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Objective: To investigate the impact of the phophodiesterase-4 inhibition (PD-4-I) with rolipram on hepatic integrity in lipopolysaccharide (LPS) induced hyperinflammation. Materials and Methods: Liver microcirculation in rats was obtained using intravital microscopy. Macrohemodynamic parameters, blood assays, and organs were harvested to determine organ function and injury. Hyperinflammation was induced by LPS and PD-4-I rolipram was administered intravenously one hour after LPS application. Cell viability of HepG2 cells was measured by EZ4U-kit based on the dye XTT. Experiments were carried out assessing the influence of different concentrations of tumor necrosis factor alpha (TNF-α) and LPS with or without PD-4-I. Results: Untreated LPS-induced rats showed significantly decreased liver microcirculation and increased hepatic cell death, whereas LPS + PD-4-I treatment could improve hepatic volumetric flow and cell death to control level whithout influencing the inflammatory impact. In HepG2 cells TNF-α and LPS significantly reduced cell viability. Coincubation with PD-4-I increased HepG2 viability to control levels. The heme oxygenase 1 (HO-1) pathway did not induce the protective effect of PD-4-I. Conclusion: Intravenous PD-4-I treatment was effective in improving hepatic microcirculation and hepatic integrity, while it had a direct protective effect on HepG2 viability during inflammation.
Background
The management of rectal cancer (RC) has substantially changed over the last decades with the implementation of neoadjuvant chemoradiotherapy, adjuvant therapy and improved surgery such as total mesorectal excision (TME). It remains unclear in which way these approaches overall influenced the rate of local recurrence and overall survival.
Methods
Clinical, histological and survival data of 658 out of 662 consecutive patients with RC were analyzed for treatment and prognostic factors from a prospectively expanded single-institutional database. Findings were then stratified according to time of diagnosis in patient groups treated between 1993 and 2001 and 2002 and 2010.
Results
The study population included 658 consecutive patients with rectal cancer between 1993 and 2010. Follow up data was available for 99.6% of all 662 treated patients. During the time period between 2002 and 2010 significantly more patients underwent neoadjuvant chemoradiotherapy (17.6% vs. 60%) and adjuvant chemotherapy (37.9% vs. 58.4%). Also, the rate of reported TME during surgery increased. The rate of local or distant metastasis decreased over time, and tumor related 5-year survival increased significantly with from 60% to 79%.
Conclusion
In our study population, the implementation of treatment changes over the last decade improved the patient’s outcome significantly. Improvements were most evident for UICC stage III rectal cancer.
Microbial spectrum and antibiotic resistance in patients suffering from penetrating Crohn's disease
(2022)
Intraabdominal abscess formation occurs in up to 30% of patients suffering from Crohn's disease (CD). While international guidelines recommend a step-up approach with a combination of empiric antibiotic therapy and percutaneous drainage to delay or even avoid surgery, evidence about microbial spectrum in penetrating ileitis is sparse. We retrospectively assessed outcomes of 46 patients with terminal penetrating Ileitis where microbial diagnostics have been performed and compared microbial spectrum and antibiotic resistance profile of CD patients with patients suffering from diverticulitis with intraabdominal abscess formation. In both groups, the most frequently isolated pathogen was the gram-negative bacterium E. coli belonging to the family of Enterobacterales. However, overall Enterobacterales were significantly more often verifiable in the control group than in CD patients. Furthermore, microbial analysis showed significant differences regarding isolation of anaerobic pathogens with decreased frequency in patients with CD. Subgroup analysis of CD patients to evaluate a potential influence of immunosuppressive therapy on microbial spectrum only revealed that Enterobacterales was less frequently detected in patients treated with steroids. Immunosuppressive therapy did not show any impact on all other groups of pathogens and did not change antibiotic resistance profile of CD patients. In conclusion, we were able to demonstrate that the microbial spectrum of CD patients does differ only for some pathogen species without increased rate of antibiotic resistance. However, the empiric antibiotic therapy for CD-associated intra-abdominal abscess remains challenging since different points such as local epidemiological and microbiological data, individual patient risk factors, severity of infection, and therapy algorithm including non-surgical and surgical therapy options should be considered before therapeutical decisions are made.
The measurement of transepithelial electrical resistance (TEER) is a common technique to determine the barrier integrity of epithelial cell monolayers. However, it is remarkable that absolute TEER values of similar cell types cultured under comparable conditions show an immense heterogeneity. Based on previous observations, we hypothesized that the heterogeneity of absolute TEER measurements can not only be explained by maturation of junctional proteins but rather by dynamics in the absolute length of cell junctions within monolayers. Therefore, we analyzed TEER in epithelial cell monolayers of Caco2 cells during their differentiation, with special emphasis on both changes in the junctional complex and overall cell morphology within monolayers. We found that in epithelial Caco2 monolayers TEER increased until confluency, then decreased for some time, which was then followed by an additional increase during junctional differentiation. In contrast, permeability of macromolecules measured at different time points as 4 kDA fluorescein isothiocyanate (FITC)-dextran flux across monolayers steadily decreased during this time. Detailed analysis suggested that this observation could be explained by alterations of junctional length along the cell borders within monolayers during differentiation. In conclusion, these observations confirmed that changes in cell numbers and consecutive increase of junctional length have a critical impact on TEER values, especially at stages of early confluency when junctions are immature.
Altered host-intestinal microbiota interactions are increasingly implicated in the metabolic benefits of Roux-en-Y gastric bypass (RYGB) surgery. We previously found, however, that RYGB-associated ileal microbiota can paradoxically impair host glycemic control when transferred to germ-free mice. Here we present complementary evidence suggesting that this could be due to the heightened development of systemic endotoxemia. Consistently, application of ileal content from RYGB-treated compared with sham-operated rats onto Caco-2 cell monolayers compromised barrier function and decreased expression of the barrier-stabilizing proteins claudin-4 and desmoglein-2. Our findings raise the possibility that RYGB-associated ileal microbiota produce and release soluble metabolites which locally increase intestinal permeability to promote systemic endotoxemia-induced insulin resistance, with potential implications for the treatment of RYGB patients who eventually relapse onto type 2 diabetes.
Early treatment with glucocorticoids could help reduce both cytotoxic and vasogenic edema, leading to improved clinical outcome after stroke. In our previous study, isosteviol sodium (STVNA) demonstrated neuroprotective effects in an in vitro stroke model, which utilizes oxygen-glucose deprivation (OGD). Herein, we tested the hypothesis that STVNA can activate glucocorticoid receptor (GR) transcriptional activity in brain microvascular endothelial cells (BMECs) as previously published for T cells. STVNA exhibited no effects on transcriptional activation of the glucocorticoid receptor, contrary to previous reports in Jurkat cells. However, similar to dexamethasone, STVNA inhibited inflammatory marker IL-6 as well as granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion. Based on these results, STVNA proves to be beneficial as a possible prevention and treatment modality for brain ischemia-reperfusion injury-induced blood–brain barrier (BBB) dysfunction.
Enteric glial cells (EGCs) of the enteric nervous system are critically involved in the maintenance of intestinal epithelial barrier function (IEB). The underlying mechanisms remain undefined. Glial cell line-derived neurotrophic factor (GDNF) contributes to IEB maturation and may therefore be the predominant mediator of this process by EGCs. Using GFAP\(^{cre}\) x Ai14\(^{floxed}\) mice to isolate EGCs by Fluorescence-activated cell sorting (FACS), we confirmed that they synthesize GDNF in vivo as well as in primary cultures demonstrating that EGCs are a rich source of GDNF in vivo and in vitro. Co-culture of EGCs with Caco2 cells resulted in IEB maturation which was abrogated when GDNF was either depleted from EGC supernatants, or knocked down in EGCs or when the GDNF receptor RET was blocked. Further, TNFα-induced loss of IEB function in Caco2 cells and in organoids was attenuated by EGC supernatants or by recombinant GDNF. These barrier-protective effects were blunted when using supernatants from GDNF-deficient EGCs or by RET receptor blockade. Together, our data show that EGCs produce GDNF to maintain IEB function in vitro through the RET receptor.
Developmentally regulated features of innate immunity are thought to place preterm and term infants at risk of infection and inflammation-related morbidity. Underlying mechanisms are incompletely understood. Differences in monocyte function including toll-like receptor (TLR) expression and signaling have been discussed. Some studies point to generally impaired TLR signaling, others to differences in individual pathways. In the present study, we assessed mRNA and protein expression of pro- and anti-inflammatory cytokines in preterm and term cord blood (CB) monocytes compared with adult controls stimulated ex vivo with Pam3CSK4, zymosan, polyinosinic:polycytidylic acid, lipopolysaccharide, flagellin, and CpG oligonucleotide, which activate the TLR1/2, TLR2/6, TLR3, TLR4, TLR5, and TLR9 pathways, respectively. In parallel, frequencies of monocyte subsets, stimulus-driven TLR expression, and phosphorylation of TLR-associated signaling molecules were analyzed. Independent of stimulus, pro-inflammatory responses of term CB monocytes equaled adult controls. The same held true for preterm CB monocytes—except for lower IL-1β levels. In contrast, CB monocytes released lower amounts of anti-inflammatory IL-10 and IL-1ra, resulting in higher ratios of pro-inflammatory to anti-inflammatory cytokines. Phosphorylation of p65, p38, and ERK1/2 correlated with adult controls. However, stimulated CB samples stood out with higher frequencies of intermediate monocytes (CD14\(^+\)CD16\(^+\)). Both pro-inflammatory net effect and expansion of the intermediate subset were most pronounced upon stimulation with Pam3CSK4 (TLR1/2), zymosan (TR2/6), and lipopolysaccharide (TLR4). Our data demonstrate robust pro-inflammatory and yet attenuated anti-inflammatory responses in preterm and term CB monocytes, along with imbalanced cytokine ratios. Intermediate monocytes, a subset ascribed pro-inflammatory features, might participate in this inflammatory state.
Loss of intestinal epithelial barrier function is a hallmark in digestive tract inflammation. The detailed mechanisms remain unclear due to the lack of suitable cell-based models in barrier research. Here we performed a detailed functional characterization of human intestinal organoid cultures under different conditions with the aim to suggest an optimized ex-vivo model to further analyse inflammation-induced intestinal epithelial barrier dysfunction. Differentiated Caco2 cells as a traditional model for intestinal epithelial barrier research displayed mature barrier functions which were reduced after challenge with cytomix (TNFα, IFN-γ, IL-1ß) to mimic inflammatory conditions. Human intestinal organoids grown in culture medium were highly proliferative, displayed high levels of LGR5 with overall low rates of intercellular adhesion and immature barrier function resembling conditions usually found in intestinal crypts. WNT-depletion resulted in the differentiation of intestinal organoids with reduced LGR5 levels and upregulation of markers representing the presence of all cell types present along the crypt-villus axis. This was paralleled by barrier maturation with junctional proteins regularly distributed at the cell borders. Application of cytomix in immature human intestinal organoid cultures resulted in reduced barrier function that was accompanied with cell fragmentation, cell death and overall loss of junctional proteins, demonstrating a high susceptibility of the organoid culture to inflammatory stimuli. In differentiated organoid cultures, cytomix induced a hierarchical sequence of changes beginning with loss of cell adhesion, redistribution of junctional proteins from the cell border, protein degradation which was accompanied by loss of epithelial barrier function. Cell viability was observed to decrease with time but was preserved when initial barrier changes were evident. In summary, differentiated intestinal organoid cultures represent an optimized human ex-vivo model which allows a comprehensive reflection to the situation observed in patients with intestinal inflammation. Our data suggest a hierarchical sequence of inflammation-induced intestinal barrier dysfunction starting with loss of intercellular adhesion, followed by redistribution and loss of junctional proteins resulting in reduced barrier function with consecutive epithelial death.
The human gastrointestinal tract is in constant contact with microbial stimuli. Its barriers have to ensure co-existence with the commensal bacteria, while enabling surveillance of intruding pathogens. At the centre of the interaction lies the epithelial layer, which marks the boundaries of the body. It is equipped with a multitude of different innate immune sensors, such as Toll-like receptors, to mount inflammatory responses to microbes. Dysfunction of this intricate system results in inflammation-associated pathologies, such as inflammatory bowel disease. However, the complexity of the cellular interactions, their molecular basis and their development remains poorly understood. In recent years, stem cell-derived organoids have gained increasing attention as promising models for both development and a broad range of pathologies, including infectious diseases. In addition, organoids enable the study of epithelial innate immunity in vitro. In this review, we focus on the gastrointestinal epithelial barrier and its regional organization to discuss innate immune sensing and development.