Identification of immune subsets with distinct lectin binding signatures using multi-parameter flow cytometry: correlations with disease activity in systemic lupus erythematosus.

Objectives: Cell surface glycosylation can influence protein-protein interactions with particular relevance to changes in core fucosylation and terminal sialylation. Glycans are ligands for immune regulatory lectin families like galectins (Gals) or sialic acid immunoglobulin-like lectins (Siglecs). This study delves into the glycan alterations within immune subsets of systemic lupus erythematosus (SLE). Methods: Evaluation of binding affinities of Galectin-1, Galectin-3, Siglec-1, Aleuria aurantia lectin (AAL, recognizing core fucosylation), and Sambucus nigra agglutinin (SNA, specific for α-2,6-sialylation) was conducted on various immune subsets in peripheral blood mononuclear cells (PBMCs) from control and SLE subjects. Lectin binding was measured by multi-parameter flow cytometry in 18 manually gated subsets of T-cells, NK-cells, NKT-cells, B-cells, and monocytes in unstimulated resting state and also after 3-day activation. Stimulated pre-gated populations were subsequently clustered by FlowSOM algorithm based on lectin binding and activation markers, CD25 or HLA-DR. Results: Elevated AAL, SNA and CD25+/CD25- SNA binding ratio in certain stimulated SLE T-cell subsets correlated with SLE Disease Activity Index 2000 (SLEDAI-2K) scores. The significantly increased frequencies of activated AALlow Siglec-1low NK metaclusters in SLE also correlated with SLEDAI-2K indices. In SLE, activated double negative NKTs displayed significantly lower core fucosylation and CD25+/CD25- Siglec-1 binding ratio, negatively correlating with disease activity. The significantly enhanced AAL binding in resting SLE plasmablasts positively correlated with SLEDAI-2K scores. Conclusion: Alterations in the glycosylation of immune cells in SLE correlate with disease severity, which might represent potential implications in the pathogenesis of SLE.

Lectin-Based Immunophenotyping and Whole Proteomic Profiling of CT-26 Colon Carcinoma Murine Model.

A murine colorectal carcinoma (CRC) model was established. CT26 colon carcinoma cells were injected into BALB/c mice's spleen to study the primary tumor and the mechanisms of cell spread of colon cancer to the liver. The CRC was verified by the immunohistochemistry of Pan Cytokeratin and Vimentin expression. Immunophenotyping of leukocytes isolated from CRC-bearing BALB/c mice or healthy controls, such as CD19+ B cells, CD11+ myeloid cells, and CD3+ T cells, was carried out using fluorochrome-labeled lectins. The binding of six lectins to white blood cells, such as galectin-1 (Gal1), siglec-1 (Sig1), Sambucus nigra lectin (SNA), Aleuria aurantia lectin (AAL), Phytolacca americana lectin (PWM), and galectin-3 (Gal3), was assayed. Flow cytometric analysis of the splenocytes revealed the increased binding of SNA, and AAL to CD3 + T cells and CD11b myeloid cells; and increased siglec-1 and AAL binding to CD19 B cells of the tumor-bearing mice. The whole proteomic analysis of the established CRC-bearing liver and spleen versus healthy tissues identified differentially expressed proteins, characteristic of the primary or secondary CRC tissues. KEGG Gene Ontology bioinformatic analysis delineated the established murine CRC characteristic protein interaction networks, biological pathways, and cellular processes involved in CRC. Galectin-1 and S100A4 were identified as upregulated proteins in the primary and secondary CT26 tumor tissues, and these were previously reported to contribute to the poor prognosis of CRC patients. Modelling the development of liver colonization of CRC by the injection of CT26 cells into the spleen may facilitate the understanding of carcinogenesis in human CRC and contribute to the development of novel therapeutic strategies.

Antimicrobial efficacy of sodium hypochlorite and hyper-pure chlorine dioxide in the depth of dentin tubules in vitro.

OBJECTIVES: The study aimed to compare the antibacterial effect of a novel disinfectant, hyper-pure chlorine dioxide (hClO2) to sodium hypochlorite (NaOCl) in various depths of dentin tubules. MATERIALS AND METHODS: The distal root of the extracted lower molars was infected artificially with Enterococcus faecalis. The control group was rinsed with saline, and the test groups were irrigated with either 5% NaOCl or 0.12% hClO2. The longitudinally split teeth were stained by viability stain. The coronal third of the root was scanned with a confocal laser scanning microscope. The fluorescent intensities were measured, and the percentage of dead bacteria was calculated at depths up to 950 µm along the dentin tubules. The effect of penetration depth, irrigants, and their interaction on antimicrobial efficacy was determined by the linear mixed model. RESULTS: The percentage of dead bacteria was higher both in the NaOCl (45.1 ± 2.3%, p < 0.01) and in the hClO2 (44.6 ± 3.8%, p < 0.01) irrigant groups compared to saline (23 ± 4.5%); however, there was no difference between them. The percentage of killed bacteria was not correlated with the depths in any group (p = 0.633). CONCLUSIONS: Our results suggest that the functional penetration depth of NaOCl is at least 2-3 times more than published to date. There is no difference in disinfection effectiveness along the dentin tubules between NaOCl and hClO2 until at least the measured 950 µm. However, both were only able to eradicate the intratubular bacteria partially. CLINICAL RELEVANCE: Hyper-pure ClO2 could be used as an alternative or final adjuvant irrigant in endodontic treatment.

Fluorescent Molecules That Help Reveal Previously Unidentified Neural Connections in Adult, Neonatal and Peripubertal Mammals.

One hundred and twenty-five years ago there was a lively discussion between Hungarian and Spanish neuroscientists on the nature of neural connections. The question was whether the neurofibrils run from one neuron to the next and connect neurons as a continuous network or the fibrils form an internal skeleton in the neurons and do not leave the cell; however, there is close contact between the neurons. About 50 years later, the invention of the electron microscope solved the problem. Close contacts between individual neurons were identified and named as synapses. In the following years, the need arose to explore distant connections between neuronal structures. Tracing techniques entered neuroscience. There are three major groups of tracers: (A) non-transsynaptic tracers used to find direct connections between two neuronal structures; (B) tracers passing gap junctions; (C) transsynaptic tracers passing synapses that are suitable to explore multineuronal circuits. According to the direction of the transport mechanism, the tracer may be ante- or retrograde. In this review, we focus on the ever-increasing number of fluorescent tracers that we have also used in our studies. The advantage of the use of these molecules is that the fluorescence of the tracer can be seen in histological sections without any other processes. Genes encoding fluorescent molecules can be inserted in various neuropeptide or neurotransmitter expressing transcriptomes. This makes it possible to study the anatomy, development or functional relations of these neuronal networks in transgenic animals.

Comparison of humoral and cellular immune responses in hematologic diseases following completed vaccination protocol with BBIBP-CorV, or AZD1222, or BNT162b2 vaccines against SARS-CoV-2.

Background: Vaccination has proven the potential to control the COVID-19 pandemic worldwide. Although recent evidence suggests a poor humoral response against SARS-CoV-2 in vaccinated hematological disease (HD) patients, data on vaccination in these patients is limited with the comparison of mRNA-based, vector-based or inactivated virus-based vaccines. Methods: Forty-nine HD patients and 46 healthy controls (HCs) were enrolled who received two-doses complete vaccination with BNT162b2, or AZD1222, or BBIBP-CorV, respectively. The antibodies reactive to the receptor binding domain of spike protein of SARS-CoV-2 were assayed by Siemens ADVIA Centaur assay. The reactive cellular immunity was assayed by flow cytometry. The PBMCs were reactivated with SARS-CoV-2 antigens and the production of activation-induced markers (TNF-α, IFN-γ, CD40L) was measured in CD4+ or CD8+ T-cells ex vivo. Results: The anti-RBD IgG level was the highest upon BNT162b2 vaccination in HDs (1264 BAU/mL) vs. HCs (1325 BAU/mL) among the studied groups. The BBIBP-CorV vaccination in HDs (339.8 BAU/mL ***p < 0.001) and AZD1222 in HDs (669.9 BAU/mL *p < 0.05) resulted in weaker antibody response vs. BNT162b2 in HCs. The response rate of IgG production of HC vs. HD patients above the diagnostic cut-off value was 100% vs. 72% for the mRNA-based BNT162b2 vaccine; 93% vs. 56% for the vector-based AZD1222, or 69% vs. 33% for the inactivated vaccine BBIBP-CorV, respectively. Cases that underwent the anti-CD20 therapy resulted in significantly weaker (**p < 0.01) anti-RBD IgG level (302 BAU/mL) than without CD20 blocking in the HD group (928 BAU/mL). The response rates of CD4+ TNF-α+, CD4+ IFN-γ+, or CD4+ CD40L+ cases were lower in HDs vs. HCs in all vaccine groups. However, the BBIBP-CorV vaccine resulted the highest CD4+ TNF-α and CD4+ IFN-γ+ T-cell mediated immunity in the HD group. Conclusion: We have demonstrated a significant weaker overall response to vaccines in the immunologically impaired HD population vs. HCs regardless of vaccine type. Although, the humoral immune activity against SARS-CoV-2 can be highly evoked by mRNA-based BNT162b2 vaccination compared to vector-based AZD1222 vaccine, or inactivated virus vaccine BBIBP-CorV, whereas the CD4+ T-cell mediated cellular activity was highest in HDs vaccinated with BBIBP-CorV.

The transcriptional control of the VEGFA-VEGFR1 (FLT1) axis in alternatively polarized murine and human macrophages.

Introduction: Macrophages significantly contribute to the regulation of vessel formation under physiological and pathological conditions. Although the angiogenesis-regulating role of alternatively polarized macrophages is quite controversial, a growing number of evidence shows that they can participate in the later phases of angiogenesis, including vessel sprouting and remodeling or regression. However, the epigenetic and transcriptional regulatory mechanisms controlling this angiogenesis-modulating program are not fully understood. Results: Here we show that IL-4 can coordinately regulate the VEGFA-VEGFR1 (FLT1) axis via simultaneously inhibiting the proangiogenic Vegfa and inducing the antiangiogenic Flt1 expression in murine bone marrow-derived macrophages, which leads to the attenuated proangiogenic activity of alternatively polarized macrophages. The IL-4-activated STAT6 and IL-4-STAT6 signaling pathway-induced EGR2 transcription factors play a direct role in the transcriptional regulation of the Vegfa-Flt1 axis. We demonstrated that this phenomenon is not restricted to the murine bone marrow-derived macrophages, but can also be observed in different murine tissue-resident macrophages ex vivo and parasites-elicited macrophages in vivo with minor cell type-specific differences. Furthermore, IL-4 exposure can modulate the hypoxic response of genes in both murine and human macrophages leading to a blunted Vegfa/VEGFA and synergistically induced Flt1/FLT1 expression. Discussion: Our findings establish that the IL-4-activated epigenetic and transcriptional program can determine angiogenesis-regulating properties in alternatively polarized macrophages under normoxic and hypoxic conditions.

[The unexplored side of creativity: malevolent creative behavior and its measuring tools].

Creativity encompasses a wide range of topics and practically all aspects of life (arts, schools, workplaces, inventions etc.). The new theme, which is a negative and malevolent type of creativity, has piqued people's interest. And, despite the fact that the number of studies on the issue has expanded in recent years, its relationship with most constructions or predictability has yet to be explored. Aggression (Meshkova, 2018; Hao et al., 2016; Harris & Reiter-Palmon, 2015; Harris, 2013; Lee & Dow, 2011), emotional intelligence (Harris & Reiter-Palmon, 2013), resilience (Wang, Wang & Chen, 2022), humor (Perchtold-Stefan et al., 2020/a), childhood neglect (Jia, Wang & Lin, 2020), and other factors have already been studied in relationship with malevolent creativity. It has also been investigated with adolescents (Wang, Wang, Chen, 2022), adults (Hao et al., 2016, 2020; Baas et al., 2019; Harris & Reiter-Palmon, 2015; Harris, 2013; Lee & Dow, 2011), and was less studied on the prison population (Meshkova et al, 2018, Bochkova, 2020). The most significant goal of this paper is to paint a complete picture of malevolent creativity, from its link to creativity to the concept's freaking out to the study of malevolent creativity's measuring stones. To accomplish so, he looks for material in the most recent professional journals and employs a critical mindset. What's crucial, and what some writers are doing already, is that they use both scientific instruments in their research, thus balancing the measuring instru ments' weaknesses (Perchtold-Stefan, 2021/a,b).

A cytotoxic survey on 2-amino-1H-imidazol based synthetic marine sponge alkaloid analogues.

Here, we describe the synthesis and biologic activity evaluation of 20 novel synthetic marine sponge alkaloid analogues with 2-amino-1H-imidazol (2-AI) core. Cytotoxicity was tested on murine 4T1 breast cancer, A549 human lung cancer, and HL-60 human myeloid leukemia cells by the resazurin assay. A total of 18 of 20 compounds showed cytotoxic effect on the cancer cell lines with different potential. Viability of healthy human fibroblasts and peripheral blood mononuclear cells upon treatment was less hampered compared to cancer cell lines supporting tumor cell specific cytotoxicity of our compounds. The most cytotoxic compounds resulted the following IC50 values 28: 2.91 µM on HL-60 cells, and 29: 3.1 µM on 4T1 cells. The A549 cells were less sensitive to the treatments with IC50 15 µM for both 28 and 29. Flow cytometry demonstrated the apoptotic effect of the most active seven compounds inducing phosphatidylserine exposure and sub-G1 fragmentation of nuclear DNA. Cell cycle arrest was also observed. Four compounds caused depolarization of the mitochondrial membrane potential as an early event of apoptosis. Two lead compounds inhibited tumor growth in vivo in the 4T1 triple negative breast cancer and A549 human lung adenocarcinoma xenograft models. Novel marine sponge alkaloid analogues are demonstrated as potential anticancer agents for further development.

Comparison of Homologous and Heterologous Booster SARS-CoV-2 Vaccination in Autoimmune Rheumatic and Musculoskeletal Patients.

Vaccination against SARS-CoV-2 to prevent COVID-19 is highly recommended for immunocompromised patients with autoimmune rheumatic and musculoskeletal diseases (aiRMDs). Little is known about the effect of booster vaccination or infection followed by previously completed two-dose vaccination in aiRMDs. We determined neutralizing anti-SARS-CoV-2 antibody levels and applied flow cytometric immunophenotyping to quantify the SARS-CoV-2 reactive B- and T-cell mediated immunity in aiRMDs receiving homologous or heterologous boosters or acquired infection following vaccination. Patients receiving a heterologous booster had a higher proportion of IgM+ SARS-CoV-2 S+ CD19+CD27+ peripheral memory B-cells in comparison to those who acquired infection. Biologic therapy decreased the number of S+CD19+; S+CD19+CD27+IgG+; and S+CD19+CD27+IgM+ B-cells. The response rate to a booster event in cellular immunity was the highest in the S-, M-, and N-reactive CD4+CD40L+ T-cell subset. Patients with a disease duration of more than 10 years had higher proportions of CD8+TNF-α+ and CD8+IFN-γ+ T-cells in comparison to patients who were diagnosed less than 10 years ago. We detected neutralizing antibodies, S+ reactive peripheral memory B-cells, and five S-, M-, and N-reactive T-cells subsets in our patient cohort showing the importance of booster events. Biologic therapy and <10 years disease duration may confound anti-SARS-CoV-2 specific immunity in aiRMDs.

Enjoying others' distress and indifferent to threat? Changes in prefrontal-posterior coupling during social-emotional processing are linked to malevolent creativity.

Malevolent creativity is characterized by malicious interpersonal goals aimed at damaging others. Neurocognitive processing patterns of negative social-emotional signals may explain variance in this disruptive phenomenon. This study examined whether individuals' brain responses to emotional expressions of others are linked to their capacity of malevolent creativity in a psychometric test. State-dependent changes of prefrontal-posterior EEG coherence were recorded while n = 60 participants listened to other people's anger, desperate crying, and laughter. These EEG measures were used to indicate affective dispositions towards emotional absorption (decreased coherence) or detachment (increased coherence) from others' emotional states. Results showed that higher malevolent creativity was reflected in relatively greater increases of EEG coherence during others' expressions of anger, and conversely, relatively greater decreases of EEG coherence during others' desperate crying. This pattern suggests that the generation of creative ideas for malicious, antisocial purposes may be partly attributed to an indifference towards others' aggression and potential retaliation, and partly to finding others' adversity rewarding on a neuronal level, increasing the quantity of ideas and the chances of hurting others. This first study linking malevolent creativity to social-emotional brain functions may offer novel insights into affective dispositions that may help understand individuals' potential for creative destruction.

Personality Traits as Predictors of Malevolent Creative Ideation in Offenders.

Malevolent creativity, which can be defined as creativity that is deliberately planned to damage others, is a concept that explains how the capacity to generate novel and effective outcomes (creativity) may, on occasion, be misapplied. The present study used 130 male inmates of the Oradea Maximum Security Penitentiary in Romania to explore the ability of a set of personality variables (the dark triad, self-efficacy, and self-esteem) to predict malevolent creative ideation. The findings indicate that Machiavellianism and self-efficacy were significant predictors of malevolent creative ideation in the form of lying, while only Machiavellianism was a significant predictor of malevolent creative ideation in the form of hurting people. In addition, the present study found significant differences among subgroups in the sample, with more experienced offenders showing higher levels of malevolent creative ideation.

Humoral and Cellular Immunogenicity and Safety of Five Different SARS-CoV-2 Vaccines in Patients With Autoimmune Rheumatic and Musculoskeletal Diseases in Remission or With Low Disease Activity and in Healthy Controls: A Single Center Study.

Background: Vaccine-induced immunity is essential for controlling the COVID-19 pandemic. Data on humoral and cellular immunogenicity and safety of different SARS-CoV-2 vaccines in patients with autoimmune rheumatic and musculoskeletal diseases (RMDs) are limited. Methods: A single center observational study evaluated the immunogenicity and safety of the two-dose regimen of the BBIBP-CorV inactivated, Gam-COVID-Vac and AZD1222 adenovirus-based, and BNT162b2 and mRNA-1273 mRNA-based vaccines in patients with RMDs (n = 89) compared with healthy controls (n = 74). Neutralizing anti-RBD (receptor binding domain) specific antibodies and SARS-CoV-2 specific T-cell response were measured one and four months after the second vaccine dose in parallel with vaccination efficacy and safety. Results: Disease-specific comparison showed that antibody response at four months was higher in spondylarthropathies compared to rheumatoid arthritis and autoimmune RMDs. Risk factors for reduced immunogenicity included longer disease duration, positive immunoserological profile and anti-CD20 therapy of patients. The rate of positive anti-RBD antibody response for healthy controls versus patients after 4 months post vaccination was 69% vs. 55% for the inactivated viral vaccine BBIBP-CorV, 97% vs. 53% for the pooled data of adenovirus vector-based vaccines Gam-COVID-Vac and AZD1222, or 100% vs. 81% for the pooled data of mRNA vaccines BNT162b2 and mRNA-1273, respectively. Patients who received the Gam-COVID-Vac or mRNA-1273 vaccines had a higher proportion of TNF-α producing CD4+ T-cells upon SARS-CoV-2 antigen stimulation compared to the inactivated viral vaccine. Conclusion: All five investigated vaccines were immunogenic in the majority of patients and healthy controls with variable antibody and T-cell response and an acceptable safety profile.

Comparative Evaluation of Body Fluid Analysis by Sysmex XN Hematology Analyzers, CellaVision, Manual Microscopy and Multicolor Flow Cytometry.

OBJECTIVE: The aim of this study was to investigate the correlation between instrumental and morphological cell differential methods in body fluid (BF) samples. METHODS: Forty ascitic (AF) and forty cerebrospinal (CSF) fluid samples were measured with Sysmex XN1000 and XN2000 instruments in BF mode. Flow cytometry (FC) was carried out with FACS Canto II. From the centrifuged cytospin preparations mononuclear (MN%) and polymorphonuclear (PMN%) cell percentages were determined using optical microscopy (OM) and a digital cell morphology system CellaVision (CV). RESULTS: Both Passing-Bablok and Bland-Altman analysis showed strong correlation between the hematology analyzers for total cell count (TC), white blood cell count (WBC), MN%, and PMN% in BFs. With slightly inferior results, all other WBC differential methods showed acceptable correlation. Passing-Bablok regression analysis yielded a slope encompassing 1.0 in all method comparisons except for three scenarios in CSF. The bias calculated with Bland-Altman plots was comprised between -6.05% and 6.05%. Strong correlations were found when comparing XN1000, XN2000, and CV to OM and FC method with linear regression analysis (r values between 0.905 and 0.984). CONCLUSION: We found strong correlation between instrumental and manual morphological WBC differential methods when testing BF samples containing no tumor cells.

α/ß-Peptides as Nanomolar Triggers of Lipid Raft-Mediated Endocytosis through GM1 Ganglioside Recognition.

Cell delivery of therapeutic macromolecules and nanoparticles is a critical drug development challenge. Translocation through lipid raft-mediated endocytic mechanisms is being sought, as it can avoid rapid lysosomal degradation. Here, we present a set of short α/ß-peptide tags with high affinity to the lipid raft-associated ganglioside GM1. These sequences induce effective internalization of the attached immunoglobulin cargo. The structural requirements of the GM1-peptide interaction are presented, and the importance of the membrane components are shown. The results contribute to the development of a receptor-based cell delivery platform.

Cervical tooth anatomy considerations for prefabricated anatomic healing abutment design: A mathematical formulation.

STATEMENT OF PROBLEM: A custom emergence profile offers the ideal horizontal dimensions for an anatomic healing abutment. However, developing such an emergence profile can be a time-consuming and complex process. PURPOSE: The purpose of this study was to develop a mathematical formula defining horizontal cervical tooth geometry to design prefabricated, tooth-specific, healing abutments. MATERIAL AND METHODS: Cone beam computed tomography (CBCT) horizontal cross sections of 989 teeth on 54 participants were measured. For anterior and premolar teeth, 2 perpendicular ellipses were fitted onto the cervical tooth cross section that was defined by 3 parameters. The lingual ellipse followed the lingual outline of the tooth, and its diameter was the largest mesiodistal diameter of the tooth (parameter "a"); its buccolingual radius became parameter "b." The buccal ellipse was perpendicular to the lingual ellipse and followed the buccal outline of the tooth. The buccolingual radius of the smaller ellipse became parameter "c." For molars, the first ellipses followed the mesial outline of the tooth, and its larger diameter (parameter "a") matched the largest buccolingual diameter of the tooth. Its smaller radius became parameter "h1." The second ellipse was parallel to the first ellipse and followed the distal outline of the tooth. Its larger diameter became parameter "b", and its mesiodistal diameter became parameter "h2". Statistical differences between parameters were evaluated by the linear mixed model (α=.05 after Bonferroni adjustment). Pairwise comparisons were made separately for each parameter of the molars and separately for each parameter for the anterior teeth plus premolars. Teeth were put into the same parameter cluster if no significant differences were found between them for a specific parameter. If neither parameter (4 for molars and 3 for the other teeth) was different for 2 teeth, they were put into the same abutment cluster. The abutment clusters determined the type of anatomic healing abutment. The areas were calculated from the developed mathematical formula by using the parameters. In addition, cervical areas of 106 randomly chosen teeth were measured directly with a photo-editing software program. A computer algorithm was used to select 5 CBCT scans from the 54 by using the simple randomization method. The agreement between the 2 methods was evaluated by Bland-Altman analysis. RESULTS: The lower and upper limits of agreement between the 2 methods were -8.57 and 7.36 mm2, respectively, with no bias (-0.61 mm2, P=.224). Significant differences were found between most parameters among the 14 tooth types (P<.001). Based on the parameters, 12 specifically distinct clusters were defined. Two tooth types were pooled into 1 abutment cluster: the maxillary first and second premolars and the mandibular first and second molars. CONCLUSIONS: The cervical tooth cross section can be accurately defined by combining 2 elliptical elements. A comprehensive array of tooth specific emergence profiles can be provided by just 12 different prefabricated abutments, designed as per the recommended parameters.

Epinephrine penetrates through gingival sulcus unlike keratinized gingiva and evokes remote vasoconstriction in human.

BACKGROUND: It has been demonstrated in non-oral tissues that the locally evoked vasoconstriction could elicit remote vasoconstriction. This study aimed to investigate the spreading vasoconstrictor effects of epinephrine in the gingiva. METHODS: Gingival blood flow (GBF) was measured by laser speckle contrast imager in 21 healthy volunteers. In group A, two wells were fabricated from orthodontic elastic ligature and placed 2 mm apically to the free gingival margin at the mid buccal line of 12 (test side) and 21 (control side) teeth. The GBF was measured in the wells and tightly apical, coronal, distal and mesial to the wells. In group B, the wells were made on the buccal surface of the same teeth, including the gingival sulcus. Four regions were selected for measurement from the gingival margin reaching the mucogingival line (coronal, midway1, midway2 and apical). After the baseline recording, 3 µg epinephrine was applied into the test, and physiological saline into the control well. The GBF was recorded for 14 min. The gingival thickness was measured with a PIROP Ultrasonic Biometer. RESULTS: In group A, the GBF did not increase or decrease after the application of epinephrine. In group B, the GBF significantly decreased in all regions of the test side and remained low for the observation period. The vasoconstriction appeared with delays in more apical regions (at min 1 in the coronal and the midway1, at min 2 in the midway2, at min 4 in the apical region). Similarly, the amount of the decrease at 14 min was the largest close to sulcus (- 53 ± 2.9%), followed by the midway1 (- 51 ± 2.8%) and midway2 (- 42 ± 4.2%) and was the lowest in the apical region (- 32 ± 5.8%). No correlation was found between GBF and gingival thickness. CONCLUSION: Epinephrine could evoke intense vasoconstriction propagating to the mucogingival junction, indicating the presence of spreading vasoconstriction in the human gingiva. The attached gingiva is impermeable to epinephrine, unlike the gingival sulcus. This trial was registered in ClinicalTrials.gov titled as Evidence of Spreading Vasoconstriction in Human Gingiva with the reference number of NCT04131283 on 16 October 2019. https://clinicaltrials.gov/show/NCT04131283.

Current State of Understanding of the Role of PACAP in the Hypothalamo-Hypophyseal Gonadotropin Functions of Mammals.

PACAP was discovered 30 years ago in Dr. Akira Arimura's laboratory. In the past three decades since then, it has become evident that this peptide plays numerous crucial roles in mammalian organisms. The most important functions of PACAP are the following: 1. neurotransmitter, 2. neuromodulator, 3. hypophysiotropic hormone, 4. neuroprotector. This paper reviews the accumulated data regarding the distribution of PACAP and its receptors in the mammalian hypothalamus and pituitary gland, the role of PACAP in the gonadotropin hormone secretion of females and males. The review also summarizes the interaction between PACAP, GnRH, and sex steroids as well as hypothalamic peptides including kisspeptin. The possible role of PACAP in reproductive functions through the biological clock is also discussed. Finally, the significance of PACAP in the hypothalamo-hypophysial system is considered and the facts missing, that would help better understand the function of PACAP in this system, are also highlighted.

Routing Nanomolar Protein Cargoes to Lipid Raft-Mediated/Caveolar Endocytosis through a Ganglioside GM1-Specific Recognition Tag.

There is a pressing need to develop ways to deliver therapeutic macromolecules to their intracellular targets. Certain viral and bacterial proteins are readily internalized in functional form through lipid raft-mediated/caveolar endocytosis, but mimicking this process with protein cargoes at therapeutically relevant concentrations is a great challenge. Targeting ganglioside GM1 in the caveolar pits triggers endocytosis. A pentapeptide sequence WYKYW is presented, which specifically captures the glycan moiety of GM1 (K D = 24 nm). The WYKYW-tag facilitates the GM1-dependent endocytosis of proteins in which the cargo-loaded caveosomes do not fuse with lysosomes. A structurally intact immunoglobulin G complex (580 kDa) is successfully delivered into live HeLa cells at extracellular concentrations ranging from 20 to 160 nm, and escape of the cargo proteins to the cytosol is observed. The short peptidic WYKYW-tag is an advantageous endocytosis routing sequence for lipid raft-mediated/caveolar cell delivery of therapeutic macromolecules, especially for cancer cells that overexpress GM1.

Altered Cell Surface N-Glycosylation of Resting and Activated T Cells in Systemic Lupus Erythematosus.

Altered cell surface glycosylation in congenital and acquired diseases has been shown to affect cell differentiation and cellular responses to external signals. Hence, it may have an important role in immune regulation; however, T cell surface glycosylation has not been studied in systemic lupus erythematosus (SLE), a prototype of autoimmune diseases. Analysis of the glycosylation of T cells from patients suffering from SLE was performed by lectin-binding assay, flow cytometry, and quantitative real-time PCR. The results showed that resting SLE T cells presented an activated-like phenotype in terms of their glycosylation pattern. Additionally, activated SLE T cells bound significantly less galectin-1 (Gal-1), an important immunoregulatory lectin, while other lectins bound similarly to the controls. Differential lectin binding, specifically Gal-1, to SLE T cells was explained by the increased gene expression ratio of sialyltransferases and neuraminidase 1 (NEU1), particularly by elevated ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 (ST6GAL1)/NEU1 and ST3 beta-galactoside alpha-2,3-sialyltransferase 6 (ST3GAL6)/NEU1 ratios. These findings indicated an increased terminal sialylation. Indeed, neuraminidase treatment of cells resulted in the increase of Gal-1 binding. Altered T cell surface glycosylation may predispose the cells to resistance to the immunoregulatory effects of Gal-1, and may thus contribute to the pathomechanism of SLE.

TMEM203 is a binding partner and regulator of STING-mediated inflammatory signaling in macrophages.

Regulation of IFN signaling is critical in host recognition and response to pathogens while its dysregulation underlies the pathogenesis of several chronic diseases. STimulator of IFN Genes (STING) has been identified as a critical mediator of IFN inducing innate immune pathways, but little is known about direct coregulators of this protein. We report here that TMEM203, a conserved putative transmembrane protein, is an intracellular regulator of STING-mediated signaling. We show that TMEM203 interacts, functionally cooperates, and comigrates with STING following cell stimulation, which in turn leads to the activation of the kinase TBK1, and the IRF3 transcription factor. This induces target genes in macrophages, including IFN-ß. Using Tmem203 knockout bone marrow-derived macrophages and transient knockdown of TMEM203 in human monocyte-derived macrophages, we show that TMEM203 protein is required for cGAMP-induced STING activation. Unlike STING, TMEM203 mRNA levels are elevated in T cells from patients with systemic lupus erythematosus, a disease characterized by the overexpression of type I interferons. Moreover, TMEM203 mRNA levels are associated with disease activity, as assessed by serum levels of the complement protein C3. Identification of TMEM203 sheds light into the control of STING-mediated innate immune responses, providing a potential novel mechanism for therapeutic interventions in STING-associated inflammatory diseases.