HEp-2 Cells Research Articles

Investigation of dense fine speckled pattern and anti-dense fine speckled 70 antibody by a single step assay

The indirect immunofluorescence (IIF) method is the gold standard for identifying anti-nuclear antibodies (ANAs). It is recommended that ANA, including the dense fine speckled (DFS) pattern, should be verified with a highly specific confirmatory test after a sensitive screening test. Although methods such as ELISA and LIA are often used to confirm the presence of anti-DFS70 antibodies, new IIF methods have been developed in recent years to prevent the difficulties in the recognition of the DFS pattern and to carry out the confirmatory test in a single step. In this study, we evaluated CytoBead (Generic Assays, Germany) test, which contained both HEp-2 cell substrate and beads coated with DFS70 antigen in one well, in comparison to the routine two-step test strategy. Five hundred forty-one samples were studied by conventional IIF assay, LIA, and CytoBead assay; 264 samples were studied by ELISA. The Bead component of the CytoBead test was found to be reliable as a confirmational test when compared with ELISA and LIA (total agreement values were 85.6% and 87.6%, respectively). The CytoBead ANA DFS70 might be a promising test in the future, allowing both screening and confirmation in a single step, saving time and being easier than two-step testing.

Biological evaluation, docking studies, and in silico ADME prediction of some pyrimidine and pyridine derivatives as potential EGFRWT and EGFRT790M inhibitors

Herein, a set of pyridine and pyrimidine derivatives were assessed for their impact on the cell cycle and apoptosis. Human breast cancer (MCF7), hepatocellular carcinoma (HEPG2), larynx cancer (HEP2), lung cancer (H460), colon cancers (HCT116 and Caco2), and hypopharyngeal cancer (FADU), and normal Vero cell lines were used. Compounds 8 and 14 displayed outstanding effects on the investigated cell lines and were further tested for their antioxidant activity in MCF7, H460, FADU, HEP2, HEPG2, HCT116, Caco2, and Vero cells by measuring superoxide dismutase (SOD), malondialdehyde content (MDA), reduced glutathione (GSH), and nitric oxide (NO) content. Besides, Annexin V-FITC apoptosis detection and cell cycle DNA index using the HEPG-2 cell line were established on both compounds as well. Furthermore, compounds 8 and 14 were assessed for their EGFR kinase (Wild and T790M) inhibitory activities, revealing eligible potential. Additionally, molecular docking, ADME, and SAR studies were carried out for the investigated candidates.

Virulence Potential and Characteristics of Multidrug-Resistant Corynebacterium amycolatum Strains Isolated from Nosocomial Infections

The present study demonstrated Corynebacterium amycolatum as an emerging MDR nosocomial pathogen with emphasis on recognition of the class A β-lactamase encoding gene. Analysis of phenotypic and genotypic features of virulence mechanisms including C. elegans survival response to infection to C. amycolatum (imipenem - resistant and susceptible) strains were also investigated. Antimicrobial resistance genes were detected by polymerase chain reaction. We also evaluated the ability of Corynebacterium amycolatum in its ability to colonize and kill or host in a C. elegans infection model system and we investigated the adherence and invasion persistence of C. amycolatum within HEp-2 and U-937 cells. This study identified two C. amycolatum strains penicillin resistant and were positive for the blagene, encoding a class A betalactamase. The ability to C. amycolatum to kill the nematode C. elegans was verified. All strains of C. amycolatum caused a decline in the survival curve of C. elegans. The interaction of nematode and C. amycolatum results in morphological changes: Dar formation and “bag of worms” in all tested strains. About U-937 macrophages assay, the data indicate that the samples of Corynebacterium amycolatum studied had greater ability to adhere to the surface in the first 3 hours of infection of the monolayers (p <0.05). According to current knowledge, this is the first identification of C. amycolatumbla gene positive. So, we suggest that medical surveillance programs should include control strategies in order to decrease potential risk factors of nosocomial infections due to C. amycolatum.

Anti-Cancer and Anti-Inflammatory Activities of a Short Molecule, PS14 Derived from the Virulent Cellulose Binding Domain of Aphanomyces invadans, on Human Laryngeal Epithelial Cells and an In Vivo Zebrafish Embryo Model.

In this study, the anti-cancer and anti-inflammatory activities of PS14, a short peptide derived from the cellulase binding domain of pathogenic fungus, Aphanomyces invadans, have been evaluated, in vitro and in vivo. Bioinformatics analysis of PS14 revealed the physicochemical properties and the web-based predictions, which indicate that PS14 is non-toxic, and it has the potential to elicit anti-cancer and anti-inflammatory activities. These in silico results were experimentally validated through in vitro (L6 or Hep-2 cells) and in vivo (zebrafish embryo or larvae) models. Experimental results showed that PS14 is non-toxic in L6 cells and the zebrafish embryo, and it elicits an antitumor effect Hep-2 cells and zebrafish embryos. Anticancer activity assays, in terms of MTT, trypan blue and LDH assays, showed a dose-dependent inhibitory effect on cell proliferation. Moreover, in the epithelial cancer cells and zebrafish embryos, the peptide challenge (i) caused significant changes in the cytomorphology and induced apoptosis; (ii) triggered ROS generation; and (iii) showed a significant up-regulation of anti-cancer genes including BAX, Caspase 3, Caspase 9 and down-regulation of Bcl-2, in vitro. The anti-inflammatory activity of PS14 was observed in the cell-free in vitro assays for the inhibition of proteinase and lipoxygenase, and heat-induced hemolysis and hypotonicity-induced hemolysis. Together, this study has identified that PS14 has anti-cancer and anti-inflammatory activities, while being non-toxic, in vitro and in vivo. Future experiments can focus on the clinical or pharmacodynamics aspects of PS14.

Increasing Prevalence of Antinuclear Antibodies in the United States.

Growing evidence suggests increasing frequencies of autoimmunity and autoimmune diseases, but findings are limited by the lack of systematic data and evolving approaches and definitions. This study was undertaken to investigate whether the prevalence of antinuclear antibodies (ANA), the most common biomarker of autoimmunity, changed over a recent 25-year span in the US. Serum ANA were measured by standard indirect immunofluorescence assays on HEp-2 cells in 13,519 participants age ≥12 years from the National Health and Nutrition Examination Survey, with approximately one-third from each of 3 time periods: 1988-1991, 1999-2004, and 2011-2012. We used logistic regression adjusted for sex, age, race/ethnicity, and survey design variables to estimate changes in ANA prevalence across the time periods. The prevalence of ANA was 11.0% (95% confidence interval [95% CI] 9.7-12.6%) in 1988-1991, 11.4% (95% CI 10.2-12.8%) in 1999-2004, and 16.1% (95% CI 14.4-18.0%) in 2011-2012 (P for trend <0.0001), corresponding to ~22.3 million, ~26.6 million, and ~41.5 million affected individuals, respectively. Among adolescents age 12-19 years, ANA prevalence increased substantially, with odds ratios of 2.07 (95% CI 1.18-3.64) and 2.77 (95% CI 1.56-4.91) in the second and third time periods relative to the first (P for trend=0.0004). ANA prevalence increased in both sexes (especially in men), older adults (age ≥50 years), and non-Hispanic white individuals. These increases in ANA prevalence were not explained by concurrent trends in weight (obesity/overweight), smoking exposure, or alcohol consumption. The prevalence of ANA in the US has increased considerably in recent years. Additional studies to determine factors underlying these increases in ANA prevalence could elucidate causes of autoimmunity and enable the development of preventative measures.

Human Bone Marrow Mesenchymal Stem Cell (hBMSCs)-Derived miR-29a-3p-Containing Exosomes Impede Laryngocarcinoma Cell Malignant Phenotypes by Inhibiting PTEN.

Although microRNA-29a-3p was reported to inhibit laryngocarcinoma progression, the potential mechanisms have not been explored clearly. Laryngocarcinoma tissues were collected for analyzing the levels of miR-29a-3p and phosphatase and tensin homolog (PTEN). The miR mimics or inhibitor was transfected into laryngocarcinoma cell lines M4E and Hep2 for the investigation of the biological functions (proliferative, invasion, migratory rates, and apoptotic rates) of this miRNA. The exosomes (Exo) from human bone marrow mesenchymal stem cells (hBMSCs) after the transfection of miR mimics/inhibitor/si-PTEN were isolated and used to stimulate M4E and Hep2 cells. The in vivo mouse model was constructed to verify our findings. The miR-29a-3p level was decreased, and PTEN level was elevated in laryngocarcinoma tissues and the cancer cell lines. MiR mimics could inhibit proliferative, invasive migratory rates while promoting apoptotic rates of M4E and Hep2 cells. MiR-29a-3p was enriched in hBMSC-derived Exo, and the Exo from miR-29a-3p mimics transfected hBMSCs could inhibit laryngocarcinoma cell malignant phenotypes in vitro and prevent tumor progression in vivo. In addition, the direct binding relationship between miR-29a-3p and PTEN in this disease was determined. In conclusion, hBMSC-derived Exo with upregulated miR-29a-3p inhibited laryngocarcinoma progression via regulating PTEN, providing a potential diagnostic and therapeutic target in this disease.

A retrospective study of prevalence and pattern of international consensus on ANA patterns among patients with hepatitis C virus infection.

BackgroundA previous study reported a 30% prevalence of various autoantibodies among patients with hepatitis C virus (HCV) infection. The International Consensus on Anti-Nuclear Antibody (ANA) Patterns was recently introduced to classify ANA patterns based on immunoassay on HEp-2 cells. There is no previous report with this newly developed classification to evaluate patients with HCV infection. The study aims to study the prevalence and pattern of ANA patterns among HCV-infected patients.MethodsWe retrospectively analyzed the medical records of patients with HCV infection from September 2020 to June 2021 at our institution. A positive ANA is defined as a titer of more than 1:320. We compared patient features among the positive and negative groups.ResultsOverall, 258 patients were enrolled—184 patients with negative ANA and 74 patients (28.7%) with positive ANA. The mean age was 67.3 in ANA positive group and 61.2 ANA negative group. Female was prominent with ANA positive and accounted for 63.5%. The most detected ANA pattern was AC-1(homogeneous) (25.9%), followed by AC-4(fine speckled) (25.2%) and AC-21(anti-mitochondrial antibody) (9.6%). In ANA positive group, we found a trend of lower HCV viral load (5.72 log10 IU/ML vs. 6.02 log10 IU/ML), lower alanine aminotransferase level (39.5 U/L vs. 44 U/L), and higher advanced fibrosis (F3 and F4) (38.5% vs. 26.1%). In addition, higher positive ANA (more than 1:640) is significantly associated with lower estimated glomerular filtration rate (eGFR) (77.76 vs. 87.94 mL/min/1.73 m2, P = 0.044).ConclusionsA high prevalence (28.7%) of ANA was found in patients with chronic hepatitis C. The presence of positive ANA is not related to the severity of their hepatic manifestation. However, higher positive ANA was significantly associated with lower eGFR.

Effect of Coated Silver Nanoparticles on Cancerous vs. Healthy Cells.

Unique properties of silver nanoparticles (NPs) ensure their wide applications, in biomedicine; for this reason, it is very important carefully to study the toxicity of such NPs. The influence of silver nanoparticles coated with natural resin (Ag NPs) on the morphological and functional features of healthy BHK-21 and cancerous Hep-2 cells were studied using fluorescence microscopy, MTT, and neutral red assays. Ag NPs induced morphological changes in both cell cultures. The modifications were dose-dependent and more pronounced with an increase in NPs concentration. The IC50 value of Ag NPs for Hep-2 cells was found to be 2.19 ± 0.22 µg/mL, whereas for BHK-21 cells it was significantly (5x) higher at 10.92 ± 2.48 µg/mL. The use of NPs at a concentration close to IC50 leads to significant increase (up to 40%) in the number of necrotic cells in cancerous cell population and a decrease in the number of mitotic cells (up to 1.3%). In noncancerous cells the cellular parameters were similar to the control cells. These data suggest that the silver nanoparticles coated with natural resin can be potentially used in cancer therapy.

Construction of Streptococcus agalactiae sialic acid mutant and evaluation of its potential as a live attenuated vaccine in Nile tilapia (Oreochromis niloticus).

This study aimed to develop a live attenuated vaccine as an effective approach to prevent streptococcosis in tilapia (Oreochromis niloticus). We eliminated the virulence factor, sialic acid (Sia) encoded by the neuA-D gene cluster from the Group B Streptococcus (Streptococcus agalactiae, GBS) strain WC1535, to construct Sia-deficient S. agalactiae (ΔSia) mutant by homologous recombination. Results showed that the ΔSia mutant had higher adherence to HEp-2 cells and lower resistance to RAW264.7 cell phagocytosis than the wild-type S. agalactiae. The virulence of the ΔSia mutant to tilapia dramatically decreased with no virulence recovery. The relative percent survivals (RPSs) were 50.00% and 54.50% at 30 days when challenged at the wild-type WC1535 doses of 1.0 × 107 and 5.0 × 107 CFU fish-1 , respectively, via intraperitoneal (IP) injection. The tilapia vaccinated via IP injection with the ΔSia mutant induced strong antibody agglutination titers. The expression of IL-1β, TNF-α, MHC-Iα, and MHC-IIβ could be enhanced in the intestine, spleen, and head kidney for tilapia administered with the ΔSia mutant. GBS Sia plays a critical role in adherence to HEp-2 cells and resistance to the immune clearance of RAW264.7 cells. Moreover, the ΔSia mutant is a safe, stable, and immunogenic live attenuated vaccine candidate to protect tilapia against GBS infection. The results offer more evidence of the importance of Sia in GBS and may be instructive in the control of tilapia streptococcosis.

Epidemiology of Human Adenovirus in Pakistani Children Hospitalized with Community-Acquired Gastroenteritis under the Age of Five Years.

Acute gastroenteritis is the major cause of morbidity and mortality among infants and children around the globe. Along with other enteropathogens, human adenovirus (HadV) is a major etiological agent associated with diarrhea in young children. However, information about the epidemiology of Adenoviruses in Pakistan is limited or has not been reported. A total of 1082 stool samples were collected from patients with acute gastroenteritis under the age of five years with symptoms of diarrhea, vomiting, nausea, and abdominal cramps who visited Benazir Bhutto Hospital Rawalpindi and Children’s hospital in Lahore of Punjab Province in Pakistan. Of this, 384 cases with no blood in their stool, negative for Rotavirus, and under the age of five years were recruited in this study. Human Adenoviruses were isolated in the human epithelial HEp-2 cell line. Furthermore, adenovirus antigen detection was carried out by an enzyme-linked immunosorbent assay (ELISA), and then all positive and negative samples were confirmed by nested PCR. After inoculating a clear stool supernatant on HEp-2 cell lines, we observed a positive cytopathic effect in 65 (16%) cases. Using an enzyme-linked immunosorbent assay, HAdV antigens were detected in 54 (14.06%) of the clear supernatant from gastroenteritis cases. However, HAdV hexon coding regions were amplified in 57 (14.80%) fecal samples, mainly from patients ≤24 months of age. The findings of this study suggest that adenovirus circulates significantly in the children population under the age of five years and may be the potential etiological factor of acute gastroenteritis in the mentioned cities. This study provides baseline data about the possible role of adenovirus in causing viral diarrhea in children. Further large-scale epidemiological surveys are recommended to better understand disease burden, etiological agents, and its clinical impact across the country.

A cell-based assay for detection of anti-fibrillarin autoantibodies with performance equivalent to immunoprecipitation.

Anti-fibrillarin autoantibodies are useful for the diagnosis and prognosis of systemic sclerosis (SSc). Anti-fibrillarin produces a clumpy nucleolar pattern in indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA). Here we develop and validate a reliable cell-based anti-fibrillarin assay (Fibrillarin/CBA) for use in clinical diagnostic laboratories. A TransMembrane Signal was fused to the human fibrillarin gene (TMS-fibrillarin). HEp-2 cells overexpressing transgenic TMS-fibrillarin at the cytoplasmic membrane were used as IFA substrate in the Fibrillarin/CBA. Sixty-two serum samples with nucleolar pattern in the HEp-2 IFA (41 clumpy; 21 homogeneous/punctate) were tested for anti-fibrillarin using Fibrillarin/CBA, immunoprecipitation (IP), line-blot and ELISA. In addition, samples from 106 SSc-patients were evaluated with Fibrillarin/CBA and the results were correlated with disease phenotypes. Thirty-eight of 41 samples with the clumpy nucleolar pattern (92.7%) were positive in the Fibrillarin/CBA, while all 21 samples with other nucleolar patterns were negative. Fibrillarin/CBA results agreed 100% with IP results. Among the 38 Fibrillarin/CBA-positive samples, only 15 (39.5%) and 11 (29%) were positive for anti-fibrillarin in line-blot and ELISA, respectively. Higher frequency of diffuse cutaneous SSc (dcSSc) phenotype (72.7% vs 36.8%; p=0.022), cardiac involvement (36.4% vs 6.5%; p=0.001) and scleroderma renal crisis (18.2% vs 3.3% p = 0.028) was observed in SSc patients with positive compared to negative Fibrillarin/CBA result. Performance of Fibrillarin/CBA in the detection of anti-fibrillarin autoantibodies was comparable to the gold standard IP. Positive Fibrillarin/CBA results correlated with disease phenotypes known to be associated with anti-fibrillarin autoantibodies, underscoring the clinical validation of this novel assay.

P37 A rare cause of autoimmune hepatitis secondary to a biological therapy

P37 A rare cause of autoimmune hepatitis secondary to a biological therapy

High prevalence of antinuclear antibodies in patients with chronic hepatitis C virus infection

BackgroundHepatitis C virus (HCV) infection is a serious public health concern due to its high prevalence and mortality rate. In chronic infection, HCV may induce autoimmune responses through the production of autoantibodies, including antinuclear antibodies (ANA).MethodsWe assessed the presence of ANA by indirect immunofluorescence using HEp-2 cells in 89 patients with chronic hepatitis C. We also collected data on epidemiological variables; clinical characteristics; and biochemical, hematological, molecular, and histopathological information from the patients to assess the impact of the presence of ANA in those patients.ResultsThe prevalence of ANA in the patients was 20.2%, which was significantly higher than that found in healthy controls (2%). However, there was no association of this marker with epidemiological, clinical-laboratory, molecular or histopathological characteristics of hepatitis C, although a slightly higher prevalence of ANA was detected in women and in patients infected with subgenotype 1a. In a specific analysis, chronic HCV patients with the “rods and rings” cytoplasmic pattern had higher degrees of hepatic fibrosis than did ANA-negative patients.ConclusionsThe results confirm a greater predisposition to the presence of ANA in patients with HCV, which may be associated with a worse prognosis, especially in the presence of the “rods and rings” cytoplasmic pattern.

Biocompatibility and Surface Roughness of Different Sustainable Dental Composite Blocks: Comprehensive In Vitro Study.

The study purposed to investigate the biocompatibility and sustainability of two computer-aided design/computer-aided manufacturing (CAD/CAM) resin-based composites compared to a resin-modified ceramic in terms of surface roughness, biofilm formation, cytotoxicity, genotoxicity, and cellular changes observed under transmission electron microscopy (TEM). Three CAD/CAM blocks were used, two resin-based composites [Brilliant Crios (BC) and Cerasmart, (CS) and one hybrid ceramic (Vita Enamic (EN)]. Each block was sectioned into 10 × 12 × 2 mm specimens, followed by finishing and polishing. Each specimen was evaluated for surface roughness using 3D optical profilometry and scanned by scanning electron microscopy. Biofilm formation and its relation to surface roughness have been investigated for all tested materials. A Hep-2 cell line was used to investigate the viability through MTT assay. The cytotoxicity of the materials was measured at 24, 48, and 168 h. The activity of P53, caspase 3, and cytochrome C was evaluated to detect the genotoxicity of different groups, followed by TEM tracking of the cellular changes. Statistical analysis was implemented by utilizing a one-way analysis of variance test. The significance was set at P ≤ 0.05. With regard to the surface roughness, no statistically significant differences were shown between groups. BC possessed the highest biofilm formation value, followed by EN and CS, with no significance between them. No correlation between surface roughness of tested materials and biofilm formation was shown. Considering viability, the highest values were recorded for EN, whereas BC showed the lowest values. P53-fold changes in EN were significantly the lowest, indicating less genotoxicity. Within the current study’s limitations, BC showed the highest biofilm formation. However, no significant surface roughness difference or correlation with biofilm formation was observed in tested materials. EN showed the lowest cytotoxicity and the highest viability. EN revealed the best compatibility performance among tested materials. On the contrary, the BC exhibited fewer preferences.

Phenotypic impacts and genetic regulation characteristics of the DNA adenine methylase gene (dam) in Salmonella Typhimurium biofilm forms

In this study, transcriptional level gene expression changes in biofilm forms of Salmonella Typhimurium ATCC 14028 and its dam mutant were investigated by performing RNAseq analysis. As a result of these analyzes, a total of 233 differentially expressed genes (DEGs) were identified in the dam mutant, of which 145 genes were downregulated and 88 genes were upregulated compared to the wild type. According to data from miRNA sequence analysis, of 13 miRNAs differentially expressed in dam mutant, 9 miRNAs were downregulated and 4 miRNAs were upregulated. These data provide the first evidence that the dam gene is a global regulator of biofilm formation in Salmonella. In addition, phenotypic analyses revealed that bacterial swimming and swarming motility and cellulose production were highly inhibited in the dam mutant. It was determined that bacterial adhesion in Caco-2 and HEp-2 cell lines was significantly reduced in dam mutant. At the end of 90 min, the adhesion rate of wild type strain was 43.3% in Caco-2 cell line, while this rate was 14.9% in dam mutant. In the HEp-2 cell line, while 45.5% adherence was observed in the wild-type strain, this rate decreased to 15.3% in the dam mutant.

HEp-2 image classification using a multi-class and multiple-binary classifier.

In medicine, identifying the indirect immunofluorescence of human epithelial type 2 (HEp-2) cells plays a decisive role in the diagnosis of autoimmune diseases. The manual interpretation of Hep-2 cell images may lead to some limitations, such as subjectivity, inconsistency and low efficiency. Therefore, it is very important to automatically identify HEp-2 images. Inspired by the outstanding performance of neural networks in image classification tasks, we propose a multi-class and multiple-binary classifier (MCMBC) for the classification of HEp-2 cells. MCMBC is an ensemble learner that contains two kinds of sub-classifiers: multi-class (MC) and multiple-binary (MB). The MC sub-classifier adopts a multi-scale convolutional neural network (MSCNN) that increases the efficiency of information transmission between layers. On the basis of classification results of the MC sub-classifier on validation sets, we can find easy-to-confuse class pairs. An easy-to-confuse class pair is two classes that are not easy to be identified from each other. The MB sub-classifiers adopt multiple-binary pre-trained VGG16 networks that are used to deal with these class pairs. The final prediction for a sample possibly belonging to an easy-to-confuse class is decided by the assembled features extracted from the last fully connected layer of MC and the output of MB sub-classifiers. To evaluate the proposed model, experiments were conducted on the ICPR 2014 Task-2 dataset. Experimental results show that MCMBC performs better than the state-of-the-art method (84.68% vs. 83.35% on the criterion of average classification accuracy (ACA) and 82.89% vs. 82.67% on the criterion of mean classification accuracy (MCA)).

Oxaliplatin Induces Immunogenic Cell Death in Human and Murine Laryngeal Cancer

Background Cisplatin resistance is observed in patients with laryngeal cancer. The present study was designed to explore the efficacy of oxaliplatin on laryngeal cancer and elucidate the underlying mechanisms. Methods Cell viability was determined by using MTT assays. Cell apoptosis was determined by using annexin V and propidium iodide (PI) staining. Flow cytometry and immunofluorescence were applied to determine the levels of calreticulin (CALR) and DiD (1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine). Flow cytometry was applied to analyze the levels of CD83, CD86, IFN-γ-producing CD8+ T cells, and CD4+CD25+FoxP3+ Tregs. The levels of adenosine triphosphate (ATP) were determined by using a chemiluminescent ATP kit and cytokines were determined by using specific enzyme-linked immunosorbent assays (ELISAs). The levels of HMGB1 were determined by using Western blot and ELISA, respectively. The xenograft animal model was constructed to evaluate the antitumor effects of oxaliplatin. Results Oxaliplatin inhibited cell growth, promoted cell apoptosis, and induced the levels of CALR, ATP, and high mobility group box protein 1 (HMGB1) in Hep-2 cells. Oxaliplatin-treated Hep-2 cells increased the intensity of DiD and the levels of CD83 and CD86 in dendritic cells (DCs), as well as induced the supernatant IL-6 and TNF-α. Oxaliplatin-treated primary laryngeal cancer cell-pulsed DCs increased the IFN-γ-producing CD8+ T cells and suppressed CD4+CD25+FoxP3+ Tregs. In vivo data showed that oxaliplatin suppressed tumor growth and increased the populations of CD86+CD80+ and CD8+CD45+ cells in the tumor tissues. Conclusion Treatment with oxaliplatin inhibited laryngeal cancer cells by inducing immunogenic cell death.

VI Brazilian consensus guidelines for detection of anti-cell autoantibodies on HEp-2 cells

BackgroundThe VI Brazilian Consensus on Autoantibodies against HEp-2 cells for determination of autoantibodies against cellular constituents on HEp-2 cells was held on September, 2019, in Fortaleza (CE, Brazil). The guidelines in this edition were formulated by the group of Brazilian experts discussing the classification of complex patterns, the classification of the nuclear discrete dots (few and multiple), the identification of the discrete fine speckled pattern (AC-4a) and improvements on the ANA report.MainbodySixteen Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of Brazil participated in the meeting. Four main topics were discussed: (1) How to classify patterns with fluorescence in more than one cell compartment considering three relevant categoris: composite patterns, mixed patterns and multiple patterns; (2) The splitting of the discrete nuclear dots pattern into the multiple discrete nuclear dots (AC-6) and few discrete nuclear dots (AC-7) patterns, respectively; (3) Inclusion of a novel nuclear pattern characterized by discrete fine speckled pattern highly associated with antibodies to SS-A/Ro60, classified as AC-4a. In addition, adjustments on the Brazilian Consensus nomenclature were implemented aiming to harmonize the designation of some patterns with the International Consensus on ANA Patterns (ICAP). Furthermore, the designations of the PCNA-like pattern (AC-13), CENP-F-like pattern (AC-14) and Topo I-like pattern (AC-29) were adjusted in accordance to ICAP. Finally, there was a recommendation for adjustment in the test report in order to address the status of nuclear envelope staining. For all topics, the aim was to establish specific guidelines for laboratories and clinicians. All recommendations were based on consensus among participants. All recommendations from the V Consensus were maintained and there was relevant progress in the BCA/HEp-2 guidelines and further harmonization with ICAP.ConclusionThe VI BCA/HEp-2 edition was successful in establishing important recommendations regarding the classification of complex patterns, in supporting the identification of a novel pattern within the AC-4 group and in the harmonization process with the ICAP terminology.

Assessment of Pharmacological Potential of Novel Exopolysaccharide Isolated from Marine Kocuria sp. Strain AG5: Broad-Spectrum Biological Investigations.

With more than 17 clinically approved Drugs and over 20 prodrugs under clinical investigations, marine bacteria are believed to have a potential supply of innovative therapeutic bioactive compounds. In the current study, Kocuria sp. strain AG5 isolated from the Red Sea was identified and characterized by biochemical and physiological analysis, and examination of a phylogenetic 16S rRNA sequences. Innovative exopolysaccharide (EPS) was separated from the AG5 isolate as a major fraction of EPS (EPSR5, 6.84 g/L−1). The analysis of EPSR5 revealed that EPSR5 has a molecular weight (Mw) of 4.9 × 104 g/mol and number average molecular weight (Mn) of 5.4 × 104 g/mol and contains sulfate (25.6%) and uronic acid (21.77%). Analysis of the monosaccharide composition indicated that the EPSR5 fraction composes of glucose, galacturonic acid, arabinose, and xylose in a molar ratio of 2.0:0.5:0.25:1.0, respectively. Assessment of the pharmacological potency of EPSR5 was explored by examining its cytotoxicity, anti-inflammatory, antioxidant, and anti-acetylcholine esterase influences. The antioxidant effect of EPSR5 was dose- and time-dependently increased and the maximum antioxidant activity (98%) was observed at 2000 µg/mL after 120 min. Further, EPSR5 displayed a significant repressive effect regarding the proliferation of HepG-2, A-549, HCT-116, MCF7, HEP2, and PC3 cells with IC50 453.46 ± 21.8 µg/mL, 873.74 ± 15.4 µg/mL, 788.2 ± 32.6 µg/mL, 1691 ± 44.2 µg/mL, 913.1 ± 38.8 µg/mL, and 876.4 ± 39.8 µg/mL, respectively. Evaluation of the inhibitory activity of the anti-inflammatory activity of EPSR5 indicated that EPSR5 has a significant inhibitory activity toward lipoxygenase (5-LOX) and cyclooxygenase (COX-2) activities (IC50 15.39 ± 0.82 µg/mL and 28.06 ± 1.1 µg/mL, respectively). Finally, ESPR5 presented a substantial hemolysis suppressive action with an IC50 of 65.13 ± 0.89 µg /mL, and a considerable inhibitory activity toward acetylcholine esterase activity (IC50 797.02 μg/mL). Together, this study reveals that secondary metabolites produced by Kocuria sp. strain AG5 marine bacteria serve as an important source of pharmacologically active compounds, and their impact on human health is expected to grow with additional global work and research.

Norsquaraine endowed with anticancer and antibacterial activities

BackgroundPhotodynamic therapy (PDT) is a method for the treatment of cancer. Furthermore, PDT can also be used for the eradication of bacteria. The photo-sensitizing drug, a.k.a photosensitizer, is critical for the success of PDT. Although norsquaraines are analogs of squaraine dyes, they are overlooked as photosensitizers. MethodsIn this work, synthesis, characterization, bioimaging and in vitro PDT applications of a new norsquaraine dye 1 were described. We also prepared nanoparticles from norsquaraine 1 and Pluronic F127 to obtain 1@F127. ResultsNorsquaraine 1 boosted the generation of reactive oxygen species over a wide range of pH (pH 8.0, 7.0, 6.0, and 2.2.). Furthermore, 1 was internalized by epidermoid laryngeal carcinoma Hep-2 (Hep-2) cells and used for fluorescence imaging. Remarkably, norsquaraine 1 destroyed most of the cancer cells (ca. 77% to 89%) after illumination with red light. Most strikingly, 1 successfully inhibited the growth of Methicillin-resistant Staphylococcus aureus (MRSA) upon illumination. Last but not least, photodynamic sterilization of tomato juice, an acidic beverage, was feasible using 1 as a photo sterilizer. Nano formulation of 1 with Pluronic F127 provided 1@F127 nanoparticles. It is lucid that 1@F127 nanoparticles permeate into Hep-2 cells and boost the generation of ROS upon illumination. ConclusionNorsquaraine 1 shows superior features as a photosensitizer pertinent to PDT in a wide range of pH. This norsquaraine is endowed with anticancer and antibacterial activities. Which should be further evaluated.