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Tuberculosis (TB) alone caused over a billion deaths in the last 200 years, making it one of the deadliest diseases to humankind. Understanding the immune mechanisms underlying protection or pathology in TB is key to uncover the much needed innovative approaches to tackle TB. The scavenger receptor cysteine-rich molecule CD5 antigen-like (CD5L) has been associated with TB, but whether and how CD5L shapes the immune response during the course of disease remains poorly understood. Here, we show an upregulation of CD5L in circulation and at the site of infection in C57BL/6 Mycobacterium tuberculosis-infected mice. To investigate the role of CD5L in TB, we studied the progression of M. tuberculosis aerosol infection in a recently described genetically engineered mouse model lacking CD5L. Despite the increase of CD5L during infection of wild-type mice, absence of CD5L did not impact bacterial burden, histopathology or survival of infected mice. Absence of CD5L associated with a modest increase in the numbers of CD4+ T cells and the expression of IFN-γ in the lungs of infected mice, with no major effect in overall immune cell dynamics. Collectively, this study confirms CD5L as a potential diagnostic biomarker to TB, showing no discernible impact on the outcome of the infection., (© 2024 John Wiley & Sons Ltd.)

Background: The success of haemodialysis (HD) critically depends on the effective use of arteriovenous fistulas (AVFs). The precise needling technique is vital to minimise complications and ensure functional vascular access., Objective: This study assesses the effectiveness of a nursing consultation protocol, which integrates physical examination (PE) with Doppler Ultrasound (DUS), in preparing patients for the first AVF needling., Design/participants: A cross-sectional analysis at a Portuguese National Health Service Hospital engaged thirty new HD patients, four HD needling experienced nurses and one HD vascular access nurse. This study examines the accuracy of PE in assessing the matured AVF by the four nurses compared to a trained vascular access nurse encompassing systematic PE and DUS., Measurements: The primary data incorporated AVF characteristics derived from PE (inspection, palpation, and auscultation) and DUS findings (vein depth, diameter, and blood flow). A secondary focus was evaluating the change in nurses' perceived needling complexity following the nursing consultation., Results: The nursing consultation significantly enhanced the identification of crucial AVF features, such as accessory veins (p = 0.002), and improved the accuracy of AVF morphology assessments. This led to identifying longer needling tracks (p = 0.031) and a higher number of safe needling points (p = 0.016). Nurses reported a notable reduction in perceived complexity and potential adverse events following this method (p = 0.027)., Conclusions: Integrating structured PE with DUS in a nursing consultation framework significantly improves the preparation for AVF needling. This approach enhances the efficiency and safety of AVF needling and boosts nurse confidence and patient care in HD settings., (© 2024 European Dialysis & Transplant Nurses Association/European Renal Care Association.)

Plastics are a cornerstone of the modern world, yet the durable material properties that we have come to depend upon have made them recalcitrant environmental pollutants. Biological solutions in the form of engineered enzymes offer low energy and sustainable approaches to recycle and upcycle plastic waste, uncoupling their production and end of life from fossil fuels and greenhouse gases. These enzymes however, encounter immense challenges acting on plastics: facing hydrophobic surfaces, molecular crowding, and high levels of substrate heterogeneity. There have been mixed reports about the benefits of fusing partner domains to polyethylene terephthalate (PET) degrading enzymes, with moderate improvements identified under specific conditions, but no clarity into the factors that underlie the mechanisms. Here, we use the SpyCatcher003:SpyTag003 technology, which demonstrates a profound 47 °C shift in T m upon irreversible complex formation, to investigate the influence of the thermal stability of the fusion partner on a range of PETases selected for their optimal reaction temperatures. We find that the thermal stability of the fusion partner does not have a positive correlation on the activity of the enzymes or their evident kinetic and thermal stabilities. Instead, it appears that the fusion to less stable SpyCatcher003 tends to increase the measured activation energy of unfolding compared to the more stable complex and wildtype enzymes. Despite this, the fusions to SpyCatcher003 do not show significantly better catalytic activity on PET films, with or without SpyTag003, and were found to be sometimes disruptive. The approach we highlight here, in using a fusion partner with controllable melting temperature, allowed us to dissect the impact of the stability of a fusion partner on enzyme properties. Although fusion stability did not appear to be coupled with identifiable trends in enzymatic activities, careful analysis of the unfolding pathways, and solid and solution activities of a wider range of enzymes may yield a more detailed understanding.

Background: T-cell membrane scaffold proteins are pivotal in T cell function, acting as versatile signaling hubs. While CD6 forms a large intracellular signalosome, it is distinguished from typical scaffolds like LAT or PAG by possessing a substantial ectodomain that binds CD166, a well-characterized ligand expressed on most antigen-presenting cells (APC), through the third domain (d3) of the extracellular region. Although the intact form of CD6 is the most abundant in T cells, an isoform lacking d3 (CD6∆d3) is transiently expressed on activated T cells. Still, the precise character of the signaling transduced by CD6, whether costimulatory or inhibitory, and the influence of its ectodomain on these activities are unclear., Methods: We expressed CD6 variants with extracellular deletions or cytosolic mutations in Jurkat cells containing eGFP reporters for NF-κB and NF-AT transcription factor activation. Cell activation was assessed by eGFP flow cytometry following Jurkat cell engagement with superantigen-presenting Raji cells. Using imaging flow cytometry, we evaluated the impact of the CD6-CD166 pair on cell adhesiveness during the antigen-dependent and -independent priming of T cells. We also examined the role of extracellular or cytosolic sequences on CD6 translocation to the immunological synapse, using immunofluorescence-based imaging., Results: Our investigation dissecting the functions of the extracellular and cytosolic regions of CD6 revealed that CD6 was trafficked to the immunological synapse and exerted tonic inhibition wholly dependent on its cytosolic tail. Surprisingly, however, translocation to the synapse occurred independently of the extracellular d3 and of engagement to CD166. On the other hand, CD6 binding to CD166 significantly increased T cell:APC adhesion. However, this activity was most evident in the absence of APC priming with superantigen, and thus, in the absence of TCR engagement., Conclusions: Our study identifies CD6 as a novel 'on/off' scaffold-receptor capable of modulating responsiveness in two ways. Firstly, and independently of ligand binding, it establishes signaling thresholds through tonic inhibition, functioning as a membrane-bound scaffold. Secondly, CD6 has the capacity for alternative splicing-dependent variable ligand engagement, modulating its checkpoint-like activity., (© 2024. The Author(s).)

Sepsis results from systemic, dysregulated inflammatory responses to infection, culminating in multiple organ failure. Here, we demonstrate the utility of CD5L for treating experimental sepsis caused by cecal ligation and puncture (CLP). We show that CD5L's important features include its ability to enhance neutrophil recruitment and activation by increasing circulating levels of CXCL1, and to promote neutrophil phagocytosis. CD5L-deficient mice exhibit impaired neutrophil recruitment and compromised bacterial control, rendering them susceptible to attenuated CLP. CD5L -/- peritoneal cells from mice subjected to medium-grade CLP exhibit a heightened pro-inflammatory transcriptional profile, reflecting a loss of control of the immune response to the infection. Intravenous administration of recombinant CD5L (rCD5L) in immunocompetent C57BL/6 wild-type (WT) mice significantly ameliorates measures of disease in the setting of high-grade CLP-induced sepsis. Furthermore, rCD5L lowers endotoxin and damage-associated molecular pattern (DAMP) levels, and protects WT mice from LPS-induced endotoxic shock. These findings warrant the investigation of rCD5L as a possible treatment for sepsis in humans., (© 2024. The Author(s).)

Background: Accurate tools to distinguish Crohn's disease [CD] from cryptoglandular disease in patients with perianal fistulas without detectable luminal inflammation on ileocolonoscopy and abdominal enterography (isolated perianal fistulas [IPF]) are lacking. We assessed the ability of video capsule endoscopy [VCE] to detect luminal inflammation in patients with IPF., Methods: We studied consecutive adults [>17 years] with IPF who were evaluated by VCE after a negative ileocolonoscopy and abdominal enterography between 2013 and 2022. We defined luminal CD by VCE as diffuse erythema, three or more aphthous ulcers, or a Lewis score greater than 135. We compared rates of intestinal inflammation in this cohort with age- and sex-matched controls without perianal fistulas, who underwent VCE for other indications. We excluded persons with pre-existing inflammatory bowel disease [IBD] and exposure to non-steroidal anti-inflammatory drugs or immunosuppressive treatments., Results: A total of 45 patients with IPF underwent VCE without complications. Twelve patients [26%] met our definition of luminal CD. Luminal CD was more common in patients with IPF than in controls [26% vs 3%; p <0.01]. Among patients with IPF, male sex (OR [odds ratio], 9.2; 95% confidence interval [CI] [1.1-79.4]), smoking (OR, 4.5; 95% CI [0.9-21.2]), abscess (OR, 6.3; 95% CI [1.5-26.8]), rectal enhancement on magnetic resonance imaging [MRI] (OR, 9.0; 95% CI [0.8-99.3]), and positive antimicrobial serology (OR, 7.1; 95% CI, [0.7-70.0]) were more common in those with a positive VCE study., Conclusions: VCE detected small intestinal inflammation suggestive of luminal CD in approximately one-quarter of patients with IPF. Larger studies are required to validate these findings., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: To evaluate the clinical response of parasacral transcutaneous electrical neural stimulation (parasacral TENS) associated with urotherapy in children with primary monosymptomatic nocturnal enuresis (PMNE) compared to urotherapy alone., Material and Methods: This prospective controlled clinical trial enrolled 72 children over 5 years of age with PMNE. Children were randomly divided into two groups, control group (CG), treated with urotherapy and scapular stimulation, and experimental group (EG), treated with urotherapy and parasacral TENS. In both groups, 20 sessions were performed, 3 times weekly, for 20 min each, with 10 Hz frequency, 700 μS pulse width and intesity determinated by the patient threshold. The percentages of dry nights were analyzed for 14 days before treatment (T0), after the 20th session (T1), 15 (T2), 30 (T3), 60 (T4), and 90 (T5) days after the end of the sessions. Patients of both groups were followed with intervals of 2 weeks in the first month and monthly for three consecutive months., Results: Twenty-eight enuretic children, 14 girls (50%) with a mean age of 9.09 ± 2.23 years completed the study. There was no difference in mean age between groups. Mean percentage of dry nights in EG at T0 was 36%, at T1 49%, at T2 54%, at T3 54%, at T4 54%, and 57% at T5; while in CG, these percentages were 28%, 39%, 37%, 35%, 36%, and 36%, respectively., Conclusions: Parasacral TENS associated with urotherapy improves the percentage of dry nights in children with PMNE, although no patient had complete resolution of symptoms in this study., (© 2023 Wiley Periodicals LLC.)

Enzyme-based depolymerization is a viable approach for recycling of poly(ethylene terephthalate) (PET). PETase from Ideonella sakaiensis (IsPETase) is capable of PET hydrolysis under mild conditions but suffers from concentration-dependent inhibition. In this study, this inhibition is found to be dependent on incubation time, the solution conditions, and PET surface area. Furthermore, this inhibition is evident in other mesophilic PET-degrading enzymes to varying degrees, independent of the level of PET depolymerization activity. The inhibition has no clear structural basis, but moderately thermostable IsPETase variants exhibit reduced inhibition, and the property is completely absent in the highly thermostable HotPETase, previously engineered by directed evolution, which simulations suggest results from reduced flexibility around the active site. This work highlights a limitation in applying natural mesophilic hydrolases for PET hydrolysis and reveals an unexpected positive outcome of engineering these enzymes for enhanced thermostability., (© 2023 The Authors. ChemSusChem published by Wiley-VCH GmbH.)

Introduction: Voiding diary (VD) is an important tool in the evaluation of children with voiding symptoms. Voiding frequency, maximal voided volume (MVV), average voided volume (AVV) and nocturnal volume (NV) can be extracted and are valuable in diagnosing and monitoring these disorders. Recently, ICCS has reduced the period of data recording on VD from 3 to 2 days.We hypothesized that one day voiding diary would be enough for guiding treatment., Materials and Methods: Children with overactive bladder (OAB) and primary monosymptomatic enuresis (PMNE) were oriented to fulfill a 3-day VD. Data obtained from VD were evaluated for the first day (1dVD), the first two days (2dVD), and all 3 days (3dVD) and compared according to the MVV, AVV, frequency, NV and expected bladder capacity (EBC). The Friedman, Student's t test and the Fisher's exact was used. ANOVA was used for multiple comparisons. We also used Pearson correlation test., Results: Ninety-eight children were included, 59 had PMNE and 30 OAB. Frequency, AVV and VN were similar regardless how many days the voiding episodes were recorded. Only MVV was higher by a mean of only 32 mL on 3dVD compared to 1dVD. A 1dVD has a sensitivity of 93,9% and a positive likelihood ratio of 2.2. As for the correlation of MVV and EBC it was observed that in 83% of children, MVV was lower than EBC. MVV corresponds to 67% and 69% of EBC in children with PMNE and OAB, respectively., Conclusion: We believe that 1dVD is sufficient to assess these children. It has a high sensitivity and good correlation to 3dVD in evaluating these children. Bladder capacity in this population, evaluated by maximum voided volume, was close to 68% of that obtained by the EBC., Competing Interests: None declared., (Copyright® by the International Brazilian Journal of Urology.)

Background: CD6 is one of many cell surface receptors known to regulate signal transduction upon T cell activation. However, whether CD6 mediates costimulatory or inhibitory signals is controversial. When T cells engage with antigen presenting cells (APCs), CD6 interacts with its ligand CD166 at the cell-cell interface while the cytosolic tail assembles a complex signalosome composed of adaptors and effector enzymes, that may either trigger activating signaling cascades, or instead modulate the intensity of signaling. Except for a few cytosolic adaptors that connect different components of the CD6 signalosome, very little is known about the mechanistic effects of the cytosolic effectors that bind CD6., Methods: Jurkat model T cells were transfected to express wild-type (WT) CD6, or a cytoplasmic truncation, signaling-disabled mutant, CD6Δcyt. The two resulting cell lines were directly activated by superantigen (sAg)-loaded Raji cells, used as APCs, to assess the net signaling function of CD6. The Jurkat cell lines were further adapted to express a FRET-based unimolecular HRas biosensor that reported the activity of this crucial GTPase at the immunological synapse., Results: We show that deletion of the cytosolic tail of CD6 enhances T-cell responses, indicating that CD6 restrains T-cell activation. One component of the CD6-associated inhibitory apparatus was found to be the GTPase activating protein of Ras (RasGAP), that we show to associate with CD6 in a phosphorylation-dependent manner. The FRET HRas biosensor that we developed was demonstrated to be functional and reporting the activation of the T cell lines. This allowed to determine that the presence of the cytosolic tail of CD6 results in the down-regulation of HRas activity at the immunological synapse, implicating this fundamental GTPase as one of the targets inhibited by CD6., Conclusions: This study provides the first description of a mechanistic sequence of events underlying the CD6-mediated inhibition of T-cell activation, involving the modulation of the MAPK pathway at several steps, starting with the coupling of RasGAP to the CD6 signalosome, the repression of the activity of Ras, and culminating in the reduction of ERK1/2 phosphorylation and of the expression of the T-cell activation markers CD69 and IL-2R α chain. Video abstract., (© 2022. The Author(s).)

Since the pioneering discoveries, by the Nobel laureates Jules Hoffmann and Bruce Beutler, that Toll and Toll-like receptors can sense pathogenic microorganisms and initiate, in vertebrates and invertebrates, innate immune responses against microbial infections, many other families of pattern recognition receptors (PRRs) have been described. One of such receptor clusters is composed by, if not all, at least several members of the scavenger receptor cysteine-rich (SRCR) superfamily. Many SRCR proteins are plasma membrane receptors of immune cells; however, a small subset consists of secreted receptors that are therefore in circulation. We here describe the first characterization of biological and functional roles of the circulating human protein SSC4D, one of the least scrutinized members of the family. Within leukocyte populations, SSC4D was found to be expressed by monocytes/macrophages, neutrophils, and B cells, but its production was particularly evident in epithelial cells of several organs and tissues, namely, in the kidney, thyroid, lung, placenta, intestinal tract, and liver. Similar to other SRCR proteins, SSC4D shows the capacity of physically binding to different species of bacteria, and this opsonization can increase the phagocytic capacity of monocytes. Importantly, we have uncovered the capacity of SSC4D of binding to several protozoan parasites, a singular feature seldom described for PRRs in general and here demonstrated for the first time for an SRCR family member. Overall, our study is pioneer in assigning a PRR role to SSC4D., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cardoso, Santos, Almeida, Sá, Pérez-Cabezas, Oliveira, Tavares and Carmo.)

Body temperature changes in laboratory mice are often assessed by invasive and stressful methods, which may confound the measurement. Infrared thermography is a possible non-invasive alternative, but the cost of standard thermal cameras, lack of dedicated software for biomedical purposes, and labour-intensiveness of thermal image analysis have limited their use. An additional limitation lies on the scarcity of research on the causing factors of differences between body surface and core body temperature. We propose a method for automatic assessment of mean body surface temperature in freely-moving mice, using dedicated software for thermal image analysis. While skin surface temperature may not necessarily be linearly correlated with core body temperature (in itself an imprecise concept), under standardized environmental conditions, such as those in which laboratory animals are kept, mean body surface temperature can provide useful information on their thermal status (i.e. deviations from normothermia, namely hypo- and hyperthermia). We developed a publicly available software that includes an imaging analysis workflow/algorithm for automatic segmentation of the pixels associated with the animal from the pixels associated with the background, removing the need for manually defining the area of analysis. A batch analysis mode is also available, for automatic and high-throughput analysis of all image files located in a folder. The software is compatible with the most widespread thermal camera manufacturer, 'FLIR Systems', as well as with the low-cost 'Thermal Expert TE-Q1' miniaturized high-resolution thermal camera used for this study. Furthermore, the software has been validated in a mouse model expressing non-transient hypothermia, where the thermal analysis results were compared with readings from implanted thermo-sensitive passive integrated transponders tags. Thermography allows for thermal assessment of laboratory animals without the effect of handling stress on their physiology or behaviour. Our automatic image analysis software also removes observer errors and bias, while speeding up the data processing., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The characterization of the architecture, structure and extracellular interactions of the CD6 glycoprotein, a transmembrane receptor expressed in medullary thymocytes and all mature T-cell populations, has been enhanced by the existence of monoclonal antibodies (mAbs) that specifically recognize the various scavenger receptor cysteine-rich (SRCR) domains of the ectodomain. Using engineered isoforms of CD6 including or excluding each of the three SRCR domains, either expressed at the membranes of cells or in soluble forms, we provide conclusive and definitive evidence that domain 2 of CD6, previously not identifiable, can be recognized by the CD6 mAbs OX125 and OX126, and that OX124 targets domain 3 and can block the interaction at the cell surface of CD6 with its major ligand CD166. Alternative splicing-dependent CD6 isoforms can now be confidently identified. We confirm that following T-cell activation there is a partial replacement of full-length CD6 by the CD6Δd3 isoform, which lacks the CD166-binding domain, and we find no evidence for the expression of other CD6 isoforms at the mRNA or protein levels., (© 2019 The Authors. Immunology Published by John Wiley & Sons Ltd.)

Introduction: Overactive bladder (OAB) is the most prevalent voiding disorder in childhood, and its main manifestation is urinary urgency. In general, urotherapy and anticholinergics are the first choices of treatment. Parasacral Transcutaneous Electrical Neural Stimulation (PTENS) was introduced as an alternative for the treatment of detrusor overactivity in children, but treatment protocols described to date require several sessions per week or long-lasting sessions, making it difficult for the child to adhere to the treatment. Thus, this study aims to evaluate the effectiveness of PTENS in single weekly sessions in the treatment of OAB in children., Study Design: This prospective, randomized controlled trial included 16 children with OAB. Children were divided into two groups: CG (urotherapy and electrical stimulation placebo) and EG (urotherapy and PTENS). For both groups, therapy was delivered in 20 weekly sessions, of duration 20 min each. Placebo electrical stimulation was done in the scapular area. The children were evaluated prior to treatment (T1), at the end of the 20 sessions (T2), and 60 days after the completion of treatment (T3), with a 3-day voiding diary, visual analogue scale (VAS), Rome III diagnostic criteria, and the Bristol Scale., Results: The groups were similar in age, gender, and ethnicity. In the initial assessment, all children, in both groups, had urgency and incontinence, 50% in each group had constipation, and enuresis was present in seven children (87.5%) in the EG and six (75%) in the CG. No differences were found between the groups regarding the volumetric measurements made in the voiding diary, urinary frequency and constipation evaluated by the Rome III criteria and the Bristol Scale. Sixty days after treatment, a significant improvement was found in the EG group (p = 0.03) regarding urgency (Table), as well as an increase in dry nights in those presenting with enuresis (p = 0.03). No difference was noted regarding urinary incontinence (Table). At the end of 20 sessions and after 60 days of treatment, those responsible for the children in the EG perceived greater improvement in symptoms measured by the VAS (p = 0.05 and 0.04, respectively)., Conclusions: Our preliminary results demonstrate that PTENS performed in single weekly sessions is effective in treating the bladder for symptoms of urinary urgency and enuresis, and in the perception of those responsible for the children. Further studies with larger populations are needed to corroborate these results., (Copyright © 2016 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Objective: Orolingual angioedema (OA) is a well known early complication of treatment with alteplase in ischemic stroke patients. Our aim was to study risk factors for OA in these patients, namely insular cortex ischemia., Methods: Retrospective case-cohort study using the prospective registry of all consecutive ischemic stroke patients submitted to intravenous thrombolysis with alteplase. Clinical data was retrieved from the registry and medical records. Two independent observers evaluated early signs of insular cortex ischemia on pre-thrombolysis computed tomography (CT) and of insular cortex infarct on early follow-up imaging. Univariate and multivariate analysis were performed to identify predictors of OA., Results: Of the 659 patients with acute ischemic stroke treated with alteplase, 32 developed OA (4.9%, 95%CI=3.3-6.6). Frequency of early signs of insular cortex ischemia on pre-thrombolysis CT and of insular cortex infarct on follow-up imaging was similar in patients with and without OA (p=0.241 and p=0.145, respectively). The only independent predictors of OA occurrence were female sex (OR=5.47, 95%CI=1.98-15.10) and angiotensin-converting enzyme inhibitor (ACE-I) use (OR=3.87, 95%CI=1.71-8.75)., Conclusions: Female sex and ACE-I use are independent risk factors for OA occurrence in ischemic stroke patients treated with alteplase. Early signs of insular cortex ischemia on pre-thrombolysis CT were not significantly associated with OA., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The scavenger receptor cysteine-rich (SRCR) family comprises a group of membrane-attached or secreted proteins that contain one or more modules/domains structurally similar to the membrane distal domain of type I macrophage scavenger receptor. Although no all-inclusive biological function has been ascribed to the SRCR family, some of these receptors have been shown to recognize pathogen-associated molecular patterns (PAMP) of bacteria, fungi, or other microbes. SSc5D is a recently described soluble SRCR receptor produced by monocytes/macrophages and T lymphocytes, consisting of an N-terminal portion, which contains five SRCR modules, and a large C-terminal mucin-like domain. Toward establishing a global common role for SRCR domains, we interrogated whether the set of five SRCR domains of SSc5D displayed pattern recognition receptor (PRR) properties. For that purpose, we have expressed in a mammalian expression system the N-terminal SRCR-containing moiety of SSc5D (N-SSc5D), thus excluding the mucin-like domain likely by nature to bind microorganisms, and tested the capacity of the SRCR functional groups to physically interact with bacteria. Using conventional protein-bacteria binding assays, we showed that N-SSc5D had a superior capacity to bind to Escherichia coli strains RS218 and IHE3034 compared with that of the extracellular domains of the SRCR proteins CD5 and CD6 (sCD5 and sCD6, respectively), and similar E. coli -binding properties as Spα, a proven PRR of the SRCR family. We have further designed a more sensitive, real-time, and label-free surface plasmon resonance (SPR)-based assay and examined the capacity of N-SSc5D, Spα, sCD5, and sCD6 to bind to different bacteria. We demonstrated that N-SSc5D compares with Spα in the capacity to bind to E. coli and Listeria monocytogenes , and further that it can distinguish between pathogenic E. coli RS218 and IHE3034 strains and the non-pathogenic laboratory E. coli strain BL21(DE3). Our work thus advocates the utility of SPR-based assays as sensitive tools for the rapid screening of interactions between immune-related receptors and PAMP-bearing microbes. The analysis of our results suggests that SRCR domains of different members of the family have a differential capacity to interact with bacteria, and further that the same receptor can discriminate between different bacteria strains and species.

In palliative care, drugs are considered futile if they do not have a short-term benefit in symptom control or quality of life. The authors examined pharmacotherapy prescribed for patients referred to palliative care to identify futile drugs. This was a retrospective analysis of patients referred over 6 months, focusing on the prescription of gastric protectants, antidiabetic agents, bisphosphonates, anticoagulants, antidementia drugs, statins, and antihypertensive agents. The sample consisted of 448 patients. Gastric protectants were prescribed futilely in 50% of cases (125/248), statins in 97% (69/71), antihypertensive agents in 27% (42/155), antidiabetic drugs in 1% (1/70), bisphosphonates in 27% (4/15), and antidementia drugs in 100% (9/9). This study reveals that many patients with advanced cancer continue to be treated with inappropriate drugs and points to the need for medical training in palliative care, drawing attention to the need for therapeutic review at each medical visit.

T lymphocytes stimulated through their antigen receptor (TCR) preferentially express mRNA isoforms with shorter 3´ untranslated regions (3´-UTRs) derived from alternative pre-mRNA cleavage and polyadenylation (APA). However, the physiological relevance of APA programs remains poorly understood. CD5 is a T-cell surface glycoprotein that negatively regulates TCR signaling from the onset of T-cell activation. CD5 plays a pivotal role in mediating outcomes of cell survival or apoptosis, and may prevent both autoimmunity and cancer. In human primary T lymphocytes and Jurkat cells we found three distinct mRNA isoforms encoding CD5, each derived from distinct poly(A) signals (PASs). Upon T-cell activation, there is an overall increase in CD5 mRNAs with a specific increase in the relative expression of the shorter isoforms. 3´-UTRs derived from these shorter isoforms confer higher reporter expression in activated T cells relative to the longer isoform. We further show that polypyrimidine tract binding protein (PTB/PTBP1) directly binds to the proximal PAS and PTB siRNA depletion causes a decrease in mRNA derived from this PAS, suggesting an effect on stability or poly(A) site selection to circumvent targeting of the longer CD5 mRNA isoform by miR-204. These mechanisms fine-tune CD5 expression levels and thus ultimately T-cell responses., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Although the relationship between enuresis and breastfeeding is still poorly documented in the literature, a possible association is speculated as both are strongly associated with children's development. Therefore, the main objective of this study was to evaluate whether there is an association between primary enuresis and the duration of exclusive breastfeeding., Material and Methods: This is an observational, case-control study, involving 200 children and adolescents from 6 to 14 years old, who were divided into two groups: the enuresis group (EG), composed of 100 children with primary enuresis; and the control group (CG) of 100 matched children without enuresis. The matching criteria were sex, age, and socioeconomic level. Adults responsible for each infant answered a structured questionnaire to identify biological and behavioral factor, as well as the duration of maternal breastfeeding. Children whose parents could not comprehend the questionnaire or children with neurological or psychiatric disorders or secondary enuresis were not included in the study., Results and Discussion: Evaluating the duration of exclusive breastfeeding, 72% of the subjects of the EG and 42% of the CG had been breastfed for less than 4 months (p < 0.001) (Figure). In bivariate analysis, there was a strong association between symptoms of enuresis with a positive family history of enuresis and duration of exclusive breastfeeding (p < 0.001), and also association with full breastfeeding duration (p = 0.044), number of children (p = 0.045), and parents' education (p = 0.045). After logistic regression, primary enuresis continued to be associated with duration of exclusive breastfeeding and family history of enuresis. The proportion of children that had been exclusively breastfed for more than 4 months was significantly higher in the CG 58% (58/100) than in the EG 28% (28/100) (p < 0.001, OR 4.35, 95% CI 1.99-9.50)., Conclusions: This study confirmed the association between primary enuresis and various factors that have already been studied, with the addition of a new factor, duration of exclusive breastfeeding for less than 4 months, which is strongly associated with primary enuresis., (Copyright © 2015 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Purpose: Some parents blame their children for bedwetting and, therefore, punish them. This study aimed to assess the rate of punishment experienced by enuretic children and associated causative factors., Materials and Methods: A total of 87 children 6 to 15 years old with monosymptomatic enuresis were assessed individually. Parents answered the questions in the tolerance scale. The forms of punishment were classified as verbal, chastisement and physical aggression. Family history of enuresis was considered only when 1 or both parents had experienced enuresis., Results: Of the 35 girls and 52 boys with a mean ± SD age of 9.3 ± 2.3 years 67 had a family history of enuresis. Of the 67 parents 57 (85.0%) had a history of being punished due to enuresis. All children experienced some sort of verbal punishment. Children who had a family history of enuresis were more prone to being punished by physical aggression than those without such a family history (32 of 67 or 47.8% vs 4 of 20 or 20%, OR 3.7, 95% CI 1.1-12.1, p = 0.03). Punishment was found 3 times more frequently in girls than in boys (20 of 35 or 57.1% vs 16 of 52 or 30.8%, OR 3.0, 95% CI 1.2-7.3). Parents of 79 of the 87 children (90.8%) had high scores on the tolerance scale regardless of the history of enuresis., Conclusions: Enuretic children are at a high risk for experiencing some kind of punishment. Children whose parents had enuresis are at risk for being physically punished. Parents should be taught about the involuntary nature of enuresis and the fact that no punishment would help improve the condition., (Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

CD6 immunotherapy to treat psoriasis and rheumatoid arthritis has reached the clinical trial stage with apparent success, and targeting CD6 with mAbs is being used in several animal models of autoimmunity and neuroinflammation with promising indications. However, the mode of action of the therapeutic CD6 mAbs is far from being understood, reflecting the uncertainties and controversy surrounding the mechanistic and biological functions of CD6. Initially regarded as a co-stimulatory receptor of T lymphocytes, recent studies suggest that CD6 can instead modulate early as well as late T cell responses. Also, opposing the contribution of CD6 adhesiveness in the establishment and stabilization of immunological synapses, the actual triggering of CD6 might induce anti-proliferative signals to the T lymphocyte. CD6 has an unusually long cytoplasmic tail and its gene undergoes peculiar patterns of activation-dependent alternative splicing that can on one hand determine whether or not the CD6 protein binds to its ligand, and on the other include or exclude intracellular sequences that may transduce positive or negative signaling. In this review we discuss the multiple aspects that determine the nature of the signals transmitted via CD6 and the context that may define a dual role for this important T cell surface molecule.

Group B Streptococcus (GBS), a commensal organism, can turn into a life-threatening pathogen in neonates and elderly, or in adults with severe underlying diseases such as diabetes. We developed a vaccine targeting the GBS glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme detected at the bacterial surface, which was proven to be effective in a neonatal mouse model of infection. Since this bacterium has emerged as an important pathogen in non-pregnant adults, here we investigated whether this vaccine also confers protection in an adult susceptible and in a diabetic mouse model of infection. For immunoprotection studies, sham or immunized adult mice were infected with GBS serotype Ia and V strains, the two most prevalent serotypes isolated in adults. Sham and vaccinated mice were also rendered diabetic and infected with a serotype V GBS strain. For toxicological (pre-clinical) studies, adult mice were vaccinated three times, with three concentrations of recombinant GAPDH adjuvanted with Allydrogel, and the toxicity parameters were evaluated twenty-four hours after the last immunization. For the stability tests, the vaccine formulations were maintained at 4°C for 6 and 12 months prior immunization. The results showed that all tested doses of the vaccine, including the stability study formulations, were immunogenic and that the vaccine was innocuous. The organs (brain, blood, heart, and liver) of vaccinated susceptible or diabetic adult mice were significantly less colonized compared to those of control mice. Altogether, these results demonstrate that the GAPDH-based vaccine is safe and stable and protects susceptible and diabetic adult mice against GBS infections. It is therefore a promising candidate as a global vaccine to prevent GBS-induced neonatal and adult diseases.

Streptococcus agalactiae (or Group B Streptococcus, GBS) is a commensal bacterium present in the intestinal and urinary tracts of approximately 30% of humans. We and others previously showed that the PI-2a pilus polymers, made of the backbone pilin PilB, the tip adhesin PilA and the cell wall anchor protein PilC, promote adhesion to host epithelia and biofilm formation. Affinity-purified PI-2a pili from GBS strain NEM316 were recognized by N-acetylneuraminic acid (NeuNAc, also known as sialic acid) specific lectins such as Elderberry Bark Lectin (EBL) suggesting that pili are sialylated. Glycan profiling with twenty different lectins combined with monosaccharide composition by HPLC suggested that affinity-purified PI-2a pili are modified by N-glycosylation and decorated with sialic acid attached to terminal galactose. Analysis of various relevant mutants in the PI-2a pilus operon by flow-cytometry and electron microscopy analyses pointed to PilA as the pilus subunit modified by glycosylation. Double labeling using PilB antibody and EBL lectin, which specifically recognizes N-acetylneuraminic acid attached to galactose in α-2, 6, revealed a characteristic binding of EBL at the tip of the pilus structures, highly reminiscent of PilA localization. Expression of a secreted form of PilA using an inducible promoter showed that this recombinant PilA binds specifically to EBL lectin when produced in the native GBS context. In silico search for potentially glycosylated asparagine residues in PilA sequence pointed to N427 and N597, which appear conserved and exposed in the close homolog RrgA from S. pneumoniae, as likely candidates. Conversion of these two asparagyl residues to glutamyl resulted in a higher instability of PilA. Our results provide the first evidence that the tip PilA adhesin can be glycosylated, and suggest that this modification is critical for PilA stability and may potentially influence interactions with the host.

Background and Purpose: Little is known on long-term follow-up after thrombolysis in ischemic stroke patients because the majority of studies evaluated outcome at 3 to 12 months. We aimed to assess 5-year outcome after intravenous thrombolysis (IVT)., Methods: Cohort study based on the prospective registry of all consecutive ischemic stroke patients submitted to IVT in our Stroke Unit. Five-year outcome, including living settings, functional outcome, stroke recurrence, and mortality, was ascertained by telephonic interviews and additional review of clinical records. Multivariate analyses were performed to identify predictors of outcome and mortality. Excellent outcome was defined as modified Rankin scale 0 to 1., Results: Five-year outcome was available for 155/164 patients submitted to IVT. At 5 years, 32.9% of patients had an excellent outcome (95% confidence interval (CI) =25.5-43.3) and mortality was 43.9% (95%CI=36.1-51.7). Increasing age (odds ratio =0.93, 95% CI =0.90-0.97) and increasing National Institute of Health Stroke Scale (NIHSS) 24 h after thrombolysis (odds ratio =0.81, 95% CI =0.74-0.90) were independently associated with a lower likelihood of an excellent 5-year outcome. Age (hazards ratio =1.07, 95% CI =1.03-1.11) and excellent functional outcome 3 months after thrombolysis (hazards ratio =0.28, 95%CI=0.12-0.66) were independently associated with mortality during follow-up., Conclusions: One third of ischemic stroke patients have excellent 5-year outcome after IVT. Younger age, lower NIHSS 24 h after IVT, and excellent 3-month functional outcome are independent predictors of excellent 5-year outcome., (© 2015 American Heart Association, Inc.)

Purpose: We determined the effectiveness of 2 methods to treat overactive bladder in children using intragroup and intergroup comparisons in a randomized clinical trial., Materials and Methods: Nine boys and 19 girls with a mean ± SD age of 6.4 ± 2.18 years were randomly divided into group 1-parasacral transcutaneous electrical stimulation with placebo drug and group 2-oxybutynin with sham scapular electrical therapy. Success was assessed by 1) the rate of complete symptom resolution, 2) a visual analog scale of 0 to 10, 3) the dysfunctional voiding score system, 4) voiding diary records, 5) Rome III criteria and 6) side effect frequency in each group., Results: A total of 13 and 15 patients were randomized to groups 1 and 2, respectively. Symptoms completely resolved in 6 patients in group 1 (46%) and 3 in group 2 (20%) (p = 0.204). A statistically significant improvement was found in the 2 groups in the dysfunctional voiding score system and voiding diary records. However, no statistically significant difference was found between the groups in the visual analog scale score, voiding frequency, and maximum and mean voided volume (p = 0.295, 0.098, 0.538 and 0.650, respectively). Constipation improved in 100% of group 1 patients but in only 55% in group 2 (p = 0.031 vs 0.073). Group 1 showed no side effects while dry mouth, hyperthermia and hyperemia developed in 58%, 25% and 50% of group 2 patients (p = 0.002, 0.096 and 0.005, respectively). Treatment was discontinued by 13.3% of patients in group 2., Conclusions: Parasacral transcutaneous electrical stimulation was as effective as oxybutynin to treat overactive bladder in children. However, transcutaneous parasacral electrical stimulation was more effective against constipation and showed no detectable side effects. Oxybutynin was more effective for decreasing voiding frequency., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Background: There is a paucity of published data regarding the quality of care of inflammatory bowel disease (IBD) in Canada. Clinical quality indicators are quantitative end points used to guide, monitor and improve the quality of patient care. In Canada, where universal health care can vary significantly among provinces, quality indicators can be used to identify potential gaps in the delivery of IBD care and standardize the approach to interprovincial management., Methods: The Emerging Practice in IBD Collaborative (EPIC) group generated a shortlist of IBD quality indicators based on a comprehensive literature review. An iterative voting process was used to select quality indicators to take forward. In a face-to-face meeting with the EPIC group, available evidence to support each quality indicator was presented by the EPIC member aligned to it, followed by group discussion to agree on the wording of the statements. The selected quality indicators were then ratified in a final vote by all EPIC members., Results: Eleven quality indicators for the management of IBD within the single-payer health care system of Canada were developed. These focus on accurate diagnosis, appropriate and timely management, disease monitoring, and prevention or treatment of complications of IBD or its therapy., Conclusions: These quality indicators are measurable, reflective of the evidence base and expert opinion, and define a standard of care that is at least a minimum that should be expected for IBD management in Canada. The next steps for the EPIC group involve conducting research to assess current practice across Canada as it pertains to these quality indicators and to measure the impact of each of these indicators on patient outcomes.

In the present work, the knowledge on target proteins of standard antibiotics was extended to antimicrobial mushroom compounds. Docking studies were performed for 34 compounds in order to evaluate their affinity to bacterial proteins that are known targets for some antibiotics with different mechanism of action: inhibitors of cell wall synthesis, inhibitors of protein synthesis, inhibitors of nucleic acids synthesis and antimetabolites. After validation of the molecular docking approach, virtual screening of all the compounds was performed against penicillin binding protein 1a (PBP1a), alanine racemase (Alr), d-alanyl-d-alanine synthetase (Ddl), isoleucyl-tRNA sinthetase (IARS), DNA gyrase subunit B, topoisomerase IV (TopoIV), dihydropteroate synthetase (DHPS) and dihydrofolate reductase (DHFR) using AutoDock4. Overall, it seems that for the selected mushroom compounds (namely, enokipodins, ganomycins and austrocortiluteins) the main mechanism of the action is the inhibition of cell wall synthesis, being Alr and Ddl probable protein targets.

Sepsis is the third most common cause of neonatal death, with Group B Streptococcus (GBS) being the leading bacterial agent. The pathogenesis of neonatal septicemia is still unsolved. We described previously that host susceptibility to GBS infection is due to early IL-10 production. In this study, we investigated whether triggering TLR2 to produce IL-10 is a risk factor for neonatal bacterial sepsis. We observed that, in contrast to wild-type (WT) pups, neonatal TLR2-deficient mice were resistant to GBS-induced sepsis. Moreover, if IL-10 signaling were blocked in WT mice, they also were resistant to sepsis. This increased survival rate was due to an efficient recruitment of neutrophils to infected tissues that leads to bacterial clearance, thus preventing the development of sepsis. To confirm that IL-10 produced through TLR2 activation prevents neutrophil recruitment, WT pups were treated with the TLR2 agonist Pam3CSK4 prior to nebulization with the neutrophil chemotactic agent LTB4. Neutrophil recruitment into the neonatal lungs was inhibited in pups treated with Pam3CSK4. However, the migration was restored in Pam3CSK4-treated pups when IL-10 signaling was blocked (either by anti-IL-10R mAb treatment or by using IL-10-deficient mice). Our findings highlight that TLR2-induced IL-10 production is a key event in neonatal susceptibility to bacterial sepsis.

Purpose: Parasacral transcutaneous electrical neural stimulation is widely used to treat hyperactive bladder in children and adults. Its use in nonmonosymptomatic enuresis has demonstrated improvement in number of dry nights. We assessed the effectiveness of parasacral transcutaneous electrical neural stimulation in the treatment of monosymptomatic primary enuresis., Materials and Methods: This prospective randomized clinical trial included 29 girls and 16 boys older than 6 years with primary monosymptomatic enuresis. Children were randomly divided into 2 groups consisting of controls, who were treated with behavioral therapy, and an experimental group, who were treated with behavioral therapy plus 10 sessions of parasacral transcutaneous electrical neural stimulation. Neural stimulation was performed with the electrodes placed in the sacral region (S2/S3). Sessions always followed the same pattern, with duration of 20 minutes, frequency of 10 Hz, a generated pulse of 700 μs and intensity determined by the sensitivity threshold of the child. Sessions were done 3 times weekly on alternate days. Patients in both groups were followed at 2-week intervals for the first month and then monthly for 6 consecutive months., Results: Rate of wet nights was 77% in controls and 78.3% in the experimental group at onset of treatment (p = 0.82), and 49.5% and 31.2%, respectively, at the end of treatment (p = 0.02). Analyzing the average rate of improvement, there was a significantly greater increase in dry nights in the group undergoing neural stimulation (61.8%) compared to controls (37.3%, p = 0.0038). At the end of treatment percent improvement in children undergoing electrical stimulation had no relation to gender (p = 0.391) or age (p = 0.911)., Conclusions: Treatment of primary monosymptomatic enuresis with 10 sessions of parasacral transcutaneous electrical neural stimulation plus behavioral therapy proved to be effective. However, no patient had complete resolution of symptoms., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Biodegradable polydioxanone stents were developed for the treatment of refractory benign esophageal strictures but have been suggested as a new therapeutic option for intestinal strictures. The primary advantage of biodegradable stents over self-expandable metallic stents is that removal is not required. There are, however, few data available on their use in the small or large bowel. We herein describe the case of a 33-year-old patient with long-standing Crohn's disease (CD) who developed a fibrotic stricture of the sigmoid too long to be amenable to balloon dilation. The use of a biodegradable polydioxanone stent was chosen to avoid surgery. Combined endoscopic and fluoroscopic placement of the stent was technically simple, safe and clinically successful, and no recurrence of obstructive symptoms occurred during a 16-mo follow-up. Further studies are needed to evaluate the long-term efficacy and safety of biodegradable stents in the treatment of intestinal strictures, particularly in the context of CD.

Glyceraldehyde 3-phosphate dehydrogenases (GAPDH) are cytoplasmic glycolytic enzymes that, despite lacking identifiable secretion signals, have been detected at the surface of several prokaryotic and eukaryotic organisms where they exhibit non-glycolytic functions including adhesion to host components. Group B Streptococcus (GBS) is a human commensal bacterium that has the capacity to cause life-threatening meningitis and septicemia in newborns. Electron microscopy and fluorescence-activated cell sorter (FACS) analysis demonstrated the surface localization of GAPDH in GBS. By addressing the question of GAPDH export to the cell surface of GBS strain NEM316 and isogenic mutant derivatives of our collection, we found that impaired GAPDH presence in the surface and supernatant of GBS was associated with a lower level of bacterial lysis. We also found that following GBS lysis, GAPDH can associate to the surface of many living bacteria. Finally, we provide evidence for a novel function of the secreted GAPDH as an inducer of apoptosis of murine macrophages.

Group B Streptococcus (GBS) is the leading cause of neonatal pneumonia, septicemia, and meningitis. We have previously shown that in adult mice GBS glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an extracellular virulence factor that induces production of the immunosuppressive cytokine interleukin-10 (IL-10) by the host early upon bacterial infection. Here, we investigate whether immunity to neonatal GBS infection could be achieved through maternal vaccination against bacterial GAPDH. Female BALB/c mice were immunized with rGAPDH and the progeny was infected with a lethal inoculum of GBS strains. Neonatal mice born from mothers immunized with rGAPDH were protected against infection with GBS strains, including the ST-17 highly virulent clone. A similar protective effect was observed in newborns passively immunized with anti-rGAPDH IgG antibodies, or F(ab')(2) fragments, indicating that protection achieved with rGAPDH vaccination is independent of opsonophagocytic killing of bacteria. Protection against lethal GBS infection through rGAPDH maternal vaccination was due to neutralization of IL-10 production soon after infection. Consequently, IL-10 deficient (IL-10(-/-)) mice pups were as resistant to GBS infection as pups born from vaccinated mothers. We observed that protection was correlated with increased neutrophil trafficking to infected organs. Thus, anti-rGAPDH or anti-IL-10R treatment of mice pups before GBS infection resulted in increased neutrophil numbers and lower bacterial load in infected organs, as compared to newborn mice treated with the respective control antibodies. We showed that mothers immunized with rGAPDH produce neutralizing antibodies that are sufficient to decrease IL-10 production and induce neutrophil recruitment into infected tissues in newborn mice. These results uncover a novel mechanism for GBS virulence in a neonatal host that could be neutralized by vaccination or immunotherapy. As GBS GAPDH is a structurally conserved enzyme that is metabolically essential for bacterial growth in media containing glucose as the sole carbon source (i.e., the blood), this protein constitutes a powerful candidate for the development of a human vaccine against this pathogen.

Certain extracellular proteins produced by several pathogenic microorganisms interfere with the host immune system facilitating microbial colonization and were thus designated virulence-associated immunomodulatory proteins. In this study, a protein with B lymphocyte stimulatory activity was isolated from culture supernatants of Streptococcus agalactiae strain NEM316. This protein, with an apparent molecular mass of 45 kDa, was identified as GAPDH by N-terminal amino acid sequencing. The gapC gene was cloned and expressed in Escherichia coli for the production of a recombinant histidyl-tagged protein. The recombinant GAPDH (rGAPDH), purified in an enzymatically active form, induced in vitro an up-regulation of CD69 expression on B cells from normal and BCR transgenic mice. In addition, rGAPDH induced an increase in the numbers of total, but not of rGAPDH-specific, splenic Ig-secreting cells in C57BL/6 mice treated i.p. with this protein. These in vitro- and in vivo-elicited B cell responses suggest that the B cell stimulatory effect of rGAPDH is independent of BCR specificity. A S. agalactiae strain overexpressing GAPDH showed increased virulence as compared with the wild-type strain in C57BL/6 mice. This virulence was markedly reduced in IL-10-deficient and anti-rGAPDH antiserum-treated mice. These results suggest that IL-10 production, which was detected at higher concentrations in the serum of rGAPDH-treated mice, is important in determining the successfulness of the host colonization by S. agalactiae and they highlight the direct role of GAPDH in this process. Taken together, our data demonstrate that S. agalactiae GAPDH is a virulence-associated immunomodulatory protein.