Production Of Factors Research Articles

Effects of aflibercept and bevacizumab on cell viability, cell metabolism and inflammation in hypoxic human Müller cells.

Anti-VEGF (vascular endothelial growth factor) drugs such as aflibercept (AFL) and bevacizumab (BVZ) inhibit pathological neo-angiogenesis and vascular permeability in retinal vascular diseases. As cytokines and growth factors are produced by Müller glial cells under stressful and pathological conditions, we evaluated the in vitro effect of AFL (Eylea®, 0.5 mg/mL) and BVZ (Avastin®, 0.5 mg/mL) on cell viability/metabolism, and cytokine/growth factor production by Müller cells (MIO-M1) under cobalt chloride (CoCl2)-induced hypoxia after 24h, 48h and 72h. Cell viability/metabolism were analyzed by Trypan Blue and MTT assays and cytokine/growth factors in supernatants by Luminex xMAP-based multiplex bead-based immunoassay. Cell viability increased with AFL at 48h and 72h and decreased with BVZ or hypoxia at 24h. BVZ-treated cells showed lower cell viability than AFL at all exposure times. Cell metabolism increased with AFL but decreased with BVZ (72h) and hypoxia (48h and72h). As expected, AFL and BVZ decreased VEGF levels. AFL increased PDGF-BB, IL-6 and TNF-α (24h) and BVZ increased PDGF-BB (72h). Hypoxia reduced IL-1β, -6, -8, TNF-α and PDGF-BB at 24h, and its suppressive effect was more prominent than AFL (EGF, PDGF-BB, IL-1β, IL-6, IL-8, and TNF-α) and BVZ (PDGF-BB and IL-6) effects. Hypoxia increased bFGF levels at 48h and 72h, even when combined with anti-VEGFs. However, the stimulatory effect of BVZ predominated over hypoxia for IL-8 and TNF-α (24h), as well as for IL-1β (72h). Thus, AFL and BVZ exhibit distinct exposure times effects on MIO-M1 cells viability, metabolism, and cytokines/growth factors. Hypoxia and BVZ decreased MIO-M1 cell viability/metabolism, whereas AFL likely induced gliosis. Hypoxia resulted in immunosuppression, and BVZ stimulated inflammation in hypoxic MIO-M1 cells. These findings highlight the complexity of the cellular response as well as the interplay between anti-VEGF treatments and the hypoxic microenvironment.

The role of the placenta-brain axis in psychoneuroimmune programming

Gestational exposures have enduring impacts on brain and neuroimmune development and function. Perturbations of pregnancy leading to placental structure/function deficits, cell stress, immune activation, and endocrine changes (metabolic, growth factors, etc.) all increase neuropsychiatric risk in offspring. The existing literature links obstetric diseases with placental involvement to offspring neuroimmune outcomes and neurodevelopmental risk. Psychoneuroimmune outcomes in offspring brain include changes to microglia, cytokine/chemokine production, cell stress, and long-term immunoreactivity. These outcomes are altered by structural, anti-angiogenic/hypoxic, inflammatory, and metabolic diseases of the placenta. This fetal programming occurs via direct placental passage or production of factors which can act directly on fetal brain substrates, or indirectly via action of circulating factors on intermediates in the placenta. Placental neuroendocrine, vascular/angiogenic, immune, and extracellular vesicular mechanisms are detailed. These mechanisms interact within various placental and pregnancy conditions. An increased understanding of the placental origins of psychoneuroimmunology will yield dividends for human health. Identifying maternal and placental biomarkers for fetal neuroimmune health may also revolutionize early diagnosis and precision psychiatry, empowering patients to make the best healthcare decisions for their families. Targeting placental mechanisms may be a valuable approach for the prevention and mitigation of intergenerational, lifelong neuropathology.

A High MCT-Based Ketogenic Diet Suppresses Th1 and Th17 Responses to Ameliorate Experimental Autoimmune Encephalomyelitis in Mice by Inhibiting GSDMD and JAK2-STAT3/4 Pathways.

Inflammation and pyroptosis play important roles in the pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). In this study, we evaluated the therapeutic potential of ketogenic diet (KD) in EAE. The administration of KD reduces demyelination and microglial activation in the spinal cord of EAE mice. Meanwhile, KD decreases the levels of Th1 and Th17 associated cytokines/transcription factors production (T-bet, IFN-γ, RORγt, and IL-17) and increases those of Th2 and Treg cytokines/transcription factors (GATA3, IL-4, Foxp3, and IL-10) in the spinal cord and spleen. Corresponding, KD reduces the expression of chemokines in EAE, which those chemokines associate with T-cell infiltration into central nervous system (CNS). In addition, KD inhibits the GSDMD activation in microglia, oligodendrocyte, CD31+ cells, CCR2+ cells, and T cells in the spinal cord. Moreover, KD significantly decreases the ratios of p-JAK2/JAK2, p-STAT3/STAT3, and p-STAT4/STAT4, as well as GSDMD in EAE mice. this study demonstrates that KD reduces the activation and differentiation of T cells in the spinal cord and spleen and prevents T cell infiltration into CNS of EAE via modulating the GSDMD and STAT3/4 pathways, suggesting that KD is a potentially effective strategy in the treatment of MS.

Bone Marrow Microenvironment Involvement in t-MN: Focus on Mesenchymal Stem Cells.

Therapy-related myeloid neoplasms (t-MN) are a late complication of cytotoxic therapy (CT) used in the treatment of both malignant and non-malignant diseases. Historically, t-MN has been considered to be a direct consequence of DNA damage induced in normal hematopoietic stem or progenitor cells (HSPC) by CT. However, we now know that treatment-induced mutations in HSC are not the only players involved in t-MN development, but additional factors may contribute to the onset of t-MN. One of the known drivers involved in this field is the bone marrow microenvironment (BMM) and, in particular, bone marrow mesenchymal stem cells (BM-MSC), whose role in t-MN pathogenesis is the topic of this mini-review. BM-MSCs, physiologically, support HSC maintenance, self-renewal, and differentiation through hematopoietic-stromal interactions and the production of cytokines. In addition, BM-MSCs maintain the stability of the BM immune microenvironment and reduce the damage caused to HSC by stress stimuli. In the t-MN context, chemo/radiotherapy may induce damage to the BM-MSC and likewise alter BM-MSC functions by promoting pro-inflammatory response, clonal selection and/or the production of factors that may favor malignant hematopoiesis. Over the last decade, it has been shown that BM-MSC isolated from patients with de novo and therapy-related MN exhibit decreased proliferative and clonogenic capacity, altered morphology, increased senescence, defective osteogenic differentiation potential, impaired immune-regulatory properties, and reduced ability to support HSC growth and differentiation, as compared to normal BM-MSC. Although the understanding of the genetic and gene expression profile associated with ex vivo-expanded t-MN-MSCs remains limited and debatable, its potential role in prognostic and therapeutic terms is acting as a flywheel of attraction for many researchers.

RESPON PETANI KARET THAILAND, INDONESIA, INDIA, DAN LAOS MENGHADAPI RENDAHNYA HARGA KARET

Thailand is the largest natural rubber producer country in the world. In several years ago, the natural rubber price shows a negative trend. This condition make a rubber smallholder face difficulties in life. This paper aims to study efforts made by rubber smallholder to face low rubber prices. Methods in this paper used descriptive analysis with literature studies of the efforts undertaken by rubber smallholder in several rubber producer countries, especially in Thailand, Indonesia, India and Laos to face low rubber price. This study show that the alternative effort undertaken by rubber smallholder to face low rubber price generally was 1) management of cost production by reduced fertilizer and others factor production which no disturbed rubber income in short term condition; 2) develop other crops by intercropping technique on rubber side line; 3) looking for side jobs such as construction laborers, public transportation drivers, opening stalls and catching fish; 4) use of loans to maintain the standard of household living; 5) implement a joint marketing system to obtain a better rubber price; 6) selling or renting old and unproductive rubber land; and 7) renew/replanting rubber plantation.

SISTEM JAJAR LEGOWO (JARWO) STUDI KASUS DESA KARANG TIRTA KECAMATAN LALAN KABUPATEN MUSI BANYUASIN

This research is purposed to know: (1) How much factor production between tabela plants system dan jarwo system is used for rice farming, (2) how much rice farming revenue between tabela plants system and with jarwo plants system, (3) know difference rice farming revenue between tabela plants system and with jarwo plants system. This research hypothesizes that the suspected rice farming revenue jarwo system is not the same as the tabela system. The sampling method in this research is used stratified random sampling, this method have take samples with observed strata or levels in the population, with the following criteria: (1) farmer who used tabela plants system in Karang Tirta village, District Lalan, Musi Banyu Asin Regency, from 664 farmers have taken 20 responded (2) farmers who used jarwo system in Karang Tirta village Laland District Musi Banyuasin Regency, from 172 farmers have taken 20 respondent. The data used is primer data dan secondary, using a list question structure (questioner). The data secondary is obtained from institutions' government, literature, and other sources which connect with this research. The result of this research is no difference significant in revenue between jarwo system and tabela system. Farmers who used tabela system have got revenue Rp. 14.417.994,-/Ha in one season, which is bigger between farmers who used jarwo system and have got average revenue RP. 13.489.051,-/Ha in one season.

Unravelling the Effect of Blood Type on FVIII:C Levels and Response to DDAVP in 20 Males with a Single Genotype (Twillingate Variant) Causing Mild Hemophilia Α

Introduction: Hemophilia A (HA) occurs secondary to >1000 variants in the F8 gene resulting in decreased/absent production of factor (F) VIII. FVIII:C (coagulant) levels in persons with mild/moderate HA are most impacted by their genotype. ABO blood type (BT) may also impact endogenous FVIII:C levels, but this impact is difficult to assess in a genetically heterogenous group of HA subjects. Canada has a population of mild-moderate HA secondary to a unique large single genotype: c.6104T>C, p.Val2035Ala. This variant arose from a founder effect that occurred about 200 years ago in Twillingate, Newfoundland. We hypothesized that subjects with the Twillingate genotype (despite having the same genotype and thus producing similar amounts of FVIII) would show different endogenous FVIII:C levels related to their BT, as BT impacts FVIII (and VWF) clearance. Our study objectives were to understand the impact of BT on endogenous FVIII:C levels whilst controlling for genotype, and to explore the impact of BT on DDAVP responsiveness. Methods: This was a retrospective, single-center cohort study. Males with a confirmed Twillingate genotype treated at our center between 1980-01-01 to 2022-03-01 were eligible. For each subject, ABO BT, VWF antigen (Ag) and activity, and FVIII:C levels were determined by chart review. We recorded the lowest FVIII:C baseline level; for subjects who had >1 FVIII:C level (done in a non-bleeding state) we also determined their mean baseline FVIII:C level. For DDAVP challenges, the age at the challenge, dose, and route of administration were recorded, as well as pre and 1-hr post FVIII:C levels. Based on 1-hr post-DDAVP FVIII:C levels (IU/mL), subjects were classified on their DDAVP response as responders (≥0.50), partial responders (0.30 to 0.49), or non-responders (<0.30). Median, IQR, mean, and SD were calculated for baseline FVIII:C and VWF:Ag levels, stratified by age (<12; ≥12 y) and by BT (O; non-O). T-tests and Mann-Whitney U (MWU) tests were used to compare normally and non-normally distributed data, respectively. The Fisher-Freeman-Halton Exact Test was used to compare responses to DDAVP between O and non-O subjects. Statistical significance was considered as p≤0.05. All analyses were performed using IBM SPSS Statistics Version 28. Results: 20 males were included: 9 were O BT and 11 were non-O BT (10 were A, 1 was B). There were statistically significant differences between the 2 ABO groups in their median lowest FVIII:C levels: 0.05 IU/mL (O BT) vs. 0.08 IU/mL (non-O BT); p=0.05 (MWU) and in mean FVIII:C levels: medians: 0.08 IU/mL (O BT) vs. 0.12 IU/mL (non-O BT); p=0.02 (t-test). Too few subjects had FVIII:C levels reported at ≥12 y of age to perform statistical analyses. Fifteen subjects (8 O BT: 7 non-O BT) underwent a DDAVP challenge; 13 performed at our center and 2 elsewhere. Median 1-hr post-DDAVP FVIII:C levels were 0.32 IU/mL (O BT) and 0.43 IU/mL (non-O BT); p=0.04 (t-test). The absolute FVIII:C increase with DDAVP was not statistically different between the 2 groups: median increase of 0.22 IU/mL (O BT) vs. 0.28 IU/mL (non-O BT); p=0.10 (t-test) nor was the FVIII:C fold increase: median increase 3.3-fold (O BT) vs. 3.3-fold (non-O BT); p=0.51 (t-test). Among O BT subjects, none were responders, 5 were partial responders and 3 were non-responders. In the non-O BT subjects, 2 were responders and 5 were partial responders. The variation in response between the 2 groups failed to achieve statistical significance (p=0.14). Given uncertainties in DDAVP challenges (e.g. dose, route of administration) done elsewhere, we also analyzed the DDAVP challenge results just for the 13 subjects from our center. The median 1-hr post-DDAVP FVIII:C levels were 0.30 IU/mL in 7 O BT subjects and 0.45 IU/mL in 6 non-O BT subjects; p=0.02 (t-test). Conclusions: This study shows that in HA subjects with an identical genotype, BT significantly impacts FVIII:C levels. Although only 1 unique genotype was studied, we believe that other hemophilic variants leading to mild HA will behave similarly. This may explain why in subjects with mild HA, there is an overrepresentation of O BT. Persons with non-O BT and HA variants that only slightly reduce FVIII production may have FVIII:C levels that are not as reduced and thus may never present with bleeding and be diagnosed with HA. In contrast, if such persons have O BT, they are likely to show much lower FVIII:C levels and thus more likely to bleed and be diagnosed with HA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Modulation of local and systemic immune responses by fermented garlic extract

Fermented garlic (FG), prepared by fermenting garlic with <em>Bacillus subtilis</em> for 3‑15 days at 20‑40˚C has been linked to immune‑boosting properties. In the present study, FG was utilized to investigate its immunomodulatory effects using RAW264.7 cells, as well as BALB/c and C3H/HeN mice. Nitric oxide (NO) and pro‑inflammatory cytokine levels were quantified in RAW264.7 cells treated with or without FG (0.016, 0.08, 0.4, 2 and 10 mg/ml), garlic extract (GE) (0.016, 0.08, 0.4, 2 and 10 mg/ml) and lipopolysaccharide (1 µg/ml). Peritoneal macrophages and splenocytes were obtained from BALB/c mice for <em>ex vivo</em> analyses. MTT and enzyme‑linked immunosorbent assays were used to determine splenocyte proliferation, natural killer (NK) cell activity and cytokine production. The intestinal immunostimulatory <em>in vitro</em> effects of FG were also investigated. In a concentration‑dependent manner, FG treatment markedly accelerated the proliferation of RAW264.7 cells, murine peritoneal macrophages and splenocytes. Moreover, FG initiated macrophage activation by significantly increasing the NO, tumor necrosis factor‑α, interleukin (IL)‑12 and IL‑6 levels in RAW264.7 cells and peritoneal macrophages. In contrast to FG, GE promoted RAW264.7 cell proliferation at lower concentrations and toxicity at higher concentrations, while no detectable NO synthesis was observed at the concentrations tested. Likewise, the FG extract at larger concentrations fortified the intestinal immune system and improved bone marrow cell proliferation by significantly elevating IL‑6 and granulocyte‑macrophage colony‑stimulating factor production in splenocytes and Peyer's patch cells of mice. In addition, the NK cell activities of splenocytes from FG‑treated mice at lower doses against YAC‑1 cells were significantly increased, which must have been fostered and activated by interferon‑γ generated by splenocytes. On the whole, these findings provide relevant <em>in vitro</em> and <em>ex vivo</em> information that describes the mechanisms through which FG can strengthen a compromised immune system by influencing local and systemic gut immune responses directly or indirectly.

The Use of Vascular Endothelial Growth Factor Inhibitors in Patients with Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) have been taking a leading position among retinal disorders, causing a persistent and significant decrease in visual functions. The base of the disease is a retinal vascular microangiopathy, which induces a tissue ischemia and excessive production of vascular endothelial growth factors. The main method of treatment for this pathology is a laser photocoagulation. However, it is not always possible to perform appropriate laser photocoagulation, by that limiting its efficacy (cases of hard cataract and hemophthalmos). In addition, this type of treatment might have a number of serious complications, such as macular oedema, recurrent vitreous hemorrhages, the occurrence of fibrosis, atrophic changes in the retina, which leads to a decline in visual acuity and contrast sensitivity, worsening the visual field. Over the past 10 years, vascular endothelial growth factor inhibitors have been introduced into clinical practice, and showed their benefits in the treatment of disorders leading to a pathological angiogenesis. But due to the limited number of systematic observations, assessing the impact of these drugs, is necessary to investigate their efficacy and safety in patients with proliferative DR, in order to develop indications for their use. The purpose of the review is evaluation of the effect of vascular endothelial growth factor inhibitors on active neovascularization in patients with proliferative diabetic retinopathy. There is a literature of international clinical guidelines, randomized clinical trials with a varying level of evidence, articles and publications by groups of authors in the article.

Enterococcus spp. in gut microbiota of patients with pulmonary tuberculosis

Aim. To defne the role of Enterococcus spp. in the gut microbiota ensemble in patients with pulmonary tuberculosis. Materials andMethods. Inoculation of intestinal cultures was performed in 64 patients with multidrug-resistant tuberculosis before the specific therapy (30 patients and 55 strains) and after 1 month of the treatment (34 patients and 63 strains). We then assessed the production of haemolysins, gelatinase, phospholipase, adhesion factors, and organic acids.Results. A high prevalence of intestinal dysbiosis (66.7%) were detected in patients before the start of anti-tuberculosis therapy. Specifc treatment was associated with a decrease in Lactobacillus spp. along with an increase in C. perfringens and Candida spp. titers as well as with higher prevalence of E. coli lac. Before the treatment, Enterococcus spp. showed pronounced adhesion properties, providing colonization of the intestinal mucosa in titers of 6 (4; 7) lg CFU/g, although not producing invasion enzymes. Upon the 1 month of anti-tuberculosis treatment, adhesion capability reduced and 36% of the strains were low-adhesive. An increase of lipase-producing strains to 18% suggested the adaptation of Enterococcus spp. to a high content of lipids and fatty acids in the intestine.Conclusion. In patients with pulmonary tuberculosis having high prevalence of intestinal dysbiosis, Enterococcus spp. are frequent symbionts well integrated into the gut microbiota.

NAD kinase promotes Staphylococcus aureus pathogenesis by supporting production of virulence factors and protective enzymes.

Nicotinamide adenine dinucleotide phosphate (NADPH) is the primary electron donor for reductive reactions that are essential for the biosynthesis of major cell components in all organisms. Nicotinamide adenine dinucleotide kinase (NADK) is the only enzyme that catalyzes the synthesis of NADP(H) from NAD(H). While the enzymatic properties and physiological functions of NADK have been thoroughly studied, the role of NADK in bacterial pathogenesis remains unknown. Here, we used CRISPR interference to knock down NADK gene expression to address the role of this enzyme in Staphylococcus aureus pathogenic potential. We find that NADK inhibition drastically decreases mortality of zebrafish infected with S. aureus. Furthermore, we show that NADK promotes S. aureus survival in infected macrophages by protecting bacteria from antimicrobial defense mechanisms. Proteome-wide data analysis revealed that production of major virulence-associated factors is sustained by NADK. We demonstrate that NADK is required for expression of the quorum-sensing response regulator AgrA, which controls critical S. aureus virulence determinants. These findings support a key role for NADK in bacteria survival within innate immune cells and the host during infection.

Effect of estrogen application timing on adipose tissue revascularization and immune regulation in rats after autologous fat transplantation

Purpose: To study the effect of timing of estrogen application on adipose tissue revascularization and immune regulation in rats given autologous fat transplantation.Methods: Ninety (90) healthy rats were selected for use in this study. The rats were randomly divided into a study group (n = 45) and control group (n = 45). After ovariectomy, the study group was given estrogen replacement therapy before autologous fat transplantation, while the control group was given estrogen replacement therapy after autologous fat transplantation. The rats were observed for 6 weeks after transplantation. Micro-vessel density, wet weight of transplanted fat, VEGF expression level, levels of M1 and M2 in macrophages, and macrophage infiltration rate were determined 40 days postautologous fat transplantation.Results: Forty days after autologous fat transplantation, microvessel density, wet weight of transplanted fat, expression level of VEGF and levels of M1 and M2 in macrophages were significantly higher in study group rats than in controls (p &lt; 0.05). There was 80 % macrophage infiltration in the study group rats, while the corresponding control value (61 %) was significantly lower (p &lt; 0.05).Conclusion: Estrogen treatment before autologous fat transplantation in rats is helpful for the revascularization of adipose tissue, enhances vascular regeneration, promotes the survival of adipose tissue after transplantation, and activates immune cells. It also promotes the production of immune factors, and improves immunoregulation in adipose tissue. Thus, this therapeutic strategy may be useful in clinical practice, but further clinical trials are required.

Abstract CT189: PRTX007, an optimized TLR7 agonist for systemic immunotherapy of cancers: Interim analysis of phase I study in healthy volunteers

Abstract Background. Toll-like receptor 7 (TLR7) agonists have long been recognized as potential systemic anti-cancer immunotherapy agents. However, clinical development has been limited by complex target-mediated pharmacology including those associated with elevated levels of proinflammatory factors, which also limit the ability to combine with immune checkpoint inhibitors (CPI). PRTX007 is an orally administered prodrug of PRX034, a novel TLR7 agonist that elicits IFN-mediated immune induction without inducing inflammation via the NF-κβ pathway. QOD dosing in primates maintains a stable immune response without immune exhaustion or toxicity. Efficacy, safety, and compatibility with immune CPI were demonstrated in a CT26 syngeneic rodent tumor model. Methods. We conducted a phase 1, single-center, prospective, randomized, double-blind placebo-controlled study of 8 single-ascending dose (SAD) and 4 multiple-ascending dose (MAD) cohorts of PRTX007 administered orally to healthy adult participants. Primary objectives were safety and tolerability. Secondary objectives were evaluation of pharmacokinetics (PK) and pharmacodynamics. This interim analysis focuses on the 8 SAD (50-600 mg) and 2 MAD (300-400 mg) cohorts, with 64 of the 84 participants in the study receiving drug. Results. Treatment-related adverse events include non-dose‒related mild headache, and minor dose-related increases in ALT (&amp;lt;2X ULN) that rapidly resolved. The PKs of PRTX007 and PRX034 are well-behaved eg, exposure increasing proportionally with PRTX007 dose. Consistent with studies in human cells in vitro and non-human primates in vivo, oral administration of PRTX007 elicits systemic TLR7-mediated immune induction without engaging NF-κβ─mediated biosynthesis of IL-6, TNFα and IL-1β in humans. A single dose of PRTX007 (300-600 mg) elicits coordinated expression of IFN-stimulated genes (ISGs) in blood without quantifiable increases in circulating IFNs. At 500 and 600 mg doses, IP-10, IL-1RA, MCP-1 and TRAIL but not IL-6, TNFα or IL-1β increase in plasma (maximal response = 4020, 1460, 650 and 360 pg/ml, respectively). When PRTX007 is administered for 2 weeks on a QOD schedule at 400 mg/dose, the degree of ISG expression progressively increases when compared to the first-dose response, (eg, ISG15 increases 2.1-fold [geometric mean] following Dose 1 vs pretreatment baseline, rising to 12.2-fold following Dose 7). This is accompanied by modest activation of circulating NK, B, and CD4+ and CD8+ T cells as measured by cell-surface markers. IL-6, TNFα and IL-1β remain unchanged from pretreatment levels. Conclusion. At the interim analysis, PRTX007 demonstrated a favorable safety profile when administered orally to all tested SAD and MAD cohorts. Dose-dependent systemic exposure was observed, as well as activation of the innate immune response, without production of proinflammatory factors. Citation Format: Charlotte Lemech, Christopher Argent, Curtis Scribner, Richard Daniels, James Richard Appleman. PRTX007, an optimized TLR7 agonist for systemic immunotherapy of cancers: Interim analysis of phase I study in healthy volunteers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT189.

Abstract SY24-03: Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy

Abstract Background: Cancer immunotherapies can lead to durable and sometimes curative responses in patients with otherwise treatment-refractory disease, and as such have revolutionized our approach to cancer therapy. Still, there are many roadblocks that must be overcome to make immunotherapies effective across a broader spectrum of patients and cancer types. Immune checkpoint blockade (ICB) therapies that are effective in some malignancies through reactivation of cytotoxic T cell immunity have failed in immunologically “cold” tumors such as pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and weak antigen presentation necessary for effective anti-tumor T cell responses. We and others have demonstrated that induction of cellular senescence and the senescence associated secretory phenotype (SASP), which leads to production of immunomodulatory cytokines, chemokines, and growth factors, can be a powerful way to not only enhance T cell infiltration into tumors but also reactivate a different type of innate, antigen-independent Natural Killer (NK) cell immunity that can mediate tumor control. Recently, we found that a combination of two RAS pathway targeting therapies- the MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib (T/P)- could induce senescence in genetically engineered mouse models of KRAS mutant lung adenocarcinoma and PDAC (Ruscetti*, Leibold*, Bott* et al. Science 2018; Ruscetti*, Morris*, Mezzadra* et al. Cell 2020). Strikingly, whereas therapy-induced senescence led to NK cell-mediated tumor regressions and long-term survival benefit in KRAS mutant lung cancer, it had little effect on NK cell immunity and tumor progression in PDAC. Here we set out to understand how the pancreas tumor microenvironment (TME) suppresses NK immunity and develop strategies to harness NK cell surveillance for immunotherapy in PDAC. Results and Discussion: We took advantage of genetically similar Kras mutant, p53 altered PDAC and lung cancer cell lines that could be transplanted into different organs of C57BL/6 mice to study whether and how the pancreas TME may impact senescence-driven immune responses. Syngeneic KRAS mutant lung and PDAC tumor cells transplanted into the pancreas of C57BL/6 mice displayed transcriptional repression and reduced chromatin accessibility of SASP transcriptional activators (NF-KB, IRFs) and factors necessary for NK cell activity (IL-15, IL-18) and chemotaxis (CCL2, CCL7, CCL8, CXCL9, CXCL10) following T/P-induced senescence, and did not undergo anti-tumor NK immune surveillance as compared to tumors propagated in the lungs and liver. Transcriptomic analysis identified induction of EZH2 and epigenetic repression of its targets, including key SASP factors and regulators, specifically in tumor cells grown in the pancreas TME. Genetic or pharmacological inhibition of EZH2 enhanced proinflammatory SASP signaling and resulted in NK cell migration and anti-tumor cytotoxicity in murine and human PDAC lines and transplanted and genetically engineered PDAC mouse models. Remarkably, tumor-intrinsic EZH2 knockdown in combination with T/P treatment led to complete tumor regressions in some PDAC-bearing mice that were reversed following NK cell depletion or suppression of key SASP factors. Pharmacological inhibition of EZH2 methyltransferase activity also enhanced NK cell infiltration and tumor control following therapy-induced senescence in transplanted and genetically engineered PDAC mouse models. Importantly, we found that an EZH2 repression signature was associated with enhanced NK cell infiltration and SASP induction in human PDAC patient samples. These results demonstrate that EZH2 mediates transcriptional repression of the proinflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK cell surveillance in PDAC to achieve immune-mediated tumor responses. In the future we plan to test these therapeutic interventions in combination with anti-PD-1/PD-L1 ICB therapy as a means to further activate both NK and T cell immunity to fully eradicate pancreatic cancer. This work highlights the importance of understanding the impact of the resident tumor microenvironment on anti-tumor immune responses to come up with new paradigms to potentiate immunotherapy in this and other immunologically “cold” tumor settings that are historically unresponsive to ICB therapy. Citation Format: Marcus Ruscetti. Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY24-03.

Abstract 1579: Increased stromal-oriented MAOB expression associated with poor clinical outcomes promotes prostate cancer progression

Abstract Background. Stromal fibroblasts, the predominant cell type within the tumor microenvironment, plays a critical regulatory role in prostate cancer (PC) by engaging in stromal-epithelial interactions to dictate cancer initiation, growth, and progression. Monoamine oxidase B (MAOB) catalyzes oxidative deamination of biogenic and dietary monoamines and generates hydrogen peroxide as a by-product, which can predispose stromal cells to a reactive, cancer-promoting state with elevated production of extracellular matrix molecules, growth factors, cytokines, and chemokines. This study investigated the clinical relevance and previously unrecognized function of stromal MAOB in PC. Methods. The stromal MAOB expression levels were determined in PC clinical specimens by immunohistochemistry and in publicly available multi-omics cancer datasets, which were associated with different clinical parameters. The human prostatic stromal fibroblast PrSC cells engineered for stable overexpression or knockdown of MAOB were co-cultured with a panel of human PC cells in 2-D or 3-D fashion, followed by examination of cancer cell behavior by cell proliferation, migration, and invasion assays. MAOB-knockdown PrSC cells were co-inoculated with luciferase-tagged PC cells as subrenal tissue recombinant xenografts into mice for assessment of MAOB’s impact on tumor growth by bioluminescence imaging. Results. MAOB expression was induced in the stroma associated with primary PC compared to normal prostatic tissues of clinical samples, which was corroborated by the same MAOB expression trend in three matched pairs of patient-derived fibroblasts. Following the analysis of three independent clinical cohorts, increased stromal MAOB levels were shown to be correlated with higher Gleason scores, castration resistance, survival, and appearance of neuroendocrine differentiation as evidenced by CHGA expression in the adjacent epithelia. Co-culturing MAOB-overexpressing PrSC cells with several human PC cell lines (C4-2, PC-3, DU145 and C4-2BENZR) accelerated cancer cell proliferation, migration, and invasion. Conversely, these cancer cell phenotypes were suppressed in the presence of MAOB-knockdown PrSC cells. Additionally, C4-2 tumor xenografts when co-inoculated with MAOB-knockdown PrSC grew to a substantially smaller size in mice, which was paralleled by reduced Ki-67 index and reactive stromal marker αSMA expression in the tumor and stromal compartments respectively, compared to controls. Conclusion. Our results suggest that the elevated MAOB expression in PC stromal cells is associated with poor clinical outcomes and further contributes to adjacent PC growth, which provides new insights into understanding the stromal support of PC pathogenesis and progression. Funding provided by the NIH/NCI grant R01CA258634 and WSU CPPS startup fund (to B.W). Citation Format: Tainjie Pu, Jing Wang, Chia-Hui Chen, Tzu-Ping Lin, Boyang Wu. Increased stromal-oriented MAOB expression associated with poor clinical outcomes promotes prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1579.

Shape, Resonant Frequency and Thermoelastic Dissipation Analysis of Free-Formed Microhemispherical Shells Based on Forming Process Modeling.

Free-form microhemispherical shell resonators have the advantages of high quality factor and mass production. The shape of microhemispherical shells created via this process is based on a single mold and is difficult to adjust, which affects the resonant frequency and quality factor. In this paper, a process analysis model is established through in-depth analysis of the process mechanism and flow of the free-forming method. Based on this model, the influence of the designed preforming parameters on the shape, resonant frequency and thermoelastic dissipation of the microhemispherical shell are analyzed in detail, providing theoretical guidance for parameter design. The results show that the depth and the ratio of internal to external pressure of the substrate’s annular groove affect the height and thickness of the microhemispherical shell, and the structural thickness affects the thickness of the microhemispherical shell; these in turn affect the resonant frequency and thermoelastic dissipation of the microhemispherical shell resonator. In addition, the inner diameter of the substrate’s annular groove mainly affects the radius of the support column of the microhemispherical shell, and the influence on the resonant frequency and thermoelastic dissipation of the resonator is relatively low.

225-OR: 5-Methoxytryptamine Improves Obesity-Related Inflammation and Insulin Resistance by Regulating M1/M2 Macrophage Polarization

In our previous study, we demonstrated that hypoglycemic agent DPP-4i changed the gut microbiota composition in diabetic patients and mice, and the DPP-4i-altered microbiome improved glucose tolerance in colonized mice. However, the mediators and their mechanisms remain to be elucidated. Comparing the intestinal metabolite profiles of HFD mice treated with or without sitagliptin (Sit) , we identified tryptophan derivative 5-methoxytryptomine (5-MT) was obviously increased after Sit treatment. In HFD and db/db mice, 5-MT treatment significantly reduced the body weight, improved glucose tolerance and insulin resistance, and ameliorated hepatic steatosis and inflammation. Drug affinity responsive target stability (DARTS) assay demonstrated the interaction between 5-MT and Aryl hydrocarbon receptor (AhR) . Specific inhibition of AhR attenuated the effects of 5-MT on improving insulin resistance. Furthermore, molecular mechanism exploration revealed that 5-MT promoted the expression and secretion of chemokine Cxcl14, which promoted macrophages maturation into alternative activation (M2-like) , subsequently inhibiting the production of inflammatory factors. These data demonstrate novel beneficial roles of 5-MT in improving obesity-related inflammation, insulin resistance and hepatic steatosis. Funding National Science Fund for Distinguished Young Scholars of China (No.81925007) , National Natural Science Foundation of China (No.81700757)

Diagnostic methods of the impact on the life’s quality of lumbar pain syndromes in workers of harmful industries

Occupational diseases associated with pain syndrome in the lower part are one of the most common causes of physical and psychological health disorders. Lower back pain (LBP) also affects labor productivity and the social sphere of life and is becoming an increasingly important factor in the growth of medical care costs. The study aims to research a comprehensive study of the impact of chronic LBP on the quality of life of workers in unfavorable conditions of metallurgical production. Experts have carried out a hygienic assessment of working conditions according to the data of the special assessment of working conditions cards, a questionnaire (a questionnaire of disorders of vital activity in Oswestrovsky's lower back pain, a questionnaire of McGill's pain) and a clinical examination of 132 employees (men) of various industries of the metallurgical enterprise. We analyzed the results of additional instrumental examination methods: radiography of the lumbar spine and electroneuromyography of the nerves of the lower extremities. Under the working conditions of workers of the leading professions of metallurgical production, combined and combined effects of harmful production factors are characteristic, the integral assessment of which corresponds to class 3.2. The severity of the labor process among employees of the studied professions was within classes 2.0.-3.1. According to the McGill questionnaire experts have found statistically significant differences in the main occupational groups: the highest pain indicators were in the group of wire-drawing workers (index - 7.5±4.9; rank - 14.6±10.2) and drivers of intra-factory vehicles (index - 6.9±4.4; rank - 15.1±11.3). After the treatment there were positive dynamics in the values of rank indicators on the affective and evaluative scales (p&lt;0.05). Researchers have identified statistically significant differences between all the studied groups (F=2.55, p=0.041), when using the Oswestrovsky questionnaire. There were degenerative-dystrophic changes of the spine on radiographs of the lumbar spine. According to the results of an electroneuromyographic study, the authors revealed signs of dysfunction at the level of segments L5-S1-2 of the lumbosacral spine. As a result of the study, we have revealed the relationship between the early detection of disorders of the musculoskeletal system with the peculiarities of the labor process and the deterioration of the quality of worker’s life in the production of hardware. To assess the intensity of LBP in workers engaged in harmful working conditions, it is possible to use the McGill and Oswestrovsky pain questionnaires. The detection of pain syndrome and early rehabilitation in people with occupational diseases of the musculoskeletal system contributes to prolonging the working age and improving the quality of life in people with physical overload in the workplace.

Peptide nano-blanket impedes fibroblasts activation and subsequent formation of pre-metastatic niche

There is evidence to suggest that the primary tumor induces the formation of a pre-metastatic niche in distal organs by stimulating the production of pro-metastatic factors. Given the fundamental role of the pre-metastatic niche in the development of metastases, interruption of its formation would be a promising strategy to take early action against tumor metastasis. Here we report an enzyme-activated assembled peptide FR17 that can serve as a “flame-retarding blanket” in the pre-metastatic niche specifically to extinguish the “fire” of tumor-supportive microenvironment adaption. We show that the in-situ assembled peptide nano-blanket inhibits fibroblasts activation, suppressing the remodeling of the metastasis-supportive host stromal tissue, and reversing vascular destabilization and angiogenesis. Furthermore, we demonstrate that the nano-blanket prevents the recruitment of myeloid cells to the pre-metastatic niche, regulating the immune-suppressive microenvironment. We show that FR17 administration effectively inhibits the formation of the pulmonary pre-metastatic niche and postoperative metastasis, offering a therapeutic strategy against pre-metastatic niche formation.

Catalpol Attenuates Pulmonary Fibrosis by Inhibiting Ang II/AT1 and TGF-β/Smad-Mediated Epithelial Mesenchymal Transition

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive and devastating chronic lung condition affecting over 3 million people worldwide with a high mortality rate and there are no effective drugs. Angiotensin II (Ang II), as a major effector peptide of the renin angiotensin aldosterone system, has been shown to act in tandem with the transforming growth factor-β (TGF-β) signaling pathway to promote the infiltration of inflammatory cells, production of reactive oxygen species (ROS) and profibrotic factors after lung injury, and to participate in the process of epithelial mesenchymal transition (EMT). Catalpol (CAT) has been shown to have anti-inflammatory and antifibrotic effects. However, the effects and mechanisms of CAT on pulmonary fibrosis are not clear.PurposeTo assess the effects and mechanisms of catalpol on bleomycin-induced pulmonary fibrosis in mice.MethodsWe used bleomycin-induced mouse model of pulmonary fibrosis to evaluate the alleviation effect of CAT at 7, 14, 28d, respectively. Next, enzyme-linked immunosorbent assay, hematoxylin-eosin staining, immunofluorescence, Masson trichrome staining and western blotting were used to study the underlying mechanism of CAT on bleomycin-induced pulmonary fibrosis.ResultsIt's demonstrated that CAT exerted a potent anti-fibrotic function in BLM-induced mice pulmonary fibrosis via alleviating inflammatory, ameliorating collagen deposition, reducing the level of Ang II and HYP and alleviating the degree of EMT. Moreover, CAT attenuate BLM-induced fibrosis by targeting Ang II/AT1 and TGF-β/Smad signaling in vivo.ConclusionCAT may serve as a novel therapeutic candidate for the simultaneous blockade of Ang II and TGF-β pathway to attenuate pulmonary fibrosis.