiological responses. Results demonstrated the presence of a recognition bias for angry facial expressions among street-children, also pinpointing a similar, although significantly less pronounced, tendency among controls. Participants’ performance was controlled for age, cognitive and educational levels and for naming skills. None of these variables influenced the recognition bias for angry facial expressions. Differently, a significant effect of heart rate on participants’ tendency to use anger label was evidenced. Taken together, these results suggest that childhood exposure to maltreatment experiences amplifies children’s “pre-existing bias” for anger labeling in forced-choice emotion recognition task. Moreover, they strengthen the thesis according to which the recognition bias for angry facial expressions is a manifestation of a functional adaptive mechanism that tunes victim’s perceptive and attentive focus on salient environmental social stimuli.     

The Association between Mycobacterium Tuberculosis Genotype and Drug Resistance in Peru

BackgroundThe comparison of Mycobacterium tuberculosis bacterial genotypes with phenotypic, demographic, geospatial and clinical data improves our understanding of how strain lineage influences the development of drug-resistance and the spread of tuberculosis.MethodsTo investigate the association of Mycobacterium tuberculosis bacterial genotype with drug-resistance. Drug susceptibility testing together with genotyping using both 15-loci MIRU-typing and spoligotyping, was performed on 2,139 culture positive isolates, each from a different patient in Lima, Peru. Demographic, geospatial and socio-economic data were collected using questionnaires, global positioning equipment and the latest national census.ResultsThe Latin American Mediterranean (LAM) clade (OR 2.4, p<0.001) was significantly associated with drug-resistance and alone accounted for more than half of all drug resistance in the region. Previously treated patients, prisoners and genetically clustered cases were also significantly associated with drug-resistance (OR''s 2.5, 2.4 and 1.8, p<0.001, p<0.05, p<0.001 respectively).ConclusionsTuberculosis disease caused by the LAM clade was more likely to be drug resistant independent of important clinical, genetic and socio-economic confounding factors. Explanations for this include; the preferential co-evolution of LAM strains in a Latin American population, a LAM strain bacterial genetic background that favors drug-resistance or the "founder effect" from pre-existing LAM strains disproportionately exposed to drugs.     

Age-Related Left Ventricular Changes and Their Association with Leukocyte Telomere Length in Healthy People

Introduction

With advancing age the left ventricle (LV) undergoes structural and functional changes, thereby creating the substrate for the development of diseases. One possible mechanism of the ageing heart is a cellular senescence. Leukocyte telomere length (LTL) is a marker of replicative ageing. The purpose of this study was to evaluate the structure and function of the LV in people of different ages free of cardiovascular diseases (CVD) and regular drug medication and to assess their relationship with LTL. We hypothesized that age-related changes in LV myocardium are associated with telomere length.

Methods

The study population consisted of 150 healthy, non-obese volunteers aged 28 to 78 years without history of CVD, significant deviations by 12-lead electrocardiogram and negative exercise test (treadmill stress test). All the participants underwent standardized transthoracic echocardiography using an available system (iE33; Philips). The LTL was measured by real-time quantitative polymerase chain reaction. We determined the relative ratio of telomere repeat copy number (T) to single-copy gene copy number (S).

Results

In the older people there was a higher wall thickness than in the younger (1.03±0.09 vs. 0.88±0.10, p<0.01), whereas LV mass index was comparable between them (85.8±15.40 vs. 83.1±11.8, p = 0.20). There was a decrease in LV dimensions with advancing age (p<0.001). Older subjects had impairment in LV relaxation. LTL was associated with decreased E/A, Em/Am ratio (β = -0.323, p = 0.0001) after adjusting for age, sex and risk factors. There is no relation between the LTL and the structure of LV.

Conclusions

Our data suggest that the ageing process leads to changes in LV structure and diastolic function and is linked with a phenotype of concentric LV remodeling. Telomere attrition is associated with age-related LV diastolic dysfunction. Telomere length appears to be a biomarker of myocardial ageing.     

Post-Operative Benefits of Animal-Assisted Therapy in Pediatric Surgery: A Randomised Study

Background

Interest in animal-assisted therapy has been fuelled by studies supporting the many health benefits. The purpose of this study was to better understand the impact of an animal-assisted therapy program on children response to stress and pain in the immediate post-surgical period.

Patients and Methods

Forty children (3–17 years) were enrolled in the randomised open-label, controlled, pilot study. Patients were randomly assigned to the animal-assisted therapy-group (n = 20, who underwent a 20 min session with an animal-assisted therapy dog, after surgery) or the standard-group (n = 20, standard postoperative care). The study variables were determined in each patient, independently of the assigned group, by a researcher unblinded to the patient’s group. The outcomes of the study were to define the neurological, cardiovascular and endocrinological impact of animal-assisted therapy in response to stress and pain. Electroencephalogram activity, heart rate, blood pressure, oxygen saturation, cerebral prefrontal oxygenation, salivary cortisol levels and the faces pain scale were considered as outcome measures.

Results

After entrance of the dog faster electroencephalogram diffuse beta-activity (> 14 Hz) was reported in all children of the animal-assisted therapy group; in the standard-group no beta-activity was recorded (100% vs 0%, p<0.001). During observation, some differences in the time profile between groups were observed for heart rate (test for interaction p = 0.018), oxygen saturation (test for interaction p = 0.06) and cerebral oxygenation (test for interaction p = 0.09). Systolic and diastolic blood pressure were influenced by animal-assisted therapy, though a higher variability in diastolic pressure was observed. Salivary cortisol levels did not show different behaviours over time between groups (p=0.70). Lower pain perception was noted in the animal-assisted group in comparison with the standard-group (p = 0.01).

Conclusion

Animal-assisted therapy facilitated rapid recovery in vigilance and activity after anaesthesia, modified pain perception and induced emotional prefrontal responses. An adaptative cardiovascular response was also present.

Trial Registration

ClinicalTrials.gov NCT02284100     

Characterisation of (R)-2-(2-Fluorobiphenyl-4-yl)-N-(3-Methylpyridin-2-yl)Propanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor

Background

Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin-2-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known.

Methodology/Principal Findings

COX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC₅₀ values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 μM; COX-2 (arachidonic acid) 20 μM; COX-2 (2-AG) 1 μM; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 μM; COX-2 (arachidonic acid) 10 μM; COX-2 (2-AG) 0.7 μM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 μM) greatly inhibited the production of prostaglandin D₂ and E₂ in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 μM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 μM).

Conclusions/Significance

Both enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the large induction of COX-2 produced by this treatment.     

Targeting the CaMKII/ERK Interaction in the Heart Prevents Cardiac Hypertrophy

Aims

Activation of Ca²⁺/Calmodulin protein kinase II (CaMKII) is an important step in signaling of cardiac hypertrophy. The molecular mechanisms by which CaMKII integrates with other pathways in the heart are incompletely understood. We hypothesize that CaMKII association with extracellular regulated kinase (ERK), promotes cardiac hypertrophy through ERK nuclear localization.

Methods and Results

In H9C2 cardiomyoblasts, the selective CaMKII peptide inhibitor AntCaNtide, its penetratin conjugated minimal inhibitory sequence analog tat-CN17β, and the MEK/ERK inhibitor UO126 all reduce phenylephrine (PE)-mediated ERK and CaMKII activation and their interaction. Moreover, AntCaNtide or tat-CN17β pretreatment prevented PE induced CaMKII and ERK nuclear accumulation in H9C2s and reduced the hypertrophy responses. To determine the role of CaMKII in cardiac hypertrophy in vivo, spontaneously hypertensive rats were subjected to intramyocardial injections of AntCaNtide or tat-CN17β. Left ventricular hypertrophy was evaluated weekly for 3 weeks by cardiac ultrasounds. We observed that the treatment with CaMKII inhibitors induced similar but significant reduction of cardiac size, left ventricular mass, and thickness of cardiac wall. The treatment with CaMKII inhibitors caused a significant reduction of CaMKII and ERK phosphorylation levels and their nuclear localization in the heart.

Conclusion

These results indicate that CaMKII and ERK interact to promote activation in hypertrophy; the inhibition of CaMKII-ERK interaction offers a novel therapeutic approach to limit cardiac hypertrophy.     

The Contrasting Role of p16Ink4A Patterns of Expression in Neuroendocrine and Non-Neuroendocrine Lung Tumors: A Comprehensive Analysis with Clinicopathologic and Molecular Correlations

Lung cancer encompasses a constellation of malignancies with no validated prognostic markers. p16^Ink4A expression has been reported in different subtypes of lung cancers; however, its prognostic value is controversial. Here, we sought to investigate the clinical significance of p16^Ink4A immunoexpression according to specific staining patterns and its operational implications. A total of 502 tumors, including 277 adenocarcinomas, 84 squamous cell carcinomas, 22 large cell carcinomas, 47 typical carcinoids, 12 atypical carcinoids, 28 large cell neuroendocrine carcinomas, and 32 small cell carcinomas were reviewed and subjected to immunohistochemical analysis for p16^Ink4A and Ki67. The spectrum of p16^Ink4A expression was annotated for each case as negative, sporadic, focal, or diffuse. Expression at immunohistochemical level showed intra-tumor homogeneity, regardless tumor histotype. Enrichments in cells expressing p16^Ink4A were observed from lower- to higher-grade neuroendocrine malignancies, whereas a decrease was seen in poorly and undifferentiated non-neuroendocrine carcinomas. Tumor proliferation indices were higher in neuroendocrine tumors expressing p16^Ink4A while non-neuroendocrine malignancies immunoreactive for p16^Ink4A showed a decrease in Ki67-positive cells. Quantitative statistical analyses including each histotype and the p16^Ink4A status confirmed the independent prognostic role of p16^Ink4A expression, being a high-risk indicator in neuroendocrine tumors and a marker of good prognosis in non-neuroendocrine lung malignancies. In this study, we provide circumstantial evidence to suggest that the routinary assessment of p16^Ink4A expression using a three-tiered scoring algorithm, even in a small biopsy, may constitute a reliable, reproducible, and cost-effective substrate for a more accurate risk stratification of each individual patient.     

Polyfunctional Specific Response to Echinococcus Granulosus Associates to the Biological Activity of the Cysts

Background

Cystic echinococcosis (CE) is a complex disease caused by Echinococcus granulosus (E.granulosus), and its immunophatogenesis is still not clearly defined. A peculiar feature of chronic CE is the coexistence of Th1 and Th2 responses. It has been suggested that Th1 cytokines are related to disease resistance, whereas Th2 cytokines are related to disease susceptibility and chronicity. The aim of this study was to evaluate, by multi-parametric flow cytometry (FACS), the presence of CE specific immune signatures.

Methodology/Principal Findings

We enrolled 54 subjects with suspected CE; 42 of them had a confirmed diagnosis, whereas 12 were classified as NO-CE. Based on the ultrasonography images, CE patients were further categorized as being in "active stages" (25) and "inactive stages" (17). The ability of CD4⁺ T-cells to produce IFN-γ, IL-2, TNF-α, Th2 cytokines or IL-10 was assessed by FACS on antigen-specific T-cells after overnight stimulation with Antigen B (AgB) of E.granulosus. Cytokine profiles were evaluated in all the enrolled subjects. The results show that none of the NO-CE subjects had a detectable AgB-specific response. Among the CE patients, the frequency and proportions of AgB-specific CD4⁺ T-cells producing IL-2⁺TNF-α⁺Th2⁺ or TNF-α⁺Th2⁺ were significantly increased in the “active stages” group compared to the “inactive stages” group. Moreover, an increased proportion of the total polyfunctional subsets, as triple-and double-functional CD4 T-cells, was found in CE patients with active disease. The response to the mitogen, used as a control stimulus to evaluate the immune competence status, was characterized by the same cytokine subsets in all the subjects enrolled, independent of CE.

Conclusions

We demonstrate, for the first time to our knowledge, that polyfunctional T-cell subsets as IL-2⁺TNF-α⁺Th2⁺ triple-positive and TNF-α⁺Th2⁺ double-positive specific T-cells associate with cyst biological activity. These results contribute to increase knowledge of CE immunophatogenesis and the disease outcome in terms of control and persistence.     

BoHV-4-Based Vector Single Heterologous Antigen Delivery Protects STAT1(-/-) Mice from Monkeypoxvirus Lethal Challenge

Monkeypox virus (MPXV) is the etiological agent of human (MPX). It is an emerging orthopoxvirus zoonosis in the tropical rain forest of Africa and is endemic in the Congo-basin and sporadic in West Africa; it remains a tropical neglected disease of persons in impoverished rural areas. Interaction of the human population with wildlife increases human infection with MPX virus (MPXV), and infection from human to human is possible. Smallpox vaccination provides good cross-protection against MPX; however, the vaccination campaign ended in Africa in 1980, meaning that a large proportion of the population is currently unprotected against MPXV infection. Disease control hinges on deterring zoonotic exposure to the virus and, barring that, interrupting person-to-person spread. However, there are no FDA-approved therapies against MPX, and current vaccines are limited due to safety concerns. For this reason, new studies on pathogenesis, prophylaxis and therapeutics are still of great interest, not only for the scientific community but also for the governments concerned that MPXV could be used as a bioterror agent. In the present study, a new vaccination strategy approach based on three recombinant bovine herpesvirus 4 (BoHV-4) vectors, each expressing different MPXV glycoproteins, A29L, M1R and B6R were investigated in terms of protection from a lethal MPXV challenge in STAT1 knockout mice. BoHV-4-A-CMV-A29LgD₁₀₆ΔTK, BoHV-4-A-EF1α-M1RgD₁₀₆ΔTK and BoHV-4-A-EF1α-B6RgD₁₀₆ΔTK were successfully constructed by recombineering, and their capacity to express their transgene was demonstrated. A small challenge study was performed, and all three recombinant BoHV-4 appeared safe (no weight-loss or obvious adverse events) following intraperitoneal administration. Further, BoHV-4-A-EF1α-M1RgD₁₀₆ΔTK alone or in combination with BoHV-4-A-CMV-A29LgD₁₀₆ΔTK and BoHV-4-A-EF1α-B6RgD₁₀₆ΔTK, was shown to be able to protect, 100% alone and 80% in combination, STAT1^(-/-) mice against mortality and morbidity. This work demonstrated the efficacy of BoHV-4 based vectors and the use of BoHV-4 as a vaccine-vector platform.