Experimental set up for the irradiation of biological samples and nuclear track detectors with UV C

Aim

In this work we present a methodology to produce an “imprint” of cells cultivated on a polycarbonate detector by exposure of the detector to UV C radiation.

Background

The distribution and concentration of ¹⁰B atoms in tissue samples coming from BNCT (Boron Neutron Capture Therapy) protocols can be determined through the quantification and analysis of the tracks forming its autoradiography image on a nuclear track detector. The location of boron atoms in the cell structure could be known more accurately by the simultaneous observation of the nuclear tracks and the sample image on the detector.

Materials and Methods

A UV C irradiator was constructed. The irradiance was measured along the lamp direction and at different distances. Melanoma cells were cultured on polycarbonate foils, incubated with borophenylalanine, irradiated with thermal neutrons and exposed to UV C radiation. The samples were chemically attacked with a KOH solution.

Results

A uniform irradiation field was established to expose the detector foils to UV C light. Cells could be seeded on the polycarbonate surface. Both imprints from cells and nuclear tracks were obtained after chemical etching.

Conclusions

It is possible to yield cellular imprints in polycarbonate. The nuclear tracks were mostly present inside the cells, indicating a preferential boron uptake.     

Occupational Exposure to Ultraviolet Radiation and Risk of Non-Melanoma Skin Cancer in a Multinational European Study

Background

Studies suggest that ambient sunlight plays an important role in the pathogenesis of non-melanoma skin cancers (NMSC). However, there is ongoing controversy regarding the relevance of occupational exposure to natural and artificial ultraviolet radiation (UV) radiation.

Objectives

We investigated potential associations between natural and artificial UV radiation exposure at work with NMSC in a case-control study conducted in Hungary, Romania, and Slovakia.

Methods

Occupational exposures were classified by expert assessment for 527 controls and 618 NMSC cases (515 basal cell carcinoma, BCC). Covariate information was collected via interview and multiple logistic regression models were used to assess associations between UV exposure and NMSC.

Results

Lifetime prevalence of occupational exposure in the participants was 13% for natural UV radiation and 7% for artificial UV radiation. Significant negative associations between occupational exposure to natural UV radiation and NMSC were detected for all who had ever been exposed (odds ratio (OR) 0.47, 95% confidence interval (CI) 0.27–0.80); similar results were detected using a semi-quantitative metric of cumulative exposure. The effects were modified by skin complexion, with significantly decreased risks of BCC among participants with light skin complexion. No associations were observed in relation to occupational artificial UV radiation exposure.

Conclusions

The protective effect of occupational exposure to natural UV radiation was unexpected, but limited to light-skinned people, suggesting adequate sun-protection behaviors. Further investigations focusing on variations in the individual genetic susceptibility and potential interactions with environmental and other relevant factors are planned.     

Localization of Oxidized Low-Density Lipoprotein and Its Relation to Plaque Morphology in Human Coronary Artery

Objectives

Oxidized low-density lipoprotein (oxLDL) plays a key role in the formation of atherosclerotic plaques. However, its localization in human coronary arterial wall is not well understood. The present study was performed to visualize deposition sites and patterns of native oxLDL and their relation to plaque morphology in human coronary artery.

Methods

Evans blue dye (EB) elicits a violet fluorescence by excitation at 345-nm and emission at 420-nm, and a reddish-brown fluorescence by excitation at 470-nm and emission at 515-nm characteristic of oxLDL only. Therefore, native oxLDL in excised human coronary artery were investigated by color fluorescent microscopy (CFM) using EB as a biomarker.

Results

(1) By luminal surface scan with CFM, the % incidence of oxLDL in 38 normal segments, 41 white plaques and 32 yellow plaques that were classified by conventional angioscopy, was respectively 26, 44 and 94, indicating significantly (p<0.05) higher incidence in the latter than the former two groups. Distribution pattern was classified as patchy, diffuse and web-like. Web-like pattern was observed only in yellow plaques with necrotic core. (2) By transected surface scan, oxLDL deposited within superficial layer in normal segments and diffusely within both superficial and deep layers in white and yellow plaques. In yellow plaques with necrotic core, oxLDL deposited not only in the marginal zone of the necrotic core but also in the fibrous cap.

Conclusion

Taken into consideration of the well-known process of coronary plaque growth, the results suggest that oxLDL begins to deposit in human coronary artery wall before plaque formation and increasingly deposits with plaque growth, exhibiting different deposition sites and patterns depending on morphological changes.     

Ultraviolet-C Irradiation: A Novel Pasteurization Method for Donor Human Milk

Background

Holder pasteurization (milk held at 62.5°C for 30 minutes) is the standard treatment method for donor human milk. Although this method of pasteurization is able to inactivate most bacteria, it also inactivates important bioactive components. Therefore, the objective of this study was to investigate ultraviolet irradiation as an alternative treatment method for donor human milk.

Methods

Human milk samples were inoculated with five species of bacteria and then UV-C irradiated. Untreated and treated samples were analysed for bacterial content, bile salt stimulated lipase (BSSL) activity, alkaline phosphatase (ALP) activity, and fatty acid profile.

Results

All five species of bacteria reacted similarly to UV-C irradiation, with higher dosages being required with increasing concentrations of total solids in the human milk sample. The decimal reduction dosage was 289±17 and 945±164 J/l for total solids of 107 and 146 g/l, respectively. No significant changes in the fatty acid profile, BSSL activity or ALP activity were observed up to the dosage required for a 5-log₁₀ reduction of the five species of bacteria.

Conclusion

UV-C irradiation is capable of reducing vegetative bacteria in human milk to the requirements of milk bank guidelines with no loss of BSSL and ALP activity and no change of FA.     

Bacterial inactivation of wound infection in a human skin model by liquid-phase discharge plasma

Background

We investigate disinfection of a reconstructed human skin model contaminated with biofilm-formative Staphylococcus aureus employing plasma discharge in liquid.

Principal Findings

We observed statistically significant 3.83-log10 (p<0.001) and 1.59-log10 (p<0.05) decreases in colony forming units of adherent S. aureus bacteria and 24 h S. aureus biofilm culture with plasma treatment. Plasma treatment was associated with minimal changes in histological morphology and tissue viability determined by means of MTT assay. Spectral analysis of the plasma discharge indicated the presence of highly reactive atomic oxygen radicals (777 nm and 844 nm) and OH bands in the UV region. The contribution of these and other plasma-generated agents and physical conditions to the reduction in bacterial load are discussed.

Conclusions

These findings demonstrate the potential of liquid plasma treatment as a potential adjunct therapy for chronic wounds.     

Sterilizing activity of second-line regimens containing TMC207 in a murine model of tuberculosis

Rationale

The sterilizing activity of the regimen used to treat multidrug resistant tuberculosis (MDR TB) has not been studied in a mouse model.

Objective and Methods

Swiss mice were intravenously inoculated with 6 log10 of Mycobacterium tuberculosis (TB) strain H37Rv, treated with second-line drug combinations with or without the diarylquinoline TMC207, and then followed without treatment for 3 more months to determine relapse rates (modified Cornell model).

Measurements

Bactericidal efficacy was assessed by quantitative lung colony-forming unit (CFU) counts. Sterilizing efficacy was assessed by measuring bacteriological relapse rates 3 months after the end of treatment.

Main Results

The relapse rate observed after 12 months treatment with the WHO recommended MDR TB regimen (amikacin, ethionamide, pyrazinamide and moxifloxacin) was equivalent to the relapse rate observed after 6 months treatment with the recommended drug susceptible TB regimen (rifampin, isoniazid and pyrazinamide). When TMC207 was added to this MDR TB regimen, the treatment duration needed to reach the same relapse rate dropped to 6 months. A similar relapse rate was also obtained with a 6-month completely oral regimen including TMC207, moxifloxacin and pyrazinamide but excluding both amikacin and ethionamide.

Conclusions

In this murine model the duration of the WHO MDR TB treatment could be reduced to 12 months instead of the recommended 18–24 months. The inclusion of TMC207 in the WHO MDR TB treatment regimen has the potential to further shorten the treatment duration and at the same time to simplify treatment by eliminating the need to include an injectable aminoglycoside.     

Prevention of UVA-Induced Oxidative Damage in Human Dermal Fibroblasts by New UV Filters,Assessed Using a Novel In Vitro Experimental System

Background

UVA rays present in sunlight are able to reach the dermal skin layer generating reactive oxygen species (ROS) responsible for oxidative damage, alterations in gene expression, DNA damage, leading to cell inflammation, photo-ageing/-carcinogenesis. Sunscreens contain UV filters as active ingredients that absorb/reflect/dissipate UV radiation: their efficiency depends on their spectral profile and photostability which should then be reflected in biological protection of underlying skin.

Methods

A set of new UV filters was synthesized, and the most photostable one was compared to BMDBM, a widely used UVA filter. Cultured human dermal fibroblasts were exposed to UVA radiation which was filtered by a base cream containing or not UV filters placed above cell culture wells. The endpoints measured were: cell viability (MTT assay), ROS generation (DCFH-DA assay), mitochondrial function (JC-1 assay), DNA integrity (Comet assay) and gene expression (MMP-1, COL1A1) by RT-qPCR.

Results

The new UV filter resulted more efficient than BMDBM in preserving cell viability, mitochondrial functionality and oxidative DNA damage, despite similar inhibition levels of intracellular ROS. Moreover, expression of genes involved in dermal photoageing were positively affected by the filtering action of the tested molecules.

Conclusions

The experimental model proposed was able to validate the efficacy of the new UV filter, taking into account important cellular events related to UV-induced intracellular oxidative stress, often underestimated in the assessments of these compounds.

General Significance

The model may be used to compare the actual biological protection of commercial sunscreens and suncare products aside from their SPF and UVA-PF values.     

Comparison of therapeutic effects between pulsed and continuous wave 810-nm wavelength laser irradiation for traumatic brain injury in mice

Background and Objective

Transcranial low-level laser therapy (LLLT) using near-infrared light can efficiently penetrate through the scalp and skull and could allow non-invasive treatment for traumatic brain injury (TBI). In the present study, we compared the therapeutic effect using 810-nm wavelength laser light in continuous and pulsed wave modes in a mouse model of TBI.

Study Design/Materials and Methods

TBI was induced by a controlled cortical-impact device and 4-hours post-TBI 1-group received a sham treatment and 3-groups received a single exposure to transcranial LLLT, either continuous wave or pulsed at 10-Hz or 100-Hz with a 50% duty cycle. An 810-nm Ga-Al-As diode laser delivered a spot with diameter of 1-cm onto the injured head with a power density of 50-mW/cm² for 12-minutes giving a fluence of 36-J/cm². Neurological severity score (NSS) and body weight were measured up to 4 weeks. Mice were sacrificed at 2, 15 and 28 days post-TBI and the lesion size was histologically analyzed. The quantity of ATP production in the brain tissue was determined immediately after laser irradiation. We examined the role of LLLT on the psychological state of the mice at 1 day and 4 weeks after TBI using tail suspension test and forced swim test.

Results

The 810-nm laser pulsed at 10-Hz was the most effective judged by improvement in NSS and body weight although the other laser regimens were also effective. The brain lesion volume of mice treated with 10-Hz pulsed-laser irradiation was significantly lower than control group at 15-days and 4-weeks post-TBI. Moreover, we found an antidepressant effect of LLLT at 4-weeks as shown by forced swim and tail suspension tests.

Conclusion

The therapeutic effect of LLLT for TBI with an 810-nm laser was more effective at 10-Hz pulse frequency than at CW and 100-Hz. This finding may provide a new insight into biological mechanisms of LLLT.     

Sentinel node biopsy in breast cancer using infrared laser system first experience with PDE camera

Background

Sentinel node biopsy (SNB) is a gold standard in staging of early breast cancer. Nowadays, routine mapping of lymphatic tract is based on two tracers: human albumin with radioactive technetium, with or without blue dye. Recent years have seen a search for new tracers to examine sentinel node as well as lymphatic network. One of them is indocyanine green (ICG) visible in infrared light.

Aim

The aim of this study is to evaluate clinical usage of ICG in comparison with standard tracer, i.e. nanocoll, in SNB of breast cancer patients.

Materials and methods

In the 1st Department of Surgical Oncology and General Surgery, Greater Poland Cancer Centre, Poznań, 13 female breast cancer patients have benn operated since September 2010. All these patients had sentinel node biopsy with nanocoll (human albumin with radioactive technetium), and with indocyanine green. The feasibility of this new method was assessed in comparison with the standard nanocoll.

Results

A lymphatic network between the place of injection of ICG and sentinel node was seen in infrared light. An area where a sentinel node was possibly located was confirmed by gamma probe. Sensitivity of this method was 100%.

Conclusion

SNB using ICG is a new, promising diagnostics technique. This procedure is not without drawbacks; nevertheless it opens new horizons in lymphatic network diagnostics.     

Characteristic male urine microbiomes associate with asymptomatic sexually transmitted infection

Background

The microbiome of the male urogenital tract is poorly described but it has been suggested that bacterial colonization of the male urethra might impact risk of sexually transmitted infection (STI). Previous cultivation-dependent studies showed that a variety of non-pathogenic bacteria colonize the urethra but did not thoroughly characterize these microbiomes or establish links between the compositions of urethral microbiomes and STI.

Methodology/Findings

Here, we used 16S rRNA PCR and sequencing to identify bacteria in urine specimens collected from men who lacked symptoms of urethral inflammation but who differed in status for STI. All of the urine samples contained multiple bacterial genera and many contained taxa that colonize the human vagina. Uncultivated bacteria associated with female genital tract pathology were abundant in specimens from men who had STI.

Conclusions

Urine microbiomes from men with STI were dominated by fastidious, anaerobic and uncultivated bacteria. The same taxa were rare in STI negative individuals. Our findings suggest that the composition of male urine microbiomes is related to STI.     

Non-Visual Effects of Light on Melatonin,Alertness and Cognitive Performance: Can Blue-Enriched Light Keep Us Alert?

Background

Light exposure can cascade numerous effects on the human circadian process via the non-imaging forming system, whose spectral relevance is highest in the short-wavelength range. Here we investigated if commercially available compact fluorescent lamps with different colour temperatures can impact on alertness and cognitive performance.

Methods

Sixteen healthy young men were studied in a balanced cross-over design with light exposure of 3 different light settings (compact fluorescent lamps with light of 40 lux at 6500K and at 2500K and incandescent lamps of 40 lux at 3000K) during 2 h in the evening.

Results

Exposure to light at 6500K induced greater melatonin suppression, together with enhanced subjective alertness, well-being and visual comfort. With respect to cognitive performance, light at 6500K led to significantly faster reaction times in tasks associated with sustained attention (Psychomotor Vigilance and GO/NOGO Task), but not in tasks associated with executive function (Paced Visual Serial Addition Task). This cognitive improvement was strongly related with attenuated salivary melatonin levels, particularly for the light condition at 6500K.

Conclusions

Our findings suggest that the sensitivity of the human alerting and cognitive response to polychromatic light at levels as low as 40 lux, is blue-shifted relative to the three-cone visual photopic system. Thus, the selection of commercially available compact fluorescent lights with different colour temperatures significantly impacts on circadian physiology and cognitive performance at home and in the workplace.     

Bioinformatics analysis of macrophages exposed to Porphyromonas gingivalis: implications in acute vs. chronic infections

Background

Periodontitis is the most common human infection affecting tooth-supporting structures. It was shown to play a role in aggravating atherosclerosis. To deepen our understanding of the pathogenesis of this disease, we exposed human macrophages to an oral bacteria, Porphyromonas gingivalis (P. gingivalis), either as live bacteria or its LPS or fimbria. Microarray data from treated macrophages or control cells were analyzed to define molecular signatures. Changes in genes identified in relevant pathways were validated by RT-PCR.

Methodology/Principal Findings

We focused our analysis on three important groups of genes. Group PG (genes differentially expressed by live bacteria only); Group LFG (genes differentially expressed in response to exposure to LPS and/or FimA); Group CG (core gene set jointly activated by all 3 stimulants). A total of 842 macrophage genes were differentially expressed in at least one of the three conditions compared to naïve cells. Using pathway analysis, we found that group CG activates the initial phagocytosis process and induces genes relevant to immune response, whereas group PG can de-activate the phagocytosis process associated with phagosome-lysosome fusion. LFG mostly affected RIG-I-like receptor signaling pathway.

Conclusion/Significance

In light of the fact that acute infections involve live bacteria while chronic infections involve a combination of live bacteria and their byproducts, group PG could represent acute P. gingivalis infection while group LFG could represent chronic P. gingivalis infection. Group CG may be associated with core immune pathways, triggered irrespective of the specific stimulants and indispensable to mount an appropriate immune response. Implications in acute vs. chronic infection are discussed.     

Impaired release of antimicrobial peptides into nasal fluid of hyper-IgE and CVID patients

Background

Patients with primary immunodeficiency (PID) often suffer from frequent respiratory tract infections. Despite standard treatment with IgG-substitution and antibiotics many patients do not improve significantly. Therefore, we hypothesized that additional immune deficits may be present among these patients.

Objective

To investigate if PID patients exhibit impaired production of antimicrobial peptides (AMPs) in nasal fluid and a possible link between AMP-expression and Th17-cells.

Methods

Nasal fluid, nasopharyngeal swabs and peripheral blood mononuclear cells (PBMCs) were collected from patients and healthy controls. AMP levels were measured in nasal fluid by Western blotting. Nasal swabs were cultured for bacteria. PBMCs were stimulated with antigen and the supernatants were assessed for IL-17A release by ELISA.

Results

In healthy controls and most patients, AMP levels in nasal fluid were increased in response to pathogenic bacteria. However, this increase was absent in patients with common variable immunodeficiency (CVID) and Hyper-IgE syndrome (HIES), despite the presence of pathogenic bacteria. Furthermore, stimulation of PBMCs revealed that both HIES and CVID patients exhibited an impaired production of IL-17A.

Conclusion

CVID and HIES patients appear to have a dysregulated AMP response to pathogenic bacteria in the upper respiratory tract, which could be linked to an aberrant Th17 cell response.     

The interplay between Entamoeba and enteropathogenic bacteria modulates epithelial cell damage

Background

Mixed intestinal infections with Entamoeba histolytica, Entamoeba dispar and bacteria with exacerbated manifestations of disease are common in regions where amoebiasis is endemic. However, amoeba–bacteria interactions remain largely unexamined.

Methodology

Trophozoites of E. histolytica and E. dispar were co-cultured with enteropathogenic bacteria strains Escherichia coli (EPEC), Shigella dysenteriae and a commensal Escherichia coli. Amoebae that phagocytosed bacteria were tested for a cytopathic effect on epithelial cell monolayers. Cysteine proteinase activity, adhesion and cell surface concentration of Gal/GalNAc lectin were analyzed in amoebae showing increased virulence. Structural and functional changes and induction of IL-8 expression were determined in epithelial cells before and after exposure to bacteria. Chemotaxis of amoebae and neutrophils to human IL-8 and conditioned culture media from epithelial cells exposed to bacteria was quantified.

Principal Findings

E. histolytica digested phagocytosed bacteria, although S. dysenteriae retained 70% viability after ingestion. Phagocytosis of pathogenic bacteria augmented the cytopathic effect of E. histolytica and increased expression of Gal/GalNAc lectin on the amoebic surface and increased cysteine proteinase activity. E. dispar remained avirulent. Adhesion of amoebae and damage to cells exposed to bacteria were increased. Additional increases were observed if amoebae had phagocytosed bacteria. Co-culture of epithelial cells with enteropathogenic bacteria disrupted monolayer permeability and induced expression of IL-8. Media from these co-cultures and human recombinant IL-8 were similarly chemotactic for neutrophils and E. histolytica.

Conclusions

Epithelial monolayers exposed to enteropathogenic bacteria become more susceptible to E. histolytica damage. At the same time, phagocytosis of pathogenic bacteria by amoebae further increased epithelial cell damage.

Significance

The in vitro system presented here provides evidence that the Entamoeba/enteropathogenic bacteria interplay modulates epithelial cell responses to the pathogens. In mixed intestinal infections, where such interactions are possible, they could influence the outcome of disease. The results offer insights to continue research on this phenomenon.     

The Added Diagnostic Value of Dynamic Contrast-Enhanced MRI at 3.0 T in Nonpalpable Breast Lesions

Objective

To investigate the added diagnostic value of 3.0 Tesla breast MRI over conventional breast imaging in the diagnosis of in situ and invasive breast cancer and to explore the role of routine versus expert reading.

Materials and Methods

We evaluated MRI scans of patients with nonpalpable BI-RADS 3–5 lesions who underwent dynamic contrast-enhanced 3.0 Tesla breast MRI. Initially, MRI scans were read by radiologists in a routine clinical setting. All histologically confirmed index lesions were re-evaluated by two dedicated breast radiologists. Sensitivity and specificity for the three MRI readings were determined, and the diagnostic value of breast MRI in addition to conventional imaging was assessed. Interobserver reliability between the three readings was evaluated.

Results

MRI examinations of 207 patients were analyzed. Seventy-eight of 207 (37.7%) patients had a malignant lesion, of which 33 (42.3%) patients had pure DCIS and 45 (57.7%) invasive breast cancer. Sensitivity of breast MRI was 66.7% during routine, and 89.3% and 94.7% during expert reading. Specificity was 77.5% in the routine setting, and 61.0% and 33.3% during expert reading. In the routine setting, MRI provided additional diagnostic information over clinical information and conventional imaging, as the Area Under the ROC Curve increased from 0.76 to 0.81. Expert MRI reading was associated with a stronger improvement of the AUC to 0.87. Interobserver reliability between the three MRI readings was fair and moderate.

Conclusions

3.0 T breast MRI of nonpalpable breast lesions is of added diagnostic value for the diagnosis of in situ and invasive breast cancer.     

Molecular Imaging of Low-density Lipoprotein in Human Coronary Plaques by Color Fluorescent Angioscopy and Microscopy

Objectives

Low-density lipoprotein (LDL) is an important risk factor for coronary artery disease. However, its localization in human coronary plaques is not well understood. The present study was performed to visualize LDL in human coronary artery wall.

Methods

(1) The fluorescence characteristic of LDL was investigated by color fluorescent microscopy (CFM) with excitation at 470-nm and emission at 515-nm using Nile blue dye (NB) as a biomarker. (2) Native LDL in 40 normal segments, 42 white plaques and 35 yellow plaques (20 with necrotic core) of human coronary arteries was investigated by color fluorescent angioscopy (CFA) and CFM.

Results

(1) NB elicited a brown, golden and red fluorescence characteristic of LDL, apolipoprotein B-100, and lysophosphatidylcholine/triglyceride, respectively. (2) The % incidence of LDL in normal segments, white, and yellow plaques was 25, 38 and 14 by CFA and 42, 42 and 14 by CFM scan of their luminal surface, respectively, indicating lower incidence (p<0.05) of LDL in yellow plaques than white plaques, and no significant differences in detection sensitivity between CFA and CFM. By CFM transected surface scan, LDL deposited more frequently and more diffusely in white plaques and yellow plaques without necrotic core (NC) than normal segments and yellow plaques with NC. LDL was localized to fibrous cap in yellow plaques with NC. Co-deposition of LDL with other lipid components was observed frequently in white plaques and yellow plaques without NC.

Conclusions

(1) Taken into consideration of the well-known process of coronary plaque growth, the results of the present study suggest that LDL begins to deposit before plaque formation; increasingly deposits with plaque growth, often co-depositing with other lipid components; and disappears after necrotic core formation. (2) CFA is feasible for visualization of LDL in human coronary artery wall.     

Nanoparticle-Encapsulated Chlorhexidine against Oral Bacterial Biofilms

Background

Chlorhexidine (CHX) is a widely used antimicrobial agent in dentistry. Herein, we report the synthesis of a novel mesoporous silica nanoparticle-encapsulated pure CHX (Nano-CHX), and its mechanical profile and antimicrobial properties against oral biofilms.

Methodology/Principal Findings

The release of CHX from the Nano-CHX was characterized by UV/visible absorption spectroscopy. The antimicrobial properties of Nano-CHX were evaluated in both planktonic and biofilm modes of representative oral pathogenic bacteria. The Nano-CHX demonstrated potent antibacterial effects on planktonic bacteria and mono-species biofilms at the concentrations of 50–200 µg/mL against Streptococcus mutans, Streptococcus sobrinus, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Enterococccus faecalis. Moreover, Nano-CHX effectively suppressed multi-species biofilms such as S. mutans, F. nucleatum, A. actinomycetemcomitans and Porphyromonas gingivalis up to 72 h.

Conclusions/Significance

This pioneering study demonstrates the potent antibacterial effects of the Nano-CHX on oral biofilms, and it may be developed as a novel and promising anti-biofilm agent for clinical use.     

Thioredoxin 80-activated-monocytes (TAMs) inhibit the replication of intracellular pathogens

Background

Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced primarily by monocytes. Trx80 induces differentiation of human monocytes into a novel cell type, named Trx80-activated-monocytes (TAMs).

Principal Findings

In this investigation we present evidence for a role of TAMs in the control of intracellular bacterial infections. As model pathogens we have chosen Listeria monocytogenes and Brucella abortus which replicate in the cytosol and the endoplasmic reticulum respectively. Our data indicate that TAMs efficiently inhibit intracellular growth of both L. monocytogenes and B. abortus. Further analysis shows that Trx80 activation prevents the escape of GFP-tagged L. monocytogenes into the cytosol, and induces accumulation of the bacteria within the lysosomes. Inhibition of the lysosomal activity by chloroquine treatment resulted in higher replication of bacteria in TAMs compared to that observed in control cells 24 h post-infection, indicating that TAMs kill bacteria by preventing their escape from the endosomal compartments, which progress into a highly degradative phagolysosome.

Significance

Our results show that Trx80 potentiates the bactericidal activities of professional phagocytes, and contributes to the first line of defense against intracellular bacteria.     

Ethanol Impairs Intestinal Barrier Function in Humans through Mitogen Activated Protein Kinase Signaling: A Combined In Vivo and In Vitro Approach

Background

Ethanol-induced gut barrier disruption is associated with several gastrointestinal and liver disorders.

Aim

Since human data on effects of moderate ethanol consumption on intestinal barrier integrity and involved mechanisms are limited, the objectives of this study were to investigate effects of a single moderate ethanol dose on small and large intestinal permeability and to explore the role of mitogen activated protein kinase (MAPK) pathway as a primary signaling mechanism.

Methods

Intestinal permeability was assessed in 12 healthy volunteers after intraduodenal administration of either placebo or 20 g ethanol in a randomised cross-over trial. Localization of the tight junction (TJ) and gene expression, phosphorylation of the MAPK isoforms p38, ERK and JNK as indicative of activation were analyzed in duodenal biopsies. The role of MAPK was further examined in vitro using Caco-2 monolayers.

Results

Ethanol increased small and large intestinal permeability, paralleled by redistribution of ZO-1 and occludin, down-regulation of ZO-1 and up-regulation of myosin light chain kinase (MLCK) mRNA expression, and increased MAPK isoforms phosphorylation. In Caco-2 monolayers, ethanol increased permeability, induced redistribution of the junctional proteins and F-actin, and MAPK and MLCK activation, as indicated by phosphorylation of MAPK isoforms and myosin light chain (MLC), respectively, which could be reversed by pretreatment with either MAPK inhibitors or the anti-oxidant L-cysteine.

Conclusions

Administration of moderate ethanol dosage can increase both small and colon permeability. Furthermore, the data indicate a pivotal role for MAPK and its crosstalk with MLCK in ethanol-induced intestinal barrier disruption.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00928733","term_id":"NCT00928733"}}NCT00928733