Pseudomonas aeruginosa and Its Bacterial Components Influence the Cytokine Response in Thymocytes and Splenocytes.

Infections with Pseudomonas aeruginosa may cause many different diseases. The spectrum of such infections in general includes inflammation and bacterial sepsis. Hospital-acquired pneumonia, naturally resistant to a wide range of antibiotics, is associated with a particularly high mortality rate in mechanically ventilated patients. The pathogenesis of P. aeruginosa is complex and mediated by several virulence factors, as well as cell-associated factors. We have previously demonstrated that stimulation with different bacteria triggers the cytokine response of thymocytes. In this study, we investigated the effect of P. aeruginosa and its different components on the cytokine production of immature and mature immune cells. We found that the induced cytokine pattern in the thymus and the spleen after infections with P. aeruginosa is primarily mediated by lipopolysaccharide (LPS) of the outer cell membrane, but other components of the bacterium can influence the cytokine secretion as well. Stimulation with heat-killed P. aeruginosa and LPS does not influence the amount of cytokine-producing CD4(+) T cells but instead suppresses the emergence of Th17 cells. However, stimulation with P. aeruginosa or its components triggers the interleukin-17 (IL-17) response both in thymocytes and in splenocytes. We conclude that infections with P. aeruginosa affect the cytokine secretion of immature and mature cells and that IL-17 and Th17 cells play only a minor role in the development of pathological systemic inflammatory disease conditions during P. aeruginosa infections. Therefore, other inflammatory immune responses must be responsible for septic reactions of the host.

Application of Deep Learning for Real-Time Ablation Zone Measurement in Ultrasound Imaging.

BACKGROUND: The accurate delineation of ablation zones (AZs) is crucial for assessing radiofrequency ablation (RFA) therapy's efficacy. Manual measurement, the current standard, is subject to variability and potential inaccuracies. AIM: This study aims to assess the effectiveness of Artificial Intelligence (AI) in automating AZ measurements in ultrasound images and compare its accuracy with manual measurements in ultrasound images. METHODS: An in vitro study was conducted using chicken breast and liver samples subjected to bipolar RFA. Ultrasound images were captured every 15 s, with the AI model Mask2Former trained for AZ segmentation. The measurements were compared across all methods, focusing on short-axis (SA) metrics. RESULTS: We performed 308 RFA procedures, generating 7275 ultrasound images across liver and chicken breast tissues. Manual and AI measurement comparisons for ablation zone diameters revealed no significant differences, with correlation coefficients exceeding 0.96 in both tissues (p < 0.001). Bland-Altman plots and a Deming regression analysis demonstrated a very close alignment between AI predictions and manual measurements, with the average difference between the two methods being -0.259 and -0.243 mm, for bovine liver and chicken breast tissue, respectively. CONCLUSION: The study validates the Mask2Former model as a promising tool for automating AZ measurement in RFA research, offering a significant step towards reducing manual measurement variability.

Bacterial flagellin and diphtheria toxin co-stimulate IL-17-producing thymocytes.

IL-17-producing thymocytes have been recently described and are believed to play a role as an immune cell population which is able to react against microbial components rapidly. For this reason, we here investigated the ability of two microbial stimulants, bacterial flagellin (a ligand for TLR5) and diphtheria toxin from Corynebacterium diphtheriae, to activate or co-activate (together with α-CD3 stimulation) thymocyte cytokine production. We find that both bacterial molecules do not induce cytokine-production by themselves, but co-activate IL-17-producing thymocytes together with α-CD3. Since diphtheria toxin is unlikely to affect mouse cells through the same mechanism as the lethal effect on human cells, our results point to an additional mechanism of diphtheria toxin to act on immune cells. However, there is no additive or super-additive effect after stimulation with diphtheria toxin combined with flagellin and α-CD3 co-activation, which suggests that microbial stimuli used in this study can only activate a limited number of IL-17 producing thymocytes.

Bile duct tissue acquisition by cholangioscopy-guided cryobiopsy technique: first-in-human application.

Video 1Case description and video in cholangioscopic view demonstrating bile duct tissue acquisition by cholangioscopy-guided cryobiopsy technique.

Effects of dimethyl fumarate on neuroprotection and immunomodulation.

BACKGROUND: Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate is a promising novel oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. These effects are presumed to originate from a combination of immunomodulatory and neuroprotective mechanisms. We aimed to clarify whether neuroprotective concentrations of dimethyl fumarate have immunomodulatory effects. FINDINGS: We determined time- and concentration-dependent effects of dimethyl fumarate and its metabolite monomethyl fumarate on viability in a model of endogenous neuronal oxidative stress and clarified the mechanism of action by quantitating cellular glutathione content and recycling, nuclear translocation of transcription factors, and the expression of antioxidant genes. We compared this with changes in the cytokine profiles released by stimulated splenocytes measured by ELISPOT technology and analyzed the interactions between neuronal and immune cells and neuronal function and viability in cell death assays and multi-electrode arrays. Our observations show that dimethyl fumarate causes short-lived oxidative stress, which leads to increased levels and nuclear localization of the transcription factor nuclear factor erythroid 2-related factor 2 and a subsequent increase in glutathione synthesis and recycling in neuronal cells. Concentrations that were cytoprotective in neuronal cells had no negative effects on viability of splenocytes but suppressed the production of proinflammatory cytokines in cultures from C57BL/6 and SJL mice and had no effects on neuronal activity in multi-electrode arrays. CONCLUSIONS: These results suggest that immunomodulatory concentrations of dimethyl fumarate can reduce oxidative stress without altering neuronal network activity.

Characterization of porcine mesenchymal stromal cells and their proliferative and osteogenic potential in long-term culture.

Background: Porcine mesenchymal stromal cells (pMSCs) are considered a valuable research model for bone tissue engineering, which requires adequate amounts of viable cells with sufficient potential for osteogenic differentiation. For isolation and expansion of these cells through long-term culture, appropriate culture conditions are needed. Objective: To study the effect of extended in vitro cultivation on pMSC proliferation and differentiation potential using different osteogenic and adipogenic induction media. Methods: pMSCs were isolated from the bone marrow of adult Göttingen minipigs, cultured, expanded to passage 20 (~160 days) and characterized by their expression of cell surface markers (wCD44, CD45, CD90, SWC9, fibronectin), alkaline phosphatase (ALP), and osteocalcin and their potential for osteogenic and adipogenic differentiation using different induction media. Results: pMSCs retained their capacity for proliferation and osteogenic differentiation, and the number of CD90-positive cells increased significantly over more than 60 population doublings. CD90 expression in uninduced cells correlated strongly with ALP expression following osteogenic induction. Medium enriched with calcium yielded a stronger osteogenic response. Conclusion: The selection of CD90-positive MSCs and adequate levels of calcium seem to enhance the osteogenic phenotype for bone tissue engineering.

The role of electrochemotherapy with intratumoral bleomycin for early tongue carcinoma.

BACKGROUND: The role of electrochemotherapy (ECT) using intratumoral bleomycin and electroporation as a first line treatment for oral tongue carcinoma has not been defined. AIMS/OBJECTIVES: To evaluate the method of ECT in oral tongue carcinoma. MATERIAL AND METHODS: Twenty-one successive patients with primary T1-T2 oral cancer predominantly of the oral tongue underwent either ECT (test; n = 9), or standard surgical resection and reconstruction (control; n = 12). Outcome variables were: local recurrence rates, 10-year-survival, adverse events, treatment cost, and quality of life. RESULTS: The 10-year local recurrence rate (44.4%) was higher and the tumour-specific survival rate (55.6%) was lower in the ECT group compared to the control group (17% and 91.6%, respectively). Postoperative haemorrhage, dysphagia, and pain were more frequent in ECT patients, treatment time was shorter, but treatment cost was higher. Quality of life was not improved by ECT. CONCLUSIONS AND SIGNIFICANCE: Our results indicate that ECT seems not as suitable for the treatment of early tongue cancer as it is for neoplastic and metastatic skin lesions and less favourable than standard surgical therapy.

Single-stage sinus augmentation with cancellous iliac bone and anorganic bovine bone in the presence of platelet-rich plasma in the miniature pig.

AIM: The aim of the present study was to evaluate the osseointegration of dental implants and bone formation in maxillary sinus grafting with autologous and anorganic bovine bone in the presence of platelet-rich plasma (PRP) in an established animal model. MATERIAL AND METHODS: We performed bilateral maxillary sinus augmentation with 50% anorganic bovine bone and simultaneously inserted a titanium screw implant in five minature pigs. Six hundred microlitre autologous PRP were added to the left side (test). The right side (no PRP) served as control. Polychrome sequential labeling was performed. The animals were sacrificed 6 weeks after surgery. Undecalcified ground sections were evaluated by microradiography, digitized histomorphometry and under fluorescent light. RESULTS: The mean bone implant content in the test and control group was 8.4% and 17.3% respectively (P=0.042). The mean height of newly formed mineralized bone in the augmented area of the test group was 3.6 mm and 5.7 mm respectively (P=0.342). In the PRP group, the mean area of newly formed bone in the base of the sinus was enhanced (75.23%) as compared to the control side (51.8%) (P=0.020(*)). Although PRP enhanced bone formation at the base of the maxillary sinus, it neither improved osseointegration of dental implants nor bone in-growth into the bone substitute under the selected experimental conditions.

Missing osteogenic effect of expanded autogenous osteoblast-like cells in a minipig model of sinus augmentation with simultaneous dental implant installation.

OBJECTIVES: With natural bovine bone mineral (BioOss) as carrier, the study aimed at investigating the effect of autogenous osteoblast-like cells on bone regeneration in an orthotopic (maxillary sinus) and an ectopic (muscle) site. MATERIALS AND METHODS: Autogenous osteoblast-like cells were isolated from iliac cancellous bone of six minipigs and expanded in an autogenous serum-supplemented osteogenic medium. After confirmation of osteogenicity, the expanded cells were precultivated on BioOss granules for 1 week. Four milliliter of cell-seeded BioOss were used for sinus augmentation of right sinus and a Straumann solid screw (dental) implant was simultaneously installed. The contralateral (control) side was filled with cell-free BioOss. Besides, 2 ml of the corresponding granules were placed in a pouch in the latissimus dorsi muscles bilaterally. Polychrome sequential labeling was performed postoperatively. Specimens were harvested at week 6. Undecalcified sections were evaluated with microradiography, fluorescence microscopy, histology and histomorphometry. RESULTS: In the test side, the coronal part of dental implant demonstrated higher bone-implant contact (BIC) than the apical part (34.88+/-28.86% vs. 16.68+/-13.80%, P=0.039), as well as higher bone density (BD) in the corresponding zone (14.88+/-6.37% vs. 11.10+/-4.54%, P=0.021). However, the test side demonstrated no advantage over control side in either BD (12.25+/-4.22% vs. 8.45+/-11.04%, P=0.473) or BIC (24.15+/-21.97% vs. 22.05+/-19.00%, P=0.270). Rare bone was formed in the muscles in both sides. CONCLUSIONS: The expanded autogenous osteoblast-like cells failed to enhance bone formation in the minipig model of sinus augmentation.

Secondary cleft nose rhinoplasty: Subjective and objective outcome evaluation.

INTRODUCTION: Secondary rhinoplasty in cleft lip and palate (CLP) is commonly the last step in a set of surgical procedures that result in a variable but typically intensive change in facial appearance. However, there is evidence that the sentiment about the aesthetic and functional outcomes between patients and surgeons is different. The present study aimed to evaluate the subjective and objective outcomes of secondary rhinoplasty in patients with CLP. MATERIAL AND METHODS: Secondary rhinoplasty was performed in 10 patients with repaired unilateral CLP via a standardized open approach. For the subjective evaluation, the patients completed the rhinoplasty outcome evaluation (ROE) questionnaire. Pre- and postoperative photographic documentation served as the basis for the objective evaluation, which included the following: (1) assessment by five specialists at craniofacial malformation consultation appointments and by three doctors in continuing education using the Asher-McDade aesthetic index (AMAI) rating, and (2) metric facial analysis to determine the nasofrontal angle and the nasolabial angle. RESULTS: Patient satisfaction was high, based on the evaluation of the ROE questionnaire. The analysis of the AMAI rating questionnaire showed no significant differences between the positive ratings of the 'experienced' and 'inexperienced' doctors. In contrast, there was an obvious and significant difference between the 'preoperative' and 'postoperative' time points for questions 1-3. The metric analysis showed statistically significant improvements of the nasolabial angle and the nasofrontal angle. The subjective and objective outcome evaluations were descriptively congruent. CONCLUSIONS: The data suggest that standardized secondary rhinoplasty in CLP leads to both a subjective and a statistically significant objective improvement of facial appearance and thus may support the psychosocial rehabilitation of affected patients. Furthermore, our results showed that the subjective and objective outcome evaluations of secondary rhinoplasty were largely compatible.

Osteoclastic bone resorption around intraosseous screws in rat and pig mandibles.

This report describes osteoclastic bone resorption around intraosseous fixation screws in rat and pig mandibles. These screws supported distraction devices and provided for neutral fixation following the distraction period. Progressive clinical instability of bicortical screws and radiographic and histologic evidence of osseous resorption were frequent findings. In rats, clinical evaluation revealed screw loss and/or loosening in 50% of the rats between 11 and 30 days of neutral fixation. Radiographic signs of resorption were apparent around 60% of the screws that were in place at the end of the observation period. The total rate of resorption or loss was 64 of 80 screws (80%). Histologic examination showed partial or extensive osteolysis around the screw holes in 87% of screws that were clinically fixed in the bone at sacrifice. In histologic sections of porcine specimens, osseous resorption around identifiable screw holes was present in 75% of the cases, and showed progressive increase in resorption with time. Osteoclastic resorption was common around bicortical screws that were evaluated after they had served for osseous stability.

Induction of pro-inflammatory cytokine production in thymocytes by the immune response modifiers Imiquimod and Gardiquimod™.

An emerging role is postulated for IL-17-producing thymocytes, which in their majority consist of IL-17-producing CD4(+) cells. For these, a specific role in the immediate defense against infectious pathogens is suggested, independent from the development of an adaptive immune response in the immune periphery. Immune response modifiers, like the TLR7 ligands Imiquimod and Gardiquimod™ are effective pharmacological therapeutics applied topically against dermal tumors and virus infections and have been demonstrated to activate immune cells. In this study, we investigated the effect of Imiquimod and Gardiquimod™ on murine thymocyte cytokine production with a particular focus on IL-17. We find that both substances dose-dependently are able to trigger IFN-γ and IL-6 production, but no IL-17 production. Moreover, a strong co-stimulating effect is detected on α-CD3-induced IFN-γ, IL-6 and IL-17 production. We conclude that Imiquimod and Gardiquimod™ are not only modifiers of the adaptive immune response, but might also have additional therapeutic potential by modifying the immune activity in the thymus.

Survival of transplanted rat bone marrow-derived osteogenic stem cells in vivo.

This study was designed to trace bone marrow-derived stromal cells (MSC) after implantation in an ectopic rat model of bone tissue engineering. MSC were isolated from adult donor rats, expanded, seeded on a hydroxyapatite/ß-tricalcium phosphate bone graft substitute (Straumann® BoneCeramic), and cultivated until confluent. Before subcutaneous implantation of seeded constructs and controls (unseeded bone graft substitute) in isogenic rats (n = 32), cells were labeled with the fluorescent dye carboxyfluoresceine-diacetate-succinimidyl-ester. Specimens were harvested at sacrifice on day 1, 3, 7, or 14 after implantation (n = 8 per group) and processed for histology (hematoxylin and eosin, CD68, 4',6-diamidino-2-phenylindol). Carboxyfluoresceine-diacetate-succinimidyl-ester-labeled transplanted cells were quantified in decalcified sections (50 fields of view per specimen) at 488 nm. Over time, transplanted cells decreased in number from 31.3 ± 2.3 (day 1) to 9.2 ± 1.1 (day 3) and 0.3 ± 0.1 (day 7) (p < 0.001). Fourteen days postimplantation MSC could no longer be identified. Additionally, starting on day 3 postimplantation, cellular disintegration was noted. Multinucleated giant cells were present in constructs and controls on day 7 and increased to day 14 postimplantation. These results indicate that ectopically transplanted MSC survive for a rather short time after implantation. Possible reasons for early cell death are discussed.

Carcinoma in situ in a lateral cervical cyst.

BACKGROUND: The development of an invasive squamous cell carcinoma within a lateral cervical cyst as a result of malignant transformation of the epithelium is considered a rare circumstance. The existence of this entity is a source of controversy in light of the differential diagnosis, which includes a cervical metastasis from an unknown primary carcinoma (CUP-syndrome). Apart from site, histologic findings, and follow-up, the principal diagnostic criterion for lateral cervical cyst carcinoma is the histomorphologic demonstration of transition of the benign epithelium into invasive squamous cell carcinoma. Although numerous case reports of this entity exist, carcinoma in situ in a lateral cervical cyst has been reported in only five cases thus far. METHOD: In this context, we present the case of a 44-year-old patient with a 7-month history of cervical swelling. RESULTS: After diagnostic extirpation of the tumor, histologic findings were consistent with a lateral branchial cyst with high-grade dysplasia and carcinoma in situ of the squamous epithelial lining. CONCLUSION: This case of a premalignant lesion within a lateral branchial cyst (ie, carcinoma in situ) supplies the "missing link" and adds credibility to the concept of primary branchiogenic carcinoma.