Alpha Production Research Articles

Lithospermum erythrorhizon Siebold & Zucc. extract reduces the severity of endotoxin-induced uveitis

BackgroundUveitis is an inflammatory eye condition that threatens vision, and effective anti-inflammatory treatments with minimal side effects are necessary to treat uveitis. PurposeThis study aimed to investigate the effects of Lithospermum erythrorhizon Siebold & Zucc. against endotoxin-induced uveitis in rat and mouse models. MethodsEndotoxin-induced uveitis models of rats and mice were used to evaluate the effects of l. erythrorhizon treatment. Clinical inflammation scores and retinal thickness were assessed in the extract of l. erythrorhizon-treated rats. Histopathological examination revealed inflammatory cell infiltration into the ciliary body. Protein concentration, cellular infiltration, and prostaglandin-E2 levels were measured in the aqueous humor of the extract of l. erythrorhizon-treated rats. Protective effects of l. erythrorhizon on the anterior segment of the eye were examined in mice with endotoxin-induced uveitis. Additionally, we investigated the effect of l. erythrorhizon on the expression of pro-inflammatory cytokines [tumor necrosis factor alpha, interleukin-6, and interleukin-8] in lipopolysaccharide-stimulated THP1 human macrophages and examined the involvement of nuclear factor kappaB/activator protein 1 and interferon regulatory factor signaling pathways. Furthermore, three components of l. erythrorhizon were identified and assessed for their inhibitory effects on LPS-induced inflammation in RAW264.7 macrophage cells. ResultsTreatment of the extract of l. erythrorhizon significantly reduced clinical inflammation scores and retinal thickening in rats with endotoxin-induced uveitis. Histopathological examination revealed decreased inflammatory cell infiltration into the ciliary body. The extract of l. erythrorhizon effectively reduced the protein concentration, cellular infiltration, and PG-E2 levels in the aqueous humor of rats with endotoxin-induced uveitis. In mice with endotoxin-induced uveitis, the extract of l. erythrorhizon demonstrated a protective effect on the anterior segment of the eye by reducing inflammation and retinal thickening. The extract of l. erythrorhizon suppressed the expression of pro-inflammatory cytokines (tumor necrosis factor alpha, interleukin-6, and interleukin-8) in lipopolysaccharide-induced inflammation in THP1 human macrophages, by modulating nuclear factor kappaB/activator protein 1 and interferon regulatory factor signaling pathways. Moreover, shikonin, acetylshikonin, and β, β-dimethylacryloylshikonin showed dose-dependent inhibition of nitric oxide, tumor necrosis factor alpha and interleukin-6 production in RAW264.7 macrophage cells. ConclusionThe extract of l. erythrorhizon is a potential therapeutic agent for uveitis management. Administration of the extract of l. erythrorhizon led to reduced inflammation, retinal thickening, and inflammatory cell infiltration in rat and mouse models of uveitis. The compounds (shikonin, acetylshikonin, and β, β-dimethylacryloylshikonin) identified in this study played crucial roles in mediating the anti-inflammatory effects of l. erythrorhizon. These findings indicate that the extract of l. erythrorhizon and its constituent compounds are promising candidates for further research and development of novel treatment modalities for uveitis.

Pseudotyping Improves the Yield of Functional SARS-CoV-2 Virus-like Particles (VLPs) as Tools for Vaccine and Therapeutic Development.

Virus-like particles (VLPs) have been proposed as an attractive tool in SARS-CoV-2 vaccine development, both as (1) a vaccine candidate with high immunogenicity and low reactogenicity and (2) a substitute for live virus in functional and neutralization assays. Though multiple SARS-CoV-2 VLP designs have already been explored in Sf9 insect cells, a key parameter ensuring VLPs are a viable platform is the VLP spike yield (i.e., spike protein content in VLP), which has largely been unreported. In this study, we show that the common strategy of producing SARS-CoV-2 VLPs by expressing spike protein in combination with the native coronavirus membrane and/or envelope protein forms VLPs, but at a critically low spike yield (~0.04-0.08 mg/L). In contrast, fusing the spike ectodomain to the influenza HA transmembrane domain and cytoplasmic tail and co-expressing M1 increased VLP spike yield to ~0.4 mg/L. More importantly, this increased yield translated to a greater VLP spike antigen density (~96 spike monomers/VLP) that more closely resembles that of native SARS-CoV-2 virus (~72-144 Spike monomers/virion). Pseudotyping further allowed for production of functional alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) SARS-CoV-2 VLPs that bound to the target ACE2 receptor. Finally, we demonstrated the utility of pseudotyped VLPs to test neutralizing antibody activity using a simple, acellular ELISA-based assay performed at biosafety level 1 (BSL-1). Taken together, this study highlights the advantage of pseudotyping over native SARS-CoV-2 VLP designs in achieving higher VLP spike yield and demonstrates the usefulness of pseudotyped VLPs as a surrogate for live virus in vaccine and therapeutic development against SARS-CoV-2 variants.

An enzyme-free method for isolating testicular macrophages from rodent models

Macrophages are the major type of immune cell in the testis of both humans and rodents. Testicular macrophages (TMs) play critical roles in maintaining the testicular microenvironment, such as Leydig cell-dependent hormone production, spermatogenesis, and immune balance. A substantial number of studies have used rodent models to investigate the functions of TMs with various methods and harvest macrophages from the testis. Studies have demonstrated that enzyme digestion, an essential part of these methods, can improve the number and purity of TMs while unavoidably altering the immunoprofile of macrophages, which is detrimental for further study in terms of immune investigation. Here, we modified the existing method of microglia isolation and set up a novel method without the enzyme digestion step to isolate TMs. According to the characteristics of testicular tissue looseness and the physical and biological characteristics of macrophages, by combining mechanical separation, gradient centrifugation, and immuno-magnetic bead selection, we can effectively avoid the enzymatic digestion of testis tissue and maintain the immune characteristics of macrophages. Additionally, we verified the purity of TM with flow cytometry (FC) at approximately 91–95%, and the production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) was lower than that isolated with enzyme digestion. In contrast to the traditional method, this novel protocol can assist those who have no convenient access to fluorescence-activated cell sorting (FACS) to isolate a sufficient number of TMs and, most importantly, avoid altering the immunoprofile of TMs without enzyme digestion.

PARP2 downregulation in T cells ameliorates lipopolysaccharide-induced inflammation of the large intestine.

T cell-dependent inflammatory response with the upregulation of helper 17 T cells (Th17) and the downregulation of regulatory T cells (Treg) accompanied by the increased production of tumor necrosis alpha (TNFa) is characteristic of inflammatory bowel diseases (IBD). Modulation of T cell response may alleviate the inflammation thus reduce intestinal damage. Poly(ADP-ribose) polymerase-2 (PARP2) plays role in the development, differentiation and reactivity of T cell subpopulations. Our aim was to investigate the potential beneficial effect of T cell-specific PARP2 downregulation in the lipopolysaccharide (LPS) induced inflammatory response of the cecum and the colon. Low-dose LPS was injected intraperitoneally to induce local inflammatory response, characterized by increased TNFa production, in control (CD4Cre; PARP2+/+) and T cell-specific conditional PARP2 knockout (CD4Cre; PARP2f/f) mice. TNFa, IL-1b, IL-17 levels were measured by ELISA, oxidative-nitrative stress was estimated by immunohistochemistry, while PARP1 activity, p38 MAPK and ERK phosphorylation, and NF-kB expression in large intestine tissue samples were examined by Western-blot. Systemic & local T cell subpopulation; Th17 and Treg alterations were also investigated using flowcytometry and immunohistochemistry. In control animals, LPS induced intestinal inflammation with increased TNFa production, while no significant elevation of TNFa production was observed in T cell-specific PARP2 knockout animals. The absence of LPS-induced elevation in TNFa levels was accompanied by the absence of IL-1b elevation and the suppression of IL-17 production, showing markedly reduced inflammatory response. The increase in oxidative-nitrative stress and PARP1-activation was also absent in these tissues together with altered ERK and NF-kB activation. An increase in the number of the anti-inflammatory Treg cells in the intestinal mucosa was observed in these animals, together with the reduction of Treg count in the peripheral circulation. Our results confirmed that T cell-specific PARP2 downregulation ameliorated LPS-induced colitis. The dampened TNFa production, decreased IL-17 production and the increased intestinal regulatory T cell number after LPS treatment may be also beneficial during inflammatory processes seen in IBD. By reducing oxidative-nitrative stress and PARP1 activation, T cell-specific PARP2 downregulation may also alleviate intestinal tissue damage.

Stress granule activation attenuates lipopolysaccharide-induced cardiomyocyte dysfunction

BackgroundSepsis is the leading cause of death in intensive care units. Sepsis-induced myocardial dysfunction, one of the most serious complications of sepsis, is associated with higher mortality rates. As the pathogenesis of sepsis-induced cardiomyopathy has not been fully elucidated, there is no specific therapeutic approach. Stress granules (SG) are cytoplasmic membrane-less compartments that form in response to cellular stress and play important roles in various cell signaling pathways. The role of SG in sepsis-induced myocardial dysfunction has not been determined. Therefore, this study aimed to determine the effects of SG activation in septic cardiomyocytes (CMs).MethodsNeonatal CMs were treated with lipopolysaccharide (LPS). SG activation was visualized by immunofluorescence staining to detect the co-localization of GTPase-activating protein SH3 domain binding protein 1 (G3BP1) and T cell-restricted intracellular antigen 1 (TIA-1). Eukaryotic translation initiation factor alpha (eIF2α) phosphorylation, an indicator of SG formation, was assessed by western blotting. Tumor necrosis factor alpha (TNF-α) production was assessed by PCR and enzyme-linked immunosorbent assays. CMs function was evaluated by intracellular cyclic adenosine monophosphate (cAMP) levels in response to dobutamine. Pharmacological inhibition (ISRIB), a G3BP1 CRISPR activation plasmid, and a G3BP1 KO plasmid were employed to modulate SG activation. The fluorescence intensity of JC-1 was used to evaluate mitochondrial membrane potential.ResultsLPS challenge in CMs induced SG activation and resulted in eIF2α phosphorylation, increased TNF-α production, and decreased intracellular cAMP in response to dobutamine. The pharmacological inhibition of SG (ISRIB) increased TNF-α expression and decreased intracellular cAMP levels in CMs treated with LPS. The overexpression of G3BP1 increased SG activation, attenuated the LPS-induced increase in TNF-α expression, and improved CMs contractility (as evidenced by increased intracellular cAMP). Furthermore, SG prevented LPS-induced mitochondrial membrane potential dissipation in CMs.ConclusionSG formation plays a protective role in CMs function in sepsis and is a candidate therapeutic target.

T cell pro-inflammatory cytokine production in young adults with major depressive disorder

Background: Major Depressive Disorder (MDD) is increasingly prevalent in young adults and is a significant contributor to disease burden worldwide. Previously we have shown that young adults with MDD produce greater T cell mitochondrial ROS compared to their heathy non-depressed counterparts. Because evidence from rodent models of stress-related mood disorders demonstrates that increased T cell mitoROS drives excessive proinflammatory cytokine production, we next sought to determine if either CD4+ (Helper) or CD8+ (cytotoxic) T cells likewise produce increased proinflammatory cytokines in young adults with MDD. We hypothesized that there will be elevated proinflammatory cytokine production in young adults with MDD compared to their healthy non-depressed counterparts. Methods: Peripheral blood mononuclear cells were isolated by density centrifugation from 5 young adults with MDD having mild to moderate symptom severity and 9 non-depressed young healthy adults. Interleukin 2 (IL-2), Interleukin 6 (IL-6), Interferon Gamma (IFN-γ), and Tumor Necrosis Factor alpha (TNF-) production were assessed by flow cytometry. All data are expressed as mean SE and group differences were assessed by unpaired T test. Results: We found that adults with MDD exhibited a greater proportion of CD8+ Interleukin 6 (IL-6) (n= 8; 5 HA, 3 MDD) (HA = 22 3.5%, MDD = 255.6%; P= 0.004) producing cells compared to healthy. There were no significant differences in the proportions of CD8+ cells producing IFN-γ (HA = 14 1.2%, MDD = 3.1%; P= 0.31), IL-2 (HA =76 3.9%, MDD = 69 6.6%; P= 0.17), and TNF- (HA = 21 2.3%, MDD = 23.5%; P= 0.11) in those with MDD compared to controls. Additionally, there were no differences in CD4+ production of these same cytokines. We also assessed phenotype of the T cells using CD27 (central vs effector) and CD45 RA (Naïve vs TEMRA). Interestingly, a greater proportion of TNF- producing CD8+ and CD4+ cells exhibited an effector memory phenotype in adults with MDD compared to controls (CD8: HA = 25.8 2.4%, MDD = 46.8 3.3%; P= 0.0001; CD4+: HA=31 2.9%, MDD = 394.3%; P= 0.05). These data provide insight into the T cell subtypes that may be driving the inflammation associated with MDD. Conclusion: These preliminary findings suggest that young adults with MDD may exhibit a proinflammatory T cell phenotype characterized by greater IL-6 and TNF- production. NIH R21 MH123928 and R00133414 (JLG), NIH K01AG061271 and R01AG060395 (DWT). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hospitalized COVID-19 patients exhibit increased numbers of low density neutrophils expressing the transcription factor ARID3a

Abstract We previously showed that individuals with systemic lupus erythematosus (SLE) and high levels of Type I inflammatory cytokines had increased numbers of low density neutrophils (LDNs) in their peripheral blood compared to healthy donors. Increased numbers of LDNs were associated with increased expression of the transcription factor ARID3a and increased interferon alpha (IFNα) production. Data from other cell types suggest that ARID3a expression occurs upstream of IFNα expression. Therefore, we sought to determine if increases in ARID3a-expressing LDNs might also occur in other severe inflammatory responses, such as infection with SARS-CoV2 where IFNα expression was known to be increased. Subjects admitted to the hospital with a confirmed diagnosis of SARS-CoV2 infection were enrolled in our IRB approved study and peripheral blood mononuclear cells from 24 patients were evaluated via flow cytometry for numbers of LDNs and ARID3a expression at the time of admission and at various time points after hospitalization. Numbers of circulating LDNs were dramatically increased in COVID patient samples at the time of hospitalization, as were percentages of those cells expressing ARID3a protein. IL-6 expression was also high in the LDNs, while IFNα expression was more variable. Over the course of two weeks, IFNα expression appeared to be associated with, or to lag just behind ARID3a expression in most patients, while IL-6 expression levels were not associated with ARID3a or IFNα expression. These data suggest that ARID3a expression is induced in LDNs in association with severe viral infection and may implicate ARID3a as a potential regulator of inflammatory pathways in infection as well as autoimmunity. Supported in part by grant NIH U19 AI06269

English

This study aims to characterize chemical groups of metabolites in extracts of Acanthospermum hispidum and Croton zambesicus, and to evaluate their anti-inflammatory activities and their immunomodulatory effect on two pro-inflammatory cytokines (TNFα and IL6). The phytochemical screening was done on the powders of both plants by staining and precipitation reactions. Anti-inflammatory activity was performed in vivo on rat paw edema induced by 2% formalin and immunomodulatory activity was performed in vitro on rat Peripheral Blood Mononuclear Cells (PBMC) stimulated by Lipopolysaccharide (LPS). The leaves of the plants contained important chemical groups such as: cachectic tannins, flavonoids, terpenes, saponosides and quinones. The ethanolic extracts of the two plants studied showed good anti-inflammatory activity by reducing the volume of the rat paw edema induced by 2% formalin and by inhibiting the production of TNFα, IL6 by PBMC of rats stimulated by LPS. Key words: Acanthospermum hispidum, Croton zambesicus, extracts, phytochemical screening, biological activities.

KDELC2 Upregulates Glioblastoma Angiogenesis via Reactive Oxygen Species Activation and Tumor-Associated Macrophage Proliferation.

Glioblastoma is notorious for its rapid progression and neovascularization. In this study, it was found that KDEL (Lys-Asp-Glu-Leu) containing 2 (KDELC2) stimulated vasculogenic factor expression and induced human umbilical vein endothelial cell (HUVEC) proliferation. The NLRP3 inflammasome and autophagy activation via hypoxic inducible factor 1 alpha (HIF-1α) and mitochondrial reactive oxygen species (ROS) production was also confirmed. The application of the NLRP3 inflammasome inhibitor MCC950 and autophagy inhibitor 3-methyladenine (3-MA) indicated that the above phenomenon activation correlated with an endothelial overgrowth. Furthermore, KDELC2 suppression decreased the endoplasmic reticulum (ER) stress factors' expression. The ER stress inhibitors, such as salubrinal and GSK2606414, significantly suppressed HUVEC proliferation, indicating that ER stress promotes glioblastoma vascularization. Finally, shKDELC2 glioblastoma-conditioned medium (CM) stimulated TAM polarization and induced THP-1 cells to transform into M1 macrophages. In contrast, THP-1 cells co-cultured with compensatory overexpressed (OE)-KDELC2 glioblastoma cells increased IL-10 secretion, a biomarker of M2 macrophages. HUVECs co-cultured with shKDELC2 glioblastoma-polarized THP-1 cells were less proliferative, demonstrating that KDELC2 promotes angiogenesis. Mito-TEMPO and MCC950 increased caspase-1p20 and IL-1β expression in THP-1 macrophages, indicating that mitochondrial ROS and autophagy could also interrupt THP-1-M1 macrophage polarization. In conclusion, mitochondrial ROS, ER stress, and the TAMs resulting from OE-KDELC2 glioblastoma cells play important roles in upregulating glioblastoma angiogenesis.

Abstract 2994: Hypoxia blocks the presentation of tumor antigens to CD8+ T cells

Abstract Tumor hypoxia is known to enhance tumor growth and immune evasion by activating angiogenic pathways supporting tumor progression and fostering suppressive phenotypes of immune cells within the tumor-microenvironment. However, the consequence of tumor hypoxia on antigen processing and presentation to surveilling CD8+ T cells is a notable knowledge gap with direct relevance towards adaptive immune responses against cancer. Here, we present evidence using the well-established OT-I transgenic model that hypoxia decreases antigen specific T cell effector responses, and importantly, that these decreases in effector responses are not attributable to any hypoxic influence on T cell function. Murine B16 melanoma cells expressing ovalbumin (OVA; MO4) or parental B16 were grown under 20% or 2% oxygen for 48 hours. These cells were then treated with 0 or 10 ng/mL recombinant murine interferon gamma (IFN-g) for 24 hours at their respective oxygen conditions. For analysis of cytotoxicity and effector responses, these cells were labeled with a fluorescence enhancing ligand (BATDA) and then incubated with OT-I T cells recognizing OVA-peptide (SIINFEKL) presented by C57BL/6 mouse MHC I (H2kb) for 4 hours at 20% oxygen. Supernatants were then tested for BATDA release using a europium solution and subsequent fluorescent signal acquisition. OT-I T cells displayed significantly more specific lysis of target MO4 cells cultured under 20% oxygen compared to those cultured under 2% oxygen (p value < 0.0001; 2-way ANOVA). Control experiments using parental B16 target cells displayed little to no specific lysis. Additionally, OT-I T cells were incubated with B16 or MO4 cells cultured as described above for 18 hours under 20% oxygen in the presence of protein transport inhibitors and analyzed for effector cytokine production. Tumor necrosis factor alpha (TNF-a) production was significantly increased in OT-I cells co-cultured with IFN-g treated MO4 target cells preconditioned in both 20% and 2% oxygen (p value < 0.05; unpaired T test), however the fold change in TNF-a production for OT-I cells incubated with 20% oxygen cultured MO4 cells was approximately twice the increase observed in OT-I cells incubated with 2% oxygen cultured MO4 cells. These differences are not attributable to altered OVA expression under hypoxia. Furthermore, IFN-g dependent up-regulation of MHC-I and immunoproteasome catalytic subunits was found to be blocked by hypoxia, suggesting that hypoxic attenuation of the MHC I pathway is responsible for observed decreases in cytotoxic and effector responses in OT-I co-culture assays. Overall, these studies suggest that tumor hypoxia blunts anti-tumor immune responses irrespective of the oxygenation status of surveilling CD8+ T cells. Citation Format: Matthew G. Smith, Heena Panchal, Adam Mailloux. Hypoxia blocks the presentation of tumor antigens to CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2994.

대추씨 추출물의 항산화 및 항염증 효과

This study was performed to evaluate the antioxidant and anti-inflammatory of methanol extract from the jujube seed. Cell viability and production of nitric oxide (NO) was determined by MTT assay and Griess assay. Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and mitogen-activated protein kinase (MAPKs) expression were detected by western blot analysis. The production of prostaglandin E₂ (PGE₂) and tumor necrosis factor alpha (TNF-α) were determined by enzyme-linked immunosorbent assay. The antioxidant effects of jujube seed were measured based on polyphenol and flavonoid contents. Jujube seed extracts decreased the production of NO, PGE₂ and TNF-α in a dose-dependent manner, and also reduced the expression of iNOS, COX-2 protein. Additionally, jujube seed extracts reduced the expression levels of inhibitor of kappa B (IkB), nuclear factor kappa B (NF-kB) and MAPKs protein. Jujube seed extracts were found to have 180.56 ㎍/㎎ and 36.67 ㎍/㎎ high polyphenol and flavonoid contents. These results provide evidence for the antioxidant and anti-inflammatory effects of jujube seed. Products made of jujube seed will be extracted and it will be confirmed to be functional materials in the future.

The Potential Role of Etanercept in the Management of Post-stroke Pain: A Literature Review.

Strokes are the second leading cause of death and disability worldwide. The brain injury resulting from stroke produces a persistent neuroinflammatory response in the brain, resulting in a spectrum of neurologic dysfunction affecting stroke survivors chronically, also known as post-stroke pain. Excess production of tumor necrosis factor alpha (TNF alpha) in the cerebrospinal fluid (CSF) of stroke survivors has been implicated in post-stroke pain. Therefore, this literature review aims to assess and review the role of perispinal etanercept in the management of post-stroke pain. Several studies have shown statistically significant evidence that etanercept, a TNFalpha inhibitor, can reduce symptoms present in post-stroke syndrome by targeting the excess TNF alpha produced in the CSF. Studies have also shown improvements in not only post-stroke pain but also in traumatic brain injury and dementia.Further research is needed to explore the effects of TNF alpha on stroke prognosis and determine the optimal frequency and duration of etanercept treatment for post-stroke pain.

Evidence for a Causal Role for Escherichia coli Strains Identified as Adherent-Invasive (AIEC) in Intestinal Inflammation.

Enrichment of adherent-invasive Escherichia coli (AIEC) has been consistently detected in subsets of inflammatory bowel disease (IBD) patients. Although some AIEC strains cause colitis in animal models, these studies did not systematically compare AIEC with non-AIEC strains, and causal links between AIEC and disease are still disputed. Specifically, it remains unclear whether AIEC shows enhanced pathogenicity compared to that of commensal E. coli found in the same ecological microhabitat and if the in vitro phenotypes used to classify strains as AIEC are pathologically relevant. Here, we utilized in vitro phenotyping and a murine model of intestinal inflammation to systematically compare strains identified as AIEC with those identified as non-AIEC and relate AIEC phenotypes to pathogenicity. Strains identified as AIEC caused, on average, more severe intestinal inflammation. Intracellular survival/replication phenotypes routinely used to classify AIEC positively correlated with disease, while adherence to epithelial cells and tumor necrosis factor alpha production by macrophages did not. This knowledge was then applied to design and test a strategy to prevent inflammation by selecting E. coli strains that adhered to epithelial cells but poorly survived/replicated intracellularly. Two E. coli strains that ameliorated AIEC-mediated disease were subsequently identified. In summary, our results show a relationship between intracellular survival/replication in E. coli and pathology in murine colitis, suggesting that strains possessing these phenotypes might not only become enriched in human IBD but also contribute to disease. We provide new evidence that specific AIEC phenotypes are pathologically relevant and proof of principle that such mechanistic information can be therapeutically exploited to alleviate intestinal inflammation. IMPORTANCE Inflammatory bowel disease (IBD) is associated with an altered gut microbiota composition, including expansion of Proteobacteria. Many species in this phylum are thought to contribute to disease under certain conditions, including adherent-invasive Escherichia coli (AIEC) strains, which are enriched in some patients. However, whether this bloom contributes to disease or is just a response to IBD-associated physiological changes is unknown. Although assigning causality is challenging, appropriate animal models can test the hypothesis that AIEC strains have an enhanced ability to cause colitis in comparison to other gut commensal E. coli strains and to identify bacterial traits contributing to virulence. We observed that AIEC strains are generally more pathogenic than commensal E. coli and that bacterial intracellular survival/replication phenotypes contributed to disease. We also found that E. coli strains lacking primary virulence traits can prevent inflammation. Our findings provide critical information on E. coli pathogenicity that may inform development of IBD diagnostic tools and therapies.

Effects of Alloferon versus Valaciclovir for Treating Chronic Epstein-Barr Virus Infection

Background and objectivesThis study aimed to analyze the influence of alloferon on the Epstein-Barr virus (EBV) DNA copy number in saliva samples and the changes of natural killer (NK) cell content, cytotoxic activity of killer cells, and production of interferon alpha and gamma in patients suffering chronic EBV infection (CEBVI) at 6 weeks after therapy completion.

Single-cell analysis reveals TLR-induced macrophage heterogeneity and quorum sensing dictate population wide anti-inflammatory feedback in response to LPS.

The role of macrophages in controlling tissue inflammation is indispensable to ensure a context-appropriate response to pathogens whilst preventing excessive tissue damage. Their initial response is largely characterized by high production of tumor necrosis factor alpha (TNFα) which primes and attracts other immune cells, thereafter, followed by production of interleukin 10 (IL-10) which inhibits cell activation and steers towards resolving of inflammation. This delicate balance is understood at a population level but how it is initiated at a single-cell level remains elusive. Here, we utilize our previously developed droplet approach to probe single-cell macrophage activation in response to toll-like receptor 4 (TLR4) stimulation, and how single-cell heterogeneity and cellular communication affect macrophage-mediated inflammatory homeostasis. We show that only a fraction of macrophages can produce IL-10 in addition to TNFα upon LPS-induced activation, and that these cells are not phenotypically different from IL-10 non-producers nor exhibit a distinct transcriptional pathway. Finally, we demonstrate that the dynamics of TNFα and IL-10 are heavily controlled by macrophage density as evidenced by 3D hydrogel cultures suggesting a potential role for quorum sensing. These exploratory results emphasize the relevance of understanding the complex communication between macrophages and other immune cells and how these amount to population-wide responses.

Treatment of dental and orthodontic complications in thalassaemia.

Thalassaemia is a quantitative abnormality of haemoglobin caused by mutations in genes controlling production of alpha or beta globins. Abnormally unpaired globin chains cause membrane damage and cell death within organ systems and destruction of erythroid precursors in the bone marrow, leading to haemolytic anaemia. The life-long management of the general health effects of thalassaemia is highly challenging, and failure to deal with dental and orthodontic complications exacerbates the public health, financial and personal burden of the condition. There is a lack of evidence-based guidelines to help care seekers and providers manage such dental and orthodontic complications. This review aimed to evaluate the available evidence on methods for treating dental and orthodontic complications in people with thalassaemia to inform future recommendations. This is an update of a Cochrane Review first published in 2019. To assess different methods for treating dental and orthodontic complications in people with thalassaemia. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Haemoglobinopathies Trials Register in September 2022, and we searched nine online databases and trials registries in January 2022. We searched the reference lists of relevant articles and reviews and contacted haematologists, experts in fields of dentistry, organisations, pharmaceutical companies and researchers working in this field. We searched for published or unpublished randomised controlled trials (RCTs) that evaluated treatment of dental and orthodontic complications in individuals diagnosed with thalassaemia, irrespective of phenotype, severity, age, sex and ethnic origin. Two review authors independently screened the 37,242 titles retrieved by the search. After deduplication, we identified two potentially relevant RCTs. On assessing their eligibility against our inclusion and exclusion criteria, we excluded one and included the other. We included one parallel-design RCT conducted in Saudi Arabia and involving 29 participants (19 males, 10 females) with thalassaemia. It aimed to assess the effectiveness of photodynamic therapy as an adjuvant to conventional full-mouth ultrasonic scaling for the treatment of gingivitis. The average age of participants was around 23 years. There is very low-certainty evidence from this trial that full-mouth ultrasonic scaling plus photodynamic therapy compared to full-mouth ultrasonic scaling alone may improve gingival index score and bleeding on probing after 12 weeks in people with thalassaemia. We found no studies that assessed other interventions for the various dental or orthodontic complications of thalassaemia. Although the included study showed greater reduction in gingivitis in the group treated with full-mouth ultrasonic scaling plus photodynamic therapy, the evidence is of very low certainty. The study had unclear risk of bias, a short follow-up period and no data on safety or adverse effects. We cannot make definitive recommendations for clinical practice based on the limited evidence of a single trial. Future studies will very likely affect the conclusions of this review. This review highlights the need for high-quality RCTs that investigate the effectiveness of various treatment modalities for dental and orthodontic complications in people with thalassaemia. It is crucial that future trials assess adverse effects of interventions.

The C-Type Lectin Receptor Dectin-2 Is a Receptor for Aspergillus fumigatus Galactomannan.

Aspergillus fumigatus is a ubiquitous environmental mold that causes significant mortality particularly among immunocompromised patients. The detection of the Aspergillus-derived carbohydrate galactomannan in patient serum and bronchoalveolar lavage fluid is the major biomarker used to detect A. fumigatus infection in clinical medicine. Despite the clinical relevance of this carbohydrate, we lack a fundamental understanding of how galactomannan is recognized by the immune system and its consequences. Galactomannan is composed of a linear mannan backbone with galactofuranose sidechains and is found both attached to the cell surface of Aspergillus and as a soluble carbohydrate in the extracellular milieu. In this study, we utilized fungal-like particles composed of highly purified Aspergillus galactomannan to identify a C-type lectin host receptor for this fungal carbohydrate. We identified a novel and specific interaction between Aspergillus galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan bound to Dectin-2 and induced Dectin-2-dependent signaling, including activation of spleen tyrosine kinase, gene transcription, and tumor necrosis factor alpha (TNF-α) production. Deficiency of Dectin-2 increased immune cell recruitment to the lungs but was dispensable for survival in a mouse model of pulmonary aspergillosis. Our results identify a novel interaction between galactomannan and Dectin-2 and demonstrate that Dectin-2 is a receptor for galactomannan, which leads to a proinflammatory immune response in the lung. IMPORTANCE Aspergillus fumigatus is a fungal pathogen that causes serious and often fatal disease in humans. The surface of Aspergillus is composed of complex sugar molecules. Recognition of these carbohydrates by immune cells by carbohydrate lectin receptors can lead to clearance of the infection or, in some cases, benefit the fungus by dampening the host response. Galactomannan is a carbohydrate that is part of the cell surface of Aspergillus but is also released during infection and is found in patient lungs as well as their bloodstreams. The significance of our research is that we have identified Dectin-2 as a mammalian immune cell receptor that recognizes, binds, and signals in response to galactomannan. These results enhance our understanding of how this carbohydrate interacts with the immune system at the site of infection and will lead to broader understanding of how release of galactomannan by Aspergillus effects the immune response in infected patients.

Rab44 Deficiency Induces Impaired Immune Responses to Nickel Allergy.

Rab44 was recently identified as an atypical Rab GTPase that possesses EF-hand and coiled-coil domains at the N-terminus, and a Rab-GTPase domain at the C-terminus. Rab44 is highly expressed in immune-related cells such as mast cells, macrophages, osteoclasts, and granulocyte-lineage cells in the bone marrow. Therefore, it is speculated that Rab44 is involved in the inflammation and differentiation of immune cells. However, little is known about the role of Rab44 in inflammation. In this study, we showed that Rab44 was upregulated during the early phase of differentiation of M1- and M2-type macrophages. Rab44-deficient mice exhibited impaired tumor necrosis factor alpha and interleukin-10 production after lipopolysaccharide (LPS) stimulation. The number of granulocytes in Rab44-deficient mice was lower, but the lymphocyte count in Rab44-deficient mice was significantly higher than that in wild-type mice after LPS stimulation. Moreover, Rab44-deficient macrophages showed impaired nickel-induced toxicity, and Rab44-deficient mice showed impaired nickel-induced hypersensitivity. Upon nickel hypersensitivity induction, Rab44-deficient mice showed different frequencies of immune cells in the blood and ears. Thus, it is likely that Rab44 is implicated in immune cell differentiation and inflammation, and Rab44 deficiency induces impaired immune responses to nickel allergies.

Advanced Glycation End Products-Induced Activation of Keratinocytes: A Mechanism Underlying Cutaneous Immune Response in Psoriasis

Psoriasis is a common inflammatory skin disease, in which epidermal keratinocytes play a vital role in its pathogenesis by acting both as the responder and as the accelerator to the cutaneous psoriatic immune response. Advanced glycation end products (AGEs) are a class of proinflammatory metabolites that are commonly accumulating in cardiometabolic disorders. Recent studies have also observed the increased level of AGEs in the serum and skin of psoriasis patients, but the role of AGEs in psoriatic inflammation has not been well investigated. In the present study, we initially detected abnormal accumulation of AGEs in epidermal keratinocytes of psoriatic lesions collected from psoriasis patients. Furthermore, AGEs promoted the proliferation of keratinocytes via upregulated Keratin 17 (K17)-mediated p27KIP1 inhibition followed by accelerated cell cycle progression. More importantly, AGEs facilitated the production of interleukin-36 alpha (IL-36α) in keratinocytes, which could enhance T helper 17 (Th17) immune response. In addition, the induction of both K17 and IL-36α by AGEs in keratinocytes was dependent on the activation of signal transducer and activator of transcription 1/3 (STAT1/3) signaling pathways. At last, the effects of AGEs on keratinocytes were mediated by the receptor for AGEs (RAGE). Taken together, these findings support that AGEs potentiate the innate immune function of keratinocytes, which contributes to the formation of psoriatic inflammation. Our study implicates AGEs as a potential pathogenic link between psoriasis and cardiometabolic comorbidities.

PD-1 AND TIM-3 BLOCKING CANNOT ENHANCE APOPTOSIS OF CHRONIC LYMPHOCYTIC LEUKEMIA CELLS INDUCED BY PERIPHERAL BLOOD CD8+ T CELLS.

Given theinvaluable success of immune checkpoint inhibitors for tumor immunotherapy, in this study, theeffect of programmed cell death 1 (PD-1)and T cell immunoglobulin-3 (TIM-3)blocking was investigated to induce apoptosis of leukemic cells by exhausted CD8+ T cells in patients with chronic lymphocytic leukemia (CLL). Peripheral blood CD8+ T cells were positively isolated from 16CLL patients using magnetic beads separation method. Isolated CD8+ T cells were treated with either blocking anti-PD-1, anti-TIM-3and isotype-matched control antibodies and then co-cultured with CLL leukemic cells as target. Thepercentage of apoptotic leukemic cells and theexpression of apoptosis-related genes were evaluated by flow cytometry and real-time polymerase chain reaction methods, respectively. Interferon gamma and tumor necrosis factor alpha concentration was also measured by ELISA. Flow cytometric analysis of apoptotic leukemic cells indicated that theblockade of PD-1and TIM-3did not significantly enhance theapoptosis of CLL cells by CD8+ T cells, which then were confirmed by analysis of BAX, BCL2and CASP3gene expression, which was similar in blocked and control groups. No significant difference was found between blocked and control groups in terms of interferon gamma and tumor necrosis factor alpha production by CD8+ T cells. We concluded that theblockade of PD-1and TIM-3is not an effective strategy to restore thefunction of CD8+ T cells in CLL patients at theearly clinical stages of thedisease. Further in vitro and in vivo studies are needed to more address theapplication of immune checkpoint blockade in CLL patients.