Monoclonal antibodies used as prophylactic,therapeutic and diagnostic agents

Monoclonal antibodies can be of mouse, part mouse part human (chimeric, humanized), or of human origin. Their preparation involves hybridoma, gene cloning, gene recombination, phage display, and gene transfection techniques. The preparation, mechanism of action, uses, and possible adverse effects of most of the available monoclonal antibodies used as prophylactic, therapeutic, and diagnostic agents are reviewed.     

Effect of opiate receptor agonists and antagonists on maternal aggression in rats

Department of Pharmacology, I. P. Pavlov First Leningrad Medical Institute. (Presented by Academy of Medical Sciences of the USSR A. V. Val'dman.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107, No. 2, pp. 207–209, February, 1989.     

Anti-inflammatory activity of small-molecule antagonists of Toll-like receptor 2 (TLR2) in mice

Toll-like receptor 2 (TLR2) is currently investigated as a potential therapeutic target in diseases with underlying inflammation like sepsis and arthritis. We reported the discovery, by virtual screening and biological testing, of eight TLR2 antagonists (AT1-AT8) which showed TLR2-inhibitory activity in human cells (Murgueitio et al., 2014). In this study, we have deepened in the mechanism of action and selectivity (TLR2/1 or TLR2/6) of those compounds in mouse primary cells and in vivo. The antagonists reduced, in a dose-dependent way the TNFα production (e.g. AT5 IC₅₀ 7.4 μM) and also reduced the nitric oxide (NO) formation in mouse bone marrow-derived macrophages (BMDM). Treatment of BMDM with the antagonists showed that downstream of TLR2, MAPKs phosphorylation and IkBα degradation was reduced. Notably, in a mouse model of tri-acylated lipopeptide (Pam3CSK4)-induced inflammation, AT5 attenuated the TNFα and IL-6 inflammatory response. Further, the effect of AT5 in the stimulation of BMDM by the endogenous alarmin HMGB1 was investigated. Our results indicate that AT4-AT7 and, particularly AT5 appear as good starting points for the development of inhibitors targeting TLR2 in inflammatory disorders.     

Effects of selective adrenergic agonists and antagonists on gastric tone in the rat

The aim of the present in vivo study was to investigate in anaesthetized rats, the effects of selective adrenergic agonists and antagonists on basal gastric tone and phasic contractions by the use of a volumetric method. L-phenylephrine, an alpha-1 agonist, induced hypertension, bradycardia and a significant gastric relaxation. Clonidine, an alpha-2 agonist, caused hypotension, bradycardia and a significant gastric contraction and a reduction of the amplitude of phasic contractions. Salbutamol, a beta-2 agonist, induced a dose-dependent tachycardia and a significant inhibition of gastric tone whereas prenalterol, a beta-1 agonist, induced tachycardia without any significant influence on gastric basal tone. Yohimbine, an alpha-2 blocker, significantly decreased gastric basal tone and reversed the inhibition of phasic contractions induced by clonidine. Prazocine, a selective alpha-1 blocker, and propranolol, a non-selective beta-blocker, had no significant influence on gastric basal tone or phasic contractions. It is concluded that sympathetic inhibition of basal gastric tone in the rat is mediated by alpha-1 and beta-2 adrenergic receptors. Activation of alpha-2 adrenergic receptors significantly increased basal gastric tone and reduced the amplitude of phasic contractions. A blockade of alpha-2 receptors significantly decreased basal gastric tone and restored the amplitude of phasic contractions.     

人TLR4的B细胞优势表位设计、合成及其免疫原性检测

目的　鉴定TLR4的优势抗原表位 ,为抗人TLR4单克隆抗体制备奠定基础。方法　分别应用Hoop&Woods亲水性参数、抗原性参数、可及性参数和抗原表位的软件分析对TLR4B细胞表位进行预测 ,最后用抗原性指数的方法进行综合评述。合成针对该表位的多肽 ,以此多肽为免疫原免疫小鼠 ,对其免疫原性进行检测。结果　预测TLR4的B细胞优势表位为 189～ 2 0 2氨基酸序列 :NH2 HKLTLRNNFDSLNV COOH ,此多肽能诱导机体产生较高的抗体滴度 ,多抗具有较高的特异性。结论　预测的TLR4短肽是B细胞的优势表位 ,以此制作抗人TLR4单克隆抗体是可行的。     

Quantification and comparison of toll-like receptor expression and responsiveness in primary and immortalized human female lower genital tract epithelia

PROBLEM: To better understand innate immune responses to sexually-transmitted infection (STI) and the appropriateness of epithelial cell (EC) models of the vaginal and cervical mucosa, quantified toll-like receptor (TLR) expression from a population of women is needed. METHOD OF STUDY: TLR gene expression was quantified in primary and immortalized endocervical, ectocervical, and vaginal EC from multiple donors. TLR bioactivity was evaluated by cytokine elaboration. RESULTS: TLR1-3 and 5-9 were expressed in each EC type with TLR2, 3, 5, 6 and CD14 expressed most abundantly. TLR4 was expressed by endocervical and vaginal EC. Agonist stimulation of TLR2, 3, 5 and 6 elicited cytokines. TLR4 and 7-9 were minimally expressed and were not consistently bioactive. Immortalized EC generally modeled primary cultures but elaborated significantly reduced cytokine levels. CONCLUSION: TLR expression patterns were remarkably conserved across the study population and evaluated tissues indicating a predictable responsiveness to STI. The results support cautious use of immortalized cells for genital tract modeling.     

Toll-like receptor (TLR) 2 and 4 mutations in periodontal disease

Toll-like receptors (TLR) are signal molecules essential for the cellular response to bacterial cell wall components. Different functional effective polymorphisms for the TLR 4 gene (Asp299Gly; Thr399Ile) and for the TLR 2 gene (Arg677Trp, Arg753Gln) have recently been described that are associated with impaired lipopolysaccharide signal transduction. A total of 122 patients with chronic periodontal disease and 122 healthy unrelated controls were genotyped for the Asp299Gly and Thr399Ile polymorphism of the TLR 4 gene and the Arg677Trp and Arg753Gln mutation of the TLR 2 gene. The mutations were identified with polymerase chain reaction followed by restriction fragment length polymorphism (RFLP) analysis. The prevalence of the Asp299Gly and the Thr399Ile mutant allele was 4.1% (10/244) and 4.5% (11/244) among periodontitis patients. For the healthy controls the prevalence was 3.3% (8/244) for the Asp299Gly (P = 0.810) and 3.7% (9/244) for the Thr399Ile mutant allele (P = 0.819). The Arg753Gln mutant allele was found in 2.9% (7/244) of the periodontitis subjects as compared to 4.1% (10/244) in the control group (P = 0.622). The Arg677Trp mutant allele was not found in any of the study subjects. Unlike in ulcerative colitis there was not observed an association between chronic periodontitis and the various mutations of the TLR 2 and 4 gene.     

Toll-like receptor (TLR) 3 immune modulation by unformulated small interfering RNA or DNA and the role of CD14 (in TLR-mediated effects)

The Toll-like receptors (TLRs) 3, 7, 8 and 9 stimulate innate immune responses upon recognizing pathogen-derived nucleic acids. TLR3 is located on the cell surface and in cellular endosomes and recognizes double-stranded viral RNA or the synthetic mimic poly rI:rC. Recently, unformulated small interfering RNA (siRNA) has been reported as ligand for surface-expressed murine TLR3. Blockage of TLR3 is achieved by single-stranded DNA. We confirm and expand the observation that poly rI:rC-mediated TLR3 immune activation is blocked in a sequence-, length-, backbone- and CpG-dependent manner. However, human TLR3 is not activated by siRNA, which may be the result of differences in the amino acid composition of the TLR3 loop 1 of mice and humans. Although CD14 was previously described as a co-receptor for murine TLR3 and other nucleic acid-recognizing TLRs, human CD14 acts only as co-receptor to human TLR9, but not TLR3, TLR7 or TLR8. We show that CD14 up-regulates the TLR9 immune response of A, B and C-class oligodeoxynucleotides but down-regulates the phosphoro-diester version of B-class oligodeoxynucleotides.     

Identification and characterization of a functional, alternatively spliced Toll-like receptor 7 (TLR7) and genomic disruption of TLR8 in chickens

Based upon the recognition of antiviral compounds and single stranded viral RNA the Toll-like receptors TLR7 and TLR8 are suggested to play a significant role in initiating antiviral immune responses. Here we report the molecular characterization of the chicken TLR7/8 loci which revealed an intact TLR7 gene and fragments of a TLR8-like gene with a 6-kilobase insertion containing chicken repeat 1 (CR1) retroviral-like insertion elements. The chicken TLR7 gene encodes a 1047-amino-acid protein with 62% identity to human TLR7 and a conserved pattern of predicted leucine-rich repeats. Highest levels of chicken TLR7 mRNA were detected in immune-related tissues and cells, especially the spleen, caecal, tonsil and splenic B cells. Alternative spliced forms of TLR7 mRNA were identified in chicken, mouse and human and expressed in similar tissues and cell types to the major form of chicken TLR7. The chicken TLR7+ HD11 cell line and fresh splenocytes produced elevated levels of interleukin-1beta (IL-1beta) mRNA after exposure to the agonists R848 and loxoribine. Interestingly, none of the TLR7 agonists stimulated increased type I interferon (IFN) mRNA whereas poly(I:C) (a TLR3 agonist) up-regulated both chicken IFN-alpha and chicken IFN-beta mRNA. In contrast, TLR7 agonists, particularly R848 and poly(U) stimulated up-regulation of chicken IL-1beta, and chicken IL-8 mRNAs more effectively than poly(I:C). Stimulation of chicken TLR7 with R848 was chloroquine sensitive, suggesting signalling within an endosomal compartment, as for mammalian TLR7. The deletion of TLR8 in galliforms, accompanied with the differential response after exposure to TLR7 agonists, offers insight into the evolution of vertebrate TLR function.     

Phosphinic acid derivatives as baclofen agonists and antagonists in the mammalian spinal cord: an in vivo study

The actions of a series of derivatives of 3-aminopropyl-phosphinic acid as baclofen agonists and antagonists have been examined on the synaptic excitation of neurones by impulses in primary afferent fibres in the lumbar spinal cords of pentobarbitone-anaesthetised cats and rats. Both the pre-and postsynaptic inhibitory actions of microelectrophoretic (-)-baclofen were reduced by similarly administered CGP 35 348, 36 742, 46 381, 52 432, 54 626 and 55 845, the latter being the most potent antagonist. None of these antagonists either decreased or increased the excitability of spinal neurones, and the inhibitory action of GABA was reduced only by local concentrations of antagonists which also reduced the action of piperidine-4-sulphonic acid, a GABA_A agonist. Although the weak inhibitory effect of 3-aminopropylphosphinic acid in both the rat and the cat was not reduced by these baclofen antagonists, the pre-and postsynaptic inhibitory effects of 3-aminopropyl-methyl-osphinic acid (CGP 35 024), which was more potent than (-)-baclofen, were reduced by the antagonists. Like (-)-baclofen, CGP 35 024 was relatively ineffective in reducing transmitter release in the cord from the terminals of excitatory spinal interneurones, the terminals of excitatory tracts in the dorsolateral funiculus and the cholinergic terminals of motor axon collaterals. In both rat and cat cords, receptors for (-)-baclofen could not be demonstrated to be activated by microelectrophoretic GABA, possibly because of the predominantly dendritic location of GABA_B receptors. Spinal pre-and postsynaptic baclofen receptors appeared to be pharmacologically similar but differed from those in the higher central nervous system of the rat, where 3-aminopropylphosphinic acid has been reported to be an effective baclofen agonist. The compounds tested, particularly CGP 55 845 and 46 381, will be of use in further investigations of the physiological relevance of baclofen receptors at central synapses where GABA may be the transmitter.     

Myocardial resistance to ischemic and reperfusion injuries under conditions of chronic administration of opioid receptor agonists and antagonists

Chronic treatment with opioid receptor ligands: nonselective peptide opioid receptor agonist dalargin (intraperitoneally in a dose of 1 mg/kg), selective nonpeptide κ-receptor agonist GR 89696 (subcutaneously in a dose of 0.03 mg/kg), nonselectrive nonpeptide antagonist quadazocine (subcutaneously in a dose of 3 mg/kg) or naltrexone (subcutaneously in a dose of 10 mg/kg) for 20 day had no effect of the incidence of ischemic ventricular arrhythmias and the size of necrotic zone after coronary occlusion and reperfusion in rats in vivo. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 145, No. 6, pp. 642–644, June, 2008     

TLR2胞外段特异性抗体对TLR2激动剂诱导的炎症和促过敏反应的抑制作用

目的:观察小鼠TLR2胞外段抗原肽特异性抗体对TLR2激动剂诱导的炎症和促过敏反应的影响。方法:用小鼠TLR2胞外段单表位抗原肽(T20)免疫动物制备抗体(T20抗体);体外观察T20抗体对PGN和LTA刺激RAW264.7细胞生成TNF-α和IL-6的影响,用ELISA法检测各组TNF-α和IL-6量;体内观察T20抗体对OVA致敏小鼠PGN致死性攻击的影响,记录各组直肠温度变化和死亡率。结果:获得小鼠TLR2特异性T20抗体;该抗体可抑制TLR2激动剂(PGN和LTA)对相应靶细胞的刺激作用,使其生成的TNF-α和IL-6显著降低;体内实验证实T20抗体对PGN加剧的过敏反应具有抑制作用。结论:T20抗体可特异结合小鼠TLR2,抑制TLR2激动剂诱导的炎症及TLR2激动剂加剧的过敏反应。     

Toll-like receptor (TLR) 4 polymorphisms are associated with a chronic course of sarcoidosis

The aetiology of sarcoidosis, an inflammatory granulomatous multi-system disorder, is unclear. It is thought to be the product of an unknown exogenous antigenic stimulus and an endogenous genetic susceptibility. Toll-like receptors (TLR) are signal molecules essential for the cellular response to bacterial cell wall components. Lipopolysaccharide (LPS), for example, binds to TLR 4. Two different polymorphisms for the TLR4 gene (Asp299Gly and Thr399Ile) have been described recently. This leads to a change in the extracellular matrix function of TLR4 and to impaired LPS signal transduction. We genotyped a total of 141 Caucasian patients with sarcoidosis and 141 healthy unrelated controls for the Asp299Gly and Thr399Ile polymorphisms in the TLR4 gene. The mutations were identified with polymerase chain reaction followed by restriction fragment length polymorphism (RFLP) analysis. Among sarcoidosis patients the prevalence for each Asp299Gly and Thr399Ile mutant allele was 15.6% (22/141). In the control group the prevalence was 5.67% (8/141) (P = 0.07). In the subgroup of patients with acute sarcoidosis there was no difference in the control group (P = 0.93), but there was a highly significant association between patients with a chronic course of sarcoidosis and TLR4 gene polymorphisms (P = 0.01).     

Endotoxin and cytokine regulation of toll-like receptor (TLR) 2 and TLR4 gene expression in murine liver and hepatocytes.

Toll-like receptor (TLR) 2 and TLR4 are members of the interleukin-1 receptor (IL-1R) family and transduce similar signals as IL-1R in response to bacteria and bacterial components. In this study, we investigated the regulation of their gene expression in murine tissues, especially in the liver and hepatocytes. When mice were administered lipopolysaccharide (LPS), TLR2 mRNA was upregulated in the brain, heart, lung, liver, and kidney. In contrast, it was downregulated in the spleen. TLR4 mRNA was decreased in the brain. In the heart and lung, it increased, and it was not affected in the liver, kidney, and spleen. TLR mRNA was further analyzed in the liver and hepatocytes. Like LPS treatment, administration of IL-1, IL-6, or tumor necrosis factor (TNF) upregulated TLR2 mRNA. However, none of them affected the TLR4 mRNA level. In primary cultured hepatocytes, TLR2 mRNA was upregulated by LPS, IL-1, or TNF but not by IL-6 or dexamethasone. None of them affected TLR4 mRNA expression. Similar responses were observed in the murine hepatoma cell line Hepa 1-6. These results suggest that in infection with gram-negative bacteria, LPS and proinflammatory cytokines differentially regulate gene expression of TLR2 and TLR4 in murine hepatocytes, which may lead to pathologic and host defense reactions in the liver.     

The role of toll-like receptor ligands/agonists in protection against genital HSV-2 infection

Control of virus replication initially depends on rapid activation of the innate immune responses. Toll-like receptor (TLR) ligands are potent inducers of innate immunity against viral infections, including herpes simplex virus (HSV). HSV-2 is currently one of the most common sexually transmitted infections in developed nations and is becoming more prevalent in adolescents. HSV-2 infects the genital mucosa and is associated with an increased risk of obtaining other sexually transmitted infections such as HIV. There is currently no vaccine available against HSV-2. In the last several years, there has been an interest in utilizing Toll-like receptor (TLR) ligands to initiate innate immune responses in order to provide an early line of defence against viral replication. This review highlights recent studies investigating the effect of various TLR ligands on genital HSV-2 infection. A considerable body of information has been published on the effect of local delivery of TLR ligands on HSV-2 replication in genital mucosa. We have outlined ligands that have a potential to provide protection against HSV-2 infection. In addition, we have presented possible mechanisms by which the local delivery of TLR ligands provides innate protection against genital HSV-2.     

Differential alteration in intestinal epithelial cell expression of toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease

Initiation and perpetuation of the inflammatory intestinal responses in inflammatory bowel disease (IBD) may result from an exaggerated host defense reaction of the intestinal epithelium to endogenous lumenal bacterial flora. Intestinal epithelial cell lines constitutively express several functional Toll-like receptors (TLRs) which appear to be key regulators of the innate response system. The aim of this study was to characterize the expression pattern of TLR2, TLR3, TLR4, and TLR5 in primary intestinal epithelial cells from patients with IBD. Small intestinal and colonic biopsy specimens were collected from patients with IBD (Crohn's disease [CD], ulcerative colitis [UC]) and controls. Non-IBD specimens were assessed by immunofluorescence histochemistry using polyclonal antibodies specific for TLR2, TLR3, TLR4, and TLR5. Primary intestinal epithelial cells (IEC) of normal mucosa constitutively expressed TLR3 and TLR5, while TLR2 and TLR4 were only barely detectable. In active IBD, the expression of TLR3 and TLR4 was differentially modulated in the intestinal epithelium. TLR3 was significantly downregulated in IEC in active CD but not in UC. In contrast, TLR4 was strongly upregulated in both UC and CD. TLR2 and TLR5 expression remained unchanged in IBD. These data suggest that IBD may be associated with distinctive changes in selective TLR expression in the intestinal epithelium, implying that alterations in the innate response system may contribute to the pathogenesis of these disorders.