Rapid and inexpensive real-time PCR for genotyping functional polymorphisms within the Toll-like receptor -2, -4, and -9 genes

Inflammation is associated with preterm birth. We previously described a mouse model of chronic inflammation-induced preterm birth after dental Porphyromonas gingivalis infection. The aim of this study was to employ this model system to investigate the mechanisms through which enhanced uterine contractility induces preterm birth. Messenger RNA (mRNA) encoding contraction-associated proteins, such as oxytocin receptors, was measured at various gestational time points by real-time polymerase chain reaction (PCR). Spontaneous and oxytocin-induced uterine contractile activity at gestational day 18 was assessed using a tissue organ bath. The expression levels of Toll-like receptor 2 (TLR2), TLR4, cyclooxygenase (COX)-2, nuclear factor-kappa B (NF-κB) p65, and p38 mitogen-activated protein kinase (MAPK) on gestational day 18 were also determined by real-time PCR or Western blotting. Messenger RNA encoding contraction-associated proteins was increased at gestational day 18, and the spontaneous contractile activity (1.6-fold greater area under the contraction curve) and sensitivity to oxytocin (EC50: 8.8 nM vs 2.2 nM) were enhanced in the P gingivalis group compared to those in the control group. In the P gingivalis group, COX-2 mRNA expression was not elevated in the placenta or myometrium but was upregulated 2.3-fold in the fetal membrane. The TLR2 mRNA levels in the fetal membrane were 2.7-fold higher in the P gingivalis group, whereas TLR4 levels were not elevated. Activation of the NF-κB p65 and p38 MAPK pathways was enhanced in the fetal membrane of the P gingivalis group. Thus, in mice with chronic dental P gingivalis infection, TLR2-induced inflammation in the fetal membrane leads to upregulation of uterine contractility, leading to preterm birth.

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Molecular Gene Expression of Toll-Like Receptors 4 & 10 in Cellular Subsets of Human Peripheral Blood among Patients with Prostatitis: Conventional, Real Time Pcr and DNA Sequencing Techniques

International Journal Of Medical Science And Clinical Invention ◽

10.18535/ijmsci/v7i11.07 ◽

2020 ◽

Vol 7 (11) ◽

pp. 5103-5110

Author(s):

Ihsan AlSaimary1 ◽

Hussein Aldhaheri ◽

Murtadha ALMusafer2

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Human Peripheral Blood ◽

Receptor Gene ◽

Gene Clusters ◽

Control Group ◽

Agarose Gel ◽

Toll Like Receptor ◽

Blood Samples ◽

Pcr Product

The Aim of this study was to determine Immunogenetic expression of Toll-like receptor gene clusters related to prostatitis, to give acknowledge about Role of TLR in prostatitis immunity in men from Basrah and Maysan provinces. A case–control study included 135 confirmed prostatitis patients And 50 persons as a control group. Data about age, marital status, working, infertility, family history and personal information like (Infection, Allergy, Steroid therapy, Residency, Smoking, Alcohol Drinking, Blood group, Body max index (BMI) and the clinical finding for all patients of Prostatitis were collected , The molecular expression study include extracting DNA from blood of Prostatitis patients , Prostitis patients and Control group by using specific primers for conventional PCR and Real Time PCR , the amplification of all extracted DNA from blood samples was preform and confirm by using electrophoresis with (100volt/30min). The amplification of all extracted DNA from blood samples was preform and confirm by using electrophoresis with (100volt/30min) , the result of this estimation revealed that the amplified DNA(PCR product) was 227bp for TLR4 on agarose gel (1%) , (50voltage for 1hour ) with a presence 100% , (PCR product) was 279bp for TLR10 on agarose gel(1%) , (50volt/1hour) with a presence 80%. The results of the present study indicate that the Toll like receptor alleles associated with risk of prostatitis.

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Quantification of interferon, interleukin, and Toll-like receptor 7 mRNA in quail splenocytes using real-time PCR

Poultry Science ◽

10.3382/ps.2012-02283 ◽

2012 ◽

Vol 91 (10) ◽

pp. 2496-2501 ◽

Cited By ~ 11

Author(s):

Y. Uno ◽

T. Usui ◽

Y. Fujimoto ◽

T. Ito ◽

T. Yamaguchi

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Toll Like Receptor

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Immunostimulant as an adjuvant therapy on Toll-like receptor concentration in children with acute otitis media

The Egyptian Journal of Otolaryngology ◽

10.1186/s43163-020-00019-z ◽

2020 ◽

Vol 36 (1) ◽

Author(s):

Ashraf S. El Hamshary ◽

Hesham A. Abdel Rahman ◽

Mohamed Aboelsoued ◽

Rasha A. Elsayed ◽

Abdelhakim F. Elwany

Keyword(s):

Adjuvant Therapy ◽

Bacterial Infection ◽

Real Time ◽

Otitis Media ◽

Real Time Pcr ◽

Acute Otitis Media ◽

Toll Like Receptor ◽

Tlr2 Expression ◽

Before And After ◽

Group B

Abstract Background Acute otitis media (AOM) is the commonest pediatric bacterial infection, affecting up to 75% of children at some time before age 5 years. AOM is among the primary reasons for antibiotic prescriptions in pediatric outpatients. This study aimed to detect the value of immunostimulant as an adjuvant therapy with antibiotics for treatment of acute otitis media in children. This study included 60 children suffering from acute otitis media; their age ranged from 3 to 5 years during the period from May 2018 to March 2019. The patients in this study were divided into 2 groups: group A included 30 patients with AOM who received amoxicillin and clavulanic acid antibiotic at attack of AOM. Group B included 30 patients with AOM who received the same antibiotic with immunostimulant (Echinacea extract) for 3 months. Samples of blood were taken from all patients to detect the level of Toll-like receptor by real-time PCR, before and after 3 months of antibiotic and immunostimulant therapy. All cases underwent assessment including complete history taking, otoscopic examination of the ear, and blood sample to detect the level of Toll-like receptor (TLR) before and after the therapy by real-time PCR. Results There was a significant (P < 0.05) decrease in the TLR2 expression in antibiotic-treated patients than its expression before treatment. On the other hand, there was a significant (P < 0.05) increase in the TLR2 expression in immunostimulant plus antibiotic-treated patients than its expression before treatment; there was a high significant (P < 0.001) increase in the expression of TLR2 in the immunostimulant plus antibiotic than the antibiotic-treated patients. Conclusion The role of antibiotics against bacterial infection causing acute otitis media can be enhanced by immunostimulant which increases the expression of Toll-like receptors which play a major role stimulating immune system to resist bacterial infection.

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A Real-Time PCR Assay for the Simultaneous Detection of Functional N284I and L412F Polymorphisms in the Human Toll-Like Receptor 3 Gene

Journal of Molecular Diagnostics ◽

10.2353/jmoldx.2010.090122 ◽

2010 ◽

Vol 12 (4) ◽

pp. 493-497 ◽

Cited By ~ 3

Author(s):

Robert A. Brown ◽

Raymund R. Razonable

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Toll Like Receptor ◽

Pcr Assay

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Toll-like receptor 5-mediated signaling enhances liver regeneration in mice

10.21203/rs.3.rs-53389/v1 ◽

2020 ◽

Author(s):

Wen Zhang ◽

Lei Wang ◽

Xue-Hua Sun ◽

Xian Liu ◽

Yang Xiao ◽

...

Keyword(s):

Liver Regeneration ◽

Real Time ◽

Real Time Pcr ◽

Early Gene ◽

Hepatocyte Proliferation ◽

Toll Like Receptor ◽

Hepatic Lipids ◽

Toll Like Receptor 5 ◽

Oil Red O

Abstract Background Toll-like receptor 5 (TLR5)-mediated pathways play critical roles in regulating hepatic immune response and show hepatoprotective effect in mouse models of liver injury. However, the role of TLR5 in experimental models of liver regeneration has not been investigated. This study aims to determine the role of TLR5 in the partial hepatectomy (PHx)-induced liver regeneration. Methods We performed 2/3 PHx in wild-type (WT) mice, TLR5 knockout mice, or TLR5 agonist CBLB502 treated mice as an established model of liver regeneration. Bacterial flagellin content was measured by ELISA, and hepatic TLR5 expression was determined by real-time PCR analyses and flow cytometry. To study the effects of TLR5 on hepatocyte proliferation, proliferating cell nuclear antigen (PCNA) and the incorporation of bromodeoxyuridine (BrdU) were analyzed by immunohistochemistry (IHC) staining. The role of TLR5 in the priming of liver regeneration was examined by real-time PCR analyses of immediate early gene mRNA levels, as well as western blot analysis of hepatic NF-κB and STAT3 activation. Cytokines and growth factors production after PHx were detected using real-time PCR analyses and cytometric bead array (CBA) assays. Oil Red O staining and hepatic lipids concentrations were analyzed to examine the effect of TLR5 on hepatic lipid accumulation after PHx. Results The bacterial flagellin content in serum and liver was increased and the hepatic TLR5 expression was significantly up-regulated in WT mice upon PHx. TLR5-deficient mice exhibited reduced numbers of BrdU- and PCNA-positive cells, suppressed immediate early gene expression, and decreased cytokines and growth factors production. Moreover, PHx-induced NF-κB and STAT3 activation was inhibited in the liver of Tlr5−/− mice compared with WT mice. Consistently, administration of CBLB502 significantly promoted PHx-mediated hepatocyte proliferation correlated with enhanced production of proinflammatory cytokines and recruitment of macrophages and neutrophils in liver. In addition, Tlr5−/− mice displayed significantly decreased hepatic lipids concentrations and Oil Red O positive areas compared with WT mice after PHx. Conclusions We reveal that TLR5 activation is involved in the initial events of liver regeneration after PHx. Our results demonstrate that TLR5 signaling positively regulates liver regeneration and suggest a potential application of TLR5 agonist in promoting liver regeneration.

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