Cytokine Signaling in Pediatric Kidney Tumor Cell Lines WT-CLS1, WT-3ab and G-401.

Renal tumors comprise ~7% of all malignant pediatric tumors. Approximately 90% of pediatric kidney tumors comprise Wilms tumors, and the remaining 10% include clear cell sarcoma of the kidney, malignant rhabdoid tumor of the kidney, renal cell carcinoma and other rare renal tumors. Over the last 30 years, the role of cytokines and their receptors has been considerably investigated in both cancer progression and anti-cancer therapy. However, more effective immunotherapies require the cytokine profiling of each tumor type and comprehensive understanding of tumor biology. In this study, we aimed to investigate the activation of signaling pathways in response to cytokines in three pediatric kidney tumor cell lines, in WT-CLS1 and WT-3ab cells (both are Wilms tumors), and in G-401 cells (a rhabdoid kidney tumor, formerly classified as Wilms tumor). We observed that interferon-alpha (IFN-α) and interferon-gamma (IFN-γ) very strongly induced the activation of the STAT1 protein, whereas IL-6 and IFN-α activated STAT3 and IL-4 activated STAT6 in all examined tumor cell lines. STAT protein activation was examined by flow cytometry and Western blot using phospho-specific anti-STAT antibodies which recognize only activated (phosphorylated) STAT proteins. Nuclear translocation of phospho-STAT proteins upon activation with specific cytokines was furthermore confirmed by immunofluorescence. Our results also showed that both IFN-α and IFN-γ caused upregulation of major histocompatibility complex (MHC) class I proteins, however, these cytokines did not have any effect on the expression of MHC class II proteins. We also observed that pediatric kidney tumor cell lines exhibit the functional expression of an additional cytokine signaling pathway, the tumor necrosis factor (TNF)-α-mediated activation of nuclear factor kappa B (NF-κB). In summary, our data show that human pediatric renal tumor cell lines are responsive to stimulation with various human cytokines and could be used as in vitro models for profiling cytokine signaling pathways.

Long-term pulmonary outcome of children with congenital diaphragmatic hernia: functional lung MRI using matrix-pencil decomposition enables side-specific assessment of lung function.

OBJECTIVES: In patients with congenital diaphragmatic hernia (CDH) the exact functional outcome of the affected lung side is still unknown, mainly due to the lack of spatially resolved diagnostic tools. Functional matrix-pencil decomposition (MP-) lung MRI fills this gap as it measures side-specific ventilation and perfusion. We aimed to assess the overall and side-specific pulmonary long-term outcomes of patients with CDH using lung function tests and MP-MRI. METHODS: Thirteen school-aged children with CDH (seven with small and six with large defect-sized CDH, defined as > 50% of the chest wall circumference being devoid of diaphragm tissue) and thirteen healthy matched controls underwent spirometry, multiple-breath washout, and MP-MRI. The main outcomes were forced expiratory volume in 1 second (FEV1), lung clearance index (LCI2.5), ventilation defect percentage (VDP), and perfusion defect percentage (QDP). RESULTS: Patients with a large CDH showed significantly reduced overall lung function compared to healthy controls (mean difference [95%-CIadjusted]: FEV1 (z-score) -4.26 [-5.61, -2.92], FVC (z-score) -3.97 [-5.68, -2.26], LCI2.5 (TO) 1.12 [0.47, 1.76], VDP (%) 8.59 [3.58, 13.60], QDP (%) 17.22 [13.16, 21.27]) and to patients with a small CDH. Side-specific examination by MP-MRI revealed particularly reduced ipsilateral ventilation and perfusion in patients with a large CDH (mean difference to contralateral side [95%-CIadjusted]: VDP (%) 14.80 [10.50, 19.00], QDP (%) 23.50 [1.75, 45.20]). CONCLUSIONS: Data indicate impaired overall lung function with particular limitation of the ipsilateral side in patients with a large CDH. MP-MRI is a promising tool to provide valuable side-specific functional information in the follow-up of patients with CDH. CLINICAL RELEVANCE STATEMENT: In patients with congenital diaphragmatic hernia, easily applicable MP-MRI allows specific examination of the lung side affected by the hernia and provides valuable information on ventilation and perfusion with implications for clinical practice, making it a promising tool for routine follow-up. KEY POINTS: • Functional matrix pencil decomposition (MP) MRI data from a small sample indicate reduced ipsilateral pulmonary ventilation and perfusion in children with large congenital diaphragmatic hernia (CDH). • Easily applicable pencil decomposition MRI provides valuable side-specific diagnostic information on lung ventilation and perfusion. This is a clear advantage over conventional lung function tests, helping to comprehensively follow up patients with congenital diaphragmatic hernia and monitor therapy effects.

Risk factors for mortality in preterm infants with necrotizing enterocolitis: a retrospective multicenter analysis.

It is difficult to predict the risk of mortality in necrotizing enterocolitis (NEC). This study aimed at identifying risk factors for severe NEC (Bell stage III) and mortality in preterm children with NEC. In this multicenter retrospective study, we analyzed multiple data from 157 premature children with confirmed NEC in the period from January 2007 to October 2018. We performed univariate, multivariate, stepwise logistic regression, and receiver operator characteristics (ROC) analyses. We were able to demonstrate that low Apgar scores (notably at 1' and 5'), low hemoglobin concentration (Hgb), and high lactate level at disease onset and during disease correlated with NEC severity and mortality (P < 0.05, respectively). Severe NEC was related to congenital heart disease (CHD - OR 2.6, CI95% 1.2-5.8, P 0.015) and patent ductus arteriosus (PDA - OR 3.3, CI95% 1.6-6.9, P 0.0012), whereas death was related to the presence of PDA (OR 5.5, CI95% 2.3-14, P < 0.001).Conclusion: Low Apgar scores, low Hgb, high lactate levels, and the presence of CHD or PDA correlated with severe NEC or mortality in children with NEC. What is Known: • It remains difficult to predict which infant that suffers from necrotizing enterocolitis at risk of death. • Several clinical and laboratory parameters tools to predict fatal outcome in NEC. What is New: • The following laboratory parameters were associated with the risk of death from NEC: Hemoglobin concentration, base excess and lactate level. • The following clinical variables were associated with the risk of death from NEC: Apgar scores, as well as the presence of congenital heart disease and patent ductus arteriosus.

Parental satisfaction with waiting time in a Swiss tertiary paediatric emergency department.

INTRODUCTION: Patient numbers in paediatric emergency departments (PED) are steadily increasing. Parental perception of waiting time and reasons for attending a PED with non-emergencies have been investigated in the UK, Australia, Korea, Canada and the USA. We sought to examine which factors influence parental satisfaction with waiting time in a tertiary Swiss PED and whether these differed from other countries. METHODS: Paper surveys were administered to parents of children presenting to our interdisciplinary PED from February to May 2015. Primary outcome was parental satisfaction with waiting time, secondary outcomes were satisfaction with treatment, parental reasons for presentation with non-emergencies, parental perception of times to triage, first physician contact and disposition from ED, level of physician training, understanding of various anticrowding strategies and comparison of perceived and true waiting times to triage and physician contact. RESULTS: 739 out of 750 surveys were returned (57 complete, 298 with 1 or 2 missing answers). Satisfaction with waiting time (on a 5-point-Likert-scale; 1 being the best possible answer) was higher in groups with shorter waiting time until triage (+0.41, p=0.001), first physician contact (+1.43, p<0.001) and discharge (+0.71, p<0.001), higher triage category urgency (+0.47, p=0.044) and available entertainment (+0.82, p<0.001). Early first physician contact (+0.33, p=0.008) and time to discharge less than 4 hours (+0.37, p<0.001) was associated with greater satisfaction with treatment (p<0.05). The most frequent reasons for presentation were parental impression that the child had an emergency (n=265, 35.9%) and referral by the family doctor (n=245, 33.2%). CONCLUSION: To counteract parental dissatisfaction associated with waiting time, we suggest the implementation of feasible measures including entertainment while waiting, early first medical review and timely discharge from the PED.

Balanced Electrolyte Solutions or Normal Saline? Resuscitative Fluid Administration Practice in Swiss Pediatric Acute Care: A Cross-Sectional Study.

INTRODUCTION: The ideal asanguineous intravenous fluid for volume resuscitation in children is controversially debated and clinical practice guidelines are scarce. Administration of large amounts of normal saline has been associated with complications including hyperchloremic acidosis, dysnatremia, neurologic damage, and fatality. AIM: We examined the current practice of intravenous fluid and blood product administration in acutely ill and injured children among pediatric acute care physicians in Switzerland. METHODS: For this descriptive, cross-sectional study, pediatric emergency departments, pediatric and neonatal intensive care units were surveyed by means of an online questionnaire. RESULTS: Sixty of 66 departments and 47 of 87 participants returned the survey. Normal saline (NS) was most commonly administered (n = 42/46, 91.3%) and twice as many times as balanced electrolyte solutions (n = 20/46, 43.5%). The mean fluid volumes ranged from 7.9 to 19.1 mL/kg. Hypertonic saline/NS were selected most often for shock with severe head injury. Half of participants administered colloids (48.9%). Packed red blood cells (97.7%) and fresh frozen plasma (88.4%) were most frequently given blood products. CONCLUSION: There is a distinct practice variation in intravenous fluid and blood product administration in children in Switzerland. Although NS is most frequently given, we observed a trend toward the use of balanced electrolyte solutions. Prospective studies are warranted to compare NS with balanced electrolyte solution (BES) in the pediatric acute care setting. We suggest that pediatric fluid administration guidelines and mass transfusion protocols are implemented to standardize this frequent intervention and minimize complications.

[Primary wound care in children]. / Kleine Wundversorgung bei Kindern.

Primary wound care in children Abstract. Successful wound care of infants and children is facilitated by good preparation and a calm atmosphere. There is not only the child as a patient but also the parents, with their fears and concerns. The physician has to take care of both of them. Parent and child should be informed about the therapeutic intervention appropriately. Reassuring of the child and distraction from the procedure are as important as the treatment itself (e. g. wound stitching or application of wound dressing). Topical anesthesia with LET Gel (lidocain, epinephrine, tetracaine), non-stinging methods to clean the wound (NaCl 0,9 % / Polyhexanid (Prontosan®) soaked swabs) and intranasal application of fentanyl / dormicum can be used to avoid fear and pain. Sedation is used deliberately in small children for wound care. Laceration wounds, mainly those affecting the scalp, chin or forehead can be treated in the emergency room without general anesthesia. Extensive wounds, burns and animal bites often require wound care under anesthesia in children.

Health-related Quality of Life After Fractures of the Lateral Third of the Clavicle in Children and Adolescents.

INTRODUCTION: The health-related quality of life after pediatric fractures of the lateral third of the clavicle is unknown. Given the trend in the literature toward surgical management of clavicular fractures in adults, we frequently receive referrals for surgical management of lateral clavicular fractures in children.Therefore, we examined (1) the health-related quality of life of patients who sustained a fracture of the lateral third of the clavicle in childhood and who were treated at a large regional pediatric trauma center, (2) if the health-related quality of life was associated with the age at the time of the injury, and (3) whether fracture and displacement patterns were associated with health-related qualify of life. METHODS: This is a retrospective case series. We were able to identify 69 patients (21 girls and 48 boys) who sustained a fracture of the lateral third of the clavicle between the period spanning from January 2004 to June 2015. These patients were asked to fill in a questionnaire that included the Quick version of the Disability of Arm, Shoulder, and Hand (DASH) (primary outcome) and the Pediatric Quality of Life Inventory (Peds-QL). Until July 2017, we were able to get a response from 56 of these patients, resulting in a follow-up of 81%. RESULTS: Treatment was conservative in all cases but one. In these cases, after a mean follow-up of 6.5 years (range, 1 to 13.2 y), the mean Quick-DASH was 1.2 (SD, 4.4) at a scale of 0 to 100, with lower values representing better quality of life. The mean physical function score of the Peds-QL was 97.8 (SD, 4.4), and the mean psychosocial function score was 91.8 (SD, 10.8), both at a scale of 0 to 100, with higher values representing better quality of life.There was no statistically significant association of age at the time of injury on the primary outcome Quick-DASH.There were no statistically significant associations between fracture or displacement patterns with regard to health-related quality of life. CONCLUSIONS: Conservative treatment of lateral clavicular fractures in children and adolescents is associated with excellent health-related quality of life, as measured with the disease-specific Quick-DASH and the non-disease-specific Peds-QL at a mean follow-up of 6.5 years (range, 1 to 13.2 y). There was no effect of age at the time of injury, and we were unable to identify associations to commonly analyzed risk factors, such as displacement patterns.As the conservative treatment of this type of fracture in children and adolescents is associated with such a good health-related quality of life, it is unlikely that these results could be further improved by surgical interventions. LEVEL OF EVIDENCE: Level IV-case series.

Randomized Controlled Trials for Arthroscopy in Degenerative Knee Disease: Was Conservative Therapy Appropriately Tried Prior to Arthroscopy?

PURPOSE: We aimed to determine if the randomized controlled trials (RCTs) evaluated in the most recent meta-analysis on arthroscopic surgery for degenerative knee arthritis included documented trials of appropriate conservative treatment prior to randomization. METHODS: We selected all RCTs of the most recent meta-analysis by Brignardello-Petersen and recorded for each RCT, if physiotherapy prior to randomization was mandatory. We compared the treatment effect of arthroscopy in studies in which physiotherapy prior to randomization was mandatory versus studies in which it was not. This review was registered in the PROSPERO database (CRD42017070091). RESULTS: Of the 13 RCTs in the meta-analysis, there were 2 in which physiotherapy prior to randomization was mandatory. In 1 additional multicenter RCT, prior conservative treatment was mentioned as mandatory in the publication, but not in the protocol. The treatment effects attributed to arthroscopy in terms of short-term pain (P = .0037), short-term function (P = .0309), and long-term function (P = .0012) were larger in studies in which prior physiotherapy was mandatory. CONCLUSIONS: Although the most recent meta-analysis claims that it is based "on patients who do not respond to conservative treatment," physiotherapy was mandatory prior to randomization only in 2 of the 13 studies. As several orthopaedic guidelines recommend that the first line of treatment in patients with degenerative arthritis of the knee should be conservative, for instance with physiotherapy, and the question of performing arthroscopy arises once conservative treatment fails, 11 of the 13 RCTs failed to adhere to these accepted guidelines. Therefore, patient selection in these 11 studies may not represent the typical indications for arthroscopy, where patients have tried conservative management prior to being offered surgery. When comparing studies where prior physiotherapy was mandatory to studies in which it was not mandatory, there were statistically significant effects favoring arthroscopy in terms of pain in the short term, and for function both in the short and the long term. These findings suggest that the treatment effects attributed to arthroscopy were higher when prior physiotherapy was mandatory. Given these findings, the external validity of most of these RCTs, and the resulting "strong recommendation against the use of arthroscopy in nearly all patients with degenerative knee disease," is called into question. LEVEL OF EVIDENCE: Level II, systematic review of Level I and II studies.

Unique Presentation of Hematometrocolpos Mimicking Cauda Equina Syndrome: Severe Back Pain and Urinary Incontinence in an Adolescent Girl.

BACKGROUND: Imperforate hymen with hematometrocolpos in adolescent females is a rare pediatric condition. Classical presentation includes abdominal pain or a pelvic mass in female patients with primary amenorrhea. Atypical complaints and reluctance among emergency physicians to perform genital examination in the emergency department or the pediatric emergency department (PED) may delay correct diagnosis. CASE REPORT: We report a unique, cauda equina syndrome-like presentation of hematometrocolpos secondary to imperforate hymen in a 13-year old, previously healthy girl with primary amenorrhea. In the PED, the unusual clinical presentation of severe back pain and urinary incontinence initially mimicked cauda equina syndrome and led to delayed correct diagnosis. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: The novelty of this case is a cauda equina-like presentation of imperforate hymen secondary to hematocolpos. This report illustrates the highly variable clinical presentation of this rare gynecological pediatric entity. It underlines the importance of considering this rare condition in the differential diagnosis of severe upper or lower back pain alongside voiding abnormalities including urinary retention and incontinence in adolescent females with primary amenorrhea. Above all, the importance of performing a thorough history and genital examination in this subgroup early in the investigation process in the PED emerges from this case. Essentially, excellent clinical judgment and genital examination by the emergency physician may minimize unnecessary radiological investigations and ultimately, accelerate correct diagnosis and expedite appropriate surgical treatment. However, not only pediatric and adult emergency physicians, but also pediatricians and general practitioners should be aware of this entity and its diverse clinical presentation.

Acute S100B in serum is associated with cognitive symptoms and memory performance 4 months after paediatric mild traumatic brain injury.

OBJECTIVE: This study explored whether acute serum marker S100B is related with post-concussive symptoms (PCS) and neuropsychological performance 4 months after paediatric mild traumatic brain injury (mTBI). RESEARCH DESIGN AND METHODS: This prospective short-term longitudinal study investigated children (aged 6-16 years) with mTBI (n = 36, 16 males) and children with orthopaedic injuries (OI, n = 27, 18 males) as a control group. S100B in serum was measured during the acute phase and was correlated with parent-rated PCS and neuropsychological performance 4 months after the injury. MAIN OUTCOMES AND RESULTS: The results revealed no between-group difference regarding acute S100B serum concentration. In children after mTBI, group-specific significant Spearman correlations were found between S100B and post-acute cognitive PCS (r = 0.54, p = 0.001) as well as S100B and verbal memory performance (r = -0.47, p = 0.006). In children after OI, there were insignificant positive relations between S100B and post-acute somatic PCS. In addition, insignificant positive correlations were found between neuropsychological outcome and S100B in children after OI. CONCLUSIONS: S100B was not specific for mild brain injuries and may also be elevated after OI. The group-specific association between S100B and ongoing cognitive PCS in children after mTBI should motivate to examine further the role of S100B as a diagnostic biomarker in paediatric mTBI.

Torsion of an Accessory Liver Lobe in a Newborn.

Accessory liver lobes are rare. We present the rare case of torsion of an accessory liver lobe in a neonate. A 13-day-old newborn presented with failure to thrive and hematemesis without fever. The initial workup with sonography, magnetic resonance imaging, and upper gastrointestinal study was suspicious of a duplication cyst, most likely in the posterior wall of the stomach. Laboratory and radiological findings were not suggesting a choledochal cyst. We performed a laparotomy with resection of the 3.2 × 2.1 × 1.1 cm mass. Intraoperatively, the cystic formation extended from of the liver bed up to the lesser curvature of the stomach. The mass was attached to the left liver lobe with fibrous bands. Histopathology revealed necrotic liver parenchyma with patent viable biliary ducts, indicative of an accessory liver lobe that underwent torsion in the perinatal period. The postoperative course and follow-up (6 months so far) were uneventful. To our knowledge, this is the youngest described patient in the literature with an accessory liver lobe torsion and the second case report concerning this entity in a neonate. It presents an extremely rare differential diagnosis in symptomatic neonates with a cystic mass in the upper abdomen.

Handlebar injuries in children.

INTRODUCTION: Handlebar injuries in children may lead to severe organ lesions despite minimal initial signs and without visible skin bruise. We present our experiences applying a diagnostic and therapeutic algorithm for blunt abdominal trauma, and present the history of two selected cases. MATERIALS AND METHODS: We retrospectively assessed the charts of children below 16 years of age, only who were observed for 24 h or more in our institution due to a handlebar injury between 2004 and 2011. All children were treated according to an institutional algorithm. RESULTS: 40 patients with a median age of 9.5 years were included. Diagnosed lesions were: ruptures of the liver (n = 6), spleen (n = 5), kidney (n = 1), and pancreas (n = 2), small bowel perforation (n = 3), and hernias of the abdominal (n = 2) or thoracic wall (n = 1). Surgical interventions were performed in 8 patients. The outcome was favorable in all the cases. Overall median hospitalization duration was 4.5 days (range 1-19 days). The overall duration between the accident and arrival at our emergency unit was 2.75 h (median, range 1-19 h). 20 children presented directly at our emergency unit after a median of 1.7 h (range 1-19.5 h). 20 children were referred by a family physician or a primary hospital after a median of 4.0 h (range 1-46 h). CONCLUSION: Handlebar injuries in children resulted in serious trunk lesions in half of the present patient series. The spectrum of injuries in handlebar accidents varies widely, especially injuries to the abdomen can unmask often only in the course. We advocate close observation of patients with thoracic and abdominal handlebar injuries which may be regarded as blunt stab wounds. An institutional algorithm for blunt abdominal trauma management is supportive for emergency care in patients with handlebar injuries.

Nurses' and physicians' reported difficulties and enablers to recognising and reporting child abuse in Swiss paediatric emergency and paediatric surgery departments - an observational study.

BACKGROUND: Under-detection and under-reporting of child abuse remains a considerable challenge in paediatric care, with a high number of cases missed each year in Switzerland and abroad. Published data regarding the obstacles and facilitators of detecting and reporting child maltreatment among paediatric nursing and medical staff in the paediatric emergency department (PED) are scarce. Despite the existence of international guidelines, the measures taken to counteract the incomplete detection of harm done to children in paediatric care are insufficient. AIM: We sought to examine up-to-date obstacles and enablers for detecting and reporting child abuse among nursing and medical staff in PED and paediatric surgery departments in Switzerland. METHODS: We surveyed 421 nurses and physicians working in PEDs and on paediatric surgical wards in six large Swiss paediatric hospitals using an online questionnaire between February 1, 2017, and August 31, 2017. RESULTS: The survey was returned by 261/421 (62.0%) respondents (complete n = 200, 76.6%; incomplete n = 61, 23.3%) with a preponderance of nurses (n = 150/261; 57.5%), 106/261 (40.6%) physicians, and 1/261 (0.4%) psychologists (n = 4/261; 1.5% missing profession). The stated obstacles to reporting child abuse were uncertainty about the diagnosis (n = 58/80; 72.5%), feeling unaccountable for notification (n = 28/80; 35%), uncertainty of whether reporting has any consequences (n = 5/80; 6.25%), lack of time (n = 4/80; 5%), forgetting to report (n = 2/80; 2.5%), and parental protection (n = 2/80; 2.5%) (unspecific answer, n = 4/80; 5%, multiple answers were possible, therefore items don not sum up to 100%). Even though most (n = 249/261 95.4%) respondents had previously been confronted with child abuse at/outside work, only 185/245 (75.5%) reported cases; significantly fewer nursing (n = 100/143, 69.9%) than medical staff (n = 83/99, 83.8%) (p = 0.013). Furthermore, significantly more nursing (n = 27/33; 81.8%) than medical staff (n = 6/33; 18.2%) (p = 0.005) reported a discrepancy between the number of suspected and reported cases (total 33/245 (13.5%). An overwhelming amount of participants were strongly interested in mandatory child abuse training (n= 226/242, 93.4%) and in the availability of standardised patient questionnaires and documentation forms (n = 185/243, 76.1%). CONCLUSION: In line with previous studies, insufficient knowledge about and lack of confidence in detecting the signs and symptoms of child abuse were the principal obstacles to reporting maltreatment. To finally address this unacceptable gap in child abuse detection, we recommend the implementation of mandatory child protection education in all countries where no such education has been implemented in addition to the introduction of cognitive aid tools and validated screening tools to increase child abuse detection rates and ultimately prevent further harm to children.

Generation of human induced pluripotent stem cell line encoding for a genetically encoded voltage indicator Arclight A242.

Genetically encoded voltage indicators (GEVIs) allow for monitoring membrane potential changes in neurons and cardiomyocytes (CMs) as an alternative to patch-clamp techniques. GEVIs facilitate non-invasive, high throughput screening of electrophysiological properties of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). A dual transgenic hiPSC line with Arclight A242 (GEVI) and an antibiotic resistance cardiac selection cassette was successfully generated from an earlier established hiPSC line MHHi001-A. After cardiac differentiation and selection, purified populations of CMs with constitutive GEVI expression can be utilized for studying cardiac development, disease modeling, and drug testing.

T lymphocytes induce human cancer cells derived from solid malignant tumors to secrete galectin-9 which facilitates immunosuppression in cooperation with other immune checkpoint proteins.

BACKGROUND: Galectin-9 is a member of the family of lectin proteins and crucially regulates human immune responses, particularly because of its ability to suppress the anticancer activities of T lymphocytes and natural killer cells. Recent evidence demonstrated that galectin-9 is highly expressed in a wide range of human malignancies including the most aggressive tumors, such as high-grade glioblastomas and pancreatic ductal adenocarcinomas, as well as common malignancies such as breast, lung and colorectal cancers. However, solid tumor cells at rest are known to secrete either very low amounts of galectin-9 or, in most of the cases, do not secrete it at all. Our aims were to elucidate whether T cells can induce galectin-9 secretion in human cancer cells derived from solid malignant tumors and whether this soluble form displays higher systemic immunosuppressive activity compared with the cell surface-based protein. METHODS: A wide range of human cancer cell lines derived from solid tumours, keratinocytes and primary embryonic cells were employed, together with helper and cytotoxic T cell lines and human as well as mouse primary T cells. Western blot analysis, ELISA, quantitative reverse transcriptase-PCR, on-cell Western and other measurement techniques were used to conduct the study. Results were validated using in vivo mouse model. RESULTS: We discovered that T lymphocytes induce galectin-9 secretion in various types of human cancer cells derived from solid malignant tumors. This was demonstrated to occur via two differential mechanisms: first by translocation of galectin-9 onto the cell surface followed by its proteolytic shedding and second due to autophagy followed by lysosomal secretion. For both mechanisms a protein carrier/trafficker was required, since galectin-9 lacks a secretion sequence. Secreted galectin-9 pre-opsonised T cells and, following interaction with other immune checkpoint proteins, their activity was completely attenuated. As an example, we studied the cooperation of galectin-9 and V-domain Ig-containing suppressor of T cell activation (VISTA) proteins in human cancer cells. CONCLUSION: Our results underline a crucial role of galectin-9 in anticancer immune evasion. As such, galectin-9 and regulatory pathways controlling its production should be considered as key targets for immunotherapy in a large number of cancers.

L-Kynurenine participates in cancer immune evasion by downregulating hypoxic signaling in T lymphocytes.

Malignant tumors often escape anticancer immune surveillance by suppressing the cytotoxic functions of T lymphocytes. While many of these immune evasion networks include checkpoint proteins, small molecular weight compounds, such as the amino acid L-kynurenine (LKU), could also substantially contribute to the suppression of anti-cancer immunity. However, the biochemical mechanisms underlying the suppressive effects of LKU on T-cells remain unclear. Here, we report for the first time that LKU suppresses T cell function as an aryl hydrocarbon receptor (AhR) ligand. The presence of LKU in T cells is associated with AhR activation, which results in competition between AhR and hypoxia-inducible factor 1 alpha (HIF-1α) for the AhR nuclear translocator, ARNT, leading to T cell exhaustion. The expression of indoleamine 2,3-dioxygenase 1 (IDO1, the enzyme that leads to LKU generation) is induced by the TGF-ß-Smad-3 pathway. We also show that IDO-negative cancers utilize an alternative route for LKU production via the endogenous inflammatory mediator, the high mobility group box 1 (HMGB-1)-interferon-gamma (IFN-γ) axis. In addition, other IDO-negative tumors (like T-cell lymphomas) trigger IDO1 activation in eosinophils present in the tumor microenvironment (TME). These mechanisms suppress cytotoxic T cell function, and thus support the tumor immune evasion machinery.

Health-Related Quality of Life after Adolescent Fractures of the Femoral Shaft Stabilized by a Lateral Entry Femoral Nail.

(1) Background: In adolescents, fractures of the femoral shaft that are not suitable for elastic-stable-intramedullary-nailing (ESIN), are challenging. We aimed to evaluate the health-related quality of life (HRQoL) and complications in adolescents treated with intramedullary rodding using the adolescent lateral trochanteric entry femoral nail (ALFN), and to assess if HRQoL was associated with additional injuries. (2) Methods: We followed-up on 15 adolescents with a diaphyseal femoral fracture who were treated with an ALFN from 2004 to 2017. Patients were asked to fill in a questionnaire that includes the iHOT, Peds-QL, and the Pedi-IKDC. (3) Results: The ALFN was used as a primary method of fixation in 13 patients, and as a fixation for failed ESIN in two cases. All 15 fractures healed radiographically. One distal locking screw broke. After a mean follow-up of 2.8 years, the mean iHOT-12 was 14.0 (SD 15.4), PedsQL-function was 85.7 (SD 19.3), PedsQL-social-score was 86.2 (SD 12.5), and the mean Pedi-IKDC was 77.2 (SD 11.3). In patients where the femoral fracture was an isolated injury, the HRQoL-scores were consistently higher compared with patients who sustained additional injures. (4) Conclusions: Treating diaphyseal fractures in adolescents with an ALFN resulted in good radiographic outcomes in all our cases. HRQoL, as measured by the iHOT, PedsQL, and Pedi-IKDC, was good to excellent; but it was consistently inferior in patients with additional injuries. These results suggest that the ALFN is a good alternative when patients are not suitable for ESIN, and that the HRQoL of adolescents who were treated with an ALFN is mainly influenced by the presence of additional injures, and less by the fracture of the femur itself.

Health-Related Quality of Life after Fractures of the Distal Forearm in Children and Adolescents-Results from a Center in Switzerland in 432 Patients.

(1) Background: We aimed to evaluate the health-related quality of life (HRQoL) in children with fractures of the distal forearm and to assess if HRQoL was associated with fracture classification; (2) Methods: We followed up on 432 patients (185 girls, 247 boys) who sustained a fracture of the distal radius or forearm from 1/2007 to 6/2007, 1/2014 to 6/2014, and 11/2016 to 10/2017. Patients filled in the Quick-DASH (primary outcome) and the Peds-QL; (3) Results: The radius was fractured in 429 and the ulna in 175 cases. The most frequent injury of the radius was a buckle fracture (51%, mean age 8.5 years), followed by a complete metaphyseal fracture (22%, 9.5 years), Salter-Harris-2 fracture (14%, 11.4 years), greenstick fracture (10%, 9.3 years), Salter-Harris-1 fracture (1%, 12.6 years), and other rare injuries. The most common treatment was closed reduction and an above-elbow cast in 138 cases (32%), followed by a cast without reduction (30%), splint (28%), and K-wire fixation and cast (9%). Definite treatment was performed initially in 95.8%, a new cast or cast wedging was performed in 1.6%, and revision surgery was performed in 2.5%. There were no open reductions and no plate fixations. After a mean follow-up of 4.2 years, patients with buckle fractures had a mean Quick-DASH of 3.3 (scale of 0-100) (complete fracture: 1.5; greenstick: 1.5; SH-1: 0.9; SH-2: 4.1; others: 0.9). The mean function score of the PedsQL ranged from 93.0 for SH-2 fractures to 97.9 for complete fractures; (4) Conclusions: In this cohort of 432 children with fractures of the distal forearm, there was equally good mean mid- and long-term HRQoL when assessed by the Quick-DASH and the PedsQL. There was a trend for children with complete metaphyseal fractures reporting better HRQoL than patients with buckle fractures or patients with Salter-Harris II fractures, however, these differences were not statistically significant nor clinically relevant.

Cytokines Activate JAK-STAT Signaling Pathway in MG-63 Cells on Titanium and Zirconia.

Although titanium has been traditionally used as the gold standard for dental implants, recent years have seen the widespread application of zirconia implants given their superiority with regards to reduced bacterial adhesion, inflammation and cellular-interaction in terms of bio-compatibility. The JAK-STAT signaling pathway plays an important role in bone remodeling and formation. The aim of the study was to investigate the activation of the JAK-STAT pathway through different cytokines in osteoblast-like cells (MG-63) on zirconia in comparison to titanium discs. IFN-γ induced the very strong activation of STAT1 protein, IFN-α activated both STAT1 and STAT3 molecules, IL-6 activated STAT3 and IL-4 induced the activation of STAT6 on both surfaces. The activation of STAT proteins was confirmed by western blot, immunofluorescence and flow cytometry using phospho-specific anti-STAT antibodies, which recognize only phosphorylated STAT proteins. The incubation of MG-63 cells with IFN-γ caused the upregulation of MHC class I and class II proteins when MG-63 cells were grown on zirconia and titanium discs. In sum, the present study shows that the JAK-STAT pathway is activated in MG-63 cells when they are incubated on titanium or zirconia surfaces.

Expression of the Immune Checkpoint Protein VISTA Is Differentially Regulated by the TGF-ß1 - Smad3 Signaling Pathway in Rapidly Proliferating Human Cells and T Lymphocytes.

Immune checkpoint proteins play crucial roles in human embryonic development but are also used by cancer cells to escape immune surveillance. These proteins and biochemical pathways associated with them form a complex machinery capable of blocking the ability of cytotoxic immune lymphoid cells to attack cancer cells and, ultimately, to fully suppress anti-tumor immunity. One of the more recently discovered immune checkpoint proteins is V-domain Ig-containing suppressor of T cell activation (VISTA), which plays a crucial role in anti-cancer immune evasion pathways. The biochemical mechanisms underlying regulation of VISTA expression remain unknown. Here, we report for the first time that VISTA expression is controlled by the transforming growth factor beta type 1 (TGF-ß)-Smad3 signaling pathway. However, in T lymphocytes, we found that VISTA expression was differentially regulated by TGF-ß depending on their immune profile. Taken together, our results demonstrate the differential biochemical control of VISTA expression in human T cells and various types of rapidly proliferating cells, including cancer cells, fetal cells and keratinocytes.