Daniel J. Berg

Depletion of Neutrophils in IL-10−/− Mice Delays Clearance of Gastric Helicobacter Infection and Decreases the Th1 Immune Response to Helicobacter

Gastric infection with Helicobacter induces a lymphocyte-rich mucosal inflammation that contains a minor population of neutrophilic granulocytes. The function of neutrophils in the local immune response to gastric Helicobacter infection remains unknown. To investigate this issue, we conducted experiments in neutrophil-depleted control wild-type (wt) and IL-10−/− mice infected with Helicobacter felis by gastric lavage. Infection of wt mice elicited a mild, focal gastritis and a Helicobacter-specific Th1 immune response. In wt mice Helicobacter colonization of the stomach was persistent and progressively increased during the 29 days of observation. Infection of IL-10−/− mice with H. felis elicited a severe chronic gastritis and a greatly enhanced Helicobacter-specific Th1 immune response, as compared with wt mice. After initial colonization, the IL-0−/− mice completely cleared Helicobacter from the stomach by day 8. The gastric inflammation in wt and IL-10−/− mice contained modest numbers of neutrophils. The intensity of gastric inflammation and the extent of Helicobacter colonization were similar in control and in neutrophil-depleted wt mice. In contrast, neutrophil depletion of Helicobacter-infected IL-10−/− mice decreased the severity of gastritis, modulated the Helicobacter-specific Th1 immune response, and delayed the clearance of bacteria from the stomach. These studies identify a role for neutrophils in the local and systemic immune response to gastric Helicobacter in IL-10−/− mice.

IL-10 Is a Central Regulator of Cyclooxygenase-2 Expression and Prostaglandin Production

IL-10 is a potent anti-inflammatory and immune regulatory cytokine. IL-10−/− mice produce exaggerated amounts of inflammatory cytokines when stimulated with LPS, indicating that endogenous IL-10 is a central regulator of inflammatory cytokine production in vivo. PGs are lipid mediators that are also produced in large amounts during the inflammatory response. To study the role of IL-10 in the regulation of PG production during the acute inflammatory response, we evaluated LPS-induced cyclooxygenase (COX) expression and PG production in wild-type (wt) and IL-10−/− mice. LPS-induced PGE2 production from IL-10−/− spleen cells was 5.6-fold greater than that from wt spleen cells. LPS stimulation resulted in the induction of COX-2 mRNA and protein in both wt and IL-10−/− spleen cells; however, the magnitude of increase in COX-2 mRNA was 5.5-fold greater in IL-10−/− mice as compared with wt mice. COX-1 protein levels were not affected by LPS stimulation in either wt or IL-10−/− mice. Neutralization of IFN-γ, TNF-α, or IL-12 markedly decreased the induction of COX-2 in IL-10−/− spleen cells, suggesting that increased inflammatory cytokine production mediates much of the COX-2 induction in IL-10−/− mice. Treatment of IL-10−/− mice with low doses of LPS resulted in a marked induction of COX-2 mRNA in the spleen, whereas wt mice had minimal expression of COX-2 mRNA. These findings indicate that, in addition to IL-10’s central role in the regulation of inflammatory cytokines, endogenous IL-10 is an important regulator of PG production in the response to LPS.

O2⋅− and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate

Pharmacological ascorbate has been proposed as a potential anti-cancer agent when combined with radiation and chemotherapy. The anti-cancer effects of ascorbate are hypothesized to involve the autoxidation of ascorbate leading to increased steady-state levels of H2O2; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study shows that alterations in cancer cell mitochondrial oxidative metabolism resulting in increased levels of O2⋅- and H2O2 are capable of disrupting intracellular iron metabolism, thereby selectively sensitizing non-small-cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox-active labile iron and H2O2. In addition, preclinical studies and clinical trials demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in GBM and NSCLC therapy.

Vascular Effects of Lipopolysaccharide Are Enhanced in Interleukin-10–Deficient Mice

BACKGROUND AND PURPOSE The role in blood vessels of interleukin-10 (IL-10), a potent anti-inflammatory cytokine, is not known. Using mice with targeted deletion of the gene for IL-10 (IL-10(-/-)), we examined the hypothesis that IL-10 is a major modulator of the vascular effects of lipopolysaccharide (LPS). Methods-We examined in vitro responses of carotid arteries obtained from wild-type (129/SvEv or C57BL/6; IL-10(+/+)) and IL-10-deficient mice 6 hours after injection of a relatively low dose of LPS (10 microgram). RESULTS Contraction of the carotid artery in response to U46619 was impaired in IL-10-deficient mice treated with LPS compared with LPS-treated controls. After LPS, U46619 (0.03 and 0.1 microgram/mL) contracted the carotid artery by 0.11+/-0.02 (mean+/-SEM) and 0.38+/-0.03 g in wild-type (n=10) and 0.03+/-0.01 and 0.19+/-0.03 g in IL-10-deficient (n=8) mice (P<0.05 versus control). Aminoguanidine, an inhibitor of inducible nitric oxide synthase (iNOS), had no significant effect on contraction of the carotid artery from LPS-treated control mice but restored contraction of the carotid artery in response to U46619 in IL-10-deficient mice to levels seen in wild-type mice. Similar findings were obtained when phenylephrine was used as a vasoconstricting agent. These findings indicate that LPS produces much greater impairment of contractile responses of the carotid artery in IL-10-deficient mice than in control mice. Impaired contractile function was eliminated by aminoguanidine, suggesting that expression of iNOS is enhanced in arteries from IL-10-deficient mice. In carotid arteries from animals injected with LPS, reverse transcription-polymerase chain reaction (RT-PCR) products for iNOS were found more frequently in IL-10-deficient mice than in wild-type mice. RT-PCR products for iNOS were not present in arteries from vehicle-treated animals (IL-10-deficient or wild-type mice). CONCLUSIONS This is the first evidence that endogenous IL-10 is a major determinant of the effects of LPS on vascular tone. The results suggest that impaired constrictor responses of the carotid artery after LPS in IL-10-deficient mice are mediated by enhanced expression of iNOS.

Role for Complement in Development of Helicobacter-Induced Gastritis in Interleukin-10-Deficient Mice

ABSTRACT The mechanisms by which the immune response can eradicate gastric Helicobacter infection are unknown. We hypothesized that Helicobacter-induced activation of the complement system could promote both inflammation and eradication of Helicobacter from the stomach. In vitro studies demonstrated that Helicobacter felis activates complement in normal mouse serum but not in serum from Rag2−/− mice, indicating that H. felis activates complement through the classical pathway. Next, we infected complement-depleted wild-type control and interleukin-10-deficient (IL-10−/−) mice with H. felis. Helicobacter infection of wild-type mice elicited a mild, focal gastritis and did not alter serum complement levels. Infection of IL-10−/− mice with H. felis elicited severe gastritis. After the initial colonization, the IL-10−/− mice completely cleared Helicobacter from the stomach by day 8. In contrast to wild-type mice, H. felis-infected IL-10−/− mice had a marked increase in serum complement levels. Complement depletion of wild-type mice did not affect the intensity of gastric inflammation or the extent of Helicobacter colonization compared to that for the wild-type control mice. In contrast, complement depletion of Helicobacter-infected IL-10−/− mice decreased the severity of gastritis, decreased the Helicobacter-induced infiltration of neutrophils into the stomach, and delayed the clearance of bacteria. In vitro studies of stimulated splenocytes and neutrophils from IL-10−/− mice produced a twofold increase in complement production compared to that for wild-type mice. Pretreatment with IL-10 inhibited this increase. These studies identify a role for complement in the local immune response to gastric Helicobacter in IL-10−/− mice and suggest a role for IL-10 in the regulation of complement production.

Increased interleukin‐10 production and Th2 skewing in the absence of 5‐lipoxygenase

Eicosanoids (prostaglandins and leukotrienes) are important mediators of inflammatory responses. These lipid mediators may also regulate the production of peptide mediators of the immune system. In this study, we investigated the effect of the absence of 5‐lipoxygenase (5‐LO)‐derived leukotrienes on interleukin (IL)‐10 production. IL‐10 is a key regulator of immune and inflammatory responses, and previous studies have suggested that prostaglandins effect their immunosuppressive functions in part by stimulation of IL‐10 production. We therefore investigated whether leukotriene production would have a similar role in regulation of IL‐10 production. We have made the striking observation that absence of 5‐LO‐derived leukotrienes results in increased IL‐10 production with a concomitant decrease in the production of pro‐inflammatory cytokines, including tumour necrosis factor (TNF)‐α and IL‐12. Moreover, T‐cell cytokine production in the absence of 5‐LO‐derived leukotrienes results in increased IL‐4 production and decreased interferon (IFN)‐γ production. This may be in part secondary to increased IL‐10 production and its effects on dendritic cell function resulting in altered T‐cell differentiation. These findings indicate that, in addition to the central role leukotrienes play in the acute inflammatory response, endogenous leukotrienes are also important regulators of inflammatory cytokine production, via regulation of IL‐10 production and in vivo differentiation of T cells.

Heme oxygenase-1 is protective against nonsteroidal anti-inflammatory drug-induced gastric ulcers.

Objectives: Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain, fever, and inflammation. Long-term use of these drugs is associated with significant gastric injury. Activated neutrophils and oxidative stress seem to play a significant role in NSAID-induced gastric mucosal damage. The objective of our study is to examine the protective effects of an antioxidant and anti-inflammatory enzyme, heme oxygenase-1 (HO-1), in NSAID-induced gastric injury. Methods: Mice were intraperitoneally injected with indomethacin (10 mg/kg) or sham. A specific inducer of HO-1, cobalt protoporphyrin (5 mg/kg), was given 24 hours before indomethacin to allow for the expression of HO-1. Controls received sham treatment. Twenty-four hours after indomethacin injection, gastric tissue damage was examined with histology. HO-1 expression was measured with immunoblot; cytokine levels were measured with enzyme-linked immunosorbent assay. Neutrophil infiltration was quantified with myeloperoxidase assay. Using electron paramagnetic resonance and desferrioxamine, we measured the labile iron pool in the mouse stomach as a marker of oxidative stress. Results: Indomethacin caused gastric inflammation and ulcers, neutrophil activation, and increased tissue expression of interleukin-6 and tumor necrosis factor-alpha in mice. Inducing HO-1 with cobalt protoporphyrin reduced gastric inflammation, number of stomach ulcers, tissue neutrophil activation, and proinflammatory cytokine expression caused by indomethacin. Conclusions: These findings suggest that the induction of an anti-inflammatory and cytoprotective enzyme HO-1 may be a strategy to overcome the gastrointestinal adverse effects limiting the use of NSAIDs.

Pharmacokinetic analysis of irinotecan plus bevacizumab in patients with advanced solid tumors

PurposeThe purpose of this study was to evaluate the effect of bevacizumab on the pharmacokinetics (PK) of irinotecan and its active metabolite. Exploratory analyses of the impact of variability in uridine diphosphate glucuronosyltransferase 1A (UGT1A) genes on irinotecan metabolism and toxicity were conducted.MethodsThis was an open-labeled, fixed-sequence study of bevacizumab with FOLFIRI (irinotecan, leucovorin, and infusional 5-fluorouracil). Pharmacokinetic assessments were conducted in cycles 1 and 3.ResultsForty-five subjects were enrolled. No difference in dose-normalized AUC0–last for irinotecan and SN-38 between irinotecan administered alone or in combination with bevacizumab was identified. Leukopenia was associated with higher exposure to both irinotecan and SN-38. UGT1A1 polymorphisms were associated with variability in irinotecan PK. Gastrointestinal toxicity was associated with UGT1A6 genotype. No other associations between UGT1A genotypes and toxicity were detected.ConclusionBevacizumab does not affect irinotecan PK when administered concurrently. A variety of pharmacogenetic relationships may influence the pharmacokinetics of irinotecan and its toxicity.

Relationship between HER-2 overexpression and brain metastasis in esophageal cancer patients

AIM To study if HER-2 overexpression by locally advanced esophageal cancers increase the chance of brain metastasis following esophagectomy. METHODS We retrospectively reviewed the medical records of esophageal cancer patients who underwent esophagectomy at University of Iowa Hospitals and Clinics between 2000 and 2010. Data analyzed consisted of demographic and clinical variables. The brain metastasis tissue was assayed for HER-2 overexpression utilizing the FDA approved DAKO Hercept Test(®). RESULTS One hundred and forty two patients were reviewed. Median age was 64 years (36-86 years). Eighty eight patients (62%) received neoadjuvant chemoradiotherapy. Pathological complete and partial responses were achieved in 17 (19%) and 71 (81%) patients. Cancer relapsed in 43/142 (30%) patients. The brain was the first site of relapse in 9/43 patients (21%, 95% CI: 10%-36%). HER-2 immunohistochemistry testing of the brain metastasis tissue showed that 5/9 (56%) cases overexpressed HER-2 (3+ staining). CONCLUSION HER-2 overexpression might be associated with increased risk of brain metastasis in esophageal cancer patients following esophagectomy. Further studies will be required to validate this observation.

Consuming a Ketogenic Diet while Receiving Radiation and Chemotherapy for Locally Advanced Lung Cancer and Pancreatic Cancer: The University of Iowa Experience of Two Phase 1 Clinical Trials

Ketogenic diets are low in carbohydrates and high in fat, which forces cells to rely more heavily upon mitochondrial oxidation of fatty acids for energy. Relative to normal cells, cancer cells are believed to exist under a condition of chronic mitochondrial oxidative stress that is compensated for by increases in glucose metabolism to generate reducing equivalents. In this study we tested the hypothesis that a ketogenic diet concurrent with radiation and chemotherapy would be clinically tolerable in locally advanced non-small cell lung cancer (NSCLC) and pancreatic cancer and could potentially exploit cancer cell oxidative metabolism to improve therapeutic outcomes. Mice bearing MIA PaCa-2 pancreatic cancer xenografts were fed either a ketogenic diet or standard rodent chow, treated with conventionally fractionated radiation (2 Gy/fraction), and tumor growth rates were assessed daily. Tumors were assessed for immunoreactive 4-hydroxy-2-nonenal-(4HNE)-modfied proteins as a marker of oxidative stress. Based on this and another previously published preclinical study, phase 1 clinical trials in locally advanced NSCLC and pancreatic cancer were initiated, combining standard radiation and chemotherapy with a ketogenic diet for six weeks (NSCLC) or five weeks (pancreatic cancer). The xenograft experiments demonstrated prolonged survival and increased 4HNE-modfied proteins in animals consuming a ketogenic diet combined with radiation compared to radiation alone. In the phase 1 clinical trial, over a period of three years, seven NSCLC patients enrolled in the study. Of these, four were unable to comply with the diet and withdrew, two completed the study and one was withdrawn due to a dose-limiting toxicity. Over the same time period, two pancreatic cancer patients enrolled in the trial. Of these, one completed the study and the other was withdrawn due to a dose-limiting toxicity. The preclinical experiments demonstrate that a ketogenic diet increases radiation sensitivity in a pancreatic cancer xenograft model. However, patients with locally advanced NSCLC and pancreatic cancer receiving concurrent radiotherapy and chemotherapy had suboptimal compliance to the oral ketogenic diet and thus, poor tolerance.