Ovine Toll-like Receptor 9 (TLR9) Gene Variation and Its Association with Flystrike Susceptibility

Toll-like receptors (TLRs) are a family of proteins that play a role in innate immune responses by recognising pathogen-associated molecular patterns derived from various microbes. Of these receptors, TLR9 recognises bacterial and viral DNA containing unmethylated cytosine-phosphate-guanine (CpG) motifs, and variation in TLR9 has been associated with resistance to various infectious diseases. Flystrike is a problem affecting the sheep industry globally and the immune response of the sheep has been suggested as one factor that influences the response to the disease. In this study, variation in ovine TLR9 from 178 sheep with flystrike and 134 sheep without flystrike was investigated using a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) approach. These sheep were collected from both commercial and stud farms throughout New Zealand and they were of 13 different breeds, cross-breds and composites. Four alleles of TLR9 were detected, including three previously identified alleles (*01, *02 and *03) and a new allele (*04). In total six single nucleotide polymorphisms (SNPs) were found. Of the three common alleles in the sheep studied, the presence of *03 was found to be associated with a reduced likelihood of flystrike being present (OR = 0.499, p = 0.024). This suggests that variation in ovine TLR9 may affect a sheep’s response to flystrike, and thus the gene may have value as a genetic marker for improving resistance to the disease.

The Potential Equivalents of TET2 Mutations

TET2 is a dioxygenase dependent on Fe2+ and a-ketoglutarate which oxidizes 5-methylcytosine (5meC) to 5-hydroxymethylcytosine (5hmeC). TET proteins successively oxidize 5mC to yield 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Among these oxidized methylcytosines, 5fC and 5caC are directly excised by thymine DNA glycosylase (TDG) and ultimately replaced with unmethylated cytosine. Mutations in TET2 have been shown to lead to a hypermethylated state of the genome and to be responsible for the initiation of the oncogenetic process, especially in myeloid and lymphoid malignancies. Nonetheless, this was also shown to be the case in other cancers. In AML, TET2 mutations have been observed to be mutually exclusive with IDH1, IDH2, and WT1 mutations, all of them showing a similar impact on the transcription profile of the affected cell. Because of this, it is possible that TET2/IDH1/2/WT1 mutated AML could be considered as having similar characteristics between each other. Nonetheless, other genes also interact with TET2 and influence its effect, thus making it possible that other signatures exist that would mimic the effect of TET2 mutations. Thus, in this review, we searched the literature for the genes that were observed to interact with TET2 and classified them in the following manner: transcription alteration, miRs, direct interaction, posttranslational changes, and substrate reduction. What we propose in the present review is the potential extension of the TET2/IDH1/2/WT1 entity with the addition of certain expression signatures that would be able to induce a similar phenotype with that induced by TET2 mutations. Nonetheless, we recommend that this approach be taken on a disease by disease basis.

Unmethylated Cytosine phosphate guanine DNA (CpG-DNA) exacerbates liver Kupffer cells (KCs) inflammation through Toll-like receptor 9 (TLR9) in diabetic rats

Recently, gut microbiota for various pathogens has attracted attention. The present study investigated the role of gut microbiota unmethylated cytosine phosphate guanine DNA (CpG-DNA) on liver Kupffer cells (KCs) inflammatory cytokine interleukin-1β (IL-1β) in diabetic rats. We induced diabetic rats models and sequenced the gut microbiota composition of fecal samples. We also applied CpG-DNA and TLR9 inhibitor on KCs to investigate the regulation of inflammatory cytokine IL-1β and Toll-like receptor 9 (TLR9) signaling pathway. We found a significant difference of gut microbiota between the control and the diabetic rats with increased Clostridium. Meanwhile, diabetes could upregulate TLR9 in KCs and increase IL-1β concentration. Furthermore, high concentration of unmethylated CpG-DNA could significantly increase IL-1β secretion while it was suppressed by TLR9 inhibitor in KCs cultured in high glucose medium. Our study suggests that unmethylated CpG-DNA, which was highly expressed in diabetic rats, activated KCs through TLR9, and induced IL-1β secretion in vitro and in vivo which plays an important role in diabetic liver inflammation. It may contribute to the progress of the diabetes.

Oligodeoxynucleotides containing unmethylated cytosine-guanine motifs are effective immunostimulants against pneumococcal meningitis in the immunocompetent and neutropenic host

Background Bacterial meningitis is a fatal disease with a mortality up to 30% and neurological sequelae in one fourth of survivors. Available vaccines do not fully protect against this lethal disease. Here, we report the protective effect of synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG ODN) against the most frequent form of bacterial meningitis caused by Streptococcus pneumoniae. Methods Three days prior to the induction of meningitis by intracerebral injection of S. pneumoniae D39, wild-type and Toll-like receptor (TLR9)−/− mice received an intraperitoneal injection of 100 μg CpG ODN or vehicle. To render mice neutropenic, anti-Ly-6G monoclonal antibody was daily administrated starting 4 days before infection with a total of 7 injections. Kaplan-Meier survival analyses and bacteriological studies, in which mice were sacrificed 24 h and 36 h after infection, were performed. Results Pre-treatment with 100 μg CpG ODN prolonged survival of immunocompetent and neutropenic wild-type mice but not of TLR9−/− mice. There was a trend towards lower mortality in CpG ODN-treated immunocompetent and neutropenic wild-type mice. CpG ODN caused an increase of IL-12/IL-23p40 levels in the spleen and serum in uninfected animals. The effects of CpG ODN on bacterial concentrations and development of clinical symptoms were associated with an increased number of microglia in the CNS during the early phase of infection. Elevated concentrations of IL-12/IL-23p40 and MIP-1α correlated with lower bacterial concentrations in the blood and spleen during infection. Conclusions Pre-conditioning with CpG ODN strengthened the resistance of neutropenic and immunocompetent mice against S. pneumoniae meningitis in the presence of TLR9. Administration of CpG ODN decreased bacterial burden in the cerebellum and reduced the degree of bacteremia. Systemic administration of CpG ODN may help to prevent or slow the progression to sepsis of bacterial CNS infections in healthy and immunocompromised individuals even after direct inoculation of bacteria into the intracranial compartments, which can occur after sinusitis, mastoiditis, open head trauma, and surgery, including placement of an external ventricular drain.

A study on the protective effects of CpG ODN-induced mucosal immunity against lung injury in a mouse ARDS model

This study aims to determine the feasibility of using oligodeoxynucleotides with unmethylated cytosine-guanine dinucleotide sequences (CpG ODN) as an immunity protection strategy for a mouse model of acute respiratory distress syndrome (ARDS). This is a prospective laboratory animal investigation. 20-week old BALB/c mice in Animal research laboratory were randomized into groups. An ARDS model was induced in mice using lipopolysaccharides. CpG ODN was intranasally and transrectally immunized before or after the 3rd and 7th day of establishing the ARDS model. Mice were euthanized on day seven after the 2nd immunization. Then, retroorbital bleeding was carried, out and the chest was rapidly opened to collect the trachea and tissues from both lungs for testing. CpG ODN significantly improved the pathologic impairment in mice lung, especially after the intranasal administration of 50 ug. This resulted in the least severe lung tissue injury. Furthermore, IL-6 and IL-8 concentrations were lower, which was second to mice treated with the rectal administration of 20 μg CpG ODN. In contrast, the nasal and rectal administration of CpG ODN in BALB/c mice before LPS immunization did not appear to exhibit any significant protective effects. In conclusion, the intranasal administration of CpG ODN may be is a potential treatment approach to ARDS. More studies are needed to further determine the protective mechanism of CpG ODN.

Detecting DNA Methylation using the Oxford Nanopore Technologies MinION sequencer

Nanopore sequencing instruments measure the change in electric current caused by DNA transiting through the pore. In experimental and prototype nanopore sequencing devices it has been shown that the electrolytic current signals are sensitive to base modifications, such as 5-methylcytosine. Here we quantify the strength of this effect for the Oxford Nanopore Technologies MinION sequencer. Using synthetically methylated DNA we are able to train a hidden Markov model to distinguish 5-methylcytosine from unmethylated cytosine in DNA. We demonstrate by sequencing natural human DNA, without any special library preparation, that global patterns of methylation can be detected from low-coverage sequencing and that the methylation status of CpG islands can be reliably predicted from single MinION reads. Our trained model and prediction software is open source and freely available to the community under the MIT license.

BAD-LAMP is a novel biomarker of nonactivated human plasmacytoid dendritic cells

The brain and dendritic cell (BAD)–associated lysosome-associated membrane protein (LAMP)–like molecule (BAD-LAMP, c20orf103, UNC-46) is a newly identified member of the family of LAMPs. BAD-LAMP expression in the mouse is confined to neurons. We demonstrate here that in humans, BAD-LAMP can specifically be found in the type I IFN-producing plasmacytoid dendritic cells (pDCs). Human BAD-LAMP is localized in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) of freshly isolated CD123+ pDCs and is rapidly lost upon activation by unmethylated cytosine-phosphate-guanine (CpG) oligonucleotides. The restricted pattern of BAD-LAMP expression allows for the rapid identification of normal and leukemic human pDCs in tissues and blood.

Impact of RASSF1A methylation on survival of stage IIIb and IV NSCLC patients treated with gemcitabine

17028 Background: The epigenetic inactivation of genes plays an important role in lung cancer carcinogenesis. We have investigated the methylation status of seven genes (APC1A, DAPK, FHIT, p14ARF, p16INK4a, RARβ, RASSF1A) in serum DNA of NSCLC patients and analyzed their potential prognostic value. Methods: Blood samples were drawn pretherapeutically. Genomic DNA was purified from serum and subjected to sodium bisulfite treatment to convert unmethylated cytosine bases. Epigenetic alteration was detected by a nested methylation-specific PCR. Results: We have investigated a group of 92 patients with histologically confirmed NSCLC (60 males, 32 females; 21 stage IIIb, 71 stage IV; median age 60.9 years, range 34–78). All patients received gemcitabine first-line alone or in combination with other drugs. The vast majority of samples (n = 87) showed at least one methylated gene. More than half of the tested genes were hypermethylated in 20 samples (21.7%). The methylation frequencies of individual genes were 30.8% (APC1A), 26.1% (DAPK), 47.3% (FHIT), 30.4% (p14ARF), 25.9% (p16INK4a), 44.6% (RARβ) and 33.3% (RASSF1A). The hypermethylation status of none of the genes altered median overall survival (11.0 ± 1.4 mo) of the total population significantly. However, patients with a methylated RASSF1A gene who showed a partial response (n = 27) survived significantly longer (33.6 ± 10.4 mo) compared to those with a wild-type allele (16.1 ± 4.7 mo, P = 0.0045). This difference was not seen in patients with stable (n = 40) or progressive (n = 20) disease. Conclusions: Our results show that the hypermethylation frequency of single genes and the accumulation of epigenetic alterations in individual samples of NSCLC patients may vary considerably. Molecular parameters such as RASSF1A hypermethylation may be useful prognostic markers in subpopulations. [Table: see text]