Endogenous and exogenous modulation of 5-lipoxygenase: impact of pregnancy, menstrual cycle and pharmacological inhibitors

This thesis comprises two parts both dealing with the modulation of 5-lipoxygenase (5-LO), the enzyme responsible for the synthesis of pro-inflammatory leukotrienes (LT). First, the impact of pregnancy was studied. LT formation was higher in blood from pregnant compared to non-pregnant females. This higher LT synthesis in blood of pregnant females is influenced by synergistic and opposite effects: (I) higher numbers of LT forming cells (“plus”), (II) lower LT formation capacity of isolated granulocytes (“minus”) and (III) upregulating effects of plasma from pregnant donors on LT formation from isolated enzyme and cells (“plus”). These results suggest that LTs might be involved in the immune regulation during pregnancy. In the second part dealing with benzoquinones as exogenous modulators of 5-LO new inhibitors were identified and their molecular mode of inhibition characterized. The natural compound embelin acted as potent inhibitor of 5-LO and microsomal prostaglandin E2 synthase-1 (IC50 = 0.06 and 0.2 µM). New targets of embelin are presented for which lower concentrations are needed for inhibition than for others described before. Besides embelin, the benzoquinone RF-Id was studied in cell-free, cellular and blood assays for its interference with LT synthesis and identified as potent 5-LO inhibitor. Mechanistic studies showed that embelin and RF-Id, though similarly structured, interfered with 5-LO in different modes. Interestingly, both inhibitors do not inhibit via redox-type 5-LO inhibition as often supposed for benzoquinones in literature. After being activated by reduction in the cell RF-Id interacts with 5-LO in a nonredox-type fashion. Structure activity relationships of 31 embelin derived benzoquinones all modified in backbone position 3 with alkyl or prenyl chains revealed distinct features for potent inhibition of 5-LO. The most potent compounds were ortho-quinones (IC50 = 0.03 to 0.06 µM) which are valuable for future pharmacological studies

Risk Factors Associated With Pouch Adenomas in Patients With Familial Adenomatous Polyposis

BACKGROUND: Restorative proctocolectomy with ileal pouch-anal reconstruction is the standard prophylactic surgical procedure for patients with familial adenomatous polyposis. However, several groups have reported the development of adenomas and even carcinomas within the ileal pouch. The predisposing factor was the time interval after pouch surgery in some studies, but it was the severity of the initial colonic disease and duodenal adenomatosis in others. OBJECTIVE: The aim of this study was to further clarify the prevalence of pouch adenomas, clinical risk factors, and a possible phenotype-genotype relation in a large population of patients with familial adenomatous polyposis, as well as to analyze pouch adenoma-free survival. DESIGN: This study was designed as a cohort study. SETTINGS: This study was conducted in a specialized outpatient clinic at the University of Heidelberg. PATIENTS: A total of 192 patients with familial adenomatous polyposis were included, and all of the available endoscopy reports after pouch surgery were screened for pouch adenomas. Additional clinical information was retrieved from the Heidelberg Polyposis Register. MAIN OUTCOME MEASURES: This present study revealed 3 main independent risk factors for the development of pouch adenomas: age <18 years at the time of IPAA, male sex, and the presence of gastric adenomas. Secondary outcome measures were adenoma progression and overall pouch adenoma-free survival. RESULTS: Pouch adenomas were detected in 46.9% of patients. Median follow-up was 12.8 years (interquartile range, 9.0-17.0 y) for patients with pouch adenomas and 7.3 years (interquartile range, 2.5-12.2 y) for those without them. Patients underwent pouch surgery at a median age of 27.5 years (range, 10.2-58.5 y), and pouch adenomas occurred a median of 8.5 years (range, 0.9-25.1 y) after surgery. Also detected were gastric adenomas in 37.2%, duodenal adenomas in 80.3%, and desmoid tumors in 24.5% of patients. Estimation of pouch adenoma-free survival revealed that, after 20 years, only approximate to 22% of patients would be free of pouch adenomas. Male sex, age 18 years at the time of pouch surgery, and gastric adenomas were found to be independent risk factors for the development of pouch adenomas in a multivariate Cox regression analysis (p = 0.0002, p = 0.0059, and p = 0.0020). No predisposing germline mutation for pouch adenoma development was detected. LIMITATIONS: Detailed information on the initial preoperative findings was not fully available, and the study was only carried out as a single-center study. CONCLUSIONS: A severe upper intestinal phenotype, male sex, and age <18 years at the time of IPAA all increase the risk for development of pouch adenomas. See Video Abstract at http://links.lww.com/DCR/A675

Laparoscopic transgastric circumferential stapler-assisted vs. endoscopic esophageal mucosectomy in a porcine model

Background and study aims Extensive endoscopic mucosal resection (EMR) for Barrett's esophagus (BE) may lead to stenosis. Laparoscopic, transgastric, stapler-assisted mucosectomy (SAM) with the retrieval of a circumferential specimen is proposed. Methods SAM was evaluated in two phases. The feasibility of SAM and the quality of specimens were assessed in eight animals. The mucosal healing was evaluated in a 6-week survival experiment comparing SAM (n = 6) with EMR (n = 6). The ratio of the esophageal lumen width (REL) at the resection level measured on fluoroscopy at 6 weeks divided by the width immediately after resection was compared. Results In all animals, a circular mucosectomy specimen was successfully obtained, with a median area of 492 mm2 (interquartile range [IQR] 426 - 573 mm2) and 941 mm2 (IQR 813 - 1209 mm2) using a 21 mm and 25 mm stapler, respectively. In the survival experiments, symptomatic stenosis developed in two animals after EMR and in none after SAM. The REL was 0.27 (0.18 - 0.39) and 0.96 (0.9 - 1.04; P < 0.0001) for EMR and SAM, respectively. Conclusions SAM provides a novel technique for en bloc mucosectomy in BE. In contrast to EMR, mucosal healing after SAM was not associated with stenosis up to 6 weeks after intervention

Immediate tumor resection in patients with locally advanced gastroesophageal adenocarcinoma with nonresponse to chemotherapy after 4 weeks of treatment versus resection after completion of chemotherapy (OPTITREAT trial, DRKS00004668): study protocol for a randomized controlled pilot trial

Background: Neoadjuvant chemotherapy is a standard of care for patients with adenocarcinoma of the esophagus and stomach in Europe, but still only 20–40 % respond to therapy and the critical issue; how to treat nonresponding patients is still unclear. So far, there is no randomized trial evaluating the impact of early termination of neoadjuvant chemotherapy and immediate tumor resection in nonresponding patients with locally advanced gastroesophageal cancer on postoperative outcome. With this exploratory pilot trial, we want to get first estimates about the effect of discontinuation of chemotherapy with the aim to plan and conduct a further definitive trial. Methods/design: OPTITREAT is designed as a single-center, randomized controlled pilot trial with two parallel study groups. Four weeks after starting neoadjuvant chemotherapy in all patients, clinical response will be assessed by endoscopy and endosonographic ultrasound. Then, nonresponding patients (n = 84) will be randomized in a 1:1 ratio to intervention group with stopping chemotherapy and immediate tumor resection or control group with completion of chemotherapy before surgery. Outcome measures are overall survival, R0 resection rate, perioperative morbidity and mortality, histopathological response, and quality of life. Statistical analysis will be based on the intention-to-treat population. Due to the study design as an explorative pilot trial, no formal sample size calculation was performed. The planned total sample size of 120 patients is considered ethical and large enough to show the feasibility and safety of the concept. First data on differences between the study groups in the defined endpoints will also be generated. Discussion: Individualized therapy is of utmost interest in the treatment of locally advanced gastroesophageal adenocarcinoma as less than half of the patients show objective response to current chemotherapy regimens. The findings of the OPTITREAT trial will help to get first data about clinical response evaluation followed by immediate tumor resection in nonresponding patients after 4 weeks of neoadjuvant chemotherapy. Based on the results of this pilot study, a future confirmatory trial will be planned to prove efficacy and evaluate significance. Trial registration: German Clinical Trial Register number: DRKS0000466

Acupuncture to improve tolerance of diagnostic esophagogastroduodenoscopy in patients without systemic sedation: results of a single-center, double-blinded, randomized controlled trial (DRKS00000164)

Background: Sedation prior to esophagogastroduodenoscopy is widespread and increases patient comfort. However, it demands additional trained personnel, accounts for up to 40% of total endoscopy costs and impedes rapid hospital discharge. Most patients lose at least one day of work. 98% of all serious adverse events occurring during esophagogastroduodenoscopy are ascribed to sedation. Acupuncture is reported to be effective as a supportive intervention for gastrointestinal endoscopy, similar to conventional premedication. We investigated whether acupuncture during elective diagnostic esophagogastroduodenoscopy could increase the comfort of patients refusing systemic sedation. Methods: We performed a single-center, double-blinded, placebo-controlled superiority trial to compare the success rates of elective diagnostic esophagogastroduodenoscopies using real and placebo acupuncture. All patients aged 18 years or older scheduled for elective, diagnostic esophagogastroduodenoscopy who refused systemic sedation were eligible; 354 patients were randomized. The primary endpoint measure was the rate of successful esophagogastroduodenoscopies. The intervention was real or placebo acupuncture before and during esophagogastroduodenoscopy. Successful esophagogastroduodenoscopy was based on a composite score of patient satisfaction with the procedure on a Likert scale as well as quality of examination, as assessed by the examiner. Results: From February 2010 to July 2012, 678 patients were screened; 354 were included in the study. Baseline characteristics of the two groups showed a similar distribution in all but one parameter: more current smokers were allocated to the placebo group. The intention-to-treat analysis included 177 randomized patients in each group. Endoscopy could successfully be performed in 130 patients (73.5%) in the real acupuncture group and 129 patients (72.9%) in the placebo group. Willingness to repeat the procedure under the same conditions was 86.9% in the real acupuncture group and 87.6% in the placebo acupuncture group. Conclusions: Esophagogastroduodenoscopy without sedation is safe and can successfully be performed in two-thirds of patients. Patients planned for elective esophagogastroduodenoscopy without sedation do not benefit from acupuncture of the Sinarteria respondens (Rs) 24 Chengjiang middle line, Pericard (Pc) 6 Neiguan bilateral, or Dickdarm (IC) 4 Hegu bilateral, according to traditional Chinese medicine meridian theory. Trial registration DRKS00000164. Registered on 10 December 2009

Phenotypic and transcriptomic analyses of seven clinical Stenotrophomonas maltophilia isolates identify a small set of shared and commonly regulated genes involved in the biofilm lifestyle

Stenotrophomonas maltophilia is one of the most frequently isolated multidrug-resistant nosocomial opportunistic pathogens. It contributes to disease progression in cystic fibrosis (CF) patients and is frequently isolated from wounds, infected tissues, and catheter surfaces. On these diverse surfaces S. maltophilia lives in single-species or multispecies biofilms. Since very little is known about common processes in biofilms of different S. maltophilia isolates, we analyzed the biofilm profiles of 300 clinical and environmental isolates from Europe of the recently identified main lineages Sgn3, Sgn4, and Sm2 to Sm18. The analysis of the biofilm architecture of 40 clinical isolates revealed the presence of multicellular structures and high phenotypic variability at a strain-specific level. Further, transcriptome analyses of biofilm cells of seven clinical isolates identified a set of 106 shared strongly expressed genes and 33 strain-specifically expressed genes. Surprisingly, the transcriptome profiles of biofilm versus planktonic cells revealed that just 9.43% ± 1.36% of all genes were differentially regulated. This implies that just a small set of shared and commonly regulated genes is involved in the biofilm lifestyle. Strikingly, iron uptake appears to be a key factor involved in this metabolic shift. Further, metabolic analyses implied that S. maltophilia employs a mostly fermentative growth mode under biofilm conditions. The transcriptome data of this study together with the phenotypic and metabolic analyses represent so far the largest data set on S. maltophilia biofilm versus planktonic cells. This study will lay the foundation for the identification of strategies for fighting S. maltophilia biofilms in clinical and industrial settings. IMPORTANCE Microorganisms living in a biofilm are much more tolerant to antibiotics and antimicrobial substances than planktonic cells are. Thus, the treatment of infections caused by microorganisms living in biofilms is extremely difficult. Nosocomial infections (among others) caused by S. maltophilia, particularly lung infection among CF patients, have increased in prevalence in recent years. The intrinsic multidrug resistance of S. maltophilia and the increased tolerance to antimicrobial agents of its biofilm cells make the treatment of S. maltophilia infection difficult. The significance of our research is based on understanding the common mechanisms involved in biofilm formation of different S. maltophilia isolates, understanding the diversity of biofilm architectures among strains of this species, and identifying the differently regulated processes in biofilm versus planktonic cells. These results will lay the foundation for the treatment of S. maltophilia biofilms

Longevity and Composition of Cellular Immune Responses Following Experimental Plasmodium falciparum Malaria Infection in Humans

Cellular responses to Plasmodium falciparum parasites, in particular interferon-gamma (IFNγ) production, play an important role in anti-malarial immunity. However, clinical immunity to malaria develops slowly amongst naturally exposed populations, the dynamics of cellular responses in relation to exposure are difficult to study and data about the persistence of such responses are controversial. Here we assess the longevity and composition of cellular immune responses following experimental malaria infection in human volunteers. We conducted a longitudinal study of cellular immunological responses to sporozoites (PfSpz) and asexual blood-stage (PfRBC) malaria parasites in naïve human volunteers undergoing single (n = 5) or multiple (n = 10) experimental P. falciparum infections under highly controlled conditions. IFNγ and interleukin-2 (IL-2) responses following in vitro re-stimulation were measured by flow-cytometry prior to, during and more than one year post infection. We show that cellular responses to both PfSpz and PfRBC are induced and remain almost undiminished up to 14 months after even a single malaria episode. Remarkably, not only ‘adaptive’ but also ‘innate’ lymphocyte subsets contribute to the increased IFNγ response, including αβT cells, γδT cells and NK cells. Furthermore, results from depletion and autologous recombination experiments of lymphocyte subsets suggest that immunological memory for PfRBC is carried within both the αβT cells and γδT compartments. Indeed, the majority of cytokine producing T lymphocytes express an CD45RO+ CD62L- effector memory (EM) phenotype both early and late post infection. Finally, we demonstrate that malaria infection induces and maintains polyfunctional (IFNγ+IL-2+) EM responses against both PfRBC and PfSpz, previously found to be associated with protection. These data demonstrate that cellular responses can be readily induced and are long-lived following infection with P. falciparum, with a persisting contribution by not only adaptive but also (semi-)innate lymphocyte subsets. The implications hereof are positive for malaria vaccine development, but focus attention on those factors potentially inhibiting such responses in the field

The trispecific DARPin ensovibep inhibits diverse SARS-CoV-2 variants

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19)