Gertrud Vieten

Obesity and the Associated Mediators Leptin, Estrogen and IGF-I Enhance the Cell Proliferation and Early Tumorigenesis of Breast Cancer Cells

Breast cancer continues to be a major cause of cancer deaths in women. Estrogen, which is also produced by the adipose tissue, is held responsible for the elevated risk of breast cancer in obese women. However, the adipose tissue secrets hormones and adipokines such as leptin and IGF-I and these substances could also contribute to an increased breast cancer risk for obese women. In this study, the impact of obesity on cell proliferation was investigated. The carcinogen 7, 12, dimethylbenz[a]anthracene (DMBA) was administered to normal weight and diet-induced obese female Sprague-Dawley rats. Cell proliferation was evaluated by immunohistological staining of BrdU-incorporation. In the mammary glands and inguinal lymphatic nodes of the obese rats, cell proliferation was significantly increased, indicating a significant influence of obesity on breast cancer. Effects of leptin, estrogen, and IGF-I on the proliferation of MCF-7 cells in vitro were assessed using an MTT assay. Cell culture experiments demonstrated a mitogenic role of these three mediators on cell proliferation. Our data demonstrate a stimulative effect of substances produced by the adipose tissue on breast cancer. Body weight specific cell proliferation suggests that obesity-related adipokines and mediators enhance cell proliferation and increase the risk for breast cancer.

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.

Mini-laparotomy and full laparotomy, but not laparoscopy, alter hepatic macrophage populations in a rat model

BackgroundImmune function is better preserved by laparoscopic versus conventional surgery. Numerous mediators of the systemic trauma response are synthesized and/or regulated by the liver. However, it has been stated that the advantages of laparoscopic surgery are no more obvious when conventional operations are performed via mini-laparotomy. We set out to compare the impact of laparoscopy and mini- and full laparotomy on the hepatic macrophage populations.MethodsMale Lewis rats were subjected to anesthesia alone (control), mini-laparotomy (1 cm), full laparotomy (7 cm), or laparoscopy for 60 min. Endpoints were the total protein in the peritoneal lavage fluid, hepatic ED-1 cells (recruited monocytes), hepatic ED-2 cells (Kupffer cells), the expression of OX-6 in the liver, and C-reactive protein (CRP) in plasma.ResultsProtein in the peritoneal lavage fluid increased significantly after all interventions. Full laparotomy was accompanied by an enhancement in ED-1-positive monocytes in the liver parenchyma compared to all other groups (p < 0.001). Mini- and full laparotomy led to an increase in ED-2-positive Kupffer cells (p < 0.001). Laparoscopy did not affect the number of monocytes/macrophages. There was no significant alteration of OX-6 expression in either group. No change in the cellular composition in the periportal fields was observed. The CRP plasma levels did not significantly differ between groups.ConclusionsLaparoscopy completely prevents hepatic macrophage populations from expansion and normal cell disposition is preserved. Laparotomy, irrespective of incision size, increases the number of Kupffer cells. Moreover, full laparotomy, but not mini-laparotomy or laparoscopy, causes an increase in hepatic monocyte recruitment. The regulating pathways after surgery differ from other immunologic challenges, such as sepsis, in which immunocompetent cells accumulate and are stimulated in the periportal fields.

METABOLISM OF SURFACTANT PHOSPHATIDYLCHOLINE MOLECULAR SPECIES IN cftr tm1HGU tm1HGU MICE COMPARED TO MF-1 MICE

In cftrtm1HGU/tm1HGU mice, an animal model designed to study pathophysiologic alterations due to the CFTR defect found in cystic fibrosis, surfactant phospholipids of bronchoalveolar lavage fluid (BALF)are increased. To study the metabolical basis of such increases, we intraperitoneally injected cftrtm1HGU/tm1HGU mice [methyl-3H]choline and measured [methyl-3In cftr(tmIHGU/m1HGU) mice, an animal model designed to study pathophysiologic alterations due to the CFTR defect found in cysticfibrosis, surfactant phospholipids of bronchoalveolar lavage fluid (BALF) are increased. To study the metabolical basis of such increases, we intraperitoneally injected cft(tm1HGU/tm1HGU) mice [methyl-3H]choline and measured [methyl-3H]choline incorporation into phosphatidylcholine (PC) molecular species of lung tissue and BALF after 1.5 to 24 hours. MF1 and MF1 x cftr(tm1HGU/tm1HGU) hybrid mice served as controls. In tissue [methyl-3H]choline incorporation into total PC was constant for 24 hours and identical in control and cftr(tmIHGU/m1HGU) mice. However, from 7.5 to 24 hours there was a shift of [methyl-3H]choline incorporation from palmitoyloleoyl-PC and palmitoyllinoleoyl-PC towards PC species enriched in surfactant, dipalmitoyl-PC, palmitoylmyristoyl-PC, and palmitoylpalmitoleoyl-PC. The relative and absolute 3H-labels of PC species were identical for cftr(tmIHGU/m1HGU) compared to control mice. In BALF [methyl-3H]choline of total PC increased from 1.5 to 24 hours (R2 > .98), mainly due to [methyl-3H]choline-labelled dipalmitoyl-PC, in all experimental groups. In BALF from cftr(tmIHGU/m1HGU) mice, the [methyl-3H]choline label of total PC and individual PC species was significantly increased over control values after 24 hours, but not after 1.5 to 6 hours. Numbers and composition of BALF cells were not different between controls and cftr(tmIHGU/m1HGU) mice. We, conclude that increased alveolar phospholipid in cftr(tmIHGU/m1HGU) mice is likely due to decreased reuptake of surfactant.

Distinct phenotypic features of neonatal murine macrophages

Neonates rely on their innate immune system. Resident tissue macrophages are considered to be initiators and regulators of the innate immune response and thus, appear to be especially important to neonates. We hypothesized that the phenotype and function of neonatal tissue macrophages differ from their adult counterparts. Peritoneal macrophages from neonatal (<24 h) and adult (6 weeks old) C57BL/6J mice were isolated and analyzed by high‐content chipcytometry. After stimulation for 6 h with LPS (0, 1, 10, 100 ng/mL), macrophage transcriptome was analyzed by microarray and cytokine release was measured using multiparametric bead assays. Antigen presenting capacity was compared by T‐cell stimulation assays. We observed that neonatal murine peritoneal macrophages are characterized by selective lack of expression of F4/80, MHC class II, and costimulatory molecules (CD80, CD86). Furthermore, we found differences in the transcriptome between neonatal and adult macrophages, unstimulated and after LPS stimulation. Although neonatal macrophages showed a significantly increased secretion of proinflammatory cytokines upon LPS stimulation, their potential to induce T‐cell proliferation was significantly reduced. In conclusion, we observed a distinct phenotype of the neonatal macrophage population. The specific functions of this macrophage population could help to understand the excessive inflammatory reactions observed in the very young.

Increased Inflammatory Reaction to Intestinal Ischemia-Reperfusion in Neonatal versus Adult Mice

AIM Neonate and preterm patients are threatened by exaggerated inflammation of the gut. This study tests the hypothesis that the neonatal gut is prone to inflammation, by comparing the inflammatory reaction of neonatal and adult murine intestine to ischemia and reperfusion. METHODS Neonatal (4 days, n=36) and adult (4 weeks, n=12) C57BL/6J mice were randomly divided between ischemia-reperfusion (IR) and untreated controls (Con). In IR animals intestinal ischemia was induced by clamping the superior mesenteric artery (30 minutes) followed by reperfusion (4 hours). After the experiment, RNA was extracted from the small intestines and the expression of the chemokines CXCL1/KC and CXCL2/MIP-2 were determined by quantitative real-time reverse transcription-polymerase chain reaction. Flow cytometry was used to analyze neutrophil influx (Live+ Ly-6G+ ) in isolated cell populations. RESULTS We observed a strong increase in all measured proinflammatory endpoints after IR in both adult and neonatal mice. However, the inflammatory reaction was significantly stronger in neonatal murine intestines, with a significantly higher increase in CXCL1/KC expression and neutrophil accumulation as compared with adults (p<0.05). CONCLUSION The intestines of neonatal mice reacted with an increased inflammatory response to the ischemic insult. This increased susceptibility could help to explain the exaggerated inflammation seen in diseases such as necrotizing enterocolitis.

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.

Dextran sodium sulfate (DSS) induces necrotizing enterocolitis-like lesions in neonatal mice

Background Necrotizing enterocolitis (NEC) is an inflammatory bowel disease of preterm human newborns with yet unresolved etiology. An established neonatal murine model for NEC employs oral administration of lipopolysaccharides (LPS) combined with hypoxia/hypothermia. In adult mice, feeding dextran sodium sulfate (DSS) represents a well-established model for experimental inflammatory bowel disease. Here we investigated the effect of DSS administration on the neonatal murine intestine in comparison with the established NEC model. Methods 3-day-old C57BL/6J mice were either fed formula containing DSS or LPS. LPS treated animals were additionally stressed by hypoxia/hypothermia twice daily. After 72 h, mice were euthanized, their intestinal tissue harvested and analyzed by histology, qRT-PCR and flow cytometry. For comparison, adult C57BL/6J mice were fed with DSS for 8 days and examined likewise. Untreated, age matched animals served as controls. Results Adult mice treated with DSS exhibited colonic inflammation with significantly increased Cxcl2 mRNA expression. In contrast, tissue inflammation in neonatal mice treated with DSS or LPS plus hypoxia/hypothermia was present in colon and small intestine as well. Comparative analysis of neonatal mice revealed a significantly increased lesion size and intestinal Cxcl2 mRNA expression after DSS exposure. Whereas LPS administration mainly induced local neutrophil recruitment, DSS treated animals displayed increased monocytes/macrophages infiltration. Conclusions Our study demonstrates the potential of DSS to induce NEC-like lesions accompanied by a significant humoral and cellular immune response in the small and large intestine of neonatal mice. The new model therefore represents a good alternative to LPS plus hypoxia/hypothermia administration requiring no additional physical stress.

IL-17A blockade or deficiency does not affect progressive renal fibrosis following renal ischaemia reperfusion injury in mice

IL‐17A contributes to acute kidney injury and fibrosis. Therefore, we asked whether IL‐17A deficiency or treatment with a IL‐17A blocking antibody impacts severe renal ischaemia reperfusion injury (IRI) and the progression to chronic kidney disease (CKD).

Long-term outcome and necessity of liver transplantation in infants with biliary atresia are independent of cytokine milieu in native liver and serum

Purpose Biliary atresia (BA) is a rare disease of unknown pathogenesis in infants characterized by an inflammatory, progressive destruction of the biliary system and deterioration of liver function. The standard treatment for BA is a Kasai‐hepatoportoenterostomy (KPE). However, liver transplantation (LTX) becomes necessary in about 50–80% of cases. Therefore, some authors advocate for primary LTX in BA, but this would require early markers to predict which children would benefit from KPE or to show rapid progression to liver cirrhosis (RLC) instead. Methods Snap‐frozen liver biopsies and sera samples of 57 infants with BA were collected during KPE. Clinical and follow‐up data were assessed via the biliary atresia and related diseases registry (BARD‐online.com). Protein‐levels of 25 pro‐ and anti‐inflammatory mediators of 49 infants were assessed via multiplex protein‐immunoassay and analyzed by t‐test as well as multidimensional principal component analysis. Results 22 different immunomodulatory mediators were detectable in livers of children with BA, while serum protein levels were very low to undetectable. Following KPE, 33 BA patients showed RLC that required early LTX, while 24 had favorable course of disease with long‐term survival with native liver (SNL). There were no significant differences between RLC and SNL in terms of local (from liver samples) nor systemic (from sera) immunomodulatory mediators. Protein levels were much lower in sera than in livers without statistical correlation. Conclusion Our data suggest that local or systemic immunomodulatory mediators are unsuitable for predicting the disease course of BA. Thus, no deduction for optimal treatment strategy can be drawn. Collectively, we conclude that in BA, the degree of inflammation and protein microenvironment in the liver at the time‐point of KPE are dismissible factors for the future course of disease. Highlights22 immunomodulatory mediators detectable in liver and sera of biliary atresia infants.No correlation of inflammatory mediators and Kasai‐procedure outcome.Immunomodulatory mediators are unsuitable predictors for biliary atresia course.