Unmethylated CpG Dinucleotides Research Articles

Lipid Nanoparticle Delivery Alters the Adjuvanticity of the TLR9 Agonist CpG by Innate Immune Activation in Lymphoid Tissue.

Pharmacological strategies to activate innate immune cells are of great relevance in the context of vaccine design and anticancer immune therapy, to mount broad immune responses able to clear infection and malignant cells. Synthetic CpG oligodeoxynucleotides (CpG-ODNs) are short single-stranded DNA molecules containing unmethylated CpG dinucleotides and a phosphorothioate backbone. Class B CpG ODNs activate robust innate immune responses through a TLR9-dependent NF-κB signaling pathway. This feature is attractive to exploit in the context of vaccine design and cancer immunotherapy. Soluble CpG-ODNs cause hepatic toxicity, which reduces its therapeutic applicability. The formulation of class B CpG ODN1826 in lipid nanoparticles (LNPs) containing an ionizable cationic lipid that complexes CpG through electrostatic interaction is reported. Upon local administration, LNP-formulated CpG drains to lymph nodes and triggers robust innate immune activation. Unformulated, soluble, CpG, by contrast, is unable to induce robust innate activation in draining lymph nodes and is distributed systemically. In a vaccination setting, LNP-formulated CpG, admixed with a protein antigen, induces higher antigen-specific antibody titers and T cell responses than antigen admixed with unformulated soluble CpG.

Impact of folic acid on regulatory B lymphocytes from patients with systemic lupus erythematosus in vitro.

Epigenetic modifications of genomes are of particular interest as numerous studies indicate the correlation between DNA methylation and the development of systemic lupus. As a major methyl group donor, folic acid is an important participant in this process. The aim of this study is to determine the effect of low or high dose folate co-culturing with peripheral blood mononuclear cells (PBMCs) on the secretion of interleukin (IL)10 from regulatory cells from lupus patients or from healthy volunteers. PBMCs from lupus patients and healthy volunteers were isolated and separated CD19+ B cell populations were cultured in the presence of 4μg/mL or of 16 μg/mL of folic acid and the DNA methylation level as well as the percentages of B lymphocytes were measured. In another experiment, PBMCs were stimulated in vitro for IL10 production with 1μg/mL recombinant human CD40L and with 2.5μg/mL unmethylated CpG dinucleotides and cultured in the presence of 4μg/mL or of 16 μg/mL of folic acid. Although co-culturing with low or high folic acid concentrations had no effect on the methylation level of B lymphocytes, particular patients showed an increase in the population of CD19+ IL10+ as well as of CD19- IL10+ cells. The observed increase may be a consequence of additional indirect or direct methylation of DNA in specific loci of the targeted cells. However, further analysis would clarify the exact mechanism of action of folate and would reveal its immunomodulating potential in this autoimmune disease.

Father-to-offspring transmission of extremely long NOTCH2NLC repeat expansions with contractions: genetic and epigenetic profiling with long-read sequencing

BackgroundGGC repeat expansions in NOTCH2NLC are associated with neuronal intranuclear inclusion disease. Very recently, asymptomatic carriers with NOTCH2NLC repeat expansions were reported. In these asymptomatic individuals, the CpG island in NOTCH2NLC is hypermethylated, suggesting that two factors repeat length and DNA methylation status should be considered to evaluate pathogenicity. Long-read sequencing can be used to simultaneously profile genomic and epigenomic alterations. We analyzed four sporadic cases with NOTCH2NLC repeat expansion and their phenotypically normal parents. The native genomic DNA that retains base modification was sequenced on a per-trio basis using both PacBio and Oxford Nanopore long-read sequencing technologies. A custom workflow was developed to evaluate DNA modifications. With these two technologies combined, long-range DNA methylation information was integrated with complete repeat DNA sequences to investigate the genetic origins of expanded GGC repeats in these sporadic cases.ResultsIn all four families, asymptomatic fathers had longer expansions (median: 522, 390, 528 and 650 repeats) compared with their affected offspring (median: 93, 117, 162 and 140 repeats, respectively). These expansions are much longer than the disease-causing range previously reported (in general, 41–300 repeats). Repeat lengths were extremely variable in the father, suggesting somatic mosaicism. Instability is more frequent in alleles with uninterrupted pure GGCs. Single molecule epigenetic analysis revealed complex DNA methylation patterns and epigenetic heterogeneity. We identified an aberrant gain-of-methylation region (2.2 kb in size beyond the CpG island and GGC repeats) in asymptomatic fathers. This methylated region was unmethylated in the normal allele with bilateral transitional zones with both methylated and unmethylated CpG dinucleotides, which may be protected from methylation to ensure NOTCH2NLC expression.ConclusionsWe clearly demonstrate that the four sporadic NOTCH2NLC-related cases are derived from the paternal GGC repeat contraction associated with demethylation. The entire genetic and epigenetic landscape of the NOTCH2NLC region was uncovered using the custom workflow of long-read sequence data, demonstrating the utility of this method for revealing epigenetic/mutational changes in repetitive elements, which are difficult to characterize by conventional short-read/bisulfite sequencing methods. Our approach should be useful for biomedical research, aiding the discovery of DNA methylation abnormalities through the entire genome.

A CpG island promoter drives the CXXC5 gene expression

CXXC5 is a member of the zinc-finger CXXC family that binds to unmethylated CpG dinucleotides. CXXC5 modulates gene expressions resulting in diverse cellular events mediated by distinct signaling pathways. However, the mechanism responsible for CXXC5 expression remains largely unknown. We found here that of the 14 annotated CXXC5 transcripts with distinct 5′ untranslated regions encoding the same protein, transcript variant 2 with the highest expression level among variants represents the main transcript in cell models. The DNA segment in and at the immediate 5′-sequences of the first exon of variant 2 contains a core promoter within which multiple transcription start sites are present. Residing in a region with high G–C nucleotide content and CpG repeats, the core promoter is unmethylated, deficient in nucleosomes, and associated with active RNA polymerase-II. These findings suggest that a CpG island promoter drives CXXC5 expression. Promoter pull-down revealed the association of various transcription factors (TFs) and transcription co-regulatory proteins, as well as proteins involved in histone/chromatin, DNA, and RNA processing with the core promoter. Of the TFs, we verified that ELF1 and MAZ contribute to CXXC5 expression. Moreover, the first exon of variant 2 may contain a G-quadruplex forming region that could modulate CXXC5 expression.

Cytosolic d-type CpG-oligonucleotides induce a type I interferon response by activating the cGAS-STING signaling pathway.

Cytosolic DNA receptor cyclic GMP-AMP (cGAMP) synthase (cGAS) has been shown to be critically involved in the detection of cytosolic, self- and non-self-DNA, initiating a type I IFN response through the adaptor protein Stimulator of Interferon Genes (STING) and interferon regulatory factor 3 (IRF3). Current studies propose that canonical binding of dsDNA by cGAS depends on DNA length, but not on base sequence. In contrast, activation of TLR9 is sequence dependent. It requires unmethylated CpG dinucleotides in microbial DNA, which is mimicked by synthetic oligodeoxynucleotides (ODN). Here, we provide evidence that d-type ODN (D-ODN), but not K-type ODN (K-ODN), bind to human cGAS and activate downstream signaling. Transfection of D-ODN into a TLR9-deficient, human monocytic cell line (THP-1) induced phosphorylation of IRF3 and secretion of IFN. This response was absent in cells with CRISPR/Cas9-mediated cGAS- or STING-deficiency. Utilizing a protein pulldown approach, we further demonstrate direct binding of D-ODN to cGAS. Induction of a type I IFN response by D-ODN was confirmed in human primary monocytes and monocyte-derived macrophages. These results are relevant to our understanding of self-nonself-discrimination by cGAS and to the pharmacologic effects of ODN, which currently are investigated in clinical studies.

A Potential Role for Mitochondrial DNA in the Activation of Oxidative Stress and Inflammation in Liver Disease

Mitochondria are organelles that are essential for cellular homeostasis including energy harvesting through oxidative phosphorylation. Mitochondrial dysfunction plays a vital role in liver diseases as it produces a large amount of reactive oxygen species (ROS), in turn leading to further oxidative damage to the structure and function of mitochondria and other cellular components. More severe oxidative damage occurred in mitochondrial DNA (mtDNA) than in nuclear DNA. mtDNA dysfunction results in further oxidative damage as it participates in encoding respiratory chain polypeptides. In addition, mtDNA can leave the mitochondria and enter the cytoplasm and extracellular environment. mtDNA is derived from ancient bacteria, contains many unmethylated CpG dinucleotide repeats similar to bacterial DNA, and thus can induce inflammation to exacerbate damage to liver cells and distal organs by activating toll-like receptor 9, inflammatory bodies, and stimulator of interferon genes (STING). In this review, we focus on the mechanism by which mtDNA alterations cause liver injuries, including nonalcoholic fatty liver, alcoholic liver disease, drug-induced liver injury, viral hepatitis, and liver cancer.

Review of CpG Island Recognition Algorithms

DNA methylation is a common epigenetic process, which exists in many eukaryotes, such as animals and plants. It has the function of regulating gene expression and affecting the stability of genetic material. The unmethylated CpG dinucleotide, known as CGI, generally presents a local aggregation pattern in the genome. Abnormal methylation of CGI is often associated with cancer and abnormal growth. Therefore, it is of great significance to accurately identify CGI and predict its methylation state. This paper summarizes the current mainstream CGI computational recognition methods, analyzes their advantages and disadvantages, and discusses the future direction of CGI recognition algorithm research.

Pro-inflammatory role of cell-free mitochondrial DNA in cardiovascular diseases.

Cardiovascular disease (CVD) is a major cause of morbidity and mortality. Inflammation contributes to the pathogenesis and progression of CVD. Circulating cell-free mitochondrial DNA (ccf-mtDNA) is that mtDNA fragments are released outside the cell and into the circulation by cell necrosis and secretion. The levels of ccf-mtDNA are increased in CVD and associated risk conditions, including hypercholesterolemia, diabetes mellitus, and arterial hypertension. MtDNA containing unmethylated CpG dinucleotides and can trigger inflammation that aggravates tissue injury by activating toll-like receptor 9, inflammasomes, and the stimulator of interferon genes pathway. Here, we review the expanding field of ccf-mtDNA-mediated inflammation and its role in the progression of CVD.

SARS-CoV-2 will constantly sweep its tracks: a vaccine containing CpG motifs in 'lasso' for the multi-faced virus.

During the current COVID-19 pandemic, the global ratio between the dead and the survivors is approximately 1 to 10, which has put humanity on high alert and provided strong motivation for the intensive search for vaccines and drugs. It is already clear that if we follow the most likely scenario, which is similar to that used to create seasonal influenza vaccines, then we will need to develop improved vaccine formulas every year to control the spread of the new, highly mutable coronavirus SARS-CoV-2. In this article, using well-known RNA viruses (HIV, influenza viruses, HCV) as examples, we consider the main successes and failures in creating primarily highly effective vaccines. The experience accumulated dealing with the biology of zoonotic RNA viruses suggests that the fight against COVID-19 will be difficult and lengthy. The most effective vaccines against SARS-CoV-2 will be those able to form highly effective memory cells for both humoral (memory B cells) and cellular (cross-reactive antiviral memory T cells) immunity. Unfortunately, RNA viruses constantly sweep their tracks and perhaps one of the most promising solutions in the fight against the COVID-19 pandemic is the creation of 'universal' vaccines based on conservative SARS-CoV-2 genome sequences (antigen-presenting) and unmethylated CpG dinucleotides (adjuvant) in the composition of the phosphorothioate backbone of single-stranded DNA oligonucleotides (ODN), which can be effective for long periods of use. Here, we propose a SARS-CoV-2 vaccine based on a lasso-like phosphorothioate oligonucleotide construction containing CpG motifs and the antigen-presenting unique ACG-containing genome sequence of SARS-CoV-2. We found that CpG dinucleotides are the most rare dinucleotides in the genomes of SARS-CoV-2 and other known human coronaviruses, and hypothesized that their higher frequency could be responsible for the unwanted increased lethality to the host, causing a ‘cytokine storm’ in people who overexpress cytokines through the activation of specific Toll-like receptors in a manner similar to TLR9-CpG ODN interactions. Interestingly, the virus strains sequenced in China (Wuhan) in February 2020 contained on average one CpG dinucleotide more in their genome than the later strains from the USA (New York) sequenced in May 2020. Obviously, during the first steps of the microevolution of SARS-CoV-2 in the human population, natural selection tends to select viral genomes containing fewer CpG motifs that do not trigger a strong innate immune response, so the infected person has moderate symptoms and spreads SARS-CoV-2 more readily. However, in our opinion, unmethylated CpG dinucleotides are also capable of preparing the host immune system for the coronavirus infection and should be present in SARS-CoV-2 vaccines as strong adjuvants.

A Method for Identification of the Methylation Level of CpG Islands From NGS Data

In the course of sample preparation for Next Generation Sequencing (NGS), DNA is fragmented by various methods. Fragmentation shows a persistent bias with regard to the cleavage rates of various dinucleotides. With the exception of CpG dinucleotides the previously described biases were consistent with results of the DNA cleavage in solution. Here we computed cleavage rates of all dinucleotides including the methylated CpG and unmethylated CpG dinucleotides using data of the Whole Genome Sequencing datasets of the 1000 Genomes project. We found that the cleavage rate of CpG is significantly higher for the methylated CpG dinucleotides. Using this information, we developed a classifier for distinguishing cancer and healthy tissues based on their CpG islands statuses of the fragmentation. A simple Support Vector Machine classifier based on this algorithm shows an accuracy of 84%. The proposed method allows the detection of epigenetic markers purely based on mechanochemical DNA fragmentation, which can be detected by a simple analysis of the NGS sequencing data.

CXXC5 as an unmethylated CpG dinucleotide binding protein contributes to estrogen-mediated cellular proliferation

Evidence suggests that the CXXC type zinc finger (ZF-CXXC) protein 5 (CXXC5) is a critical regulator/integrator of various signaling pathways that include the estrogen (E2)-estrogen receptor α (ERα). Due to its ZF-CXXC domain, CXXC5 is considered to be a member of the ZF-CXXC family, which binds to unmethylated CpG dinucleotides of DNA and through enzymatic activities for DNA methylation and/or chromatin modifications generates a chromatin state critical for gene expressions. Structural/functional features of CXXC5 remain largely unknown. CXXC5, suggested as transcription and/or epigenetic factor, participates in cellular proliferation, differentiation, and death. To explore the role of CXXC5 in E2-ERα mediated cellular events, we verified by generating a recombinant protein that CXXC5 is indeed an unmethylated CpG binder. We uncovered that CXXC5, although lacks a transcription activation/repression function, participates in E2-driven cellular proliferation by modulating the expression of distinct and mutual genes also regulated by E2. Furthermore, we found that the overexpression of CXXC5, which correlates with mRNA and protein levels of ERα, associates with poor prognosis in ER-positive breast cancer patients. Thus, CXXC5 as an unmethylated CpG binder contributes to E2-mediated gene expressions that result in the regulation of cellular proliferation and may contribute to ER-positive breast cancer progression.

Toll-like receptor 9 expressed in proximal intestinal enteroendocrine cells detects bacteria resulting in secretion of cholecystokinin

Toll-like receptors (TLRs) play a key role in the recognition of microbes via detection of specific and conserved microbial molecular features. TLRs, mainly expressed in immune cells, interact with intestinal microbiome. Little is known about mechanism(s) of sensing of bacteria by the intestinal surface enteroendocrine cells (EECs). We show here that TLR9 is expressed by the EECs of proximal intestine in a range of species and is co-expressed with the satiety hormone cholecystokinin (CCK). CCK secreted in excess induces emesis (vomiting). Using an EEC model cell line, STC-1, we demonstrate that in response to the TLR9 agonist, DNA containing unmethylated CpG dinucleotide motifs, STC-1 cells secrete CCK and that this secretion is inhibited by specific inhibitors of TLR9. Exposure of STC-1 cells to heat-inactivated pathogenic bacteria, Escherichia coli O55/H7, Shigella flexneri 2457T, Salmonella typhimurium ST4/74, and non-pathogenic Lactobacillus amylovorus GRL1112, results to an increase in CCK secretion compared to untreated control. The magnitudes of CCK release are higher in response to pathogenic bacteria and lowest in response to the non-pathogenic L. amylovorus. The pathogenic strains not only have substantially bigger genomes than L. amylovorus, they also have significantly higher numbers/frequency of RR/CG/YY stimulatory CpG hexamers in their genomic DNA. Pathogen-induced excessive secretion of the gut hormone CCK, provoking emesis can serve as a protective mechanism against development of enteric infections.

The discovery of potent immunostimulatory CpG-ODNs widely distributed in bacterial genomes.

Oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG-ODN) can be specifically recognized by Toll-like receptor 9 (TLR9), provoking innate immune responses. Designed according to this structural feature, many synthetic phosphorothioate CpG-ODNs successfully activate macrophages. However, it is difficult to find potent stimulatory CpG-DNA fragments in microbial genomes. Therefore, whether microbial CpG-DNA substantially contributes to infectious and immune diseases remains controversial. In this study, high-throughput scanning was carried out for thousands of bacterial genomes with bioinformatics tools to comprehensively evaluate the distribution of CpG-DNA fragments. A random sampling test was then performed to verify their immunostimulatory properties by experiments in vitro and in vivo. Natural TLR9-dependent and potent stimulatory CpG-DNA fragments were found in microbial genomes. Interestingly, highly conserved stimulatory CpG-DNA fragments were found in 16S and 23S rDNA sequences with multiple copies, while others were species-specific. Additionally, we found that the reported active motifs were mostly non-stimulatory in natural CpG fragments. This evidence indicates that the previous structural descriptions of functional CpG-ODNs are incomplete. Our study has assessed the distribution of microbial CpG-DNA fragments, and identified natural stimulatory CpG-DNA fragments. These findings provide a deeper understanding of CpG-ODN structures and new evidence for microbial DNA inflammatory function and pathogenicity.

Evaluation of suitable antigens and adjuvant concentration for sublingual immunization to prevent periodontal disease

AbstractThe oral cavity is dominated by both mucosal and systemic immune systems, due to the presence of immunoglobulin (Ig)A antibodies (Abs) in saliva and IgG Abs in gingival crevicular fluid. Transmucosally administered vaccines allowing the development of both systemic and local immune responses are thus optimal for preventing oral infections. We assessed the capacity of a mucosal vaccine targeting recombinant HSP60 from Porphyromonas gingivalis (GroEL) to induce protective mucosal immune responses. As the basic experiment, optimal concentrations of GroEL as antigen (Ag) and synthetic oligonucleotides containing unmethylated CpG dinucleotide (CpG ODN) as a mucosal adjuvant for sublingual immunization were examined in mice. GroEL‐specific immune responses were observed in sublingual immunization with a combination of GroEL plus cholera toxin or GroEL plus CpG ODN. The optimal concentrations of GroEL and CpG ODN as antigen and adjuvant in sublingual immunization were 3 and 25 μg per mouse, respectively. These results suggest that the sublingual administration of GroEL plus CpG ODN is effective for inducing mucosal and systemic Ag‐specific Ab responses.

TLR9 limits enteric antimicrobial responses and promotes microbiota-based colonisation resistance during Citrobacter rodentium infection.

Mammalian cells express an array of toll-like receptors to detect and respond to microbial pathogens, including enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC). These clinically important attaching and effacing (A/E) pathogens infect the apical surface of intestinal epithelial cells, causing inflammation as well as severe diarrheal disease. Because EPEC and EHEC are human-specific, the related murine pathogen Citrobacter rodentium has been widely used to define how hosts defend against A/E pathogens. This study explored the role of TLR9, a receptor that recognises unmethylated CpG dinucleotides present in bacterial DNA, in promoting host defence against C.rodentium. Infected Tlr9-/- mice suffered exaggerated intestinal damage and carried significantly higher (10-100fold) pathogen burdens in their intestinal tissues as compared with wild type (WT) mice. C.rodentium infection also induced increased antimicrobial responses, as well as hyperactivation of NF-κB signalling in the intestines of Tlr9-/- mice. These changes were associated with accelerated depletion of the intestinal microbiota in Tlr9-/- mice as compared with WT mice. Notably, antibiotic-based depletion of the gut microbiota in WT mice prior to infection increased their susceptibility to the levels seen in Tlr9-/- mice. Our results therefore indicate that TLR9 signalling suppresses intestinal antimicrobial responses, thereby promoting microbiota-mediated colonisation resistance against C.rodentium infection.

CpG Methylation Inhibits Prolidase Promoter Driven Transcription

Prolidase, a cytosolic exopeptidase, and a member of the matrix metalloproteinase (MMP) family, is encoded by the PEPD gene in humans. Since Prolidase is the only enzyme which can cleave dipeptides containing C‐terminal proline, or hydroxyproline, it plays an essential role in protein metabolism, collagen turnover, and matrix remodeling. Prolidase is essential in several physiological and pathological processes such as wound healing, inflammation, angiogenesis, cell proliferation, and carcinogenesis. Surprisingly, little is known about the molecular and cellular regulation of PEPD gene expression. In initial studies, we observed a dense CG‐rich portion around the transcription start site, suggesting a possible role of promoter methylation in the transcriptional regulation of PEPD gene. To study the transcriptional regulation of PEPD gene, we cloned and characterized the human PEPD promoter. The PEPD promoter was amplified from the human genome via PCR and inserted into a luciferase reporter construct. As demonstrated in our results, increase in luciferase activity was obtained after transfection of the PEPD luciferase reporter in HEK293T, indicating the successful construction of the luciferase reporter. Next, we performed in vitro methylation of the PEPD promoter using SssI methyltransferase which methylates DNA CpG islands. Methylation was confirmed by BstUI digestion that cleaves at unmethylated CpG dinucleotides but not at methylated CpG. The CpG methylation significantly inhibited PEPD promoter activity in comparison to the unmethylated construct. Taken together, we believe, these results will generate new knowledge on the molecular regulation of prolidase and provide better insight in our understanding of regulation of prolidase expression in various physiological and pathological conditions.Support or Funding InformationSupported by National Institutes of Health (NIH) Grants DA037779 and MD007586 (to Jui Pandhare) and DA024558, DA30896, DA033892, DA021471, AI22960 and MD007586 (to Chandravanu Dash). Ireti Eni‐Aganga is supported by RISE Grant. The work is also supported by the RCMI Grant G12MD007586, the Vanderbilt CTSA Grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 Grant MD007593 from NIMHD/NIH, and the Tennessee Center for AIDS Research (P30 AI110527).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Paying the Toll for Inflammation

Paying the Toll for Inflammation

ON THE PROBLEM OF DEVELOPMENT OF THE UNIVERSAL IMMUNOTHERAPEUTIC ANTICANCER VACCINE

The authors of this paper theoretically substantiated the cancer treatment method, using in situ activation of dendritic cells with intratumoral injection of two molecular “danger signals” of bacterial origin – plasmid DNA containing unmethylated CpG-dinucleotides and cyclic diguanosine monophosphate (cyclo-diGMP). Based on literature data it might be presumed that this procedure is capable to release from the dying cancer cells a large number of tumor-associated mutant proteins, to recruit effector immune cells into the tumor bed, to activate dendritic cells and as a result to induce a potent anti-cancer T-cellular immune response leading to elimination of both primary solid tumors and possible metastases.

Methyl-CpG binding domain protein 1 regulates localization and activity of Tet1 in a CXXC3 domain-dependent manner.

Cytosine modifications diversify and structure the genome thereby controlling proper development and differentiation. Here, we focus on the interplay of the 5-methylcytosine reader Mbd1 and modifier Tet1 by analyzing their dynamic subcellular localization and the formation of the Tet oxidation product 5-hydroxymethylcytosine in mammalian cells. Our results demonstrate that Mbd1 enhances Tet1-mediated 5-methylcytosine oxidation. We show that this is due to enhancing the localization of Tet1, but not of Tet2 and Tet3 at heterochromatic DNA. We find that the recruitment of Tet1 and concomitantly its catalytic activity eventually leads to the displacement of Mbd1 from methylated DNA. Finally, we demonstrate that increased Tet1 heterochromatin localization and 5-methylcytosine oxidation are dependent on the CXXC3 domain of Mbd1, which recognizes unmethylated CpG dinucleotides. The Mbd1 CXXC3 domain deletion isoform, which retains only binding to methylated CpGs, on the other hand, blocks Tet1-mediated 5-methylcytosine to 5-hydroxymethylcytosine conversion, indicating opposite biological effects of Mbd1 isoforms. Our study provides new insights on how cytosine modifications, their modifiers and readers cross-regulate themselves.

HYPOGLYCEMIC POTENTIAL OF PROBIOTIC DNA LOADED CHITOSAN NANOPARTICLES: AN IN VIVO STUDY

The rise in infectious diseases as well as noninfectious immune related disorders causes millions of premature deaths worldwide and demands the need for the development of efficient immunomodulators. Consumer awareness about the harmful effects of chemical drugs raised a need to search for natural/ alternative therapies for the treatment of diseases. Immunotherapy is one of the alternative ways of modification of diseases. Some bacterial cell components such as peptidoglycans, lipoteichoic acid, secreted soluble substances, genomic DNA, etc, play role in immunomodulation responses. Structural difference between bacterial and eukaryotic DNA apparently account for the ability of bacterial DNA to serve as an immune activating agent, because of its high content of unmethylated CpG dinucleotides. Probiotic DNA’s efficacy could be further enhanced by loading it onto nanoparticles. Nanoparticle-assisted delivery may be a promising approach to alleviate the problem of instability and degradation of DNA. The present study was conducted to prepare and characterize the probiotic DNA loaded chitosan nanoparticles (DLCNP) and to find out the anti-diabetic potential of probiotic bacterial DNA Lactobacillus acidophilus NCDC 343 (LA DNA). Therapeutic efficacy of Probiotic DNA loaded with Chitosan Nanoparticles was evaluated in treating diabetic Balb/c mice. DNA loaded Chitosan Nanoparticles proved as effective anti-diabetic agents and in addition, the bioactivity of probiotic DNA was improved through nano delivery. Keywords: Probiotics, Probiotic DNA, Chitosan-DNA Nanoparticles, Immunotherapy, Anti-diabetic/ Hypoglycemic activity