Toll-like receptor 2 is expressed on the intestinal M cells in swine

The Toll-like receptor (TLR) 2 binds a wide variety of microbial cell wall components. In this study, we investigated the expression pattern of TLR2 in adult swine gut-associated lymphoid tissues using real-time quantitative PCR, Western blotting, immunohistochemistry, and flow cytometric analysis. The mRNA for TLR2 was preferentially expressed in the mesenteric lymph nodes (MLNs) and Peyer's patches (Pps) of adult swine. Expression in these two tissues was approximately 15- and 9-fold higher than that of spleen, respectively. Western blotting further confirmed that the TLR2 protein was highly expressed in the MLNs and Pps. Interestingly, TLR2-expressing cells were found not only in immune cells, such as T cells and B cells, but also in membranous (M) cells. In addition, double immunostaining for TLR2 and cytokeratin 18 revealed that TLR2 was strongly expressed not only in the cytoplasm but also in the apical membrane of the pocket-like M cells. These results indicate that TLR2 on the MLNs and Pps enable the host defense to respond to a variety of cell wall components. Furthermore, the potential function of TLR2 as a pattern recognition receptor and its cellular distribution suggest that TLR2 plays an important role in ligand-specific transcytosis and transport in M cells.     

Evaluation of lactic acid bacterial strains of boza for their exopolysaccharide and enzyme production as a potential adjunct culture

Boza is a non-alcoholic beverage obtained from fermented cereals. Thirteen lactic acid bacteria (LAB), previously isolated from boza were identified and evaluated to determine the various technological properties for selecting appropriate strains as adjunct culture in boza. Each isolate was checked for purity, Gram-stained and tested for the catalase and oxidase activity and then subjected to identification by polymerase chain reaction (PCR) with partial 16S rRNA gene sequencing. The tests for carbohydrate fermentation and enzyme profiles were carried out with the API 50 CHL and API ZYM galleries, respectively. Exopolysaccharide (EPS) production of strains was determined by Fourier transform infrared spectroscopy (FT-IR) and quantified by the phenol sulphuric acid method. To our best knowledge, this is the first study reporting on Lactococcus garvieae (E32), Pediococcus parvulus (E42) and Streptococcus macedonicus (A15) in boza. All strains, except S. macedonicus (A15) produced EPS. Leuconostoc citreum (E55) and Lactococcus lactis (A47) were the highest EPS producing strains, yielding 2.39 ± 0.49 and 1.98 ± 0.23 g/L of EPS, respectively. Lactobacillus paracasei (D41), Lactobacillus plantarum (B2), Lactococcus lactis (F39) and among low-EPS producing strains Lactobacillus coryniformis (C55), L. paracasei (E8), and P. parvulus (E42) were evaluated to be promising candidates as potential adjunct culture in boza. The variety of enzyme production was also concern. Lc. garvieae (E32) was found to produce the largest variety of enzymes among the strains. FT-IR spectroscopy can be used for the assessment of EPS production by microorganisms reliably and accurately.     

Direct Toll-like receptor 2 mediated co-stimulation of T cells in the mouse system as a basis for chronic inflammatory joint disease

The pathogenesis of chronic inflammatory joint diseases such as adult and juvenile rheumatoid arthritis and Lyme arthritis is still poorly understood. Central to the various hypotheses in this respect is the notable involvement of T and B cells. Here we develop the premise that the nominal antigen-independent, polyclonal activation of preactivated T cells via Toll-like receptor (TLR)-2 has a pivotal role in the initiation and perpetuation of pathogen-induced chronic inflammatory joint disease. We support this with the following evidence. Both naive and effector T cells express TLR-2. A prototypic lipoprotein, Lip-OspA, from the etiological agent of Lyme disease, namely Borrelia burgdorferi, but not its delipidated form or lipopolysaccharide, was able to provide direct antigen-nonspecific co-stimulatory signals to both antigen-sensitized naive T cells and cytotoxic T lymphocyte (CTL) lines via TLR-2. Lip-OspA induced the proliferation and interferon (IFN)-γ secretion of purified, anti-CD3-sensitized, naive T cells from C57BL/6 mice but not from TLR-2-deficient mice. Induction of proliferation and IFN-γ secretion of CTL lines by Lip-OspA was independent of T cell receptor (TCR) engagement but was considerably enhanced after suboptimal TCR activation and was inhibitable by monoclonal antibodies against TLR-2.     

The protective effect of trefoil factor 3 on the intestinal tight junction barrier is mediated by toll-like receptor 2 via a PI3K/Akt dependent mechanism

Trefoil factor peptides are highly conserved secreted molecules characterized by heat and enzymatic digestion resistance. Intestinal trefoil factor 3 (TFF3) protects and repairs the gastrointestinal mucosa and restores normal intestinal permeability, which is dependent on the integrity of the tight junction (TJ) barrier and the TJ associated proteins claudin-1, zona occludens-1 (ZO-1) and occludin. Despite the important role of intestinal barrier dysfunction in the pathogenesis of inflammatory bowel diseases, the underlying mechanisms and associated molecules remain unclear. In the present study, we show that TFF3 and toll-like receptor 2 (TLR2) are functionally linked and modulate intestinal epithelial permeability via a mechanism that involves the PI3K/Akt pathway. We used the Caco-2 cell model to show that TLR2 and TFF3 inhibit the IL-1β induced increase in permeability and release of proinflammatory cytokines, and that this effect is mediated by activation of PI3K/Akt signaling. TLR2 silencing downregulated the expression of TFF3 and overexpression of TLR2 and TFF3 increased the levels of phospho-Akt. TFF3 overexpression significantly upregulated the expression of ZO-1, occludin and claudin-1 and this effect was abrogated by inhibition of the PI3K/Akt pathway. Taken together, our results indicate that TLR2 signaling selectively enhances intestinal TJ barrier integrity through a mechanism involving TFF3 and the activation of the PI3K/Akt pathway.     

Toll-like receptor 9-mediated cytosolic phospholipase A2 activation regulates expression of inducible nitric oxide synthase

Although CpG containing DNA is an important regulator of innate immune responses via toll-like receptor 9 (TLR9), excessive activation of this receptor is detrimental to the host. Here, we show that cytosolic phospholipase A₂ (cPLA₂) activation is important for TLR9-mediated inducible nitric oxide synthase (iNOS) expression. Activation of TLR9 signaling by CpG induces iNOS expression and NO production. Inhibition of TLR9 blocked the iNOS expression and NO production. The CpG also stimulates cPLA₂-hydrolyzed arachidonic acid (AA) release. Inhibition of cPLA₂ activity by inhibitor attenuated the iNOS expression by CpG response. Additionally, knockdown of cPLA₂ protein by miRNA also suppressed the CpG-induced iNOS expression. Furthermore, the CpG rapidly phosphorylates three MAPKs and Akt. A potent inhibitor for p38 MAPK or Akt blocked the CpG-induced AA release and iNOS expression. These results suggest that TLR9 activation stimulates cPLA₂ activity via p38 or Akt pathways and mediates iNOS expression.     

A microsatellite polymorphism in intron 2 of human Toll-like receptor 2 gene: functional implications and racial differences

The human Toll-like receptor 2 (TLR2) mediates responses of both innate and adaptive immunity to Gram-positive bacteria, including mycobacteria. We sought functional polymorphisms in the 5'-untranslated region (UTR) of TLR2. We found a highly polymorphic (GT)n dinucleotide repeat 100 bp upstream of the TLR2 translational start site in intron 2. The numbers of GT repeats varied from 12 to 28. There were significant differences in allele distribution between African Americans and Caucasians (P=0.008) and between African Americans and Koreans (P=0.0003). The promoter activities of recombinant promoter-intron2/reporter constructs including the shortest [GT)n=12] or longest [(GT)n=28] alleles were significantly more stimulated when exposed to 200 IU ml(-1) of interferon-gamma than when exposed to 100 IU ml(-1) of GM-CSF (P<==0.03). Since TLR2 plays a critical role in the human innate immune response, this functional microsatellite polymorphism may be important in the pathogenesis of infectious and inflammatory diseases.     

Simple synthetic toll-like receptor 2 ligands

Stimulation of toll-like receptor 2 (TLR2) by bacterial lipoproteins induces fast non-specific immune responses against pathogens followed by slow but specific adaptive immune responses. Development of synthetic TLR2 agonists/antagonists would be useful in the prevention of different infectious and immunologic disorders. The current study reports synthesis and TLR2 activity of two simple TLR2 ligands, which feature minimal structural requirement for TLR2 activity (two long lipid chains) and stimulate agonistic activity at nanomolar concentration.     

Synthesis,structure-activity relationships and preliminary mechanism study of N-benzylideneaniline derivatives as potential TLR2 inhibitors

Toll-like receptor 2 (TLR2) can recognize pathogen-associated molecular patterns to defense against invading organisms and has been represents an attractive therapeutic target. Until today, none TLR2 small molecule antagonist have been developed in clinical trial. Herein, we designed and synthesized 50?N-benzylideneaniline compounds with the help of CADD. And subsequent in vitro studies leading to the optimized compound SMU-A0B13 with most potent inhibitory activity to TLR2 (IC₅₀=18.21?±?0.87?μM). Preliminary mechanism studies indicated that this TLR2 inhibitor can work through the NF-κB signaling pathway with high specificity and low toxicity, and can also efficiently downregulate inflammatory cytokines, such as SEAP, TNF-α and NO in HEK-Blue hTLR2, human PBMC and Raw 264.7 cell lines. Additionally, the docking situation also indicate SMU-A0B13 can well bind to the TLR2-TIR (PDB: 1FYW) active domain, which probably explains the bioactivity.     

The diacylated lipopeptide FSL-1 enhances phagocytosis of bacteria by macrophages through a Toll-like receptor 2-mediated signalling pathway

A significant amount of evidence has been accumulated to show that Toll-like receptors (TLRs) function as sensors for microbial invasion. However, little is known about how signalling triggered by TLRs leads to the phagocytosis of pathogens. This study was designed to determine whether stimulation of TLR2 mainly with the lipopeptide FSL-1 plays a role in the phagocytosis of pathogens by macrophages. FSL-1 enhanced the phagocytosis of Escherichia coli to a markedly greater extent than it did that of Staphylococcus aureus, but did not enhance the phagocytosis of latex beads. FSL-1 stimulation resulted in enhanced phagocytosis of bacteria by macrophages from TLR2(+/+) mice but not by those from TLR2(-/-) mice. Chinese hamster ovary cells stably expressing TLR2 failed to phagocytose these bacteria, but the cells expressing CD14 did. FSL-1 induced upregulation of the expression of phagocytic receptors, including MSR1, CD36, DC-SIGN and Dectin-1 in THP-1 cells. Human embryonic kidney 293 cells transfected with DC-SIGN and MSR1 phagocytosed these bacteria. These results suggest that the FSL-1-induced enhancement of phagocytosis of bacteria by macrophages may be explained partly by the upregulation of scavenger receptors and the C-type lectins through TLR2-mediated signalling pathways, and that TLR2 by itself does not function as a phagocytic receptor.     

Guanosine analog in the pyrido[2,3-d]pyrimidine ring system As a potential Toll-like receptor agonist

The synthesis of a guanosine analog in the pyrido[2,3-d]pyrimidine ring system has been accomplished by glycosylation of the preformed aromatic heterocyclic base, which was prepared in 2 steps by condensation of methyl acrylate with guanidine carbonate and methyl cyanoacetate in the presence of sodium methoxide, followed by dehydrogenation. The analog was evaluated in vitro for its ability to modulate the innate immune response by acting as an agonist or as an antagonist of Toll-like receptor (TLR) signaling by measuring cytokine induction or inhibition of induction, respectively, in mouse bone marrow-derived macrophages. Despite its structural similarity to 7-thia-8-oxoguanosine, a known TLR7 agonist, the analog was found to antagonize TLR7-induced cytokine induction in this cell-based assay.     

Pharmacophore mapping and in silico screening to identify new potent leads for A2A adenosine receptor as antagonists

A_2A adenosine receptor (AR) antagonists play an important role in neurodegenerative diseases like Parkinson’s disease. A 3D-QSAR study of A_2A AR antagonists, was taken up to design best pharmacophore model. The pharmacophoric features (ADHRR) containing a hydrogen bond acceptor (A), a hydrogen bond donor (D), a hydrophobic group (H) and two aromatic rings (R), is projected as the best predictive pharmacophore model. The QSAR model was further treated as a template for in silico search of databases to identify new scaffolds. The binding patterns of the leads with A_2A AR are analysed using docking studies and novel potent ligands of A_2A AR are projected.     

Adenosine A2A and A2B receptor expression in neuroendocrine tumours: potential targets for therapy

The clinical management of neuroendocrine tumours is complex. Such tumours are highly vascular suggesting tumour-related angiogenesis. Adenosine, released during cellular stress, damage and hypoxia, is a major regulator of angiogenesis. Herein, we describe the expression and function of adenosine receptors (A(1), A(2A), A(2B) and A(3)) in neuroendocrine tumours. Expression of adenosine receptors was investigated in archival human neuroendocrine tumour sections and in two human tumour cell lines, BON-1 (pancreatic) and KRJ-I (intestinal). Their function, with respect to growth and chromogranin A secretion was carried out in vitro. Immunocytochemical data showed that A(2A) and A(2B) receptors were strongly expressed in 15/15 and 13/18 archival tumour sections. Staining for A(1) (4/18) and A(3) (6/18) receptors was either very weak or absent. In vitro data showed that adenosine stimulated a three- to fourfold increase in cAMP levels in BON-1 and KRJ-1 cells. The non-selective adenosine receptor agonist (adenosine-5'N-ethylcarboxamide, NECA) and the A(2A)R agonist (CGS21680) stimulated cell proliferation by up to 20-40% which was attenuated by A(2B) (PSB603 and MRS1754) and A(2A) (SCH442416) receptor selective antagonists but not by the A(1) receptor antagonist (PSB36). Adenosine and NECA stimulated a twofold increase in chromogranin A secretion in BON-1 cells. Our data suggest that neuroendocrine tumours predominantly express A(2A) and A(2B) adenosine receptors; their activation leads to increased proliferation and secretion of chromogranin A. Targeting adenosine signal pathways, specifically inhibition of A(2) receptors, may thus be a useful addition to the therapeutic management of neuroendocrine tumours.     

New integration vector using a cellulase gene as a screening marker for Lactobacillus

The new integration vector for Lactobacillus, pJC4, was developed using the extracellular endoglucanase A gene (celA) of Clostridium thermocellum as a screening marker. pJC4 was transformed into four Lactobacillus species, Lb. johnsonii, Lb. gasseri, Lb. bulgaricus, and Lb. plantarum. In each species, the pJC4 integrants were easily and accurately detected by the appearance of a clear halo on a cellulase screening plate without any false transformants. Polymerase chain reaction and Southern hybridization indicated that all transformants with clear halos contained pJC4 in their chromosomal DNAs. The celA gene could be a useful screening marker for other lactic acid bacteria.     

Modulation of interleukin 2 high-affinity binding by lymphocyte-derived tetrahydrobiopterin: pterins as potential participants in the control of interleukin 2 receptor assembly

In this report, we have examined whether (6R)-tetrahydrobiopterin (H4biopterin) modulates the binding of interleukin 2 to high-affinity sites of the cloned mouse cytotoxic T-lymphocyte clone CTLL-2. Scatchard plot analysis of the equilibrium binding data reveals increased affinity when the cells are exposed simultaneously to interleukin 2 and to the pterin. The Kd values are statistically significantly reduced from 1.4 x 10(-11) M to 0.78 x 10(-11) M interleukin 2. The dissociation kinetics of the ligand were followed at 4 degrees C after equilibrium binding under high-affinity conditions (1.2 x 10(-10) M interleukin 2). In the presence of H4 biopterin, the dissociation rate constant (k-1) decreases from 6.2 x 10(-3) min-1 to 3.0 x 10(-3) min-1 and the half-time for dissociation increases from 106.8 min to 218.0 min. As a third approach interleukin 2 was bound to the surface of cells under high-affinity conditions by incubation in the cold and the internalization kinetics upon warming were determined. Sigmoidal-shaped kinetics of endocytosis in control cells indicate that the internalization rates increase only gradually. The presence of H4 biopterin causes an apparent immediate transition from higher-order kinetics to a linear response so that maximum internalization rates are reached immediately upon warming. The data show that lymphocyte-derived H4 biopterin in vitro at concentrations ranging from 2-8 x 10(-7) M modulates interleukin 2 high-affinity binding and that H4 biopterin potentially participates in the control of interleukin 2 receptor assembly.     

An efficient biocatalytic system for floxuridine biosynthesis based on Lactobacillus animalis ATCC 35046 immobilized in Sr-alginate

An efficient and green immobilized biocatalyst is herein reported to obtain 5-fluorouracil-2′-deoxyriboside (5FUradRib), an antimetabolite known as Floxuridine, used in gastrointestinal cancer treatment.Alginate is a natural polysaccharide used in the pharmaceutical industry due to its physicochemical properties, biocompatibility and non-toxicity. Multivalent cations, exposure time and cross-linking solution concentration were optimized, being Sr²⁺, 2 h and 0.2 M the best immobilization conditions. Furthermore, compression strength, swelling ratio and fracture frequency were evaluated, improving the mechanical stability of the biocatalyst favoring a future scale-up.On the other hand, the reaction parameters for 5FUradRib biosynthesis were optimized in order to obtain an immobilized biocatalyst with enhanced activity. Thus, Lactobacillus animalis ATCC 35046 immobilized in Sr-alginate showed yields of 96% at short reaction times.The obtained biocatalyst was stable for more than 25 days in storage conditions (4 °C) and could be reused at least 10 times without loss of its activity.Additionally, Sr-alginate biocatalyst stability was evaluated in different organic solvents to obtain hydrophobic compounds such as 5-bromouracil-2′-deoxyriboside (5BrUradRib), an effective radiosensitizing agent used in anti-cancer therapy, being hexane the best co-solvent.Finally, a smooth, cheap and environmentally friendly method to obtain anti-cancer drugs was developed in this study.     

Immunochemistry for oestrogen receptor,progesterone receptor and HER2 on cell blocks in primary breast carcinoma

K. Kumar S, N. Gupta, A. Rajwanshi, K. Joshi and G. Singh Immunochemistry for oestrogen receptor, progesterone receptor and HER2 on cell blocks in primary breast carcinoma Objective: Steroid receptors and human epidermal growth receptor 2 (HER2) have been used for predicting response to treatment in breast cancers. Fine needle aspiration cytology can provide highly cellular material and can be used for such analysis. The present study was undertaken to assess the reliability of oestrogen and progesterone receptor (ER, PR) status and HER2 as demonstrated by immunochemistry (IHC) on cell blocks from breast carcinoma cases, in comparison with histological sections. Methods: IHC for ER, PR and HER2 was performed on cell blocks and their corresponding tissue sections of 50 primary pre‐chemotherapy breast carcinomas. Positivity for ER and PR was scored according to the Allred scoring system. Strong membranous positivity in more than 30% of tumour cells was considered positive for HER2. The tumours were classified as luminal A, luminal B, HER2‐over‐expressing and triple negative on the basis of ER, PR and HER2 status and results on cell blocks compared with histological sections. Results: Correlation between immunostaining on cell blocks and the corresponding tumour tissues revealed a concordance rate for ER, PR and HER2 of 90% [Correlation coefficient (r) = 0.79], 94% (r = 0.86) and 90% (r = 0.76), respectively. Including five cases in which cell blocks were either ER or PR positive, 43/50 cases (86.0%) could be correctly classified on cell block immunostaining alone. The main reasons for seven discordant cases included technical errors (sampling error and staining error) and interpretational error in HER2 evaluation on cell blocks; the core biopsy was inadequate in one, and apparently false negative for HER2 in another. Conclusion: Cell blocks are useful in the assessment of hormone receptor status and HER2 by IHC, especially in cases of locally advanced breast cancer for planning neoadjuvant chemotherapy. It is highly recommended to have good quality cell blocks and quality control of their interpretation.     

The human bitter taste receptor hTAS2R39 is the primary receptor for the bitterness of theaflavins

We purified several hundred mgs of four major theaflavins (theaflavin, theaflavin-3-O-gallate, theaflavin-3′-O-gallate, and theaflavin-3,3′-O-digallate). Among the 25 hTAS2Rs expressed in HEK293T cells, hTAS2R39 and hTAS2R14 were activated by theaflavins. Both hTAS2R39 and hTAS2R14 responded to theaflavin-3′-O-gallate. In addition, hTAS2R39 was activated by theaflavin and theaflavin-3,3′-O-gallate, but not by theaflavin-3-O-gallate. In contrast, hTAS2R14 responded to theaflavin-3-O-gallate.     

Protease-activated receptor 1 as a potential therapeutic target for COVID-19

Acute respiratory disease caused by a novel coronavirus (SARS-CoV-2) has spread all over the world, since its discovery in 2019, Wuhan, China. This disease is called COVID-19 and already killed over 1 million people worldwide. The clinical symptoms include fever, dry cough, dyspnea, headache, dizziness, generalized weakness, vomiting, and diarrhea. Unfortunately, so far, there is no validated vaccine, and its management consists mainly of supportive care. Venous thrombosis and pulmonary embolism are highly prevalent in patients suffering from severe COVID-19. In fact, a prothrombotic state seems to be present in most fatal cases of the disease. SARS-CoV-2 leads to the production of proinflammatory cytokines, causing immune-mediated tissue damage, disruption of the endothelial barrier, and uncontrolled thrombogenesis. Thrombin is the key regulator of coagulation and fibrin formation. In severe COVID-19, a dysfunctional of physiological anticoagulant mechanisms leads to a progressive increase of thrombin activity, which is associated with acute respiratory distress syndrome development and a poor prognosis. Protease-activated receptor type 1 (PAR1) is the main thrombin receptor and may represent an essential link between coagulation and inflammation in the pathophysiology of COVID-19. In this review, we discuss the potential role of PAR1 inhibition and regulation in COVID-19 treatment.     

Identification of serotonin 2A receptor as a novel HCV entry factor by a chemical biology strategy

Hepatitis C virus(HCV)is a leading cause of liver disease worldwide.Although several HCV protease/polymerase inhibitors were recently approved by U.S.FDA,the combination of antivirals targeting multiple processes of HCV lifecycle would optimize anti-HCV therapy and against potential drug-resistanee.Viral entry is an essential target step for antiviral development,but FDA-approved HCV entry inhibitor remains exclusive.Here we identify serotonin 2A receptor(5-HT2aR)is a HCV entry factor amendable to therapeutic intervention by a chemical biology strategy.The silencing of 5-HT2aR and clinically available 5-HT2aR antagonist suppress cell culture-derived HCV(HCVcc)in different liver cells and primary human hepatocytes at late endocytosis process.The mechanism is related to regulate the correct plasma membrane localization of claudin 1(CLDN1).Moreover,phenoxybenzamine(PBZ),an FDAapproved 5-HT2aR antagonist,inhibits all major HCV genotypes in vitro and displays synergy in combination with clinical used anti-HCV drugs.The impact of PBZ on HCV genotype 2a is documented in immune-competent humanized transgenic mice.Our results not only expand the understanding of HCV entry,but also present a promising target for the invention of HCV entry inhibitor.