CD68-positive Cells Research Articles

Macrophage Activation in Synovitis and Osteoarthritis of Temporomandibular Joint and Its Relationship with the Progression of Synovitis and Bone Remodeling

This study investigates the regulatory mechanisms of synovial macrophages and their polarization in the progression of temporomandibular joint osteoarthritis (TMJOA). Macrophage depletion models were established by intra-articular injection of clodronate liposomes and unloaded liposomes. TMJOA was induced by intra-articular injection of 50 μL Complete Freund's Adjuvant and the surgery of disc perforation. The contralateral joint was used as the control group. The expression of F4/80, CD86, and CD206 in the synovium was detected by immunofluorescence staining analysis. Hematoxylin and eosin staining and TMJOA synovial score were detected to show the synovial changes in rat joints after TMJOA induction and macrophage depletion. Changes in rat cartilage after TMJOA induction and macrophage depletion were shown by safranin fast green staining. The bone-related parameters of rats' joints were evaluated by micro-computed tomography analysis. The TMJOA model induced by Complete Freund's Adjuvant injection and disc perforation aggravated synovial hyperplasia and showed a significant up-regulation of expression of F4/80-, CD86-, and CD206-positive cells. F4/80, CD86, and CD206 staining levels were significantly decreased in macrophage depletion rats, whereas the synovitis score further increased and cartilage and subchondral bone destruction was slightly aggravated. Macrophages were crucially involved in the progression of TMJOA, and macrophage depletion in TMJOA synoviocytes promoted synovitis and cartilage destruction.

Inhibition of mucus secretion by niclosamide and benzbromarone in airways and intestine

The Ca2+ activated Cl− channel TMEM16A (anoctamin 1; ANO1) is expressed in secretory epithelial cells of airways and intestine. Previous studies provided evidence for a role of ANO1 in mucus secretion. In the present study we investigated the effects of the two ANO1-inhibitors niclosamide (Niclo) and benzbromarone (Benz) in vitro and in vivo in mouse models for cystic fibrosis (CF) and asthma. In human CF airway epithelial cells (CFBE), Ca2+ increase and activation of ANO1 by adenosine triphosphate (ATP) or ionomycin was strongly inhibited by 200 nM Niclo and 1 µM Benz. In asthmatic mice airway mucus secretion was inhibited by intratracheal instillation of Niclo or Benz. In homozygous F508del-cftr mice, intestinal mucus secretion and infiltration by CD45-positive cells was inhibited by intraperitoneal injection of Niclo (13 mg/kg/day for 7 days). In homozygous F508del-cftr rats intestinal mucus secretion was inhibited by oral application of Benz (5 mg/kg/day for 60 days). Taken together, well tolerated therapeutic concentrations of niclosamide and benzbromarone corresponding to plasma levels of treated patients, inhibit ANO1 and intracellular Ca2+ signals and may therefore be useful in inhibiting mucus hypersecretion and mucus obstruction in airways and intestine of patients suffering from asthma and CF, respectively.

Expression of placental CD146 is dysregulated by prenatal alcohol exposure and contributes in cortical vasculature development and positioning of vessel-associated oligodendrocytes.

Recent data showed that prenatal alcohol exposure (PAE) impairs the "placenta-brain" axis controlling fetal brain angiogenesis in human and preclinical models. Placental growth factor (PlGF) has been identified as a proangiogenic messenger between these two organs. CD146, a partner of the VEGFR-1/2 signalosome, is involved in placental angiogenesis and exists as a soluble circulating form. The aim of the present study was to investigate whether placental CD146 may contribute to brain vascular defects described in fetal alcohol spectrum disorder. At a physiological level, quantitative reverse transcription polymerase chain reaction experiments performed in human placenta showed that CD146 is expressed in developing villi and that membrane and soluble forms of CD146 are differentially expressed from the first trimester to term. In the mouse placenta, a similar expression pattern of CD146 was found. CD146 immunoreactivity was detected in the labyrinth zone and colocalized with CD31-positive endothelial cells. Significant amounts of soluble CD146 were quantified by ELISA in fetal blood, and the levels decreased after birth. In the fetal brain, the membrane form of CD146 was the majority and colocalized with microvessels. At a pathophysiological level, PAE induced marked dysregulation of CD146 expression. The soluble form of CD146 decreased in both placenta and fetal blood, whereas it increased in the fetal brain. Similarly, the expression of several members of the CD146 signalosome, such as VEGFR2 and PSEN, was differentially impaired between the two organs by PAE. At a functional level, targeted repression of placental CD146 by in utero electroporation (IUE) of CRISPR/Cas9 lentiviral plasmids resulted in (i) a decrease in cortical vessel density, (ii) a loss of radial vascular organization, and (iii) a reduced density of oligodendrocytes. Statistical analysis showed that the more the vasculature was impaired, the more the cortical oligodendrocyte density was reduced. Altogether, these data support that placental CD146 contributes to the proangiogenic "placenta-brain" axis and that placental CD146 dysfunction contributes to the cortical oligo-vascular development. Soluble CD146 would represent a promising placental biomarker candidate representative of alcohol-induced neurovascular defects in neonates, as recently suggested by PlGF (patents WO2016207253 and WO2018100143).

Evaluating the State of Glomerular Disease by Analyzing Urinary Sediments: mRNA Levels and Immunofluorescence Staining for Various Markers.

Renal biopsy is the gold standard for making the final diagnosis and for predicting the progression of renal disease, but monitoring disease status by performing biopsies repeatedly is impossible because it is an invasive procedure. Urine tests are non-invasive and may reflect the general condition of the whole kidney better than renal biopsy results. We therefore investigated the diagnostic value of extensive urinary sediment analysis by immunofluorescence staining for markers expressed on kidney-derived cells (cytokeratin: marker for tubular epithelial cells, synaptopodin: marker for podocytes, claudin1: marker for parietal epithelial cells, CD68: marker for macrophages (MΦ), neutrophil elastase: marker for neutrophils). We further examined the expression levels of the mRNAs for these markers by real-time reverse transcription polymerase chain reaction. We also examined the levels of mRNAs associated with the M1 (iNOS, IL-6) and M2 (CD163, CD204, CD206, IL-10) MΦ phenotypes. Evaluated markers were compared with clinical and histological findings for the assessment of renal diseases. Claudin1- and CD68-positive cell counts in urinary sediments were higher in patients with glomerular crescents (especially cellular crescents) than in patients without crescents. The relative levels of mRNA for CD68 and the M2 MΦ markers (CD163, CD204, CD206, and IL-10) in urinary sediments were also higher in patients with glomerular crescents. These data suggest that immunofluorescence staining for claudin1 and CD68 in urinary sediments and the relative levels of mRNA for CD68 and M2 MΦ markers in urinary sediments are useful for evaluating the state of glomerular diseases.

Lethal severe fever with thrombocytopenia syndrome virus infection causes systemic germinal centre failure and massive T cell apoptosis in cats.

Severe fever with thrombocytopenia syndrome (SFTS) is a fatal viral disease characterized by high fever, thrombocytopenia, leukopenia, and multi-organ haemorrhage. Disruption of the humoral immune response and decreased lymphocyte numbers are thought to contribute to the disease severity. These findings have been obtained through the analysis of peripheral blood leukocytes in human patients, whereas analysis of lymph nodes has been limited. Thus, in this study, we characterized the germinal centre response and apoptosis in the lymph nodes of cats with fatal SFTS, because SFTS in cats well mimics the pathology of human SFTS. Lymph node tissue sections collected during necropsy from seven fatal SFTS patients and five non-SFTS cases were used for histopathological analysis. Additionally, lymph node tissue sections collected from cats with experimental infection of SFTS virus (SFTSV) were also analysed. In the lymphoid follicles of cats with SFTS, a drastic decrease in Bcl6- and Ki67-positive germinal centre B cells was observed. Together, the number of T cells in the follicles was also decreased in SFTS cases. In the paracortex, a marked increase in cleaved-caspase3 positivity was observed in T cells. These changes were independent of the number of local SFTS virus-positive cell. Furthermore, the analysis of cats with experimental SFTSV infection revealed that the intrafollicular Bcl6- and CD3-positive cell numbers in cats with low anti-SFTSV antibody production were significantly lower than those in cats with high anti-SFTSV antibody production. These results suggest that dysfunction of the humoral response in severe SFTS was caused by the loss of germinal centre formation and massive apoptosis of T cells in the lymph nodes due to systemically circulating viruses.

Dual soluble epoxide hydrolase inhibitor - farnesoid X receptor agonist interventional treatment attenuates renal inflammation and fibrosis.

Renal fibrosis associated with inflammation is a critical pathophysiological event in chronic kidney disease (CKD). We have developed DM509 which acts concurrently as a farnesoid X receptor agonist and a soluble epoxide hydrolase inhibitor and investigated DM509 efficacy as an interventional treatment using the unilateral ureteral obstruction (UUO) mouse model. Male mice went through either UUO or sham surgery. Interventional DM509 treatment (10mg/kg/d) was started three days after UUO induction and continued for 7 days. Plasma and kidney tissue were collected at the end of the experimental protocol. UUO mice demonstrated marked renal fibrosis with higher kidney hydroxyproline content and collagen positive area. Interventional DM509 treatment reduced hydroxyproline content by 41% and collagen positive area by 65%. Renal inflammation was evident in UUO mice with elevated MCP-1, CD45-positive immune cell positive infiltration, and profibrotic inflammatory gene expression. DM509 treatment reduced renal inflammation in UUO mice. Renal fibrosis in UUO was associated with epithelial-to-mesenchymal transition (EMT) and DM509 treatment reduced EMT. UUO mice also had tubular epithelial barrier injury with increased renal KIM-1, NGAL expression. DM509 reduced tubular injury markers by 25-50% and maintained tubular epithelial integrity in UUO mice. Vascular inflammation was evident in UUO mice with 9 to 20-fold higher ICAM and VCAM gene expression which was reduced by 40-50% with DM509 treatment. Peritubular vascular density was reduced by 35% in UUO mice and DM509 prevented vascular loss. Interventional treatment with DM509 reduced renal fibrosis and inflammation in UUO mice demonstrating that DM509 is a promising drug that combats renal epithelial and vascular pathological events associated with progression of CKD.

MASQUELET TECHNIQUE USING A MOUSE FEMUR CRITICAL-SIZED BONE DEFECT MODEL: CHARACTERIZATION OF MACROPHAGE EXPRESSION IN INDUCED MEMBRANE

The Masquelet technique is a variable method for treating critical-sized bone defects, but there is a need to develop a technique for promoting bone regeneration. In recent studies of bone fracture healing promotion, macrophage-mesenchymal stem cell (MSC) cross-talk has drawn attention. This study aimed to investigate macrophage expression in the induced membrane (IM) of the Masquelet technique using a mouse critical-sized bone defect model.The study involved a 3-mm bone defect created in the femur of mice and fixed with a mouse locking plate. The Masquelet (M) group, in which a spacer was inserted, and the Control (C) group, in which the defect was left intact, were established. Additionally, a spacer was inserted under the fascia of the back (B group) to form a membrane due to the foreign body reaction. Tissues were collected at 1, 2, and 4 weeks after surgery (n=5 in each group), and immunostaining (CD68, CD163: M1, M2 macrophage markers) and RT-qPCR were performed to investigate macrophage localization and expression in the tissues.The study found that CD68-positive cells were present in the IM of the M group at all weeks, and RT-qPCR showed the highest CD68 expression at 1 week. In addition, there was similar localization and expression of CD163. The C group showed lower expression of CD68 and CD163 than the M group at all weeks. The B group exhibited CD68-positive cells in the fibrous capsule and CD163-positive cells in the connective tissue outside the capsule, with lower expression of both markers compared to the M group at all weeks.Macrophage expression in IM in M group had different characteristics compared to C group and B group. These results suggest that the IM differs from the fibrous capsules due to the foreign body reaction, and the macrophage-MSC cross-talk may be involved in Masquelet technique.

VNS improves VSMC metabolism and arteriogenesis in infarcted hearts through m/n-AChR-Akt-SDF-1α in adult male rats

Vagal nerve stimulation (VNS) provides a novel therapeutic strategy for injured hearts by activating cholinergic anti-inflammatory pathways. However, little information is available on the metabolic pattern and arteriogenesis of VSMCs after MI. VNS has been shown to stimulate the expression of CPT1α, CPT1β, Glut1, Glut4 and SDF-1α in coronary VSMCs, decreasing the number of CD68-positive macrophages while increasing CD206-positive macrophages in the infarcted hearts, leading to a decrease in TNF-α and IL-1β accompanied by a reduced ratio of CD68- and CD206-positive cells, which were dramatically abolished by atropine and mecamylamine in vivo. Knockdown of SDF-1α substantially abrogated the effect of VNS on macrophagecell alteration and inflammatory factors in infarcted hearts. Mechanistically, ACh induced SDF-1α expression in VSMCs in a dose-dependent manner. Conversely, atropine, mecamylamine, and a PI3K/Akt inhibitor completely eliminated the effect of ACh on SDF-1α expression. Functionally, VNS promoted arteriogenesis and improved left ventricular performance, which could be abolished by Ad-shSDF-1α. Thus, VNS altered the VSMC metabolism pattern and arteriogenesis to repair the infarcted heart by inducing SDF-1α expression, which was associated with the m/nAChR-Akt signaling pathway.

The Essential Role of Angiogenesis in Adenosine 2A Receptor Deficiency-mediated Impairment of Wound Healing Involving c-Ski via the ERK/CREB Pathways.

Adenosine receptor-mediated signaling, especially adenosine A2A receptor (A2AR) signaling, has been implicated in wound healing. However, the role of endothelial cells (ECs) in A2AR-mediated wound healing and the mechanism underlying this effect are still unclear. Here, we showed that the expression of A2AR substantially increased after wounding and was especially prominent in granulation tissue. The delaying effects of A2AR knockout (KO) on wound healing are due mainly to the effect of A2AR on endothelial cells, as shown with A2AR-KO and EC-A2AR-KO mice. Moreover, the expression of c-Ski, which is especially prominent in CD31-positive cells in granulation tissue, increased after wounding and was decreased by both EC-A2AR KO and A2AR KO. In human microvascular ECs (HMECs), A2AR activation induced EC proliferation, migration, tubule formation and c-Ski expression, whereas c-Ski depletion by RNAi abolished these effects. Mechanistically, A2AR activation promotes the expression of c-Ski through an ERK/CREB-dependent pathway. Thus, A2AR-mediated angiogenesis plays a critical role in wound healing, and c-Ski is involved mainly in the regulation of angiogenesis by A2AR via the ERK/CREB pathway. These findings identify A2AR as a therapeutic target in wound repair and other angiogenesis-dependent tissue repair processes.

Retinal response to systemic inflammation differs between sexes and neurons.

Neurological dysfunction and glial activation are common in severe infections such as sepsis. There is a sexual dimorphism in the response to systemic inflammation in both patients and animal models, but there are few comparative studies. Here, we investigate the effect of systemic inflammation induced by intraperitoneal administration of lipopolysaccharide (LPS) on the retina of male and female mice and determine whether antagonism of the NLRP3 inflammasome and the extrinsic pathway of apoptosis have protective effects on the retina. A single intraperitoneal injection of LPS (5 mg/kg) was administered to two months old C57BL/6J male and female mice. Retinas were examined longitudinally in vivo using electroretinography and spectral domain optical coherence tomography. Retinal ganglion cell (RGC) survival and microglial activation were analysed in flat-mounts. Retinal extracts were used for flow cytometric analysis of CD45 and CD11b positive cells. Matched plasma and retinal levels of proinflammatory cytokines were measured by ELISA. Retinal function and RGC survival were assessed in animals treated with P2X7R and TNFR1 antagonists alone or in combination. In LPS-treated animals of both sexes, there was transient retinal dysfunction, loss of vision-forming but not non-vision forming RGCs, retinal swelling, microglial activation, cell infiltration, and increases in TNF and IL-1β. Compared to females, males showed higher vision-forming RGC death, slower functional recovery, and overexpression of lymphotoxin alpha in their retinas. P2X7R and TNFR1 antagonism, alone or in combination, rescued vision-forming RGCs. P2X7R antagonism also rescued retinal function. Response to treatment was better in females than in males. Systemic LPS has neuronal and sex-specific adverse effects in the mouse retina, which are counteracted by targeting the NLRP3 inflammasome and the extrinsic pathway of apoptosis. Our results highlight the need to analyse males and females in preclinical studies of inflammatory diseases affecting the central nervous system.

MACULOPAPULAR CUTANEOUS MASTOCYTOSIS HARBORING A KIT MUTATION (ASP419DEL): A CASE REPORT

A 2-year-old, male patient presented with an 18-month history of scattered, brown macules and nodules up to 2 cm in size on his trunk and extremities. These macules were accompanied by pruritus and were positive for Darier's sign. A skin biopsy of a brown macule on the left thigh revealed a dense accumulation of CD117-positive, round or oval cells with amphophilic cytoplasm within the upper to middle dermis. The patient was otherwise healthy and had normal laboratory and imaging test results. Sequence analysis of genomic DNA from a skin biopsy demonstrated the presence of an Asp419del mutation in exon 8 of the KIT gene. Based on these findings, maculopapular cutaneous mastocytosis (MPCM) was diagnosed. The patient received H 1-antihistamine. Although the pruritus resolved, the brown macules remained for one year after the initial treatment. To the best of our knowledge, only three cases of cutaneous mastocytosis (CM) with an Asp419del mutation, including the present case, have been reported in the Japanese literature to date; moreover, while the previous two cases were of DCM, the present case was the first instance of MPCM. Normally, the symptoms of childhood-onset MPCM are dormant until puberty. However, a recent study reported that many MPCM patients may experience persistent or exacerbated symptoms. The present study therefore evaluated 53 Japanese cases of childhood onset MPCM with a KIT gene mutation and discussed the patients' clinical outcomes.

Spatial distributions of CD8 and Ki67 cells in the tumor microenvironment independently predict breast cancer-specific survival in patients with ER+HER2- and triple-negative breast carcinoma.

Breast cancer (BC) presents diverse malignancies with varying biological and clinical behaviors, driven by an interplay between cancer cells and tumor microenvironment. Deciphering these interactions is crucial for personalized diagnostics and treatment. This study explores the prognostic impact of tumor proliferation and immune response patterns, assessed by computational pathology indicators, on breast cancer-specific survival (BCSS) models in estrogen receptor-positive HER2-negative (ER+HER2-) and triple-negative BC (TNBC) patients. Whole-slide images of tumor surgical excision samples from 252 ER+HER2- patients and 63 TNBC patients stained for estrogen and progesterone receptors, Ki67, HER2, and CD8 were analyzed. Digital image analysis (DIA) was performed for tumor tissue segmentation and quantification of immunohistochemistry (IHC) markers; the DIA outputs were subsampled by hexagonal grids to assess the spatial distributions of Ki67-positive tumor cells and CD8-positive (CD8+) cell infiltrates, expressed as Ki67-entropy and CD8-immunogradient indicators, respectively. Prognostic models for BCSS were generated using multivariable Cox regression analysis, integrating clinicopathological and computational IHC indicators. In the ER+HER2- BC, multivariable Cox regression revealed that high CD8+ density within the tumor interface zone (IZ) (HR: 0.26, p = 0.0056), low immunodrop indicator of CD8+ density (HR: 2.93, p = 0.0051), and low Ki67-entropy (HR: 5.95, p = 0.0.0061) were independent predictors of better BCSS, while lymph node involvement predicted worse BCSS (HR: 3.30, p = 0.0013). In TNBC, increased CD8+ density in the IZ stroma (HR: 0.19, p = 0.0119) and Ki67-entropy (HR: 3.31, p = 0.0250) were independent predictors of worse BCSS. Combining these independent indicators enhanced prognostic stratification in both BC subtypes. Computational biomarkers, representing spatial properties of the tumor proliferation and immune cell infiltrates, provided independent prognostic information beyond conventional IHC markers in BC. Integrating Ki67-entropy and CD8-immunogradient indicators into prognostic models can improve patient stratification with regard to BCSS.

Vitamin D3 exacerbates steatosis while calcipotriol inhibits inflammation in non-alcoholic fatty liver disease in Sod1 knockout mice: a comparative study of two forms of vitamin D.

The role of vitamin D (VD) in non-alcoholic fatty liver disease (NAFLD) remains controversial, possibly due to the differential effects of various forms of VD. In our study, Sod1 gene knockout (SKO) mice were utilized as lean NAFLD models, which were administered 15 000 IU VD3 per kg diet, or intraperitoneally injected with the active VD analog calcipotriol for 12 weeks. We found that VD3 exacerbated hepatic steatosis in SKO mice, with an increase in the levels of Cd36, Fatp2, Dgat2, and CEBPA. However, calcipotriol exerted no significant effect on hepatic steatosis. Calcipotriol inhibited the expression of Il-1a, Il-1b, Il-6, Adgre1, and TNF, with a reduction of NFκB phosphorylation in SKO mice. No effect was observed by either VD3 or calcipotriol on hepatocyte injury and hepatic fibrosis. Co-immunofluorescence stains of CD68, a liver macrophage marker, and VDR showed that calcipotriol reduced CD68 positive cells, and increased the colocalization of VDR with CD68. However, VD3 elevated hepatocyte VDR expression, with no substantial effect on the colocalization of VDR with CD68. Finally, we found that VD3 increased the levels of serum 25(OH)D3 and 24,25(OH)2D3, whereas calcipotriol decreased both. Both VD3 and calcipotriol did not disturb serum calcium and phosphate levels. In summary, our study found that VD3 accentuated hepatic steatosis, while calcipotriol diminished inflammation levels in SKO mice, and the difference might stem from their distinct cellular selectivity in activating VDR. This study provides a reference for the application of VD in the treatment of lean NAFLD.

Tranexamic Acid Ameliorates Skin Hyperpigmentation by Downregulating Endothelin-1 Expression in Dermal Microvascular Endothelial Cells.

Although reports suggest that tranexamic acid (TXA) has clinical benefits for melasma patients by oral, intralesional and topical treatment, the optimal route of TXA therapy and the underlying mechanism involved remain poorly defined. To compare the skin lightening effect between oral TXA and topical TXA and to dissect the molecular mechanisms using ultraviolet B (UVB)-induced hyperpigmentation mouse model, ex vivo cultured human skin explant, and cultured melanocytes (MCs) and endothelial cells. Melanin content and cluster of differentiation 31 (CD31)-positive cell numbers were measured in tail skins from UVB-irradiated mice treated by intragastral or topical TXA using immunofluorescent and Fontana-Masson staining. The conditioned medium (CM) was harvested from human umbilical vein endothelial cells treated with or without 3 mM TXA and was used to treat MCs for 48 hours. mRNA and protein levels of tyrosinase and microphthalmia-associated transcription factor were measured using quantitative real-time reverse transcription polymerase chain reaction and western blotting assays. HMB45- and CD31-positive cell numbers as well as melanin content were also examined in ex vivo cultured human skin explants. The hyperpigmented phenotype were significantly mitigated in UVB-irradiated tail skin plus intragastral TXA-treated mice compared with mice treated with UVB only or with UVB plus topical TXA. CD31-positive cell numbers correlated with the anti-melanogenic activity of TXA therapy. The data from cultured cells and skin tissues showed that suppression of endothelin-1 (ET-1) in vascular endothelial cells by TXA reduced melanogenesis and MC proliferation. Oral TXA outperforms topical TXA treatment in skin lightening, which contributes to suppression of ET-1 in dermal microvascular endothelial cells by TXA.

Expression of CD31, CD34, and smooth muscle actin (SMA) in endothelial cells of dental pulp vessels.

The dental pulp is a highly vascularized and innervated loose connective tissue surrounded by hard dental tissues - enamel and dentine. With the primary dentin formation and the closure of the root apex, the conditions in the dental pulp change and pulp tissue compliance are reduced. Endothelial cells of pulpal blood vessels are highly differentiated and are capable of adaptation to changes in the environment. We aimed to evaluate the phenotypic plasticity of endothelial cells of pulpal blood vessels in permanent premolars with open (N = 6) or closed root apex (N = 30). The pulp tissue was stained with hematoxylin-eosin (HE) for the histological analysis, and immunohistochemically for a cluster of differentiation 31 (CD31), a cluster of differentiation 34 (CD34), and for smooth muscle actin (SMA) to detect vessels with CD31, CD34, and SMA positive endothelial cells. We used the student's t-test and Pearson correlation test for the statistical analysis. We found a significantly higher percentage of the vessels with CD31 (P = 0.005) and CD34 (P = 0.000) positive endothelial cells in the group of teeth with closed root apex compared to the group with open apex. A significant positive correlation between SMA positive and CD31 positive vessels (P = 0.003) and between CD31 positive and CD34 positive vessels (P = 0.031) was also found. We conclude that the endothelial cells of dental pulp vessels express a small amount of CD31, but have a pronounced expression of SMA and CD34, which indicates their progenitor potential and contractile ability.

POSSIBLE CAUSES OF CHANGES IN SEROTONIN CONTENT IN THE LIVER OF RATS IN CONDITIONS OF CHRONIC INTAKE OF SILICON WITH DRINKING WATER

The paper presents data on changes in the intensity of serotonin luminescence in the liver structures of rats who were in an experiment with the intake of silicon with drinking water for two and nine months. The possible causes of identified changes are also considered and discussed. The aim of the study was to correlate the serotonin status of the liver with the number of immunocompetent cells (eosinophils, mast cells, CD68–positive cells) and with peripheral blood indicators in different periods of the experiment with the intake of silicon with drinking water. Material and methods. The rats were divided into two groups: the control group (n = 20) received bottled drinking water, the experimental group (n = 20) received the same water, but supplemented with Na2SiO3*9H2O at the concentration of 10 mg/l in terms of silicon. The animals had free access to a water source. Blood was taken from the caudal vein for analysis before the start of the experiment, as well as in 2 and 9 months after its start. After two and nine months, the rats were withdrawn from the experiment. Fresh frozen slices with a thickness of 10 microns were prepared from one part of the liver to perform the Falk–Hillarp reaction, the other part of the liver was placed in a 10% formalin solution for subsequent paraffin embedding. After deparaffination, the sections were processed by various methods for counting immunocompetent cells: by hematoxylin and eosin (eosinophils), by Unna's toluidine blue (mast cells), by indirect immunohistochemical method (CD68-positive cells). The differences in all cases were considered statistically significant at p < 0.05. Results and their discussion. An increase in the intensity of serotonin luminescence was found in the studied liver structures (hepatocytes, central vein membranes, luminescent granular cells and their microenvironment) in rats of the experimental group. At this, an increase in their intensity by 2-3 times was recorded in the liver of rats who were in the experiment for 9 months. It was revealed that the number of platelets, erythrocytes, hemoglobin, leukocytes, a mixture of monocytes, eosinophils, basophils and immature cells in the blood of rats treated with silicon with drinking water at a concentration of 20 mg/ l is inversely dependent on the duration of the experiment. At both terms, an increase in the number of eosinophils in the portal zones was observed, as well as an increase in the area of mast cells. After 2 months of the experiment, the number of CD68-positive cells and mast cells increased in the liver of rats of the experimental group, and after 9 months their number equalized and decreased, respectively. Attention is drawn to the participation of the above-mentioned immunocompetent cells in serotonin metabolism. Serotonin involvement in the processes of physiological and reparative liver regeneration is discussed. Conclusions. Changes in the number and areas of immunocompetent cells (eosinophils, mast cells, macrophages) in the liver of rats making the experimental groups, as well as a decrease in the platelet content in their blood are associated with an increase in the intensity of serotonin luminescence in the liver structures containing it.

Endothelial to mesenchymal transition is an active process in smokers and patients with early COPD contributing to pulmonary arterial pathology.

We have previously reported pulmonary arterial remodelling in smokers and patients with early COPD, which can be attributed to endothelial to mesenchymal transition (EndMT). In this study, we aimed to evaluate if EndMT is an active mechanism in smokers and COPD. Immunohistochemical staining for the EndMT biomarkers CD31, N-cadherin, vimentin and S100A4 was done on lung resection tissue from 49 subjects. These comprised 15 nonsmoker controls (NC), six normal lung function smokers (NLFS), nine patients with small airway disease (SAD), nine current smokers with mild-moderate COPD (COPD-CS) and 10 ex-smokers with COPD (COPD-ES). Pulmonary arteries were analysed using Image ProPlus software v7.0. We noted reduced junctional CD31+ endothelial cells (p<0.05) in the intimal layer of all smoking groups compared to NC. We also observed increased abundance of the mesenchymal markers N-cadherin (p<0.05) and vimentin (p<0.001) in all smoking groups and across all arterial sizes versus NC, except for N-cadherin in large arteries in COPD-CS. The abundance of S100A4 correlated with arterial thickness (small: r=0.29, p=0.05; medium: r=0.33, p=0.03; large: r=0.35, p=0.02). Vimentin in the small arterial wall negatively correlated with forced expiratory volume in 1 s/forced vital capacity (r= -0.35, p=0.02) and forced expiratory flow rate at 25-75% of forced vital capacity (r= -0.34, p=0.03), while increased cytoplasmic CD31 abundance in the intimal layer of medium and large arteries negatively correlated with predicted diffusing capacity of the lung for carbon monoxide (medium: r= -0.35, p=0.04; large: r= -0.39, p=0.03). This is the first study showing the acquisition of mesenchymal traits by pulmonary endothelial cells from NLFS, SAD and mild-moderate COPD patients through EndMT. This informs on the potential early origins of pulmonary hypertension in smokers and patients with early COPD.

Comparative Study of Immunodeficient Rat Strains in Engraftment of Human-Induced Pluripotent Stem Cell-Derived Airway Epithelia.

The airway epithelia (AE) play a role in the clearance of foreign substances through ciliary motility and mucus secreted. We developed an artificial trachea that is made of collagen sponges and polypropylene mesh for the regeneration of the tracheal defect, and it was used for a clinical study. Then, a model in which the luminal surface of an artificial trachea was covered with a human-induced pluripotent stem cell-derived AE (hiPSC-AE) was transplanted into the tracheal defect of nude rats to promote epithelialization. In the future, this model was expected to be applied to research on infectious diseases and drug discovery as a trachea-humanized rat model. However, at present, sufficient engraftment has not been achieved to evaluate functional recovery in transplanted cells. Therefore, this study focused on immunosuppression in recipient rats. Nude rats lack T cell function and are widely used for transplantation experiments; however, more severe immunosuppressed recipients are preferred for xenotransplantation. Several strains of immunodeficient rats were created as rats that exhibit more severe immunodeficiency until now. In this study, to establish a trachea-humanized rat model in which human AE function can be analyzed to improve engraftment efficiency, engraftment efficiency in nude rats and X-linked severe combined immunodeficiency (X-SCID) rats following hiPSC-AE transplantation was compared. In the analysis of the proportion of engrafted cells in total cells at the graft site, the engraftment efficiency of epithelial cells tended to be high in X-SCID rats, although no statistical difference was found between the two groups, whereas the engraftment efficiency of mesenchymal cells was higher in X-SCID rats. Furthermore, the number of immune cells that accumulated in the grafts showed that a pan T cell marker, that is, CD3-positive cells, did not differ between the two strains; however, CD45-positive cells and major histocompatibility complex (MHC) class II-positive cells significantly decreased in X-SCID rats. These results indicate that X-SCID rats are more useful for the transplantation of hiPSC-AE into the tracheae to generate trachea-humanized rat models.

CD68 in Cerebral Aneurysms of Smokers and Nonsmokers: An Immunohistochemical Analysis.

There is some evidence indicating that inflammation of the aneurysmal wall is related to aneurysmal growth and rupture. The presence of CD68 may indicate greater inflammatory activity. The objective of this study is to evaluate CD68 immunoexpression in surgically resected brain aneurysms and its association with smoking. The resected brain aneurysmal walls after microsurgical clipping were envoyed to immunohistochemistry investigation. The objective was to evaluate the expression of CD68 and CD34 antibodies. The associations between inflammatory markers, smoking, and rupture were tested using Fischer's exact test. CD68 immunoexpression in the tunica media was associated with larger aneurysms: 7.0 mm (7.0-9.0 mm) versus 5.0 mm (3.5-5 mm; p = 0.011). There was no statistically significant association between smoking and CD68 expression in the tunica media (p = 0.234) or in either the tunica media or the tunica intima (p = 0.628). There was also no statistically significant association between hemorrhagic presentation of the aneurysm and CD68 expression in the tunica media (p = 0.689) or in either the tunica media or the tunica intima (p = 0.348). Therefore, the presence of CD68-positive cells in the aneurysmal walls indicates an association with size, especially if the tunica media is exclusively compromised (p = 0.011). Immunohistochemistry investigation for CD68 antibodies was used to determine histiocytic infiltration. Adequately powered studies are necessary to further investigate the association between CD68-positive cells and both smoking history and hemorrhagic presentation of aneurysms.

Myeloid deletion of talin-1 reduces mucosal macrophages and protects mice from colonic inflammation

The intestinal immune response is crucial in maintaining a healthy gut, but the enhanced migration of macrophages in response to pathogens is a major contributor to disease pathogenesis. Integrins are ubiquitously expressed cellular receptors that are highly involved in immune cell adhesion to endothelial cells while in the circulation and help facilitate extravasation into tissues. Here we show that specific deletion of the Tln1 gene encoding the protein talin-1, an integrin-activating scaffold protein, from cells of the myeloid lineage using the Lyz2-cre driver mouse reduces epithelial damage, attenuates colitis, downregulates the expression of macrophage markers, decreases the number of differentiated colonic mucosal macrophages, and diminishes the presence of CD68-positive cells in the colonic mucosa of mice infected with the enteric pathogen Citrobacter rodentium. Bone marrow-derived macrophages lacking expression of Tln1 did not exhibit a cell-autonomous phenotype; there was no impaired proinflammatory gene expression, nitric oxide production, phagocytic ability, or surface expression of CD11b, CD86, or major histocompatibility complex II in response to C. rodentium. Thus, we demonstrate that talin-1 plays a role in the manifestation of infectious colitis by increasing mucosal macrophages, with an effect that is independent of macrophage activation.