CD69 on Tumor-Infiltrating Cells Correlates With Neuroblastoma Suppression by Simultaneous PD-1 and PD-L1 Blockade.

INTRODUCTION: Tumor-infiltrating cells play an important role in tumor immunology, and tumor-infiltrating lymphocytes (TILs) are critical in antitumor reaction related to immune checkpoint inhibition targeting programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1). METHODS: In nude mice, which are immune deficient because they lack T cells, and inbred A/J mice, which are syngeneic to neuroblastoma cells (Neuro-2a) and have normal T cell function, we investigated the importance of T lymphocytes in immune checkpoint inhibition in mouse neuroblastoma and analyzed the immune cells in the tumor microenvironment. Then, we subcutaneously injected mouse Neuro-2ainto nude mice and A/J mice, administered anti-PD-1 and anti-PD-L1 antibodies by intraperitoneal injection, and evaluated tumor growth. At 16 d after Neuro-2a cells injection, mice were euthanized, tumors and spleens were harvested, and immune cells were analyzed by flow cytometry. RESULTS: The antibodies suppressed tumor growth in A/J but not in nude mice. The co-administration of antibodies did not affect regulatory T cells (culster of differentiation [CD]4+CD25+FoxP3+ cells) or activated CD4+ lymphocytes (expressing CD69). No changes in activated CD8+ lymphocytes (expressing CD69) were observed in spleen tissue. However, increased infiltration of activated CD8+ TILs was seen in tumors weighing less than 300 mg, and the amount of activated CD8+ TILs was negatively correlated with tumor weight. CONCLUSIONS: Our study confirms that lymphocytes are essential for the antitumor immune reaction induced by blocking PD-1/PD-L1 and raises the possibility that promoting the infiltration of activated CD8+ TIL into tumors may be an effective treatment for neuroblastoma.

Thoracoscopic removal with fluoroscopic radiographic guidance of thoracoamniotic shunting catheters in newborns.

Fetal thoracoamniotic shunting (TAS), which drains pleural effusion, is a treatment for severe primary fetal pleural effusion. While TAS is an effective treatment, its complications include bleeding and the catheter becoming dislodged, and also penetrating the thoracic cavity or chest wall. Catheters dislodged into the thoracic cavity in TAS can be removed by thoracoscopy. However, if there are adhesions in the thoracic cavity, finding the TAS catheter with a thoracoscope can be difficult. We used fluoroscopic radiography in addition to a thoracoscope to remove a TAS catheter in four patients. A 5-mm trocar was inserted into the thoracic cavity, and a 2.7-mm scope and 3-mm forceps were inserted into the trocar. We searched for TAS catheters using a thoracoscope and fluoroscopic radiography. If there are adhesions in the thoracic cavity and removing the TAS catheter is difficult, the combined use of a thoracoscope and fluoroscopic radiography may prove helpful.

Expression of programmed cell death-1 on neuroblastoma cells in TH-MYCN transgenic mice.

BACKGROUND: Immunotherapy may improve the poor prognosis of high-risk neuroblastoma. Programmed cell death-1 (PD-1) is expressed in several cancers. The tyrosine hydroxylase MYCN (TH-MYCN) transgenic mouse model is widely used in neuroblastoma research, but detailed information on its immunological background is lacking. Therefore, we studied the immunological tumor microenvironment and tumor cell surface antigen expression in homozygote and hemizygote mice and effects of antibody therapy against PD-1. METHODS: CD4, CD8, CD11b, and CD11c expression in immune cells from retroperitoneal lymph nodes and spleen was analyzed by flow cytometry. Tumor cell surface antigen expression was confirmed, and data from homozygote and hemizygote mice were compared. Effects of anti-PD-1 antibody were evaluated. RESULTS: CD4-, CD8-, CD11b-, and CD11c-positive cells were not significantly different in homozygote and hemizygote mice, and CD11b- and CD11c-positive cells were identified in the tumor microenvironment in both. Tumor cells expressed PD-1, and anti-PD-1 antibody had anti-tumor effects and significantly reduced the percentage of living tumor cells in cultures after 2 h. CONCLUSION: The immunological background is similar in homozygote and hemizygote TH-MYCN transgenic mice, and both have PD-1-positive tumor cells. Anti-PD-1 antibody suppresses tumor growth. This mouse model may be a useful for studying immunotherapy of neuroblastoma.

Female pediatric inguinal hernia: uterine deviation toward the hernia side.

PURPOSE: We aimed to clarify the frequency and the clinical significance of deviation of the uterus in female pediatric inguinal hernia. METHODS: We retrospectively evaluated the data of 94 female pediatric inguinal hernia cases that were treated by laparoscopic percutaneous extraperitoneal closure. We assessed for correlations between uterine deviation and age, body weight, the size of the hernia orifice, and the presence of contralateral processus vaginalis (PV) patency. RESULTS: Eighty-four of 94 cases were diagnosed with unilateral inguinal hernia. A total of 62 (73.8%) of these had uterine deviation to the hernia side (Group D); 22 (26.2%) had no deviation to the hernia side (Group N) (P < 0.001). Group D cases were significantly younger than those in Group N (P = 0.0351). There was no difference in body weight, size of the hernia orifice, or contralateral PV patency between the two groups. CONCLUSION: The incidence of uterine deviation toward the hernia side was statistically significant. It is important to recognize that female pediatric inguinal hernia repair carries an increased risk of ovarian and fallopian tube damage, because these appendages are close to the hernia orifice as a result of the uterine deviation.

Immune Checkpoint Inhibition Followed by Tumor Infiltration of Dendritic Cells in Murine Neuro-2a Neuroblastoma.

BACKGROUND: Most tumors responding to immunotherapy with monoclonal antibodies targeting programmed cell death protein1 (PD1) and programmed death ligand-1 (PD-L1) show surface expression of PD-L1. Neuroblastoma has been reported to show low PD-L1 surface expression. METHODS: The effect of immune checkpoint inhibitor on mouse neuroblastoma was investigated, and host immune cells were analyzed in the tumor microenvironment. Expression of co-stimulatory molecules by Neuro-2a mouse neuroblastoma cells was analyzed using flow cytometer. Neuro-2a cells were inoculated subcutaneously into A/J mice, followed by intraperitoneal injection of antibodies targeting PD-1 and PD-L1. Mice were sacrificed for the measurement of tumor weights on day 14 following tumor inoculation, and tumor-infiltrating cells were analyzed using a flow cytometer. RESULTS: Dim expression of PD-L1 was observed on the cell surface of cultured Neuro-2a cells. Growth of subcutaneous tumors was significantly suppressed, and PD-L1-expressing tumor cells were depleted by the antibody treatment. We confirmed that Neuro-2a cells opsonized by the anti-PD-L1 antibody were phagocytosed in the in vitro setting. In the treated tumor microenvironments, CD8α+ lymphocyte and CD11c+ MHC II+ cells were significantly accumulated in comparison with the control group. These CD11c+ MHC II+ cells expressed CD80, CD86, CD14, and CD40, but not CD205, PD-L1, or CTLA4. PD-1 expression was detected dimly. Immune suppressive effects of CD11b+Gr-1+ myeloid-derived suppressor cells by the administration of anti-PD-1 and PD-L1 antibodies were not observed in spleen, regional lymph nodes, or tumor microenvironment. CONCLUSIONS: Our findings raise the possibility that co-administration of anti-PD-1 and anti-PD-L1 antibodies have a synergistic effect on inhibition of tumor growth and could be an effective therapy against neuroblastoma with dim expression of PD-L1.

Acute acalculous cholecystitis with eosinophilic infiltration.

We report a case of acute acalculous cholecystitis with eosinophilic infiltration. A previously healthy 6-year-old boy was referred with right abdominal pain. Imaging demonstrated marked thickening of the gallbladder wall and peri-cholecystic effusion. Acute acalculous cholecystitis was diagnosed. Symptoms persisted despite conservative treatment, therefore cholecystectomy was performed. Pathology indicated infiltration of eosinophils into all layers of the gallbladder wall. The postoperative course was uneventful and the patient has had no further symptoms. Eosinophilic cholecystitis is acute acalculous cholecystitis with infiltration of eosinophils. The causes include parasites, gallstones, allergies, and medications. In addition, it may be seen in conjunction with eosinophilic gastroenteritis, eosinophilic pancreatitis, or both. An allergic reaction to abnormal bile is thought to be the underlying cause. The present case did not fulfill the diagnostic criteria of eosinophilic cholecystitis, but this may have been in the process of developing.

Phagocytosis of bafilomycin A1-treated apoptotic neuroblastoma cells by bone marrow-derived dendritic cells initiates a CD8α+ lymphocyte response to neuroblastoma.

This study aimed to determine whether bafilomycin A1 (Baf-A1), a vacuolar H-ATPase inhibitor, could promote an immune response after the induction of apoptosis in mouse neuroblastoma cells. Mouse neuro-2a cells were cultured in a medium containing Baf-A1, and apoptosis was evaluated by flow cytometry. To examine the influence in the phagocytic cell, CD11b spleen cells or bone marrow-derived dendritic cells (BM-DCs) were cocultured with Baf-A1-treated neuro-2a. Interferon-γ (IFN-γ) production was used as an index of the immune response, and CDDP was used as the negative control. When CD8α cells were cocultured with CD11b cells and Baf-A1-treated neuro-2a cells in the presence of CpG-oligodeoxynucleotide (CpG-ODN) (a toll-like receptor 9 [TLR-9] agonist), CD8α lymphocyte proliferation and secretion of IFN-γ were observed. Phagocytosis of apoptotic cells by BM-DCs was maximal after simultaneous stimulation with CpG-ODN and lipopolysaccharide (LPS; a TLR-4 agonist). IFN-γ secretion was maximal when Baf-A1-treated neuro-2a cells and CD8α lymphocytes were cocultured with BM-DCs and stimulated with CpG-ODN. In contrast, IFN-γ production was not increased when the cells were cultured with LPS. When cells were stimulated with both CpG-ODN and LPS, promotion of IFN-γ production by CpG-ODN was suppressed. Induction of apoptosis by Baf-A1 could possibly enhance antitumor immunity in patients receiving chemotherapy for neuroblastoma. Stimulation of BM-DCs with a TLR-9 agonist could promote antitumor activity after Baf-A1 treatment.

Thoracoscopy-assisted removal of a thoracoamniotic shunt double-basket catheter dislodged into the fetal thoracic cavity: report of three cases.

The indications for and timing of surgical removal of a dislodged thoracoamniotic shunt double-basket catheter are not established, and the side effects of the dislodged into the thoracic cavity remain controversial. The double-basket catheter was designed to reduce the incidence of catheter dislodgement; however, we have encountered four cases of thoracoamniotic shunt double-basket catheter dislodgement into the fetal thorax. The dislodged shunt catheters were removed safely with thoracoscopic assistance within several days of birth, when additional treatments for pleural effusion were needed, such as thoracic drainage tube insertion and adhesion treatment of the thorax. We report the clinical courses of three of these cases of thoracoamniotic shunt tube dislocation. By waiting several days postnatally for stabilization of respiratory and circulatory status and the effective use of thoracoscopic assistance, the dislodged catheter was safely removed from the neonatal thorax. The accumulation of case reports will help establish suitable treatments, and their indication, for a dislodged thoracoamniotic shunt catheter within the fetal thoracic cavity.

[Comparison of the chemoimmunotherapeutic effect of doxorubicin and bafilomycin-A1 in mouse neuroblastoma cells].

The aim of this study was to compare the ability of the drugs doxorubicin and Bafilomycin-A1(Baf-A1)to promote an immune reaction following the induction of cell death in a mouse neuroblastoma model. Neuro-2a cells were cultured in medium containing doxorubicin or Baf-A1. Bone marrow-derived dendritic cells(BM-DCs)were co-cultured with neuro-2A cells that were grown in doxorubicin- or Baf-A1-containing media, and phagocytosis of neuro-2a cells by the BN-DCs was evaluated. Additionally, dead neuro-2a cells were co-cultured with CD8a + lymphocytes and BM-DCs, and the proliferation of CD8a + cells was evaluated. Interferon-g(IFN-g)production was used as an indexof the immune response. Dead neuro-2a cells treated with doxorubicin were phagocytosed effectively compared to the cells treated with Baf-A1. However, phagocytosis of cells treated with Baf-A1 was promoted after stimulation with CpG oligodeoxynucleotide (CpG-ODN). When CD8a + cells were co-cultured with BM-DCs and doxorubicin-treated neuro-2a cells, CD8a + lymphocyte proliferation was observed. There was no statistical difference in IFN-g secretion between the doxorubicin-treated and Baf-A1-treated cells. However, after stimulation by CpG-ODN, IFN-g production was more effectively observed in the Baf-A1-treated cells. Induction of cell death by doxorubicin or Baf-A1 could possibly enhance antitumor immunity in patients receiving chemotherapy for neuroblastoma. Selection of anti-tumor agents and stimulation of BM-DCs with a toll-like receptor (TLR) agonist is considered important in promoting antitumor activity after chemotherapy.

Two cases of right congenital diaphragmatic hernia with stable respiratory condition: Two case reports.

INTRODUCTION: Right congenital diaphragmatic defect (CDH) has been reported poor prognosis. However, laterality of the defect as the prognostic factor is recent controversial topic. We experienced two cases of right CDH with relatively stable respiratory condition and good clinical course. PRESENTATION OF CASES: Case 1 was a girl diagnosed with right CDH by fetal ultrasonography and delivered by planned caesarian section at 37 weeks, 3 days. Fetal MRI showed liver herniation into the right thoracic cavity. High frequency oscillatory ventilation with nitric oxide gas was administered until day 5. At surgery on day 8, we found defects in the right posterolateral diaphragm and sac herniation of the right side of the liver into the right thoracic cavity. The postsurgical course was uneventful, and she was discharged on day 41. Case 2 was a girl with suspected congenital jejunal atresia after fetal ultrasonography detected polyhydramnios and dilatation. She was delivered by normal vaginal delivery at 38 weeks, 5 days, and thoraco-abdominal X ray showed right CDH but no small intestinal atresia. Surgery performed on day 3 found liver herniation into the diaphragmatic defect. Subsequently, bacterial infection occurred and was treated with the antibiotics, but her respiratory condition remained stable. She was discharged on day 49. DISCUSSION: The volume of herniated abdominal organs is affected by the presence of a hernia sac or the size of the diaphragmatic defect. CONCLUSION: The size of diaphragmatic defect, but not the laterality of the diaphragmatic defect, may be an important prognostic factor in right CDH.

A rare case of extremely low birth weight infant with Beckwith-Wiedemann syndrome.

INTRODUCTION: Beckwith-Wiedemann syndrome (BWS) manifests distinctive features, such as macroglossia, overgrowth, and abdominal wall defects. In this report, we describe a case of BWS in an extremely low birth weight infant diagnosed at three months after birth because of the intensive care for low birth weight. PRESENTATION OF CASE: A female infant was delivered at 24 weeks and 6 days of gestation with a weight of 845 g. After birth, significant small intestinal intra-umbilical prolapse was observed, and abdominal wall closure using a sutureless method was performed on day zero. Careful neonatal management was performed; however, an episode of bloody stools led to a diagnosis of intestinal volvulus due to intestinal malrotation. At 119 days of age, the Ladd procedure was performed. Notably, during anaesthesia induction, features suggestive of BWS were observed, leading to its diagnosis. DISCUSSION: Early diagnosis of BWS is vital because of its association with tumors. However, because she was an extremely low birth weight infant who required oral intubation and supine management for respiratory control, nevus flammeus and macroglossia were not observed. Therefore, BWS was not diagnosed for approximately three months after birth. It is important to recognize that omphalocele in extremely low birth weight infants is a risk factor for delayed diagnosis of BWS. CONCLUSION: Timely diagnosis of BWS is critical because of its association with tumors and varied clinical presentations. Early screening, especially for tumors, and awareness among surgical practitioners can aid in timely interventions and improved patient outcomes.

A rare case of oesophageal atresia complicated by duodenal atresia and anorectal malformation successfully treated with multi-stage surgery.

INTRODUCTION: Oesophageal atresia, duodenal atresia, and anorectal malformations are rare. This report describes a case of an infant with these three conditions treated using a multi-stage surgical procedure. PRESENTATION OF CASE: A male infant was delivered via caesarean section at 34 weeks and 4 days of gestation, weighing 1709 g. Radiography at birth showed a coil-up of the gastrointestinal tube in the oesophagus, a double bubble sign; the patient was subsequently diagnosed with gross type C oesophageal atresia with duodenal atresia. A gastrostomy was performed at day 0. Oesophago-oesophageal anastomosis was performed after tracheoesophageal fistula and blind-end dissection. A duodeno-duodenal diamond-shaped anastomosis was performed, and a tube enterostomy was created from the gastric area near gastrostomy as a trans-anastomotic feeding tube. A colostomy was performed in the descending colon owing to a non-rotation-type anomaly of intestinal malrotation. After other multi-stage surgeries and weight gain, posterior sagittal anorectoplasty was performed at age 1 year 2 months. DISCUSSION: Triple atresia (TA), characterized by triumvirate oesophageal atresia, duodenal atresia, and anorectal malformations, remains a clinical puzzle. Notably, standardized therapeutic guidelines for managing TA are lacking. The complexity of this constellation of anomalies necessitates astute diagnostic acumen and strategic treatment planning. CONCLUSION: Our patient showed a favourable clinical course with an accurate and timely diagnosis, serving as an experience for an innovative multi-stage therapeutic strategy. Our case showed the appropriate challenges of TA while illuminating the potential for successful outcomes through meticulous clinical management.

Neonatal inguinal hernias containing the uterus: a case report on changes in uterine position.

A female newborn weighing 542 g and delivered at 27 weeks gestation presented with bilateral inguinal hernias while in the neonatal intensive care unit. Ultrasonography confirmed herniation of the uterus into the right inguinal hernia without signs of incarceration. Due to the absence of complications, she was discharged and scheduled for follow-up at the outpatient clinic. At 11 months of age, a subsequent ultrasonography showed only omental herniation, with no evidence of uterine prolapse. When she reached 1 year of age, a laparoscopic percutaneous extraperitoneal closure procedure was performed. During the surgery, it was observed that the uterus and fallopian tubes were located near the hernia orifice, but no clear prolapse was detected. The procedure concluded safely with successful high ligation. It has noted that in cases of uterine prolapse hernias, the uterus tends to recede as the child grows, which supports the decision to delay surgery for improved safety.

Minimally invasive fenestration for congenital hepatic cyst in infant.

A 19-year-old woman underwent prenatal ultrasonography, which confirmed the presence of an isolated cystic mass in the upper abdominal cavity of a fetus. A female infant weighing 3085 g was delivered at 36 weeks' gestation. Ultrasonography and computed tomography examination revealed a clear unilocular cyst and occupying the right side of the abdomen. The infant's respiratory status was unstable, and she fed poorly, owing to compression by the hepatic cyst. We performed ultrasound-guided aspiration of a hepatic cyst at 15 days old, but it rapidly re-grew. Therefore, we performed laparoscopic findings and fenestration of the hepatic cyst via an umbilical arc incision and the cyst wall was excised at 43 days old. The histopathological diagnosis was mesothelial cell-derived hepatic cyst. Three years after the operation, no recurrence has been observed. Hepatic cyst fenestration by umbilical incision can be performed safely in infants and it is a cosmetically superior method.

Gallbladder volvulus in a child with mild clinical presentation.

Gallbladder volvulus in children is rare. Pre-operative diagnosis is considered difficult because of the nonspecific symptoms and inflammatory blood analysis findings. Sometimes diagnosis is confirmed at laparotomy. Many reports mention that the chief complaints of this disease are sudden and severe abdominal pain. We report a case of gallbladder volvulus in a boy with mild clinical symptoms and laboratory data of nonspecific inflammation. A reconstructed coronal CT abdominal view showed clearly the gallbladder torsion. Laparoscopic cholecystectomy was performed and postoperative course was uneventful. Recent reports have suggested the effectiveness of MRI. This case highlights the utility of a reconstructed coronal view of abdominal CT in successful pre-operative diagnosis for gallbladder volvulus in children.

Erratum: Ex vivo induction of antitumor DEC-205+ CD11c+ cells in a murine neuroblastoma model by co-stimulation with doxorubicin, lipopolysaccharide and interleukin-4.

[This corrects the article DOI: 10.3892/br.2015.546.].

Chemoimmunotherapeutic effect of combined treatment with ex vivo generated antigen-presenting immune cells and conventional antitumor agents in a mouse neuroblastoma model.

PURPOSE: Combining antitumor immunotherapy with conventional intensive multimodal therapy may be considered for advanced neuroblastoma. We investigated combination therapy with ex vivo generated immunostimulatory cells and intraperitoneal doxorubicin. METHODS: Immunogenic death of neuro-2a neuroblastoma cells was induced by doxorubicin or cisplatin (negative control). Mouse bone marrow cells were cultured with granulocyte-macrophage colony-stimulating factor, followed by addition of doxorubicin-killed neuro-2a cells with or without interleukin-4 and/or CpG-oligodeoxynucleotide to induce immunostimulatory cells. CD8α+ lymphocytes were cocultured with killed neuro-2a cells and immunostimulatory cells, and interferon-γ was measured in the supernatant. Furthermore, female A/J mice were injected with viable neuro-2a cells, followed by immunostimulatory cells and doxorubicin. Then intraabdominal tumor nodules were evaluated. RESULTS: Bone marrow-derived immunostimulatory cells only promoted interferon-γ production by CD8α+ lymphocytes after first being stimulated by doxorubicin-killed neuro-2a cells and interleukin-4, followed by CpG-oligodeoxynucleotide. These cells had a surface antigen expression profile compatible with activated dendritic cells and suppressed tumors in mice intravenously injected with neuro-2a cells. Despite a similar surface antigen profile, the in vivo antitumor effect was stronger after injection of immunostimulatory cells induced by doxorubicin-killed neuro-2a cells compared with cells induced by cisplatin-killed neuro-2a cells. Moreover, interferon-γ production was greater when CD8α+ lymphocytes were cocultured with doxorubicin-killed neuro-2a cells and immunostimulatory cells rather than with cisplatin-killed cells. CONCLUSION: Cells with antitumor activity can be induced from bone marrow cells. Combining such cells with doxorubicin may activate antitumor immunity in tumor-bearing mice. Interactions between induced immunostimulatory cells and conventional chemotherapy might be important for antitumor immunity.