Value of a café-au-lait macules screening clinic: Experience from The Hospital for Sick Children in Toronto.

BACKGROUND/OBJECTIVES: Café-au-lait macules (CALMs) are a characteristic feature of neurofibromatosis type 1 (NF1), but also occur in other genetic disorders. Differential diagnosis of CALMs remains challenging and can be stressful for families. We sought to examine the role of an established CALMs screening clinic in diagnosing CALMs-related disorders. METHOD: We retrospectively reviewed patients seen between July 2012 and January 2019 in a CALMs screening clinic at The Hospital for Sick Children, a tertiary pediatric hospital in Toronto, Canada. Pediatric patients were referred because of multiple CALMs or suspected NF1. Selection was based on a chronological referral sample with no exclusions. A pediatric dermatologist examined all patients for CALMs and NF1 manifestations. Genetic testing was offered to confirm a clinical diagnosis or when clinical findings were inconclusive. RESULTS: Three hundred patients, of which 152 (50.7%) were female and had a mean age of 5.6 ± 4.8 years were seen during the study period. NF1 was diagnosed in 76 (25.3%) patients, mosaic NF1 in 38 (12.7%) patients, and 8 (2.7%) patients received other genetic diagnoses. One hundred and twelve (37.3%) patients were diagnosed with isolated CALMs not associated with an underlying genetic disease. Furthermore, 36 (12%) of our patients did not have CALMs. CONCLUSIONS: The CALMs screening clinic aided in the early diagnosis of genetic disorders such as NF1 and distinguished CALMs from other hyperpigmented lesions. We encourage the adoption of this clinic model in referral centers to streamline and optimize care of patients with presumptive diagnosis of CALMs.

Nerve-associated transient receptor potential ion channels can contribute to intrinsic resistance to bacterial adhesion in vivo.

The cornea of the eye differs from other mucosal surfaces in that it lacks a viable bacterial microbiome and by its unusually high density of sensory nerve endings. Here, we explored the role of corneal nerves in preventing bacterial adhesion. Pharmacological and genetic methods were used to inhibit the function of corneal sensory nerves or their associated transient receptor potential cation channels TRPA1 and TRPV1. Impacts on bacterial adhesion, resident immune cells, and epithelial integrity were examined using fluorescent labeling and quantitative confocal imaging. TRPA1/TRPV1 double gene-knockout mice were more susceptible to adhesion of environmental bacteria and to that of deliberately-inoculated Pseudomonas aeruginosa. Supporting the involvement of TRPA1/TRPV1-expressing corneal nerves, P. aeruginosa adhesion was also promoted by treatment with bupivacaine, or ablation of TRPA1/TRPV1-expressing nerves using RTX. Moreover, TRPA1/TRPV1-dependent defense was abolished by enucleation which severs corneal nerves. High-resolution imaging showed normal corneal ultrastructure and surface-labeling by wheat-germ agglutinin for TRPA1/TRPV1 knockout murine corneas, and intact barrier function by absence of fluorescein staining. P. aeruginosa adhering to corneas after perturbation of nerve or TRPA1/TRPV1 function failed to penetrate the surface. Single gene-knockout mice showed roles for both TRPA1 and TRPV1, with TRPA1-/- more susceptible to P. aeruginosa adhesion while TRPV1-/- corneas instead accumulated environmental bacteria. Corneal CD45+/CD11c+ cell responses to P. aeruginosa challenge, previously shown to counter bacterial adhesion, also depended on TRPA1/TRPV1 and sensory nerves. Together, these results demonstrate roles for corneal nerves and TRPA1/TRPV1 in corneal resistance to bacterial adhesion in vivo and suggest that the mechanisms involve resident immune cell populations.

Protein-Protein Interaction Inhibitor of SRPKs Alters the Splicing Isoforms of VEGF and Inhibits Angiogenesis.

Serine-arginine (SR) protein kinases (SRPKs) regulate the functions of the SR-rich splicing factors by phosphorylating multiple serines within their C-terminal arginine-serine-rich domains. Dysregulation of these phosphorylation events has been implicated in many diseases, suggesting SRPKs are potential therapeutic targets. In particular, aberrant SRPK1 expression alters the balances of proangiogenic (VEGF165) and antiangiogenic (VEGF165b) splicing isoforms of the key angiogenesis factor, vascular endothelial growth factor (VEGF), through the phosphorylation of prototypic SR protein SRSF1. Here, we report a protein-protein interaction (PPI) inhibitor of SRPKs, docking blocker of SRPK1 (DBS1), that specifically blocks a conserved substrate docking groove unique to SRPKs. DBS1 is a cell-permeable inhibitor that effectively inhibits the binding and phosphorylation of SRSF1 and subsequently switches VEGF splicing from the proangiogenic to the antiangiogenic isoform. Our findings thus provide a new direction for the development of SRPK inhibitors through targeting a unique PPI site to combat angiogenic diseases.

Hidradenitis Suppurativa in the Pediatric Population: An International, Multicenter, Retrospective, Cross-sectional Study of 481 Pediatric Patients.

Importance: Hidradenitis suppurativa (HS) in pediatric patients has been understudied. Increased awareness and recognition of HS prevalence in children demand efforts to better understand this condition. Objective: To describe the demographics, clinical features, treatment, associated comorbidities, and outcomes in a large cohort of pediatric patients with HS. Design, Setting, and Participants: International, multicenter, retrospective medical record review of pediatric patients (aged 1-18 years) with a clinical diagnosis of HS carried out in 10 dermatology clinics across the US, Canada, Israel, Australia, and Italy from January 1996 to January 2017. Main Outcomes and Measures: Patient demographics, clinical features, severity, associated comorbidities, and treatments in pediatric patients with HS. Results: This cross-sectional study included 481 patients diagnosed with HS. Overall, 386 (80%) were girls. The mean (SD) age of disease onset was 12.5 (2.9) years, and the mean (SD) age at diagnosis was 14.4 (3.5) years. Family history of HS was present in 111 of 271 (41%) patients. First signs/symptoms reported at disease onset were cyst/abscess in 229 of 481 (48%), pain/tenderness in 118 of 481 (25%), and papules/pustules in 117 of 481 (24%). At initial dermatologic assessment, 233 of 481 (48%) patients already had evidence of skin scarring. Disease severity (Hurley staging) was documented in 288 of 481 (60%) patients (47% stage 1, 45% stage 2 and 8% stage 3). Comorbid conditions were reported in 406 of 481 (85%) patients, the most common being obesity (263/406 [65%]) and acne vulgaris (118/406 [29%]). Complications occurred in 378 of 481 (79%) patients, the most common of which were scars or contractures (301/378 [80%]). Conclusions and Relevance: The findings of this study indicate that there is a gap in recognizing and diagnosing pediatric HS. Pediatric patients with HS are likely to present with other comorbidities. Prospective observational and interventional studies are needed to better understand clinical course and optimal treatments for pediatric HS.

Resistance of the murine cornea to bacterial colonization during experimental dry eye.

The healthy cornea is remarkably resistant to infection, quickly clearing deliberately inoculated bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus. Contrasting with the adjacent conjunctiva and other body surfaces, it also lacks a resident viable bacterial microbiome. Corneal resistance to microbes depends on intrinsic defenses involving tear fluid and the corneal epithelium. Dry eye, an ocular surface disease associated with discomfort and inflammation, can alter tear fluid composition and volume, and impact epithelial integrity. We previously showed that experimentally-induced dry eye (EDE) in mice does not increase corneal susceptibility to P. aeruginosa infection. Here, we explored if EDE alters corneal resistance to bacterial colonization. EDE was established in mice using scopolamine injections and dehumidified air-flow, and verified by phenol-red thread testing after 5 and 10 days. As expected, EDE corneas showed increased fluorescein staining versus controls consistent with compromised epithelial barrier function. Confocal imaging using mT/mG knock-in mice with red-fluorescent membranes revealed no other obvious morphological differences between EDE corneas and controls for epithelium, stroma, and endothelium. EDE corneas were imaged ex vivo and compared to controls after alkyne-functionalized D-alanine labeling of metabolically-active colonizing bacteria, or by FISH using a universal 16S rRNA gene probe. Both methods revealed very few viable bacteria on EDE corneas after 5 or 10 days (median of 0, upper quartile of ≤ 1 bacteria per field of view for each group [9-12 eyes per group]) similar to control corneas. Furthermore, there was no obvious difference in abundance of conjunctival bacteria, which included previously reported filamentous forms. Thus, despite reduced tear flow and apparent compromise to corneal barrier function (fluorescein staining), EDE murine corneas continue to resist bacterial colonization and maintain the absence of a resident viable bacterial microbiome.

Contact lens-related corneal infection: Intrinsic resistance and its compromise.

Contact lenses represent a widely utilized form of vision correction with more than 140 million wearers worldwide. Although generally well-tolerated, contact lenses can cause corneal infection (microbial keratitis), with an approximate annualized incidence ranging from ~2 to ~20 cases per 10,000 wearers, and sometimes resulting in permanent vision loss. Research suggests that the pathogenesis of contact lens-associated microbial keratitis is complex and multifactorial, likely requiring multiple conspiring factors that compromise the intrinsic resistance of a healthy cornea to infection. Here, we outline our perspective of the mechanisms by which contact lens wear sometimes renders the cornea susceptible to infection, focusing primarily on our own research efforts during the past three decades. This has included studies of host factors underlying the constitutive barrier function of the healthy cornea, its response to bacterial challenge when intrinsic resistance is not compromised, pathogen virulence mechanisms, and the effects of contact lens wear that alter the outcome of host-microbe interactions. For almost all of this work, we have utilized the bacterium Pseudomonas aeruginosa because it is the leading cause of lens-related microbial keratitis. While not yet common among corneal isolates, clinical isolates of P. aeruginosa have emerged that are resistant to virtually all currently available antibiotics, leading the United States CDC (Centers for Disease Control) to add P. aeruginosa to its list of most serious threats. Compounding this concern, the development of advanced contact lenses for biosensing and augmented reality, together with the escalating incidence of myopia, could portent an epidemic of vision-threatening corneal infections in the future. Thankfully, technological advances in genomics, proteomics, metabolomics and imaging combined with emerging models of contact lens-associated P. aeruginosa infection hold promise for solving the problem - and possibly life-threatening infections impacting other tissues.

Venous Thrombosis in Acquired Hemophilia: The Complex Management of Competing Pathologies.

Introduction Venous thrombosis is rare in the setting of factor VIII (FVIII) deficiency. Cases of deep vein thrombosis (DVT) have been described in hemophiliacs after recent major surgery, or in association with the administration of FVIII concentrate and activated prothrombin complex concentrates, but occurrence of spontaneous DVT is even more uncommon. Aim We describe the challenging management of extensive DVT in a patient with acquired hemophilia A with concurrent hemorrhagic manifestations and review similar published cases. Methods We summarize a series of 10 cases with the following demographics: 6 males and 4 females; median age at presentation of 65 (21-80); mean inhibitor titer of 68.5 Bethesda Units (BU 1.9 to BU 350). Results Four cases were idiopathic and six had associated conditions (cancer [two cases], recent pregnancy [two cases], and recent surgery [two cases]). Three cases had an inferior vena cava filter inserted for acute lower limb DVT/pulmonary embolism. Inhibitor eradication was achieved with high-dose steroids with or without cyclophosphamide, and adjunct Rituximab administration was used in three cases. One patient received concurrent therapeutic plasma exchange (TPE). Inhibitor eradication was fastest with concurrent TPE at 6 days (range: 6-733 days). The 30-day survival was 90%. Conclusions There was adequate response of inhibitors to immunosuppression with steroids and cyclophosphamide therapy. For more refractory disease, Rituximab is emerging as a beneficial and cost-effective adjunct with better rates of complete remission, and the threshold for its use may be lowered in this complex cohort with dual competing pathologies.

DMBT1 inhibition of Pseudomonas aeruginosa twitching motility involves its N-glycosylation and cannot be conferred by the Scavenger Receptor Cysteine-Rich bacteria-binding peptide domain.

The scavenging capacity of glycoprotein DMBT1 helps defend mucosal epithelia against microbes. DMBT1 binding to multiple bacterial species involves its conserved Scavenger Receptor Cysteine-Rich (SRCR) domains, localized to a 16-mer consensus sequence peptide, SRCRP2. Previously, we showed that DMBT1 bound Pseudomonas aeruginosa pili, and inhibited twitching motility, a pilus-mediated movement important for virulence. Here, we determined molecular characteristics required for twitching motility inhibition. Heat-denatured DMBT1 lost capacity to inhibit twitching motility and showed reduced pili binding (~40%). Size-exclusion chromatography of Lys-C-digested native DMBT1 showed that only high-Mw fractions retained activity, suggesting involvement of the N-terminal containing repeated SRCR domains with glycosylated SRCR-Interspersed Domains (SIDs). However, individual or pooled consensus sequence peptides (SRCRPs 1 to 7) showed no activity and did not bind P. aeruginosa pili; nor did recombinant DMBT1 (aa 1-220) or another SRCR-rich glycoprotein, CD163. Enzymatic de-N-glycosylation of DMBT1, but not de-O-glycosylation, reduced its capacity to inhibit twitching motility (~57%), without reducing pili binding. Therefore, DMBT1 inhibition of P. aeruginosa twitching motility involves its N-glycosylation, its pili-binding capacity is insufficient, and it cannot be conferred by the SRCR bacteria-binding peptide domain, either alone or mixed with other unlinked SRCRPs, suggesting an additional mechanism for DMBT1-mediated mucosal defense.

Distinct mechanisms govern the phosphorylation of different SR protein splicing factors.

Serine-arginine (SR) proteins are essential splicing factors containing a canonical RNA recognition motif (RRM), sometimes followed by a pseudo-RRM, and a C-terminal arginine/serine-rich (RS) domain that undergoes multisite phosphorylation. Phosphorylation regulates the localization and activity of SR proteins, and thus may provide insight into their differential biological roles. The phosphorylation mechanism of the prototypic SRSF1 by serine-arginine protein kinase 1 (SRPK1) has been well-studied, but little is known about the phosphorylation of other SR protein members. In the present study, interaction and kinetic assays unveiled how SRSF1 and the single RRM-containing SRSF3 are phosphorylated by SRPK2, another member of the SRPK family. We showed that a conserved SRPK-specific substrate-docking groove in SRPK2 impacts the binding and phosphorylation of both SR proteins, and the localization of SRSF3. We identified a nonconserved residue within the groove that affects the kinase processivity. We demonstrated that, in contrast to SRSF1, for which SRPK-mediated phosphorylation is confined to the N-terminal region of the RS domain, SRSF3 phosphorylation sites are spread throughout its entire RS domain in vitro Despite this, SRSF3 appears to be hypophosphorylated in cells at steady state. Our results suggest that the absence of a pseudo-RRM renders the single RRM-containing SRSF3 more susceptible to dephosphorylation by phosphatase. These findings suggest that the single RRM- and two RRM-containing SR proteins represent two subclasses of phosphoproteins in which phosphorylation statuses are maintained by unique mechanisms, and pose new directions to explore the distinct roles of SR proteins in vivo.

A novel murine model for contact lens wear reveals clandestine IL-1R dependent corneal parainflammation and susceptibility to microbial keratitis upon inoculation with Pseudomonas aeruginosa.

PURPOSE: Contact lens wear carries a risk of complications, including corneal infection. Solving these complications has been hindered by limitations of existing animal models. Here, we report development of a new murine model of contact lens wear. METHODS: C57BL/6 mice were fitted with custom-made silicone-hydrogel contact lenses with or without prior inoculation with Pseudomonas aeruginosa (PAO1-GFP). Contralateral eyes served as controls. Corneas were monitored for pathology, and examined ex vivo using high-magnification, time-lapse imaging. Fluorescent reporter mice allowed visualization of host cell membranes and immune cells. Lens-colonizing bacteria were detected by viable counts and FISH. Direct-colony PCR was used for bacterial identification. RESULTS: Without deliberate inoculation, lens-wearing corneas remained free of visible pathology, and retained a clarity similar to non-lens wearing controls. CD11c-YFP reporter mice revealed altered numbers, and distribution, of CD11c-positive cells in lens-wearing corneas after 24 h. Worn lenses showed bacterial colonization, primarily by known conjunctival or skin commensals. Corneal epithelial cells showed vacuolization during lens wear, and after 5 days, cells with phagocyte morphology appeared in the stroma that actively migrated over resident keratocytes that showed altered morphology. Immunofluorescence confirmed stromal Ly6G-positive cells after 5 days of lens wear, but not in MyD88 or IL-1R gene-knockout mice. P. aeruginosa-contaminated lenses caused infectious pathology in most mice from 1 to 13 days. CONCLUSIONS: This murine model of contact lens wear appears to faithfully mimic events occurring during human lens wear, and could be valuable for experiments, not possible in humans, that help solve the pathogenesis of lens-related complications.

Audiovisual interventions for parental preoperative anxiety: A systematic review and meta-analysis.

OBJECTIVE: To systematically review and meta-analyze studies assessing the effectiveness of audiovisual (AV) interventions aimed at reducing anxiety in parents whose children are undergoing elective surgery. METHODS: A comprehensive search of multiple electronic databases was performed. A narrative synthesis of findings and random-effects meta-analyses were used to summarize the results. Our primary outcome was parental anxiety. Secondary outcomes included children's preoperative anxiety and postoperative outcomes; parental satisfaction, knowledge, and need for anesthesia information. Risk of bias was appraised within and across studies. RESULTS: Our search yielded 723 studies and 11 were included. A Standardized Mean Difference (SMD) of -0.53 (95% CI [-0.91, -0.15], p < .01) was found between parental anxiety scores in AV interventions and control groups. In terms of children's preoperative anxiety, there was a SMD of -0.59 (95% CI [-1.11, -0.07], p < .05) between children's anxiety scores in AV intervention and nonintervention participants. Furthermore, AV interventions were shown to shorten the recovery time for children undergoing surgery (SMD = -0.21; 95% CI [-0.39, -0.02], p = .03) but did not lead to improvements on other postoperative outcomes. CONCLUSIONS: These findings suggest that AV interventions have modest, positive effects on both parental and children's preoperative anxiety. Although a statistically significant medium size effect was detected, the clinical significance of this finding requires further exploration. Further research aimed at developing better AV interventions to help guide future practice is warranted. (PsycINFO Database Record

IL-1R and MyD88 Contribute to the Absence of a Bacterial Microbiome on the Healthy Murine Cornea.

Microbial communities are important for the health of mucosal tissues. Traditional culture and gene sequencing have demonstrated bacterial populations on the conjunctiva. However, it remains unclear if the cornea, a transparent tissue critical for vision, also hosts a microbiome. Corneas of wild-type, IL-1R (-/-) and MyD88 (-/-) C57BL/6 mice were imaged after labeling with alkyne-functionalized D-alanine (alkDala), a probe that only incorporates into the peptidoglycan of metabolically active bacteria. Fluorescence in situ hybridization (FISH) was also used to detect viable bacteria. AlkDala labeling was rarely observed on healthy corneas. In contrast, adjacent conjunctivae harbored filamentous alkDala-positive forms, that also labeled with DMN-Tre, a Corynebacterineae-specific probe. FISH confirmed the absence of viable bacteria on healthy corneas, which also cleared deliberately inoculated bacteria within 24 h. Differing from wild-type, both IL-1R (-/-) and MyD88 (-/-) corneas harbored numerous alkDala-labeled bacteria, a result abrogated by topical antibiotics. IL-1R (-/-) corneas were impermeable to fluorescein suggesting that bacterial colonization did not reflect decreased epithelial integrity. Thus, in contrast to the conjunctiva and other mucosal surfaces, healthy murine corneas host very few viable bacteria, and this constitutive state requires the IL-1R and MyD88. While this study cannot exclude the presence of fungi, viruses, or non-viable or dormant bacteria, the data suggest that healthy murine corneas do not host a resident viable bacterial community, or microbiome, the absence of which could have important implications for understanding the homeostasis of this tissue.

Quantification of Bacterial Twitching Motility in Dense Colonies Using Transmitted Light Microscopy and Computational Image Analysis.

A method was developed to allow the quantification and mapping of relative bacterial twitching motility in dense samples, where tracking of individual bacteria was not feasible. In this approach, movies of bacterial films were acquired using differential interference contrast microscopy (DIC), and bacterial motility was then indirectly quantified by the degree to which the bacteria modulated the intensity of light in the field-of-view over time. This allowed the mapping of areas of relatively high and low motility within a single field-of-view, and comparison of the total distribution of motility between samples.

Patterns of Care Related to Post-Operative Radiotherapy for Patients with Prostate Cancer among Canadian Radiation Oncologists and Urologists.

The American Society for Radiation Oncology (ASTRO) and American Urological Association (AUA) developed post-prostatectomy radiotherapy (RT) guidelines to aid patient counseling on adjuvant (ART) and salvage radiotherapy (SRT). Our study compared how aware and compliant Canadian radiation oncologists and urologists are to these guidelines. Our online survey was distributed through the Canadian Association of Radiation Oncology (CARO) and Canadian Urology Association (CUA) to radiation oncologists and urologists that treat prostate cancer. We used Wilcoxon rank-sum test and Chi-square test to compare radiation oncologists and urologists. P values for significant findings are reported. A total of 128 participants responded the survey, 52 radiation oncologists, and 76 urologists. The majority (82%) of radiation oncologists had read these guidelines, compared to only 49% of urologists (p < 0.001). Radiation oncologists were more likely to recommend ART >50% for adverse pathological findings post-radical prostatectomy compared to urologists (76 vs. 51%, p = 0.011). Urologists were more likely to monitor their patient's PSA level post-prostatectomy compared to radiation oncologists (93 vs. 77%, p = 0.016). Post-thematic analysis of open-ended questions revealed that urologists rarely refer patients to radiation oncologists for ART, with radiation oncologists confirming that they rarely receive referrals. This study demonstrates the low compliance to ASTRO/AUA guidelines. While radiation oncologists were more aware and compliant to guidelines, urologists were significantly more likely to monitor their patient's PSA. This study highlighted the need for better communication between urologists and radiation oncologists, especially in referrals for ART, to facilitate treatment delivery that is concordant with ASTRO/AUA guidelines.

Fourteen-month-old infants use interpersonal synchrony as a cue to direct helpfulness.

Musical behaviours such as dancing, singing and music production, which require the ability to entrain to a rhythmic beat, encourage high levels of interpersonal coordination. Such coordination has been associated with increased group cohesion and social bonding between group members. Previously, we demonstrated that this association influences even the social behaviour of 14-month-old infants. Infants were significantly more likely to display helpfulness towards an adult experimenter following synchronous bouncing compared with asynchronous bouncing to music. The present experiment was designed to determine whether interpersonal synchrony acts as a cue for 14-month-olds to direct their prosocial behaviours to specific individuals with whom they have experienced synchronous movement, or whether it acts as a social prime, increasing prosocial behaviour in general. Consistent with the previous results, infants were significantly more likely to help an experimenter following synchronous versus asynchronous movement with this person. Furthermore, this manipulation did not affect infant's behaviour towards a neutral stranger, who was not involved in any movement experience. This indicates that synchronous bouncing acts as a social cue for directing prosociality. These results have implications for how musical engagement and rhythmic synchrony affect social behaviour very early in development.