Mathematical models disentangle the role of IL-10 feedbacks in human monocytes upon proinflammatory activation.

Inflammation is one of the vital mechanisms through which the immune system responds to harmful stimuli. During inflammation, proinflammatory and anti-inflammatory cytokines interplay to orchestrate fine-tuned and dynamic immune responses. The cytokine interplay governs switches in the inflammatory response and dictates the propagation and development of the inflammatory response. Molecular pathways underlying the interplay are complex, and time-resolved monitoring of mediators and cytokines is necessary as a basis to study them in detail. Our understanding can be advanced by mathematical models that enable to analyze the system of interactions and their dynamical interplay in detail. We, therefore, used a mathematical modeling approach to study the interplay between prominent proinflammatory and anti-inflammatory cytokines with a focus on tumor necrosis factor and interleukin 10 (IL-10) in lipopolysaccharide-primed primary human monocytes. Relevant time-resolved data were generated by experimentally adding or blocking IL-10 at different time points. The model was successfully trained and could predict independent validation data and was further used to perform simulations to disentangle the role of IL-10 feedbacks during an acute inflammatory event. We used the insight to obtain a reduced predictive model including only the necessary IL-10-mediated feedbacks. Finally, the validated reduced model was used to predict early IL-10-tumor necrosis factor switches in the inflammatory response. Overall, we gained detailed insights into fine-tuning of inflammatory responses in human monocytes and present a model for further use in studying the complex and dynamic process of cytokine-regulated acute inflammation.

Therapeutic plasma exchange for the treatment of refractory necrotizing autoimmune myopathy.

INTRODUCTION: Necrotizing autoimmune myopathy (NAM) is strongly associated with pathognomonic autoantibodies targeting 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) or signal recognition particle (SRP), whose levels in turn are correlated with serum creatine kinase (CK) and necrosis. Thus, NAM may be amenable to therapeutic plasma exchange (TPE) to remove pathogenic antibodies and improve patient symptoms. METHODS: A retrospective case series and literature review of patients presenting with NAM and undergoing treatment with TPE was performed. Clinical data including patient demographics, symptoms, physical exam findings, muscle biopsy, lower extremity imaging, prior therapy, and duration from diagnosis to TPE initiation were collected retrospectively for adult patients with NAM treated with TPE after failing to respond to immunomodulatory therapy. Laboratory data including change in CK levels and myositis-specific antibody titers from baseline were measured in some patients. RESULTS: Six patients (median age at diagnosis 52.5 years, interquartile range [IQR] 35.8-64.5 years, four male/two female) underwent a median of 7.5 (IQR: 5-10) TPE procedures with 5% albumin as replacement. All patients exhibited a statistically significant reduction in CK level from pre-TPE baseline (range: 43.0%-58.7% reduction). Responses in this cohort were best in patients with antibodies targeting HMGCR and SRP, which are most strongly associated with NAM. These results compare favorably to a literature review of NAM patients (n = 19) treated with TPE, who also exhibited positive clinical and laboratory responses across varying treatment lengths. CONCLUSION: TPE can play a role in the management of NAM, particularly in patients with HMGCR or SRP antibodies who are refractory to pharmacologic immunosuppression.

A rapid, point-of-care red blood cell agglutination assay detecting antibodies against SARS-CoV-2.

The COVID-19 pandemic has caused significant morbidity and mortality. There is an urgent need for serological tests to detect antibodies against SARS-CoV-2, which could be used to assess past infection, evaluate responses to vaccines in development, and determine individuals who may be protected from future infection. Current serological tests developed for SARS-CoV-2 rely on traditional technologies such as enzyme-linked immunosorbent assays (ELISA) and lateral flow assays, which have not scaled to meet the demand of hundreds of millions of antibody tests so far. Herein, we present an alternative method of antibody testing that depends on one protein reagent being added to patient serum/plasma or whole blood with direct, visual readout. Two novel fusion proteins, RBD-2E8 and B6-CH1-RBD, were designed to bind red blood cells (RBCs) via a single-chain variable fragment (scFv), thereby displaying the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on the surface of RBCs. Mixing mammalian-derived RBD-2E8 and B6-CH1-RBD with convalescent COVID-19 patient serum and RBCs led to visible hemagglutination, indicating the presence of antibodies against SARS-CoV-2 RBD. B6-CH1-RBD made in bacteria was not as effective in inducing agglutination, indicating better recognition of RBD epitopes from mammalian cells. Given that our hemagglutination test uses methods routinely used in hospital clinical labs across the world for blood typing, we anticipate the test can be rapidly deployed at minimal cost. We anticipate our hemagglutination assay may find extensive use in low-resource settings for detecting SARS-CoV-2 antibodies.

A Hemagglutination-Based Semiquantitative Test for Point-of-Care Determination of SARS-CoV-2 Antibody Levels.

Serologic point-of-care tests to detect antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are an important tool in the COVID-19 pandemic. The majority of current point-of-care antibody tests developed for SARS-CoV-2 rely on lateral flow assays, but these do not offer quantitative information. To address this, we developed a novel antibody test leveraging hemagglutination, employing a dry card format currently used for typing ABO blood groups. Two hundred COVID-19 patient and 200 control plasma samples were reconstituted with O-negative red blood cells (RBCs) to form whole blood and added to dried viral-antibody fusion protein, followed by a stirring step and a tilting step, 3-min incubation, and a second tilting step. The sensitivities of the hemagglutination test, Euroimmun IgG enzyme-linked immunosorbent assay (ELISA), and receptor binding domain (RBD)-based CoronaChek lateral flow assay were 87.0%, 86.5%, and 84.5%, respectively, using samples obtained from recovered COVID-19 individuals. Testing prepandemic samples, the hemagglutination test had a specificity of 95.5%, compared to 97.3% and 98.9% for the ELISA and CoronaChek, respectively. A distribution of agglutination strengths was observed in COVID-19 convalescent-phase plasma samples, with the highest agglutination score (4) exhibiting significantly higher neutralizing antibody titers than weak positives (2) (P < 0.0001). Strong agglutinations were observed within 1 min of testing, and this shorter assay time also increased specificity to 98.5%. In conclusion, we developed a novel rapid, point-of-care RBC agglutination test for the detection of SARS-CoV-2 antibodies that can yield semiquantitative information on neutralizing antibody titer in patients. The 5-min test may find use in determination of serostatus prior to vaccination, postvaccination surveillance, and travel screening.

Endoscopic-mediated, biliary hydrodynamic injection mediating clinically relevant levels of gene delivery in pig liver.

BACKGROUND AND AIMS: Gene therapy could provide curative therapies to many inherited monogenic liver diseases. Clinical trials have largely focused on adeno-associated viruses (AAVs) for liver gene delivery. These vectors, however, are limited by small packaging size, capsid immune responses, and inability to redose. As an alternative, nonviral, hydrodynamic injection through vascular routes can successfully deliver plasmid DNA (pDNA) into mouse liver but has achieved limited success in large animal models. METHODS: We explored hydrodynamic delivery of pDNA through the biliary system into the liver of pigs using ERCP and a power injector to supply hydrodynamic force. Human factor IX (hFIX), deficient in hemophilia B, was used as a model gene therapy. RESULTS: Biliary hydrodynamic injection was well tolerated without significant changes in vital signs, liver enzymes, hematology, or histology. No off-target pDNA delivery to other organs was detected by polymerase chain reaction. Immunohistochemistry revealed that 50.19% of the liver stained positive for hFIX after hydrodynamic injection at 5.5 mg pDNA, with every hepatic lobule in all liver lobes demonstrating hFIX expression. hFIX-positive hepatocytes were concentrated around the central vein, radiating outward across all 3 metabolic zones. Biliary hydrodynamic injection in pigs resulted in significantly higher transfection efficiency than mouse vascular hydrodynamic injection at matched pDNA per liver weight dose (32.7%-51.9% vs 18.9%, P < .0001). CONCLUSIONS: Biliary hydrodynamic injection using ERCP can achieve higher transfection efficiency into hepatocytes compared with AAVs at magnitudes of less cost in a clinically relevant human-sized large animal. This technology may serve as a platform for gene therapy of human liver diseases.

Isohemagglutinin titering performed on an automated solid-phase and hemagglutinin-based analyzer is comparable to results obtained by manual gel testing.

BACKGROUND: Isohemagglutinins (anti-A and anti-B) mediate hemolytic transfusion reactions, antibody-mediated rejection of solid-organ transplants, and delayed engraftment after stem cell transplant. However, quantification of isohemagglutinins is often labor intensive and operator dependent, limiting availability and interfacility comparisons. We evaluated an automated, solid-phase and agglutination-based antibody titer platform versus manual gel testing. STUDY DESIGN AND METHODS: Plasma samples were obtained from 54 randomly selected patients. Titers were determined by our laboratory's standard assay (manual dilution followed by manual gel testing) and were compared to results obtained on a fully automated blood bank analyzer (Galileo NEO, Immucor). The analyzer determined immunoglobulin G (IgG) antibodies using solid-phase and immunoglobulin M (IgM) antibodies by direct hemagglutination. RESULTS: Isohemagglutinin titers obtained by manual gel versus the automated assay generally (>80%) agreed within one doubling dilution, and always (100%) agreed within two dilutions. Among O samples, the gel titer and the highest titer obtained with the automated assay (either IgG or IgM) were similar in paired, nonparametric analysis (p = 0.06 for anti-A; p = 0.13 for anti-B). Gel titers from group A and group B patients were slightly higher than the highest titer obtained using the automated assay (p = 0.04 for group A; p = 0.009 for group B), although these differences were within the accepted error of measurement. CONCLUSION: Manual and automated methodologies yielded similar isohemagglutinin titers. Separate quantification of IgM and IgG isohemagglutinins via automated titration may yield additional insight into hemolysis, graft survival after ABO-incompatible transplantation, and red blood cell engraftment after ABO-incompatible stem cell transplant.

HBsAg-redirected T cells exhibit antiviral activity in HBV-infected human liver chimeric mice.

BACKGROUND: Chronic hepatitis B virus (HBV) infection remains incurable. Although HBsAg-specific chimeric antigen receptor (HBsAg-CAR) T cells have been generated, they have not been tested in animal models with authentic HBV infection. METHODS: We generated a novel CAR targeting HBsAg and evaluated its ability to recognize HBV+ cell lines and HBsAg particles in vitro. In vivo, we tested whether human HBsAg-CAR T cells would have efficacy against HBV-infected hepatocytes in human liver chimeric mice. RESULTS: HBsAg-CAR T cells recognized HBV-positive cell lines and HBsAg particles in vitro as judged by cytokine production. However, HBsAg-CAR T cells did not kill HBV-positive cell lines in cytotoxicity assays. Adoptive transfer of HBsAg-CAR T cells into HBV-infected humanized mice resulted in accumulation within the liver and a significant decrease in plasma HBsAg and HBV-DNA levels compared with control mice. Notably, the fraction of HBV core-positive hepatocytes among total human hepatocytes was greatly reduced after HBsAg-CAR T cell treatment, pointing to noncytopathic viral clearance. In agreement, changes in surrogate human plasma albumin levels were not significantly different between treatment and control groups. CONCLUSIONS: HBsAg-CAR T cells have anti-HBV activity in an authentic preclinical HBV infection model. Our results warrant further preclinical exploration of HBsAg-CAR T cells as immunotherapy for HBV.

In vivo reduction of hepatitis B virus antigenemia and viremia by antisense oligonucleotides.

BACKGROUND & AIMS: Current treatment of chronic hepatitis B virus infection (CHB) includes interferon and nucleos(t)ide analogues, which generally do not reduce HBV surface antigen (HBsAg) production, a constellation that is associated with poor prognosis of CHB. Here we evaluated the efficacy of an antisense approach using antisense oligonucleotide (ASO) technology already in clinical use for liver targeted therapy to specifically inhibit HBsAg production and viremia in a preclinical setting. METHODS: A lead ASO was identified and characterized in vitro and subsequently tested for efficacy in vivo and in vitro using HBV transgenic and hydrodynamic transfection mouse and a cell culture HBV infection model, respectively. RESULTS: ASO treatment decreased serum HBsAg levels ⩾2 logs in a dose and time-dependent manner; HBsAg decreased 2 logs in a week and returned to baseline 4 weeks after a single ASO injection. ASO treatment effectively reduced HBsAg in combination with entecavir, while the nucleoside analogue alone did not. ASO treatment has pan-genotypic antiviral activity in the hydrodynamic transfection system. Finally, cccDNA-driven HBV gene expression is ASO sensitive in HBV infected cells in vitro. CONCLUSION: Our results demonstrate in a preclinical setting the efficacy of an antisense approach against HBV by efficiently reducing serum HBsAg (as well as viremia) across different genotypes alone or in combination with standard nucleoside therapy. Since the applied antisense technology is already in clinical use, a lead compound can be rapidly validated in a clinical setting and thus, constitutes a novel therapeutic approach targeting chronic HBV infection.

Carbon monoxide releasing molecule-2 (CORM-2) inhibits growth of multidrug-resistant uropathogenic Escherichia coli in biofilm and following host cell colonization.

BACKGROUND: Increased resistance to antimicrobial agents is a characteristic of many bacteria growing in biofilms on for example indwelling urinary catheters or in intracellular bacterial reservoirs. Biofilm-related infections caused by multidrug-resistant bacteria, such as extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae, are a major challenge. The aim of this study was to investigate if a carbon monoxide-releasing molecule (CORM-2) has antibacterial effects against ESBL-producing uropathogenic E. coli (UPEC) in the biofilm mode of growth and following colonization of host bladder epithelial cells. RESULTS: The effect of CORM-2 was examined on bacteria grown within an established biofilm (biofilm formed for 24 h on plastic surface) by a live/dead viability staining assay. CORM-2 (500 µM) exposure for 24 h killed approximately 60 % of the ESBL-producing UPEC isolate. A non-ESBL-producing UPEC isolate and the E. coli K-12 strain TG1 were also sensitive to CORM-2 exposure when grown in biofilms. The antibacterial effect of CORM-2 on planktonic bacteria was reduced and delayed in the stationary growth phase compared to the exponential growth phase. In human bladder epithelial cell colonization experiments, CORM-2 exposure for 4 h significantly reduced the bacterial counts of an ESBL-producing UPEC isolate. CONCLUSION: This study shows that CORM-2 has antibacterial properties against multidrug-resistant UPEC under biofilm-like conditions and following host cell colonization, which motivate further studies of its therapeutic potential.

Clinical outcomes of hepatitis B virus coinfection in a United States cohort of hepatitis C virus-infected patients.

UNLABELLED: The effect of hepatitis B virus (HBV) coinfection in patients with hepatitis C virus (HCV) remains unclear. We used the National Veterans Affairs HCV Clinical Case Registry to identify patients with confirmed HCV viremia during 1997-2005. We defined HBV coinfection as a positive test for hepatitis B surface antigen, HBV DNA, or hepatitis B e antigen. We defined cirrhosis and hepatocellular carcinoma (HCC) based on the validated ICD-9 codes and determined mortality through the end of 2009. We performed Cox proportional hazard regression analyses to examine the effect of HBV coinfection stratified by HBV DNA status (positive or negative) on the risk of cirrhosis, HCC, and death adjusting for patients' age, gender, race, human immunodeficiency virus (HIV) infection, alcohol or drug use, Deyo Score, and antiviral treatment. Among 99,548 patients with HCV infection, 1,370 patients (1.4%) had HBV coinfection. Of the coinfected patients, 677 (49.4%) patients had at least one HBV DNA test done and 303 patients (44.7%) tested positive for HBV DNA. The incidence rates of cirrhosis, HCC, and death were significantly higher in patients with HBV coinfection and detectable HBV DNA compared to HCV monoinfection (36.8, 6.9, and 41.7 versus 17.4, 3.6, and 31.4 per 1,000 person-years, respectively; P < 0.05 for all comparisons). After adjustment for demographic, clinical, and treatment factors, patients with detectable HBV DNA had a significantly higher risk for cirrhosis (hazard ratio [HR] = 1.89 95% confidence interval [CI] = 1.46-2.45), HCC (HR = 2.12, 95% CI = 1.26-3.60), and death (HR = 1.62, 95% CI = 1.33-1.99) compared to HCV monoinfected patients. There were no differences in the risk of cirrhosis, HCC, or overall mortality between coinfected patients with undetectable HBV DNA and those with HCV monoinfection (HR = 1.18, 95% CI = 0.90-1.55; 1.54, 95% CI = 0.93-2.56; 1.08, 95% CI = 0.88-1.33, respectively). CONCLUSION: We found that while only a small number of HCV patients were coinfected with HBV, patients with documented HBV viremia were at a significantly higher risk for cirrhosis, HCC, and overall death than HCV monoinfected patients. Absence of HBV replication was associated with a clinical course similar to that of HCV monoinfected patients.

Ceftibuten-induced filamentation of extended spectrum beta lactamase (ESBL)-producing uropathogenic Escherichia coli alters host cell responses during an in vitro infection.

Inadequate and delayed antibiotic treatment of extended spectrum beta-lactamase (ESBL)-producing isolates have been associated with increased mortality of affected patients. The purpose of this study was to compare the host response of human renal epithelial cells and polymorphonuclear leucocyte (PMN) cells when infected by ESBL-producing uropathogenic Escherichia coli (UPEC) isolates in the presence or absence of ineffective antibiotics. The renal epithelial cell line A498 and PMN cells were stimulated with ESBL-producing UPEC isolates in the presence or absence of three different antibiotics (trimetoprim, ceftibuten and ciprofloxacin). Host cell responses were evaluated as release of cytokines (IL-6, IL-8), reactive oxygen species (ROS), ATP and endotoxins. Bacterial morphology and PMN phagocytosis were evaluated by microscopy. In the presence of ceftibuten, 2 out of 3 examined ESBL-isolates changed their morphology into a filamentous form. The presence of ceftibuten enhanced IL-6, IL-8 and ROS-production from host cells, but only from cells stimulated by the filamentous isolates. The bacterial supernatant and not the filamentous bacteria per se was responsible for the increased release of IL-6, IL-8 and ROS. Increased endotoxin and ATP levels were found in the bacterial supernatants from filamentous isolates. Apyrase decreased IL-6 secretion from A498 cells and polymyxin B abolished the increased ROS-production from PMN cells. PMN were able to inhibit the bacterial growth of some ESBL-isolates in the presence of ceftibuten. In conclusion, antibiotic-induced filamentation of ESBL-producing UPEC isolates and the associated release of ATP and endotoxins can alter the host cell response in the urinary tract.

Multiresistant uropathogenic extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli are susceptible to the carbon monoxide releasing molecule-2 (CORM-2).

Carbon monoxide (CO) releasing molecules (CO-RMs) have been shown to inhibit growth of commensal Escherichia coli (E. coli). In the present study we examined the effect of CORM-2 on uropathogenic E. coli (UPEC) that produces extended-spectrum ß-lactamase (ESBL). Viability experiments showed that CORM-2 inhibited the growth of several different ESBL-producing UPEC isolates and that 500 µM CORM-2 had a bactericidal effect within 4 h. The bactericidal effect of CORM-2 was significantly more pronounced than the effect of the antibiotic nitrofurantoin. CORM-2 demonstrated a low level of cytotoxicity in eukaryotic cells (human bladder epithelial cell line 5637) at the concentrations and time-points where the antibacterial effect was obtained. Real-time RT-PCR studies of different virulence genes showed that the expression of capsule group II kpsMT II and serum resistance traT was reduced and that some genes encoding iron acquisition systems were altered by CORM-2. Our results demonstrate that CORM-2 has a fast bactericidal effect against multiresistant ESBL-producing UPEC isolates, and also identify some putative UPEC virulence factors as targets for CORM-2. CO-RMs may be candidate drugs for further studies in the field of finding new therapeutic approaches for treatment of uropathogenic ESBLproducing E. coli.

IL-8 and global gene expression analysis define a key role of ATP in renal epithelial cell responses induced by uropathogenic bacteria.

The recent recognition of receptor-mediated ATP signalling as a pathway of epithelial pro-inflammatory cytokine release challenges the ubiquitous role of the TLR4 pathway during urinary tract infection. The aim of this study was to compare cellular responses of renal epithelial cells infected with uropathogenic Escherichia coli (UPEC) strain IA2 to stimulation with ATP-γ-S. A498 cells were infected or stimulated in the presence or absence of apyrase, that degrades extracellular ATP, or after siRNA-mediated knockdown of ATP-responding P2Y2 receptors. Cellular IL-8 release and global gene expression were analysed. Both IA2 and A498 cells per se released ATP, which increased during infection. IA2 and ATP-γ-S caused a ∼5-fold increase in cellular release of IL-8 and stimulations performed in the presence of apyrase or after siRNA knockdown of P2Y2 receptors resulted in attenuation of IA2-mediated IL-8 release. Microarray results show that both IA2 and ATP-γ-S induced marked changes in gene expression of renal cells. Thirty-six genes were in common between both stimuli, and many of these are key genes belonging to classical response pathways of bacterial infection. Functional analysis shows that 88 biological function-annotated cellular pathways were identical between IA2 and ATP-γ-S stimuli. Results show that UPEC-induced release of IL-8 is dependent on P2Y2 signalling and that cellular responses elicited by UPEC and ATP-γ-S have many identical features. This indicates that renal epithelial responses elicited by bacteria could be mediated by bacteria- or host-derived ATP, thus defining a key role of ATP during infection.

Faecal biomarkers for diagnosis and prediction of disease course in treatment-naïve patients with IBD.

BACKGROUND: Faecal biomarkers can be used to assess inflammatory bowel disease (IBD). AIM: To explore the performance of some promising biomarkers in diagnosing and predicting disease course in IBD. METHODS: We included 65 patients with treatment-naïve, new-onset Crohn's disease (CD), 90 with ulcerative colitis (UC), 67 symptomatic controls (SC) and 41 healthy controls (HC) in this prospective observational study. We analysed faecal samples for calprotectin (FC), myeloperoxidase (MPO), human neutrophil lipocalin (HNL), eosinophil cationic protein ECP and eosinophil-derived neurotoxin (EDN) and compared markers among groups. We assessed the diagnostic capability of biomarkers with receiver operating characteristic curves. Clinical disease course was determined for each patient with IBD and analysed the association with biomarkers by logistic regression. RESULTS: All markers were elevated at inclusion in patients with IBD compared with HC (p < 0.001) and SC (p < 0.001). FC (AUC 0.85, 95% CI: 0.79-0.89) and MPO (AUC 0.85, 95% CI: 0.80-0.89) showed the highest diagnostic accuracy in distinguishing IBD from SC. The diagnostic ability of biomarkers differed between IBD subtypes with the highest performance for FC and MPO in CD. The diagnostic accuracy was further improved by combining FC and MPO (p = 0.02). Levels of FC, MPO and HNL at inclusion were predictive of an aggressive disease course with MPO showing the strongest association (p = 0.006). CONCLUSIONS: This study provides new insight into the diagnostic and prognostic capability of neutrophil and eosinophil biomarkers in IBD and suggests that MPO, alone or in combination with FC, may add to the diagnostic power of faecal biomarkers.

Identification and validation of a blood- based diagnostic lipidomic signature of pediatric inflammatory bowel disease.

Improved biomarkers are needed for pediatric inflammatory bowel disease. Here we identify a diagnostic lipidomic signature for pediatric inflammatory bowel disease by analyzing blood samples from a discovery cohort of incident treatment-naïve pediatric patients and validating findings in an independent inception cohort. The lipidomic signature comprising of only lactosyl ceramide (d18:1/16:0) and phosphatidylcholine (18:0p/22:6) improves the diagnostic prediction compared with high-sensitivity C-reactive protein. Adding high-sensitivity C-reactive protein to the signature does not improve its performance. In patients providing a stool sample, the diagnostic performance of the lipidomic signature and fecal calprotectin, a marker of gastrointestinal inflammation, does not substantially differ. Upon investigation in a third pediatric cohort, the findings of increased lactosyl ceramide (d18:1/16:0) and decreased phosphatidylcholine (18:0p/22:6) absolute concentrations are confirmed. Translation of the lipidomic signature into a scalable diagnostic blood test for pediatric inflammatory bowel disease has the potential to support clinical decision making.

The periodontal pathogen Porphyromonas gingivalis changes the gene expression in vascular smooth muscle cells involving the TGFbeta/Notch signalling pathway and increased cell proliferation.

BACKGROUND: Porphyromonas gingivalis is a gram-negative bacterium that causes destructive chronic periodontitis. In addition, this bacterium is also involved in the development of cardiovascular disease. The aim of this study was to investigate the effects of P. gingivalis infection on gene and protein expression in human aortic smooth muscle cells (AoSMCs) and its relation to cellular function. RESULTS: AoSMCs were exposed to viable P. gingivalis for 24 h, whereafter confocal fluorescence microscopy was used to study P. gingivalis invasion of AoSMCs. AoSMCs proliferation was evaluated by neutral red assay. Human genome microarray, western blot and ELISA were used to investigate how P. gingivalis changes the gene and protein expression of AoSMCs. We found that viable P. gingivalis invades AoSMCs, disrupts stress fiber structures and significantly increases cell proliferation. Microarray results showed that, a total of 982 genes were identified as differentially expressed with the threshold log2 fold change > |1| (adjust p-value <0.05). Using bioinformatic data mining, we demonstrated that up-regulated genes are enriched in gene ontology function of positive control of cell proliferation and down-regulated genes are enriched in the function of negative control of cell proliferation. The results from pathway analysis revealed that all the genes belonging to these two categories induced by P. gingivalis were enriched in 25 pathways, including genes of Notch and TGF-beta pathways. CONCLUSIONS: This study demonstrates that P. gingivalis is able to invade AoSMCs and stimulate their proliferation. The activation of TGF-beta and Notch signaling pathways may be involved in the bacteria-mediated proliferation of AoSMCs. These findings further support the association between periodontitis and cardiovascular diseases.

Smoking during pregnancy is associated with the placental proteome.

Maternal smoking during pregnancy (MSDP) is a significant risk factor for the development of foetal, neonatal, and childhood morbidities. We hypothesized that infants exposed to MSDP have a distinct proteomic expression in their term placentas compared to infants without such an exposure. A total of 39 infants exposed (cord blood cotinine levels of >1 ng/mL) and 44 infants not exposed to MSDP were included in the study. Women with chronic disease, body mass index of > 30, or a history of uterine surgery were excluded. Total proteome abundance was analysed with quantitative mass spectrometry. For univariate analysis of differences in placental protein levels between groups, ANOVA with multiple testing corrections by the Benjamini-Hochberg method was used. For multivariate analysis, we used principal component analysis, partial least squares, lasso, random forest, and neural networks. The univariate analyses showed four differentially abundant proteins (PXDN, CYP1A1, GPR183, and KRT81) when heavy and moderate smoking groups were compared to non-smokers. With the help of machine learning, we found that an additional six proteins (SEPTIN3, CRAT, NAAA, CD248, CADM3, and ZNF648) were discriminants of MSDP. The placental abundance of these ten proteins together explained 74.1% of the variation in cord blood cotinine levels (p = 0.002). Infants exposed to MSDP showed differential abundance of proteins in term placentas. We report differential placental abundance of several proteins for the first time in the setting of MSDP. We believe that these findings supplement the current understanding of how MSDP affects the placental proteome.

Glimepiride Compared to Liraglutide Increases Plasma Levels of miR-206, miR-182-5p, and miR-766-3p in Type 2 Diabetes Mellitus: A Randomized Controlled Trial.

BACKGRUOUND: Diabetes is a chronic disease with several long-term complications. Several glucose-lowering drugs are used to treat type 2 diabetes mellitus (T2DM), e.g., glimepiride and liraglutide, in which both having different modes of action. Circulating microRNAs (miRNAs) are suggested as potential biomarkers that are associated with the disease development and the effects of the treatment. In the current study we evaluated the effect of glimepiride, liraglutide on the expression of the circulating miRNAs. METHODS: The present study is a post hoc trial from a previously randomized control trial comparing liraglutide versus glimepiride both in combination with metformin in subjects with T2DM, and subclinical heart failure. miRNAs were determined in the subjects' serum samples with next generation sequencing. Expression patterns of the circulating miRNAs were analyzed using bioinformatic univariate and multivariate analyses (clinical trial registration: NCT01425580). RESULTS: Univariate analyses show that treatment with glimepiride altered expression of three miRNAs in patient serum, miR-206, miR-182-5p, and miR-766-3p. Both miR-182-5p and miR-766-3p were also picked up among the top contributing miRNAs with penalized regularised logistic regressions (Lasso). The highest-ranked miRNAs with respect to Lasso coefficients were miR-3960, miR-31-5p, miR-3613-3p, and miR-378a-3p. Liraglutide treatment did not significantly influence levels of circulating miRNAs. CONCLUSION: Present study indicates that glucose-lowering drugs differently affect the expression of circulating miRNAs in serum in individuals with T2DM. More studies are required to investigate possible mechanisms by which glimepiride is affecting the expression of circulating miRNAs.