Anandamide prodrugs. 1. Water-soluble phosphate esters of arachidonylethanolamide and R-methanandamide.

Phosphate esters of arachidonylethanolamide (AEA) and R-methanandamide were synthesized and evaluated as water-soluble prodrugs. Various physicochemical properties (pK(a), partition coefficient, aqueous solubility) were determined for the synthesized phosphate esters. The chemical stability of phosphate esters was determined at pH 7.4. In vitro enzymatic hydrolysis rates were determined in 10% liver homogenate, and in a pure enzyme-containing (alkaline phosphatase) solution at pH 7.4. The intraocular pressure (IOP) lowering properties of R-methanandamide phosphate ester were tested on normotensive rabbits. The phosphate promoiety increased the aqueous solubility of the parent compounds by more than 16500-fold at pH 7.4. Phosphate esters were stable in buffer solutions, but rapidly hydrolyzed to their parent compounds in alkaline phosphatase solution (t(1/2)<15 s) and liver homogenate (t(1/2)=8-9 min). The phosphate ester of R-methanandamide reduced IOP in rabbits. These results indicate that the phosphate esters of AEA and R-methanandamide are useful water-soluble prodrugs.     

Intracellular Reservoir of Streptococcus pyogenes In Vivo: A Possible Explanation for Recurrent Pharyngotonsillitis

Numerous theories have been presented that attempt to explain the frequent recurrences of pharyngotonsillitis caused by Streptococcus pyogenes; these recurrences occur after seemingly adequate antibiotic treatment. We previously have demonstrated that S pyogenes can survive for up to 7 days intracellularly in immortalized human respiratory epithelial cells grown in an antibiotic supplemented medium. Viable S pyogenes were externalized and established an extracellular infection, whenever the extracellular antibiotic was removed. We have investigated the presence of intracellular S pyogenes in two in vivo studies using respiratory epithelial cells collected from patients with tonsillitis and the tonsils of asymptomatic carriers. Electron microscopy and immunohistochemistry demonstrated intracellular S pyogenes in pharyngeal epithelial cells in 13 of 14 patients with tonsillitis (93%). Furthermore, intracellular S pyogenes were found in macrophage-like cells in eight (73%) and in epithelial cells in four (36%) tonsils from 11 asymptomatic S pyogenes carriers. These in vivo data strongly support the hypothesis that intracellular S pyogenes can constitute a reservoir of bacteria with the potential to cause reinfections     

Chlamydial antibodies and risk of prostate cancer.

OBJECTIVE: We assessed the risk of prostate cancer by exposure to Chlamydia trachomatis. METHOD: Seven hundred thirty eight cases of prostate cancer and 2,271 matched controls were identified from three serum sample banks in Finland, Norway, and Sweden by linkage to the population based cancer registries. RESULTS: A statistically significant inverse association (odds ratio, 0.69; 95% confidence interval, 0.51-0.94) was found. It was consistent by different serotypes and there was a consistent dose-response relationship. CONCLUSION: C. trachomatis infection is not likely to increase the risk of prostate cancer. Whether the inverse relationship is true or due to difficulties in measuring the true exposure in prostatic tissue by serology, confounders or other sources of error remain open.     

Effects of Upper Body Eccentric versus Concentric Strength Training and Detraining on Maximal Force,Muscle Activation,Hypertrophy and Serum Hormones in Women

Effects of eccentric (ECC) versus concentric (CON) strength training of the upper body performed twice a week for 10 weeks followed by detraining for five weeks on maximal force, muscle activation, muscle mass and serum hormone concentrations were investigated in young women (n = 11 and n = 12). One-repetition bench press (1RM), maximal isometric force and surface electromyography (EMG) of triceps brachii (TB), anterior deltoid (AD) and pectoralis major (PM), cross-sectional area (CSA) of TB (Long (LoH) and Lateral Head (LaH)) and thickness of PM, as well as serum concentrations of free testosterone, cortisol, follicle-stimulating hormone, estradiol and sex hormone-binding globulin were measured. ECC and CON training led to increases of 17.2 ± 11.3% (p < 0.001) and 13.1 ± 5.7% (p < 0.001) in 1RM followed by decreases of -6.6 ± 3.6% (p < 0.01) and -8.0 ± 4.5% (p < 0.001) during detraining, respectively. Isometric force increased in ECC by 11.4 ± 9.6 % (p < 0.05) from week 5 to 10, while the change in CON by 3.9±6.8% was not significant and a between group difference was noted (p < 0.05). Maximal total integrated EMG of trained muscles increased only in the whole subject group (p < 0.05). CSA of TB (LoH) increased in ECC by 8.7 ± 8.0% (p < 0.001) and in CON by 3.4 ± 1.6% (p < 0.01) and differed between groups (p < 0.05), and CSA of TB (LaH) in ECC by 15.7 ± 8.0% (p < 0.001) and CON by 9.7 ± 6.6% (p < 0.001). PM thickness increased in ECC by 17.7 ± 10.9% (p < 0.001) and CON by 14.0 ± 5.9% (p < 0.001). Total muscle sum value (LoH + LaH + PM) increased in ECC by 12.4 ± 6.9% (p < 0.001) and in CON by 7.1 ± 2.9% (p < 0.001) differing between groups (p < 0.05) and decreased during detraining in ECC by -6.5 ± 4.3% (p < 0.001) and CON by -6.1 ± 2.8% (p < 0.001). The post detraining combined sum value of CSA and thickness was in ECC higher (p < 0.05) than at pre training. No changes were detected in serum hormone concentrations, but baseline free testosterone levels in the ECC and CON group combined correlated with changes in 1RM (r = 0.520, p < 0.016) during training. Large neuromuscular adaptations of the upper body occurred in women during ECC, and CON training in 10 weeks. Isometric force increased only in response to ECC, and total muscle sum value increased more during ECC than CON training. However, no changes occurred in serum hormones, but individual serum-free testosterone baseline concentrations correlated with changes in 1RM during strength training in the entire group. Both groups showed significant decreases in neuromuscular performance and muscle mass during detraining, while post detraining muscle sum value was only in ECC significantly higher than at pre training. Key points

• Maximal eccentric and concentric strength training in women for 10 weeks (only twice a week) led to great gains in dynamic strength of upper body.
• Upper body muscle mass increased more after eccentric than concentric strength training.
• During detraining for 5 weeks both groups showed decreases in strength and muscle mass.
• However, post-detraining combined sum muscle mass value was only in ECC higher than at pre-training.

Key words: Muscle force, training, EMG, mass, females, testosterone     

Molecular alterations in pediatric brainstem gliomas

1 Background

Diffuse intrinsic pontine gliomas (DIPGs) have a dismal prognosis. Previously, diagnosis was based on a typical clinical presentation and magnetic resonance imaging findings. After the start of the era of biopsies, DIPGs bearing H3 K27 mutations have been reclassified into a novel entity, diffuse midline glioma, based on the presence of this molecular alteration. However, it is not well established how clinically diagnosed DIPG overlap with H3 K27‐mutated diffuse midline gliomas, and whether rare long‐term survivors also belong to this group.

2 Methods

We studied tumor samples obtained at diagnosis or upon autopsy from 23 children, including two long‐term survivors. Based on clinical, radiological, and histological findings, all tumors were previously diagnosed as DIPGs. All samples were analyzed for genetic alterations by next‐generation sequencing (NGS) and for protein expression by immunohistochemistry (IHC).

3 Results

H3 K27 was mutated in NGS or IHC in 20 patients, excluding both long‐term survivors. One of these long‐term survivors harbored a mutation in IDH1, formerly considered to be an alteration absent in pediatric diffuse brainstem gliomas. Other altered genes in NGS included TP53 (10 patients), MET and PDGFRA (3 patients each), VEGFR and SMARCA4 (2 patients each), and PPARγ, PTEN and EGFR in 1 patient, respectively. IHC revealed cMYC expression in 15 of 24 (63%) of all samples, exclusively in the biopsies.

4 Conclusions

Eighty‐seven percent of the tumors formerly diagnosed as DIPGs could be reclassified as H3 K27‐mutated diffuse midline gliomas. Both long‐term survivors lacked this alteration. Contrary to former conceptions, IDH1 mutations may occur also in pediatric brainstem gliomas.     

Celastrol regulates innate immunity response via NF-κB and Hsp70 in human retinal pigment epithelial cells

Elevated nuclear factor kappa B (NF-κB) activity and interleukin-6 (IL-6) secretion participates in the pathology of several age and inflammatory-related diseases, including age-related macular degeneration (AMD), in which retinal pigment epithelial cells are the key target. Recent findings reveal that heat shock protein 70 (Hsp70) may affect regulation of NF-κB. In the current study, effects of Hsp70 expression on NF-κB RelA/p65 activity were evaluated in human retinal pigment epithelial cells (ARPE-19) by using celastrol, a novel anti-inflammatory compound. Anti-inflammatory properties of celastrol were determined by measuring expression levels of IL-6 and endogenous NF-κB levels during lipopolysaccharide (LPS) exposure by using enzyme-linked immunosorbent assays (ELISA). Cell viability was measured by MTT and LDH assay, and Hsp70 expression levels were analyzed by Western blotting. ARPE-19 cells were transfected with hsp70 small interfering RNA (siRNA) in order to attenuate Hsp70 expression and activity of NF-κB RelA/p65 was measured using NF-κB consensus bound ELISA.Simultaneous exposures to LPS and celastrol reduced IL-6 expression levels as well as activity of phosphorylated NF-κB at serine 536 (Ser536) in ARPE-19 cells when compared to LPS exposure alone. In addition, inhibition of NF-κB RelA/p65 activity by celastrol was attenuated when Hsp70 response was silenced by siRNA. Favorable anti-inflammatory concentrations of celastrol showed no signs of cytotoxic response. Our findings reveal that celastrol is a novel plant compound which suppresses innate immunity response in human retinal pigment epithelial cells via NF-κB and Hsp70 regulation, and that Hsp70 is a critical regulator of NF-κB.     

Physiological instability is linked to mortality in primary central nervous system lymphoma: A case–control fMRI study

Primary central nervous system lymphoma (PCNSL) is an aggressive brain disease where lymphocytes invade along perivascular spaces of arteries and veins. The invasion markedly changes (peri)vascular structures but its effect on physiological brain pulsations has not been previously studied. Using physiological magnetic resonance encephalography (MREG_BOLD) scanning, this study aims to quantify the extent to which (peri)vascular PCNSL involvement alters the stability of physiological brain pulsations mediated by cerebral vasculature. Clinical implications and relevance were explored. In this study, 21 PCNSL patients (median 67y; 38% females) and 30 healthy age‐matched controls (median 63y; 73% females) were scanned for MREG_BOLD signal during 2018–2021. Motion effects were removed. Voxel‐by‐voxel Coefficient of Variation (CV) maps of MREG_BOLD signal was calculated to examine the stability of physiological brain pulsations. Group‐level differences in CV were examined using nonparametric covariate‐adjusted tests. Subject‐level CV alterations were examined against control population Z‐score maps wherein clusters of increased CV values were detected. Spatial distributions of clusters and findings from routine clinical neuroimaging were compared [contrast‐enhanced, diffusion‐weighted, fluid‐attenuated inversion recovery (FLAIR) data]. Whole‐brain mean CV was linked to short‐term mortality with 100% sensitivity and 100% specificity, as all deceased patients revealed higher values (n = 5, median 0.055) than surviving patients (n = 16, median 0.028) (p < .0001). After adjusting for medication, head motion, and age, patients revealed higher CV values (group median 0.035) than healthy controls (group median 0.024) around arterial territories (p ≤ .001). Abnormal clusters (median 1.10 × 10⁵mm³) extended spatially beyond FLAIR lesions (median 0.62 × 10⁵mm³) with differences in volumes (p = .0055).