TLR4 Gene Research Articles

Association between several immune response-related genes and the effectiveness of biological treatments in patients with moderate-to-severe psoriasis.

Biological therapies are safer and more effective against psoriasis than conventional treatments. Even so, 30-50% of psoriatic patients show an inadequate response, which is associated with individual genetic heterogeneity. Pharmacogenetic studies have identified several single nucleotide polymorphisms (SNPs) as possible predictive and prognostic biomarkers for psoriasis treatment response. The objective of this study was to determine the link between several SNPs and the clinical response to biological therapies in patients with moderate-severe psoriasis. A set of 21 SNPs related to psoriasis and/or other immunological diseases were selected and analysed from salivary samples of patients (n = 88). Treatment effectiveness and patient improvement was assessed clinically through Relative Psoriasis Area and Severity Index (PASI), also called 'PASI response', as well as absolute PASI. Associations between SNPs and PASI factors were assessed at 3 and 12 months for every treatment category of IL-17, IL-23, IL-12&23 and TNF-α inhibitors. Multivariate correlation analysis and Fisher's exact test were used to analyse the relationship between SNPs and therapy outcomes. Several SNPs located in the TLR2, TLR5, TIRAP, HLA-C, IL12B, SLC12A8, TNFAIP3 and PGLYRP4 genes demonstrated association with increased short and long-term therapy-effectiveness rates. Most patients achieved values of PASI response ≥75 or absolute PASI<1, regardless of the biological treatment administered. In conclusion, we demonstrate a relationship between different SNPs and both short- and especially long-term effectiveness of biological treatment in terms of PASI. These polymorphisms may be used as predictive markers of treatment response in patients with moderate-to-severe psoriasis, providing personalized treatment.

Activation of murine macrophages by membrane proteins from Tritrichomonas foetus grown on iron- and calcium-rich conditions.

Tritrichomonas foetus is a protozoan parasite that causes a venereal disease in cattle limiting reproduction by abortions and sterility. The immune response against this parasite is poorly understood. Since the iron and calcium ions are important regulators of the microenvironment of the urogenital tract in cattle, we decided to evaluate the role of these divalent cations on the antigenicity of membrane proteins of T. foetus on macrophage activation as one of the first inflammatory responses towards this pathogen. Colorimetric methods and ELISA were used to detect the nitric oxide and oxygen peroxide production and expression of cytokines in culture supernatant from macrophage incubated with membrane proteins from T. foetus cultured in iron- and calcium-rich conditions. qRT-PCR assays were used to evaluate the transcript expression of genes involved in the inflammatory response on the macrophages. The membrane proteins used for in vitro stimulation caused the up-regulation of the iNOS and NOX-2 genes as well as the generation of NO and H2 O2 in murine macrophages on a dependent way of the metal concentrations. Additionally, after stimulation, macrophages showed a considerable rise in pro-inflammatory cytokines and a downregulation of anti-inflammatory cytokines, as well as up-regulation in the transcription of the TLR4 and MyD88 genes. These data suggest that membrane proteins of T. foetus induced by iron and calcium can activate an inflammatory specific macrophage response via TLR4/MyD88 signalling pathway.

Heat-inactivated mycobacteria activate the toll-like receptor 2 and 4 pathways in the zebrafish model of tuberculosis

Based on previous evidence demonstrating the efficacy of inactivated mycobacteria for the control of fish mycobacteriosis, we explored the protective efficacy of two inactivated Mycobacterium bovis administered via parenteral and mucosal routes against Mycobacterium marinum infection mimicking natural conditions in the zebrafish model of tuberculosis. Although we did not observe a clear effect of any of the immunostimulants on mycobacterial burden, the results showed a significant increase in TLR2 and TLR4 gene expression levels in fishes parenterally immunized with inactivated Bacillus Calmette-Guérin (BCG). Our findings demonstrated that the TLR2 and the TLR4 signaling pathways are involved in the immune response elicited by inactivated mycobacteria in the zebrafish model of tuberculosis and support the use of inactivated mycobacteria in vaccine formulations for the control of mycobacteriosis.

Examining the level of inflammatory cytokines TNF-α and IL-8 produced by osteoblasts differentiated from dental pulp stem cells.

The use of dental pulp stem cells (DPSCs) in clinical applications instead of bone marrow stem cells is a very promising method capable of significantly changing the future of medical treatment. If further studies prove that DPSCs and the cells differentiated from them do not stimulate the immune system, these cells can be used more reliably in treatment of autoimmune diseases. In this research, we examined the isolated DPSCs and differentiated osteoblasts from them in medium without inflammatory stimulants in terms of TLR3 and TLR4 gene expression and inflammatory cytokines, including TNF-α and IL-8 using qRT-PCR, and measured the concentration of inflammatory cytokines IL-8 and TNF-α produced by these two types of cells through ELISA. The obtained results showed that the expression level of inflammatory cytokines IL-8 and TNF-α in differentiated osteoblasts is significantly different as compared with DPSCs. However, no significant difference was observed in TLR-4 expression between two groups. An increase in TNF-α expression level was found to directly correlate with an increase in the expression of IL-8. The concentration of cytokine TNF-α in osteoblasts was significantly higher than that of IL-8 in DPSCs. In comparison to DPSCs, osteoblast cells first lead to inflammatory responses. These responses reduce overtime. However, DPSCs retain their immunomodulatory properties and do not show inflammatory responses.

The association of TLR4 gene polymorphisms with the severity of peritonitis in acute inflammatory diseases of the abdominal cavity organs.

Aim: To determine the role of TLR4 gene polymorphisms as risk factors for peritonitis severity in patients undergoing surgery for acute inflammatory diseases of the abdominal cavity. Materials and Methods: The study included 139 patients who were operated on for acute abdominal diseases (acute appendicitis and cholecystitis, perforated gastric or duodenal ulcer, etc.). Depending on the number of points on the modified APACHE II scale, patients were divided into two groups: Group 1 - 1-3 points (63 patients, 45.3%) and Group 2 - 4 or more points (76 patients, 54.7%). Polymorphisms rs1927911, rs2149356 and rs4986790 were determined by polymerase chain reaction. Results: The rs1927911 polymorphism of the TLR4 gene was protective for the development of peritonitis (according to the allelic model, OR 0.48; 95% CI 0.27-0.84; p=0.015). Regression analysis revealed a reduced (p=0.015) risk of severe peritonitis in rs1927911 A/A or G/A genotype carriers (OR 0.42; 95% CI 0.21-0.84) compared with G/G genotype carriers. There was no effect on the severity of peritonitis of TLR4 polymorphisms rs2149356 and rs4986790. There was a tendency to increase the frequency of the mutant G rs4986790 allele in patients with severe peritonitis (χ2=2.17; p<0.001). The analysis of the association of TLR4 gene polymorphisms with the phenotype of patients showed that carriers of mutant homozygotes and heterozygotes in the presence of severe peritonitis were older, had a tendency to coagulopathy, higher leukocytosis and leukocyte clotting rate. Conclusions: Thus, the importance of TLR in the development of severe peritonitis was confirmed and the protective role of the rs1927911 promoter polymorphism was established.

Prevalence of rs4986790 (Asp299Gly) polymorphism of the TLR-4 gene in patients with acute brucellosis depending on the damage to the hepatobiliary system

Prevalence of rs4986790 (Asp299Gly) polymorphism of the TLR-4 gene in patients with acute brucellosis depending on the damage to the hepatobiliary system

Impact of the mare colostral immunoglobulins on the expression of TLR3, TLR4, and TLR7 in foals

AbstractColostrum contains substances such as hormones, enzymes, polyamides, nucleic acid derivatives, and amino acid derivatives and also includes immunomodulating substances, proline‐rich polypeptides, immunoglobulins (Ig) as well as bacteriostatic compounds. The aim of the study was to evaluate the impact of colostral Ig on the level of expression of selected Toll‐like receptor genes (TLR3, TLR4, and TLR7). The experiments were conducted on 25 Polish Pony foals. Blood samples were collected according to the following scheme: before first suckling, at the 1st, 3rd, 5th, 10th, 20th, and 30th, day of age. Colostrum was obtained before the first suckling, 24 hr, and 3 days after the delivery. Gene expression analyses were performed on Illumina Eco using a commercial kit TaqMan®MGB probes. The quality of colostrum was assessed via refractometer and BRIX value. The total Ig level was calculated from the spectrophotometric method. IgG levels were measured using ELISA assay. The quality of colostrum significantly correlated with TLR4 and TLR7 expression. Foals that ingested colostrum with low Ig content displayed a higher level of TLR4 gene expression, while in the case of TLR7 the opposite trend was shown. This result indicates that colostrum may play an important role in shaping the mechanisms of building the immune system's response to bacterial pathogens.

Genetic Selection Pressure on TLR9 Gene Increases Individual Susceptibility to Systemic Inflammatory Disease: A Case Study of Kano State Population

Genetic Selection Pressure on TLR9 Gene Increases Individual Susceptibility to Systemic Inflammatory Disease: A Case Study of Kano State Population

Impact of TLR9 gene Polymorphism on Severity of Helicobacter pylori Infection and Its Association with Virulence genes

Impact of TLR9 gene Polymorphism on Severity of Helicobacter pylori Infection and Its Association with Virulence genes

Synonymous variant of TLR7 at restriction site rs864058 identified in Covid 19 Pakistani patients.

TLR7, the receptor accountable for immune response to RNA viruses, has been studied extensively to identify its variants related to the severity of Covid-19 in different populations worldwide. However, the genotype of Pakistani population is still unknown. This study aimed to determine the TLR7 genotypes and their relation with severity in our population. This cross sectional study collected data on 151 Covid-19 positive patients (aged 18-80 years), from June 2022 to May 2023, after an informed consent, from Ziauddin University and Hospital. Prior to that approval from ethics review committee was taken. The demographic variables and comorbidities were recorded along with health status till LAMA (Leave Against Medical Advise), recovery or death. The DNA was extracted from collected blood samples, PCR and Sanger sequencing was done for identification of TLR7 variants. SPSS was used for data analyses and Chi-Square for categorical variables. P-values of <0.05 was considered significant. Out of 151 patients' sequencing was done for 59 samples. The restriction site, rs864058 of TLR7 gene, identified G/A and G/G variants. This missense variant of TLR7 identified at rs864058 of TLR7 gene, has not been previously reported in population control databases. The genotype G/G was main variant of 49 (83%) patients, whereas, G/A was found in 10 (17%). Majority, 25 (51%) of patients with mild covid-19 had GG genotype but results were not significant (P=0.684). Among female patients the main genotype was GA 8 (80%) while male had G/G 29 (59.2%) with significant results (P=0.024). Since G/G genotype was the major genotype, high percentage was found in hypertensives [20 (40.8%)], Diabetics [13 (26.5%)], depression [24 (49%)] and pneumonia patients [20 (40.8%)]. However, significant association (P=0.023) was only found with pneumonia. Males, in majority had severe [17 (68%)] infection and death [40 (26.4%)], whereas, females had mild [14 (25%)] with [12 (7.9%)] deaths. A variant rs864058 "G/A" of TLR7, in relation to covid-19 were found in our population. Males were found more at risk of morbidity and mortality due to covid-19. Larger studies are required to further confirm these results.

Comparison of NOD-like and Toll-like receptors gene expression levels in blood monocytes of colon cancer patients.

Colon cancer is one of the most common cancers affecting many people worldwide. This disease can be treated if diagnosed in the early stages. Therefore, with the hypothesis that the level of expression of inflammatory genes in peripheral blood monocytes of patients with colon cancer is different from that of healthy people, this research was done to find out the role of inflammation in the development of colon cancer by relying on its immunopathological profile to help diagnose it in the early stages. In this case-control study, the expression levels of TLR4, TLR2, NLRP3, and NOS2 genes in 15 patients with confirmed stage II colon cancer were determined by the TNM method. Also, 15 healthy people referred for this cancer screening were selected as the control group. First, RNA was extracted from the blood monocytes of two groups, and after making cDNA, the comparison was created using the qPCR method. In this study, the β-actin gene was used as a reference gene. The expression levels of TLR2 and TLR4 at the mRNA level were significantly lower in colon cancer patients compared to the healthy control group (P<0.05). The expression level of NLRP3 in the group of colon cancer patients showed a relative increase. Still, it was not significant, while the expression level of the NOS2 gene in the group of colon cancer patients increased significantly compared to the healthy control group (P<0.05). Considering the significant changes in TLR4, TLR2, and NOS2 gene expression in monocytes of patients with grade II colon cancer and the role of inflammatory reactions in the development of this cancer, these findings can be used to diagnose and determine the prognosis. However, this requires further studies.

Study the mRNA level of IL-27/IL-27R pathway molecules in kidney transplant rejection.

Renal transplantation stands as the sole remedy for individuals afflicted with end-stage renal diseases, and safeguarding them from transplant rejection represents a vital, life-preserving endeavor posttransplantation. In this context, the impact of cytokines, notably IL-27, assumes a critical role in managing immune responses aimed at countering rejection. Consequently, this investigation endeavors to explore the precise function of IL-27 and its associated cytokines in the context of kidney transplant rejection. The study involved the acquisition of blood samples from a cohort of participants, consisting of 61 individuals who had undergone kidney transplantation (comprising 32 nonrejected patients and 29 rejected patients), and 33 healthy controls. The expression levels of specific genes were examined using SYBR Green Real-time PCR. Additionally, the evaluation encompassed the estimation of the ROC curve, the assessment of the relationship between certain blood factors, and the construction of protein-protein interaction networks for the genes under investigation. Significant statistical differences in gene expression levels were observed between the rejected group and healthy controls, encompassing all the genes examined, except for TLR3 and TLR4 genes. Moreover, the analysis of the Area Under the Curve (AUC) revealed that IL-27, IL-27R, TNF-α, and TLR4 exhibited greater significance in discriminating between the two patient groups. These findings highlight the potential importance of IL-27, IL-27R, TNF-α, and TLR4 as key factors for distinguishing between individuals in the rejected group and those in the healthy control group. In the context of kidney rejections occurring within the specific timeframe of 2 weeks to 2 months post-transplantation, it is crucial to emphasize the significance of cytokines mRNA level, including IL-27, IL-27R, TNF-α, and TLR4, in elucidating and discerning the diverse immune system responses. The comprehensive examination of these cytokines' mRNA level assumes considerable importance in understanding the intricate mechanisms underlying kidney rejection processes during this critical period.

Genetic predictors of abdominal complications after cardiac surgery: a prospective study

Introduction: The prediction of postoperative abdominal complications is a continuing concern in the cardiac surgery. Identifying genetic predictors will allow a better understanding of the pathophysiological mechanisms underlying postoperative disorders and their prevention.Objective: To identify genetic predictors of abdominal complications after heart surgery.Methods: A prospective study was carried out at the Federal Center for Cardiovascular Surgery (Chelyabinsk) involving 72 patients with abdominal complications and 102 controls without such complications. The distribution of genotypes was assessed for adherence to the Hardy-Weinberg distribution using the exact method according to the SNP-HWE algorithm in the PLINK package (version 1.9). To identify marker genes associated with the risk of abdominal complications, 6 models were evaluated for each polymorphic gene: allelic, codominant, dominant, recessive, overdominant, and log-additive. To analyze the SNPs of the IL6 (rs1800795), TNF (rs1800629), and SERPINE1 (rs1799768) genes, real-time polymerase chain reaction (PCR) was performed on a Rotor-Gene 6000 detecting cycler (Corbett Research Pty Ltd., Mortlake, Australia) using primers and probes manufactured by Syntol CJSC (Moscow, Russia) according to the manufacturer's instructions. To analyze the SNPs of the IL1B (rs1143634), CXCL8 (rs4073), IL10 (rs1800872), APOE (rs429358), FABP2 (rs1799883), NOS3 (rs1799983), VEGFA (rs699947) genes, real-time PCR was performed on a DTprime 4 analyzer (DNA-Technology, Moscow, Russia) using primers and probes manufactured by TestGene LLC (Ulyanovsk, Russia) according to the manufacturer's instructions. The determination of the SNP of the TLR3 gene (rs3775291) relied on real-time PCR according to melting curves using TestGene LLC reagent kits on a DTprime 4 device.Results: Two genes, IL1B rs1143634 and FABP2 rs1799883, were found to be associated with the risk of abdominal complications. Following the adjustment for multiple comparisons, only FABP2 rs1799883 remained statistically significant. The rs1799883 polymorphism of the FABP2 gene can be considered a predictor of the risk of abdominal complications and can be evaluated within the codominant and dominant models, which exhibit similar p-values (0.003 and 0.002, respectively). Overall, the analysis of the obtained data suggests that the risk of abdominal complications is associated with the presence of the mutant allele C in the genome, likely due to its dominance. Specifically, in the dominant model, the odds ratio was 3.53 (95% CI 1.62 to 7.70).Conclusion: The most significant markers of the risk of abdominal complications were the IL1B (rs1143634) and FABP2 (rs1799883) genes, of which FABP2 (rs1799883) remained statistically significant after adjusting for multiple comparisons. Its polymorphism as a predictor of abdominal complications can be evaluated within the codominant and dominant models. Received 13 June 2023. Revised 28 November 2023. Accepted 29 November 2023. Funding: The study was supported by the Russian Science Foundation, project No. 22-25-20016, https://rscf.ru/project/22-25-20016/ Conflict of interest: The authors declare no conflict of interest. Contribution of the authorsConception and study design: O.S. Abramovskikh, A.A. Fokin, D.V. BelovData collection and analysis: Yu.V. LoginovaStatistical analysis: M.A. ZotovaDrafting the article: D.V. BelovCritical revision of the article: Yu.V. LoginovaFinal approval of the version to be published: D.V. Belov, O.S. Abramovskikh, M.A. Zotova, Yu.V. Loginova, A.A. Fokin

Association of Variants in Innate Immune Genes TLR4 and TLR5 with Reproductive and Milk Production Traits in Czech Simmental Cattle.

Bovine genes TLR4 and TLR5, which encode antibacterial toll-like receptors, were screened for polymorphisms in Czech Red Pied (Czech Simmental) cattle to identify variants associated with reproduction, udder health, and milk production traits. Variants were discovered by hybrid resequencing of 164 bulls using HiSeq X-Ten and PacBio technologies and then individually genotyped. Nominal p-values < 0.05 for associations were detected in 18 combinations between 14 polymorphisms and 15 traits using one-way analysis of variance (ANOVA). The TLR4 variants g.610C>T (rs43578094) and g.10310T>G (rs8193072) in reference AC000135.1 were strictly associated with the index of early reproductive disorders and maternal calving ease, respectively, at false discovery rate (FDR) < 0.05. A highly permissive false discovery rate cutoff of 0.6 separated seventeen combinations in both genes comprising eight positives. In the case of the TLR4 variant g.9422T>C (rs8193060), indications were obtained for the association with as many as four reproductive traits: incidence of cystic ovaries, early reproductive disorders, calving ease, and production longevity. The permissive FDR interpretation for the TLR5 data indicated associations with cyst incidence and early reproduction disorders with maternal calving ease. Moreover, three TLR5 polymorphisms correlated with milk production traits. The discrepancy of the observed associations with the predicted impacts of the SNPs on protein function points to the role of haplotypes. Nevertheless, this question should be resolved on a larger scale. The observed associations are endorsed by independent evidence from the published functional roles in other species and by the published QTL mapping data.

Inflammatory response modulation by epinephrine and norepinephrine

Relevance. Inflammation is a defense response of an organism to a pathogen. It appears in order to maintain homeostasis and is regulated by the immune, nervous, and endocrine systems. The hormones epinephrine and norepinephrine are produced in the adrenal medulla and in the brain, and are universal messengers that trigger the transmission of nerve impulses at synapses, and also have a receptor-mediated effect on immunocompetent cells. The aim of this study was to investigate adrenergic pathway regulation of inflammation on the neutrophil granulocytes in the presence of activators of innate immunity receptors. Materials and Methods. Neutrophil granulocytes were obtained from peripheral blood of healthy volunteers in a density gradient of Histopaque 1077 and Histopaque 1119 (Sigma Aldrich, Steinheim, Germany), and cultured in the presence of LPS, GMDP, epinephrine and norepinephrine. The amount of human neutrophil peptides 1-3 (HNP1-3) was examined using an enzyme-linked immunosorbent assay; the gene expression of TLR4, NOD2, ATF3 and A20 was determined using RT-PCR. Results and Discussion. Norepinephrine (noradrenaline) was found to decrease the synthesis of human neutrophils peptides 1-3 (HNP 1-3 defensins, alone and in the combination with agonists of TLR4 and NOD2 receptors - LPS and GMDP respectively. It was found out that there was no a statistically significant effect of epinephrine (adrenaline) on the production of HNP 1-3, including when combined with LPS and GMDP. As a result of the study, an increase in the levels of expression of the genes TLR4, NOD2 and regulator of inflammatory reactions A20 both in LPS- and GMDP- induced neutrophil culture were uncovered, while ATF3 was increased only in LPS-induced neutrophil culture. Epinephrine demonstrated the absence of a statistically significant effect on the expression of the studied genes. While norepinephrine significantly increased the expression of A20 genes. Conclusion. The data obtained shows that norepinephrine can reduce the synthesis of HNP 1-3, including the one induced by LPS and GMDP. Moreover, the ability of norepinephrine to induce the expression of A20 may play a significant role in modulation of inflammation.

Study of polymorphism of toll like receptor-4 gene in crossbred cattleand its association with somatic cell count

The present study was undertaken to study the TLR4 gene polymorphism in exon 3 and its association with somatic cell count in HF×Gir cows using PCR-RFLP. Sixty crossbred cows (40 affected and 20 normal) between 3rd to 5th lactation were selected from Bombay Gowrakshak Mandali, Betegaon, Maharashtra. The confirmation of mastitis was done with the help of California MastitisTest (CMT) and the blood samples of 40 affected animals were collected aseptically along with 20 normal animals. The mean of somatic cell score in normal cows ranged between 2.86 to 2.99 × 105 cells/ml, whereas for affected animal the range of somatic cell score was 3.45 to 5.72 × 105 cells/ml. The genomic DNA was isolated and exon 3 of TLR4 gene was successfully amplified by PCR using a specific primer. The PCR product was digested with HaeIII restriction enzyme. The PCR-RFLP of exon 3 with HaeIII enzyme revealed two genotypes ‘BB’ and ‘AB’ with band pattern 49 bp, 98 bp &amp; 196 bp (BB) and 49 bp, 98 bp, 196 bp &amp; 245 bp (AB), respectively. The mean of somatic cell score estimated for animals with genotypes ‘BB’ and ‘AB’ was 3.99±0.13 and 3.66±0.03, respectively. The twomean ‘t’ test analysis showed non-significant difference between somatic cell count with genotypes ‘BB’ and ‘AB’. It indicated that there was no association between the genotypes of exon 3 of TLR4 gene with somatic cell count in crossbred cows under present study.

Candidate Gene Analysis of Genetic Resistance to Gastrointestinal Nematodes in Sheep Through Association of Single Nucleotide Polymorphism with Phenotypic Traits

Background: A study was executed to analyze the genetic resistance to gastrointestinal nematodes through the association of single nucleotide polymorphism markers in Toll-like receptor genes with phenotypic indicator traits in Kilakarsal and Vembur sheep breeds. Methods: The phenotypic traits for analyzing the gastrointestinal nematode infection namely FEC, change in PCV and change in body weight were recorded. The SNP markers in TLR3, TLR5, TLR6, TLR9 and TLR10 genes were employed for genotyping. Competitive allele-specific PCR-based endpoint genotyping was used to type the SNPs. The gene and genotype frequencies were estimated by using the PEAS software program. A complete fixed effects model was utilized for analysis of the association of various genotypes at each SNP with phenotypic indicator traits. Result: The global minor allele frequency of different polymorphic SNP loci ranged from 0.06 to 0.48 with a mean of 0.23, signifying their fitness for the association study. The effect of the farm had no significant influence on FEC and change in body weight, however, had a significant effect on change in PCV (P less than 0.05). No significant difference was detected between the sexes with FEC, change in PCV and change in body weight. The TT genotype in the TLR9_1769_CT locus showed the lowest least-squares mean FEC. The remaining 22 SNP loci showed no significant difference (P greater than 0.05) with mean FEC. Association of 23 TLR SNP genotypes with change in PCV and change in body weight revealed no significant effect (P greater than 0.05).

The Thymic Hormone Thymosin-1α Reduces the Pro-Inflammatory Response of Raw 264.7 Cells Induced by Endotoxin

—The aim of this work was to study the effects of thymosin-1 alpha (Tα1) on the anti-inflammatory response of RAW 264.7 macrophages cultured in the presence of lipopolysaccharide (LPS) from the walls of gram-negative bacteria. As well, we evaluated production of pro-inflammatory cytokines and the activity of the NF-κB and SAPK/JNK signaling pathways. In addition, the level of expression of a number of genes that regulate cell apoptosis, as well as the activity of receptors involved in the pro-inflammatory response, was determined. First, the addition of Tα1 normalized the level of cytokine production to varying degrees, with a particularly noticeable effect on IL-1β and IL-6. Second, the addition of Tα1 normalized the activity of the NF-κB and SAPK/JNK signaling cascades and the expression of the Tlr4 gene. Third, Tα1 significantly reduced p53 and the activity of the P53 gene, which is a marker of cell apoptosis. Fourth, it was shown that the increase in Ar-1 gene expression under the influence of LPS was significantly reduced using Tα1. Thus, it was found that the presence of Tα1 in the RAW 264.7 cell culture medium significantly reduced the level of the pro-inflammatory response of cells.

Design, synthesis and anti-rheumatoid arthritis activity of target TLR4 inhibitors

A series of 1-(2-oxocyclohexyl)butane-1, 3-dione derivatives were designed and synthesized as TLR4 inhibitors by modifying the core structure of the lead compound ((6, 8-dioxo-1, 2, 3, 4, 6, 7, 8, 8a-octahydronaphthalen-2-yl) carbamate)). In vitro, compound 3p significantly inhibited the proliferation of rat synovial cells, inhibited the proliferation of LPS-induced RAW264.7 cells, and inhibited TLR4 activity, with IC50 values of 1.21 ± 0.09 μM, 0.73 ± 0.05 μM and 0.43 ± 0.03 μM, respectively, which was superior to the positive control methotrexate. In vivo anti-rheumatoid arthritis evaluation, compound 3p can significantly inhibit the inflammatory factors TNF-α, IL-1β and IL-6, so as to achieve the therapeutic purpose. In the preliminary mechanism study, compound 3p has obvious regulatory effects on the abnormal increase of TLR4, JAK2 and STAT3 protein and gene expression related to inflammatory signaling pathway in RAW264.7 cells. In summary, this study aims to develop more effective candidates for rheumatoid arthritis.

Clinical Characteristics, Management, and Allogeneic Hematopoietic Cell Transplantation of Patients with Toll-like Receptor 8 Gain-of-Function Mutations

Toll-like receptor 8 gain-of-function (TLR8 GOF) is a recently described inborn error of immunity due to germline or somatic mutations in the TLR8 gene and characterized by severe neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases, bone marrow failure. Treatment of TLR8 GOF has been challenging to date, and guidance for optimal therapy is lacking. We describe the clinical characteristics and management of 10 patients with TLR8 GOF variants, including the first female patient and further clinical outcome on the 6 patients initially reported with this disease with a focus on the role of allogeneic hematopoietic cell transplantation (HCT). All but one patient were male ranging from 0.5 months to 28 years at age of symptom onset. Eight patients had somatic mutations with variant allele frequencies of 7-26%; two patients had germline mutations. The 9 male patients all had severe neutropenia and varying degrees of anemia and/or thrombocytopenia. Oral ulcers, recurrent infections, hepatosplenomegaly, and hypogammaglobulinemia were common. Immune phenotype was variable, ranging from severe lymphopenia to marked T cell lymphoproliferation. Several patients had an inverted CD4:CD8 T cell ratio and skewing of T cell subsets to the terminal effector memory phenotype. Increased double-negative T cells were also observed. All patients had low class switched memory B cells. Bone marrows were hypo- to hypercellular with marked myeloid hypoplasia, and in most cases, with increased T cells, large granular lymphocytes, and/or lymphoid aggregates. Neutropenia was initially responsive to granulocyte colony stimulating factor (GCSF) +/- steroids in several patients but ultimately become refractory to therapy. Immunomodulatory agents and chemotherapy were unsuccessful. Six patients underwent allogeneic HCT; two patients died at 8 and 18 years without definitive therapy; and one patient was lost to follow-up. The female patient had germline disease and presented with pure red blood cell aplasia at 2 weeks of life and is scheduled to undergo HCT. Transplant data were available for 5 of 6 patients who underwent HCT. Age at transplant was 3 to 21 years. One patient who received a graft from a 7/8 HLA-matched unrelated donor developed severe veno-occlusive disease and secondary graft failure. The patient underwent a second transplant and ultimately died. Another patient received a matched sibling donor HCT while critically ill from Candida lusitaniae and mucormycosis infection with associated multisystem organ failure. The patient engrafted with 100% donor chimerism but died on day +15 post-HCT. The remaining 3 patients are alive and well 8 months to 2 years post-HCT. Two patients received grafts from HLA-matched donors (1 sibling and 1 unrelated) with reduced intensity conditioning and cyclophosphamide, mycophenolate mofetil and vorinostat for graft-versus-host disease (GVHD) prophylaxis. The third patient underwent a haploidentical HCT with myeloablative conditioning and post-transplant cyclophosphamide. Post-HCT complications included acute skin and/or gastrointestinal GVHD that was responsive to therapy in all patients, and severe lung GVHD in one patient. There were no major infectious complications. All three patients had full donor chimerism in myeloid and T cells with complete resolution of disease phenotype at last follow-up. The patient for whom detailed transplant data is not available received a haploidentical HCT. He developed mixed myeloid chimerism for which he received multiple donor lymphocyte infusions and was alive at last contact &amp;gt;3 years post-HCT. TLR8 GOF should be considered in male and female patients with severe, unexplained neutropenia refractory to GCSF, and particular care should be taken when investigating for mutations given the disease-causing variant allele frequencies as low as 7%. Neutropenia and immune dysregulation were refractory to all therapies in our cohort, and TLR8 GOF was fatal in two patients who did not receive definitive therapy. Allogeneic HCT is curative and should be considered without delay for medically suitable patients.