Mycobacterium avium paratuberculosis Infection Suppresses Vitamin D Activation and Cathelicidin Production in Macrophages through Modulation of the TLR2-Dependent p38/MAPK-CYP27B1-VDR-CAMP Axis.

BACKGROUND: Vitamin D plays a vital role in modulating both innate and adaptive immune systems. Therefore, vitamin D deficiency has been associated with higher levels of autoimmune response and increased susceptibility to infections. CYP27B1 encodes a member of the cytochrome P450 superfamily of enzymes. It is instrumental in the conversion of circulating vitamin D (calcifediol) to active vitamin D (calcitriol). This is a crucial step for macrophages to express Cathelicidin Anti-microbial Peptide (CAMP), an anti-bacterial factor released during the immune response. Our recent study indicated that a Crohn's disease (CD)-associated pathogen known as Mycobacterium avium paratuberculosis (MAP) decreases vitamin D activation in macrophages, thereby impeding cathelicidin production and MAP infection clearance. The mechanism by which MAP infection exerts these effects on the vitamin D metabolic axis remains elusive. METHODS: We used two cell culture models of THP-1 macrophages and Caco-2 monolayers to establish the effects of MAP infection on the vitamin D metabolic axis. We also tested the effects of Calcifediol, Calcitriol, and SB203580 treatments on the relative expression of the vitamin D metabolic genes, oxidative stress biomarkers, and inflammatory cytokines profile. RESULTS: In this study, we found that MAP infection interferes with vitamin D activation inside THP-1 macrophages by reducing levels of CYP27B1 and vitamin D receptor (VDR) gene expression via interaction with the TLR2-dependent p38/MAPK pathway. MAP infection exerts its effects in a time-dependent manner, with the maximal inhibition observed at 24 h post-infection. We also demonstrated the necessity to have toll-like receptor 2 (TLR2) for MAP infection to influence CYP27B1 and CAMP expression, as TLR2 gene knockdown resulted in an average increase of 7.78 ± 0.88 and 13.90 ± 3.5 folds in their expression, respectively. MAP infection also clearly decreased the levels of p38 phosphorylation and showed dependency on the p38/MAPK pathway to influence the expression of CYP27B1, VDR, and CAMP which was evident by the average fold increase of 1.93 ± 0.28, 1.86 ± 0.27, and 6.34 ± 0.51 in their expression, respectively, following p38 antagonism. Finally, we showed that calcitriol treatment and p38/MAPK blockade reduce cellular oxidative stress and inflammatory markers in Caco-2 monolayers following macrophage-mediated MAP infection. CONCLUSIONS: This study characterized the primary mechanism by which MAP infection leads to diminished levels of active vitamin D and cathelicidin in CD patients, which may explain the exacerbated vitamin D deficiency state in these cases.

AMS-U-Net: automatic mass segmentation in digital breast tomosynthesis via U-Net.

Purpose: The objective of this study was to develop a fully automatic mass segmentation method called AMS-U-Net for digital breast tomosynthesis (DBT), a popular breast cancer screening imaging modality. The aim was to address the challenges posed by the increasing number of slices in DBT, which leads to higher mass contouring workload and decreased treatment efficiency. Approach: The study used 50 slices from different DBT volumes for evaluation. The AMS-U-Net approach consisted of four stages: image pre-processing, AMS-U-Net training, image segmentation, and post-processing. The model performance was evaluated by calculating the true positive ratio (TPR), false positive ratio (FPR), F-score, intersection over union (IoU), and 95% Hausdorff distance (pixels) as they are appropriate for datasets with class imbalance. Results: The model achieved 0.911, 0.003, 0.911, 0.900, 5.82 for TPR, FPR, F-score, IoU, and 95% Hausdorff distance, respectively. Conclusions: The AMS-U-Net model demonstrated impressive visual and quantitative results, achieving high accuracy in mass segmentation without the need for human interaction. This capability has the potential to significantly increase clinical efficiency and workflow in DBT for breast cancer screening.

From peripheral finger-derived pulse waveforms to aortic pressure waveform features: an application of a generalized transfer function.

OBJECTIVE: Aortic (central) pressure features are associated with cardiovascular complications and can be algorithmically derived from non-invasive peripheral arterial waveforms. This has conventionally been performed with a pressure waveform (i.e., tonometry or oscillometry) rather than with the optical-based sensor (photoplethysmography (PPG)) that is predominantly used in wearable health devices. Extraction of aortic features from a peripheral PPG waveform has yet to be investigated. This study aims to compare aortic features extracted from peripheral arterial waveforms acquired with different sensor modalities using the same transfer function. DESIGN AND METHOD: Radial tonometry (reference), finger volume-clamped PPG (Penáz) and fingertip PPG waveforms were measured in participants (n=29, 36±16 years, 15 female) under baseline conditions. Waveforms were converted into an aortic pressure waveform using the transfer function. Waveform features were extracted from the converted waveform. Extracted features were compared with correlation plots and a Bland-Altman analysis. RESULTS: Aortic pressure features extracted from a finger using the Penáz technique were comparable to radial tonometry derived features. Aortic features extracted from a fingertip waveform were more variable in comparison to radial tonometry-derived features. CONCLUSIONS: Aortic (central) pressure waveform features contain valuable haemodynamic information and have the capacity to be easily and conveniently implemented in wearable health devices. Future use of these features in wearable health devices incorporating PPG requires the development, and/or, optimization of a unique transfer function to more accurately represent the aortic pressure waveform for cardiovascular assessment.Clinical Relevance- Aortic pressure features might be used in wearable health devices following the development of a unique transfer function for optical-transduced peripheral vascular signals.

Comparison of effects of peripheral vasculature on tonometric radial pulse and cuff-based brachial pulse waveform as used in estimation of central aortic pressures.

OBJECTIVE: Aortic pressure estimation requires reliable peripheral pulse waveform acquisition. The peripheral waveform can change with local vascular effects that can be independent of aortic pressure. This study quantifies the effects of peripheral vasculature changes on radial and brachial waveforms. DESIGN AND METHOD: In 20 subjects (37± 15 years, 7 female), brachial volumetric displacement (cuff-based) and radial tonometry waveforms were simultaneously measured whilst a cuff around the hand on the same arm was inflated to induce transmural pressures of -60, -30, -15, 0, 15 and 30 mmHg, altering local peripheral resistance and compliance by graded arterial wall unloading. Aortic blood pressure (BP), augmentation index (AIx) and ejection duration were calculated from the measurements using a generalized transfer function. The parameters under unloaded conditions were compared to baseline measurements. RESULTS: Brachial systolic and diastolic BP did not change throughout the experiment. Altering peripheral resistance and compliance did not significantly change calculated aortic BP values, although changes were nominally greater for radial (maximum +8±1 mmHg) compared to brachial (maximum +2±1 mmHg) waveforms. AIx at 0 mmHg transmural pressure (maximum arterial wall unloading) was higher when derived from radial waveforms (+24±3%, p<0.001) but not when derived from brachial waveforms. CONCLUSIONS: Localized changes in peripheral resistance and compliance affect tonometer acquired radial waveforms but not volumetric displacement acquired brachial pressure waveforms, as judged by computed central aortic augmentation pressure parameters. This suggests aortic pressure estimation from the brachial cuff waveform is less sensitive to peripheral vasculature disturbances that alter the peripheral arterial pulse morphology.

Folate and Vitamin B12 Deficiency Exacerbate Inflammation during Mycobacterium avium paratuberculosis (MAP) Infection.

Folate and vitamin B12 deficiency is highly prevalent among Crohn's disease (CD) patients. Furthermore, CD pathology can be mediated by Mycobacterium avium subsp. paratuberculosis (MAP) infection. However, the direct effect of folate (B9) and cobalamin (B12) deficiency during MAP infection remains uncharacterized. This study investigates how folate and B12 deficiency impedes macrophage apoptosis and exacerbates the inflammation in macrophages infected with MAP isolated from CD patients. Accordingly, we measured folate and B12 in ex vivo plasma samples collected from CD patients with or without MAP infection (N = 35 per group). We also measured the expression of the pro-inflammatory cytokines IL-1ß and TNF-α, cellular apoptosis and viability markers, and bacterial viability in MAP-infected macrophages cultured in folate and B12 deficient media. We determined that MAP-positive CD patients have significantly lower plasma folate and B12 in comparison to MAP-negative CD patients [414.48 ± 94.60 pg/mL vs. 512.86 ± 129.12 pg/mL, respectively]. We further show that pro-inflammatory cytokines IL-1ß and TNF-α are significantly upregulated during folate and vitamin B12 deprivation following MAP infection by several folds, while supplementation significantly reduces their expression by several folds. Additionally, depletion of folate, B12, and folate/B12 following MAP infection, led to decreased macrophage apoptosis from 1.83 ± 0.40-fold to 1.04 ± 0.08, 0.64 ± 0.12, and 0.45 ± 0.07 in folate-low, B12-low, and folate/B12-low cells, respectively. By contrast, folate and folate/B12 supplementation resulted in 3.38 ± 0.70 and 2.58 ± 0.14-fold increases in infected macrophages. Interestingly, changes in overall macrophage viability were only observed in folate-high, folate/B12-high, and folate/B12-low media, with 0.80 ± 0.05, 0.82 ± 0.02, and 0.91 ± 0.04-fold changes, respectively. Incubation of Caco-2 intestinal epithelial monolayers with supernatant from infected macrophages revealed that folate/B12 deficiency led to increased LDH release independent of oxidative stress. Overall, our results indicate that folate and B12 are key vitamins affecting cell survival and inflammation during MAP infection.

Attenuation of Excess TNF-α Release in Crohn's Disease by Silencing of iRHOMs 1/2 and the Restoration of TGF-ß Mediated Immunosuppression Through Modulation of TACE Trafficking.

TNFα converting enzyme (TACE) is a transmembrane metalloprotease that sheds an assortment of signaling receptors, cytokines, growth factors, and pro-inflammatory mediators. In Crohn's disease (CD), TACE activity is upregulated, resulting in a marked increase of TNFα secretion and inflammation. Although treatment of CD with TNFα monoclonal antibodies is beneficial, many patients are at risk for acquiring opportunistic infections, and the treatment efficacy of TNFα monoclonal antibodies typically decreases over time. This study investigated an alternative approach for mitigating TNFα release by knocking down TACE membrane translocation in macrophages via inhibitory rhomboid proteins 1 and 2 (iRHOMs 1/2) siRNA treatment. First we measured TGFßRII shedding in ex vivo plasma samples collected from CD patients and healthy control subjects (N=40 per group). Then, we measured TGFßRII shedding and the expression and production of TGFß ligand, TNFα, IL-6, IL-1ß, IL-10, and total versus membranous TACE in vitro with THP-1 derived macrophage infected with Mycobacterium avium subspecies paratuberculosis (MAP), a highly studied CD-related pathogen. We determined that TGFßRII shedding was significantly higher in CD patients compared to healthy controls [515.52 ± 54.23 pg/mL vs 310.81 ± 43.16 pg/mL, respectively], and MAP-infected CD plasma samples had significantly more TGFßRII shedding (601.83 ± 49.56 pg/mL) than MAP-negative CD samples (430.37 ± 45.73 pg/mL). Moreover, we also determined that TACE production; TGFß ligand expression and production; and TGFßRII shedding were also higher in MAP-infected THP-1 macrophages. Nevertheless, once we transfected the MAP infected macrophages with iRHOM siRNA, TACE production and membrane localization were significantly decreased, resulting in a significant decrease in TGFßRII shedding; an increase in Smad3 phosphorylation; a decrease in the expression and production of pro-inflammatory cytokines; and a decrease in the expression and production of stricture-associated factor, plasminogen activator inhibitor-1 (PAI-1). Our data clearly demonstrates that the regression of TACE trafficking, via iRHOM 1/2 silencing, significantly reduces the release of TNFα and restores the immunosuppressive capabilities of TGFß signaling, which ultimately reverses inflammatory tissue damage. Accordingly, this study may provide a framework for the creation of newer, safer therapeutic options designed to treat inflammatory autoimmune diseases such as CD and rheumatoid arthritis.

Plasma miRNA Profile of Crohn's Disease and Rheumatoid Arthritis Patients.

Crohn's disease (CD) and rheumatoid arthritis (RA) are immune mediated inflammatory diseases. Several studies indicate a role for microRNAs (miRNAs) in the pathogenesis of a variety of autoimmune diseases, including CD and RA. Our study's goal was to investigate circulating miRNAs in CD and RA patients to identify potential new biomarkers for early detection and personalized therapeutic approaches for autoimmune diseases. For this study, subjects with CD (n = 7), RA (n = 8) and healthy controls (n = 7) were recruited, and plasma was collected for miRNA sequencing. Comparison of the expression patterns of miRNAs between CD and healthy patients identified 99 differentially expressed miRNAs. Out of these miRNAs, 4 were down regulated, while 95 were up regulated. Comparison of miRNAs between RA and healthy patients identified 57 differentially expressed miRNAs. Out of those, 12 were down regulated, while 45 were up regulated. For all the miRNAs down regulated in CD and RA patients, 420 GO terms for biological processes were similarly regulated between both groups. Therefore, the identification of new plasma miRNAs allows the emergence of new biomarkers that can assist in the diagnosis and treatment of CD and RA.

Cathelicidin Mediates an Anti-Inflammatory Role of Active Vitamin D (Calcitriol) During M. paratuberculosis Infection.

Vitamin D is a key regulator in calcium and phosphorus metabolism which are essential for maintaining bone health. Recent reports also showed a role for vitamin D in immune regulation which may be linked to vitamin D deficiency in autoimmune disorders including inflammatory diseases and Crohn's disease (CD). This study examines the role of vitamin D deficiency in the regulation of Cathelicidin Antimicrobial Peptide (CAMP) in CD-like macrophages. The latter includes macrophages infected with Mycobacterium avium subsp. paratuberculosis (MAP) isolated from CD patient. Initially, we measured cathelicidin and calcitriol in ex vivo plasma samples from CD patients with or without MAP infection (N=40 per group). We also measured the expression and production of CAMP/LL-37, TNF-α, IL-1ß, IL-10, cellular oxidative stress markers, and bacterial viability following treatment of MAP-infected macrophages with four different forms of vitamin D (D2, D3, calcifediol, and calcitriol). From these studies, we determined that LL-37 and calcitriol were significantly lower in CD samples from MAP-positive patients [155.55 ± 49.77 ng/mL and 51.48 ± 31.04 pg/mL, respectively] compared to MAP-negative patients [193.01 ± 78.95 ng/mL and 272.36 ± 94.77 pg/mL, respectively]. Moreover, calcitriol and calcifediol upregulated CAMP expression by nearly 5-fold and 3-fold, respectively. However, following MAP infection, only calcitriol increased CAMP by 3-folds. Both calcitriol and LL-37 reduced intracellular MAP viability by ~3 folds and inhibited TNF-α and IL-1ß expression and production in these cells. Treating co-culture of Caco-2 monolayers and MAP-infected macrophages with LL-37 or calcitriol have shown a reduction in NOX-1 expression and DHE signal, in addition to a higher NADPH/NADPt ratio. Notably, calcitriol's anti-inflammatory effects were lost upon CAMP knockdown by CAMP-siRNA transfection. Altogether, the data indicate that MAP infection and burden is significant in CD by disrupting the conversion of calcifediol to calcitriol and downregulation of CAMP expression leading to vitamin D deficiency.

Extra-Pulmonary Complications in SARS-CoV-2 Infection: A Comprehensive Multi Organ-System Review.

Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is typically presented with acute symptoms affecting upper and lower respiratory systems. As the current pandemic progresses, COVID-19 patients are experiencing a series of nonspecific or atypical extra-pulmonary complications such as systemic inflammation, hypercoagulability state, and dysregulation of the renin-angiotensin-aldosterone system (RAAS). These manifestations often delay testing, diagnosis, and the urge to seek effective treatment. Although the pathophysiology of these complications is not clearly understood, the incidence of COVID-19 increases with age and the presence of pre-existing conditions. This review article outlines the pathophysiology and clinical impact of SARS-CoV-2 infection on extra-pulmonary systems. Understanding the broad spectrum of atypical extra-pulmonary manifestations of COVID-19 should increase disease surveillance, restrict transmission, and most importantly prevent multiple organ-system complications.

Challenges Presented by Cuffless Measurement of Blood Pressure if Adopted for Diagnosis and Treatment of Hypertension.

The global health burden presented by hypertension is providing increased motivation for improved means of collection of blood pressure (BP) data. A growing area of research and commercial activity is the use of wearable devices to provide BP data using non-invasive cuffless techniques. The accelerated progress in recent years, particularly relating to connectivity of smartphone technology, has promoted the availability of consumer devices that provide values of BP. The main types of devices are wrist-worn, watch-type devices with sensors that typically record a photoplethysmography (PPG) signal, sometimes also with an electrocardiography (ECG) signal. The general underlying concept of the cuffless BP measurement in most device types is the association of BP and the travel time of the arterial pulse between two locations, determined from the time delay between the ECG and PPG signals. Other methods may involve additional analysis of the PPG waveform features. Experimental data are presented to illustrate the challenges presented by cuffless BP techniques in obtaining reliable BP measurements when the change in BP is caused by different stimuli affecting cardiac and vascular mechanisms. These effects influence the association of the measured and physiological BP change, thus presenting significant challenges and potential limitations in the use of cuffless BP devices for the diagnosis and treatment of hypertension.

Enteropathogenic infections modulate intestinal serotonin transporter (SERT) function by activating Toll-like receptor 2 (TLR-2) in Crohn's disease.

Serotonin (5-hydroxytryptamine [5-HT]) is an intestinal neuromodulator that regulates several essential enteric physiological functions such as absorption or secretion of fluids, and peristaltic reflexes. Availability of the intestinal 5-HT is dependent on serotonin transporter (SERT), which uptakes 5-HT and facilitates its degradation. Interestingly, Toll-like receptor 2 (TLR-2) is co-localized with 5-HT, which suggests a possible impact of neuroendocrine cells in the inflammatory response through TLR-2 activation. Serum 5-HT levels were measured in 80 Crohn's disease (CD) patients and 40 healthy control subjects. Additionally, fully differentiated Caco-2 monolayers were infected with Mycobacteria paratuberculosis (MAP), L. monocytogenes, or M. smegmatis in the presence of exogenous 5-HT at different concentrations. Cells were subsequently harvested and used for measuring SERT activity, RNA isolation followed by RT-PCR, protein quantification, and tissue damage markers (DHE, LDH, GSH and MDA). TLR-2 intracellular signaling pathways were assessed by pre-incubating Caco-2 monolayers with selective blockers of ERK, cAMP/PKA, p38 MAPK, and 5-HT3 receptors. MAP-infected CD patients (N = 40) had higher serum 5-HT levels (462.95 ± 8.55 ng/mL, N = 40) than those without MAP infection (385.33 ± 10.3 ng/mL, N = 40). TLR-2 activation by enteropathogenic bacteria inhibited SERT activity in the presence of exogenous 5-HT by up to 50%. These effects were increasing gradually over 72 h, and MAP infection had the greatest effect on SERT inhibition when cells were exposed to 5-HT in a concentration dependent manner. Additionally, inhibition of SERT activity was accompanied with higher levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-8) and oxidative stress markers (DHE, LDH and MDA), whereas SERT expression and protein level were downregulated. Consequently, inhibition of TLR-2 and p38 MAPK signaling or blocking 5-HT3 receptors restored SERT activity and reduced the production of pro-inflammatory cytokines, as reflected by the downregulation of oxidative stress and tissue damage markers. The involvement of TLR-2 in the intestinal pathology might be concluded not only from its innate immune role, but also from its effect on modulating the intestinal serotonergic response. Ultimately, regulating the intestinal serotonergic system can be therapeutically exploited to mitigate other enteropathogenic infections, which will help in understanding the gut-microbiome-brain connection.

Modulation of PTPN2/22 Function by Spermidine in CRISPR-Cas9-Edited T-Cells Associated with Crohn's Disease and Rheumatoid Arthritis.

Crohn's Disease (CD) and Rheumatoid Arthritis (RA) share some single nucleotide polymorphisms (SNPs) in protein tyrosine phosphatase non-receptor types 2 and 22 (PTPN2/22). Recently, we reported that clinical samples from CD and RA patients associated with PTPN2:rs478582 or PTPN22:rs2476601 genotypes were linked to overactive immune response and exacerbation of inflammation. Here, we investigated in vitro the effects of these SNPs in Jurkat T-cells using CRISPR-Cas9. All cells were evaluated for PTPN22/22 loss of function and effects on cell response. We measured gene expression via RT-qPCR and cytokines by ELISA. We also measured cell proliferation using a BrdU labeling proliferation ELISA, and T-cell activation using CD-25 fluorescent immunostaining. In PTPN2 SNP-edited cells, PTPN2 expression decreased by 3.2-fold, and proliferation increased by 10.2-fold compared to control. Likewise, expression of PTPN22 decreased by 2.4-fold and proliferation increased by 8.4-fold in PTPN22 SNP-edited cells. IFN-γ and TNF-α secretions increased in both edited cell lines. CD25 expression (cell activation) was 80.32% in PTPN2 SNP-edited cells and 85.82% in PTPN22 SNP-edited cells compared to 70.48% in unedited Jurkat T-cells. Treatment of PTPN2 and PTPN22-edited cells with a maximum 20 µM spermidine restored PTPN2/22 expression and cell response including cell proliferation, activation, and cytokines secretion. Most importantly, the effect of spermidine on edited cells restored normal expression and secretion of IFN-γ and TNF-α. The data clearly demonstrated that edited SNPs in PTPN2 or PTPN22 were associated with reduced gene expression, which resulted in an increase in cell proliferation and activation and overactive immune response. The data validated our earlier observations in CD and RA clinical samples. Surprisingly, spermidine restored PTPN2/22 expression in edited Jurkat T-cells and the consequent beneficial effect on cell response and inflammation. The study supports the use of polyamines dietary supplements for management of CD and in RA patients.

Coronavirus Disease 2019 (COVID-19) Diagnostic Tools: A Focus on Detection Technologies and Limitations.

The ongoing coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a severe threat to human health and the global economy and has resulted in overwhelming stress on health care systems worldwide. Despite the global health catastrophe, especially in the number of infections and fatalities, the COVID-19 pandemic has also revolutionized research and discovery with remarkable success in diagnostics, treatments, and vaccine development. The use of many diagnostic methods has helped establish public health guidelines to mitigate the spread of COVID-19. However, limited information has been shared about these methods, and there is a need for the scientific community to learn about these technologies, in addition to their sensitivity, specificity, and limitations. This review article is focused on providing insights into the major methods used for SARS-CoV-2 detection. We describe in detail the core principle of each method, including molecular and serological approaches, along with reported claims about the rates of false negatives and false positives, the types of specimens needed, and the level of technology and the time required to perform each test. Although this study will not rank or prioritize these methods, the information will help in the development of guidelines and diagnostic protocols in clinical settings and reference laboratories.

Assessment of Central Arterial Hemodynamics in Children: Comparison of Noninvasive and Invasive Measurements.

BACKGROUND: In adults, central systolic blood pressure (cSBP) and augmentation index (cAIx) are independently associated with cardiovascular events and mortality. There is increasing interest in central hemodynamic indices in children. We aimed to assess the accuracy of current techniques against invasive intra-aortic measurements in children. METHODS: Intra-aortic pressure waveforms were recorded with simultaneous brachial, radial, and carotid waveforms in 29 children (6.7 ± 3.9 years old) undergoing cardiac catheterization. Adult and age-appropriate transfer functions (TFs) (brachial adult: b-aTF; radial adult: r-aTF; radial for 8-year-old children: TF8; and radial for 14-year-old children: TF14) were used to synthesize central aortic waveforms from peripheral waveforms calibrated either to invasively or noninvasively recorded BP. Central hemodynamic indices were measured by pulse wave analysis. RESULTS: cSBP measured from invasively calibrated r-aTF (ß = 0.84; intraclass correlation coefficient = 0.91; mean error ± SDD = -1.0 ± 5.0 mm Hg), TF8 (ß = 0.78; intraclass correlation coefficient = 0.84; mean error ± SDD = 4.4 ± 5.6 mm Hg), and TF14 (ß = 0.82; intraclass correlation coefficient = 0.90; mean error ± SDD = 2.0 ± 4.7 mm Hg)-synthesized central waveforms correlated with and accurately estimated intra-aortic cSBP measurements, while noninvasively calibrated waveforms did not. cAIx derived from TF-synthesized central waveforms did not correlate with intra-aortic cAIx values, and degree of error was TF-dependent. CONCLUSIONS: The currently available r-aTF accurately estimates cSBP with invasive pulse pressure calibration, while. Age-appropriate TFs do not appear to provide additional benefit. Accuracy of cAIx estimation appears to be TF dependent.

Anti-MAP Triple Therapy Supports Immunomodulatory Therapeutic Response in Crohn's Disease through Downregulation of NF-κB Activation in the Absence of MAP Detection.

We previously reported that the triple antibiotic formulation, known as anti-MAP therapy, exhibits unique synergistic antimicrobial activity and should be effective for treatment of Crohn's disease (CD) associated with Mycobacterium avium subspecies paratuberculosis (MAP). The absence of MAP detection in some CD cases may be linked to poor diagnostics or lack of association with the disease. To understand the therapeutic response of some CD patients to anti-MAP therapy in absence of MAP detection, we investigated the immunomodulatory potency of anti-MAP therapy and its major ingredients, clarithromycin (CLA) and rifabutin (RIF), in THP-1, Caco-2, and Jurkat T-cells. Anti-MAP formulation at 2.0 µg/mL decreased MAP viability in macrophages by 18-fold over 72 h. Additionally, M1/M2 macrophage polarization ratio was reduced by 6.7-fold, and expression and protein levels of TNF-α and IL-6 were reduced by 2.9-fold, whereas IL-10 increased by 5.0-fold in these cells. Mechanistically, the effect of anti-MAP formulation on NF-κB p65 activation was dose-dependent and decreased to 13.4% at 2.0 µg/mL. Most importantly, anti-MAP therapy also reversed pro-inflammatory response in lipopolysaccharide (LPS)-induced macrophages, which shows that the anti-inflammatory effect of the treatment is not just due to a decrease in MAP viability. To study the anti-cytotoxic effects of anti-MAP therapy in Caco-2 monolayers infected with MAP or treated with dextran sodium sulfate (DSS), we showed a 45% decrease in lactate dehydrogenase (LDH) activity and an 84% increase in glutathione (GSH) activity, which supports anti-apoptotic activity of the drug. In Jurkat T-cells, anti-MAP therapy decreased T-cell proliferation by 4.8-fold following treatment with phytohemagglutinin (PHA) and by 2.9-fold with MAP purified protein derivative (PPD). Overall, the data demonstrate that anti-MAP therapy plays a significant role in modulating and eliciting a protective immune response in macrophages, endothelial cells, and T lymphocytes, even in absence of infection. This may explain the therapeutic response of some CD patients to treatment, even in absence of MAP detection, infection, or total eradication. The study supports anti-MAP therapy as an alternate treatment option in CD patients, especially in absence of reliable MAP diagnostics.

Divergent Effect of Cigarette Smoke on Innate Immunity in Inflammatory Bowel Disease: A Nicotine-Infection Interaction.

Cigarette smoke (CS) has adverse effects in patients with Crohn's disease (CD), an inflammatory bowel disease (IBD) that has been associated with microbial infection, immuno-dysregulation, and mucosal dysfunction. However, CS seems to provide relief and protection to patients with another IBD known as ulcerative colitis (UC). These two subsets are featured as M1- and M2-mediated responses, respectively. Nicotine is the most active, addictive, and studied ingredient in CS. The mechanism of how nicotine and/or other CS ingredients induce pro-inflammatory or anti-inflammatory phenotypes in IBD patients remains under investigation. Our most recent in vitro nicotine study provided significant insights toward understanding the contradictory effects of nicotine on IBD patients, and it elucidated the mechanistic role of α7nAChR in modulation of macrophages in tobacco smokers. Shifting the beneficial effect of nicotine to a harmful outcome in CD patients was linked to a nicotine-microbe interaction that supports a microbial etiology in CD pathogenesis. Among the most debated pathogens in CD etiology is Mycobacterium avium subspecies paratuberculosis (MAP). Other studies associated nicotine with upregulation of miR-124 expression in macrophages, which led to anti-inflammatory response. This review discusses published work on the role of nicotine in modulation of the innate immune response and subsequent signaling in macrophages in IBD subsets.

Validation of a cuff-based device for measuring carotid-femoral pulse wave velocity in children and adolescents.

Carotid-femoral pulse wave velocity is associated with arterial stiffness in major elastic arteries, and predicts future cardiovascular events. However, little is known about carotid-femoral pulse wave velocity as a marker of vascular health in children. Semi-automated cuff-based devices for assessing pulse wave velocity are increasingly popular, although these utilize an algorithm developed and validated in adults. Physiological differences between adults and children may, however, reduce the accuracy of cuff-based methods. We sought to determine the accuracy of a cuff-based pulse wave velocity device in healthy children, and determine whether a novel age-appropriate algorithm increases accuracy. We recruited 29 healthy children between the ages of 2 and 20 years. Pulse wave velocity was measured both by using a tonometer on the carotid artery and an inflated cuff on the thigh, and using a tonometer on both the carotid artery and femoral artery as a reference standard. Accuracy of the cuff-based device with its standard algorithm developed in adults, and a novel age-appropriate algorithm corrected for physiological differences in leg pulse wave velocity was assessed with Regression analysis and Bland-Altman plots. Cuff-based device estimates of pulse wave velocity had excellent agreement to the reference standard (Δ = -0.26 ms-1 [SD 0.44]). The novel age-appropriate algorithm improved the accuracy of the cuff-based method (Δ = 0.02 ms-1 [SD 0.44]). The cuff-based semi-automatic approach estimates carotid-femoral pulse wave velocity with excellent agreement to the reference standard. However, adjusting the algorithm for known differences in leg pulse wave velocity further improves the accuracy of cuff-based measurement in children and adolescents.

Polymorphisms in TNF Receptor Superfamily 1B (TNFRSF1B:rs3397) are Linked to Mycobacterium avium paratuberculosis Infection and Osteoporosis in Rheumatoid Arthritis.

We previously discovered that single nucleotide polymorphisms (SNPs) in PTPN2/22 (T-cell negative-regulators) occur in 78% of rheumatoid arthritis (RA), along with Mycobacterium avium paratuberculosis (MAP) infection in 33% of patients. In Crohn's disease, we reported that SNPs in TNFα and receptors (TNFRSF1A/TNFRSF1B) benefited intracellular MAP-survival, increased infection, and elevated inflammatory response mimicking the poor response to anti-TNFα treatment in some patients. Here, we studied the frequency and effects of SNPs in TNFα/TNFRSF1A/TNFRSF1B in RA including gene expression, MAP infection, and osteoporosis marker levels in blood (54 RA and 48 healthy controls). TNFα:rs1800629 (GA) was detected in 19/48 (40%) RA and 8/54 (15%) controls (p-value < 0.05, odds ratio (OR) = 3.6, 95% CI: 1.37-9.54). TNFRS1B:rs3397 (CT) was detected in 21/48 (44%) RA and 10/54 (19%) controls (p-value < 0.05, OR = 4.43, 95% CI: 1.73-11.33). In RA, rs3397 downregulated TNFRSF1B expression (CC > CT (0.34 ± 0.14) and CC > TT (0.27 ± 0.12)), compared to wildtype CC (0.51 ± 0.17), p-value < 0.05. MAP DNA was detected significantly in 17/48 (35.4%) RA compared to 11/54 (20.4%) controls (p-value < 0.05, OR = 2.14, 95% CI: 1.12-5.20). The average osteocalcin level was significantly lower (p-value < 0.05) in RA (2.70 ± 0.87 ng/mL), RA + MAP (0.60 ± 0.31 ng/mL), RA + TNFRSF1B:rs3397 (TT) (0.67 ± 0.35 ng/mL), compared to the healthy control (5.31 ± 1.39 ng/mL), and MAP-free RA (3.85 ± 1.31 ng/mL). Overall, rs3397 appears to downregulate TNFRSF1B, increase MAP infection, worsen inflammation, and cause osteocalcin deficiency and possibly osteoporosis in RA.

Genetic polymorphisms in tumour necrosis factor receptors (TNFRSF1A/1B) illustrate differential treatment response to TNFα inhibitors in patients with Crohn's disease.

BACKGROUND: Monoclonal antibodies inhibiting tumour necrosis factor-α (TNFα) signalling pathway (anti-TNFα) have been widely used in Crohn's disease (CD). However, treatment response varies among patients with CD and the clinical outcome is dependent on single nucleotide polymorphisms (SNP) in TNFα receptor superfamily 1A and 1B (TNFRSF1A/1B). METHODS: We tested nine SNPs in TNFα, TNFRSF1A and TNFRSF1B by TaqMan genotyping from peripheral blood samples of 104 subjects. Additionally, we quantified the effects of these SNPs on their corresponding gene expression by RT-PCR and susceptibility to Mycobacterium avium subsp paratuberculosis (MAP) infection by IS900 nested PCR. RESULTS: Four SNPs (TNFα:rs1800629, TNFRSF1A:rs767455, TNFRSF1B:rs1061624 and TNFRSF1B:rs3397) were over-represented significantly (p<0.05) among patients with CD compared with healthy controls. The TNFRSF1A:rs767455 GG genotype was found in 15/54 patients with CD (28%), while it was only found in 2/50 healthy controls (4%) (OR 9.2, 95% CI 1.98 to 42.83). The TNFRSF1B:rs3397 TT genotype was found in 15/54 patients with CD (28%) compared with (4/50) healthy controls (8%) (OR 4.4, 95% CI 1.36 to 14.14). Furthermore, the SNPs TNFRSF1A:rs767455 and TNFRSF1B:rs3397 were associated with downregulating their corresponding genes significantly (p<0.05). MAP infection was predominantly found among patients with CD in comparison to healthy controls (57% vs 8%, respectively), which was also dependent on the SNPs TNFRSF1A:rs767455 and TNFRSF1B:rs3397. Our SNP haplotype analysis of TNFRSF1A:rs767455 and TNFRSF1B:rs3397 indicates that the G-T haplotype is significantly distributed among patients with CD (46%) and MAP infection susceptibility is also associated with this specific haplotype (31%). CONCLUSION: The SNPs TNFRSF1A:rs767455 and TNFRSF1B:rs3397, which are known to affect anti-TNFα clinical outcome in CD, were associated with lower corresponding gene expression and higher MAP infection susceptibility.

Intra-arterial analysis of the best calibration methods to estimate aortic blood pressure.

OBJECTIVE: Estimation of aortic blood pressure (BP) requires peripheral BP waveform calibration. Mean arterial pressure (MAP)/DBP calibration is purported to estimate aortic BP more accurately than SBP/DBP calibration. However, this is based on inaccurate cuff calibration. Thus, direct comparisons of each calibration method using intra-arterial BP are required to confirm this, and was the aim of this study. METHODS: Ascending aortic, brachial and radial artery intra-arterial BPs were measured among 107 patients (61.9 ±â€Š10.0 years, 70% men) undergoing coronary angiography. Radial waveforms were calibrated with brachial SBP/DBP and brachial MAP/DBP to directly test the accuracy of estimated aortic SBP (derived using a commercial device) from each calibration compared with intra-arterial aortic SBP. Estimated aortic BP accuracy from aortic MAP/DBP, brachial and radial SBP/DBP calibrations of peripheral waveforms was also tested (six calibration methods in total; all using intra-arterial BP). RESULTS: Estimated aortic SBP from brachial MAP/DBP calibration of radial waveforms had a significantly smaller mean difference than from brachial SBP/DBP calibration (-0.7 ±â€Š7.5 mmHg versus -6.9 ±â€Š7.3 mmHg, P < 0.0001 for difference). Of the other calibration methods, estimated aortic SBP was most accurate from aortic MAP/DBP calibration of radial waveforms (-1.8 ±â€Š5.0 mmHg, P = 0.00023). However, for all calibration methods, aortic-to-brachial artery and/or brachial-to-radial artery SBP amplification had a major influence on estimated aortic SBP. CONCLUSION: Brachial and aortic MAP/DBP were confirmed as the best calibration methods to estimate aortic SBP, but irrespective of calibration method, accuracy was significantly influenced by the level of SBP amplification. Thus, improved accuracy of estimated aortic SBP should be possible by closer consideration of SBP amplification or individual waveform characteristics that differ according to the level of SBP amplification.