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

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

Activation of immune evasion machinery is a part of the process of malignant transformation of human cells.

Malignant transformation of human cells is associated with their re-programming which results in uncontrolled proliferation and in the same time biochemical activation of immunosuppressive pathways which form cancer immune evasion machinery. However, there is no conceptual understanding of whether immune evasion machinery pathways and expression of immune checkpoint proteins form a part of the process of malignant transformation or if they are triggered by T lymphocytes and natural killers (NK) attempting to attack cells which are undergoing or already underwent malignant transformation. To address this fundamental question, we performed experimental malignant transformation of BEAS-2B human bronchial epithelium cells and RC-124 non-malignant human kidney epithelial cells using bracken extracts containing carcinogenic alkaloid called ptaquiloside. This transformation led to a significant upregulation of cell proliferation velocity and in the same time led to a significant upregulation in expression of key immune checkpoint proteins - galectin-9, programmed death ligand 1 (PD-L1), indoleamine 2,3-dioxygenase (IDO1). Their increased expression levels were in line with upregulation of the levels and activities of HIF-1 transcription complex and transforming growth factor beta type 1 (TGF-β)-Smad3 signalling pathway. When co-cultured with T cells, transformed epithelial cells displayed much higher and more efficient immune evasion activity compared to original non-transformed cells. Therefore, this work resolved a very important scientific and clinical question and suggested that cancer immune evasion machinery is activated during malignant transformation of human cells regardless the presence of immune cells in microenvironment

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

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-β1, 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

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

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