Immunohistological Analysis of Lichen Sclerosus of the Foreskin in Pediatric Age: Could It Be Considered a Premalignant Lesion?

BACKGROUND: A major worry of juvenile penile LS is potential malignant degeneration to spinocellular carcinoma (SCC) in adulthood. LS is characterized by increased CD8+ and CD57+ cells, dermal sclerosis, epidermal atrophy, and hyperkeratosis. p53 and Ki67 are reliable premalignant markers. Our aim was to define the LS immunohistochemical profile of foreskin in children, focusing on tissue immune response and cell proliferation. METHODS: Thirty specimens of foreskins removed from pediatric patients during circumcision were included: six from ritual operation (A), twelve from phimosis (B), and twelve from phimosis with LS (C). Formalin-fixed paraffin-embedded sections were stained for histomorphology and immunohistochemistry. A quantitative evaluation for CD8, CD57, p53, and Ki-67 and a statistical analysis were performed. RESULTS: As compared to groups A and B, the samples from group C patients showed an acanthotic epidermis, a dermal band of lymphoid infiltrate with a significant enhancement of CD8+ CD57+ lymphocytes, and a keratinocytic hyperplasia with an overexpression of Ki67+ and p53+ cells. CONCLUSIONS: Immunohistological findings confirmed an immune reaction and proliferative behavior in juvenile LS of foreskin. We believe that radical circumcision should be the first treatment of choice in pediatric patients with clinical suspicious of LS for the potential risk of transformation to SCC in adulthood.

CO2 pneumoperitoneum effects on proliferation and apoptosis in two different neuroblastoma cell lines.

PURPOSE: The proto-oncogene MYCN is considered a transcription factor involved in the regulation of neuroblastoma (NB) cell biology. Since minimally invasive-surgery represents a debated treatment of NB, we investigated CO2 effects on proliferative activity and apoptotic pathway in two NB cell lines, SH-SY5Y (MYCN-non-amplified) and IMR-32 (MYCN-amplified). METHODS: SH-SY5Y and IMR-32 were exposed to CO2 (100%) at a pressure of 15 mmHg for 4 h and then moved to normal condition for 24 h. Cell proliferation, caspase 3 activity and transcript levels of BAX, BCL-2, cyclin B, cyclin D and MMP-2 were evaluated. RESULTS: CO2 exposure caused a decrease in cell proliferation associated to increases in BAX/BCL-2 ratio and caspase 3 activity in SH-SY5Y, while opposite effects have been found in IMR-32. CO2 exposure induced a decrease of cyclin B1 in SH-SY5Y, while an increase in cyclin B1 and D1 was observed in IMR-32. A slight up-regulation of MMP-2 expression in SH-SY5Y and a significant increase of 2.2 folds in IMR-32 was observed (p < 0.05). CONCLUSIONS: Our results suggest that CO2 exposure may cause different effects on various NB cell lines, likely due to MYCN amplification status. Further in vitro and in vivo studies are needed to highlight the role of laparoscopy on NB behaviour.

Hypoxia-Dependent Expression of TG2 Isoforms in Neuroblastoma Cells as Consequence of Different MYCN Amplification Status.

Transglutaminase 2 (TG2) is a multifunctional enzyme and two isoforms, TG2-L and TG2-S, exerting opposite effects in the regulation of cell death and survival, have been revealed in cancer tissues. Notably, in cancer cells a hypoxic environment may stimulate tumor growth, invasion and metastasis. Here we aimed to characterize the role of TG2 isoforms in neuroblastoma cell fate under hypoxic conditions. The mRNA levels of TG2 isoforms, hypoxia-inducible factor (HIF)-1α, p16, cyclin D1 and B1, as well as markers of cell proliferation/death, DNA damage, and cell cycle were examined in SH-SY5Y (non-MYCN-amplified) and IMR-32 (MYCN-amplified) neuroblastoma cells in hypoxia/reoxygenation conditions. The exposure to hypoxia induced the up-regulation of HIF-1α in both cell lines. Hypoxic conditions caused the up-regulation of TG2-S and the reduction of cell viability/proliferation associated with DNA damage in SH-SY5Y cells, while in IMR-32 did not produce DNA damage, and increased the levels of both TG2 isoforms and proliferation markers. Different cell response to hypoxia can be mediated by TG2 isoforms in function of MYCN amplification status. A better understanding of the role of TG2 isoforms in neuroblastoma may open new venues in a diagnostic and therapeutic perspective.

CO2 Pneumoperitoneum Effects on Molecular Markers of Tumor Invasiveness in SH-SY5Y Neuroblastoma Cells.

INTRODUCTION: CO2 pneumoperitoneum can influence the biological behavior of neuroblastoma (NB). Angiogenesis and genetic features are responsible for malignant phenotype of this tumor. We examined the CO2 effects on N-Myc, vascular endothelial growth factor (VEGF), and matrix metalloproteinase-2 (MMP-2) expression as critical biomarkers of tumor invasiveness, in NB cells without N-Myc amplification. MATERIALS AND METHODS: SH-SY5Y cells were exposed to CO2 (100%) at 15 mm Hg pressure for 4 hours and then moved to normal condition for 24 hours. Control cells were incubated with 5% CO2 for the same time. In control and CO2-exposed cells, the messenger ribonucleic acid (mRNA) levels of hypoxia-inducible factor (HIF)-1α, HIF-2α, VEGF-A, and MMP-2 were quantified by real-time polymerase chain reaction. N-Myc expression was evaluated by Western blot analysis. RESULTS: The exposure to 15 mm Hg CO2 (100%) for 4 hours induced an increase in HIF-1α, but not in HIF-2α, mRNA levels. No differences were observed in N-Myc expression between exposed and control cells at each incubation time. Similarly, no significant differences were found for VEGF-A and MMP-2 transcript levels. In CO2 exposed cells, we observed only a slight increase in both VEGF-A and MMP-2 mRNA levels after 4 and 24 hours in comparison to controls. CONCLUSION: In our study, the hypoxic environment induced by CO2 exposure does not affect the expression of critical biomarkers of NB aggressiveness, such as N-Myc, VEGF, and MMP-2, in human SH-SY5Y NB cells without N-Myc amplification. These data suggest that CO2 pneumoperitoneum might not adversely impact NB cell invasiveness; however, it is necessary to evaluate these effects in others in vitro and in vivo models.

Premedication with melatonin vs midazolam: efficacy on anxiety and compliance in paediatric surgical patients.

Preoperative anxiety is a major problem in paediatric surgical patients. Melatonin has been used as a premedicant agent and data regarding effectiveness are controversial. The primary outcome of this randomized clinical trial was to evaluate the effectiveness of oral melatonin premedication, in comparison to midazolam, in reducing preoperative anxiety in children undergoing elective surgery. As secondary outcome, compliance to intravenous induction anaesthesia was assessed. There were 80 children undergoing surgery randomly assigned, 40 per group, to receive oral midazolam (0.5 mg/kg, max 20 mg) or oral melatonin (0.5 mg/kg, max 20 mg). Trait anxiety of children and their mothers (State-Trait Anxiety Inventory) at admission, preoperative anxiety and during anaesthesia induction (Modified Yale Pre-operative Anxiety Scale), and children's compliance with anaesthesia induction (Induction Compliance Checklist) were all assessed. Children premedicated with melatonin and midazolam did not show significant differences in preoperative anxiety levels, either in the preoperative room or during anaesthesia induction. Moreover, compliance during anaesthesia induction was similar in both groups. CONCLUSIONS: This study adds new encouraging data, further supporting the potential use of melatonin premedication in reducing anxiety and improving compliance to induction of anaesthesia in children undergoing surgery. Nevertheless, further larger controlled clinical trials are needed to confirm the real effectiveness of melatonin as a premedicant agent in paediatric population. What is Known: • Although midazolam represents the preferred treatment as a premedication for children before induction of anaesthesia, it has several side effects. • Melatonin has been successfully used as a premedicant agent in adults, while data regarding effectiveness in children are controversial. What is New: • In this study, melatonin was as effective as midazolam in reducing children's anxiety in both preoperative room and at induction of anaesthesia.

Melatonin versus midazolam premedication in children undergoing surgery: A pilot study.

AIM: Melatonin has been proposed as a premedication alternative to midazolam, preceding anaesthesia induction. However, to our knowledge, data concerning interaction between melatonin and intravenous anaesthetic drugs in children are not available. The aim of this prospective, randomized, double-blind pilot study was to investigate the possible effect of melatonin premedication, in comparison to midazolam, on the required infusion of propofol in children undergoing surgery. As a secondary outcome, the effect of oral melatonin on the preoperative sedation level and on the post anaesthesia recovery score was evaluated. METHODS: Children between the age of 5 and 14 years, scheduled for elective surgery, were prospectively enrolled between January 2012 and December 2013, and randomly assigned to two groups based on whether they received oral melatonin (0.5 mg/kg) or oral midazolam (0.5 mg/kg) premedication before induction of anaesthesia with propofol. Degree of sedation before and after anaesthesia was also evaluated. RESULTS: Ninety-two patients were studied, 46 for each group. We found that oral administration of melatonin significantly reduced doses of propofol required for induction of anaesthesia in paediatric patients, more than midazolam (P < 0.001). No statistically significant differences were found in the pre- and post-anaesthesia sedation score (P = 0.387 and P = 0.525, respectively) between the two groups. CONCLUSIONS: The present study demonstrates that melatonin enhances the potency of propofol also in paediatric patients. Moreover, considering the paediatric level of sedation, melatonin was equally as effective as midazolam. These data support the use of melatonin as a premedicant in paediatric surgical patients.

Surgical stress after open and transumbilical laparoscopic-assisted appendectomy in children.

INTRODUCTION: The transumbilical laparoscopic-assisted appendectomy (TULAA) effects on the surgical stress response in children have not been studied. Our aim is to investigate the stress response in TULAA. METHODS: A total of 35 children underwent the appendectomy by open approach (OA) or TULAA approach. Interleukins (ILs)-6, -18, and -10 were measured before (T0), at the beginning (T1a), and at the end of surgery (T1b) and 24 hours after (T2). RESULTS: An increase in IL-6 levels at T1b and T2 and in IL-18 at T2 was observed after OA. A significant increase of both IL-6 and IL-18 were observed at T2 but values were less compared with OA (11.6 ± 4.4 vs. 31.9 ± 8.9 pg/mL for IL-6, p = 0.0006; 145.6 vs. 174.9 pg/mL for IL-18, p = NS). CONCLUSIONS: A significant reduction in the postoperative cytokines in TULAA group suggests that this approach causes less surgical trauma in children.

In vitro CO2-induced ROS production impairs cell cycle in SH-SY5Y neuroblastoma cells.

PURPOSE: We evaluated in vitro the role of CO(2)-induced oxidative stress on the expression of proteins involved in cell-cycle regulation of neuroblastoma cells. METHODS: SH-SY5Y cells were exposed to CO(2) at 15 mmHg pressure (100 %) for 4 h and then moved to normal condition for 24 h. Control cells were maintained in 5 % CO(2) for the same time. ROS production was determined by fluorescent staining with H2DCF-DA. DNA damage was measured by COMET assay. p53 protein expression was analyzed by western blot and confocal laser scanning microscopy was used to evaluate its sub-cellular localization. Cyclin expression was quantified by real-time PCR and western blot. Cell-cycle analysis was performed by FACS. RESULTS: CO(2) incubation was associated with an increase in ROS production (p < 0.01), cell DNA damage mainly after 24 h (12 % increase of tail DNA content and 4-fold increase of tail length) and a significant up-regulation in p53 expression at 24 h with an intense nuclear staining. In CO(2)-treated cells, we observed an S-phase arrest in correlation with a reduction of cyclin B1 expression. CONCLUSIONS: In vitro-simulated pneumoperitoneum environment with CO(2) induces oxidative stress and cell DNA damage, leading to p53 up-regulation involved in cell-cycle arrest of neuroblastoma cells.

Thoracoscopy versus thoracotomy for esophageal atresia and tracheoesophageal fistula repair: review of the literature and meta-analysis.

INTRODUCTION: The thoracoscopic approach to esophageal atresia (EA) with tracheoesophageal fistula (TOF) represents a challenging procedure whose real benefits remains unclear. Our purpose is to identify, through a meta-analysis, clinical evidence of the reliability of the thoracoscopic repair (TR) for EA/TOF compared with the open repair. MATERIALS AND METHODS: Defined PubMed search, with analysis of intraoperative and postoperative complications after open or thoracoscopic primary anastomosis for EA/TOF. RESULTS: Five articles met the criteria of meta-analysis, being comparative studies between TR and conventional open repair (COR), although they were retrospective. One article was excluded because it was available only in Japanese. We observed a slight prevalence, statistically insignificant, of the intraoperative and postoperative complication rate for TR: odds ratio (OR) 1.29. Excluding the conversion rate, the meta-analysis between the complication rate for TR and COR did not show a significant difference (OR 0.64). Anastomosis's leaks and strictures considered together did not show a significant difference between the two techniques, p = not significant and OR of 0.56. Similar results were observed analyzing the single outcome of leaks and strictures; the meta-analysis did not show any significant differences with an OR, respectively, of 1.05 and 0.43. CONCLUSIONS: The effectiveness of the endoscopic technique for EA/TOF repair is indicated with outcomes not different from open surgery. A randomized controlled trial is needed in this field to indicate which procedure is superior, open or TR.

Long-term follow-up of neonatally diagnosed primary megaureter: rate and predictors of spontaneous resolution.

OBJECTIVE: Primary megaureter (PM) represents 6-10% of all antenatal displaced urinary malformations. Spontaneous resolution of PM is a well-known event. This long-term follow-up study evaluated the incidence and rate of resolution of PM. Some predictive factors were revised, based on morphological classification and scintigraphic pattern. MATERIAL AND METHODS: Sixty neonates with PM were followed. The diagnosis was confirmed by ultrasound examination and (99m)Tc-DTPA diuretic renal scan. All the observed patients underwent antibiotic prophylaxis. All conservatively treated children were followed from 6 months to 15 years. Follow-up consisted of monthly urine cultures, renal ultrasound and DTPA diuretic renography. Hydroureteronephrosis was considered to have resolved when a retrovesical cross-sectional diameter of ureter less than 6 mm was found. RESULTS: In total, 72 PM were identified in this series. At the end of the follow-up period, 38 PM (52.8%) had resolved, in 18 PM (25%) ureteral dilatation persisted and 16 PM (22.2%) required a surgical procedure. The median age at resolution was significantly affected by presenting hydronephrosis grade and cross-sectional diameter at diagnosis, but not by gender. The (99m)Tc-DTPA renogram results showed no functional impairment in resolved and persisting cases, even after long-term observation. CONCLUSIONS: The data show that 22% of neonatal PM require surgical treatment. Poor drainage on (99m)Tc-DTPA scan, grade IV-V hydronephrosis and ureteric diameter more than 15.0 mm were statistically significant and independent predictive factors for surgery. The time to spontaneous resolution in neonatally diagnosed PM may exceed 3.6 years, after which recovery is rare.

CO2 pneumoperitoneum impact on early liver and lung cytokine expression in a rat model of abdominal sepsis.

BACKGROUND: Experimental evidence suggests that laparoscopy could have reduced inflammatory sequelae compared with laparotomy following abdominal surgery for peritonitis. The aim of the present study is to investigate the possible beneficial effects of CO(2) insufflation on liver and lung expression of proinflammatory cytokines during sepsis. METHODS: Cecal ligation and puncture (CLP) was induced in Sprague-Dawley rats, and 6 h later rats were randomly subjected to CO(2) pneumoperitoneum (5-7 mmHg) or to laparotomy for 1 h. At the end of the CO(2) pneumoperitoneum or laparotomy procedures, animals were sacrificed, and liver and lung were removed and stored for molecular and histological analysis. RESULTS: Liver and lung expression of proinflammatory cytokines was significantly reduced in animals subjected to CO(2) pneumoperitoneum compared with laparotomy. In particular, tumor necrosis factor-alpha (TNF-α) protein expression was significantly reduced (p < 0.05) following CO(2) pneumoperitoneum compared with laparotomy procedures. Interleukin (IL)-6 protein expression was accordingly, markedly reduced (p < 0.05) following CO(2) pneumoperitoneum. Histological analysis showed a reduced inflammatory infiltrate in liver and lung from animals subjected to CO(2) pneumoperitoneum compared with laparotomy. CONCLUSIONS: Our results support the hypothesis that laparoscopic procedures reduce the inflammatory cascade, following peritoneal sepsis, via reduced expression of proinflammatory cytokines.

Peroxisome proliferator activated receptor beta/delta activation prevents extracellular regulated kinase 1/2 phosphorylation and protects the testis from ischemia and reperfusion injury.

PURPOSE: Testicular torsion is a medical emergency that requires immediate diagnosis and treatment to avoid subsequent testicular injury and infertility. PPARs are a family of nuclear hormone receptors belonging to the steroid receptor superfamily. Three PPAR isotypes (alpha, beta/delta and gamma) encoded by separate genes and showing different tissue distribution patterns have been identified. PPARbeta/delta is expressed in testis and its role is largely unknown. We tested whether pharmacological activation of PPARbeta/delta might protect the testis from ischemia and reperfusion injury. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were subjected to 1-hour testicular ischemia, followed by 24 hours of reperfusion. Sham testicular ischemia-reperfusion rats served as controls. The animals were randomized to receive immediately after detorsion 1) L-165,041 (4 mg/kg intraperitoneally), a potent agonist of PPARbeta/delta, 2) GW9662 (Calbiochem(R)) (4 mg/kg intraperitoneally), an antagonist of PPAR, 3) L-165,041 (4 mg/kg intraperitoneally) plus GW9662 (4 mg/kg intraperitoneally) concomitantly or 4) vehicle (1 ml/kg 10% dimethyl sulfoxide/NaCl solution). We evaluated testicular extracellular signal regulated kinase, tumor necrosis factor-alpha and interleukin-6 by Western blot. We also investigated PPARbeta/delta activation by Western blot, mRNA expression and organ damage. RESULTS: Testicular ischemia-reperfusion injury caused a significant increase in extracellular signal regulated kinase, tumor necrosis factor-alpha and interleukin-6 expression in each testis. Furthermore, histological examination revealed marked damage. L-165,041 administration increased the PPARbeta/delta message and protein, inhibited extracellular signal regulated kinase, tumor necrosis factor-alpha and interleukin-6 expression, and decreased histological damage. Concomitant administration of GW9662 reversed the protection exerted by PPARbeta/delta agonist. CONCLUSIONS: These findings indicate that PPARbeta/delta agonists might be an attractive therapeutic candidate for managing testicular torsion.

Scintigraphic evaluation of colonic motility in patients with anorectal malformations and constipation.

BACKGROUND AND PURPOSE: Constipation is one of the major sequelae in patients after correction of anorectal anomalies (ARAs). The aim of the present work has been to assess the colonic transit time, using radioisotope scintigraphy, in patients operated for ARA and experiencing constipation in the follow-up. The results were compared with transit time from children with true functional constipation. METHODS: Twelve or 32 patients operated for ARA during the period 1994-2003 experienced mild or severe constipation (6 with high or intermediate form of ARA and 6 with low type) at follow-up. The mean age of this group was 5.8 years. Eighteen patients, mean age 6.7 years, with true functional constipation were studied as well. Colonic transit times were investigated using radioisotope scintigraphy. Normal values for colonic transit time were derived from historical controls. Radioisotope diethylenetriamine pentaacetic acid labelled with indium 111 was administered orally to determine a segmental colonic transit. Images of the abdomen have been taken at 6, 24, 48, and again at 72 hours, if radioactivity was not cleared from the colon. To quantify colonic transit, we calculated the geometric centre (GC) dividing the colon into anatomic regions. RESULTS: According to normal controls, 2 different type of delayed transit can be observed: (a) slow-transit constipation if GC at 48 hours is less than 4.1; (b) functional rectosigmoid obstruction (FRSO) if GC at 48 hours is 4.1 or more but less than 6.1 at 72 hours. Patients with functional constipation were divided into 2 groups: (a) slow-transit constipation in 12 patients with a GC at 48 hours of 3.7 +/- 0.5; (b) FRSO in 6 patients with a GC of 4.7 +/- 0.04 and 5.02 at 48 and 72 hours, respectively. Patients operated for high ARA had values characteristic of FRSO with GC at 48 hours of 5.1 +/- 0.8 and 4.75 +/- 0.5 at 72 hours. In low ARA, the transit times were similar to the ones observed in patients with high ARA at 48 hours with a GC of 4.9 +/- 0.5. CONCLUSIONS: Patients with ARA frequently have functional sequelae in the postoperative period such as constipation. According to our results, constipation seems to be secondary to segmental motility disorders limited to the rectosigmoid area, similar to constipated children with FRSO. No evidence of more generalised motility disturbance, as previously postulated, could be recorded.