Autophagy and ubiquitin-dependent proteolysis processes in left ventricular mass loss in pulmonary arterial hypertension.

The goal of this study was to evaluate the intensity of autophagy and ubiquitin-dependent proteolysis processes occurring in myocardium of left ventricle (LV) in subsequent stages of pulmonary arterial hypertension (PAH) to determine mechanisms responsible for LV mass loss in a monocrotaline-induced PAH rat model. LV myocardium samples collected from 32 Wistar rats were analyzed in an early PAH group (n = 8), controls time-paired (n = 8), an end-stage PAH group (n = 8), and their controls (n = 8). Samples were subjected to histological analyses with immunofluorescence staining, autophagy assessment by western blotting, and evaluation of ubiquitin-dependent proteolysis in the LV by immunoprecipitation of ubiquitinated proteins. Echocardiographic, hemodynamic, and heart morphometric parameters were assessed regularly throughout the experiment. Considerable morphological and hemodynamic remodeling of the LV was observed over the course of PAH. The end-stage PAH was associated with significantly impaired LV systolic function and a decrease in LV mass. The LC3B-II expression in the LV was significantly higher in the end-stage PAH group compared to the early PAH group (p = 0.040). The measured LC3B-II/LC3B-I ratios in the end-stage PAH group were significantly elevated compared to the controls (p = 0.039). Immunofluorescence staining showed a significant increase in the abundance of LC3 puncta in the end-stage PAH group compared to the matched controls. There were no statistically significant differences in the levels of expression of all ubiquitinated proteins when comparing both PAH groups and matched controls. Autophagy may be considered as the mechanism behind the LV mass loss at the end stage of PAH.

Anabolic Steroids Activate the NF-κB Pathway in Porcine Ovarian Putative Stem Cells Independently of the ZIP-9 Receptor.

Boldenone (Bdn) and nandrolone (Ndn) are anabolic androgenic steroids (AASs) that, as our previous studies have shown, may increase the risk of neoplastic transformation of porcine ovarian putative stem cells (poPSCs). The NF-κB pathway may be important in the processes of carcinogenesis and tumour progression. Therefore, in this work, we decided to test the hypothesis of whether Bdn and Ndn can activate the NF-κB pathway by acting through the membrane androgen receptor ZIP-9. For this purpose, the expression profiles of both genes involved in the NF-κB pathway and the gene coding for the ZIP-9 receptor were checked. The expression and localization of proteins of this pathway in poPSCs were also examined. Additionally, the expression of the ZIP-9 receptor and the concentration of the NF-κB1 and 2 protein complex were determined. Activation of the NF-κB pathway was primarily confirmed by an increase in the relative abundances of phosphorylated forms of RelA protein and IκBα inhibitor. Reduced quantitative profiles pinpointed not only for genes representing this pathway but also for unphosphorylated proteins, and, simultaneously, decreased concentration of the NF-κB1 and 2 complex may indicate post-activation silencing by negative feedback. However, the remarkably and sustainably diminished expression levels noticed for the SLC39A9 gene and ZIP-9 protein suggest that this receptor does not play an important role in the regulation of the NF-κB pathway.

Omalizumab in the treatment of Morbihan syndrome in an adolescent girl - case report and literature review.

Morbihan syndrome (MS) is characterized by solid facial edema, usually related to rosacea or acne vulgaris. The facial edema deforms the patient's features, can impair peripheral vision, and affects quality of life. Its pathophysiology remains unclear. The disease usually has a slow and chronic course. MS most commonly affects middle-aged Caucasian men with rosacea and is rare in people below 20 years of age. MS is a diagnosis of exclusion. There is no standard treatment for MS, though systemic isotretinoin and antihistamines are mainly used. We present the case of an adolescent girl with MS nonresponding to 19 months of isotretinoin treatment with add-on antihistamines. Therapy with monthly administration of omalizumab (anti-IgE) for 6 months was an effective therapeutic option, improving the quality of life. Our case is the second description of omalizumab use in Morbihan syndrome, the first in an adolescent.

Omalizumab in the treatment of Morbihan syndrome in an adolescent girl – case report and literature review

Morbihan syndrome (MS) is characterized by solid facial edema, usually related to rosacea or acne vulgaris. The facial edema deforms the patient’s features, can impair peripheral vision, and affects quality of life. Its pathophysiology remains unclear. The disease usually has a slow and chronic course. MS most commonly affects middle-aged Caucasian men with rosacea and is rare in people below 20 years of age. MS is a diagnosis of exclusion. There is no standard treatment for MS, though systemic isotretinoin and antihistamines are mainly used. We present the case of an adolescent girl with MS nonresponding to 19 months of isotretinoin treatment with add-on antihistamines. Therapy with monthly administration of omalizumab (anti-IgE) for 6 months was an effective therapeutic option, improving the quality of life. Our case is the second description of omalizumab use in Morbihan syndrome, the first in an adolescent. (AU)

Cytosolic nucleic acid sensing and mitochondrial transcriptomic changes as early triggers of metabolic disease in db/db mice.

Animal models of diabetes, such as db/db mice, are a useful tool for deciphering the genetic background of molecular changes at the initial stages of disease development. Our goal was to find early transcriptomic changes in three tissues involved in metabolism regulation in db/db mice: adipose tissue, muscle tissue and liver tissue. Nine animals (three per time point) were studied. Tissues were collected at 8, 12 and 16 weeks of age. Transcriptome-wide analysis was performed using mRNA-seq. Libraries were sequenced on NextSeq (Illumina). Differential expression (DE) analysis was performed with edgeR. The analysis of the gene expression profile shared by all three tissues revealed eight upregulated genes (Irf7, Sp100, Neb, Stat2, Oas2, Rtp4, H2-T24 and Oasl2) as early as between 8 and 12 weeks of age. The most pronounced differences were found in liver tissue: nine DE genes between 8 and 12 weeks of age (Irf7, Ly6a, Ly6g6d, H2-Dma, Pld4, Ly86, Fcer1g, Ly6e and Idi1) and five between 12 and 16 weeks of age (Irf7, Plac8, Ifi44, Xaf1 and Ly6a) (adj. p-value < 0.05). The mitochondrial transcriptomic profile also changed with time: we found two downregulated genes in mice between 8 and 12 weeks old (Ckmt2 and Cox6a2) and five DE genes between 12 and 16 weeks of age (Mavs, Tomm40L, Mtfp1, Ckmt2 and Cox6a2). The KEGG pathway analysis showed significant enrichment in pathways related to the autoimmune response and cytosolic DNA sensing. Our results suggest an important involvement of the immunological response, mainly cytosolic nucleic acid sensing, and mitochondrial signalling in the early stages of diabetes and obesity.

Osteopontin expression and the effect of anti-VLA-4 mAb treatment in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis.

INTRODUCTION: Osteopontin (OPN) is involved in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). The aim of this study was to investigate the expression of OPN in spinal cords of mice in the successive phases of EAE, to compare it with the density of inflammatory cells, oligodendrocytes and with the expression of interleukin (IL)-17A and to assess the effect of anti-α4ß1 integrin (VLA-4) treatment. MATERIAL AND METHODS: Experimental autoimmune encephalomyelitis (EAE) mice were injected with anti-VLA-4 antibodies or, as treatment control, with immunoglobulin G (IgG). Spinal cords were sectioned and immunostained for OPN, CD45 (overall leukocytes), CD3 (T cells), Iba1 (activated macrophages/microglia), IL-17A, and CNP1 (oligodendrocytes). Microscopic images were analysed and the percentage of immunopositive areas encompassing the whole spinal cord cross-sectional area were assessed in images for each antigen. RESULTS: Osteopontin was expressed by inflammatory cells and by a minority of neurons and blood vessels. Most of the studied parameters followed the temporal pattern of clinical scores: increase in the peak phase and decrease in the chronic phase. Only OPN and IL-17A remained at a high level in the chronic phase, while CNP1 expression gradually decreased in the successive phases. Anti-VLA-4 treatment lowered the expression of the studied antigens in the peak and chronic phases with the exception of oligodendrocyte marker CNP1 which in both phases showed an increased expression. CONCLUSIONS: Involvement of OPN is particularly significant in advanced EAE. Anti-VLA-4 treatment not only inhibits migration of myelin-reactive T cells, but also downregulates OPN and inhibits loss of oligodendrocytes.

Skin prick tests are not useful for the qualification for venom immunotherapy in children.

Background: The basis for qualification for venom immunotherapy (VIT) is the fulfilment of both the clinical and immunological criteria. Diagnostic tests that confirm the immunological criterion of an IgE-mediated sensitization include skin prick tests (SPT), intradermal tests (IDT), and serum specific IgE (sIgE) for the culprit venom. Objective: This study aimed to assess the usefulness of SPT as the immunological marker in the diagnosis of insect venom sensitization in children with history of systemic reaction (SR) to insect sting evaluated by means of I-IV-grades Mueller's scale. There are no such studies in children. Methods: This cross-sectional study sample consisted of 416 children aged 3-18 years (mean age 10.6 ± 3.8), 76% males, all with the history of a systemic reaction (SR) after a Hymenoptera sting (48% of grade III/IV according to Mueller scale), diagnosed between 1999 and 2019 in the tertiary referral centre. The standard diagnostic tests were used. Specificity, sensitivity, and positive and negative predictive values were computed to assess the diagnostic properties of the clinical tests to distinguish between mild and severe SR. To assess the relative value of an individual test in predicting the qualification to VIT we incorporated the Shapley value (SV). Results: Positive SPT results were found in up to no more than 3% of children; among them less than 1% had only positive SPT and were negative for sIgE and IDT. Approximately 85% of the children had detectable venom sIgE, followed by positive IDT (75%). Almost 70% of children had positive both sIgE and IDT results. In children with grade III/IV reaction, about 80% of children had positive results of both of these tests. sIgE and IDT had sensitivity >0.80, whereas SPT had high specificity (>0.97) in differentiating between mild and severe SR. Relative value of diagnostic tests in predicting qualification to VIT varied between venoms. Bee venom IDT had higher SV (0.052) than sIgE (0.041). In contrast, wasp venom sIgE had higher SV (0.075) than IDT (0.035). Conclusion: SPTs are not an useful immunological marker of venom sensitization in children, and eliminating SPT does not result in a loss of diagnostic accuracy. Limiting diagnostics to venom sIgE and IDT would shorten the procedure and reduce costs. Future studies are needed to determine if venom sIgE as the first line diagnostic test, with IDT added only if the venom sIgE is undetectable, is an optimal diagnostic process.

Stinging insect identification in children with Hymenoptera venom allergy and their parents.

Introduction: The treatment of Hymenoptera venom allergy (HVA) is based among others on the proper identification of the culprit insect. Aim: To assess the accuracy in identifying stinging insects by children with HVA and their parents. Material and methods: Participants were recruited from a paediatric medical centre. The data on their demographics, sting history and ability of insect identification (based on pictures) were obtained using a questionnaire. The study sample consisted of 102 children with HVA and their parents as well as 98 children without HVA and their parents. Results: The rates of subjects correctly identifying insects in the groups were 91.2%, 92.5%, 78.8%, 82.4%, respectively. When compared to children with HVA, those without HVA were less likely to correctly identify the bee, bumblebee and hoverfly. In this group, the correct identification of the wasp was more common among children living in the countryside. The correct identification of the bee and bumblebee by children without HVA was more common among children living in the city. Conclusions: Some children with HVA and their parents cannot correctly identify stinging insects despite previous life-threatening allergic reactions. The ability to identify stinging insects may depend on the HVA diagnosis and place of residence.

Penetration of the posterior interosseous nerve fibers into the dorsal capsule of the wrist - a new perspective on wrist innervation.

The dorsal capsule of the wrist and the DCSS may play a significant role in the conduction of nerve signals transmitted from proprioceptors present in SL to PIN, which is located above the dorsal capsule. Hence, this study aimed to determine if nerve fibers of PIN penetrate inside the dorsal capsule. The dorsal capsules of the wrist were dissected from both sides from 15 cadavers. Eventually, 30 dorsal capsules were dissected. It can be concluded that the PIN nerve fibers penetrate the dorsal capsule of the wrist, as the penetration was noticeable in every part evaluated. The present study proves that afferent fibers from the mechanoreceptors of the SLIL potentially pass through the DCSS and subsequently through the dorsal capsule of the wrist to the PIN. This knowledge can surely be of great use for hand surgeons that perform procedures on the dorsal wrist.

Adhesion Molecule Profile and the Effect of Anti-VLA-4 mAb Treatment in Experimental Autoimmune Encephalomyelitis, a Mouse Model of Multiple Sclerosis.

In the course of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), the infiltration of lymphocytes and other inflammatory cells across the blood-brain barrier is associated with interactions between adhesion molecules expressed by infiltrating cells and vascular endothelium. Monoclonal antibodies (mAb) against the α4 subunit of α4-ß1 integrin (VLA-4) show beneficial effects in both MS and EAE. (1) Background: The aim of this study was to examine the expression of selected adhesion molecules: VLA-4, VCAM-1, LFA-1, ICAM-1 and PECAM-1 in the successive phases of EAE and the effect of anti-VLA-4 mAb treatment on that expression. (2) Methods: EAE was induced in C57BL/6 mice by immunization with MOG35-55 peptide. The animals were killed in three successive phases of the disease: onset (day 13), peak (day 18) and chronic (day 28). Frozen sections of the lumbar spinal cord were examined by quantitative immunofluorescence microscopy. The expression of the studied molecules was quantified as the percentage of the cross-sectioned spinal cord lesion area occupied by immunopositive structures. (3) Results: The expression of the studied molecules showed two temporal patterns: (1) an increase in the onset phase, a maximum in the peak phase and a decrease in the chronic phase, which corresponded to the temporal pattern of the clinical score, the number of lesions and the inflammation level (ICAM-1, LFA-1 and PECAM-1), and (2) an increase in the peak phase and no significant change or further increase in the chronic phase (VCAM-1, VLA-4). Among the molecules studied, ICAM-1 and LFA-1 exhibited the highest expression levels in the peak phase of EAE. Anti-VLA-4 mAb inhibited the expression of not only VLA-4 but also other adhesion molecules. (4) Conclusions: The interactions of adhesion molecules governing the migration of leukocytes across the blood-brain barrier change in the successive phases of EAE. The therapeutic mechanism of anti-VLA-4 mAb treatment seems to include a complex influence on a variety of adhesion molecules expressed by infiltrating cells and vascular endothelium.

Interdisciplinary Methods for Zoonotic Tissue Acellularization for Natural Heart Valve Substitute of Biomimetic Materials.

The goal of this work was to create a bioactive tissue-based scaffold using multi-disciplinary engineering materials and tissue engineering techniques. Materials & methods: Physical techniques such as direct laser interference lithography and proton radiation were selected as alternative methods of enzymatic and chemical decellularization to remove cells from a tissue without degradation of the extracellular matrix nor its protein structure. This study was an attempt to prepare a functional scaffold for cell culture from tissue of animal origin using new physical methods that have not been considered before. The work was carried out under full control of the histological and molecular analysis. Results & conclusions: The most important finding was that the physical methods used to obtain the decellularized tissue scaffold differed in the efficiency of cell removal from the tissue in favour of the laser method. Both the laser method and the proton method exhibited a destructive effect on tissue structure and the genetic material in cell nuclei. This effect was visible on histology images as blurred areas within the cell nucleus. The finite element 3D simulation of decellularization process of the three-layer tissue of animal origin sample reflected well the mechanical response of tissue described by hyperelastic material models and provided results comparable to the experimental ones.

Vitamin D receptor gene polymorphism influence on lumbar intervertebral disc degeneration.

Intervertebral disc (IVD) degeneration is a multifaceted pathology that is the main morphological cause of lower back pain. This study aimed to determine the link between the vitamin D receptor gene single nucleotide polymorphisms (SNPs) and degenerative processes of the lumbar spine. The complete lumbar spinal columns were collected from 100 Caucasian cadavers via ventral dissection. The specimens for the histological analysis were harvested from the L5/S1 IVDs and endplates. Then, the tissues were cut into slices, inserted into paraffin blocks, and stained. The histology was evaluated according to the Boos' protocol. Moreover, TaqI(rs731236), FokI(rs2228570), and ApaI(rs7975232) genotyping were performed. Lastly, the histological scores for different genotypes were analyzed. The overall Boos' score in the study group was 12.49. It consisted of a mean IVD score of 7.46 and endplate score of 5.39. The determination of the SNPs was successful in 99 specimens and had a distribution of all alleles in accordance with the Hardy-Weinberg equilibrium. No significant differences in overall histological degeneration scores were found between samples from donors with different genotypes. However, in subgroup analysis of specific regions on the IVD, the significant difference was found in posterior inner anulus fibrosus for ApaI. The results of this study suggest that one must be careful when interpreting the results of the clinical and/or radiological studies on vitamin D receptor gene polymorphisms and lumbar spine degeneration risk, because such a relationship, if present, is likely to be very subtle.

Obesity aggravates contact hypersensitivity reaction in mice.

BACKGROUND: Obesity is associated with chronic, low-grade inflammation in tissues and predisposes to various complications, including inflammatory skin diseases. However, the link between obesity and contact hypersensitivity (CHS) is not fully understood. OBJECTIVES: We sought to determine the influence of obesity on T helper 1 (Th1)-mediated CHS. METHODS: The activity/phenotype/cytokine profile of the immune cells was tested in vivo and in vitro. Using quantitative polymerase chain reaction (qPCR) and fecal microbiota transplantation (FMT), we tested the role of a high-fat diet (HFD)-induced gut microbiota (GM) dysbiosis in increasing the effects of CHS. RESULTS: Exacerbated CHS correlates with an increased inflammation-inducing GM in obese mice. We showed a proinflammatory milieu in the subcutaneous adipose tissue of obese mice, accompanied by proinflammatory CD4+ T cells and dendritic cells in skin draining lymph nodes and spleen. Obese interleukin (IL)-17A-/-B6 mice are protected from CHS aggravation, suggesting the importance of IL-17A in CHS aggravation in obesity. CONCLUSIONS: Obesity creates a milieu that induces more potent CHS-effector cells but does not have effects on already activated CHS-effector cells. IL-17A is essential for the pathogenesis of enhanced CHS during obesity. Our study provides novel knowledge about antigen-specific responses in obesity, which may help with the improvement of existing treatment and/or in designing novel treatment for obesity-associated skin disorders.

Is the clinical manifestation of anaphylaxis in children influenced by the trigger of reaction?

INTRODUCTION: The number of anaphylaxis diagnoses in children is rising, being still based on the clinical picture. AIM: To determine whether triggers of anaphylaxis influence its clinical characteristics in children and adolescents. MATERIAL AND METHODS: The study group included 114 children (5 months-17 years, mean age: 8.0 ±4.8 years), (66%: boys) with the episode of anaphylaxis up to 1 year back. Medical data were entered to the NORA Registry by means of a validated structured on-line questionnaire. RESULTS: Three most frequent triggers of anaphylaxis were: insect venom (47.4%), food (35.1%), drugs (5.3%), with a predominance of food (egg white, cow's milk, nuts and peanuts) in the 0-6 years age group, while insect venom (bee predominance) in the 7-17 years age group (p = 0.016). Clinical manifestations differed between food vs. venom allergic reactions and presented as gastro-intestinal (GI) (61.4%) (p = 0.004), respiratory (RS) (93.9%) (p = 0.036), and cardiovascular (CVS) (74.6%) (p = 0.022) symptoms. Among objective symptoms, vomiting was the most common symptom in the 0-2 years age group (47.1%) (p = 0.006), while hypotension in those aged 7-12 years (40%) (p = 0.010). Severity of symptoms evaluated as Mueller's grade (IV - 74.5%) and as Ring and Messmer's grade (III - 65.8%), depended on the trigger (p = 0.028, p = 0.029, respectively). Life-threatening symptoms occurred in 26 children (fall of the blood pressure - 22%, loss of consciousness - 4.4%). CONCLUSIONS: The clinical manifestation of anaphylaxis in children is both trigger and age dependent, irrespective of the gender. A typical patient with food anaphylaxis was younger, presenting predominantly GI symptoms, while a typical patient with venom anaphylaxis was older, with mostly cardiovascular symptoms.

Microanatomy of the myocardial extensions of the pulmonary valve in light of modern catheter ablation methodology.

INTRODUCTION: The muscular sleeves (or myocardial extensions) derived from the right ventricle infundibulum myocardium are considered the true anatomic substrate for right ventricular outflow tract arrhythmias. METHODS: Pulmonary valve specimens obtained from 65 donors (24.6% females, mean age 45.9 ± 15.8 years) were investigated micro-anatomically. Specimens were histologically processed, stained with Masson's Trichrome, and examined under a light microscope. RESULTS: The myocardial extensions were present in the left anterior pulmonary valve sinus in 86.2% of cases, in the right anterior sinus in 89.2% of cases and in 90.7% of cases in the posterior sinus (p = .699). In 69.2% of examined hearts, the myocardial extensions were present in all sinuses. The mean height of the extensions was 4.12 ± 1.76 (left anterior) versus 3.69 ± 1.47 (right anterior) versus 4.28 ± 1.73 mm (posterior) (p = .137). The myocardial extensions occupied an average of 28.9 ± 10.4% of the left anterior sinus, 26.7 ± 11.2% of the right anterior sinus, and 31.9 ± 11.3% of the posterior sinus (p = .044). Sleeves extending beyond the fibro-arterial transition zone were present in at least one sinus in 33.8% of hearts (in 7.7% (5/65) of the left and right anterior sinuses and 21.5% (14/65) of posterior sinus, p = .021). CONCLUSIONS: The myocardial extensions of the pulmonary valve are common anatomical entities. Although the length of the myocardial sleeves is similar in all pulmonary valve sinuses, their relative extent is greatest in the posterior sinus. Long sleeves that spread beyond the fibro-arterial transition zone were observed in one-third of hearts, predominantly in the posterior sinus. Myocardial and fibrous tissue layer thicknesses varied considerably.

Intervention in anaphylaxis: the experience of one paediatric centre based on NORA reports.

Introduction: Intramuscular adrenaline administration is the primary intervention in anaphylaxis. Aim: To analyse the data on intervention in children admitted due to anaphylaxis to the tertiary paediatric centre and compare them to the data from the Network for Online-Registration of Anaphylaxis. Material and methods: A validated structured on-line questionnaire was used to collect data concerning the first and second-line intervention in anaphylaxis. The study was conducted in cooperation with the European Anaphylaxis Registry. Results: The study group comprised 114 children (76 boys, 66.87%) aged 5 months-17 years with the predominance of moderate-to-severe anaphylaxis (grade III in Ring and Messmer's, and grade IV in Mueller's scale). In 103 (90.4%) children the first line of medical intervention was provided by medical staff. In the first-line intervention 39 (34.8%) children were given adrenaline. Five (4.4%) children were given the second dose of adrenaline and were admitted to the intensive care unit. In the second-line intervention adrenaline was given to 12 (15.6%) children. In one third it was at least the second reaction to the same trigger. Children treated with adrenaline were older (9.3 ±4.8 years), in comparison to those not treated (7.3 ±4.6 years, p = 0.034). Directly after the episode of anaphylaxis the children got the prescription for the adrenaline autoinjector in 35.1%, emergency training in 7.9%, and counselling on the avoidance of the anaphylaxis trigger in 30.7%. Grade III R&M reaction increased 3-fold the odds of AAI prescription (95% CI: 1.08-8.15). Conclusions: There is a strong need to continue education on proper management of anaphylaxis in children.

Circadian Changes of Dendritic Spine Geometry in Mouse Barrel Cortex.

The circadian rhythmicity changes the density and shape of dendritic spines in mouse somatosensory barrel cortex, influencing their stability and maturation. In this study, we analyzed the main geometric parameters of dendritic spines reflecting the strength of synapses located on these spines under light/dark (12:12) and constant darkness conditions, in order to distinguish between endogenously regulated and light-driven parameters. Using morphological analysis of serial electron micrographs, as well as three-dimensional reconstructions, we found that the light induces elongation of single-synapse spine necks and increases in the diameter of double-synapse spine necks, increasing and decreasing the isolation of synapses from the parent dendrite, respectively. During the subjective night of constant darkness, we observed an enlargement of postsynaptic density area in inhibitory synapses and an increase in the number of polyribosomes inside double-synapse spines. The results show that both endogenous effect (circadian clock/locomotor activity) and light affect the morphological parameters of single- and double-synapse spines in the somatosensory cortex: light reduces the efficiency of excitatory synapses on single-synapse spines, increases the effect of synaptic transmission in double-synapse spines, and additionally masks the endogenous clock-driven enlargement of inhibitory synapses located on double-synapse spines. This indicates a special role of double-synapse spines and their inhibitory synapses in the regulation of synaptic transmission during both circadian and diurnal cycles in the mouse somatosensory cortex.

Identification of CD34+/PGDFRα+ Valve Interstitial Cells (VICs) in Human Aortic Valves: Association of Their Abundance, Morphology and Spatial Organization with Early Calcific Remodeling.

Aortic valve interstitial cells (VICs) constitute a heterogeneous population involved in the maintenance of unique valvular architecture, ensuring proper hemodynamic function but also engaged in valve degeneration. Recently, cells similar to telocytes/interstitial Cajal-like cells described in various organs were found in heart valves. The aim of this study was to examine the density, distribution, and spatial organization of a VIC subset co-expressing CD34 and PDGFRα in normal aortic valves and to investigate if these cells are associated with the occurrence of early signs of valve calcific remodeling. We examined 28 human aortic valves obtained upon autopsy. General valve morphology and the early signs of degeneration were assessed histochemically. The studied VICs were identified by immunofluorescence (CD34, PDGFRα, vimentin), and their number in standardized parts and layers of the valves was evaluated. In order to show the complex three-dimensional structure of CD34+/PDGFRα+ VICs, whole-mount specimens were imaged by confocal microscopy, and subsequently rendered using the Imaris (Bitplane AG, Zürich, Switzerland) software. CD34+/PDGFRα+ VICs were found in all examined valves, showing significant differences in the number, distribution within valve tissue, spatial organization, and morphology (spherical/oval without projections; numerous short projections; long, branching, occasionally moniliform projections). Such a complex morphology was associated with the younger age of the subjects, and these VICs were more frequent in the spongiosa layer of the valve. Both the number and percentage of CD34+/PDGFRα+ VICs were inversely correlated with the age of the subjects. Valves with histochemical signs of early calcification contained a lower number of CD34+/PDGFRα+ cells. They were less numerous in proximal parts of the cusps, i.e., areas prone to calcification. The results suggest that normal aortic valves contain a subpopulation of CD34+/PDGFRα+ VICs, which might be involved in the maintenance of local microenvironment resisting to pathologic remodeling. Their reduced number in older age could limit the self-regenerative properties of the valve stroma.

Combination of ERK2 and STAT3 Inhibitors Promotes Anticancer Effects on Acute Lymphoblastic Leukemia Cells.

BACKGROUND/AIM: Deregulated activation of signaling through the RAS/RAF/mitogen-activated protein kinase/extracellular signal-regulated kinase (RAS/RAF/MEK/ERK) and signal transducer and activator of transcription (STAT) pathways is involved in numerous hematological malignancies, making it an attractive therapeutic target. This study aimed to assess the effect of the combination of ERK2 inhibitor VX-11e and STAT3 inhibitor STA-21 on acute lymphoblastic leukemia cell lines REH and MOLT-4. MATERIALS AND METHODS: REH and MOLT-4 cell lines were cultured with each drug alone and in combination. Cell viability, ERK activity, cell cycle distribution, apoptosis and oxidative stress induction were assessed by flow cytometry. Protein levels of STAT3, phospho-STAT3, protein tyrosine phosphatase 4A3 (PTP4A3), survivin, p53 and p21 were determined by western blotting. RESULTS: VX-11e in combination with STA-21 significantly inhibited cell viability, induced G0/G1 cell-cycle arrest, enhanced production of reactive oxygen species, and induced apoptosis. These effects were associated with an increased level of p21 protein in REH cells and with reduced levels of phopho-STAT3, survivin and PTP4A3 proteins in MOLT-4 cells. CONCLUSION: Our findings provide a rationale for combined inhibition of RAS/RAF/MEK/ERK and STAT3 pathways in order to enhance anticancer effects against acute lymphoblastic leukemia cells.

Targeting the MAPK/ERK and PI3K/AKT Signaling Pathways Affects NRF2, Trx and GSH Antioxidant Systems in Leukemia Cells.

The mitogen-activated protein kinase (MAPK)/extracellular signal kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signal transduction pathways have been implicated in the pathogenesis of leukemia. The aim of this study was to investigate the effect of the combination of ERK1/2 inhibitor AZD0364 and PI3K inhibitor ZSTK474 on acute lymphoblastic leukemia (ALL) REH, MOLT-4, acute myeloid leukemia (AML) MOLM-14, and chronic myeloid leukemia (CML) K562 cell lines. To evaluate the interactions of the drugs, cells were treated for 48 h with AZD0364 or ZSTK474 alone and in combination at fixed ratios. The combinatorial effects of both inhibitors were synergistic over a wide range of concentrations in REH, MOLT-4, and MOLM-14 cell lines. However, in K562 cells, the effects were found to be antagonistic. Furthermore, AZD0364 and ZSTK474 significantly decreased both ERK1/2 and AKT activation in REH, MOLT-4, and MOLM-14 cells. The results showed that incubation with both AZD0364 and ZSTK474 inhibited cell viability, increased reactive oxygen species (ROS) production, and induced apoptosis in leukemia cells. We observed that combined treatment with AZD0364 and ZSTK474 affected nuclear factor-κB (NF-κB) and antioxidant protein levels: NF-E2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), thioredoxin (Trx), thioredoxin reductase (TrxR), and the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio. These effects were accompanied with decreased antiapoptotic survivin protein level. However, distinct cell line dependent effects were observed. In conclusion, the combination of AZD0364 and ZSTK474 can exert a synergistic anticancer effect in ALL and AML cells, which is associated with the induction of oxidative stress and the involvement of cellular antioxidant defense mechanisms.