Soluble factors derived from neuroblastoma cell lines suppress dendritic cell differentiation and activation.

Dendritic cells (DC) play a key role in the initiation of both antitumor immunity and immunological tolerance. It has been demonstrated that exposure to soluble factors produced by tumor cells modulates DC functions and induces tolerogenic DC differentiation. In this study, we investigated the effects of neuroblastoma cell line-derived soluble factors on DC differentiation. Monocytes isolated from healthy volunteers were incubated with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor in the presence of culture supernatants from neuroblastoma cell lines. The culture supernatants from neuroblastoma cell lines, such as NLF and GOTO, partially blocked both downregulation of CD14 and upregulation of CD1a, and dramatically decreased IL-12 and tumor necrosis factor (TNF)-α production from mature DC, while no effect of SH-SY5Y cell supernatant was noted. In addition, IL-6 and IL-10 production from monocytes was increased by the supernatants of NLF and GOTO cells at 24 hours after incubation. Furthermore, we evaluated DC functions through stimulation of invariant natural killer T (iNKT) cells. α-Galactosylceramide-pulsed DC co-cultured with supernatants of NLF cells were unable to sufficiently stimulate iNKT cells. The decreased ability of iNKT cells to produce interferon (IFN)-γ after stimulation with neuroblastoma cell line supernatant-cultured DC was reversed by addition of IL-12. CD40 expression and IL-12 production in NLF-sup-treated DC were increased by addition of exogenous IFN-γ. These results indicate that tolerogenic DC are induced in the neuroblastoma tumor microenvironment and attenuate the antitumor effects of iNKT cells. Interactions between iNKT cells and αGalCer-pulsed DC have the potential to restore the immunosuppression of tolerogenic DC through IFN-γ production.

Frequency and proliferative response of circulating invariant natural killer T cells in pediatric patients with malignant solid tumors.

BACKGROUND: Invariant natural killer T (iNKT) cells play an important role in tumor immunity, enhancing both innate and acquired immunity. We have previously shown the enhancement of antibody-dependent cellular cytotoxicity against neuroblastoma by activated iNKT cells. As a first step towards clinical application, we studied the frequency and proliferative response of circulating iNKT cells in children with and without cancer. METHODS: Blood samples were collected from 10 patients with pediatric malignant solid tumors and 11 patients with non-neoplastic diseases (control). The frequency of circulating iNKT cells was quantified by flow cytometry. Whole peripheral blood mononuclear cells were then stimulated with α-galactosylceramide (α-GalCer) for 7 days, and the expansion rate of the iNKT-cell fraction was assessed. RESULTS: The frequency of iNKT cells in the patients of the cancer and control group did not differ to a statistically significant extent. The iNKT-cell population increased after α-GalCer stimulation in all cases. The iNKT cells of patients who had undergone intensive chemotherapy also had the potential to expand in vitro. CONCLUSIONS: Unlike adult cancer patients, the numbers of circulating iNKT cells were not decreased in pediatric cancer patients. α-GalCer stimulation induced a proliferative response in all of the patients.

Invariant natural killer T infiltration in neuroblastoma with favorable outcome.

BACKGROUND: Tumor immunity has been suggested to play a key role in clinical and biological behavior of neuroblastomas. Given that CD1-restricted invariant natural killer T (iNKT) cells enhance both innate and acquired tumor immunity, we investigated the expression of the iNKT-cell-specific T-cell receptor Vα24-Jα18 in neuroblastoma tissues and its correlation with clinical and biological characteristics. METHODS: Using real- time quantitative PCR, we quantified the expression of Vα24-Jα18 in untreated tumor samples from 107 neuroblastoma cases followed in our institution and analyzed the correlation between the presence of infiltrated iNKT cells and clinical characteristics or patients' outcome. RESULTS: Vα24-Jα18 receptor was detected in 62 untreated cases (57.9%). The expression was significantly higher in stages 1, 2, 3, or 4S (P = 0.0099), in tumors with low or intermediate risk (P = 0.0050), with high TrkA expression (P = 0.0229), with favorable histology (P = 0.0026), with aneuploidy (P = 0.0348), and in younger patients (P = 0.0036). The overall survival rate was significantly higher in patients with iNKT-cell infiltration (log-rank; P = 0.0089). CONCLUSIONS: Since tumor-infiltrating iNKT cells were predominantly observed in neuroblastomas undergoing spontaneous differentiation and/or regression, we suggest that iNKT cells might play a key role in these processes.

[A Case of Jejunal Ectopic Pancreatic Cancer with Small Bowel Obstruction].

Were port a caseof an 82-year-old man who presented with vomiting. Computed tomography(CT)revealed a jejunum tumor and small bowel obstruction. Enteroscopy revealed a protruded lesion and biopsy indicated adenocarcinoma. PET-CT revealed nothing without jejunal tumor. Therefore, with a preoperative diagnosis of primary small bowel cancer, we performed operation. Surgery indicated peritoneal disseminations and a jejunal tumor 40 cm distal from the ligament of Treitz, and we performed small bowel partial resection. Pathological examination revealed adenocarcinoma originating from a Heinrich type I ectopic pancreas in the jejunum. Ectopic pancreatic cancer in the jejunum is rare, and we review case reports in the literature.

Antibody-dependent cellular cytotoxicity toward neuroblastoma enhanced by activated invariant natural killer T cells.

Anti-ganglioside GD2 antibodies mainly work through antibody-dependent cellular cytotoxicity (ADCC) and have demonstrated clinical benefit for children with neuroblastoma. However, high-risk neuroblastoma still has a high recurrence rate. For further improvement in patient outcomes, ways to maximize the cytotoxic effects of anti-GD2 therapies with minimal toxicity are required. Activated invariant natural killer T (iNKT) cells enhance both innate and type I acquired anti-tumor immunity by producing several kinds of cytokines. In this report, we investigated the feasibility of combination therapy using iNKT cells and an anti-GD2 antibody. Although some of the expanded iNKT cells expressed natural killer (NK) cell markers, including FcγR, iNKT cells were not directly associated with ADCC. When co-cultured with activated iNKT cells, granzyme A, granzyme B and interferon gamma (IFNγ) production from NK cells were upregulated, and the cytotoxicity of NK cells treated with anti-GD2 antibodies was increased. Not only cytokines produced by activated iNKT cells, but also NK-NKT cell contact or NK cell-dendritic cell contact contributed to the increase in NK cell cytotoxicity and further IFNγ production by iNKT cells and NK cells. In conclusion, iNKT cell-based immunotherapy could be an appropriate candidate for anti-GD2 antibody therapy for neuroblastoma.

Blockade of programmed death-1/programmed death ligand pathway enhances the antitumor immunity of human invariant natural killer T cells.

The role of invariant natural killer T (iNKT) cells in antitumor immunity has been studied extensively, and clinical trials in patients with advanced cancer have revealed a prolonged survival in some cases. In recent years, humanized blocking antibodies against co-stimulatory molecules such as PD-1 have been developed. The enhancement of T cell function is reported to improve antitumor immunity, leading to positive clinical effects. However, there are limited data on the role of PD-1/programmed death ligand (PDL) molecules in human iNKT cells. In this study, we investigated the interaction between PD-1 on iNKT cells and PDL on antigen-presenting cells (APCs) in the context of iNKT cell stimulation. The blockade of PDL1 at the time of stimulation resulted in increased release of helper T cell (Th) 1 cytokines from iNKT cells, leading to the activation of NK cells. The direct antitumor function of iNKT cells was also enhanced after stimulation with anti-PDL1 antibody-treated APCs. According to these results, we conclude that the co-administration of anti-PDL1 antibody and alpha-galactosylceramide (αGalCer)-pulsed APCs enhances iNKT cell-mediated antitumor immunity.

Optimal surgical treatment and urological outcomes in boys with pelvic and urogenital rhabdomyosarcomas and soft tissue sarcomas.

BACKGROUND: Soft tissue sarcomas (STS) of pelvic origin in boys often involve the urogenital organs. The optimal extensiveness of radical surgery has long been an issue of discussion, since exenterative surgeries result in severe urogenital adverse effects. We conducted a retrospective review of patients with pelvic STS treated in two regional center hospitals and assessed the radicality of surgery and the functional outcome of the bladder. PATIENTS: Medical records and surgical reports of nine cases (embryonal rhabdomyosarcoma 6, malignant triton tumor 2, suspected rhabdomyosarcoma 1) treated within 1997-2012 were reviewed. Site of origin was prostate in seven, retroperitoneal in two. Average follow-up period was 96 months. TREATMENT AND OUTCOME: All cases were subjected to neoadjuvant chemotherapy. Response was PR in four, SD in two, and PD in two. Radical surgery resulted in gross total resection in eight, and partial resection in one. Three underwent cystoprostatectomy, two cases underwent prostatectomy, and bladder-preserving tumor resection was carried out in four cases. At the last follow-up, three retained a functional bladder. Two required augmentation cystoplasty with intestinal conduits. CONCLUSIONS: The majority of the on-going clinical trials recommend conservative surgery for bladder/prostate rhabdomyosarcoma, and to preserve the bladder function particularly in chemosensitive tumors. Some other groups, however, advocate the importance of radical surgery to prevent local relapse. These reports include heterogenous group of patients in the cohort, and therefore it is difficult to draw a conclusion of which approach truly contributes to the survival of the patients better. Future studies should evaluate bladder and sexual function objectively to establish reliable evidence regarding the benefit and adverse effects of different surgical approaches. These data would be informative to optimize the treatment balance for children with pelvic rhabdomyosarcomas.

Larger Physique as a Risk Factor for Infantile Appendicitis: A Retrospective Study.

The clinical features and risk factors of acute appendicitis in infants are unclear. Our aim was to evaluate the association between anthropometrics and the occurrence of infantile appendicitis. This was a retrospective study of infants (<6 years of age) and school-age children (6-10 years of age) of Asian ethnicity who required hospitalization for appendicitis at our two participating institutions between 2004 and 2018. The Z-score for height, body weight, and body mass index (BMI) was compared between the two groups, as well as between patients presenting with perforated and non-perforated appendicitis. The analysis included data from 73 infants and 362 school-age children. Z-scores were greater in infants than in school-age children for height (0.37 versus -0.03, p = 0.003) and body weight (0.12 versus -0.36, p = 0.023), with no between-group difference for the Z-score of BMI. There was no difference in Z-scores for height, weight, and BMI between the perforated and non-perforated appendicitis infant groups. Infants presenting with acute appendicitis were characterized by a larger physique but with normal proportion. This trend was not observed in school-age children. Therefore, larger infants presenting with abdominal pain should be screened for appendicitis.

Src kinase family inhibitor PP2 induces aggregation and detachment of neuroblastoma cells and inhibits cell growth in a PI3 kinase/Akt pathway-independent manner.

PURPOSE: Neuroblastoma (NB) is one of the most common extracranial solid tumors in children and is known for its clinical and biological heterogeneity. The aim of this study is to reveal the functional role of src family kinases in the biological behavior of NB by inhibiting their kinase activities with a specific inhibitor, PP2 (4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine). METHODS: NB cell lines (SH-SY5Y, IMR32, RT-BM-1, CHP134, NLF, and LA-N-5) were treated with 0.1-10 µM of PP2. Morphological changes, cell growth, and cell death were assessed, as well as all-trans retinoic acid (ATRA)-induced neuronal differentiation and epidermal growth factor (EGF)-induced proliferation. RESULTS: At 24 h after PP2 treatment, NB cell lines showed drastic cell aggregation. PP2 also inhibited cell growth of NB in a dose-dependent manner. Apoptosis was detected in these cells. ATRA-induced neuronal differentiation of RT-BM-1 was not affected by PP2. PP2 reduced the proliferative effect of EGF. EGF-induced rapid activation of Akt, which was not blocked by PP2 treatment, suggesting that the cellular events triggered by PP2 were independent to PI3 kinase/Akt signaling pathway. CONCLUSION: Our data suggests that src family kinases promote cell survival/proliferation and reduces cell aggregation of NBs. Src family kinase inhibitors may be good candidates for a novel molecular target therapy.

Evidence for viral infection as a causative factor of human biliary atresia.

OBJECTIVES: To explore the evidence for viral infections triggering human biliary atresia (BA) by reviewing archival original articles that analyzed human samples via polymerase chain reaction (PCR) experiments, considering the recent experimental trend of extensive use of rotaviral BA animal models. METHODS: A PubMed search retrieved original articles that reported the results of PCR experiments for detecting viral DNA or RNA in patient samples as proof of past infection. Search terms included the often-debated DNA or RNA viruses and BA. Special focus was directed toward PCR analyses that targeted reovirus and rotavirus, where PCR accuracy, specimen characteristics and their interpretations were compared. RESULTS: Nineteen studies were conducted on 16 different kinds of viruses using PCR, with 5 studies on reovirus, 3 on rotavirus, 10 on cytomegalovirus, 5 on Epstein-Barr virus, 4 on parvovirus B19, and so on. Among the papers suggesting a possible viral link to only BA, there was no study on reovirus, 1 on rotavirus, 3 on cytomegalovirus, 1 on EB virus, and 1 on papillomavirus. Of the 6 PCR studies on Reoviridae, 3 on reovirus and 2 on rotavirus were evaluated rigorously for experimental accuracy, including their sensitivity. Two research groups analyzed preoperative stool samples in addition to generic hepatobiliary tissue obtained at surgery. Sample collection timing varied widely, with storage period prior to PCR experimentation not revealed in most reports on Reoviridae. CONCLUSION: Although a considerable number of PCR studies have sought to clarify a viral role in the pathogenesis of BA using human samples, the findings have been contradictory and have not succeeded in achieving an obvious differentiation between causative and accidental infection of the focused virus. Reproducible and convincing evidence for a causative Reoviridae infection has been lacking based on objective data from highly sensitive PCR experiments. Even though the possibility remains of viral disappearance at the timing of collection, to avoid further ambiguous interpretations of PCR results, rigorous and meticulous collection of large numbers of specimens at carefully planned timing, along with a strictly adjusted and finely tuned PCR system, is strongly recommended for obtaining more reliable and consistent results.

Risk Factors for Intestinal Obstruction After Ladd Procedure.

Intestinal obstruction is a common complication after Ladd procedure. Ninety-three cases who had undergone the Ladd procedure between 1977 and 2013 treated at our own institution were retrospectively reviewed to identify the causes and risk factors for intestinal obstruction. The Ladd procedure has been performed without any intestinal fixing. Of the 87 cases who survived to discharge, intestinal obstruction was observed in 22 (25.3%). Among the cases with intestinal obstruction, 13 (59.1%) showed intestinal ischemia at the initial operation; this incidence was notably high, although it is low when only those cases with another concurrent surgical digestive disease are considered. All cases of intestinal obstruction were caused not by recurrent volvulus, but by adhesion between the intestine and the mesentery. Intestinal fixing is not required to prevent recurrent volvulus, but it is important to achieve adequate widening of the mesenteric base. The risk of intestinal obstruction after the Ladd procedure, on the other hand, is high. Moreover, patients with intestinal ischemia have an increased risk of intestinal obstruction.

Hybrid video-assisted thoracoscopic surgery lobectomy of fissureless congenital cystic adenomatoid malformation: a case report.

INTRODUCTION: Thoracoscopic lobectomy for congenital pulmonary airway malformation has been indicated from the neonatal period to adolescence. However, it is difficult to approach the pulmonary artery for lobectomy in congenital lung malformations with incomplete or absent interlobar fissures. Multidetector computed tomographic images and computed tomography pulmonary angiography gave us helpful information before the operation. We performed thoracoscopic lobectomy for congenital pulmonary airway malformations with absent interlobar fissures and adhesions in accordance with information from multidetector computed tomographic images. CASE PRESENTATION: A 14-year-old Japanese girl received a diagnosis of congenital pulmonary airway malformation when she presented with pneumonia. Using multidetector computed tomography and three-dimensional reconstruction provides meticulous characterization of the anatomy in pediatric patients. We confirmed that her left A4+5 artery arose from her left pulmonary artery medial to A6. Her left pulmonary artery was divided just proximal to the A6 origin before the lobes were separated safely. We took advantage of using a stapler to divide the fissureless thick parenchyma. Perioperative diagnosis was congenital cystic adenomatoid malformation. CONCLUSIONS: We used preoperative multidetector computed tomography to outline the bronchovascular anatomy and guide hybrid video-assisted thoracoscopic surgery for a congenital cystic adenomatoid malformation in a fissureless left lung.