A large and diverse brain organoid dataset of 1,400 cross-laboratory images of 64 trackable brain organoids.

Brain organoids represent a useful tool for modeling of neurodevelopmental disorders and can recapitulate brain volume alterations such as microcephaly. To monitor organoid growth, brightfield microscopy images are frequently used and evaluated manually which is time-consuming and prone to observer-bias. Recent software applications for organoid evaluation address this issue using classical or AI-based methods. These pipelines have distinct strengths and weaknesses that are not evident to external observers. We provide a dataset of more than 1,400 images of 64 trackable brain organoids from four clones differentiated from healthy and diseased patients. This dataset is especially powerful to test and compare organoid analysis pipelines because of (1) trackable organoids (2) frequent imaging during development (3) clone diversity (4) distinct clone development (5) cross sample imaging by two different labs (6) common imaging distractors, and (6) pixel-level ground truth organoid annotations. Therefore, this dataset allows to perform differentiated analyses to delineate strengths, weaknesses, and generalizability of automated organoid analysis pipelines as well as analysis of clone diversity and similarity.

Tyrosine hydroxylase variants influence protein expression, cellular localization, stability, enzymatic activity and the physical interaction between tyrosine hydroxylase and GTP cyclohydrolase 1.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis catalyzing the tetrahydrobiopterin (BH4 )-dependent hydroxylation of tyrosine to L-DOPA. Here, we analyzed 25 TH variants associated with various degrees of dopa-responsive dystonia and evaluate the effect of each variant on protein stability, activity and cellular localization. Furthermore, we investigated the physical interaction between TH and human wildtype (wt) GTP cyclohydrolase 1 (GTPCH) and the effect of variants on this interaction. Our in vitro results classify variants according to their resistance to proteinase K digestion into three groups (stable, intermediate, unstable). Based on their cellular localization, two groups of variants can be identified, variant group one with cytoplasmic distribution and variant group two forming aggregates. These aggregates do not correlate with loss of enzymatic activity but nevertheless might be a good target for molecular chaperones. Unfortunately, no obvious correlation between the half-life of a variant and its enzymatic activity or between solubility, stability and enzymatic activity of a given variant could be found. Excitingly, some variants disrupt the physical interaction between TH and human wildtype GTPCH, thereby interfering with enzymatic activity and offering new druggable targets for therapy. Taken together, our results highlight the importance of an in-depth molecular analysis of each variant in order to be able to classify groups of disease variants and to find specific therapies for each subgroup. Stand-alone in silico analyses predict less precise the effect of specific variants and should be combined with other in vitro analyses in cellular model systems.

An AI-based segmentation and analysis pipeline for high-field MR monitoring of cerebral organoids.

Cerebral organoids recapitulate the structure and function of the developing human brain in vitro, offering a large potential for personalized therapeutic strategies. The enormous growth of this research area over the past decade with its capability for clinical translation makes a non-invasive, automated analysis pipeline of organoids highly desirable. This work presents a novel non-invasive approach to monitor and analyze cerebral organoids over time using high-field magnetic resonance imaging and state-of-the-art tools for automated image analysis. Three specific objectives are addressed, (I) organoid segmentation to investigate organoid development over time, (II) global cysticity classification and (III) local cyst segmentation for organoid quality assessment. We show that organoid growth can be monitored reliably over time and cystic and non-cystic organoids can be separated with high accuracy, with on par or better performance compared to state-of-the-art tools applied to brightfield imaging. Local cyst segmentation is feasible but could be further improved in the future. Overall, these results highlight the potential of the pipeline for clinical application to larger-scale comparative organoid analysis.

Intradermal testing and serum allergen-specific IgE-testing in cats with naturally occurring feline bronchial disease.

OBJECTIVES: While feline asthma (FA) is considered to be of allergic origin, the etiology of feline chronic bronchitis (CB) to date is unknown. Aim of the study was to compare the results of intradermal testing (IDT) and serum testing for allergen-specific immunoglobulin E (SAT) in cats diagnosed with FA and CB. MATERIAL AND METHODS: Twenty-seven client-owned cats with clinical signs, suggestive of feline inflammatory bronchial disease (FBD) were prospectively enrolled in the study. Patients were assigned to 3 groups based on results of bronchoalveolar-lavage-fluid (BALF)-cytology: FA (n=8), CB (n=10), or cats with a physiological BALF cytology (PB; n=9). A standardized IDT for 27 allergens was performed in all cats. In addition, allergen-specific IgE was measured in serum samples using an FcεRIα-ELISA. The number of positive reactions in both tests was compared between groups, and agreement between test results of both tests was evaluated. RESULTS: Regarding the number of positive reactions, no statistically significant difference was detected between groups in IDT (p=0.65) and SAT (p=0.51). When comparing the 2 test systems, a weak correlation was found for the allergens Tyrophagus putrescentiae (k=0.256), Dermatophagoides farinae (k=0.276), and rye (k=0.273). The most commonly observed reactions were to house dust mites, storage mites, rye and nettle in IDT and to sheep sorrel, storage mites, and house dust mites in SAT. CONCLUSION AND RELEVANCE: IDT and SAT in cats with feline inflammatory bronchial disease (FBD) cannot be used interchangeably for allergen detection. Sensitization to environmental allergens can occur in cats with and without airway inflammation. Therefore, a positive test result should always be assessed in context with clinical signs and allergen exposure.

Immunoadsorption in a dog with severe immune mediated hemolytic anemia.

Immune mediated hemolytic anemia (IMHA) is a life-threatening disease with severe, acute hemolysis as a result of an autoimmune response directed against erythrocyte surface antigens. In veterinary medicine, IMHA is usually treated with immunosuppressants and often multiple blood transfusions. In human medicine, immunoadsorption (IA) is an established therapy for antibody removal in immune-mediated diseases. A female, spayed, five-year-old, 28 kg Entlebucher Mountain dog was presented with regenerative anemia and positive autoagglutination diagnosed as immune-mediated hemolytic anemia to the veterinary emergency service. Conventional treatment consisting immunosuppression with prednisolone and mycophenolate failed to improve hemolysis. As hematocrit dropped daily, multiple blood transfusions of blood group DEA 1 negative were required. IA was initiated at day 3 with COM.TEC and ADAsorb platforms and a LIGASORBstaphylococcus antitoxin A column. IA with citrate anticoagulation was performed over the treatment time of 77 minutes with a blood flow of 50 mL/min. Total plasma volume of 1.6 L was processed. Complications consisted of vomitus and lid swelling, shivering, excessive clotting in the tubing after a calcium bolus and hypotension. After IA, hemolysis stopped immediately, plasma concentrations of immunoglobulin G, immunoglobulin M and bilirubin decreased, and hematocrit remained stable. The dog was discharged without further hemolysis 4 days after immunoadsorption with immunosuppressive therapy. IA is a promising adjunctive therapy in severe cases of canine IMHA, but it cannot be concluded to which degree IA or concurrent immunosuppression contributed to cessation of hemolysis in the present case.

Improvement of in vitro potency assays by a resting step for clinical-grade chimeric antigen receptor engineered T cells.

BACKGROUND: Chimeric antigen receptor engineered T (CAR-T) cell therapy is a promising approach currently revolutionizing the field of cancer immunotherapy. However, data concerning clinical-grade CAR-T cell stability and functionality after months of cryopreservation have not been released by companies so far. To investigate the effect of cryopreservation on CAR-T cells and to further optimize the potency assays, we performed this study. METHODS: A third generation of CD19 CAR-T cells was manufactured according to Good Manufacturing Practice (GMP) requirements, which is applied to patients in an ongoing clinical phase 1 study. Quality control tests for sterility, endotoxin and mycoplasma were performed for each batch. Stability in terms of viability, recovery, transduction efficiency and functional capacity was determined using microscopy, multiparametric flow cytometry as well as chromium-51 release tests. RESULTS: Up to 90days of cryopreservation had no influence on viability, recovery and transduction efficiency of CAR-T cells. However, higher cell concentration for cryopreservation could alter the cell viability and recovery but not the transduction efficiency. Moreover, directly after thawing, both the quantity and quality of the functionality of CAR-T cells were transiently hampered by the negative effects of cryopreservation. Notably, the impaired functionality could be fully restored and even strengthened after an overnight resting process. DISCUSSION: Cryopreservation is a challenge for the functional activity of CAR-T cells. However, CAR-T cells regain their potency by overnight incubation at 37°C, which mimics the clinical application setting. Therefore, an overnight resting step should be included in in vitro potency assays.

Cardiopulmonary response during whole-body vibration training in patients with severe COPD.

Several studies in patients with chronic obstructive pulmonary disease (COPD) have shown that whole-body vibration training (WBVT) has beneficial effects on exercise capacity. However, the acute cardiopulmonary demand during WBVT remains unknown and was therefore investigated in this study. Ten patients with severe COPD (forced expiratory volume in 1 s: 38±8% predicted) were examined on two consecutive days. On day one, symptom-limited cardiopulmonary exercise testing was performed on a cycle ergometer. The next day, six bouts of repeated squat exercises were performed in random order for one, two or three minutes either with or without WBVT while metabolic demands were simultaneously measured. Squat exercises with or without WBVT induced comparable ventilatory efficiency (minute ventilation (VE)/carbon dioxide production (V'CO2 ): 38.0±4.4 with WBVT versus 37.4±4.1 without, p=0.236). Oxygen uptake after 3 min of squat exercises increased from 339±40 mL·min-1 to 1060±160 mL·min-1 with WBVT and 988±124 mL min-1 without WBV (p=0.093). However, there were no significant differences between squat exercises with and without WBVT in oxygen saturation (90±4% versus 90±4%, p=0.068), heart rate (109±13 bpm versus 110±15 bpm, p=0.513) or dyspnoea (Borg scale 5±2 versus 5±2, p=0.279). Combining squat exercises with WBVT induced a similar cardiopulmonary response in patients with severe COPD compared to squat exercises without WBVT. Bearing in mind the small sample size, WBVT might be a feasible and safe exercise modality even in patients with severe COPD.

Myofibroblasts have an impact on expression, dimerization and signaling of different ErbB receptors in OSCC cells.

INTRODUCTION: Receptors of the ErbB family belong to the key players in cancer development and are targets of several therapeutic approaches. Their functional dependency on the tumor microenvironment, especially on CAFs is albeit still poorly understood. Our objective was to investigate the impact of CAF secretome on ErbB receptor expression and signaling behavior in OSCC. METHODS: Stimulation of PE/CA-PJ15 OSCC cells with conditioned media of TGF-ß1-activated fibroblasts was used as model system for CAF to cancer cell communication. Thereby costimulation with inhibitors against matrix metalloproteinases (MMPs), epidermal growth factor receptor (EGFR), MAPK/ERK kinase (MEK), phosphoinositide-3 kinase (PI3-K), signal transducer and activator of transcription 3 (Stat3) or knockdown of Her3 by siRNA was utilized for detailed investigation of the expression, dimerization and signaling pattern of ErbB in western blot and coimmunoprecipitation. RESULTS: Our results show that soluble factors in activated fibroblast secretome stimulate metalloproteinase activity in the membrane of cancer cells. Thereby ligands are released that activate EGFR and subsequently upregulates EGFR expression via the STAT3 pathway. Simultaneously, the expression of PKCɛ was enhanced via a PI3-kinase/Akt-mediated pathway and a negative feedback regulation loop on EGFR downstream signaling generated. Furthermore, the activated fibroblasts secretome stimulated the highly oncogenic hetero-dimerization between HER3 and p95HER2. That protein association is inversely dependent on the expression level of HER3. CONCLUSIONS: Our results demonstrate that the activated fibroblasts secretome can induce a counterbalanced regulation of protein expression, downstream signaling and the dimerization patterns of different ErbB receptor subtypes in the cancer cell. Thus, the combinatorial targeting of CAFs and selective ErbB receptor subtype inhibitors may provide a useful approach in cancer therapy.

Bevacizumab-Induced Inhibition of Angiogenesis Promotes a More Homogeneous Intratumoral Distribution of Paclitaxel, Improving the Antitumor Response.

The antitumor activity of angiogenesis inhibitors is reinforced in combination with chemotherapy. It is debated whether this potentiation is related to a better drug delivery to the tumor due to the antiangiogenic effects on tumor vessel phenotype and functionality. We addressed this question by combining bevacizumab with paclitaxel on A2780-1A9 ovarian carcinoma and HT-29 colon carcinoma transplanted ectopically in the subcutis of nude mice and on A2780-1A9 and IGROV1 ovarian carcinoma transplanted orthotopically in the bursa of the mouse ovary. Paclitaxel concentrations together with its distribution by MALDI mass spectrometry imaging (MALDI MSI) were measured to determine the drug in different areas of the tumor, which was immunostained to depict vessel morphology and tumor proliferation. Bevacizumab modified the vessel bed, assessed by CD31 staining and dynamic contrast enhanced MRI (DCE-MRI), and potentiated the antitumor activity of paclitaxel in all the models. Although tumor paclitaxel concentrations were lower after bevacizumab, the drug distributed more homogeneously, particularly in vascularized, non-necrotic areas, and was cleared more slowly than controls. This happened specifically in tumor tissue, as there was no change in paclitaxel pharmacokinetics or drug distribution in normal tissues. In addition, the drug concentration and distribution were not influenced by the site of tumor growth, as A2780-1A9 and IGROV1 growing in the ovary gave results similar to the tumor growing subcutaneously. We suggest that the changes in the tumor microenvironment architecture induced by bevacizumab, together with the better distribution of paclitaxel, may explain the significant antitumor potentiation by the combination.

Targeted delivery of interleukin-10 to chronic cardiac allograft rejection using a human antibody specific to the extra domain A of fibronectin.

BACKGROUND AND AIMS: Management of chronic rejection is challenging since there are not sufficient preventive or therapeutic strategies. The rejection process leads to overexpression of ED-A(+) fibronectin (ED-A(+) Fn). The human antibody F8, specific to ED-A(+) Fn, may serve as a vehicle for targeted delivery of bioactive payloads, e.g. interleukin 10 (IL-10). The aim of this study was to investigate the therapeutic effects of the fusion protein F8-interleukin-10 (F8-IL10) in the process of chronic rejection development. METHODS: A heterotopic rat heart transplantation model was used to induce chronic rejection. For therapeutic interventions, the immunocytokines F8-humanIL10 (DEKAVIL), F8-ratIL10 as well as KSF-humanIL10 (irrelevant antigen-specificity) were used. Treatment was performed weekly for 10 weeks starting at day 7 after transplantation (1mg/animal). RESULTS: In the cardiac allografts, treatment with F8-huIL10 or F8-ratIL10 was associated with increased heart weights, a higher grade of chronic rejection, increased CIF, higher protein expression levels of alpha-smooth muscle actin (α-SMA), an augmented infiltration with inflammatory cells (CD4+, CD8+ and CD68+ cells) and higher serum levels of brain natriuretic peptide (BNP) compared to the control groups. CONCLUSIONS: All observed treatment effects are transplantation-specific since the F8 antibody is specific to ED-A(+) Fn that is not expressed in healthy hearts. A clear targeting effect of F8-huIL10 as well as F8-ratIL10 could be proven. Against that background, a further study is needed to address the question, if F8-IL10 treatment is capable to reduce CAV and CIF starting at a time point when chronic rejection has fully developed (therapeutic approach).