Tilivalline- and Tilimycin-Independent Effects of Klebsiella oxytoca on Tight Junction-Mediated Intestinal Barrier Impairment

Klebsiella oxytoca causes antibiotic-associated hemorrhagic colitis and diarrhea. This was attributed largely to its secreted cytotoxins tilivalline and tilimycin, inductors of epithelial apoptosis. To study whether Klebsiella oxytoca exerts further barrier effects, T84 monolayers were challenged with bacterial supernatants derived from tilivalline/tilimycin-producing AHC6 or its isogeneic tilivalline/tilimycin-deficient strain Mut-89. Both preparations decreased transepithelial resistance, enhanced fluorescein and FITC-dextran-4kDa permeabilities, and reduced expression of barrier-forming tight junction proteins claudin-5 and -8. Laser scanning microscopy indicated redistribution of both claudins off the tight junction region in T84 monolayers as well as in colon crypts of mice infected with AHC6 or Mut-89, indicating that these effects are tilivalline/tilimycin-independent. Furthermore, claudin-1 was affected, but only in a tilivalline/tilimycin-dependent manner. In conclusion, Klebsiella oxytoca induced intestinal barrier impairment by two mechanisms: the tilivalline/tilimycin-dependent one, acting by increasing cellular apoptosis and a tilivalline/tilimycin-independent one, acting by weakening the paracellular pathway through the tight junction proteins claudin-5 and -8

Successful Use of Interferon-Gamma for Refractory Disseminated Candida Tropicalis Infection in a Pediatric Patient with Acute Myeloid Leukemia and Heterozygous CARD9 Mutation

Invasive fungal infections remain among the most frequent severe and fatal complications of oncology, despite effective antifungal agents. Genetic aberrations may additionally increase the individual patient’s susceptibility to invasive mycosis, with the need for an intensified treatment, such as adjunctive immunotherapy combined with standard antifungal medication. Interferon-gamma (IFN-γ) as an immunostimulatory agent may represent a promising surrogate to reactivate the cytotoxic activity of macrophages in patients with ‘blind spots’. We report the case of a pediatric patient with AML and disseminated candida tropicalis infection, bearing a heterozygous CARD9 mutation, who was treated successfully with combined IFN-γ and conventional antifungal therapy

Campylobacter concisus Impairs Sodium Absorption in Colonic Epithelium via ENaC Dysfunction and Claudin-8 Disruption

The epithelial sodium channel (ENaC) can increase the colonic absorptive capacity for salt and water. Campylobacter concisus is a common pathogenic epsilonproteobacterium, causing enteritis and diarrhea. It can induce barrier dysfunction in the intestine, but its influence on intestinal transport function is still unknown. Therefore, our study aimed to characterize C. concisus effects on ENaC using the HT-29/B6-GR/MR (epithelial cell line HT-29/B6 transfected with glucocorticoid and mineralocorticoid receptors) cell model and mouse colon. In Ussing chambers, C. concisus infection inhibited ENaC-dependent Na+ transport as indicated by a reduction in amiloride-sensitive short circuit current (-55%, n = 15, p < 0.001). This occurred via down-regulation of β- and γ-ENaC mRNA expression and ENaC ubiquitination due to extracellular signal-regulated kinase (ERK)1/2 activation, predicted by Ingenuity Pathway Analysis (IPA). In parallel, C. concisus reduced the expression of the sealing tight junction (TJ) protein claudin-8 and induced claudin-8 redistribution off the TJ domain of the enterocytes, which facilitates the back leakage of Na+ ions into the intestinal lumen. In conclusion, C. concisus caused ENaC dysfunction via interleukin-32-regulated ERK1/2, as well as claudin-8-dependent barrier dysfunction-both of which contribute to Na+ malabsorption and diarrhea

Roles for ADAM17 in TNF-R1 Mediated Cell Death and Survival in Human U937 and Jurkat Cells

Signaling via death receptor family members such as TNF-R1 mediates pleiotropic biological outcomes ranging from inflammation and proliferation to cell death. Pro-survival signaling is mediated via TNF-R1 complex I at the cellular plasma membrane. Cell death induction requires complex IIa/b or necrosome formation, which occurs in the cytoplasm. In many cell types, full apoptotic or necroptotic cell death induction requires the internalization of TNF-R1 and receptosome formation to properly relay the signal inside the cell. We interrogated the role of the enzyme A disintegrin and metalloprotease 17 (ADAM17)/TACE (TNF-α converting enzyme) in death receptor signaling in human hematopoietic cells, using pharmacological inhibition and genetic ablation. We show that in U937 and Jurkat cells the absence of ADAM17 does not abrogate, but rather increases TNF mediated cell death. Likewise, cell death triggered via DR3 is enhanced in U937 cells lacking ADAM17. We identified ADAM17 as the key molecule that fine-tunes death receptor signaling. A better understanding of cell fate decisions made via the receptors of the TNF-R1 superfamily may enable us, in the future, to more efficiently treat infectious and inflammatory diseases or cancer

Critical evaluation of molecular tumour board outcomes following 2 years of clinical practice in a Comprehensive Cancer Centre

Recently, molecular tumour boards (MTBs) have been integrated into the clinical routine. Since their benefit remains debated, we assessed MTB outcomes in the Comprehensive Cancer Center Ostbayern (CCCO) from 2019 to 2021. Methods and results In total, 251 patients were included. Targeted sequencing was performed with PCR MSI-evaluation and immunohistochemistry for PD-L1, Her2, and mismatch repair enzymes. 125 treatment recommendations were given (49.8%). High-recommendation rates were achieved for intrahepatic cholangiocarcinoma (20/30, 66.7%) and gastric adenocarcinoma (10/16, 62.5%) as opposed to colorectal cancer (9/36, 25.0%) and pancreatic cancer (3/18, 16.7%). MTB therapies were administered in 47 (18.7%) patients, while 53 (21.1%) received alternative treatment regimens. Thus 37.6% of recommended MTB therapies were implemented (47/125 recommendations). The clinical benefit rate (complete + partial + mixed response + stable disease) was 50.0% for MTB and 63.8% for alternative treatments. PFS2/1 ratios were 34.6% and 16.1%, respectively. Significantly improved PFS could be achieved for m1A-tier-evidence-based MTB therapies (median 6.30 months) compared to alternative treatments (median 2.83 months; P = 0.0278). Conclusion The CCCO MTB yielded a considerable recommendation rate, particularly in cholangiocarcinoma patients. The discrepancy between the low-recommendation rates in colorectal and pancreatic cancer suggests the necessity of a weighted prioritisation of entities. High-tier recommendations should be implemented predominantly

Granulocyte transfusions made with modified fluid gelatin in pediatric and adolescent patients with prolonged neutropenia

Background Granulocyte transfusions (GT) are used to treat progressive systemic or local infections in prolonged neutropenic patients with antibiotic or antifungal resistance. Granulocytes are most commonly collected from whole blood by apheresis using hydroxyethyl starch (HES) as the red blood cell (RBC) sedimentation agent. This is the first study on the safety and efficacy of transfusing granulocytes collected with modified fluid gelatin (MFG) instead of HES to pediatric patients. Methods Clinical data from 46 pediatric and adolescent patients receiving at least one MFG-based granulocyte transfusion and in total 295 granulocyte concentrates from July 2013 to August 2019 at our local university medical center were evaluated retrospectively. Results Forty-one patients (89%) survived at least 21 days after their last granulocyte transfusion. These survivors had lower CRP values and higher leukocyte counts after GT than non-survivors (mean delta of −5.34 mg/dl vs. –11.99 mg/dl and + 0.62 × 103/μl vs. +0.18 × 103/μl of all GT, respectively). The neutrophil corrected count increment (CCI) was 68.72 mm2/ml in survivors versus 28.00 mm2/ml in non-survivors. There were no major or severe adverse events. Conclusion This study suggests that modified fluid gelatin is a safe and effective alternative to hydroxyethyl starch for the collection of granulocytes for transfusion to prolonged neutropenic patients with progressive systemic or local infections refractory to antibiotic or antifungal therapy