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Descriptive histopathology of mouse models of pneumonia is essential in assessing the outcome of infections, molecular manipulations, or therapies in the context of whole lungs. Quantitative comparisons between experimental groups, however, have been limited to laborious stereology or ill-defined scoring systems that depend on the subjectivity of a more or less experienced observer. Here, we introduce self-learning digital image analyses that allow us to transform optical information from whole mouse lung sections into statistically testable data. A pattern-recognition-based software and a nuclear count algorithm were adopted to quantify user-defined pathologies from whole slide scans of lungs infected with Streptococcus pneumoniae or influenza A virus compared with PBS-challenged lungs. The readout parameters \"relative area affected\" and \"nuclear counts per area\" are proposed as relevant criteria for the quantification of lesions from hematoxylin and eosin-stained sections, also allowing for the generation of a heat map of, for example, immune cell infiltrates with anatomical assignments across entire lung sections. Moreover, when combined with immunohistochemical labeling of marker proteins, both approaches are useful for the identification and counting of, for example, immune cell populations, as validated here by direct comparisons with flow cytometry data. The solutions can easily and flexibly be adjusted to specificities of different models or pathogens. Automated digital analyses of whole mouse lung sections may set a new standard for the user-defined, high-throughput comparative quantification of histological and immunohistochemical images. Still, our algorithms established here are only a start, and need to be tested in additional studies and other applications in the future.

Background Ulcerative colitis (UC) is a chronic inflammatory bowel disease with significant morbidity and mortality. Although the precise cause remains unknown, disturbances in the intestinal microbial community have been linked to its pathogenesis. Randomized controlled trials in UC and relapsing Clostridioides difficile infection (CDI) have established fecal microbiota (FM) transfer (FMT) as an effective therapy. In this context, preliminary results indicated that the transfer of sterile fecal microbiota filtrates (<0.2 μm; FMF, FMFT) of donor stool also drives gastrointestinal microbiota changes and eliminates symptoms in CDI patients. However, along with the success of FMT, regulatory agencies issued safety alerts following reports of serious adverse events due to transmission of enteric pathogens through FMT. To reduce this risk, we established an extensive test protocol for our donors and quarantine regulations for the produced capsules, but alternative concepts are desirable. Methods Our project is a randomized, controlled, longitudinal, prospective, three-arm, multicenter, double-blind study to determine the safety and efficacy of repeated long-term, multi-donor FM or FMF transfers compared to placebo using oral, frozen capsules in 174 randomized patients with mild to moderate active UC. The primary outcome will be clinical remission at week 12. Discussion This proposal aims to examine (a) the efficacy of encapsulated transfer of FM and FMF as a therapy for mild to moderate UC, (b) the short- and long-term safety of FMT and FMFT in patients with UC, and (c) the microbial and immunologic changes that occur after FMT and FMFT to help understand how and why it affects inflammatory bowel disease. Trial registration ClinicalTrials.gov NCT03843385 . DRKS (Deutsches Register für Klinische Studien) DRKS00020471

Background and objective: Current guidelines recommend chest tube (CT) drainage as the initial treatment of secondary spontaneous pneumothorax (SSP). Surgery should be considered in cases of persistent air leak or recurrent disease. Video-assisted thoracoscopic surgery (VATS) is nowadays an established surgical treatment for complicated spontaneous pneumothorax. However, reports on VATS-bullectomy with partial pleurectomy (VBPP) for treatment of secondary spontaneous pneumothorax (SSP) are limited. The primary aim of this study was to evaluate and compare the clinical outcomes of patients with secondary pneumothorax treated either by VBPP or CT drainage in our institution. Secondly, we assessed underlying clinical parameters to identify potential risk factors for SSP recurrence. Materials and Methods: Eighty-two patients were included in this study. Long-term recurrence rates and potential risk factors for SSP recurrence were analyzed. Results: Thirty-six patients (43.9%) underwent VBPP, whereas 46 (56.1%) patients subsequently underwent CT treatment. During a median follow-up period of 76.5 months, VBPP patients experienced a significantly low recurrence rate compared to CT patients (VBPP vs. CT: 16.7% vs. 41.3%; p = 0.016). However, VBPP was associated with a higher complication rate and significantly longer length of hospital stay (LOS). Male sex (male vs. female: p = 0.021) and CT treatment (VBPP vs. CT: p < 0.001) were identified as potential risk factors for SSP recurrence. Conclusions: VBPP is a suitable surgical treatment for SSP. However, prolonged LOS and possible complications should be discussed prior to VBPP.

Background Two-port VATS (2-P-VATS) and three-port VATS (3-P-VATS) are well-established techniques for surgical therapy of primary spontaneous pneumothorax (PSP). However, comparisons of both techniques in terms of postoperative outcome and recurrence are limited. Methods From January 2010 to March 2020, we retrospectively reviewed data of 58 PSP patients who underwent VATS in our institution. For statistical analysis, categorical and continuous variables were compared by chi-square test or Fisher’s exact test and the Student´s t-test, respectively. Twenty-eight patients underwent 2-P-VATS and 30 were treated with 3-P-VATS. Operation time, length of hospital stay (LOS), total dose of analgesics per stay (opioids and non-opioids), duration of chest tube drainage, pleurectomy volume (PV), postoperative complications and recurrence rates were compared between both groups . Results Clinical and surgical characteristics including mean age, gender, Body-Mass-Index (BMI), pneumothorax size, smoking behaviour, history of contralateral pneumothorax, side of pneumothorax, pleurectomy volume and number of resected segments were similar in both groups. The mean operation time, LOS and total postoperative opioid and non-opioid dose was significantly higher in the 3-P-VATS group compared with the 2-P-VATS group. Despite not being statistically significant, duration of chest tube was longer in the 3-P-VATS group compared with the 2-P-VATS group. In terms of postoperative complications, the occurrence of hemothorax was significantly higher in the 3-P-VATS group (3-P-VATS vs. 2-P-VATS; p = 0.001). During a median follow-up period of 61.6 months, there was no significant statistical difference in recurrence rates in both groups (2/28 (16.7%) vs. 5/30 (7.1%); p = 0.274). Conclusion Our data demonstrate that 2-P-VATS is safer and effective. It is associated with reduced length of hospital stay and decreased postoperative pain resulting in less analgesic use.

Background: Video-assisted thoracoscopic surgery (VATS) with bullectomy and partial pleurectomy (VBPP) is an increasingly used and well-established surgical treatment for primary spontaneous pneumothorax (PSP). However, reports on its effectiveness and long-term outcomes are limited. The aim of this study was to assess and compare long-term recurrence rates following VBPP and chest tube (CT) treatment and to identify potential risk factors for disease recurrence in patients with PSP. Methods: A total of 116 patients treated either by VBPP or CT were included in this study. Long-term recurrence rates and associations between clinical parameters and recurrence of pneumothorax were analyzed. Results: Sixty-two patients (53.4%) underwent VBPP, whereas 54 (46.6%) patients underwent CT treatment only. During a median follow-up period of 76.5 months, VBPP patients experienced a significantly lower recurrence rate compared to CT patients (6/62 vs. 35/54; p < 0.0001). CT treatment (VBPP vs. CT; p < 0.001) and a large initial pneumothorax size (Collins < 4 vs. Collins ≥ 4; p = 0.018) were independent risk factors for pneumothorax recurrence. Conclusion: VBPP is an effective and safe surgical treatment for PSP. Therefore, patients with a large pneumothorax size might benefit from VBPP, as they are at high risk for disease recurrence.

Background: Two-port (2P) and three-port (3P) video-assisted thoracoscopic surgery (VATS) are well-established surgical methods for the treatment of complicated spontaneous pneumothorax (SP). However, a comparison between both techniques, in terms of clinical outcomes in patients with secondary spontaneous pneumothorax (SSP), is unreported. The aim of this study was to evaluate and compare postoperative pain, as well as clinical outcome, following 2P and 3P VATS for SSP in our institution. Methods: Between January 2008 and December 2020, we retrospectively analyzed the data of 115 SSP patients treated by VATS in our institution. Fifty-two patients underwent 2P-VATS, while 63 patients were treated by 3P-VATS. The total dose of analgesic use per stay (opioid and non-opioid), length of hospital stay (LOS), operation time, total area of pleurectomy, recurrence rates and postoperative complications were compared between both groups. Results: The 3P-VATS group had a significantly higher total dose of analgesic use compared with the 2P-VATS patients. The LOS and mean operation time were significantly shorter in the 2P-VATS group. A larger area of pleurectomy was resected using 3P-VATS compared to 2P-VATS. The postoperative complications and recurrence of SSP during a median follow-up period of 76.5 months were similar in both groups. Conclusion: 2P-VATS is a safe surgical technique. It is associated with a short LOS and less postoperative pain, and, thus, low analgesic use.

Background: Video-assisted thoracoscopic surgery (VATS) with partial pleurectomy is an established treatment for primary spontaneous pneumothorax (PSP). However, postoperative pulmonary function and health-related quality of life (HR-QoL) after VATS–bullectomy with partial pleurectomy (VBPP) have not been elucidated. Methods: Eligible patients were assessed for HR-QoL using the Short-Form 36 (SF-36) health survey. Pulmonary function (PF) was evaluated by spirometry. We compared the results of the VBPP cohort with the German national norms, and with a similar cohort of patients successfully treated by chest tube (CT) only. Results: A total of 25 VBPP patients completed the SF-36 health survey, of whom 15 presented for PF assessment. Between the VBPP and CT groups, the mean forced vital capacity (FVC), forced expiratory volume in one second (FEV1), and FEV1/FVC ratio were not statistically significantly different. However, in both groups, FVC, FEV1, and FEV1/FVC were above the lower limit of normal (LLN), suggesting no restrictive or obstructive patterns. Compared with the sex- and age-matched normal German population, patients who underwent VBPP displayed a similar physical component summary score and a significantly decreased mental component summary score. Interestingly, comparison of the SF-36 domains between the VBPP and CT groups showed no statistical difference. Conclusion: VBPP is a suitable surgical treatment for PSP, with no apparent adverse impacts on pulmonary or physical function. However, psychological distress and measures to counteract its impact should be considered.

ObjectiveBleeding ulcers and erosions are hallmarks of active ulcerative colitis (UC). However, the mechanisms controlling bleeding and mucosal haemostasis remain elusive.DesignWe used high-resolution endoscopy and colon tissue samples of active UC (n = 36) as well as experimental models of physical and chemical mucosal damage in mice deficient for peptidyl-arginine deiminase-4 (PAD4), gnotobiotic mice and controls. We employed endoscopy, histochemistry, live-cell microscopy and flow cytometry to study eroded mucosal surfaces during mucosal haemostasis.ResultsErosions and ulcerations in UC were covered by fresh blood, haematin or fibrin visible by endoscopy. Fibrin layers rather than fresh blood or haematin on erosions were inversely correlated with rectal bleeding in UC. Fibrin layers contained ample amounts of neutrophils coaggregated with neutrophil extracellular traps (NETs) with detectable activity of PAD. Transcriptome analyses showed significantly elevated PAD4 expression in active UC. In experimentally inflicted wounds, we found that neutrophils underwent NET formation in a PAD4-dependent manner hours after formation of primary blood clots, and remodelled clots to immunothrombi containing citrullinated histones, even in the absence of microbiota. PAD4-deficient mice experienced an exacerbated course of dextrane sodium sulfate-induced colitis with markedly increased rectal bleeding (96 % vs 10 %) as compared with controls. PAD4-deficient mice failed to remodel blood clots on mucosal wounds eliciting impaired healing. Thus, NET-associated immunothrombi are protective in acute colitis, while insufficient immunothrombosis is associated with rectal bleeding.ConclusionOur findings uncover that neutrophils induce secondary immunothrombosis by PAD4-dependent mechanisms. Insufficient immunothrombosis may favour rectal bleeding in UC.

Integrin-containing focal adhesions transmit extracellular signals across the plasma membrane to modulate cell adhesion, signalling and survival. Although integrins are known to undergo continuous endo/exocytic traffic, the potential impact of endocytic traffic on integrin-induced signals is unknown. Here, we demonstrate that integrin signalling is not restricted to cell–ECM adhesions and identify an endosomal signalling platform that supports integrin signalling away from the plasma membrane. We show that active focal adhesion kinase (FAK), an established marker of integrin–ECM downstream signalling, localizes with active integrins on endosomes. Integrin endocytosis positively regulates adhesion-induced FAK activation, which is early endosome antigen-1 and small GTPase Rab21 dependent. FAK binds directly to purified endosomes and becomes activated on them, suggesting a role for endocytosis in enhancing distinct integrin downstream signalling events. Finally, endosomal integrin signalling contributes to cancer-related processes such as anoikis resistance, anchorage independence and metastasis. Ivaska and colleagues report that endocytosed integrins are able to signal from endosomes in an FAK-dependent manner. They further show that endosomal integrin signalling can promote anoikis resistance and lung colonization in cancer cells.

Background Matrix metalloproteinase 12 (MMP12) is a macrophage-secreted protein that is massively upregulated as a pro-inflammatory factor in metabolic and vascular tissues of mice and humans suffering from cardiometabolic diseases (CMDs). However, the molecular mechanisms explaining the contributions of MMP12 to CMDs are still unclear. Methods We investigated the impact of MMP12 deficiency on CMDs in a mouse model that mimics human disease by simultaneously developing adipose tissue inflammation, insulin resistance, and atherosclerosis. To this end, we generated and characterized low-density lipoprotein receptor (Ldlr)/Mmp12-double knockout (DKO) mice fed a high-fat sucrose- and cholesterol-enriched diet for 16–20 weeks. Results DKO mice showed lower cholesterol and plasma glucose concentrations and improved insulin sensitivity compared with LdlrKO mice. Untargeted proteomic analyses of epididymal white adipose tissue revealed that inflammation- and fibrosis-related pathways were downregulated in DKO mice. In addition, genetic deletion of MMP12 led to alterations in immune cell composition and a reduction in plasma monocyte chemoattractant protein-1 in peripheral blood which indicated decreased low-grade systemic inflammation. Aortic en face analyses and staining of aortic valve sections demonstrated reduced atherosclerotic plaque size and collagen content, which was paralleled by an improved relaxation pattern and endothelial function of the aortic rings and more elastic aortic sections in DKO compared to LdlrKO mice. Shotgun proteomics revealed upregulation of anti-inflammatory and atheroprotective markers in the aortas of DKO mice, further supporting our data. In humans, MMP12 serum concentrations were only weakly associated with clinical and laboratory indicators of CMDs. Conclusion We conclude that the genetic deletion of MMP12 ameliorates obesity-induced low-grade inflammation, white adipose tissue dysfunction, biomechanical properties of the aorta, and the development of atherosclerosis. Therefore, therapeutic strategies targeting MMP12 may represent a promising approach to combat CMDs.