Decreased expression of Toll-like receptor 4 and 5 during progression of prostate transformation in transgenic adenocarcinoma of mouse prostate mice

Chronic inflammation has been considered an important risk factor for development of prostate cancer. Toll-like receptors (TLRs) recognize microbial moieties or endogenous molecules and play an important role in the triggering and promotion of inflammation. In this study, we examined whether expression of TLR4 and TLR5 was associated with progression of prostate transformation in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. The expression of TLR4 and TLR5 was evaluated by immunohistochemisty in formalin-fixed paraffin-embedded prostate tissue from wild-type (WT) and TRAMP mice. Normal prostate tissue from WT mice showed strong expression of TLR4 and TLR5. However, TLR4 expression in the prostate tissue from TRAMP mice gradually decreased as pathologic grade became more aggressive. TLR5 expression in the prostate tissue from TRAMP mice also decreased in low-grade prostate intraepithelial neoplasia (PIN), high-grade PIN and poorly differentiated adenocarcinoma. Overall, our results suggest that decreased expression of TLR4 and TLR5 may contribute to prostate tumorigenesis.     

Development of a simple typing method for the ovine Toll-like receptor 4 gene

Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS) from Gram-negative bacteria, as well as a number of other ligands. Genetic variation in the TLR gene has been associated with altered immune responses to pathogens and results in variation in disease susceptibility. The objective of this work was to develop a simple and rapid genotyping system for ovine TLR4 and of a sensitivity that would allow detection of allelic variation in this gene. While variation in exon 3 of the ovine TLR4 gene has been described previously, we describe here an improved genotyping method. This method could not only reveal the four alleles that have been reported previously, but also revealed a further three new alleles of this gene in a population of 1670 New Zealand sheep. This improved genotyping method will be useful in understanding innate immune responses in individual sheep and could also be a useful tool for large-scale immune studies in sheep production systems.     

Expression profiles of antiviral response genes in chicken bursal cells stimulated with Toll-like receptor ligands

Cells of the adaptive immune system express Toll-like receptors (TLRs) and are able to respond to TLR ligands. With this in mind, the goal of the current study was to determine the expression of antiviral response genes in the cells of the chicken bursa of Fabricius (BF) to stimulation with TLR ligands. We investigated initially the response of bursal B cells to CpG-ODN, lipopolysaccharide (LPS) and poly(I:C) treatment. The expression level of type I interferons (IFNs) and interferon regulatory factor 7 (IRF7) did not differ between CpG-ODN and LPS treated groups compared to the non-stimulated cells. Poly(I:C) was the only TLR ligand, which has induced significant expression of antiviral innate immune response genes from bursal cells. Further in vitro and in vivo studies need to examine the efficacy of these antiviral responses against avian viruses.     

Induction of Toll-like receptor 4 signaling in avian macrophages inhibits infectious laryngotracheitis virus replication in a nitric oxide dependent way

LPS is one of the pathogen associated molecular patterns that activates Toll-like receptor 4 (TLR4) signaling pathway eliciting antiviral host responses in mammals although information on such responses in avian species is scarce. Our objectives were to characterize the LPS induced innate responses particularly the expression of LPS receptors (TLR4, CD14) in avian macrophages and observe whether TLR4 mediated induction of NO can elicit antiviral response against infectious laryngotracheitis virus (ILTV) replication. We found that LPS was capable of inducing the expression of TLR4, CD14 and NO production but not the type 1 interferons in an avian macrophage cell line, MQ-NCSU. We also showed that TLR4 mediated NO production can lead to antiviral response against ILTV replication when MQ-NCSU cells were treated with LPS and the resultant supernatant was then transferred to ILTV replicating cells to assess antiviral activity. Antiviral activity of NO was blocked by a selective inhibitor, S-methylisothiourea sulfhate that inhibits inducible NO synthase. This observation confirms that the antiviral activity is positively correlated with NO production. The data show that LPS can be a potential innate immune stimulant that can be used against ILTV infection in chickens that require further evaluation in vivo.     

Toll-like receptor expression in the nervous system of bovine alpha-herpesvirus-infected calves

In this study, the expression levels of viral Toll-like receptors (TLRs) in the nervous system of bovine herpesvirus type 5 (BoHV-5)-infected calves were investigated. A significant increase in the expression of TLRs 3 and 7–9 was found in the anterior cerebral cortex during acute infection and viral reactivation. In the trigeminal ganglia, only TLR9 expression was significantly affected. The magnitude of the increase was lower in BoHV-1-infected calves, suggesting that a restricted immune response might protect against exacerbated inflammatory responses in the brain. This work describes, for the first time, the involvement of TLRs 3 and 7–9 in the recognition of BoHV in the bovine nervous system, indicating that the expression of these receptors might be associated with the development of neurological disease. Modulation of the signalling pathways mediated by TLRs might provide an effective approach to control the neuro-immune response to BoHV-5, which may be responsible for neurological lesions.     

Cetacean Toll-like receptor 4 and myeloid differentiation factor 2, and possible cetacean-specific responses against Gram-negative bacteria

Toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (MD-2) are essential for recognizing the lipopolysaccharides (LPS) of Gram-negative bacteria. We determined the sequences of cDNAs encoding TLR4 and MD-2 from cetaceans and generated three-dimensional (3D) models for a better understanding of their modes of interaction and LPS recognition. The 3D reconstructions showed that cetacean TLR4 and MD-2 formed a horseshoe-like structure comprised of parallel β-strands and a β-cup structure consisting of two anti-parallel β-sheets, respectively. The (TLR4-MD-2)₂ duplex-heterodimer was shown to form a symmetrical structure. Comparison with the interfaces of the complexes in other mammals revealed that cetacean TLR4s have some amino acid residue substitutions involved in duplex-heterodimer formation and in species variation for LPS recognition. These substitutions in the changed amino acid residues may alter the interaction among TLR4, MD-2, and LPS and modify the TLR4/MD-2 immunological responses.     

脂多糖对小鼠乳腺组织中Toll-like Receptor4表达的影响

将40只雌性ICR小鼠,受孕后随机分为试验组和对照组。雌鼠产后10-11d,试验组经第4对乳头灌注LPS,对照组灌注生理盐水,分别于灌注后不同时间采集样本,组织学分析乳腺病理变化;分析对各组乳腺组织中TLR4和TNF-α mRNA表达变化。组织学结果显示,灌注1．5h后乳腺组织中炎性细胞增多,6、12h乳腺腺泡内有大量的炎性细胞浸润,腺泡结构崩解;6h TLR4 mRNA表达极显著高于对照组（P〈0．01）;4个试验组中TNF-αmRNA表达极显著高于对照组（P〈0．01）。试验结果表明LPS能够增强TLR4和TNF-α mRNA表达。     

Advanced molecular immunoassay system for immunobiotic lactic acid bacteria using a transfectant of Toll-like receptor 2

Toll‐like receptor 2 (TLR2) is a receptor for a variety of microbial components, and it also mediates activation signals in the cell relating to the innate immune system. In order to evaluate the precise molecular immunoregulation by various strains of lactic acid bacteria (LAB) via TLR2, the swine TLR2 (sTLR2)‐expressing transfectant was constructed using human embryonic kidney (HEK) 293 cells. It is demonstrated that intact immunobiotic LAB can induce immune responses through TLR2, and that different nuclear factor‐κB (NF‐κB) activities of various strains can be accurately detected by sTLR2‐expressing HEK293 cells. Furthermore, cellular activation of NF‐κB via TLR2 is reflected in enhanced binding and uptake of LAB. The sTLR2‐expressing HEK293 cells were also useful for characterizing the expression pattern of type I helper T (Th1) and type II helper T (Th2) cytokines by the stimulation of immunobiotic LAB. These results suggest that sTLR2‐expressing HEK293 cells may be useful in certain molecular immunoassay systems for producing new physiologically functional foods with intestinal immunomodulatory abilities, such as the maintenance of Th1/Th2 polarization.     

A clinical association between Toll-like receptor 2 Arg753Gln polymorphism with recurrent cystic echinococcosis in postsurgery patients: A case control study

Recurrence of hydatid cysts in cystectomy patients has dramatically remained a serious concern within the surgical community. Predisposing factors for recurrence of hydatid cysts remained to be identified. Toll-like receptor (TLR) plays a pivotal role in bridging between acquired and innate immunity in cystic echinococcosis (CE) infection. 117 CE patients including 66 acute hydatidosis (AH; primary infection) and 51 recurrent hydatidosis (RH; chronic infection), and 117 ethnically matched healthy control (HC) were investigated from endemic regions of Iran in the period of 2015–2018. CE patients were definitely confirmed using histopathological and immunological assays. Genotyping of TLR2 Arg753Gln was carried out by restriction fragment length polymorphism and sequencing. The homozygous mutant-type TLR2 Gln/Gln (A/A) was represented to be associated with the occurrence of RH (P = 0.04) and conferred a 9 fold risk for susceptibility, while the heterozygous mutant-type TLR2 Arg/Gln (G/A) indicated a tendency to be associated with the occurrence of RH (P = 0.07). There was no discrepancy in the frequency of TLR2 Arg753Gln haplotypes between AH patients and HC individuals (P = 0.09). The mutant allele A was observed to be a risk factor for susceptibility to RH patients. Our results point to a clinical association between TLR2 Arg753Gln haplotypes with RH in postoperative patients. It can be inferred that allele G may lead to protection against the CE, while mutant allele A may be a diagnostic hallmark in the screening of RH susceptibility. Nevertheless, further studies with a larger sample size of different ethnic populations are required to authenticate this association.     

Development of immune assay system for both CpG and non-CpG DNA from lactic acid bacteria using a transfectant of swine Toll-like receptor 9

Bacterial DNA is expected to be a potent immune stimulating agent to Toll‐like receptor (TLR) 9 expressed cells such as macrophages, monocytes, B lymphocytes, NK cells and dendritic cells. In the present study, we constructed a transfectant of swine TLR9 with mammalian cells. We demonstrated that the transfectant, recognizing both CpG and non‐CpG oligonucleotides from lactic acid bacteria, induced NF‐κB activation by gene reporter assay. These findings indicate that the swine TLR9 transfectant will be highly useful for the screening of immunostimulatory DNA from lactic acid bacteria.     

黑皮质素4型受体在绵羊下丘脑区域的定位

黑皮质素4型受体(melanocortin 4 receptor,MC4R)作为下丘脑参与能量调控过程中的关键性受体,在动物能量代谢调节中被广泛关注。目前,关于该受体在绵羊下丘脑中分布区域的相关报道较少。针对试验过程中MC4R免疫荧光结果不稳定,分布区域不明显的特点,本研究在改进现有MC4R免疫荧光试验方法基础上,对MC4R在绵羊下丘脑的分布区域进行了观察。结果显示,分别经过4%多聚甲醛长时间固定(6 d),Triton X-100(pH≈8.68)长时间通透(2 d),以及3%过氧化氢和0.2 mol/L甘氨酸+0.3%SDS+20%甲醇的预处理后,可以获得稳定且饱满的MC4R免疫荧光结果。MC4R主要分布在绵羊下丘脑的室旁核(arcuate nucleus,ARC)区域,但在弓状核区域并无类似分布。     

荧光定量RT-PCR检测雏鸡感染NDV强毒后胸腺和法氏囊中TLR2与TLR4 mRNA的表达

Toll样受体2(TLR2)和Toll样受体4(TLR4)在识别病原微生物过程中发挥重要作用。为了定量检测TLR2、TLR4 mRNA表达水平,研究病原与机体的相互作用,本研究建立了检测鸡TLR2、TLR4 mRNA表达水平的SYBR GreenⅠ荧光定量RT-PCR(RRT-PCR)方法,检测了新城疫病毒强毒(vNDV)感染SPF雏鸡后36h、48h时胸腺、法氏囊中TLR2、TLR4 mRNA表达量变化。结果显示该方法特异性好,RRT-PCR产物分别在85.5℃、83.3℃出现单特异峰,对TLR2、TLR4的扩增效率分别为105.91%和95.30%,相关系数分别为0.9980、0.9996,最低检测限分别为108拷贝/反应和461拷贝/反应。感染后36h vNDV显著抑制TLR2、TLR4基因在法氏囊、胸腺中的表达;感染后48h时,法氏囊、胸腺中TLR2基因的表达水平显著升高,胸腺中TLR4基因的表达显著升高,而法氏囊中TLR4基因的表达仍处于抑制状态。本研究证明TLR2和TLR4参与了鸡体对NDV的感染应答。     

The role of Corynebacterium pseudotuberculosis lung lesions in the transmission of this bacterium to other sheep

SUMMARY: :Five groups of 5 shorn and 5 unshorn caseous lymphadenitis (CLA)-free Merino wether weaners were each placed in feedlot pens with 6 Merino ewes, 2 or more of which had CLA lung lesions but no discharging superficial lesions. The sheep were kept together for 5 months.
Twenty-eight per cent of the shorn weaners and 20% of the unshorn weaners developed antibodies to Corynebacterium pseudotuberculosis. At slaughter, 8% of the shorn weaners and 12% of the unshorn weaners had CLA lesions in either lungs, lymph nodes or both. In the absence of contact with CLA-infected ewes, a control group of 5 shorn and 5 unshorn weaners failed to develop antibodies to C. pseudotuberculosis or CLA lesions in the same period. This showed that sheep with CLA abscesses in the lungs but no discharging superficial abscesses were a source of C. pseudotuberculosis infection to other sheep. Aust Vet J. 64: 261–263     

不同日龄鸡法氏囊中TLR15、LY86和TRIM39 mRNA表达水平

本研究通过荧光定量PCR方法检测了TLR15、LY86和TRIM39 mRNA在不同日龄鸡法氏囊中的变化。结果显示,10日龄时,TLR15 mRNA的相对表达量显著高于18胚龄时的,10日龄和30日龄时的表达量基本相同;出壳后,LY86 mRNA的表达水平呈下降趋势,30日龄时的表达量极显著低于18胚龄时的;而TRIM39 mRNA在出壳后表达量逐渐增加,30日龄时的表达量极显著高于18胚龄时的。本研究结果表明,TLR15、LY86和TRIM39在鸡法氏囊发育过程中发挥着重要作用。     

Concurrent response to challenge infection with Cryptosporidium parvum in immunosuppressed C57BL/6N mice

We investigated the response to challenge infection with Cryptosporidium parvum oocysts in immunosuppressed C57BL/6N mice. In the primary infection, fecal oocyst shedding and parasite colonization were greater in immunosuppressed mice than in nonimmunosuppressed mice. Compared with primary infection, challenge infection with C. parvum didn''t show any oocyst shedding and parasite colonization. Especially, oocyst shedding and parasite colonization from the mice infected with heat-killed oocysts were not detected. After challenge infection with C. parvum oocysts, however, these mice were shedding small numbers of oocysts and parasite colonization. Except normal control and uninfected groups, the antibody titers of other groups appear similar. Based on the fecal oocyst shedding, parasite colonization of ilea, and antibody titers in the mice, these results suggest that the resistance to challenge infection with C. parvum in immunosuppressed C57BL/6N mice has increased.     

Exendin-4 improves resistance to Listeria monocytogenes infection in diabetic db/db mice

The incidence of diabetes mellitus is increasing among companion animals. This disease has similar characteristics in both humans and animals. Diabetes is frequently identified as an independent risk factor for infections associated with increased mortality. In the present study, homozygous diabetic (db/db) mice were infected with Listeria (L.) monocytogenes and then treated with the anti-diabetic drug exendin-4, a glucagon-like peptide 1 analogue. In aged db/db mice, decreased CD11b⁺ macrophage populations with higher lipid content and lower phagocytic activity were observed. Exendin-4 lowered high lipid levels and enhanced phagocytosis in macrophages from db/db mice infected with L. monocytogenes. Exendin-4 also ameliorated obesity and hyperglycemia, and improved ex vivo bacteria clearance by macrophages in the animals. Liver histology examined during L. monocytogenes infection indicated that abscess formation was much milder in exendin-4-treated db/db mice than in the control animals. Moreover, mechanistic studies demonstrated that expression of ATP binding cassette transporter 1, a sterol transporter, was higher in macrophages isolated from the exendin-4-treated db/db mice. Overall, our results suggest that exendin-4 decreases the risk of infection in diabetic animals by modifying the interaction between intracellular lipids and phagocytic macrophages.     

禽传染性喉气管炎病毒体内外感染模型中鸡Toll样受体21mRNA转录水平的研究

为研究病毒与机体的相互作用,本研究参考GenBank中鸡Toll样受体21 (ChTLR21)的基因序列设计实时定量PCR特异性引物,以鸡核糖体蛋白L4 (RPL4)为内参基因,建立检测ChTLR21 mRNA相对转录水平的实时定量PCR方法,分析ChTLR21在禽传染性喉气管炎病毒(ILTV)感染的鸡胚成纤维细胞(CEF)中和感染SPF雏鸡免疫器官脾脏、法氏囊和胸腺组织中的转录水平.结果显示:ILTV感染CEF后2h、4h、8h和18h时间点ChTLR21 mRNA转录水平分别为未感染对照细胞的1 540.53、0.98、1.19和3.70倍.但仅在ILTV感染2h时引起ChTLR21转录水平升高,与对照组相比差异显著(P＜0.05).ILTV感染SPF雏鸡后6h、24 h和30 h脾脏中ChTLR21 mRNA转录水平分别为未感染对照的56.34、59.85和3.61倍;法氏囊中分别为0.03、25.98和3.08倍;胸腺中分别为2.52、50和7.32倍.在感染初期,脾脏中ChTLR21转录量显著升高,随后有所降低,但均高于未感染对照(p＜0.05);法氏囊中仅在感染6h时呈显著抑制(p＜0.01);胸腺中呈波动性转录水平升高,但与对照组无统计学差异(p＞0.05).本研究证明ChTLR21参与了鸡体对ILTV感染的应答,并在体内外感染模型中呈现不同的表达规律.     

猪附红细胞体对不同宿主红细胞的体外感染试验

本试验在猪附红细胞体体外培养的基础上,用猪附红细胞体的阳性血液体外感染家兔、昆明小白鼠、犬、羊、牛及人的健康红细胞。结果表明,上述宿主的健康红细胞均可不同程度地发生感染,其中体外感染家兔红细胞,在第36小时感染率最高,为45.0%;感染昆明小白鼠红细胞,在第24小时感染率最高,为40.3%;感染人红细胞,在第48小时感染率最高,为30.0%,呈现轻度感染;而对其他宿主红细胞,呈现一过性感染。