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.

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 inhibitor VX-11e and voreloxin synergistically enhances anti-proliferative and pro-apoptotic effects in leukemia cells.

ERK1/2 inhibitors are new promising anticancer drugs. The aim of this study was to investigate the effect of the combination of ERK2 inhibitor VX-11e and voreloxin on MOLM-14, K562, REH and MOLT-4 leukemia cell lines. We found that VX-11e alone and in combination with voreloxin significantly decreased ERK activation in all cell lines tested. To evaluate the interactions of the drugs, cells were treated for 24 h with VX-11e or voreloxin alone and in combination at fixed ratios based on IC50 values. The combinatorial effects of both drugs were synergistic over a wide range of concentrations in MOLM-14, REH and MOLT-4 cell lines. In K562 cells, three effects were found to be additive, one antagonistic and only one synergistic. The results showed that incubation with both VX-11e and voreloxin inhibited the growth of leukemia cells, affected cell cycle and induced apoptosis. Furthermore, the molecular mechanism of these effects might be attributed to an increased expression of p21 and a decreased expression of survivin and NF-κB in all cell lines tested except from K562 cells. In conclusion, combination of VX-11e and voreloxin can exert a synergistic anticancer effect in leukemia cells.

Endothelial injury is closely related to osteopontin and TNF receptor-mediated inflammation in end-stage renal disease.

BACKGROUND: Endothelial dysfunction, inflammation and active mineralization are key processes involved in cardiovascular burden in end stage renal disease (ESRD). Serum (soluble) thrombomodulin (sTM) is an established marker of endothelial injury. PATIENTS: 80 patients in ESRD were recruited consecutively. Baseline distribution of sex, age, main comorbidities and Framingham score was similar. A biochemical panel including sTM, intact PTH (iPTH), interleukin-6 (IL-6), pentraxin 3 (PTX3), fibroblast growth factor 23 (FGF-23), osteopontin (OPN), osteoprotegerin (OPG), osteocalcin (OC), osteonectin (ON), soluble tumor necrosis factor receptor type 2 (TNFR2), transforming growth factor-ß (TGF-ß), hepatocyte growth factor (HGF), vascular endothelial growth factor receptor type 2 (sVEGFR2) and stromal cell-derived factor 1α (SDF1α) was investigated in each patient. Samples obtained while establishing haemodialysis (HD) access were stained for radial artery calcifications (RACs) with Alizarin red and examined histologically. RESULTS: After adjustment for HD status, sTM showed a significant positive correlation with serum creatinine, TNFR2, OPN, HGF, SDF1α, sVEGFR2, Pi, iPTH, FGF-23, OPG, OC and ON. In forward stepwise multiple regression, serum creatinine, TNFR2, and OPN were identified as significant, independent predictors of sTM. Grades 1-3 of RACs correlated with sTM (R = 0.50, p = 0.017), while grade 3 RACs were significantly associated with higher sTM (p = 0.02) than less advanced lesions. CONCLUSION: Among novel renal and cardiovascular biomarkers, OPN and TNFR2 are closely related to sTM. This may link endothelial damage, vascular remodeling and inflammation. Progression of RAC parallels a presumed compensatory rise in sTM, reflecting endothelial injury. sTM has an intricate role in endothelial function and potential clinical and prognostic applications.

Complex regional pain syndrome: diagnosis and treatment at the very onset as the key to success? A case report with implications for first contact doctors.

The case report describes a 67-year-old man who suffered from a minor left ankle injury. Physical examination on day 12 revealed swelling of the foot, erythema on its dorsal surface as well as elevated temperature, hyperesthesia, hyperalgesia and allodynia of that area. The treatment included local application of dexamethasone and oral administration of meloxicam. Within a week the symptoms disappeared and one-year follow-up did not show their recurrence. The presented symptoms allowed diagnosis of the earliest stage of complex regional pain syndrome (CRPS), which may be a disabling and difficult to treat adverse event. This report suggests that immediately introduced simple anti-inflammatory therapy may bring a quick and permanent recovery. Hence, first contact physicians should advise the patient to report such symptoms as burning pain of the injured area lasting for a few days and, if CRPS suspicion is justified by the results of physical examination, they should apply an anti-inflammatory treatment immediately.

Effect of Associative Learning on Memory Spine Formation in Mouse Barrel Cortex.

Associative fear learning, in which stimulation of whiskers is paired with mild electric shock to the tail, modifies the barrel cortex, the functional representation of sensory receptors involved in the conditioning, by inducing formation of new inhibitory synapses on single-synapse spines of the cognate barrel hollows and thus producing double-synapse spines. In the barrel cortex of conditioned, pseudoconditioned, and untreated mice, we analyzed the number and morphological features of dendritic spines at various maturation and stability levels: sER-free spines, spines containing smooth endoplasmic reticulum (sER), and spines containing spine apparatus. Using stereological analysis of serial sections examined by transmission electron microscopy, we found that the density of double-synapse spines containing spine apparatus was significantly increased in the conditioned mice. Learning also induced enhancement of the postsynaptic density area of inhibitory synapses as well as increase in the number of polyribosomes in such spines. In single-synapse spines, the effects of conditioning were less pronounced and included increase in the number of polyribosomes in sER-free spines. The results suggest that fear learning differentially affects single- and double-synapse spines in the barrel cortex: it promotes maturation and stabilization of double-synapse spines, which might possibly contribute to permanent memory formation, and upregulates protein synthesis in single-synapse spines.

Circadian rhythmicity of synapses in mouse somatosensory cortex.

The circadian rhythmicity displayed by motor behavior of mice: activity at night and rest during the day; and the associated changes in the sensory input are reflected by cyclic synaptic plasticity in the whisker representations located in the somatosensory (barrel) cortex. It was not clear whether diurnal rhythmic changes in synapse density previously observed in the barrel cortex resulted from changes in the activity of the animals, from daily light/dark (LD) rhythm or are driven by an endogenous clock. These changes were investigated in the barrel cortex of C57BL/6 mouse strain kept under LD 12 : 12 h conditions and in constant darkness (DD). Stereological analysis of serial electron microscopic sections was used to assess numerical density of synapses. In mice kept under LD conditions, the total density of synapses and the density of excitatory synapses located on dendritic spines was higher during the light period (rest phase). In contrast, the density of inhibitory synapses located on dendritic spines increased during the dark period (activity phase). Under DD conditions, the upregulation of the inhibitory synapses during the activity phase was retained, but the cyclic changes in the density of excitatory synapses were not observed. The results show that the circadian plasticity concerns only synapses located on spines (and not those on dendritic shafts), and that excitatory and inhibitory synapses are differently regulated during the 24 h cycle: the excitatory synapses are influenced by light, whilst the inhibitory synapses are driven by the endogenous circadian clock.

Cardiovascular risk in chronic kidney disease patients: intima-media thickness predicts the incidence and severity of histologically assessed medial calcification in radial arteries.

BACKGROUND: The objective of the study was to determine the relationship between common carotid artery intima-media thickness (CCA-IMT) and histologically assessed calcification of radial artery in relation to clinical features and laboratory markers of bone and mineral metabolism, inflammation, and oxidative stress in patients with stage 5 chronic kidney disease (CKD). METHODS: The study comprised 59 patients (36 hemodialyzed, 23 predialysis). CCA-IMT was measured by ultrasonography; the biochemical parameters examined were assessed using routine laboratory methods, ELISA micro-plate immunoassays and spectrophotometry. Fragments of radial artery obtained during creation of hemodialysis access were cryosectioned and stained for calcifications using von Kossa method and alizarin red. RESULTS: Glucose, osteoprotegerin, pentraxin 3 and Framingham risk score significantly correlated with CCA-IMT. In multiple regression analysis, OPG positively predicted CCA-IMT. Radial artery calcifications were found in 34 patients who showed higher CCA-IMT (0.98 ± 0.13 vs 0.86 ± 0.14 mm; P = 0.006). Higher CCA-IMT values were also associated with more advanced calcifications. CCA-IMT and the presence of plaques in common carotid artery were positive predictors of radial artery calcifications, independent of dialysis status, Framingham risk score, CRP and Ca x Pi [OR for calcifications 2.19 (1.08-4.45) per 0.1 mm increase in CCA-IMT]. The presence of radial artery calcifications was a significant predictor of mortality, independent of dialysis status and Framingham risk score [HR 3.16 (1.03-9.64)]. CONCLUSIONS: In CKD patients, CCA-IMT examination can be used as a surrogate measure to assess the incidence and severity of arterial medial calcification which is associated with poor clinical outcome in these patients.

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.

Vascular architecture of human uterine cervix visualized by corrosion casting and scanning electron microscopy.

BACKGROUND: In contrast to the uterine corpus, the vascular architecture of the human cervix has been the subject of only a few studies, mostly dealing with the ectocervical mucosal vessels. This study presents the vascular system of the cervical wall surrounding the endocervical canal visualized by the best currently available technique, corrosion casting combined with scanning electron microscopy. METHODS: Uteri collected at autopsy (n= 20) were perfused via afferent vessels with fixative followed by Mercox resin and corroded after polymerization of the resin. The obtained vascular casts of the cervix visualizing all vessels including capillaries were examined in the scanning electron microscope. RESULTS: The vascular system of the cervix was nearly completely replicated in only two (10%) of the samples. In the wall of the cervix, four distinct vascular zones surrounding the endocervical canal were observed: (i) the outer zone containing larger vessels, arteries and veins of 0.3-1 mm diameter; (ii) the zone containing arterioles and venules; (iii) the zone of endocervical mucosal capillaries showing a very high density, parallel arrangement and relatively few interconnections and (iv) the innermost, subepithelial zone containing small veins running along the endocervical canal. CONCLUSIONS: Despite the loss of the delicate ectocervical mucosal vessels from the cast during the corrosion step, we have successfully visualized the majority of the cervical vasculature. The vascular pattern of the human cervix, especially that of the endocervical mucosa, may facilitate the adaptation of the cervical vasculature to the extensive remodeling of the cervix during parturition.

Development of rat tibia innervation: colocalization of autonomic nerve fiber markers with growth-associated protein 43.

Development of autonomic innervation of the tibia was investigated in rat fetuses on gestational days (GD) 17-21 and in juvenile animals on postnatal days (PD) 1-28. Double immunofluorescence combined with confocal microscopy was applied to study colocalization of neuronal growth- associated protein 43 (GAP-43) and panneuronal marker protein gene product 9.5 (PGP) with markers of the autonomic nervous system: neuropeptide Y (NPY) and dopamine beta-hydroxylase (DbetaH) for adrenergic, as well as vasoactive intestinal polypeptide (VIP) and vesicular acetylcholine transporter (VAChT) for cholinergic fibers. The first GAP-43-immunoreactive (GAP-IR) nerve fibers were seen on GD17 in the perichondrium of the proximal epiphysis. Further GAP- and PGP-IR innervation appeared in the perichondrium/periosteum of the diaphysis and in the distal epiphysis (GD19), then in the bone marrow and in the intercondylar eminence (GD21). On PD1, NPY-IR and DbetaH-IR fibers appeared within the diaphyseal periosteum and on PD4 within the bone marrow. From PD14, GAP-43 immunoreactivity of NPY-positive fibers decreased. From PD7 on, NPY-IR fibers were observed in cartilage canals of both epiphyses and in the intercondylar eminence. In secondary ossification centers, NPY-IR fibers were seen from PD10, and in the bone marrow of the epiphyses from PD14. First VIP-IR and VAChT-IR fibers were observed on PD4 within the periosteum, bone marrow and patellar ligament. From PD10 on, VIP-positive fibers were seen in the intercondylar eminence, and from PD14 in secondary ossification centers. GAP-43 proved to be superior to PGP 9.5 as marker of growing nerve fibers, mostly due to its earlier appearance. The presence of specific nerve fibers may suggest possible involvement of autonomic innervation in regulation of bone development.

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.

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.

Interplay of nitric oxide metabolites and markers of endothelial injury, inflammation, and vascular disease in the spectrum of advanced chronic kidney disease.

BACKGROUND: Chronic kidney disease is linked to cardiovascular morbidity; therefore, relevant biomarkers are widely investigated. AIMS: We aimed to assess the relationship between nitric oxide (as measured by its metabolites, NOx), a key endothelial molecule, with markers of endothelial dysfunction, inflammation, antioxidant status, and mineral disorders as well as histologically assessed vascular calcification in uremic and hemodialysis patients with chronic kidney disease. METHODS: Plasma and serum samples were obtained from 62 patients with renal failure. NOx was assessed by the Griess method, while the other biomarkers were measured by the immunoenzymatic assay. Morphological analysis of arterial calcification was performed in a blinded, semiquantitative manner. Common carotid intima­media thickness and atherosclerotic plaques were assessed by ultrasonography. RESULTS: In the simple analysis, NOx levels correlated positively with the parameters of renal function, mineral metabolism, endothelial injury, and inflammation. NOx predicted carotid intima­media thickness in simple (P = 0.014) and multiple analysis (P = 0.036) adjusted for the Framingham risk score, C­reactive protein, serum creatinine, and parathormone. The occurrence of atherosclerotic plaques in the common carotid artery was correlated with higher NOx concentrations (P = 0.021). CONCLUSIONS: In chronic renal failure, NOx is associated with surrogate markers of atherosclerosis, even after adjustment for traditional cardiovascular risk factors, inflammation, and renal function, but not with the presence or grade of medial arterial calcification. Endothelial injury, inflammation, and mineral metabolism markers are associated with NOx levels, though a causal link requires further study.

Development of mature lamellar bone with a hematopoietic compartment in an aortic valve homograft.

Two foci of mature lamellar bone with features of remodeling and with an adjacent hematopoietic compartment were revealed for the first time in an aortic valve homograft by hematoxylin and eosin staining and polarized light microscopy. The valve had been obtained originally from a 52-year-old non-beating-heart donor and implanted as 'fresh antibiotic-preserved' into the left ventricular outflow tract of a 21-year-old man, but was explanted after six years due to valvular insufficiency. The areas close to bone showed the presence of cells resembling osteoblasts, osteoclasts and degenerating chondrocytes. Von Kossa staining disclosed a small area of dystrophic calcification in the vicinity of one bone fragment, whereas the second fragment was accompanied by only weak, diffuse calcification. These findings shows that the formation of ectopic mature bone with secondary development of the hematopoietic compartment can occur in a relatively short time, and suggest that initiators of the process may be present in the grafted valve.

Giant schwannoma of the cheek--a comprehensive histological and immunohistochemical description of a rare tumour.

Schwannoma is a benign tumour originating from Schwann cells forming sheaths of peripheral nerves. Its location in the oral cavity, and particularly in the cheek, is very rare. A large tumour (5 cm) was surgically removed from the left cheek of a fifty-five-year-old man and pathological examination revealed schwannoma with Antoni A and B patterns. The tumour was investigated using immunofluorescence and histochemical stainings. It showed positive immunostaining for S-100, PGP 9.5, NSE, collagen IV, laminin, merosin and vimentin. No immunofluorescence for GFAP, NPY and CGRP was observed. Cells of the macrophage family (CD68-immunopositive) were scattered in the connective tissue. Neither B (CD20+) nor T (CD3+, CD8+) lymphocytes were found. The capillary network was revealed by CD34 immunostaining. SMA immunoreactivity was observed in walls of larger blood vessels but not in tumour cells. The tumour contained numerous mast cells visualized by thionin staining and an abundance of collagen fibres revealed by picrosirius red.

Circadian clock regulates the shape and content of dendritic spines in mouse barrel cortex.

Circadian rhythmicity affects neuronal activity induced changes in the density of synaptic contacts and dendritic spines, the most common location of synapses, in mouse somatosensory cortex. In the present study we analyzed morphology of single- and double-synapse spines under light/dark (12:12) and constant darkness conditions. Using serial electron micrographs we examined the shape of spines (stubby, thin, mushroom) and their content (smooth endoplasmic reticulum, spine apparatus), because these features are related to the maturation and stabilization of spines. We observed significant diurnal and circadian changes in the shape of spines that are differentially regulated: single-synapse spines remain under circadian clock regulation, while changes of double-synapse spines are driven by light. The thin and mushroom single-synapse spines, regardless of their content, are more stable comparing with the stubby single-synapse spines that show the greatest diversity. All types of double-synapse spines demonstrate a similar level of stability. In light/dark regime, formation of new mushroom single-synapse spines occurs, while under constant darkness new stubby single-synapse spines are formed. There are no shape preferences for new double-synapse spines. Diurnal and circadian alterations also concern spine content: both light exposure and the clock influence translocation of smooth endoplasmic reticulum from dendritic shaft to the spine. The increasing number of mushroom single-synapse spines and the presence of only those mushroom double-synapse spines that contain spine apparatus in the light phase indicates that the exposure to light, a stress factor for nocturnal animals, promotes enlargement and maturation of spines to increase synaptic strength and to enhance the effectiveness of neurotransmission.

Proteoglycan/glycosaminoglycan and collagen content in the arterial wall of patients with end-stage renal disease: new indicators of vascular disease.

INTRODUCTION: The prevalence of cardiovascular (CV) comorbidity in patients with chronic kidney disease (CKD) is high, particularly in end­stage renal disease (ESRD). There is an ongoing search for novel biomarkers of CV disease in this population. OBJECTIVES: We aimed to investigate the associations of matrix proteoglycans (PGs) and glycosaminoglycans (GAGs), collagen, and arterial calcifications with selected serum and plasma markers of endothelial dysfunction, inflammation, oxidative stress, and bone turnover in patients with ESRD. PATIENTS AND METHODS: We enrolled 47 adult patients (32 men) with stage 5 CKD. The following parameters were investigated: fibrinogen, soluble thrombomodulin (sTM), plasminogen activator inhibitor 1 (PAI­1), stromal cell­derived factor 1α (SDF­1α), calcium (Ca), phosphate (Pi), intact parathormone, interleukin 6, high­sensitivity C­reactive protein (hs­CRP), ferric reducing ability of plasma, 2,2­diphenyl­1­picrylhydrazyl scavenging, ferric reducing ability of ascorbate in plasma, fetuin­A, fibroblast growth factor 23, osteopontin, osteoprotegerin, osteocalcin, transforming growth factor ß (TGF­ß), hepatocyte growth factor, secreted protein acidic and rich in cysteine, as well as matrix metalloproteinase 2. Radial artery specimens were stained with alizarin red for calcifications, alcian blue for PGs and GAGs, and sirius red for collagen. RESULTS: We observed positive correlations between PG and GAG, collagen, and calcification staining. The most intense (grade 3) alcian blue staining was significantly correlated with diabetes as well as higher levels of Ca × Pi product, hs­CRP, fibrinogen, SDF­1α, PAI­1, and sTM. However, PAI­1 was the only significant predictor of grade 3 alcian blue staining in a multiple logistic regression model adjusted for hemodialysis, Ca× Pi product, and hs­CRP levels. CONCLUSIONS: Coagulation disorders and endothelial dysfunction are the hallmarks of ESRD. The levels of SDF­1α, PAI­1, sTM, and fibrinogen may be novel predictors of early vascular wall alterations and may serve as CV risk markers.

Incidence of pituitary necrotic lesions in autopsy material.

Pituitary necrosis is mostly a result of pituitary haemorrhage or infarct. The frequency of pituitary necrosis in the Polish population has not yet been investigated. Hence, the aim of this study was to estimate the incidence of pituitary necrotic lesions in forensic autopsy material and to assess possible correlations of pituitary necrosis with sex, age, other pituitary pathologies, endocrine disorders and atherosclerosis. Serial sections of 100 human pituitary glands stained with hematoxylin-eosin were examined microscopically. Pituitary necrosis was found in 19 cases (19%), all of them in persons aged > 40 years. The majority of the lesions had relatively large size, occupying 10-50% of the gland. According to family interviews, none of the subjects manifested any clinical symptoms related to pituitary insufficiency, hence al the detected cases can be regarded as subclinical. There was no association of pituitary necrosis occurrence with sex, other pituitary pathologies found upon autopsy, endocrine diseases or cause of death. Only correlations with age and atherosclerosis were statistically significant. This study has shown that subclinical pituitary necrosis is a relatively frequent phenomenon in elderly persons, probably resulting from age-related deterioration in the vascular status.

Immunohistochemical analysis of spinal cord components in mouse model of experimental autoimmune encephalomyelitis.

INTRODUCTION: Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for studying immunopathology of multiple sclerosis (MS) because it repeats the hallmarks of the human disease, such as focal inflammation and demyelination of the central nervous system, subsequently leading to axonal and neuronal loss. The interrelationships, timing and sequence of different pathological processes that lead to histologically observed lesions in SM are still incompletely understood. MATERIAL AND METHODS: EAE was induced in female C57Bl/6 mice by active immunization with MOG35-55 antigen. Development of the neurological symptoms in the animals was monitored and on that basis spinal cords were collected in three successive phases of the disease (onset, peak, chronic). Total leukocytes, T cells, macrophages/microglia, oligodendrocytes, damaged axons and surviving neuronal cell bodies were visualized using appropriate immunohistochemical markers and their density was quantitatively assessed using image analysis software. RESULTS: The density of all studied cells except neurons was significantly higher in EAE mice than in the control mice. The density of total leukocytes, T cells, and damaged axons increased from the onset to the peak phase and decreased in the chronic phase to reach values lower than those in the peak phase. The density of macrophages/microglia increased in the peak phase and remained at the elevated level in the chronic phase. Oligodendrocytes showed the highest density in the onset phase and gradually decreased afterwards. The density of neuronal cell bodies decreased only in the chronic phase of the disease. CONCLUSIONS: In mouse model of EAE, inflammatory cells predominate in the early phases of the disease. This study shows for the first time that inflammation precedes oligodendrocyte death and neuronal loss and that macrophages/ microglia are the only cells persisting in large numbers in the chronic phase of the disease, probably because of the switch from proinflammatory to anti-inflammatory phenotype.