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.

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.

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.

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.

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.

School-age structural and functional MRI and lung function in children following lung resection for congenital lung malformation in infancy.

BACKGROUND: The management of asymptomatic congenital lung malformations is debated. Particularly, there is a lack of information regarding long-term growth and development of the remaining lung in children following lung resection for congenital lung malformations. In addition to conventional pulmonary function tests, we used novel functional magnetic resonance imaging (MRI) methods to measure perfusion and ventilation. OBJECTIVE: To assess functionality of the remaining lung expanded into the thoracic cavity after resection of congenital lung malformations. MATERIALS AND METHODS: A prospective, cross-sectional pilot study in five children who had surgery for congenital lung malformations during infancy. Participants had structural and functional MRI as well as spirometry, body plethysmography and multiple breath washout at school age. RESULTS: Structural MRI showed an expansion of the remaining lung in all cases. Fractional ventilation and relative perfusion of the expanded lung were locally decreased in functional MRI. In all other parts of the lungs, fractional ventilation and relative perfusion were normal in all children. There was an association between overall impairment of perfusion and elevated lung clearance index. The results of spirometry and body plethysmography varied between patients, including normal lung function, restriction and obstruction. CONCLUSION: Fractional ventilation and relative perfusion maps from functional MRI specifically locate impairment of the remaining lung after lung resection. These changes are not captured by conventional measures such as structural MRI and standard pulmonary function tests. Therefore, following lung resection for congenital lung malformation, children should be investigated more systematically with functional lung MRI.

Lack of Mucosal Cholinergic Innervation Is Associated With Increased Risk of Enterocolitis in Hirschsprung's Disease.

BACKGROUND & AIMS: Hirschsprung's disease (HSCR) is a congenital intestinal motility disorder defined by the absence of enteric neuronal cells (ganglia) in the distal gut. The development of HSCR-associated enterocolitis remains a life-threatening complication. Absence of enteric ganglia implicates innervation of acetylcholine-secreting (cholinergic) nerve fibers. Cholinergic signals have been reported to control excessive inflammation, but the impact on HSCR-associated enterocolitis is unknown. METHODS: We enrolled 44 HSCR patients in a prospective multicenter study and grouped them according to their degree of colonic mucosal acetylcholinesterase-positive innervation into low-fiber and high-fiber patient groups. The fiber phenotype was correlated with the tissue cytokine profile as well as immune cell frequencies using Luminex analysis and fluorescence-activated cell sorting analysis of colonic tissue and immune cells. Using confocal immunofluorescence microscopy, macrophages were identified in close proximity to nerve fibers and characterized by RNA-seq analysis. Microbial dysbiosis was analyzed in colonic tissue using 16S-rDNA gene sequencing. Finally, the fiber phenotype was correlated with postoperative enterocolitis manifestation. RESULTS: The presence of mucosal nerve fiber innervation correlated with reduced T-helper 17 cytokines and cell frequencies. In high-fiber tissue, macrophages co-localized with nerve fibers and expressed significantly less interleukin 23 than macrophages from low-fiber tissue. HSCR patients lacking mucosal nerve fibers showed microbial dysbiosis and had a higher incidence of postoperative enterocolitis. CONCLUSIONS: The mucosal fiber phenotype might serve as a prognostic marker for enterocolitis development in HSCR patients and may offer an approach to personalized patient care and new therapeutic options.

Cholinergic Signaling Attenuates Pro-Inflammatory Interleukin-8 Response in Colonic Epithelial Cells.

Infants affected by Hirschsprung disease (HSCR), a neurodevelopmental congenital disorder, lack ganglia of the intrinsic enteric nervous system (aganglionosis) in a variable length of the colon, and are prone to developing severe Hirschsprung-associated enterocolitis (HAEC). HSCR patients typically show abnormal dense innervation of extrinsic cholinergic nerve fibers throughout the aganglionic rectosigmoid. Cholinergic signaling has been reported to reduce inflammatory response. Consequently, a sparse extrinsic cholinergic innervation in the mucosa of the rectosigmoid correlates with increased inflammatory immune cell frequencies and higher incidence of HAEC in HSCR patients. However, whether cholinergic signals influence the pro-inflammatory immune response of intestinal epithelial cells (IEC) is unknown. Here, we analyzed colonic IEC isolated from 43 HSCR patients with either a low or high mucosal cholinergic innervation density (fiber-low versus fiber-high) as well as from control tissue. Compared to fiber-high samples, IEC purified from fiber-low rectosigmoid expressed significantly higher levels of IL-8 but not TNF-α, IL-10, TGF-ß1, Muc-2 or tight junction proteins. IEC from fiber-low rectosigmoid showed higher IL-8 protein concentrations in cell lysates as well as prominent IL-8 immunoreactivity compared to IEC from fiber-high tissue. Using the human colonic IEC cell line SW480 we demonstrated that cholinergic signals suppress lipopolysaccharide-induced IL-8 secretion via the alpha 7 nicotinic acetylcholine receptor (a7nAChR). In conclusion, we showed for the first time that the presence of a dense mucosal cholinergic innervation is associated with decreased secretion of IEC-derived pro-inflammatory IL-8 in the rectosigmoid of HSCR patients likely dependent on a7nAChR activation. Owing to the association between IL-8 and enterocolitis-prone, fiber-low HSCR patients, targeted therapies against IL-8 might be a promising immunotherapy candidate for HAEC treatment.

Risk Factors for Necrotizing Enterocolitis in Infants With Patent Arterial Duct. A Retrospective Matched Paired Analysis.

Background: The development of necrotizing enterocolitis (NEC) in neonates with patent ductus arteriosus (PDA) is not well-understood. Our aim was to find risk factors for NEC in children with a significant PDA and to assess differences in mortality and duration of hospital stay between patients with PDA and those with PDA and NEC. Methods: We performed a retrospective single center case control study including infants with PDA scheduled for treatment. We compared multiple patient data between patients with PDA and those with PDA and NEC from 2004 to 2018 using 1:2 and 1:1 matching. Results: We used 1:2 matching with 26 NEC patients (cases) and 52 PDA patients without NEC (controls) and 1:1 matching with 5 NEC patients and 5 PDA patients without NEC. NEC patients had lower Apgar score (1'), more congenital malformations, more suspected sepsis, less hypotension, higher minimum platelet count and higher CRP-values during the week before NEC (P < 0.05, respectively). The mortality was higher in NEC cases [29% (9/31)] compared to the control patients [2% (1/57), P < 0.001]. Lower Apgar score (1') was correlated with an increased risk of NEC stage III. Hypotension was inversely correlated with the odds of NEC (OR 0.3). Conclusions: NEC increased mortality in infants with PDA. Hypotension did not increase the risk of NEC in infants with PDA. Routine clinical parameters were not able to predict NEC in infants who suffer from PDA.

Chromosomal Heterogeneity of the G-401 Rhabdoid Tumor Cell Line: Unusual Partial 7p Trisomy.

Rhabdoid tumor is a very aggressive and hardly curable pediatric malignancy. It commonly starts in the kidneys but also can occur in the brain, liver, and other organs. The treatment of this tumor usually involves a combination of surgery, radiation, and chemotherapy. Because this tumor is rare, there is still limited experience with a defined standard of care. Cytogenetic analysis is an important routine method to monitor chromosomal aberrations. We have analyzed metaphases of the G-401 rhabdoid tumor cell line. In these cells we have observed metaphases with derivative chromosome 12 arising from partial trisomy 7p. With increasing passage number the numbers of metaphases having this derivative chromosome 12 were found to be higher. In passage number 2 only one metaphase had this pathological chromosome 12. By passage number 10 and passage number 15 about 25 and 95% of this derivative chromosome 12 were found, respectively. We were able to subclone G-401 cells by limiting dilutions and successfully separated cells having apparently normal karyotypes from cells having derivative chromosome 12. Using the cell proliferation assay we showed that clones possessing the derivative chromosome 12 grew more rapidly than clones with normal chromosomes. The cell cycle analysis confirmed this observation. Overall, in this study we describe for the first time a 7p triplication in a rare rhabdoid tumor of kidney. Both types of clones described in this study could be used as a preclinical model to study the involvement of partial chromosome 7 alterations in the development of rhabdoid tumors.

The Tim-3-Galectin-9 Pathway and Its Regulatory Mechanisms in Human Breast Cancer.

Human cancer cells operate a variety of effective molecular and signaling mechanisms which allow them to escape host immune surveillance and thus progress the disease. We have recently reported that the immune receptor Tim-3 and its natural ligand galectin-9 are involved in the immune escape of human acute myeloid leukemia (AML) cells. These cells use the neuronal receptor latrophilin 1 (LPHN1) and its ligand fibronectin leucine rich transmembrane protein 3 (FLRT3, and possibly other ligands) to trigger the pathway. We hypothesized that the Tim-3-galectin-9 pathway may be involved in the immune escape of cancer cells of different origins. We found that studied breast tumors expressed significantly higher levels of both galectin-9 and Tim-3 compared to healthy breast tissues of the same patients and that these proteins were co-localized. Increased levels of LPHN2 and expressions of LPHN3 as well as FLRT3 were also detected in breast tumor cells. Activation of this pathway facilitated the translocation of galectin-9 onto the tumor cell surface, however no secretion of galectin-9 by tumor cells was observed. Surface-based galectin-9 was able to protect breast carcinoma cells against cytotoxic T cell-induced death. Furthermore, we found that cell lines from brain, colorectal, kidney, blood/mast cell, liver, prostate, lung, and skin cancers expressed detectable amounts of both Tim-3 and galectin-9 proteins. The majority of cell lines expressed one of the LPHN isoforms and FLRT3. We conclude that the Tim-3-galectin-9 pathway is operated by a wide range of human cancer cells and is possibly involved in prevention of anti-tumor immunity.

A Swiss database and biobank to better understand and manage congenital lung anomalies.

Congenital lung anomalies are a group of rare malformations, often diagnosed during the prenatal period. Guidelines on how to manage these patients are currently under debate, especially with regard to prophylactic surgery in asymptomatic patients, or how to proceed with conservative follow-up. Currently, there is no clear consensus on management strategies. A Swiss congenital lung anomaly national database and biobank was created in 2016 to enable data recording and collection of surgical lung samples in order to help define the most appropriate management strategies. This national observational cohort study represents an important step towards a better understanding of the pathophysiology and clinical course of the diseases included under congenital lung anomalies, especially in the context of a small country like Switzerland.

Congenital Heart Disease Increases Mortality in Neonates With Necrotizing Enterocolitis.

Background: Studies on the influence of congenital heart disease (CHD) on neonates with necrotizing enterocolitis (NEC) have produced varied results. We therefore examined the influence of CHD on NEC outcomes. Methods: We carried out a retrospective single-center study including infants with confirmed NEC, treated between 2004 and 2017. We excluded patients with isolated patent ductus arteriosus or pulmonary hypertension (n = 45) and compared outcomes of patients with hemodynamically relevant CHD (n = 38) and those without CHD (n = 91). Results: Patients with CHD were more mature than those without CHD [gestational age, median, 95% confidence interval (CI95), 37.1, 34.5-37.2w, vs. 32.6, 31.9-33.3w; P < 0.01]. The presence of CHD did not influence the frequencies of severe disease (overall 21% Bell stage III), nor surgical interventions (overall 30%), the occurrence of intestinal complications (overall 13%), nor the duration of hospitalization (overall 38 days in survivors). The overall mortality as well as NEC-related mortality was increased with the presence of CHD, being 50% (19 out of 38) and 13% (5 out of 38), respectively, when compared to patients without CHD, being 8% (7 out of 91) and 3% (3 out of 91). The presence of CHD and of advanced NEC stage III were independent predictors of NEC-associated fatalities with multivariable odds ratios (CI95) of 7.0, 1.3-39.5 for CHD, and of 3.4, 1.6-7.5 for stage III disease. Conclusions: While some outcome parameters in neonates with NEC remained unaffected by the presence of CHD, the mortality risk for patients with CHD was seven times higher than without CHD.

Outcome in neonates with necrotizing enterocolitis and patent ductus arteriosus.

BACKGROUND: There is no agreement of the influence of patent ductus arteriosus (PDA) on outcomes in patients with necrotizing enterocolitis (NEC). In this study, we assessed the influence of PDA on NEC outcomes. METHODS: A retrospective study of 131 infants with established NEC was performed. Outcomes (death, disease severity, need for surgery, hospitalization duration), as well as multiple clinical parameters were compared between NEC patients with no congenital heart disease (n=102) and those with isolated PDA (n=29). Univariate, multivariate and stepwise logistic regression analyses were performed. RESULTS: Birth weight and gestational age were significantly lower in patients with PDA [median (95% CI): 1120 g (1009-1562 g), 28.4 wk (27.8-30.5 wk)] than in those without PDA [median (95% CI): 1580 g (1593-1905 g), 32.4 wk (31.8-33.5 wk); P<0.05]. The risk of NEC-attributable fatality was higher in NEC patients with PDA (35%) than in NEC patients without PDA (14%)[univariate odds ratio (OR)=3.3, 95% CI: 1.8-8.6, P<0.05; multivariate OR=2.4, 95% CI: 0.82-2.39, P=0.111]. Significant independent predictors for non-survival within the entire cohort were advanced disease severity stage III (OR=27.9, 95% CI: 7.4-105, P<0.001) and birth weight below 1100 g (OR=5.7, 95% CI: 1.7-19.4, P<0.01). CONCLUSIONS: In patients with NEC, the presence of PDA is associated with an increased risk of death. However, when important differences between the two study groups are controlled, only birth weight and disease severity may independently predict mortality.

One-stage laparoscopic surgery for inspissated bile syndrome: case report and review of surgical techniques.

Inspissated bile syndrome in a 6 week old boy was unresponsive to oral ursodesoxycholic acid. Intraoperative cholangiography revealed complete obstruction of the common bile duct. Therefore, the gallbladder fundus was pulled out through a laparoscopy port site and sutured to the fascia. A catheter was positioned into the infundibulum for irrigation with ursodesoxycholic acid. At day 8 complete resolution of the plug and free passage of contrast medium into the duodenum was documented radiologically. The catheter was removed, skin closed spontaneously without a second surgery for closure of the gall bladder.

Dissection of the appendix with ultrasound-activated scalpel: an experimental study in pediatric laparoscopic appendectomy.

BACKGROUND: The aim of this study was to examine mechanical, microbiologic, and morphologic changes of the appendicle rim to assess if it is appropriate to dissect the appendix with the ultrasound-activated scalpel (UAS) during laparoscopic appendectomy. MATERIALS AND METHODS: After laparoscopic resection of the appendix, using conventional Roeder slings, we investigated 50 appendicle rims with an in vitro procedure. The overall time of dissection of the mesoappendix with UAS was noted. Following removal, the appendix was dissected in vitro with the UAS one cme from the resection rim. Seal-burst pressures were recorded. Bacterial cultures of the UAS-resected rim were compared with those of the scissors resected rim. Tissue changes were quantified histologically with hematoxylin and eosin (HE) stains. RESULTS: The average time to dissect the mesoappendix was 228 seconds (25-900). Bacterial culture growths were less in the UAS-resected probes (7 versus 36 positive probes; (p > 0.01). HE-stained tissues revealed mean histologic changes in the lamina propria muscularis externa of 2 mm depth. The seal-burst pressure levels of the appendicle lumen had a mean of 420 mbar. Seal-burst pressures and depths of histologic changes were not dependent on the different stages of appendicitis investigated, gender, or age groups. Seal-burst pressure levels were not related to different depths of tissue changes (P = 0.64). CONCLUSIONS: The UAS is a rapid instrument for laparoscopic appendectomy and appears to be safe with respect to stability, sterility and tissue changes. It avoids complex time consuming instrument change manoeuvres and current transmission, which may induce intra- and postoperative complications. Our results suggest that keeping a safety margin of at least 5 mm from the bowel would be sufficient to avoid thermal damage.