Simultaneous Blocking Research Articles

Unsupervised detection of large-scale weather patterns in the northern hemisphere via Markov State Modelling: from blockings to teleconnections

Detecting recurrent weather patterns and understanding the transitions between such regimes are key to advancing our knowledge of the low-frequency variability of the atmosphere and have important implications in terms of weather and climate-related risks. We adopt an analysis pipeline inspired by Markov State Modelling and detect in an unsupervised manner the dominant winter mid-latitude Northern Hemisphere weather patterns in the Atlantic and Pacific sectors. The daily 500 hPa geopotential height fields are first classified in about 200 microstates. The weather dynamics are then represented on the basis of these microstates and the slowest decaying modes are identified from the spectral properties of the transition probability matrix. These modes are defined on the basis of the nonlinear dynamical processes of the system and not as tentative metastable states, as often done in Markov state analysis. When focusing on a shifting longitudinal window of 60∘, we find that the longitude-dependent estimate of the longest relaxation time is smaller where stronger baroclinic activity is found. In the Atlantic and Pacific sectors slow relaxation processes are mainly related to transitions between blocked regimes and zonal flow. We also find strong evidence of a dynamical regime associated with the simultaneous Atlantic-Pacific blocking. When the analysis is performed on a broader geographical region of the Atlantic sector, we discover that the slowest relaxation modes of the system are associated with transitions between dynamical regimes that resemble teleconnection patterns like the North Atlantic Oscillation and weather regimes like the Scandinavian and Greenland blocking, yet have a much stronger dynamical foundation than classical methods based e.g. on EOF analysis. Our method clarifies that, as a result of the lack of a time-scale separation in the atmospheric variability of the mid-latitudes, there is no clear-cut way to represent the atmospheric dynamics in terms of few, well-defined modes of variability. The approach proposed here can be seamlessly applied across different regions of the globe for detecting regional modes of variability, and has a great potential for intercomparing climate models and for assessing the impact of climate change on the low-frequency variability of the atmosphere.

Abstract 6484: Overcoming resistance to EGFR-TKI in NSCLC by simultaneously inhibition of anti-apoptotic proteins

Abstract Lung cancer is the second most common cancer worldwide with 1.8 million deaths in 2020 being the leading cause of cancer related death. With 86% of cases, non-small cell lung cancer (NSCLC) is the most prominent subtype. The survival rate improved over the past 20 years by application of targeted therapies. Driver mutations in protein kinases such as the epidermal growth factor receptor (EGFR) are frequent and have evolved as therapeutic targets in anti-cancer therapy. Inhibitors of receptor tyrosine kinase (RTKIs), prominently EGFR-TKIs, are effective in anti-cancer therapy but tumor evolution inevitably generates resistance. Thus, strategies for the treatment of patients with acquired resistance to RTKIs remains an urgent need. Resistance in solid tumors is overcome by a combination of bortezomib with the BH3-mimetic ABT-199 (Muenchow et al. 2020). Also, overcoming ALK-TKI resistance in NSCLC was described by Tanimoto et al. (2021) by proteasome inhibiton that mediates induction of NOXA. Furthermore, the novel described BCL-2 independent activity of ABT-199 to transactivate NOXA expression (Weller et al. 2022) represents a generally active principle by combining drugs with independent mechanism of action. We hypothesize, that combined treatment of EGFR-TKI Osimertinib (OSI) with BH3-mimetics enhances sensitivity to apoptosis induction. To explore this hypothesis, we investigated cell death induction by combined administration of OSI and BH3-mimetics or PIs in NSCLC differing in their mutational status. In addition, we analyzed the expression of BCL-2 family proteins and their relevance and regulation during RTKI-induced cell death by RNA sequencing and Western Blot. These analyses will be performed in cellular model systems of NSCLC with specific resistance mediating mutations of EGFR or activated parallel survival pathways. We found that BCL-2 inhibitors enhance efficacy of OSI in NSCLC, resulting in synergistic cell death induction. Additionally, the sensitivity to BCL-2 inhibitors tends to increase in OSI-resistant NSCLC. Western Blot analysis and RNA sequencing data of OSI-resistant NSCLC identified BCL-2 overexpression and an EMT signature as resistance-mediating mechanisms, which can be overcome by combination of OSI with BCL-2 inhibitors. Indeed, our data suggest that combination of OSI with BCL-2 inhibitors disrupts resistance to OSI in NSCLC. We propose that BCL-2 inhibitors sensitize OSI-resistant NSCLC to effectively induce cell death. Thus, studying the molecular mechanism of synergistic efficacy of RTKI/BCL-2 inhibitor combination therapies, should prompt future efforts in the clinical evaluation of combinatorial regimens. We hypothesize that a general therapeutic concept which relies on simultaneous blocking of driver mutations and survival-promoting BCL-2 proteins represents a generally active principle of a tumor-agnostic mechanism of action. Citation Format: Sandra Weller, Tobias Beigl, Frank Essmann, Annika Harsch, Hans-Georg Kopp, Tom Ekstrom. Overcoming resistance to EGFR-TKI in NSCLC by simultaneously inhibition of anti-apoptotic proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6484.

Androgen Receptor/AP-1 Activates UGT2B15 Transcription to Promote Esophageal Squamous Cell Carcinoma Invasion.

Esophageal squamous cell carcinoma (ESCC) is an aggressive epithelial malignancy with poor prognosis. Interestingly, ESCC is strongly characterized by a male-predominant propensity. Our previous study showed that androgen receptor (AR) orchestrated a transcriptional repression program to promote ESCC growth, but it remains unclear whether AR can also activate oncogenic signaling during ESCC progression. In this study, by analyzing our previous AR cistromes and androgen-regulated transcriptomes, we identified uridine diphosphate glucuronosyltransferase family 2 member B15 (UGT2B15) as a bona fide target gene of AR. Mechanistically, AP-1 cofactors played important and collaborative roles in AR-mediated UGT2B15 upregulation. Functional studies have revealed that UGT2B15 promoted invasiveness in vitro and lymph node metastasis in vivo. UGT2B15 was partially responsible for the AR-induced invasive phenotype in ESCC cells. Importantly, simultaneous blocking of AP-1 and AR resulted in stronger inhibition of cell invasiveness compared to inhibiting AP-1 or AR alone. In conclusion, our study reveals the molecular mechanisms underlying the AR-driven ESCC invasion and suggests that the AR/AP1/UGT2B15 transcriptional axis can be potentially targeted in suppressing metastasis in male ESCC patients.

ALDOB/KAT2A interactions epigenetically modulate TGF-β expression and T cell functions in hepatocellular carcinogenesis.

Crosstalk between tumor cells and immune cells enables tumor cells to escape immune surveillance and dictate responses to immunotherapy. Previous studies have identified that downregulation of the glycolytic enzyme fructose-1,6-bisphosphate aldolase B (ALDOB) in tumor cells orchestrated metabolic programming to favor hepatocellular carcinogenesis (HCC). However, it remains elusive whether and how ALDOB expression in tumor cells affects the tumor microenvironment in HCC. We found that ALDOB downregulation was negatively correlated with CD8+ T cell infiltration in human HCC tumor tissues but in a state of exhaustion. Similar observations were made in mice with liver-specific ALDOB knockout or in subcutaneous tumor models with ALDOB knockdown. Moreover, ALDOB deficiency in tumor cells upregulates TGF-β expression, thereby increasing the number of Treg cells and impairing the activity of CD8+ T cells. Consistently, a combination of low ALDOB and high TGF-β expression exhibited the worst overall survival for HCC patients. More importantly, the simultaneous blocking of TGF-β and PD-1 with antibodies additively inhibited tumorigenesis induced by ALDOB deficiency in mice. Further mechanistic experiments demonstrated that ALDOB enters the nucleus and interacts with lysine acetyltransferase 2A (KAT2A), leading to inhibition of H3K9 acetylation and thereby suppressing TGFB1 transcription. Consistently, inhibition of KAT2A activity by small molecule inhibitors suppressed TGF-β and HCC. Our study has revealed a novel mechanism by which a metabolic enzyme in tumor cells epigenetically modulates TGF-β signaling, thereby enabling cancer cells to evade immune surveillance and affect their response to immunotherapy.

Synovial macrophages of rheumatoid arthritic mice protectively responded by altered M1/M2 differentiation after antibody blocking of TNFR1 and IL-1R.

Rheumatoid arthritis (RA) primarily affecting the synovial tissue, has emerged as a major concern leading to the pressing need to develop effective treatment strategies. In the affected synovial tissue, resident macrophages play a pivotal role in the pathogenesis of RA. TNF-α and IL-1β released from pro-inflammatory M1 synovial macrophages are the master regulators of chronic joint inflammation. In this study collagen-induced rheumatoid arthritis model was developed in mice and post isolation, macrophages were subjected to administration with neutralizing antibodies IL1R and TNFR1 either alone or in combination. Flow cytometric analysis followed by Western blots, ROS, and IL-1β, TNF-α release assays were performed. Outcomes suggested that post-dual blockade of IL1R and TNFR1 arthritic synovial macrophages showed a shifting of the M1 towards the anti-inflammatory M2 phenotype. Moreover, the switch towards the M2 phenotype might be responsible for decreased levels of IL-1β,TNF-α, and ROS and simultaneous elevation in the activity of antioxidant enzymes like SOD, CAT, and GPX content in the isolated macrophages. Simultaneous blocking of both IL1R and TNFR1 also showed a sharp reduction in the expression of NF-κB and SAPK-JNK. The elevated arginase and GRX activity further confirmed the polarization towards M2. Moreover, bioinformatics analysis was performed,and it was found that blocking TNFR1 with an antibody could hamper the binding of TNF to TNFR1 in the TNF-TNFR1 pathway. Thus, it may be inferred that dual blockade of IL1R and TNFR1 and a suitable antibody blocking of TNFR1 might be alternative therapeutic approaches for the regulation of RA-induced inflammation in the future.

SiRNAs based gene silencing of potato virus Y by simultaneous blocking of HC-Pro and NIa

Potato virus Y (PVY) is one of the most dangerous pathogens that cause serious damage to crop production. Fighting plant viral infections is crucial to limit these damages and save millions of dollars for farmers. Gene suppression of selected virus genes is one of the modern, innovative and effective approaches to block viral replication and infection. The virus, in turn, has developed its own strategy to avoid this gene suppression through mutations in the attacked genes of the virus genome. For this reason, blocking several important viral genes simultaneously led to better results in blocking viral replication. In this study, we achieved an epigenetic control of viral infection of PVY N/NTN of potato plants cultivar Agria by applying a combination of ds/siRNAs specific for two different conservative regions from the PVY genome, CI-6K2-NIa and HC-Pro. The potato plants that received the ds/siRNAs remained virus-free, suggesting that the CI-6K2-NIa and HC-Pro regions could serve as effective targets for control of PVY.

Titania Thin Film Coated Glass for Simultaneous Ammonia Degradation and UV Light Blocking Layer in Photovoltaics

In this work, we have investigated the potential dual application of TiO2 thin films as a photocatalyst for ammonia degradation, and as a UV light blocking layer in c-Si photovoltaics. For this purpose, we deposited a series of TiO2 thin films on a glass substrate by reactive magnetron sputtering and analysed the influence of the deposition parameters (O2/Ar working gas content and pressure) on the structural, optical and photocatalytic properties. All samples are nanocrystalline anatase TiO2 and have a uniform surface (RMS roughness < 5 nm) in a wide range of magnetron sputtering deposition parameters. They are transparent in the Vis/NIR spectral range and strongly absorb light in the UV range above the optical bandgap energy (3.3 eV), which makes them suitable for the use as UV blocking layers and photocatalysts. The photocatalytic properties were studied in a mini-photocatalytic wind tunnel reactor by examining ammonia degradation. A kinetic study was performed to estimate the reaction rate constants for all samples. The intrinsic reaction rate constant confirmed the crucial role of surface morphology in ammonia decomposition efficiency.

Upconversion Nanocrystals with High Lanthanide Content: Luminescence Loss by Energy Migration versus Luminescence Enhancement by Increased NIR Absorption

AbstractLanthanide‐doped upconversion nanoparticles (UCNPs) have attracted a lot of interest due to their benefits in biological applications: They are not suffering from intermittence and provide nearly background‐free luminescence. The progress in synthesis nowadays enables access to complex core‐shell particles of controlled size and composition. Nevertheless, the frequently used doping ratio dates back to where mostly core‐only particles of relatively large size have been studied. Especially at low power excitation as needed in biology, a decrease in particle size leads to a drastic decrease in the upconversion efficiency. An enhancement strategy based on an increased absorption rate of near‐infrared light provided by an increase of the sensitizer content, together with the simultaneous blocking of the energy migration pathways to the particle surface, is presented. NaYbF4(20%Er) particles of 8.5 nm diameter equipped with an about 2 nm thick NaYF4 shell show significantly enhanced upconversion luminescence in the red (660 nm) compared to the most commonly used particles with only 20% Yb3+ and 2% Er3+. The impact of size, composition, and core‐shell architecture on photophysical properties are studied. The findings demonstrate that an increase in doping rates enables the design of small, bright UCNPs useful for biological applications.

Continued androgen signalling inhibition improves cabazitaxel efficacy in prostate cancer.

Background: The androgen receptor (AR) pathway is a key driver of neoplastic behaviour in the different stages of metastatic prostate cancer (mPCa). Targeting the AR therefore remains the cornerstone for mPCa treatment. We have previously reported that activation of AR signalling affects taxane chemo-sensitivity in preclinical models of castration resistant PCa (CRPC). Here, we explored the anti-tumour efficacy of the AR targeted inhibitor enzalutamide combined with cabazitaxel.Methods: We used the AR positive CRPC model PC346C-DCC-K to assess the in vitro and in vivo activity of combining enzalutamide with cabazitaxel. Subsequent validation studies were performed using an enzalutamide resistant VCaP model. To investigate the impact of AR signalling on cabazitaxel activity we used quantitative live-cell imaging of tubulin stabilization and apoptosis related nuclear fragmentation.Findings: Enzalutamide strongly amplified cabazitaxel anti-tumour activity in the patient-derived xenograft models PC346C-DCC-K (median time to humane endpoint 77 versus 48 days, P<0.0001) and VCaP-Enza-B (median time to humane endpoint 80 versus 53 days, P<0.001). Although enzalutamide treatment by itself was ineffective in reducing tumour growth, it significantly suppressed AR signalling in PC346C-DCC-K tumours as shown by AR target gene expression. The addition of enzalutamide enhanced cabazitaxel induced apoptosis as shown by live-cell imaging (P<0.001).Interpretation: Our study demonstrates that cabazitaxel efficacy can be improved by simultaneous blocking of AR signalling by enzalutamide, even if AR targeted treatment no longer affects tumour growth. These findings support clinical studies that combine AR targeted inhibitors with cabazitaxel in CRPC.

Differentiated Approach to Surgical Treatment of Blount's Disease and Rickets-Like Diseases

Relevance. Blount's disease and vitamin-D-dependent rickets (VDDR) are nosologically different skeletal diseases in childhood. Considering the similarity of the clinical and radiological picture, orthopedists and pediatricians make mistakes in diagnostics, which leads to wrong treatment tactics.&#x0D; Objective: to establish clear indications for surgical treatment of Blount's disease and rickets-like diseases.&#x0D; Materials and Methods. Surgical treatment of 13 patients with varus lower extremity deformities with vitamin-D-dependent rickets and 29 patients with Blount's disease was carried out; 26 and 42 surgical interventions were made, respectively. The patients' age ranged from 6 months to 14 years.&#x0D; Results. Multilevel osteotomies with blocking and growing intramedullary rods should be performed for the treatment of rickety deformities. Applied plates are used only in single-level corrective osteotomies. The use of eight-shaped plates is indicated in the process of the growth zone functioning. In Blount's disease, we recommend: (1) blocking growth zones in patients with 1-4 degrees of deformity, II-VI stages according to Langenskiöld, and skeletal maturity of 23 to 33 points; (2) semicircular dissection in patients with 1-2 degrees of deformity, I-II stages according to Langenskiöld, and skeletal maturity of 18 to 23 points; (3) corrective osteotomies combined with simultaneous blocking of growth zones in patients with 3-4 degrees of deformity, V-VI stages according to Langenskiöld, and skeletal maturity of 30 to 33 points.&#x0D; Conclusions. The presented laboratory diagnostics enables differentiate diagnostics of the etiological affiliation of the varus deformity of the lower leg. If rickets is detected, pathogenetic conservative treatment should be performed; in case of Blount's disease, surgical treatment should be started using minimally invasive methods. In the absence of positive dynamics in case of rickets, guided growth should be applied. In case of multiplanar deformities, one should apply the methods of corrective and reinforced surgical treatment: multilevel osteotomies with a blocking intramedullary rod after the closure of the growth zones and osteotomy with a telescopic intramedullary rod in case of preserved growth zone. If Blount's disease is diagnosed early, timely and correctly, growth zone blocking is the method of choice. In case of recurrence, corrective osteotomies with blockage of growth zones should be performed.

Adaptable peptide-based therapeutics modulating tumor microenvironment for combinatorial radio-immunotherapy

Radiotherapy is one of the conventional tumor treatments, while its abscopal therapeutic efficacy is severely hampered by the immunosuppressive tumor microenvironment. To address this challenge, we herein report on the morphology-adaptable peptide-based therapeutics for efficiently reversing the immunosuppression in the combinatorial radio-immunotherapy through simultaneous checkpoint blocking and induction of immunogenic cell death. The peptide-based therapeutics were created via co-assembling a pentapeptide containing a 4-amino proline residue with its derivatives containing IDO-1 inhibitor NLG919. The resulting therapeutics underwent pH-adaptable morphological transformation between nanofibrils and nanoparticles and released NLG919 upon GSH cleavage. In vivo studies confirmed that the pH-adaptable morphologies of the therapeutics facilitated their tumor accumulation and retention at tumor sites compared to morphology-persistent counterparts, thus resulting in efficient delivery of IDO-1 inhibitors. Simultaneously treating the tumor-bearing mice with the therapeutics and external γ-ray radiation boosted the tumor immunogenicity via inducing ICD cascade of the tumor cells and reverse the immunosuppressive tumor microenvironment due to the inhibition of IDO-1 for depletion of tryptophan. Our findings strongly demonstrate that the morphology-adaptable peptide-based therapeutics exhibit the capability to reverse the immunosuppressive tumor microenvironment during irradiation, thus providing a new strategy for the combinatorial radio-immunotherapy.

Effects of indoleamine 2, 3-dioxygenase (IDO) silencing on immunomodulatory function and cancer-promoting characteristic of adipose-derived mesenchymal stem cells (ASCs).

Indoleamine 2, 3-dioxygenase (IDO) catabolizes tryptophan, mediates immunomodulatory functions, and is released by stromal cells such as mesenchymal stem cells. The aims of this study were to investigate the effects of IDO silencing on immunosuppressive function of adipose-derived mesenchymal stem cells (ASCs), T cells phenotype, and the proliferation/migration of tumor cells. ASCs isolated from adipose tissues of healthy women were transfected with IDO-siRNA. Galectin-3, transforming growth factor-β1, hepatocyte growth factor, and interleukin-10 as immunomodulators were measured in ASCs using qRT-PCR. T cells phenotype, interferon-γ, and interleukin-17 expression were evaluated in peripheral blood lymphocytes (PBLs) cocultured with IDO silenced-ASCs by flow cytometry and qRT-PCR, respectively. Scratch assay was applied to assess the proliferation/migration of MDA-MB-231 cell line. Galectin-3 was upregulated (p ˂ 0.05) while hepatocyte growth factor was downregulated (p ˂ 0.05) in IDO-silenced ASCs compared to control groups. Regulatory T cells were inhibited in PBLs cocultured with IDO-silenced ASCs; also T helper2 was decreased in PBLs cocultured with IDO-silenced ASCs relative to the scramble group. IDO-silenced ASCs caused interferon-γ overexpression but interleukin-17 downregulation in PBLs. The proliferation/migration of MDA-MB-231 was suppressed after exposing to condition media of IDO-silenced ASCs compared with condition media of untransfected (p < 0.01) and scramble-transfected ASCs (p < 0.05). The results exhibited the weakened capacity of IDO-silenced ASCs for suppressing the immune cells and promoting the tumor cells' proliferation/migration. IDO suppression may be utilized as a strategy for cancer treatment. Simultaneous blocking of immunomodulators along with IDO inhibitors may show more effects on boosting the efficiency of immune-based cancer therapies.

Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes.

CRISPR-Cas9-based genome-editing is a highly efficient and cost-effective method to generate zebrafish loss-of-function alleles. However, introducing patient-specific variants into the zebrafish genome with CRISPR-Cas9 remains challenging. Targeting options can be limited by the predetermined genetic context, and the efficiency of the homology-directed DNA repair pathway is relatively low. Here, we illustrate our efficient approach to develop knock-in zebrafish models using two previously variants associated with hereditary sensory deficits. We employ sgRNA-Cas9 ribonucleoprotein (RNP) complexes that are micro-injected into the first cell of fertilized zebrafish eggs together with an asymmetric, single-stranded DNA template containing the variant of interest. The introduction of knock-in events was confirmed by massive parallel sequencing of genomic DNA extracted from a pool of injected embryos. Simultaneous morpholino-induced blocking of a key component of the non-homologous end joining DNA repair pathway, Ku70, improved the knock-in efficiency for one of the targets. Our use of RNP complexes provides an improved knock-in efficiency as compared to previously published studies. Correct knock-in events were identified in 3–8% of alleles, and 30–45% of injected animals had the target variant in their germline. The detailed technical and procedural insights described here provide a valuable framework for the efficient development of knock-in zebrafish models.

Discovery of small molecule acting as multitarget inhibitor of colorectal cancer by simultaneous blocking of the key COX-2, 5-LOX and PIM-1 kinase enzymes

Colorectal cancer (CRC) is the second cause of cancer death worldwide. Inhibitors of COX-2, 5-LOX and PIM-1 kinase were very effective in the treatment and prevention of CRC in mouse models in vivo. Furthermore, thymol was confirmed to inhibit CRC cell proliferation in cancer cell lines and inhibitory activity against COX-2 and 5-LOX. On the other hand, 4-thiazolidinone pharmacophore was incorporated in the structures of various reported COX-2, 5-LOX and PIM kinase inhibitors. Consequently, the aim of the present investigation was to combat CRC by synthesis and biological evaluation of new thymol – 4-thiazolidinone hybrids as multitarget anticancer agents that could inhibit the key COX-2, 5-LOX and PIM-1 kinase enzymes simultaneously. Compounds 5a-d and 5g displayed inhibitory activity against COX-2 nearly equal to Celecoxib with high selectivity index (SI). Moreover, compounds 5b-e showed 5-LOX inhibitory activity nearly equal to the reference Quercetin while compounds 5a, 5f and 5g elicited inhibitory activity slightly lower than Quercetin. Furthermore, in vivo formalin-induced paw edema test revealed that, compounds 5a, 5c, 5f and 5g showed higher % inhibition than Celecoxib and compounds 5a, 5f and 5g showed higher % inhibition than Diclofenac sodium. In addition, compounds 5a-c, 5e-g showed in vivo superior gastrointestinal safety profile as Celecoxib in fasted rats. Besides, compounds 5d, 5e and 5g exhibited the highest activity against human CRC cell lines (Caco-2 and HCT-116) at doses less than their EC100 on normal human cells. Furthermore, compounds 5e and 5g induced apoptosis-dependent death by above 50% in the treated CRC cell lines. Moreover, compounds 5e and 5g induced caspase activation by >50% in human CRC. Also, compounds 5d, 5e and 5g showed in vitro inhibitory activity against both PIM-1\\2 kinases comparable to the reference Staurosporine. In silico docking studies were concordant with the biological results. In conclusion, compound 5g, of simple chemical structure, achieved the target goal of inhibiting three targets leading to inhibition of human CRC cell proliferation. It inhibited the target key enzymes COX-2, 5-LOX and PIM-1\\2 kinase in vitro. Besides, it revealed in vitro inhibition of cell proliferation in cancer cell lines via activation of caspase 3\\7 dependent-apoptosis in human CRC cell lines. In addition, it elicited in vivo anti-inflammatory activity in formalin-induced paw edema test and in vivo oral safety in gastric ulcerogenic activity test.

TLR4 and TNFR1 blockade dampen M1 macrophage activation and shifts them towards an M2 phenotype.

The Gram-negative bacterial lipopolysaccharide (LPS)-induced sepsis has emerged as major concern worldwide due to the pressing need to develop its effective treatment strategies which is not available yet. LPS is the major causative agent in the pathogenesis of septic shock. In macrophages, LPS interacts with cell surface TLR4 leading to reactive oxygen species (ROS), TNF-α, IL-1β production, oxidative stress and markedly activated the MAPKs and NF-kB pathway. Post cell isolation, the macrophages were subjected to administration with neutralizing antibodies to TLR4 and TNFR1 either alone or in combination prior to LPS challenge. Subsequently, we performed flow cytometric analysis along with Western blots, reactive oxygen species production, and TNF-α, IL-1β release. Outcomes suggested that the dual blockade of TLR4 and TNFR1 was indeed beneficial in shifting the LPS-induced M1 polarization towards M2. Both TLR4 and TNFR1 exhibited dependency during LPS stimulation. Furthermore, the switch towards the M2 phenotype might be responsible for the decreased levels of TNF-α, IL-1β, NO, and superoxide anion and the simultaneous elevation in the activity level of anti-oxidant enzymes like SOD, CAT (catalase), and GSH content in the isolated peritoneal macrophages. Simultaneous blocking of both TLR4 and TNFR1 also showed reduced expression of NF-kB, JNK, and COX-2 by promoting TNFR2-mediated TNF-α signaling. The increased arginase activity further confirmed the polarization towards M2. Thus it may be inferred that dual blockade of TLR4 and TNFR1 might be an alternative therapeutic approach for regulating of sepsis in future.

Recapitulating monocyte extravasation to the synovium in an organotypic microfluidic model of the articular joint

The synovium of osteoarthritis (OA) patients can be characterized by an abnormal accumulation of macrophages originating from extravasated monocytes. Since targeting monocyte extravasation may represent a promising therapeutic strategy, our aim was to develop an organotypic microfluidic model recapitulating this process. Synovium and cartilage were modeled by hydrogel-embedded OA synovial fibroblasts and articular chondrocytes separated by a synovial fluid channel. The synovium compartment included a perfusable endothelialized channel dedicated to monocyte injection. Monocyte extravasation in response to chemokines and OA synovial fluid was quantified. The efficacy of chemokine receptor antagonists, RS-504393 (CCR2 antagonist) and Cenicriviroc (CCR2/CCR5 antagonist) in inhibiting extravasation was tested pre-incubating monocytes with the antagonists before injection. After designing and fabricating the chip, culture conditions were optimized to achieve an organotypic model including synovial fibroblasts, articular chondrocytes, and a continuous endothelial monolayer expressing intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. A significantly higher number of monocytes extravasated in response to the chemokine mix (p < 0.01) and OA synovial fluid (p < 0.01), compared to a control condition. In both cases, endothelium pre-activation enhanced monocyte extravasation. The simultaneous blocking of CCR2 and CCR5 proved to be more effective (p < 0.001) in inhibiting monocyte extravasation in response to OA synovial fluid than blocking of CCR2 only (p < 0.01). The study of extravasation in the model provided direct evidence that OA synovial fluid induces monocytes to cross the endothelium and invade the synovial compartment. The model can be exploited either to test molecules antagonizing this process or to investigate the effect of extravasated monocytes on synovium and cartilage cells.

Phosphoproteomic Characterization of Primary AML Samples and Relevance for Response Toward FLT3-inhibitors.

Phosphoproteomic Characterization of Primary AML Samples and Relevance for Response Toward FLT3-inhibitors.

Abstract 526: Novel mechanisms of Neuropilin-1 inhibition result in improved tumor growth inhibition in vivo

Abstract Background Cygnal Therapeutics, pioneer in exoneuronal biology, has identified the neuronal protein, Neuropilin-1 (NRP1) as a critical regulator of tumor growth and anti-tumor immunity. NRP1 is a co-receptor that complexes with diverse ligands and their cognate receptors. As such, it plays a role in multiple different biological processes, including axon guidance and angiogenesis. NRP1 contains two CUB domains (a1 and a2) involved in binding the ligand Semaphorin3A (SEMA3A), two Factor V/VIII domains (b1 and b2) involved in VEGF ligand binding and one MAM domain (c domain). While functional antibodies with anti-tumor activity have been generated against the SEMA3A and VEGF binding domains, targeting other domains of NRP1 has not been well-studied. For example, the c-domain of NRP1 has been implicated in the dimerization of NRP1, a prerequisite for functionality. Thus c-domain blockers might inhibit multiple signaling pathways required for tumor growth. Alternatively, binders targeting the interface of two different domains might allow for simultaneous blocking of multiple ligands. We therefore hypothesized that these novel binders to alternative regions of NRP1 such as the b1/b2 interface and the c-domain would have improved and significant anti-tumor activity. Methods Antibodies with diverse binding characteristics were tested for in vivo anti-tumor activity in multiple syngeneic models, including anti-PD1 non-responsive models. Ability of these of the anti-NRP1 antibodies to elicit an adaptive immune response was evaluated. Results Novel binders to alternative regions of NRP1 such as domain interfaces and the c-domain resulted in significant tumor growth inhibition in multiple syngeneic models. Additionally, the NRP1 antagonists induced a robust CD8 T cell response, indicating immune-stimulatory role of these antibodies. Conclusions Cygnal has developed fully human monoclonal antibodies against previously untargeted domains of NRP1. These novel domain binders show remarkable inhibition of tumor growth in multiple syngeneic models and offer a novel means of therapeutic intervention in patients. Citation Format: Shalini Sethumadhavan, Eric Zhu, Aaron Fulgham, Mandana Abbassi, Ameya Apte, Tiffany Liao, Chengfeng Merriman, Katherine Molloy, Caitlin Stein, Amber Hanna, James Geoghegan, Bianka Prinz, Hongyue Dai, Jonathan Hurov, Pearl Huang, Daniel Blom. Novel mechanisms of Neuropilin-1 inhibition result in improved tumor growth inhibition in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 526.

Change one category at a time: Sequence effects beyond interleaving and blocking.

Research on study sequences has not considered the cross-classification of to-be-learned categories. In two experiments, we utilized cross-classified exemplars, which simultaneously belonged to categories of two orthogonal dimensions. Experiment 1 addressed the question of how interleaving one category dimension while simultaneous blocking another category dimension affects the induction of the simultaneously blocked category dimension. Experiment 2 examined our proposed mechanism by manipulating the degree of change (one-category vs. cross-category change) and the frequency of change (high vs. low) in the presentation sequence of exemplars with cross-classified characteristics. In Experiment 1, sequences that combined interleaving one dimension while blocking the other dimension were superior to sequences that provided no comparison opportunities when classifying both interleaved and blocked categories. This revealed a carry-over effect of interleaving: blocked and interleaved categories were equally well classified. Our findings are incompatible with the discriminative contrast hypothesis and the attentional bias framework, where interleaving is not assumed to support within-category comparisons. We explain the results according to the principle of change one category at a time (COCAT). Interleaving exemplars on one category dimension, but blocking them on another category dimension, enables learners to reliably map the distinctive features onto the covarying categories and the shared features onto the constant category. In contrast, there is a risk of confounding common characteristics when no comparison opportunities are given. Likewise, pure interleaving impedes category induction by confounding changing characteristics. Accordingly, Experiment 2 demonstrated that as long as a sequence implemented the COCAT principle, learners accurately identify diagnostic feature sets. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Early phase studies of JAK1 selective inhibitors in rheumatoid arthritis.

The first approved Janus kinase (JAK) inhibitors for treatment of RA targeted more than one JAK molecule. Although this brings an advantage of simultaneous blocking of more cytokines involved in RA, it may also carry an increased risk of toxicity. Subsequently, more selective JAK inhibitors were developed with the aim of improving the safety–efficacy profile and to further increase drug maintenance. With this proposal, early phase trials of selective JAK1 inhibitors, namely upadacitinib, filgotinib and itacitinib, were initiated in recent years to identify the efficacy and adverse effects of these agents and to define their potential role in treatment of inflammatory and autoimmune diseases. Early phase (Phase I–II) studies of upadacitinib and filgotinib provided evidence for efficacy and safety of the selective JAK1 inhibitors in refractory populations of RA patients and allowed informed selection of the appropriate dose by balancing the optimal benefit–risk profile for further evaluation in the later successfully performed Phase III trials. Although itacitinib also demonstrated a good efficacy and safety in a Phase II trial in RA patients, it is mainly in development for haematologic and oncologic conditions.