Enhanced immunogenic potential of cancer immunotherapy antibodies in human IgG1 transgenic mice.

ABBREVIATIONS: ADA Anti-Drug Antibodies; BCR B Cell Receptor; BId Idiotype-specific B Cell; BiTE Bispecific T cell Engager; BMC Bone Marrow Chimeric Mice; BSA Bovine Serum Albumin; CDR Complementary Determining Region; CEA Carcinoembryonic Antigen; CIT Cancer Immunotherapy; CitAbs Cancer Immunotherapy Antibodies; DC Dendritic Cell; ELISA Enzyme-Linked Immunosorbent Assay; FcRn Neonatal Fc Receptor; FcyR Fc gamma Receptor; GM-CSF Granulocyte-Macrophage Colony Stimulating Factor; gMFI Geometric Mean Fluorescence Intensity; H Heavy Chain; IC Immune Complex; Id Idiotype; IgA Immunoglobulin alpha; IgG1 Immunoglobulin gamma 1; IL-2 Interleukin 2; IL-2R Interleukin 2 Receptor; IL2v Interleukin 2 Variant; IVIG1 Intravenous Immunoglobulin 1; KLH Keyhole Limpet Hemocyanin; L Light Chain; MAPPs MHC-associated Peptide Proteomics; MHC Major Histocompatibility Complex; PBMC Peripheral Blood Mononuclear Cells; PBS Phosphate Buffered Saline; SHM Somatic Hypermutation; scFv Single-chain Variable Fragment; TCR T cell Receptor; TFc Fc-specific T cell; TId Id-specific T cell; UV Ultraviolet; V Variable.

A Novel In Vitro Method to Assess the Microbial Barrier Function of Tissue Adhesives Using Bioluminescence Imaging Technique.

Background. Tissue glues can minimize treatment invasiveness, mitigate the risk of infection, and reduce surgery time; ergo, they have been developed and used in surgical procedures as wound closure devices beside sutures, staples, and metallic grafts. Regardless of their structure or function, tissue glues should show an acceptable microbial barrier function before being used in humans. This study proposes a novel in vitro method using Escherichia coli Lux and bioluminescence imaging technique to assess the microbial barrier function of tissue glues. Different volumes and concentrations of E. coli Lux were applied to precured or cured polyurethane-based tissue glue placed on agar plates. Plates were cultured for 1 h, 24 h, 48 h, and 72 h with bioluminescence signal measurement subsequently. Herein, protocol established a volume of 5 µL of a 1 : 100 dilution of E. coli Lux containing around 2 × 107 CFU/mL as optimal for testing polyurethane-based tissue glue. Measurement of OD600nm, determination of CFU/mL, and correlation with the bioluminescence measurement in p/s unit resulted in a good correlation between CFU/mL and p/s and demonstrated good reproducibility of our method. In addition, this in vitro method could show that the tested polyurethane-based tissue glue can provide a reasonable barrier against the microbial penetration and act as a bacterial barrier for up to 48 h with no penetration and up to 72 h with a low level of penetration through the material. Overall, we have established a novel, sensitive, and reproducible in vitro method using the bioluminescence imaging technique for testing the microbial barrier function of new tissue glues.

A Dual Protective Effect of Intestinal Remote Ischemic Conditioning in a Rat Model of Total Hepatic Ischemia.

The present study aimed to investigate the effects of intestinal remote ischemic preconditioning (iRIC) on ischemia-reperfusion injury (IRI) and gut barrier integrity in a rat model of total hepatic ischemia (THI). Male Wistar rats (n = 50; 250-300 g) were randomly allocated into two experimental groups: RIC/Control. Thirty minutes of THI was induced by clamping the hepatoduodenal ligament. iRIC was applied as 4-min of ischemia followed by 11-min of reperfusion by clamping the superior mesenteric artery. Animals were sacrificed at 1, 2, 6, 24 h post-reperfusion (n = 5/group/timepoint). RIC of the gut significantly improved microcirculation of the ileum and the liver. Tissue ATP-levels were higher following iRIC (Liver: 1.34 ± 0.12 vs. 0.97 ± 0.20 µmol/g, p = 0.04) and hepatocellular injury was reduced significantly (ALT: 2409 ± 447 vs. 6613 ± 1117 IU/L, p = 0.003). Systemic- and portal venous IL-6 and TNF-alpha levels were markedly lower following iRIC, demonstrating a reduced inflammatory response. iRIC led to a structural and functional preservation of the intestinal barrier. These results suggest that iRIC might confer a potent protection against the detrimental effects of THI in rats via reducing IRI and systemic inflammatory responses and at the same time by mitigating the dramatic consequences of severe intestinal congestion and bacterial translocation.

Advantages of laparoscopic compared to conventional surgery are not related to an innate immune response of peritoneal immune activation: an animal study in rats.

PURPOSE: Laparoscopic surgery (LS) has proved superior compared to conventional surgery (CS) regarding morbidity, length of hospital stay, rate of wound infection and time until recovery. An improved preservation of the postoperative immune function is assumed to contribute to these benefits though the role of the local peritoneal immune response is still poorly understood. Our study investigates the peritoneal immune response subsequent to abdominal surgery and compares it between laparoscopic and conventional surgery to find an immunological explanation for the clinically proven benefits of LS. METHODS: Wistar rats (N = 140) underwent laparoscopic cecum resection (LCR; N = 28), conventional cecum resection (CCR; N = 28), laparoscopic sham operation (LSO; N = 28), conventional sham operation (CSO; N = 28), or no surgical treatment (CTRL; N = 28). Postoperatively, peritoneal lavages were performed, leukocytes isolated and analyzed regarding immune function and phagocytosis activity. RESULTS: Immune function was inhibited postoperatively in animals undergoing LCR or CCR compared to CTRL reflected by a lower TNF-α (CTRL 3956.65 pg/ml, LCR 2018.48 pg/ml (p = 0.023), CCR 2793.78 pg/ml (n.s.)) and IL-6 secretion (CTRL 625.84 pg/ml, LCR 142.84 pg/ml (p = 0.009), CCR 169.53 pg/ml (p = 0.01)). Phagocytosis was not affected in rats undergoing any kind of surgery compared to CTRL. Neither cytokine secretion nor phagocytosis activity differed significantly between laparoscopic and conventional surgery. CONCLUSIONS: According to our findings the benefits associated with LS compared to CS cannot be explained by differences in the postoperative peritoneal innate immune response. Further studies are needed to elucidate the causes for a more favorable postoperative outcome in patients after LS compared to CS.

Gut microbial translocation corrupts myeloid cell function to control bacterial infection during liver cirrhosis.

OBJECTIVE: Patients with liver cirrhosis suffer from increased susceptibility to life-threatening bacterial infections that cause substantial morbidity. METHODS: Experimental liver fibrosis in mice induced by bile duct ligation or CCl4 application was used to characterise the mechanisms determining failure of innate immunity to control bacterial infections. RESULTS: In murine liver fibrosis, translocation of gut microbiota induced tonic type I interferon (IFN) expression in the liver. Such tonic IFN expression conditioned liver myeloid cells to produce high concentrations of IFN upon intracellular infection with Listeria that activate cytosolic pattern recognition receptors. Such IFN-receptor signalling caused myeloid cell interleukin (IL)-10 production that corrupted antibacterial immunity, leading to loss of infection-control and to infection-associated mortality. In patients with liver cirrhosis, we also found a prominent liver IFN signature and myeloid cells showed increased IL-10 production after bacterial infection. Thus, myeloid cells are both source and target of IFN-induced and IL-10-mediated immune dysfunction. Antibody-mediated blockade of IFN-receptor or IL-10-receptor signalling reconstituted antibacterial immunity and prevented infection-associated mortality in mice with liver fibrosis. CONCLUSIONS: In severe liver fibrosis and cirrhosis, failure to control bacterial infection is caused by augmented IFN and IL-10 expression that incapacitates antibacterial immunity of myeloid cells. Targeted interference with the immune regulatory host factors IL-10 and IFN reconstitutes antibacterial immunity and may be used as therapeutic strategy to control bacterial infections in patients with liver cirrhosis.

Establishing a biomarker for postoperative ileus in humans - Results of the BiPOI trial.

AIMS: Postoperative ileus (POI) is a frequent complication after abdominal surgery, resulting from an inflammation of the muscularis externa (ME). So far no valid biomarker for occurrence, duration or intensity of POI exists. Extravasation of monocytes and neutrophils from blood circulating into the postoperative ME is well known as a hallmark of POI. In a previous study we demonstrated that a low abundant subset of TH1 cells, activated by IL-12, can be detected in the peripheral blood of a small subset of patients in response to abdominal surgery. The aim of the present study was to investigate if these specific TH1 cells, IL-12 or circulating leukocyte levels could act as a valid marker for POI occurrence. MAIN METHODS: At different time points, blood samples of patients undergoing abdominal or extraabdominal surgery were collected. Serum levels of IL-12 or TH1 cells as well as neutrophils and monocytes were analyzed. Data were compared between both groups and correlated with clinical signs of POI. KEY FINDINGS: Time until first flatus and defecation as well as solid food tolerances are delayed after abdominal compared to extraabdominal surgery. Circulating IL-12 levels and numbers of TH1 cells, neutrophils and monocytes did not differ between both groups. SIGNIFICANCE: While previous experiments indicated that specific TH1 cells play a crucial role in POI dissemination, our present data from a larger human cohort demonstrate that they do not seem to be suitable to distinguish between abdominal and extraabdominal surgery. Furthermore neither TH1 cells nor leukocytes or serum IL-12 levels are appropriate biomarkers for POI in a clinical setting.

Development of a standardized laparoscopic caecum resection model to simulate laparoscopic appendectomy in rats.

BACKGROUND: Laparoscopic appendectomy (LA) has become one of the most common surgical procedures to date. To improve and standardize this technique further, cost-effective and reliable animal models are needed. METHODS: In a pilot study, 30 Wistar rats underwent laparoscopic caecum resection (as rats do not have an appendix vermiformis), to optimize the instrumental and surgical parameters. A subsequent test study was performed in another 30 rats to compare three different techniques for caecum resection and bowel closure. RESULTS: Bipolar coagulation led to an insufficiency of caecal stump closure in all operated rats (Group 1, n = 10). Endoloop ligation followed by bipolar coagulation and resection (Group 2, n = 10) or resection with a LigaSure™ device (Group 3, n = 10) resulted in sufficient caecal stump closure. CONCLUSIONS: We developed a LA model enabling us to compare three different caecum resection techniques in rats. In conclusion, only endoloop closure followed by bipolar coagulation proved to be a secure and cost-effective surgical approach.

Postoperative ileus involves interleukin-1 receptor signaling in enteric glia.

BACKGROUND & AIMS: Postoperative ileus (POI) is a common consequence of abdominal surgery that increases the risk of postoperative complications and morbidity. We investigated the cellular mechanisms and immune responses involved in the pathogenesis of POI. METHODS: We studied a mouse model of POI in which intestinal manipulation leads to inflammation of the muscularis externa and disrupts motility. We used C57BL/6 (control) mice as well as mice deficient in Toll-like receptors (TLRs) and cytokine signaling components (TLR-2(-/-), TLR-4(-/-), TLR-2/4(-/-), MyD88(-/-), MyD88/TLR adaptor molecule 1(-/-), interleukin-1 receptor [IL-1R1](-/-), and interleukin (IL)-18(-/-) mice). Bone marrow transplantation experiments were performed to determine which cytokine receptors and cell types are involved in the pathogenesis of POI. RESULTS: Development of POI did not require TLRs 2, 4, or 9 or MyD88/TLR adaptor molecule 2 but did require MyD88, indicating a role for IL-1R1. IL-1R1(-/-) mice did not develop POI; however, mice deficient in IL-18, which also signals via MyD88, developed POI. Mice given injections of an IL-1 receptor antagonist (anakinra) or antibodies to deplete IL-1α and IL-1ß before intestinal manipulation were protected from POI. Induction of POI activated the inflammasome in muscularis externa tissues of C57BL6 mice, and IL-1α and IL-1ß were released in ex vivo organ bath cultures. In bone marrow transplantation experiments, the development of POI required activation of IL-1 receptor in nonhematopoietic cells. IL-1R1 was expressed by enteric glial cells in the myenteric plexus layer, and cultured primary enteric glia cells expressed IL-6 and the chemokine monocyte chemotactic protein 1 in response to IL-1ß stimulation. Immunohistochemical analysis of human small bowel tissue samples confirmed expression of IL-1R1 in the ganglia of the myenteric plexus. CONCLUSIONS: IL-1 signaling, via IL-1R1 and MyD88, is required for development of POI after intestinal manipulation in mice. Agents that interfere with the IL-1 signaling pathway are likely to be effective in the treatment of POI.

Expression of type I interferon by splenic macrophages suppresses adaptive immunity during sepsis.

Early during Gram-negative sepsis, excessive release of pro-inflammatory cytokines can cause septic shock that is often followed by a state of immune paralysis characterized by the failure to mount adaptive immunity towards secondary microbial infections. Especially, the early mechanisms responsible for such immune hypo-responsiveness are unclear. Here, we show that TLR4 is the key immune sensing receptor to initiate paralysis of T-cell immunity after bacterial sepsis. Downstream of TLR4, signalling through TRIF but not MyD88 impaired the development of specific T-cell immunity against secondary infections. We identified type I interferon (IFN) released from splenic macrophages as the critical factor causing T-cell immune paralysis. Early during sepsis, type I IFN acted selectively on dendritic cells (DCs) by impairing antigen presentation and secretion of pro-inflammatory cytokines. Our results reveal a novel immune regulatory role for type I IFN in the initiation of septic immune paralysis, which is distinct from its well-known immune stimulatory effects. Moreover, we identify potential molecular targets for therapeutic intervention to overcome impairment of T-cell immunity after sepsis.

Effective collaboration between marginal metallophilic macrophages and CD8+ dendritic cells in the generation of cytotoxic T cells.

The spleen is the lymphoid organ that induces immune responses toward blood-borne pathogens. Specialized macrophages in the splenic marginal zone are strategically positioned to phagocytose pathogens and cell debris, but are not known to play a role in the activation of T-cell responses. Here we demonstrate that splenic marginal metallophilic macrophages (MMM) are essential for cross-presentation of blood-borne antigens by splenic dendritic cells (DCs). Our data demonstrate that antigens targeted to MMM as well as blood-borne adenoviruses are efficiently captured by MMM and exclusively transferred to splenic CD8(+) DCs for cross-presentation and for the activation of cytotoxic T lymphocytes. Depletion of macrophages in the marginal zone prevents cytotoxic T-lymphocyte activation by CD8(+) DCs after antibody targeting or adenovirus infection. Moreover, we show that tumor antigen targeting to MMM is very effective as antitumor immunotherapy. Our studies point to an important role for splenic MMM in the initial steps of CD8(+) T-cell immunity by capturing and concentrating blood-borne antigens and the transfer to cross-presenting DCs which can be used to design vaccination strategies to induce antitumor cytotoxic T-cell immunity.