Intestinal Microbiota in Patients with Spinal Cord Injury

Human intestinal flora comprises thousands of bacterial species. Growth and composition of intestinal microbiota is dependent on various parameters, including immune mechanisms, dietary factors and intestinal motility. Patients with spinal cord injury (SCI) frequently display neurogenic bowel dysfunction due to the absence of central nervous system control over the gastrointestinal system. Considering the bowel dysfunction and altered colonic transit time in patients with SCI, we hypothesized the presence of a significant change in the composition of their gut microbiome. The objective of this study was to characterize the gut microbiota in adult SCI patients with different types of bowel dysfunction. We tested our hypothesis on 30 SCI patients (15 upper motor neuron [UMN] bowel syndrome, 15 lower motor neuron [LMN] bowel syndrome) and 10 healthy controls using the 16S rRNA sequencing. Gut microbial patterns were sampled from feces. Independent of study groups, gut microbiota of the participants were dominated by Blautia, Bifidobacterium, Faecalibacterium and Ruminococcus. When we compared all study groups, Roseburia, Pseudobutyrivibrio, Dialister, Marvinbryantia and Megamonas appeared as the genera that were statistically different between groups. In comparison to the healthy group, total bacterial counts of Pseudobutyrivibrio, Dialister and Megamonas genera were significantly lower in UMN bowel dysfunction group. The total bacterial count of Marvinbryantia genus was significantly lower in UMN bowel dysfunction group when compared to the LMN group. Total bacterial counts of Roseburia, Pseudobutyrivibrio and Megamonas genera were significantly lower in LMN bowel dysfunction group when compared to healthy groups. Our results demonstrate for the first time that butyrate-producing members are specifically reduced in SCI patients when compared to healthy subjects. The results of this study would be of interest since to our knowledge, microbiome-associated studies targeting SCI patients are non-existent and the results might help explain possible implications of gut microbiome in SCI

Sentetik cpg oligodeoksinükleotid/katyonik pettid komplekslerinin immün stimülan özelliklerinin belirlenmesi

TÜBİTAK SBAG15.12.2013Yapısal olarak birbirinden farklı CpG motifleri içeren sentetikoligodeoksinükleotidler (ODN) insan immün sistem hücrelerini de farklı şekillerdeuyarmaktadır. Aşı adjuvantı/immünoterapötik ajan olarak klinik denemelere girmişolan K-tipi ODN’ler güçlü B hücre etkinleştirici ve plasmasitoid dendritik hücre(pDC) olgunlaştırıcı, TNF üretici özelliğe sahiptir. Buna karşı, D-tipi ODN’lerpDC’lerden yüksek miktarda IFN üretimine yol açmaktadırlar. Bu aktivite, DODN’lerin G-quadruplex temelli nanometre ebatında parçacıklar oluşturabilmeözelliğine dayalıdır ve bu sebeple üretimlerinde yaşanan sorunlar bu ODN tipininklinik denemelere ulaşmasını engellemektedir. D-ODN aktivitesini tekrarlayabilenve klinik gelişime müsait yeni ajanlar oluşturabilmek amacıyla bu projede K-tipiODN’leri katyonik peptitlerle kompleksleyip kararlı nanoyapılar oluşturmayıhedefledik. Bu projede esnek olmayan ikincil yapıya sahip kısa bir CpG ODNsekansının HIV virüsünden köken alan katyonik Tat peptidi(47-57) ilekomplekslendiğinde nükleazlara dirençli nanohalkalar oluşturduğugösterilmektedir. Nanohalkaların hücre içine alımı arttırdığı ve ODN’i erkenendozomlara hedefleyerek pDC’lerden yüksek miktarda IFN salımına yol açtığıbulunmuştur. Konvansiyonel K tipi ODN’lerle karşılaştırıldığında nanohalkalarınŞap aşısına karşı Th1 ağırlıklı immün yanıt oluşturdukları ve terapötik tümör aşısıadjuvantı olarak Ovalbümin (OVA) proteini ifade eden EG.7 timoma tümörütaşıyan C57BL/6 farelerde daha üstün anti-tümör immünite tetiklediklerigösterilmektedir. Bu sonuçlar, nanohalkaların D-ODN aktivitesini tekrarlayabilenyeni ajanlar olarak klinik uygulamalarda yerini alabileceğine işaret etmektedir.Anahtar kelimeler: TLR9, CpG oligodeoksinükleotidler, katyonik peptidler,ODN-peptid nanoparçacıkları, aşı adjuvantı, anti-kanser ajanStructurally distinct classes of synthetic oligonucleotides (ODN) expressing CpGmotifs differentially activate human immune cells. K-type ODN that progressedinto human clinical trials as vaccine adjuvants/immunotherapeutic agents arestrong activators of B cells and trigger plasmacytoid dendritic cells (pDCs) todifferentiate and produce TNF. In contrast, D-type ODN stimulate large amountsof IFN secretion from pDCs. This activity depends on the ability of D-ODN toadopt nanometre sized G-quadruplex-based structures, complicating theirmanufacturing and hampering their progress into the clinic. In search of a DODN substitute, we attempted to multimerize K-ODN into stable nanostructuresusing cationic peptides. Here we show that a short ODN with a rigid secondarystructure uniquely forms nuclease resistant nanorings following condensationwith the HIV-derived peptide Tat(47-57). The nanorings enhanced cellularinternalization, targeted the ODN to early endosomes and induced a robust IFNresponse from pDCs. Compared to the conventional K-type ODN, nanoringsboosted Th-1 mediated immune responses in mice immunized with theinactivated Foot and Mouth Disease Virus vaccine and generated superior antitumor immunity when used as a therapeutic tumor vaccine adjuvant in C57BL/6mice bearing ovalbumin (OVA)-expressing EG.7 thymoma tumors. These resultssuggest that the nanorings can act as D-ODN surrogates and may find a nichefor further clinical applications.Keywords: TLR9, CpG oligodeoxynucleotides, cationic peptides, ODNpeptide nanoparticles, vaccine adjuvant, anti-cancer agentStructurally distinct classes of synthetic oligonucleotides (ODN) expressing CpG motifs differentially activate human immune cells. K-type ODN that progressed into human clinical trials as vaccine adjuvants/immunotherapeutic agents are strong activators of B cells and trigger plasmacytoid dendritic cells (pDCs) to differentiate and produce TNF. In contrast, D-type ODN stimulate large amounts of IFN secretion from pDCs. This activity depends on the ability of D-ODN to adopt nanometre sized G-quadruplex-based structures, complicating their manufacturing and hampering their progress into the clinic. In search of a DODN substitute, we attempted to multimerize K-ODN into stable nanostructures using cationic peptides. Here we show that a short ODN with a rigid secondary structure uniquely forms nuclease resistant nanorings following condensation with the HIV-derived peptide Tat(47-57). The nanorings enhanced cellular internalization, targeted the ODN to early endosomes and induced a robust IFN response from pDCs. Compared to the conventional K-type ODN, nanorings boosted Th-1 mediated immune responses in mice immunized with the inactivated Foot and Mouth Disease Virus vaccine and generated superior antitumor immunity when used as a therapeutic tumor vaccine adjuvant in C57BL/6 mice bearing ovalbumin (OVA)-expressing EG.7 thymoma tumors. These results suggest that the nanorings can act as D-ODN surrogates and may find a niche for further clinical applications

Bakterilerden salgılanan nanokeseciklerin immün stimülan özelliklerinin belirlenmesi ve immünoterapötik uygulamaları

TÜBİTAK SBAG15.10.201

Repetitive elements in mammalian telomeres suppress bacterial DNA-induced immune activation.

Bacterial DNA contains immunostimulatory CpG motifs that trigger an innate immune response capable of promoting host survival following infectious challenge. Yet CpG-driven immune activation may also have deleterious consequences, ranging from autoimmune disease to death. We find that repetitive elements present at high frequency in mammalian telomeres, but rare in bacteria, down-regulate CpG-induced immune activation. Suppressive activity correlates with the ability of telomeric TTAGGG repeats to form G-tetrads. Colocalization of CpG DNA with Toll-like receptor 9 in endosomal vesicles is disrupted by these repetitive elements, although cellular binding and uptake remain unchanged. These findings are the first to establish that specific host-derived molecules can down-regulate the innate immune response elicited by a TLR ligand

Effect of suppressive DNA on CpG-induced immune activation.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs stimulate a strong innate immune response. This stimulation can be abrogated by either removing the CpG DNA or adding inhibitory/suppressive motifs. Suppression is dominant over stimulation and is specific for CpG-induced immune responses (having no effect on LPS- or Con A-induced activation). Individual cells noncompetitively internalize both stimulatory and suppressive ODN. Studies using ODN composed of both stimulatory and suppressive motifs indicate that sequence recognition proceeds in a 5'-->3' direction, and that a 5' motif can block recognition of immediately 3' sequences. These findings contribute to our understanding of the immunomodulatory activity of DNA-based products and the rules that govern immune recognition of stimulatory and suppressive motifs

Beyond the jab: A need for global coordination of pharmacovigilance for COVID-19 vaccine deployment Comment

Commentary - No abstract available

İki Farklı Tip Tlr9 Agonisti İle Uyarılan Hücrelerde Etkinleşen Sinyal İletim Yolakla

Bu projede CAL-1 insan plazmasitoid dendritik hücre hattı kullanılarak iki farklı tip CpG ODN ile (K ve D tipleri, Bkz Tablo I) hücreler uyarılacak, bu hücrelerdeki sinyal ileti yolaklarında rol alan transkripsiyon faktörleri florokromla (Alexa flor488, fikoeritrin gibi) işaretli antikorlar yardımıyla incelenecektir. Kısaca, bu hücrelerin yüzeyi önce i) bu hücrelere spesifik olan anti-BDCA-2 ve anti- CD123 florokromlu antikorlarla boyanacak, ii) hücreler permiabilize edildikten sonra hücre içi değişik transkripsiyon faktörleri ise üçüncü boyayı içeren fosfo-spesifik antikorla boyanacaktır. Araştırmada öncelikle şu iki transkripsiyon faktörünün incelenmesi planlanmıştır: STAT-1 ve p38. Her iki faktörün fosfo-spesifik florokromlu antikorları akım sitometrik analiz için mevcuttur. Bu yöntemle pDC lerdeki hücre içi sinyal iletiminin gelişimi zamana karşı haritalanacaktır. Projenin ilk altı ayında kimyasal ve teçhizat alımları tamamlanacak, projede çalışacak lisans/yüksek lisans öğrencisi alınıp eğitilecek ve yeni kurmakta olduğumuz laboratuarın altyapısı kısmen tamamlanacaktır. İkinci altı ayda i) optimizasyon deneyleri, ii) değişik ODN lerin zaman ve iii) konsantrasyona bağlı TLR9 ‘un yönlendirdiği sinyal yolaklarının aktivasyon kinetikleri deneysel olarak araştırılacaktır