Home-based color card screening for biliary atresia: the first steps for implementation of a nationwide newborn screening in Germany.

INTRODUCTION: Biliary atresia is a rare neonatal disease and the most common indication for pediatric liver transplantation. Kasai portoenterostomy is the initial treatment, aiming to prevent liver transplantation. Beyond age at Kasai, few prognostic factors are known. Multiple countries have established screening methods to reduce the age at Kasai and recent analysis shows significant better outcomes for screening cohorts. In 2016, we established a decentralized stool color card screening in Lower Saxony and we present our first 2 years of experiences. METHODS: In cooperation with a major German health insurance company and the Medical Association of Lower Saxony, we established the screening project, printed 120,000 color cards, and distributed them to all maternity hospitals. Program advertises were printed in newspapers and medical journals. After the first year, the project was evaluated. Thirty maternity hospitals and local practitioners were contacted via telephone, Internet, intranet, and pediatric journals. RESULTS: One out of seventy-six maternity hospitals (1.3%) refused to participate in the screening. 30 hospitals (40%) were contacted and 93.5% of the interviewed staff reported that stool color cards were handed out regularly and discussed with the parents. Only 20% of local practitioners assessed neonatal cholestasis to be a relevant problem during daily practice, and 55% regarded a stool color card screening to be useful. CONCLUSIONS: In the second year, we extended the screening project to outpatient maternity clinics. Based on the responses of local practitioners, we regard the voluntary screening as insufficient and we have contacted the Federal Joint Committee for the initiation of a nationwide obligatory stool color card screening.

Arterioportal shunting, splanchnic capillary perfusion, and the effects of colloids during capnoperitoneum in neonatal and adolescent pigs.

BACKGROUND: Clinical and experimental data indicate that neonates are sensitive to the CO2 pneumoperitoneum. An impaired splanchnic perfusion during laparoscopy in adults has been reported. We recently confirmed that intravenous colloids improve macrocirculatory function in neonates. We aimed to determine the impact of CO2 pneumoperitoneum on the perfusion of splanchnic organs in the young including effects of colloid application. METHODS: Male piglets (n = 25) were divided into four groups: (1) neonatal controls, (2) neonates with crystalloid restitution, (3) neonates with colloidal restitution, and (4) adolescents with crystalloid restitution. Animals were ventilated and subjected to a 3-h, 10 mmHg CO2 pneumoperitoneum followed by 2 h resuscitation. Hepatic, splanchnic, and arteriovenous shunt perfusion was assessed via central and portal venous catheters. Capillary organ flow was detected by fluorescent microspheres. The rate of bile flow was measured. RESULTS: The neonatal crystalloid group showed a significant decrease in the intestinal capillary perfusion at the end of the recovery period. This was not detectable in the adolescent and colloid group. There was a significant increase in microcirculatory arterioportal shunt flow during the CO2 pneumoperitoneum in both neonatal groups but not in the sham and adolescent groups (p < 0.05). Hepatic arterial perfusion increased after insufflation in all groups and dropped during capnoperitoneum to levels of about 70% baseline. There was no significant impairment of splanchnic perfusion or bile flow as a result of the pneumoperitoneum in all groups. CONCLUSIONS: Capillary perfusion of the abdominal organs was stable during capnoperitoneum and recovery in adolescents and neonates with colloid restitution, but not with crystalloid restitution. Significant arterioportal shunting during capnoperitoneum could affect hepatic microcirculation in neonates. Our data confirm that moderate pressure capnoperitoneum has no major effect on the perfusion of abdominal organs in neonates with adequate substitution.

Liver fibrosis during the development of biliary atresia: Proof of principle in the murine model.

BACKGROUND: The murine model of biliary atresia (BA) is used for examining the pathogenesis of BA. The aim of the study was description of the morphological features and illustrating the detailed development of fibrosis using the Biliary Atresia Research Consortium (BARC) system. METHODS: Neonatal mice were injected intraperitoneally with rhesus rotavirus (RRV) strain (N=17). Healthy mice were the control group (N=29). All mice were sacrificed at 7 or 14days after birth. Two pathologists examined the morphological features using the BARC system; CK19, αSMA and collagen type I were assessed by immunohistochemistry. RESULTS: In RRV mice, portal fibrous expansion with focal bile duct proliferation and strong portal cellular infiltrate was found in contrast to healthy mice. In RRV mice, CK19 bile duct staining was significantly less or absent (p<0.01), with stronger portal staining of collagen type I (p=0.02). Expansion of staining for αSMA was more in RRV mice (p<0.01), but αSMA portal staining was stronger in healthy mice (p=0.02). CONCLUSIONS: The morphological features observed in the murine model of BA correspond with the BA characteristics according to the BARC criteria. Fibrosis is an important feature of the model. Therefore, this murine model is useful for investigating the pathogenesis of BA.

Neonatal murine macrophages show enhanced chemotactic capacity upon toll-like receptor stimulation.

BACKGROUND: The neonatal surgical patient is threatened by exuberant inflammatory reactions. Neonatal macrophages are key players in this process. We investigated the ability of neonatal macrophages to initiate a local inflammatory reaction upon exposure to different bacterial or viral ligands to toll-like receptors (TLRs). METHODS: Peritoneal wash outs from neonatal (<24 h) and adult (42 days) C57BL/6J mice were gained by peritoneal lavages. In a first set of experiments, macrophages were purified and stimulated for 6 h by four different TLR ligands. mRNA was extracted for transcriptome analysis. In a second set of experiments, lipopolysaccharide was applied into peritoneal cavities. After 6 h of incubation, the cellular composition of the inflamed cavities was evaluated by cytological staining as well as chipcytometry. RESULTS: Neonatal murine peritoneal macrophages differed significantly in the expression of pro- and anti-chemotactic genes. Functional assignment of these genes revealed enhanced chemotactic potential of neonatal macrophages and was confirmed by a higher influx of pro-inflammatory cells into neonatal peritoneal cavities. CONCLUSION: Neonatal peritoneal macrophages demonstrated an enhanced chemotactic potential upon stimulation with four TLR ligands. This was associated with an increased influx of inflammatory cells to the peritoneal cavity. This might contribute to the strong inflammatory responses of neonates and preterms.

Biliary atresia: lessons learned from the voluntary German registry.

INTRODUCTION: Aim of the study was to carry out a 5-year survey of German patients with biliary atresia (BA) and to launch a discussion regarding the feasibility of voluntary registries in unregulated healthcare systems. METHODS: A retrospective analysis of German BA patients born between 2001 and 2005, based on data collected from the voluntary European Biliary Atresia Registry (EBAR), was carried out and supplemented by data from all BA patients who underwent liver transplantation at the only 4 pediatric transplantation centers (pLTx) in Germany which are so far not registered at EBAR. Survival rates were calculated using Kaplan-Meier analysis and compared by Cox regression to determine the predictive value of age at surgery and the influence of the center size (fewer or more than 5 patients/study period) on overall survival and survival with native liver. RESULTS: A critical review of the 148 German EBAR charts revealed that 11 patients (7.4%) had no biliary atresia. The remaining 137 patients from EBAR together with 46 BA patients who underwent LTx without prior registration at EBAR were evaluated with a median follow-up of 39 months (range: 25-85 months). 29 hospitals performed a total of 159 Kasai procedures, but only 7 centers treated 5 or more patients (116 patients, range: 5-68), and 22 hospitals performed less than 5 KP (43 patients, range: 1-4). Primary LTx was performed in 21 patients (11.5%) and 3 patients died without surgical intervention. 16 patients were lost to follow-up (8.7%). Overall survival after 2 years was 83.3% (139 patients), including 105 patients (63%) who had undergone LTx and 34 patients (20.3%) with native liver. 28 patients died (16.7%), 8 after LTx (5.8%). The experience of the center was the only factor with a significant predictive value for jaundice-free survival with native liver (p=0.001). CONCLUSION: 25% of all German BA patients were not registered at EBAR, and 29 clinics were involved in the surgical management of BA patients. Therefore a new approach consisting of an internet-based decentralized registry for rare neonatal liver diseases is outlined which could improve the future management of patients with BA. The centralization of such patients at experienced centers with higher caseloads is necessary in Germany and would improve the outcome of patients with biliary atresia.

Portal venous gas detected by ultrasound differentiates surgical NEC from other acquired neonatal intestinal diseases.

BACKGROUND: The definite clinical diagnosis of acquired neonatal intestinal diseases (ANID) is a challenge, usually met by applying Bell's or, more recently, Gordon's classification. Both classifications incorporate radiological pneumatosis intestinalis (PI) as a cornerstone of the NEC diagnosis. However, PI may be absent or difficult to identify by abdominal X-ray. Portal venous gas detected by ultrasound (PVG-US) has been proposed as another characteristic of NEC, but its incidence in other entities of ANID remains unknown. OBJECTIVE: To determine whether PVG-US and Gordon's classification can help to differentiate between NEC and other ANID, especially SIP. METHODS: Retrospective analysis of the data of 83 infants, who underwent laparotomy for a clinical diagnosis of ANID was performed. The results of PVG-US and other markers of ANID were compared with the operative result, defined as the gold standard for diagnosis. RESULTS: The NEC diagnosis was confirmed in 28/83 infants and PVG-US was present in 23 (82%) of those patients prior to operation. PVG-US was detected in 2 patients without NEC (one volvulus, one SIP), resulting in an 82% sensitivity and a 96% specificity. The sensitivity and specificity of radiological PI for patients with NEC was lower (75 and 91%), but the combination of PVG-US and radiological PI increased the sensitivity for NEC detection to 89%. Gordon's classification had a sensitivity of 93% and a specificity of 92% for NEC diagnosis. CONCLUSION: Screening for PVG-US and Gordon's classification are valid tools to differentiate between NEC and other ANID including SIP. Although an effect of these proposed diagnostic tools on treatment regimen and operative management has yet to be verified, the improvement in diagnosing ANID is certainly valuable.

Preconceptional oral vaccination prevents experimental biliary atresia in newborn mice.

INTRODUCTION: Biliary atresia (BA) in humans resembles BA induced in Balb/c-mice by Rhesus Rotavirus (RRV). In mice, susceptibility to BA is ascribed to the lack of maternally derived immune protection. This study investigated whether vaccination of dams against RRV protected their offspring from developing BA. MATERIALS AND METHODS: Before mating, female mice were vaccinated orally with RotaTeq or Rotarix. Pups (n=243) from both test groups and a control group were intraperitoneally infected with RRV. Sacrifice of the animals was scheduled for days 7, 14 and 21 after infection. Then, gross and mircoscopia findings of the liver and the hepatoduodenal ligament gave evidence of BA, and hepatic viral load was tested by virus-specific real-time PCR, as well as plaque forming units. RESULTS: Two weeks after infection, the incidence of cholestasis was 100% in controls, 77% in pups of RotaTeq-vaccinated dams, and 56% in pups of Rotarix-vaccinated dams. However, in contrast to controls (incidence of BA: 82%) most pups in the test groups recovered (incidence of BA in pups of RotaTeq-vaccinated dams 11%; incidence of BA in pups of Rotarix-vaccinated dams 3%). Hepatic viral load was identical at various time-points in all pups, suggesting that differences in RRV clearance did not underlie this effect. CONCLUSION: In this mouse model, oral vaccination with RotaTeq and Rotarix prevented most RRV-induced BA. This provides a new approach to a better understanding of both the pathomechanism of BA development and the capabilities of the innate immune system. It also suggests a first approach for prophylaxis against BA.

CO2 modulates the inflammatory cytokine release of primary human pleural macrophages.

BACKGROUND: It is well known that CO (2) used during laparoscopy affects the peritoneal surface and local inflammatory response, including the inflammatory reactivity of peritoneal macrophages. However, little is known about the local effects of CO (2) during thoracoscopy. In a previous study we have shown that in healthy adolescents, macrophages are the dominant cell population on the pleural surface. Therefore, we examined the effects of CO (2) on the inflammatory response of primary human pleural macrophages. METHODS: Human primary macrophages were harvested lavage from healthy adolescents undergoing elective surgery for pectus bar correction (n=8). After purification and 24 h resting, cells were incubated for 2 h in 100% CO (2), 5% CO (2) or 95% inert helium with 5% CO (2) as hypoxic control. After incubation cells were stimulated with LPS for 4 h and 24 h. The release of TNF-alpha, IL-8, IL-6, IL-10 and IL-1 beta were determined by ELISA. RESULTS: CO (2), but not hypoxia, induced a significant reduction in the release of TNF-alpha and IL-8 as well as a significant increase in the release of IL-10 and IL-1 beta within the first 4 h after incubation. The levels of IL-6 and the release of cytokines at 24 h after incubation were not significantly affected. CONCLUSIONS: CO (2) directly modulates the immediate inflammatory response of pleural macrophages. Therefore, CO (2) insufflation during thoracoscopy could lower the local stress response, but does not appear to have a lasting effect.

Cardiovascular responses to prolonged carbon dioxide pneumoperitoneum in neonatal versus adolescent pigs.

BACKGROUND: Long-lasting minimally invasive procedures are increasingly used for children. However, the hemodynamic effects of a prolonged carbon dioxide (CO(2)) pneumoperitoneum (PP) on small infants are poorly understood and may differ from the effects on adolescents. This study aimed to detect and compare these effects in neonatal and adolescent pigs. METHODS: Six neonatal pigs weighing 5.6-6.4 kg and six adolescent pigs weighing 51-57 kg underwent a prolonged CO(2) PP of 180 min. Five neonatal pigs weighing 5.7-6.6 kg underwent sham PP as an additional control group. All the animals received crystalloid electrolyte solution at 10 ml/kg/h during the experiments. After CO(2) decompression, all the animals were monitored for a further 120 min. The end points of the study were mean arterial pressure (MAP), central venous pressure (CVP), and cardiac index (CI). The parameters were assessed after a 60-min resting phase (i.e., after initial placement of catheters in the jugular vein, the carotid artery, and the femoral artery four times during CO(2) PP and three times afterward. RESULTS: The comparison of neonates and adolescent pigs showed that neonates had a significantly more pronounced decrease in MAP during CO(2) PP (88.1 +/- 2.7% of baseline vs 95.1 +/- 1.6%; p < 0.05) and the recovery period (71 +/- 5.1% vs 86.4 +/- 1.4%; p < 0.05). Differences in CVP changes between the neonatal and adolescent pigs during and after CO(2) PP were not significant. For the neonates, the decrease in CI was most pronounced during the recovery period after decompression of the CO(2) PP and significantly less than in adolescents (84.3 +/- 3.3% of baseline vs 97.4 +/- 4.5%; p < 0.05). The neonates with sham PP did not show any significant MAP, CVP, or CI changes during the experiments. CONCLUSIONS: A prolonged CO(2) PP induces hypotension and cardiac depression in neonatal but not in adolescent pigs. Thus, intensive monitoring during prolonged laparoscopic procedures and particularly afterward may be mandatory for neonates.

Penile block is associated with less urinary retention than caudal anesthesia in distal hypospadia repair in children.

BACKGROUND: Caudal anesthesia (CA) is widely recommended due to excellent analgesia in distal hypospadia repairs, but its potential side effect on urinary retention interferes with patient comfort. To objective is to determine the impact of CA versus penile block (PB) on postoperative micturition. METHODS: Sixty distal hypospadia repairs performed over a 48-month period were analyzed concerning the impact of applicated analgesia on postoperative micturition. Inclusion criteria were Mathieu or Snodgrass procedures, use of a non-dribbling urethral stent, CA or PB. Endpoint was first postoperative micturation (<6 h), or treatment of delayed postoperative micturation (>6 h). RESULTS: CA was used in 27 and PB in 33 cases. Compared groups had similar age (mean: 31 months, range 12-68 vs. 28 months, range: 14-145), weight (mean: 13 kg, range 9-18 vs. 15 kg, range 8-59), operation duration (mean 61 min, range 30-105 vs. mean 67 min, range 35-120) and surgical technique (Mathieu/Snodgrass: 7/20 CA vs. 19/14 PB). Micturation was significantly less impaired in the PB than CA group (5/33 vs. 15/27; p < 0.05). Delayed or non-micturation was successfully treated in 4 and 14 patients, respectively, by cholinergic agonists. One patient of each group required a suprapubic drain on the first operative day after unsuccessful medical treatment. CONCLUSIONS: In our series, children undergoing distal hypospadia repair experienced significantly less impaired micturition when using penile block instead of caudal anesthesia. We recommend penile block as the first choice perioperative analgesia, when spontaneous postoperative micturition must be guaranteed.

Endoloop closure of the urinary bladder is safe and efficient in female piglets undergoing transurethral NOTES nephrectomy.

INTRODUCTION: Recently, we showed that various procedures for rigid Natural Orifice Transluminal Endoscopic Surgery (NOTES) can be successfully performed in piglets. However, the safety and effectiveness of endoscopic closure of the urinary bladder remains to be determined before introducing transurethral NOTES in children. Our study was designed to investigate the safety and impermeability of the urinary bladder after endoscopic closure in transurethrally assisted nephrouretectomy in piglets. MATERIAL AND METHODS: Five female piglets (mean weight 14.5 kg; range: 14-15) underwent right-sided transurethral nephroureterectomy using a hybrid technique with one 15 mm trocar placed umbilically and one 3 mm trocar placed transvesically. Hilar dissection was performed with a 5 mm Endoligasure vessel sealing device. After umbilical retrieval of the resected kidney, the urinary bladder was closed by an Endoloop via an umbilical "two in one system" with the assistance of a 2 mm transurethrally placed endoscopic clamp. Endpoints of the study were total operation time, duration of cystoscopy and transurethral trocar positioning, duration of nephrectomy, duration of bladder closure and determination of bladder impermeability. Intraoperatively, bladder filling was performed with normal saline (ml), while bursting pressure (mmHg) was measured by filling the harvested bladder with air. RESULTS: All nephroureterectomies were successfully performed with negligible blood loss. Mean total operation time was 46 min (range: 35-69). Cystoscopy and transurethral trocar positioning took 14 min (range: 9-23), and nephrectomy took 28 min (range: 18-48). Mean duration of bladder closure was 4 min (range: 2-6). Mean volume of intraoperative bladder filling was 94 ml (range: 80-100), indicating adequate capacity after closure with Endoloops. Post-mortem bladder impermeability was confirmed by a mean air filling bursting pressure of 88 mmHg (range: 70-140) indicating sufficient bladder closure. CONCLUSION: A safe urinary bladder closure is mandatory for transurethral rigid NOTES procedures and can be adequately achieved with Endoloops.

Laparoscopically assisted ventriculoperitoneal shunt placement in infants with previous multiple abdominal operations.

BACKGROUND: The insertion of a ventriculoperitoneal shunt (VPS) is the treatment of choice in patients with hydrocephalus. However, VPS placement may be difficult in patients with extensive adhesions following prior abdominal interventions. Laparoscopic placement of the abdominal part of the VPS allows controlled adhesiolysis in combination with an optimal shunt placement in patients with a body weight above 5 kg. We investigated the feasibility and safety of laparoscopic VPS placement in young infants who had undergone abdominal operations. PATIENTS AND METHODS: In our institution, 6 children with prior laparotomies (range: 2-9; median 3) received a VPS catheter between 2004 and 2008. The median age was 9 months (range: 2 months-4 years) and the median body weight was 4.5 kg (3.5-8.2 kg). All procedures were laparoscopically assisted and performed simultaneously by an interdisciplinary neurosurgical and a pediatric surgical team. RESULTS: Median operating time was 63 min (35-100 min). In all cases, correct placement of the shunt with sufficient drainage was achieved. Enteral feeding was started on the day of operation in all patients. Median follow-up was 10 months (range: 2 months-3 years). There were no complications except in one patient, who developed shunt dysfunction 4 weeks postoperatively and underwent a laparoscopic shunt revision. CONCLUSION: In our series laparoscopically assisted VPS insertion in low-weight children who had undergone repeated prior abdominal surgery was feasible and had a low complication rate. We recommend laparoscopically assisted VPS insertion in small infants to avoid the complications of alternative techniques, such as open techniques or ventriculoatrial shunt.

Laparoscopy for suspected appendicitis in children: May a macroscopically normal appendix be left in situ?

BACKGROUND/PURPOSE: An appendix which appears macroscopically normal is found in 10%-20% of laparoscopic explorations for suspected appendicitis. The appropriate surgical strategy for these cases is a matter of debate. We analysed a consecutive series of children in whom an inconspicuous appendix was left in situ. MATERIAL AND METHODS: Laparoscopic exploration was performed in 188 consecutive children with suspected appendicitis and an expected need for immediate operation from 2002 to 2006. Our concept included laparoscopic appendectomy in patients with macroscopic signs of inflammation. Normal appearing appendices were left in situ. All patients with a remaining appendix underwent follow-up. Major endpoints were defined as postoperative complications, re-operations for abdominal symptoms, hospital admissions and consultations with medical doctors during the follow-up period. In addition, other symptoms and well-being were assessed. RESULTS: The appendix appeared macroscopically normal in 21 (11%) of the 188 patients (mean age 11.7 years (+/-4.2); 11 f, 10 m), and was therefore left in situ. The immediate postoperative course was uneventful in all patients with a mean hospital stay of 2.7 (+/-1.2) days. During the follow-up period (mean of 25 (+/-17) months), 18 patients (86%) did not or only rarely (< or = 2 times) consult a medical doctor for abdominal symptoms. Three patients (14%) reported more than 2 consultations. No patient was readmitted to hospital or operated for acute appendicitis. At the last follow-up, 20 patients (95%) were entirely symptom-free. One patient complained about persisting right lower quadrant pain, but refused further diagnostic procedures or interventions. CONCLUSION: According to our results, a macroscopically inconspicuous appendix may be left in situ in patients undergoing laparoscopy for suspected appendicitis. However, this conclusion is based on a small number of patients and larger series are mandatory.

Carbon dioxide, hypoxia and low pH lead to overexpression of c-myc and HMGB-1 oncogenes in neuroblastoma cells.

PURPOSE: It has been reported that CO (2) gas, used to establish a pneumoperitoneum during laparoscopy, affects the behavior of tumor cells. The proto-oncogenes C-MYC and HMGB-1 mediate aggressive behavior of neuroblastomas. We studied whether exposure to CO (2), hypoxia or acidosis affects the expression of C-MYC and HMGB-1 in neuroblastoma cells. METHODS: SH-SY5Y cells were incubated with 100% CO (2), 95% helium/5% CO (2) or pH 6.2 for 2 h. The expression of C-MYC and HMGB-1 was measured by Western blot test immediately, 3 h and 6 h after incubation. Additionally, we measured apoptosis after incubation using fluorometric measurements of caspase 3 and 7 activity. RESULTS: C-myc (160+/-26%, p=0.007 and 138+/-16% vs. control, p=0.04) and HMGB-1 proteins (140+/-13% and 136+/-11%, both p=0.037) were found to be significantly upregulated 6 h after incubation with CO (2) and helium. There was early upregulation of both oncogenes 3 h after CO (2) incubation (251+/-79%, p=0.04 and 292+/-136, p=0.037). Correspondingly, pH 6.2 led to significant overexpression. Levels of apoptosis were reduced. CONCLUSIONS: Exposures mimicking conditions of CO (2) pneumoperitoneum lead to significant overexpression of C-MYC and HMGB-1 in neuroblastoma cells with decreased apoptosis. These results point to a negative influence and potentially increased malignancy of tumor cells.

[Predictive parameters in children with biliary atresia]. / Prognostische Marker bei Kindern mit Gallengangatresie.

Most children with biliary atresia require liver transplantation, and only about 20% survive in the long term with their native livers. Prognostic factors that determine disease progression are still lacking. This retrospective survey of 85 BA patients from 1993 to 2003 was aimed to evaluate prognostic factors using the log rank test. After 5 years 40% of the patients are alive with their native livers (35/85), 26 of them with normal bilirubin (31%). Age at Kasai operation (P=0.46), degree of liver fibrosis (P=0.95), and all laboratory test results before Kasai failed to correlate with outcome. Normal levels of bilirubin 3, 6, and 12 months after Kasai and of aspartate aminotransferase with gammaGT after 6 months are associated with survival with native liver. In conclusion our data demonstrate that a lack of predictive factors must prevent primary liver transplantation in BA patients.

Functional Mx protein does not prevent experimental biliary atresia in Balb/c mice.

Biliary atresia (BA) is a rare cholestatic disease, which manifests itself in the form of inflammation of the liver and bile ducts in newborns, with an unknown etiology and a poor outcome. Mx proteins, which are mediators of innate, antiviral resistance induced by type I interferon, were recently detected in the livers of children with BA. Therefore, the aim of this study was to examine whether the expression of Mx protein could affect the course of experimental BA in mice. A total of 185 newborn Balb/c mice (expressing dysfunctional Mx protein) and Balb/c-Mx+-A2G mice (with functional Mx protein) were intraperitoneally infected with rhesus rotavirus (RRV) or injected with saline solution as controls. They were sacrificed if they showed signs of cholestasis or at three weeks after infection. The expression of hepatic Mx protein was detected by immunostaining (POX) and the hepatic virus load was determined. There was no significant difference in the incidence of cholestasis between wild-type Balb/c mice and Balb/c-Mx+-A2G mice (67 % vs. 65 %). However, Mx protein was highly expressed in Balb/c-Mx+-A2G mice with BA phenotype, but not in wild type Balb/c mice or disease-free Balb/c-Mx+-A2G mice despite RRV infection. The difference in the hepatic virus load was not statistically significant in mice with BA. In conclusion, Mx protein does not prevent newborn Balb/c mice from developing biliary atresia after RRV infection. However, the expression of Mx protein is independent of the hepatic virus load and could be used as a marker of BA in humans, as well as in the RRV model.

CO2 pneumoperitoneum increases survival in mice with polymicrobial peritonitis.

PURPOSE: Laparoscopic techniques are commonly used in patients with bacterial peritonitis. CO2 is known to suppress local and systemic inflammatory responses. Nonetheless, an active immune system is needed to contain bacterial contamination of the abdominal cavity. Therefore, we investigated the early and late effects of CO2 pneumoperitoneum on the ability of mice to overcome polymicrobial peritonitis. MATERIAL AND METHODS: Male C57/B6 mice were subjected to pneumoperitoneum with CO2 or helium, or underwent a midline laparotomy. In a first set, changes of arterial blood gases were monitored. In further experiments, polymicrobial peritonitis was induced after 1 h of pneumoperitoneum/laparotomy by cecal ligation and puncture. In a second set of experiments polymicrobial peritonitis was induced 4 h prior to exposure to pneumoperitoneum/laparotomy. After the interventions, survival rates (early survival: 6 to 48 h; late survival > 48 h) were monitored for 7 days. RESULTS: There was no significant effect of pneumoperitoneum or laparotomy on arterial blood gas parameters. CO2 pneumoperitoneum significantly reduced the early (6 to 48 h) mortality of subsequent peritonitis after CO2 pneumoperitoneum compared to laparotomy (2/20 vs. 9/25; p < 0.05). The protective effect did not reach significance after 7 days (late mortality). The application of a helium peritoneum did not show any beneficial effect. Application of a CO2 pneumoperitoneum during polymicrobial peritonitis significantly reduced overall mortality (p < 0.05) compared to laparotomy. CONCLUSIONS: The modulation of immune responses by CO2, but not helium pneumoperitoneum, has a significant positive impact on survival during abdominal sepsis in a mouse model. Thus, application of a CO2 pneumoperitoneum may be beneficial in conditions with bacterial contamination of the abdominal cavity.

Laparoscopic transperitoneal nephrectomy is feasible in the first year of life and is not affected by kidney size.

PURPOSE: We analyzed the feasibility of laparoscopic nephroureterectomy in children younger than 1 year, with regard to size of impaired kidney. MATERIALS AND METHODS: A total of 40 consecutive children underwent transperitoneal laparoscopic nephrectomy during a 4-year period. Of the patients 19 (48%) were younger than 1 year and were analyzed in detail. Nine of these patients (47.4%) had a multicystic dysplastic kidney, 9 (47.4%) had reflux nephropathy and 1 (5.3%) had obstructive nephropathy. The duration of operation, reasons for conversion, and intraoperative and postoperative complications were prospectively documented. RESULTS: Mean operative time was 133 minutes (range 60 to 240), and did not differ significantly between patients up to age 12 months compared to children 1 year and older (126 vs 148 minutes, NS). Nephroureterectomy was completed laparoscopically in 17 of 19 children (89%) up to age 12 months vs 20 of 21 (95%) 1 year and older (NS). In 1 child younger than 1 year suture dislocation at the renal artery required laparoscopic resuturing. No further complications were seen. In children younger than 1 year the mean operating time was not significantly different for resection of multicystic dysplastic kidney (8 patients, 113 minutes) compared to reflux nephropathy (9, 134 minutes, NS). Mean operating time did not differ significantly for kidney volumes less than 10 cc (8 patients, 119 minutes) compared to kidney volumes greater than 10 cc (9, 129 minutes, NS). CONCLUSIONS: The feasibility of transperitoneal laparoscopic nephroureterectomy in children younger than 1 year is excellent. The duration of operation is not affected by patient age, underlying disease or kidney size.

Carbon dioxide differentially affects the cytokine release of macrophage subpopulations exclusively via alteration of extracellular pH.

BACKGROUND: The improved outcome after endoscopic surgery has been attributed to less surgical trauma. However, the underlying mechanisms are not fully understood, and direct effects of CO2 used for pneumoperitoneum, cellular acidification, and/or the lack of air contamination have been postulated to additionally modulate immune functions during endoscopic surgery. We investigated the effects of CO2 incubation, extracellular acidification, and air contamination on the inflammatory response of two distinct macrophage populations. METHODS: R2 and NR 8383 rat macrophage cell lines were used. Interleukin-6 (IL-6) and nitric oxide after lipopolysaccharide (LPS) stimulation were determined in these sets of experiments: incubation in 100% CO2, 5% CO2, and room air for 2h; incubation at pH 7.4, 6.5, and 5.5 for 2 h in 5% CO2; and incubation in 100% CO2, 5% CO2 and room air in fixed pH 6.3. The extracellular pH was monitored during incubation. We determined the alteration of intracellular pH in cells subjected to extracellular acidification by fluorescence microscopy. RESULTS: Extracellular pH decreased to 6.3 during 100% CO2 incubation. IL-6 release was reduced after CO2 incubation in NR 8383 cells and increased in R2 cells (p < 0.05). It was not altered by air incubation. Decreasing the extracellular pH to 6.5 mimicked the effects of CO2 and a decrease to 5.5 suppressed IL-6 release in both cell lines. In fixed pH at 6.3, CO2 and air incubation had no effect. CO2 and pH had no impact on nitric oxide release and vitality. Intracellular pH decreased with extracellular acidification without significant difference between the two cell lines. CONCLUSIONS: A decrease in extracellular pH during incubation in CO2 differentially affects IL-6 release in macrophage subpopulations. This may explain contradictory results in the literature. Moreover, we demonstrated that air contamination does not affect macrophage cytokine release. The decrease in extracellular pH is the primary underlying mechanism of the alteration of macrophage cytokine release after CO2 incubation, and it appears that the ability to maintain intracellular pH is not determined by the effects of CO2 or extracellular acidification.