Immunofluorescence Investigations Research Articles

Characteristic terpenylated coumarins from Ferula ferulaeoides as potential inhibitors on overactivation of microglia

A total of 37 characteristic terpenylated coumarins (1–25), including 17 undescribed compounds (1–5, 6a/6b, 7–10, 11a/11b–13a/13b), have been isolated from the root of Ferula ferulaeoides. Meanwhile, twelve pairs of enantiomers (6a/6b, 11a/11b–15a/15b, 17a/17b, 18a/18b, 20a/20b–22a/22b, and 25a/25b) were chirally purified. The structures of these new compounds were elucidated using HRESIMS, UV, NMR, and calculated 13C NMR with a custom DP4 + analysis. The absolute configurations of all the compounds were determined for the first time using electronic circular dichroism (ECD). Then, their inhibitory effects on nitric oxide (NO) production were evaluated with LPS-induced BV-2 microglia. Compared with the positive control minocycline (IC50 = 59.3 μM), ferulaferone B (2) exhibited stronger inhibitory potency with an IC50 value of 12.4 μM. The immunofluorescence investigation indicated that ferulaferone B (2) could inhibit Iba-1 expression in LPS-stimulated BV-2 microglia.

Abstract 4121: Unveiling discordant views of a novel drug's influence on cell motility from proteomics and immunofluorescence perspectives

Abstract Introduction: Metastasis is a leading cause of death in patients with solid tumors. Our group has discovered KBU2046, a selective inhibitor of cell motility (Nature Communications 2018). We conducted a time-course immunofluorescent staining to investigate the impact of KBU2046 on the cell adhesion and motility components of human prostate cancer cells. Our objective was to compare our findings with our proteomic analysis results in order to gain insights into the novel molecular mechanisms of KBU2046 in modulating cell motility. Experimental Procedures: Proteomics analysis. We treated PC3 cells with KBU2046 or vehicle (N=3/group) and quantified protein expression using Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS). In separate experiments (N=4), we performed LC-MS/MS after Tandem Mass Tag (TMT) labeling on isolated membrane fractions. We used bioinformatics tools, including Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Ingenuity Pathway Analysis (IPA), and protein-protein interaction (PPI) network analysis, to characterize significantly differentially expressed proteins upon KBU2046 treatment and tried to verify by Western blots. Immunofluorescence (IF) staining. Following treatment of PC3, 1532CPTX, and 1532NPTX cells with KBU2046 or vehicle at various intervals, cells were stained for β1, β5 integrin, actin, and DAPI. Images were acquired by a Zeiss LSM 800 Confocal Laser Scanning Microscope. Results: The proteomics analysis demonstrated that KBU2046 was having a major impact on cell motility processes, consistent with our prior functional studies. Despite conducting Western blot analysis, we could not confirm the candidate proteins linked to cell adhesion. Using IF, we examined changes in cell adhesion proteins over time in response to KBU2046 treatment. The time-course IF investigation revealed that KBU2046 affects adhesion proteins kinetically by altering their subcellular localization over time rather than altering their total expression level. This finding corroborates KBU2046's known effects on cell motility, demonstrating that a kinetic-based analysis yields important new information not directly represented in a single time point screen, although functionally related. Conclusions: Our study underscores the importance of integrating proteomic and imaging methodologies to procure a holistic comprehension of the molecular mechanisms that govern cell behavior, especially those that relate to highly dynamic processes. Through these methodologies, we have obtained a more profound understanding of how cells regulate motility and how that process can be therapeutically modulated. Citation Format: Weining Chen, Nicholas Woods, Henry Chun Hin Law, Fangfang Qiao, Sankarasubramanian Jagadesan, Chittibabu Guda, Raymond Bergan. Unveiling discordant views of a novel drug's influence on cell motility from proteomics and immunofluorescence perspectives [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4121.

Unravelling the impact of COVID-19 on pregnancy: In aspect of placental histopathology and umbilical cord macrophage immunoactivity with neonatal outcomes

COVID-19 has turned into a global pandemic since it was first detected in 2019, causing serious public health problems. Our objective was to investigate the impact of COVID-19 on pregnant women and newborns, who belong to the vulnerable segments of society. Our study involved the histopathological examination of placentas and umbilical cords from two groups of pregnant women. Group I consisted of pregnant women who had never tested positive for COVID-19 during their pregnancy (n: 20). Group II consisted of pregnant women who had contracted COVID-19, exhibited moderate and mild symptoms, and recovered from the disease before giving birth (n: 23). Furthermore, we employed immunofluorescence techniques to detect macrophage activity in the umbilical cord. Prenatal assessments were based on maternal complete blood counts and coagulation assays (n:40 in both groups). Newborn conditions were evaluated using birth weight, height, head circumference, and APGAR (n:40 in both groups). Our analyses reveal that COVID-19 causes placental and umbilical cord inflammation and maternal and foetal vascular malperfussion. Our immunofluorescence investigations demonstrate a notable increase in macrophage numbers and the macrophage-to-total cell ratio within the COVID-19 group. In this aspect, this study provides the initial report incorporating macrophage activity results from Warton's jelly in pregnants who have recovered from COVID-19. We have also ascertained that COVID-19 abbreviates gestation periods and concurrently diminishes maternal haemoglobin concentrations. Consequently, COVID-19 with mild and moderate symptoms during pregnancy, causes significant changes to the placenta and umbilical cord, but propitiously does not cause a significant difference in the neonatal outcomes.

Titanium Dioxide Nanoparticles: Effects on Development and Male Reproductive System.

Titanium dioxide nanoparticles (TiO2-NPs) are used intensively. Thanks to their extremely small size (1-100 nm), TiO2-NPs are more absorbable by living organisms; consequently, they can cross the circulatory system and then be distributed in various organs including the reproductive organs. We have evaluated the possible toxic effect of TiO2-NPs on embryonic development and the male reproductive system using Danio rerio as an organism model. TiO2-NPs (P25, Degussa) were tested at concentrations of 1 mg/L, 2 mg/L, and 4 mg/L. TiO2-NPs did not interfere with the embryonic development of Danio rerio, however, in the male gonads the TiO2-NPs caused an alteration of the morphological/structural organization. The immunofluorescence investigation showed positivity for biomarkers of oxidative stress and sex hormone binding globulin (SHBG), both confirmed by the results of qRT-PCR. In addition, an increased expression of the gene responsible for the conversion of testosterone to dihydrotestosterone was found. Since Leydig cells are mainly involved in this activity, an increase in gene activity can be explained by the ability of TiO2-NPs to act as endocrine disruptors, and, therefore, with androgenic activity.

Exosomes from circ-Astn1-modified adipose-derived mesenchymal stem cells enhance wound healing through miR-138-5p/SIRT1/FOXO1 axis regulation

Wound healing impairment is a dysfunction induced by hyperglycemia and its effect on endothelial precursor cells (EPCs) in type 2 diabetes mellitus. There is increasing evidence showing that exosomes (Exos) derived from adipose-derived mesenchymal stem cells (ADSCs) exhibit the potential to improve endothelial cell function along with wound healing. However, the potential therapeutic mechanism by which ADSC Exos contribute to wound healing in diabetic mice remains unclear.To reveal the potential therapeutic mechanism of ADSC Exos in wound healing in diabetic mice.Exos from ADSCs and fibroblasts were used for high-throughput RNA sequencing (RNA-Seq). ADSC-Exo-mediated healing of full-thickness skin wounds in a diabetic mouse model was investigated. We employed EPCs to investigate the therapeutic function of Exos in cell damage and dysfunction caused by high glucose (HG). We utilized a luciferase reporter (LR) assay to analyze interactions among circular RNA astrotactin 1 (circ-Astn1), sirtuin (SIRT) and miR-138-5p. A diabetic mouse model was used to verify the therapeutic effect of circ-Astn1 on Exo-mediated wound healing.High-throughput RNA-Seq analysis showed that circ-Astn1 expression was increased in ADSC Exos compared with Exos from fibroblasts. Exos containing high concentrations of circ-Astn1 had enhanced therapeutic effects in restoring EPC function under HG conditions by promoting SIRT1 expression. Circ-Astn1 expression enhanced SIRT1 expression through miR-138-5p adsorption, which was validated by the LR assay along with bioinformatics analyses. Exos containing high concentrations of circ-Astn1 had better therapeutic effects on wound healing in vivo compared to wild-type ADSC Exos. Immunofluorescence and immunohistochemical investigations suggested that circ-Astn1 enhanced angiopoiesis through Exo treatment of wounded skin as well as by suppressing apoptosis through promotion of SIRT1 and decreased forkhead box O1 expression.Circ-Astn1 promotes the therapeutic effect of ADSC-Exos and thus improves wound healing in diabetes via miR-138-5p absorption and SIRT1 upregulation. Based on our data, we advocate targeting the circ-Astn1/miR-138-5p/SIRT1 axis as a potential therapeutic option for the treatment of diabetic ulcers.

ALKBH5 Drives Immune Suppression Via Targeting AXIN2 to Promote Colorectal Cancer and Is a Target for Boosting Immunotherapy

Immune checkpoint blockade therapy benefits only a small subset of patients with colorectal cancer (CRC), and identification of CRC-intrinsic events modulating immune checkpoint blockade efficacy is an unmet need. Wefound that AlkB homolog 5 (ALKBH5), an RNA N6-methyladenosineeraser, drives immunosuppression and is a molecular target to boost immune checkpoint blockade therapy in CRC. Clinical significance of ALKBH5 was evaluated in human samples (n= 205). Function of ALKBH5 was investigated in allografts, CD34+ humanized mice, and Alkbh5 knockin mice. Immunity change was determined by means of flow cytometry, immunofluorescence, and functional investigation. Methylated RNA immunoprecipitation sequencing and RNA sequencing were used to identify ALKBH5 targets. Vesicle-like nanoparticle-encapsulated ALKBH5-small interfering RNA was constructed for targeting ALKBH5 invivo. High ALKBH5 expression predicts poor prognosis in CRC. ALKBH5 induced myeloid-derived suppressor cell accumulation but reduced natural killer cells and cytotoxic CD8+ T cells to induce colorectal tumorigenesis in allografts, CD34+ humanized mice, and intestine-specific Alkbh5 knockin mice. Mechanistically, AXIN2, a Wnt suppressor, was identified as a target of ALKBH5. ALKBH5 binds and demethylates AXIN2 messenger RNA, which caused its dissociation from N6-methyladenosine reader IGF2BP1 and degradation, resulting in hyperactivated Wnt/β-catenin. Subsequently, Wnt/β-catenin targets, including Dickkopf-related protein 1 (DKK1) were induced by ALKBH5. ALKBH5-induced DKK1 recruited myeloid-derived suppressor cells to drive immunosuppression in CRC, and this effect was abolished by anti-DKK1 invitro and invivo. Finally, vesicle-like nanoparticle-encapsulated ALKBH5-small interfering RNA, or anti-DKK1 potentiated anti-PD1 treatment in suppressing CRC growth by enhancing antitumor immunity. This study identified an ALKBH5-N6-methyladenosine-AXIN2-Wnt-DKK1 axis in CRC, which drives immune suppression to facilitate tumorigenesis. Targeting of ALKBH5 is a promising strategy for sensitizing CRC to immunotherapy.

ZO-1 expression in normal human macula densa: Immunohistochemical and immunofluorescence investigations.

The macula densa (MD) is an anatomical structure having a plaque shape, placed in the distal end of thick ascending limb of each nephron and belonging to juxtaglomerular apparatus (JGA). The aim of the present investigation is to investigate the presence of ZO-1, a specific marker of tight juncions (TJs), in MD cells. Six samples of normal human renal tissue were embedded in paraffin for ZO-1 expression analysis by immunohistochemical and immunofluorescence techniques. We detected ZO-1 expression in the apical part of cell membrane in MD cells by immunohistochemistry. In addition, ZO-1 and nNOS expressions (a specific marker of MD) were colocalized in MD cells providing clear evidence of TJs presence in normal human MD. Since ZO-1 is responsible for diffusion barrier formation, its presence in the MD supports the existence of a tubulomesangial barrier that ensures a regulated exchange between MD and JGA effectors in renal and glomerular haemodynamic homeostasis.

Experimental Induction of Intracranial Aneurysms in Rats: A New Model Utilizing a Genetic Modification within the EDNRA Gene.

The rupture of an intracranial aneurysm (IA) leads to life-threatening subarachnoid hemorrhage. Aside from well-established risk factors, recently published genome-wide association studies of IA revealed the strong association of a common variant near the endothelin receptor type A (EDNRA) gene with IA risk. However, the role of EDNRA in the pathogenesis of IA remains unclear. The aim of this study was to investigate the influence of a genetic modification within the EDNRA gene on IA pathogenesis in a novel in vivo model. Adult wild-type Sprague–Dawley rats (WT rats) and genetically modified rats (EDNRA rats) were used for the induction of IA using arterial hypertension (HT). Animals were stratified into four groups: WT rats without (WT_CTL) and with induction of HT (WT + HT), as well as EDNRA rats without (EDNRA_CTL) and with induction of HT (EDNRA + HT). Blood pressure (BP) was observed for 12 weeks. After the observation period, cerebral arteries were analyzed for morphological (i.e., aneurysmal) changes as well as histological and functional changes by immunofluorescence and functional investigation. In the groups of rats with induction of HT, BP was higher in EDNRA + HT compared with that in WT + HT. No IAs were observed in WT_CTL and EDNRA_CTL but were found in WT + HT and EDNRA + HT. There was no histological difference in the immunofluorescence of EDNRA between all groups. Contractility and potency of endothelin-1 differed between the groups in functional investigation. In summary, we created a new model that is suitable for further studies for better understanding of the role of EDNRA in IA pathogenesis.

Enhanced Extracellular Matrix Deposition on Titanium Implant Surfaces: Cellular and Molecular Evidences.

The surface structure of the titanium dental implants can modulate the activity of mesenchymal stem cells in order to promote the upregulation of osteoblastic related genes and the release of extracellular matrix (ECM) components. The present work was focused on the in vitro evaluation of the interaction of human periodontal ligament stem cells (hPDLSCs) and two different implant titanium surfaces topography (CTRL and TEST). This study was aimed at analyzing the cytotoxicity of the dental implant surfaces, the cellular adhesion capacity, and the improvement in the release of ECM molecules in an in vitro model. These parameters were carried out by means of the microscopic evaluation, viability assays, immunofluorescence, Western blot and RT-PCR investigations. The knowledge of the cell/implant interaction is essential for implant healing in order to obtain a more performing surfaces that promote the ECM release and provide the starting point to initiate the osseointegration process.

DMD – BIOMARKERS & OUTCOME MEASURES: P.125 Combined protein and transcript studies identify periostin as a DMD tissue and blood marker protein

Muscular dystrophies (MDs) represent a group of genetically caused muscle wasting diseases. Hereby, Duchenne muscular dystrophy (DMD) represents the most frequent form and is caused by hemizygous mutations within the X-chromosomal encoded DMD gene. Generally, MDs are characterized by myofiber dropout due to cellular necrosis, inflammation, fatty cell replacement and alterations in extracellular matrix (ECM) composition leading to fibrosis. ECM-alterations are modulated by altered expression of a variety of proteins including TGF-β, a proinflammatory cytokine transforming growth factor, which in turn profoundly influences ECM composition through pathological increased secretion of profibrotic proteins including the matricellular protein periostin. Notably, increase and concomitant secretion of periostin was shown in the δ-sarcoglycan null mouse model of MD, enhancing disease progression. Postn gene deletion in these mice altered TGF-β signalling resulting in enhanced tissue regeneration with reduced levels of fibrosis suggesting periostin as a MD determinant. We hypothesized that periostin might also represent a biomarker for DMD. To test this assumption, targeted-proteomic and transcriptomic, immunohistochemical, immunofluorescence and ELISA-based investigations have been carried out on dystrophin-deficient skeletal muscle and blood samples derived from DMD-patients with different states of disease progression. Our combined studies revealed correlated increase of POSTN-transcripts and the corresponding protein in quadriceps muscle upon disease progression independent of TGF-β expression along with altered abundances of periostin in DMD-blood samples. Our study presents periostin as a novel DMD tissue and blood marker protein involved in muscle cell vulnerability and restructuring of the surrounding mesenchyme.

Oxygen self-enriched nanoplatform combined with US imaging and chemo/photothermal therapy for breast cancer

Oxygen-saturated perfluorohexane-cored, cisplatin (Pt)-decorated hollow gold nanospheres (Pt-HAuNS-PFH@O2) have been synthesized for ultrasound (US) imaging-guided tumor treatment depending on chemo/photothermal therapy, relief of hypoxia, and photothermal induced US contrast signal. Both NIR laser-induced hyperthermia generation by gold nanospheres and acidity triggered release of Pt resulted in high toxicity after internalization by breast cancer cells. According to ex vivo immunofluorescence investigation and in vivo pharmacodynamic studies on MDA-MB-231 tumor bearing mice, the susceptibility of tumors to Pt-HAuNS-PFH@O2 was improved by the relief of hypoxia. In addition, US imaging under different conditions verified the amplified US contrast property of Pt-HAuNS-PFH@O2 by the heat-dependent liquid–gas conversion of PFH. Overall, Pt-HAuNS-PFH@O2 can be promisingly used as an oxygen self-enriched nanoplatform for US imaging-guided chemo/photothermal therapy.

Significant decrease in the viability and tumor stem cell marker expression in tumor cell lines treated with curcumin

Lung cancer, colon cancer and Leukemia are among the ten most common cancers worldwide with high incidence of recurrence, metastasis and chemoresistance. Limitations of therapy have been attributed to cancer stem cells (CSCs). The natural multi-targeting agent curcumin (turmeric) has shown significant anti-cancer effects. In this study the authors investigated the effect of curcumin on cancer cell viability, proliferation and suppression of CSCs markers (CD44, CD133, ALDH1) in vitro. Additionally, as limitations for curcumin application arise from its poor bioavailability, the stability of curcumin in different media conditions was investigated.The effect of curcumin on three cancer cell lines (A549, HCT116, 183E95) was investigated with MTT-assay, Flowcytometry, Immunofluorescence and western blotting. Stability of curcumin (diferuloylmethane) and curcuminoids (bisdemetoxycurcumin; desmetoxycurcumin) was determined by HPLC technique in A549 cells in different culture systems (culture medium with/without 10 % FCS, culture medium and cells with/without 10 % FCS) every three hours for over 24 h.Curcumin suppressed cell viability and proliferation as well as expression of CD44 in a time- and dose-dependent manner in all cell lines. Exemplary immunofluorescent and western blot investigation of HCT116 demonstrated that curcumin down-regulated CD44, CD133 and ALDH1 time-dependently. Stability of curcumin was markedly prolonged in a medium supplemented with 10 % FCS independent of application to cell cultures. The most stable curcuminoid over time was bisdemetoxycurcumin and the least stable was Curcumin.The results underline the immense potential of curcumin as an anti-cancer agent by targeting CSCs and further demonstrate that adequate media composition can significantly enhance curcumin stability in vitro.

Molecular mechanisms underlying the effects of temperature increase on Mytilus sp. and their hybrids at early larval stages

The present work aims to investigate the effects of water temperature increase on Mytilus galloprovincilis and Mytilus edulis pure larvae (PG, PE) and their hybrids (HFG, HFE). D-larvae were maintained at 18 °C or exposed to a higher temperature of 22 °C for 48 h. Initially, Embryotoxicity test was evaluated. Second, a transcriptomic analysis using a recently developed microarray platform was applied to determine the main biological processes involved in early life stages responses to temperature increase. Finally, an immunofluorescence investigation was performed to bridge the gap between transcriptomic regulation and the real changes at cellular/tissue levels. Embryotoxicity test revealed a higher sensitivity of M. edulis (PE) D-larvae as well as hybrids from females M. edulis (HFE) to temperature increase, with the highest rate of larval malformations. Transcriptomic results indicated a lack of an adequate heat shock protein (Hsp) response in PE and HFE larvae (the high expression was observed in PG larvae); the differential expression of gene involved in translation, energy metabolism and oxidative stress response may contribute to explain the observed complex alterations in the studied conditions. As revealed by immunohistochemistry, cytoskeleton proteins changes associated with a drastic decrease of Histidine-Rich Glycoprotein (HRG) may elucidate the larval abnormalities in shell development observed for PE and HFE larvae. Overall, the results indicate that each type of pure larva (PG and PE) and their respective female hybrid (HFG and HFE) react similarly to the temperature increase. Our data should be carefully considered in view of the water temperature increase in marine ecosystems and especially for the mussel’s species in confluence zones.

Cryo-Gel embedding compound for renal biopsy biobanking

Optimal preservation and biobanking of renal tissue is vital for good diagnostics and subsequent research. Optimal cutting temperature (OCT) compound is a commonly used embedding medium for freezing tissue samples. However, due to interfering polymers in OCT, analysis as mass spectrometry (MS) is difficult. We investigated if the replacement of OCT with Cryo-Gel as embedding compound for renal biopsies would enable proteomics and not disturb other common techniques used in tissue diagnostics and research. For the present study, fresh renal samples were snap-frozen using Cryo-Gel, OCT and without embedding compound and evaluated using different techniques. In addition, tissue samples from normal spleen, skin, liver and colon were analyzed. Cryo-Gel embedded tissues showed good morphological preservation and no interference in immunohistochemical or immunofluorescent investigations. The quality of extracted RNA and DNA was good. The number of proteins identified using MS was similar between Cryo-Gel embedded samples, samples without embedding compound and OCT embedded samples. However, polymers in the OCT disturbed the signal in the MS, while this was not observed in the Cryo-Gel embedded samples. We conclude that embedding of renal biopsies in Cryo-Gel is an excellent and preferable alternative for OCT compound for both diagnostic and research purposes, especially in those cases where proteomic analysis might be necessary.

Identification of linear polyubiquitin chain immunoreactivity in tau pathology of Alzheimer’s disease

Characteristic neuropathological structures observed in Alzheimer’s disease, such as neurofibrillary tangles and dystrophic neurites of senile plaques, are universally ubiquitinated. Eight linkage types of polyubiquitin chains are known, and each type of chain exerts different intracellular action. Among them, linear polyubiquitin chain was recently reported to be associated with the pathomechanism of neuronal cell death. We therefore generated a novel antibody that specifically recognizes linear polyubiquitin chain. We immunohistochemically examined the brains of patients with Alzheimer’s disease. A subset of neurofibrillary tangles, dystrophic neurites of senile plaques, and neuropil threads were evident to be immunopositive for linear polyubiquitin chains, but their number was fewer than those recognized by the antibody against K48-linked polyubiquitin chains. Double immunofluorescence investigation showed that in certain neurofibrillary tangles, a part of K48-linked polyubiquitin-immunopositive structures were immunolabeled for linear polyubiquitin chains. Our results imply that linear polyubiquitination follows K48-linked polyubiquitination in abnormally accumulated tau proteins in Alzheimer’s disease, and imply involvement in its neurodegeneration.

Herpes simplex virus: A seed or sequelae to pemphigus?

Pemphigus vulgaris is a vesiculobullous lesion in which autoantibodies are produced against desmoglein-3 antigens. Despite having an autoimmune etiology, herpes simplex virus (HSV) is found to be an additional aggravating factor for this disease. Sometimes, there can be a coexistence between the two entities. Here, a unique case of a 65-year-old female patient who presented with the chief complaint of ulcers in the oral cavity for the past 2–3 months is discussed. Histopathologic and immunofluorescence investigations were suggestive of pemphigus, but the cytopathologic smears manifested characteristics indicative of HSV infection. The existence of a viral infection with the known case of pemphigus poses a potential difficulty in the classical treatment of the latter. This case report throws light on the interesting coexistence of these two disease processes, thus highlighting the pathogenesis and treatment plan which may have to be considered in such controversial cases.

Linear IgA bullous dermatosis

Aim: to present a clinical case of linear IgA bullous dermatosis.Materials and methods. A 44 years old patient complaining of skin rashes on the trunk and extremities, accompanied by severe itching was examined. We carried out a morphological investigation of biopsy samples derived from the lesion and apparently unaffected skin areas using the method of indirect immunofluorescence.Results. The patient having rashes in the form of multiple vesicles and small bubbles with a tight cover, which had been grouped into figures resembling pearl necklaces, demonstrated the presence of focal subepidermal cracks (subepidermal bubble in one location), as well as a linear deposition of IgA along the epidermal basement membrane. According to the clinical picture and following the histological and immunofluorescent investigation of skin biopsies, the patient was diagnosed with linear IgA bullous dermatosis. Lesion regression was achieved as a result of systemic therapy with prednisolone at a dose of 50 mg per day.Conclusion. The diagnosis of linear IgA bullous dermatosis should be made on the basis of skin biopsy investigation by the method of indirect immunofluorescence. Systemic glucocorticosteroids are seen as an effective approach to the treatment of such patients.

BML-111 Reduces Neuroinflammation and Cognitive Impairment in Mice With Sepsis via the SIRT1/NF-κB Signaling Pathway.

Sepsis is a life-threatening state of organ dysfunction caused by infection and which can induce severe neurological disorders that lead to neuroinflammation and cognitive impairment. Inflammation has been reported to cause neuronal apoptosis in sepsis, which can finally lead to cognitive impairment. Previous studies have suggested that BML-111 can exhibit anti-inflammatory and proresolution activities. Additionally, silent information regulator 1 (SIRT1) can inhibit the NF-κB signaling pathway in an inflammation state. However, the role of the SIRT1/NF-κB signaling pathway in the protective effects of BML-111 against sepsis-induced neuroinflammation and cognitive impairment remains unclear. This study aimed to determine the effects of BML-111 on neuroinflammation and cognitive impairment induced by sepsis. Male C57BL/6J mice were subjected to cecal ligation and puncture (CLP) or a sham operation. BML-111 was administered via intracerebroventricular injection (0.1 mg/kg) immediately after CLP. Boc-2 (50 μg/kg) was administered intracerebroventricularly 30 min before CLP, and EX527 (10 μg) was administered every 2 days for a total of three times before CLP, also intracerebroventricularly. Some of the surviving mice underwent open-field, novel-object-recognition, and fear-conditioning behavioral tests at 7 days after surgery. Some of the other surviving mice were killed at 24 h after surgery to assess synaptic damage (PSD95 and Synapsin1), markers of inflammation [tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β], cytoplasmic p65, nuclear p65, Ac- NF-κB and SIRT1. At 48 h after CLP, TUNEL and glia-activation by immunofluorescence investigations were performed on a separate cohort of surviving animals. The results suggested that sepsis resulted in cognitive impairment, which was accompanied by the decreased the expression of PSD95 and Synapsin1, increased amount of TUNEL-positive cells and the activation of glias, increased production of TNF-α and IL-1β, increased expression of nuclear p65, Ac- NF-κB, and decreased expression of SIRT1 and cytoplasmic p65. It is especially notable that these abnormalities could be reduced by BML-111 treatment. EX527, an SIRT1 inhibitor, abolished the effects of BML-111. These results demonstrate that BML-111 can reduce the neuroinflammation and cognitive impairment induced by sepsis via SIRT/NF-κB signaling pathway.

Biological effects of Salvia Officinalis leaf extract on murine myeloma cells

Background: In the Mediterranean region, Salvia officinalis plant is widely used and routinely added to food for a traditional culinary utilization, and moreover, from long time, it is recognized to possess pharmacological activities. Objectives: In the present study, we investigated the biological properties of S. officinalis leaf ethanol extracts and a thin layer chromatography-isolated spot on murine myeloma cells. Materials and Methods: 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide tests were carried out to determinate the EC50 of both the leaf extracts and the isolated spot; the isolated spot was also investigated by liquid chromatography–mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). Electron microscopy and immunofluorescence were used for morphological studies on treated P3X murine myeloma cells. Results: LC-MS and NMR techniques identified methyl carnosate, a methyl derivative of carnosic acid, as the main component of spot B. Moreover, scanning electron microscopy, transmission electron microscopy, and immunofluorescence investigations carried out on murine myeloma cells treated for 20 h with EC50 values of spot B revealed some changes both in the cellular morphology and in the microtubular array. Conclusions: The present studies indicate that S. officinalis extracts have biological effects on murine myeloma cells and identify methyl carnosate as an interesting molecule for further investigations on human cell lines and possibly on cancer prevention. Abbreviations used: EC50: Effective concentration 50, LC-MS: Liquid chromatography–mass spectrometry, NMR: Nuclear magnetic resonance, TLC: Thyn layer chromatography, MTT: 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide, RF: Retention factor, MS/Ms: Tandem mass spectrometry, dqf-COSY: double quantum filter COSY

Cathepsin G in the immune defense of the human duodenum: New sources for biosynthesis

Proteases play a key role in the physiological processes of the small intestine, supporting its normal physiological functions as a part of the digestive system, in which hydrolysis and assimilation of nutrients are implemented. A high concentration of antigens in the intestinal lumen activates immunity and stimulates a chronic weakly expressed inflammatory response in a normal gastrointestinal tract (GIT). Cathepsin G, a serine protease controlling the functional state of immune cells, directly participates in the complicated system for the regulation of balance between physiological and pathological inflammations. To determine the role of cathepsin G in the small intestine, an immunofluorescent investigation of biopsies from the human duodenal mucosa were investigated using the confocal immunofluorescence microscopy method and human antibodies to cathepsin G. It has been shown for the first time that cathepsin G, which was regarded conventionally as one of the effectors of the inflammatory process, is a constitutive enzyme of the human duodenum and is constantly present in its normal mucosa. The new cell sources for the cathepsin G biosynthesis identified: intraepithelial lymphocytes (IELs), lamina propria lymphocytes, CD14-positive intestinal macrophages, and Paneth cells, which are specialized epitheliocytes of intestinal glands. Our data on the cathepsin G expression by immunocytes and Paneth cells in the duodenum allow us to attribute cathepsin G to the main proteases of intestinal immunity, which indicates the important role of this enzyme in the regulation of human GIT functions.