Inhibition Of Cytokines Research Articles

Antiangiogenic Effects of Infliximab in Chick Chorioallantoic Membrane Model

Introduction: TNF-α plays a key role in initiating pathologic angiogenesis, however, literature on the effects of infliximab on angiogenesis is limited.
 Objective: The study aimed to investigate the effects of infliximab on angiogenesis in an in vivo chick chorioallantoic membrane (CAM) model.
 Methods: The study was approved by the local ethics committee on animal experimentation. Thirty fertilized specific pathogen-free eggs were incubated and kept under appropriate temperature and humidity control. On the third day of the incubation, infliximab (1 µmol) dissolved in phosphate-buffered saline in the first group, phosphate-buffered saline (negative control) (0.1 ml) in the second group, anti-VEGF (positive control) (1 µmol) in the third group were administered by injection. On the eighth day of incubation, the vascular structures of the CAMs were macroscopically examined, and standard digital photographs were taken. The digital images were analyzed, and data including mean vessel density, thickness, and number were compared between groups. P < 0.05 was considered statistically significant.
 Results: Angiogenesis was significantly reduced in the anti-VEGF and infliximab groups compared to the saline-only group. Vessel thickness, vessel number, and vessel density were significantly less in the infliximab and anti-VEGF groups compared to the saline-only group (p=0.034, 0.029, 0.024, respectively).
 Conclusions: Infliximab showed promising antiangiogenic effects in the chick CAM model. Thus, infliximab could be a treatment agent in pathological processes in which angiogenesis is responsible. The antiangiogenic effect of infliximab could be due to the inhibition of various angiogenesis-related cytokines and adhesion molecules. Further studies are needed to clarify the exact mechanisms.

6-Formylindolo[3,2-b]carbazole, a potent ligand for the aryl hydrocarbon receptor, attenuates concanavalin-induced hepatitis by limiting T-cell activation and infiltration of proinflammatory CD11b+ Kupffer cells.

FICZ (6-formylindolo[3,2-b]carbazole) is a potent aryl hydrocarbon receptor agonist that has a poorly understood function in the regulation of inflammation. In this study, we investigated the effect of aryl hydrocarbon receptor activation by FICZ in a murine model of autoimmune hepatitis induced by concanavalin A. High-throughput sequencing techniques such as single-cell RNA sequencing and assay for transposase accessible chromatin sequencing were used to explore the mechanisms through which FICZ induces its effects. FICZ treatment attenuated concanavalin A-induced hepatitis, evidenced by decreased T-cell infiltration, decreased circulating alanine transaminase levels, and suppression of proinflammatory cytokines. Concanavalin A revealed an increase in natural killer T cells, T cells, and mature B cells upon concanavalin A injection while FICZ treatment reversed the presence of these subsets. Surprisingly, concanavalin A depleted a subset of CD55+ B cells, while FICZ partially protected this subset. The immune cells showed significant dysregulation in the gene expression profiles, including diverse expression of migratory markers such as CCL4, CCL5, and CXCL2 and critical regulatory markers such as Junb. Assay for transposase accessible chromatin sequencing showed more accessible chromatin in the CD3e promoter in the concanavalin A-only group as compared to the naive and concanavalin A-exposed, FICZ-treated group. While there was overall more accessible chromatin of the Adgre1 (F4/80) promoter in the FICZ-treated group, we observed less open chromatin in the Itgam (CD11b) promoter in Kupffer cells, supporting the ability of FICZ to reduce the infiltration of proinflammatory cytokine producing CD11b+ Kupffer cells. Taken together, these data demonstrate that aryl hydrocarbon receptor activation by FICZ suppresses liver injury through the limitation of CD3+ T-cell activation and CD11b+ Kupffer cell infiltration.

Neuroinflammation and Acetylcholinesterase Inhibitory Potentials of a Spiroketal-Enol Ether Polyyne Isolated from Artemisia pallens Wall. ex DC.

Artemisia pallens Wall. ex DC (Asteraceae) is cultivated for the production of high-value essential oil from its aerial biomass. In this study, the chemical composition of the root (crop-residue) essential oil was investigated for the first time, using column-chromatography, GC-FID, GC-MS, LC-QTOF, and NMR techniques, which led to the identification of twenty constituents, with isolation of (E)-2-(2',4'-hexadiynylidene)-1,6-dioxaspiro [4.5]dec-3-ene (D6). The D6 was evaluated in vitro for neuroinflammation and acetylcholinesterase inhibitory potential. It showed inhibition of neuroinflammation in a concentration-dependent manner with significant inhibition of pro-inflammatory cytokines (TNF-α and IL-6) in LPS-stimulated BV2 microglial cells. D6 did not have any significant effect on the viability of the cells at the therapeutic concentrations. D6 also has shown acetylcholinesterase inhibitory potential (51.90±1.19 %) at the concentration of log 106 nM. The results showed that D6 has a potential role in the resolution of neuroinflammation, and its acetylcholinesterase inhibitory potential directs further investigation of its role in the management of Alzheimer's disease-related pathogenesis.

Melittin promotes dexamethasone in the treatment of adjuvant rheumatoid arthritis in rats.

Background: Rheumatoid arthritis (RA) is an erosive-destructive inflammation of the joints, and the chronic, long-term stiffness and deformation induced by RA are some of the symptoms of arthritis that are difficult to treat. Dexamethasone (DEX) and melittin (MLT) are two interesting anti-inflammatory substances, both of which possess anti-inflammatory effects exerted through the suppression of the immune system. The purpose of this study was to explore the role of MLT in the treatment of RA by DEX as well as to clarify the influence of MLT on the efficacy and side effects of DEX. Method: The rats were injected with Complete Freund's Adjuvant (CFA) to induce arthritis, followed by treatment with different doses of DEX and/or MLT. The relevant indexes of paw inflammation were determined, and the appetite, growth status, arthritis status, cytokine levels, and organ coefficient of the rats were evaluated. In addition, the paraffin sections of the joint tissues were prepared to analyze the pathological changes. Result: DEX exhibited side effects, notably hindering feed intake and growth, and inducing immune organ lesions in the rats. MLT significantly reduced the side effects of DEX and promoted its efficacy. DEX in combination with MLT demonstrated a synergistic efficacy in RA treatment, showing advantages in detumescence reduction, pro-inflammatory cytokine inhibition, and joint internal pathological improvement. Conclusion: Thus, MLT promoted the efficacy of DEX in adjuvant RA treatment in rats, offering an approach to reduce the use dosage and side effects of DEX.

TGFβR1 inhibition drives hepatocellular carcinoma proliferation through induction of toll-like-receptor signalling.

Transforming growth factor (TGF)-β and toll-like receptors (TLRs) have been shown to independently modulate the proliferation of hepatocellular carcinoma (HCC). Since a direct cross-talk between these two signalling pathways in HCC has not been clearly described before, we aimed here to explore the possibility of such interaction. A human HCC tissue array (n = 20 vs. four control samples), human HCC samples (n = 10) and steatohepatitis-driven murine HCC samples (control, NASH and HCC; n = 6/group) were immunostained for TGFβR1, pSMAD2, TRAF6, IRAK1 and PCNA. The results were confirmed by immunoblotting. Effects of constant activation of the SMAD pathway by constitutive expression of ALK5 or knockdown of mediators of TLR signalling, IRAK1 and MyD88, on HCC proliferation, were investigated in the HCC cell line (HUH-7) after treatment with TGFβ1 cytokine or TGFβR1 kinase inhibitor (LY2157299) using PCNA and MTS assay. TGFβR1 expression is decreased in human and murine HCC and associated with downregulated pSMAD2, but increased IRAK1, TRAF6 and PCNA staining. TGFβR1 kinase inhibition abolished the cytostatic effects of TGFβ1 and led to the induction of IRAK1, pIRAK1 and elevated mRNA levels of TLR-9. Overexpression of ALK5 and knockdown of MyD88 or IRAK1 augmented the cytostatic effects of TGFβ1 on HUH-7. In another epithelial HCC cell line, that is, HepG2, TGFβR1 kinase inhibitor similarly elevated cellular proliferation. There is a balance between the canonical SMAD-driven tumour-suppressing arm and the non-canonical tumour-promoting arm of TGFβ signalling. Disruption of this balance, by inhibition of the canonical pathway, induces HCC proliferation through TLR signalling.

Mechanisms underlying immunosuppression by regulatory cells.

Regulatory cells, such as regulatory T cells (Tregs), regulatory B cells (Bregs), and myeloid-derived suppressor cells (MDSCs), play a crucial role in preserving immune tolerance and controlling immune responses during infections to prevent excessive immune activation. However, pathogens have developed strategies to hijack these regulatory cells to decrease the overall effectiveness of the immune response and persist within the host. Consequently, therapeutic targeting of these immunosuppressive mechanisms during infection can reinvigorate the immune response and improve the infection outcome. The suppressive mechanisms of regulatory cells are not only numerous but also redundant, reflecting the complexity of the regulatory network in modulating the immune responses. The context of the immune response, such as the type of pathogen or tissue involved, further influences the regulatory mechanisms involved. Examples of these immunosuppressive mechanisms include the production of inhibitory cytokines such as interleukin 10 (IL-10) and transforming growth factor beta (TGF-β) that inhibit the production of pro-inflammatory cytokines and dampen the activation and proliferation of effector T cells. In addition, regulatory cells utilize inhibitory receptors like cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) to engage with their respective effector cells, thereby suppressing their function. An alternative approach involves the modulation of metabolic reprogramming in effector immune cells to limit their activation and proliferation. In this review, we provide an overview of the major mechanisms mediating the immunosuppressive effect of the different regulatory cell subsets in the context of infection.

Sex-dependent improvement in traumatic brain injury outcomes after liposomal delivery of dexamethasone in mice.

Traumatic brain injury (TBI) can have long-lasting physical, emotional, and cognitive consequences due to the neurodegeneration caused by its robust inflammatory response. Despite advances in rehabilitation care, effective neuroprotective treatments for TBI patients are lacking. Furthermore, current drug delivery methods for TBI treatment are inefficient in targeting inflamed brain areas. To address this issue, we have developed a liposomal nanocarrier (Lipo) encapsulating dexamethasone (Dex), an agonist for the glucocorticoid receptor utilized to alleviate inflammation and swelling in various conditions. In vitro studies show that Lipo-Dex were well tolerated in human and murine neural cells. Lipo-Dex showed significant suppression of inflammatory cytokines, IL-6 and TNF-α, release after induction of neural inflammation with lipopolysaccharide. Further, the Lipo-Dex were administered to young adult male and female C57BL/6 mice immediately after controlled cortical impact injury (a TBI model). Our findings demonstrate that Lipo-Dex can selectively target the injured brain, thereby reducing lesion volume, cell death, astrogliosis, the release of pro-inflammatory cytokines, and microglial activation compared to Lipo-treated mice in a sex-dependent manner, showing a major impact only in male mice. This highlights the importance of considering sex as a crucial variable in developing and evaluating new nano-therapies for brain injury. These results suggest that Lipo-Dex administration may effectively treat acute TBI.

Leaf extracts of eight selected southern African medicinal plants modulate pro-inflammatory cytokine secretion in LPS-stimulated RAW 264.7 macrophages

This study investigates the anti-inflammatory properties of extracts prepared from the leaves of eight southern African medicinal plants used traditionally to treat inflammation and pain. The inhibitory effect of aqueous and ethanol extracts on the release of pro-inflammatory cytokines was determined in lipopolysaccharide (LPS) stimulated and unstimulated RAW 264.7 murine macrophage cells. The levels of interleukin (IL)-1β, IL-6, tumour necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein (MIP)-2 release were determined using cytokine multiplex-bead assays. The ethanol extracts of Melianthus comosus Vahl (commonly known as honey flower), Tetradenia riparia (Hochst.) Codd (misty plume bush) and Warburgia salutaris (G. Bertol.) Chiov. (pepper-bark tree), demonstrated the most significant inhibitory activity, with over 50-fold inhibition of IL-1β, IL-6 and TNF-α levels in LPS-stimulated RAW 264.7 macrophages. The aqueous extract of M. comosus also significantly inhibited the secretion of all the tested cytokines and chemokines. Phytochemical investigation of M. comosus ethanol leaf extract using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) led to the detection of crassolide, deoxylimonoic acid D-ring-lactone, 2-hydroxynonanoic acid and 5-noniloxytryptamine. To the best of our knowledge, the cytokine inhibition properties of most of the medicinal plants screened in this study are reported for the first time. Our results support the use of southern African medicinal plants as anti-inflammatory remedies and provide an insight into the immunomodulatory mechanisms of action.

Pathophysiology and Management of Refractory Rheumatoid Arthritis: A Narrative Literature Review

Refractory rheumatoid arthritis is rheumatoid arthritis that fails to achieve low levels of disease activity using 2-3 DMARDS treatment and at least one other first-line bDMARD. The definition of refractory action is based on the number of failed DMARDs, the scale of the problem, and risk factors for a refractory disease course. Until now, there has been no further research discussing the pathophysiology of refractory RA, but it is known that TNF-α, IL-6, and IL-1 are the most important mediators that enable cell migration and inflammation in RA. IL-6 in particular, acts directly on neutrophils, which then contribute to inflammation and joint destruction by secreting proteolytic enzymes. The main goal to be achieved in RA is remission, low disease activity and an alternative goal for those who fail to achieve therapy targets. Currently, there are several bDMARD agents that can be used as therapy for refractory RA, namely TNF alfa inhibitors, B cell depletion agents, T cell activity inhibitors, and cytokine inhibitors.

Cytokines and Regulating Epithelial Cell Division.

Physiologically, cytokines play an extremely important role in maintaining cellular and subcellular homeostasis, as they interact almost with every cell in the organism. Therefore, cytokines play a significantly critical role in the field of pathogenic pharmacological therapy of different types of pathologies. Cytokine is a large family containing many subfamilies and can be evaluated into groups according to their action on epithelial cell proliferation; stimulatory include transforming growth factor-α (TGF-α), Interlukine-22 (IL-22), IL-13, IL-6, IL-1RA and IL-17 and inhibitory include IL-1α, interferon type I (IFN type I), and TGF-β. The balance between stimulatory and inhibitory cytokines is essential for maintaining normal epithelial cell turnover and tissue homeostasis. Dysregulation of cytokine production can contribute to various pathological conditions, including inflammatory disorders, tissue damage, and cancer. Several cytokines have shown the ability to affect programmed cell death (apoptosis) and the capability to suppress non-purpose cell proliferation. Clinically, understanding the role of cytokines' role in epithelial tissue is crucial for evaluating a novel therapeutic target that can be of use as a new tactic in the management of carcinomas and tissue healing capacity. The review provides a comprehensive and up-to-date synthesis of current knowledge regarding the multifaceted effects of cytokines on epithelial cell proliferation, with a particular emphasis on the intestinal epithelium. Also, the paper will highlight the diverse signaling pathways activated by cytokines and their downstream consequences on epithelial cell division. It will also explore the potential therapeutic implications of targeting cytokine- epithelial cell interactions in the context of various diseases.

Basella alba L. (Malabar Spinach) as an Abundant Source of Betacyanins: Identification, Stability, and Bioactivity Studies on Natural and Processed Fruit Pigments.

The antioxidant and anti-inflammatory activities of acylated and decarboxylated gomphrenins, as well as Basella alba L. fruit extract, were investigated in relation to gomphrenin, known for its high biological potential. The most abundant natural acylated gomphrenins, namely, 6'-O-E-caffeoyl-gomphrenin (malabarin) and 6'-O-E-4-coumaroyl-gomphrenin (globosin), were isolated from B. alba extract for the studies. In addition, controlled thermal decarboxylation of gomphrenin in the purified B. alba extract at 65-75 °C resulted in the formation of the most prevalent decarboxylated products, including 17-decarboxy-gomphrenin and 2,17-bidecarboxy-gomphrenin, along with their isoforms. The structures of the decarboxylated pigments were confirmed by NMR analyses. Exploring the matrix effect on pigment reactivity revealed a tremendous increase in the stability of all betacyanins after the initial stage of extract purification using a cation exchanger under various conditions. This indicates the removal of a substantial portion of the unfavorable matrix from the extract, which presumably contains reactive species that could otherwise degrade the pigments. Furthermore, the high concentration of citrates played a significant role in favoring the formation of 2-decarboxy-gomphrenin to a considerable extent. In vitro screening experiments revealed that the tested compounds demonstrated strong anti-inflammatory properties in lipopolysaccharide (LPS)-activated human macrophages. This effect encompassed the selective inhibition of cytokine and chemokine release from activated macrophages, modulation of the chemotactic activity of immune cells, and the regulation of tissue remodeling mediators' release.

Abstract B036: Micromechanical regulation of tumor immune microenvironment

Abstract Although the prognosis of primary breast cancer (BC) is considerably good, the prognosis of therapy-resistant, metastatic BC remains rather poor, thus being the major cause of BC-related deaths. Due to the undifferentiated and heterogenous nature of this disease, immunotherapies have been among the most promising therapeutic strategies for metastatic BC. However, in recent clinical trials, immunotherapies have had a poor success rate for breast cancer. Therefore, new therapeutic strategies and more profound knowledge of BC-specific immune-evasion mechanisms are urgently needed. We have developed a patient-derived explant model (PDEC), that allows us to cultivate patient-derived primary tumor samples. Moreover, the model also maintains alive the tumor resident immune cells, hence providing us with a unique platform for researching immunological drugs in vivo. In our model, tumor cells and tumor-derived immune cells are embedded in a 3D scaffold matrix with altering biological and physical properties. By altering the material in which the tumor cells are embedded we can also research the effect of microenvironmental factors such as stiffness and growth factors on the tumor cells. Microenvironmental stiffness is a well-known regulator of BC identity; in stiff 3D microenvironment breast cancer cells maintain their original identity, while in a softer microenvironment, they dedifferentiate towards more undifferentiated, therapy-resistant, and metastasis-mimicking subtype (Munne et al, 2021). In this study, we observed for the first time how the alterations in breast tumor microenvironmental stiffness also alter the phenotype in the breast tumor immune microenvironment concomitantly. In the soft microenvironment, the cytokine profile is polarized towards more tumor suppressing via upregulation of TGFB and downregulation of IL1B. Moreover, immune response and antigen presentation are heavily downregulated. Lastly, macrophages are depolarized from M1-like toward more tumor-promoting. This effect is induced and mediated by tumor cells and fresh immune cells embedded in similar 3D conditions that fail to secrete tumor suppressive cytokines or silence antitumor immunity. Manipulation of the tumor cell identity and tumor immune microenvironment via alteration of 3D matrices provides us a unique perspective for observing new methods of how metastatic breast cancer creates immune evasion. Moreover, the model allows us to develop novel treatments against metastatic, theraphy resistant breast cancer and test them easily in ex vivo conditions. Citation Format: Aino Peura, Rita Turpin, Maria Salmela, Ruixian Liu, Piia Mikkonen, Juha Klefström, Pauliina M Munne. Micromechanical regulation of tumor immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B036.

T helper cell responses to Opisthorchis viverrini infection associate with host susceptibility.

Opisthorchis viverrini infection is endemic in the lower Mekong subregion. The liver is an organ that worms are drawn to and cause damage. However, the immune-related susceptibility in the liver is poorly understood. In this study, we investigated T helper (Th) cell responses in the liver of BALB/c mice and golden Syrian hamsters during 2-28days post-infection (DPI). We found that Th cell responses were distinct between mice and hamsters in terms of dynamics and polarization. Mice exhibited the early induction of Th1, Th2, Th17, and regulatory T (Treg) cells responses after the presence of O. viverrini worms at 2 DPI. In hamsters, the late induction of Th1/Th17, downregulation of Th2/Treg responses and early elevation of suppressive cytokine interleukin (IL)-10 were found together with swift reduction of Th cell numbers. Interestingly, expressions of IL-4 (Th2 functional cytokine) and Foxp3 (Treg lineage) were completely different between mice and hamsters which elevated in mice but suppressed in hamsters. These results suggest that early induction and well-regulation are related to host resistance. In contrast, late induction of Th cell response might allow immature worms to develop in the host. Our findings provide a greater understanding in Th cell response-related susceptibility in O. viverrini infection which would be targeting immunity for the development of immune-based intervention such as vaccine.

Infectious bursal disease virus VP5 triggers host shutoff in a transcription-dependent manner.

Viruses have evolved intricate mechanisms to evade host antiviral responses and exploit cellular resources by manipulating the expression profile of host genes. During infection, viruses encode proteins with shutoff activity to globally inhibit host protein synthesis, which is an effective strategy for immune evasion. In this study, compelling evidence shows that infectious bursal disease virus (IBDV) infection triggers the suppression of host protein synthesis. Furthermore, using both in vitro and in vivo viral infection models, we have identified that IBDV specifically impedes the transcription of host genes via the shutoff activity of viral VP5, simultaneously conferring advantages to IBDV infection in these circumstances. The proposed mechanism suggests that VP5 competitively binds to RanBP1, disrupting the RanGDP/GTP gradient. This disruption interferes with cellular nucleocytoplasmic transport, impairing the nuclear import of proteins bearing nuclear localization signals. The nuclear transport of pivotal transcriptional regulatory factors, such as p65 and IFN regulatory factor 7, is also compromised, leading to the inhibition of pro-inflammatory cytokines and interferon expression. This newly discovered strategy employed by IBDV enables them to manipulate host gene expression, providing novel insights into how viruses evade host immune responses and establish infections.IMPORTANCEViruses manipulate host processes at various levels to regulate or evade both innate and adaptive immune responses, promoting self-survival and efficient transmission. The "host shutoff," a global suppression of host gene expression mediated by various viruses, is considered a critical mechanism for evading immunity. In this study, we have validated the presence of host shutoff during infectious bursal disease virus (IBDV) infection and additionally uncovered that the viral protein VP5 plays a pivotal role in inhibiting the overall synthesis of host proteins, including cytokines, through a transcription-dependent pathway. VP5 competitively binds with RanBP1, leading to disruption of the Ran protein cycle and consequently interfering with nucleocytoplasmic transport, which ultimately results in the suppression of host gene transcription. These findings unveil a novel strategy employed by IBDV to evade host innate immunity and rapidly establish infection. This study also suggests a novel supplement to understanding the pathway through which viruses inhibit host protein synthesis.

Macrophages enhance regeneration of lateral line neuromast derived from interneuromast cells through TGF-β in zebrafish.

Macrophages play a pivotal role in the response to injury, contributing significantly to the repair and regrowth of damaged tissues. The external lateral line system in aquatic organisms offers a practical model for studying regeneration, featuring interneuromast cells connecting sensory neuromasts. Under normal conditions, these cells remain dormant, but their transformation into neuromasts occurs when overcoming inhibitory signals from Schwann cells and posterior lateral line nerves. The mechanism enabling interneuromast cells to evade inhibition by Schwann cells remains unclear. Previous observations suggest that macrophages physically interact with neuromasts, nerves, and Schwann cells during regeneration. This interaction leads to the regeneration of neuromasts in a subset of zebrafish with ablated neuromasts. To explore whether macrophages achieve this effect through secreted cytokines, we conducted experiments involving tail amputation in zebrafish larvae and tested the impact of cytokine inhibitors on neuromast regeneration. Most injured larvae remarkably regenerated a neuromast within 4 days post-amputation. Intriguingly, removal of macrophages and inhibition of the anti-inflammatory cytokine transforming growth factor-beta (TGF-β) significantly delayed neuromast regeneration. Conversely, inhibition of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) had minor effects on the regeneration process. This study provides insights into how macrophages activate interneuromast cells, elucidating the pathways underlying neuromast regeneration.

DISEASE MODELING IN INFLAMMATORY BOWEL DISEASE (IBD) USING PATIENT-DERIVED EXPLANTS (PDE) REVEALS CHEMOKINES AS A RELEVANT TARGET OF JAK INHIBITION

Abstract Despite a recent explosion of approved drugs for IBD that primarily target cytokines and T-cell trafficking, therapeutic response rates remain low (30-50 %), underscoring the need for more efficacious treatments with fewer side effects. From a drug development perspective, it takes over 10 years for a new drug to reach the market with >90% attrition rates. The realization that drug testing in animal models does not always translate to humans, particularly for complex diseases of high heterogeneity like IBD, leaving a significant gap in pre-clinical drug evaluation. Technologies such as organoids or “gut on a chip” attempted to fulfill this gap, but among their limitations are lack of tissue complexity and an externally induced, and often biased, “disease state” that fails to capture patient heterogeneity and underlying disease mechanisms. We developed a Therapeutic Response Predictor Assay for IBD (TheRPA _ibd) which is based on establishing short term explant cultures from the patient’s diseased tissue (PDE). PDEs can be treated with various candidate drugs or their combinations and inform whether an individual patient is a responder or non-responder to the particular treatment. TheRPA_ibd has been clinically validated for anti-TNF with a predictive accuracy of 0.87 (p<0.0001). TheRPA_ibd was used to comparatively evaluate 6 JAK inhibitors (JAKi) with different JAK isoform selectivity (tofacitinib, baricitinib, upadacitinib, ritlecitinib, filgotinib, and peficitinib). PDEs were derived from mucosal inflamed biopsies obtained during colonoscopy or surgical resection (26 CD/11 UC). JAKi responses were evaluated using targeted and bulk gene expression, antibody arrays and multiplex ELISA. Our analysis, in addition to previously described cytokines, identified several chemokines as downstream targets of JAK inhibition: CXCL1; CXCL2; CXCL3; CXCL5; CXCL8; CXCL9; CXCL10; CXCL11; CXCL16; CCL10; CCL20. Of clinical importance, interrogation of publicly available gene expression IBD datasets confirmed a robust dysregulation of those targets in both UC and CD. Such findings open new avenues for exploring chemokine-driven pathways as potential therapeutic targets in IBD and potentially additional inflammatory conditions. From a mechanistic perspective, JAK1 or JAK3 selective inhibitors appeared to lack broader cytokine and chemokine inhibition compared to pan-JAKi. Notably, as a target, TYK2 was found to be critical for maximizing the anti-inflammatory effects of JAKi. The PDE-based assay (TheRPA) not only enables selection of patient-tailored treatments but also as a “clinical trial in a dish” platform can efficiently contribute to candidate drug de-risking and prioritization. As a proof of concept, using this technology we have been able to demonstrate efficacy in CD of certain JAK inhibitors prior to their FDA approval for this indication.

P128 Salt-Inducible Kinase 2 is a promising therapeutic target in patients with ulcerative colitis

Abstract Background Salt-Inducible Kinases (SIKs) are a family of 3 serine/threonine kinases which represent promising therapeutic targets in a number of inflammatory diseases including ulcerative colitis (UC). Their catalytic activity is highly phosphoregulated in response to changes in cellular homeostasis. Inactivation of SIKs results in proinflammatory cytokine downregulation and the concomitant upregulation of anti-inflammatory IL-10. Of the three isozymes, SIK2 is the most active in myeloid cells and is thought to be the key driver of IL-10 production. Selective pharmacological inhibition of SIKs has thus far proved challenging: a number of SIK inhibitors have previously been developed and whilst they have demonstrated biological activity in UC, dosing has been limited due to potential side effects underlining the need for improved selectivity. We hypothesized that more selective inhibition of SIK2 represents a promising approach in ulcerative colitis. Methods We sought to examine the effects of inhibiting SIK2 with a potent small molecule in both in vitro cell cultures and in ex vivo biopsies from UC patients. CD14+ monocytes were isolated from donor PBMCs and differentiated into hMDMs (monocyte-derived macrophages) and moDCs (monocyte-derived DCs). Cultures were treated with compound followed by LPS stimulation for 4 hours (hMDMs) or maturation for 48 hours (moDCs). To recapitulate the disease microenvironment ex vivo, a colonic explant culture model was used: colonic biopsies were collected from patients with active (Mayo 1 /2) and inactive UC and cultured in the presence of compound or control for 24-48h. Supernatants were assayed for cytokine and chemokine release. Cells or biopsies were lysed for qPCR. Surface markers were assessed by flow cytometry Results Using more SIK2-selective small molecules, we show that SIK inhibition modulates inflammatory and immunometabolic pathways in human myeloid cells. Furthermore, SIK2 inhibition drives a tolerogenic phenotype in dendritic cells, as demonstrated by suppression of proinflammatory cytokines and chemokines such as TNFα and the CC/CXC chemokine family as well as downregulation of activation markers such as CD80 and 83; functionally these modulated DCs can suppress Th1 CD4 T cell IFNγ production. Modulation of cytokine and chemokine production by SIK2 inhibition was also observed in colonic explant cultures from UC patients with a reduction in levels of TNFα and the CC chemokine family. Conclusion In both myeloid cellular cultures and UC biopsies, SIK2 inhibition modulates an inflammatory environment into a pro-resolution state supporting the therapeutic potential of SIK2 inhibition in UC.

Recombinant Newcastle disease viruses expressing immunological checkpoint inhibitors induce a pro-inflammatory state and enhance tumor-specific immune responses in two murine models of cancer.

Tumor microenvironments are immunosuppressive due to progressive accumulation of mutations in cancer cells that can drive expression of a range of inhibitory ligands and cytokines, and recruitment of immunomodulatory cells, including myeloid-derived suppressor cells (MDSC), tumor-associated macrophages, and regulatory T cells (Tregs). To reverse this immunosuppression, we engineered mesogenic Newcastle disease virus (NDV) to express immunological checkpoint inhibitors anti-cytotoxic T lymphocyte antigen-4 and soluble programmed death protein-1. Intratumoral administration of recombinant NDV (rNDV) to mice bearing intradermal B16-F10 melanomas or subcutaneous CT26LacZ colon carcinomas led to significant changes in the tumor-infiltrating lymphocyte profiles. Vectorizing immunological checkpoint inhibitors in NDV increased activation of intratumoral natural killer cells and cytotoxic T cells and decreased Tregs and MDSCs, suggesting induction of a pro-inflammatory state with greater infiltration of activated CD8+ T cells. These notable changes translated to higher ratios of activated effector/suppressor tumor-infiltrating lymphocytes in both cancer models, which is a promising prognostic marker. Whereas all rNDV-treated groups showed evidence of tumor regression and increased survival in the CT26LacZ and B16-F10, only treatment with NDV expressing immunological checkpoint blockades led to complete responses compared to tumors treated with NDV only. These data demonstrated that NDV expressing immunological checkpoint inhibitors could reverse the immunosuppressive state of tumor microenvironments and enhance tumor-specific T cell responses.

Terpenes and cannabidiol against human corona and influenza viruses–Anti-inflammatory and antiviral in vitro evaluation

The activity of the terpenes and Cannabidiol (CBD) against human coronavirus (HCoV) strain OC43 and influenza A (H1N1) was evaluated in human lung fibroblasts (MRC-5 cells). Also, we examined whether these ingredients inhibit pro-inflammatory cytokines in peripheral blood mononuclear cells (PBMC). The tested preparations exhibited both anti-inflammatory and antiviral effects. The combination of terpenes was effective against both HCoV-OC43 and influenza A (H1N1) virus. The addition of CBD improved the antiviral activity in some, but not all cases. This variation in activity may suggest an antiviral mechanism. In addition, there was a strong correlation between the quantitative results from a cell-viability assay and the cytopathic effect after 72 h, as observed under a microscope. The anti-inflammatory properties of terpenes were demonstrated using a pro-inflammatory cytokine-inhibition assay, which revealed significant cytokine inhibition and enhanced by the addition of CBD.

Insights into the mechanisms of diabetic wounds: pathophysiology, molecular targets, and treatment strategies through conventional and alternative therapies.

Diabetes mellitus is a prevalent cause of mortality worldwide and can lead to several secondary issues, including DWs, which are caused by hyperglycemia, diabetic neuropathy, anemia, and ischemia. Roughly 15% of diabetic patient's experience complications related to DWs, with 25% at risk of lower limb amputations. A conventional management protocol is currently used for treating diabetic foot syndrome, which involves therapy using various substances, such as bFGF, pDGF, VEGF, EGF, IGF-I, TGF-β, skin substitutes, cytokine stimulators, cytokine inhibitors, MMPs inhibitors, gene and stem cell therapies, ECM, and angiogenesis stimulators. The protocol also includes wound cleaning, laser therapy, antibiotics, skin substitutes, HOTC therapy, and removing dead tissue. It has been observed that treatment with numerous plants and their active constituents, including Globularia Arabica, Rhus coriaria L., Neolamarckia cadamba, Olea europaea, Salvia kronenburgii, Moringa oleifera, Syzygium aromaticum, Combretum molle, and Myrtus communis, has been found to promote wound healing, reduce inflammation, stimulate angiogenesis, and cytokines production, increase growth factors production, promote keratinocyte production, and encourage fibroblast proliferation. These therapies may also reduce the need for amputations. However, there is still limited information on how to prevent and manage DWs, and further research is needed to fully understand the role of alternative treatments in managing complications of DWs. The conventional management protocol for treating diabetic foot syndrome can be expensive and may cause adverse side effects. Alternative therapies, such as medicinal plants and green synthesis of nano-formulations, may provide efficient and affordable treatments for DWs.