RESUMO
BACKGROUND: Formalin-fixed paraffin embedded (FFPE) tissues may provide an exciting resource to study microbial associations in human disease, but the use of these low biomass specimens remains challenging. We aimed to reduce unintentional bacterial interference in molecular analysis of FFPE tissues and investigated the feasibility of conducting quantitative polymerase chain reaction (qPCR) and 16S rRNA amplicon sequencing using 14 colorectal cancer, 14 normal adjacent and 13 healthy control tissues. RESULTS: Bacterial contaminants from the laboratory environment and the co-extraction of human DNA can affect bacterial analysis. The application of undiluted template improves bacterial DNA amplification, allowing the detection of specific bacterial markers (Escherichia coli and Faecalibacterium prausnitzii) by qPCR. Nested and non-nested PCR-based 16S rRNA amplicon sequencing approaches were employed, showing that bacterial communities of tissues and paired paraffin controls cluster separately at genus level on weighted Unifrac in both non-nested (R2 = 0.045; Pr(> F) = 0.053) and nested (R2 = 0.299; Pr(> F) = 0.001) PCR datasets. Nevertheless, considerable overlap of bacterial genera within tissues was seen with paraffin, DNA extraction negatives (non-nested PCR) or PCR negatives (nested PCR). Following mathematical decontamination, no differences in α- and ß diversity were found between tumor, normal adjacent and control tissues. CONCLUSIONS: Bacterial marker analysis by qPCR seems feasible using non-normalized template, but 16S rRNA amplicon sequencing remains challenging. Critical evaluation of laboratory procedures and incorporation of positive and negative controls for bacterial analysis of FFPE tissues are essential for quality control and to account for bacterial contaminants.
Assuntos
Bactérias/isolamento & purificação , Neoplasias Colorretais/microbiologia , Formaldeído/química , Bactérias/classificação , Bactérias/genética , Neoplasias Colorretais/patologia , DNA Bacteriano/genética , Microbioma Gastrointestinal/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Inclusão em Parafina , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Manejo de EspécimesAssuntos
Disbiose/complicações , Microbioma Gastrointestinal/imunologia , Hidradenite Supurativa/imunologia , Pele/imunologia , Adulto , Estudos de Casos e Controles , Disbiose/diagnóstico , Disbiose/imunologia , Disbiose/microbiologia , Fezes/microbiologia , Feminino , Voluntários Saudáveis , Hidradenite Supurativa/microbiologia , Hidradenite Supurativa/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Pele/patologia , Adulto JovemRESUMO
Primary sclerosing cholangitis (PSC) is a common indication for liver transplantation (LT). Up to 25% of patients experience recurrence of PSC (rPSC) after LT, which is associated with significant morbidity and mortality. To date, it is not possible to predict which patients are at risk for rPSC. The aetiology of PSC is complex and is speculated to involve translocation of intestinal bacteria to the liver, because of its frequent co-occurrence with inflammatory bowel diseases (IBD). Here, we investigate whether the mucosal intestinal microbiome of PSC patients (n = 97) at time of first LT can identify those patients who will develop rPSC. 16S gene sequencing of bacterial DNA isolated from formalin-fixed paraffin-embedded biopsies showed that PSC patients with Crohn's disease (n = 15) have a reduced microbial diversity and that inflammation of the mucosa is associated with beta-diversity changes and feature differences. No differences in alpha- or beta diversity were observed between patients with rPSC (n = 14) and without rPSC (n = 83). However, many over-represented bacterial features were detected in patients with rPSC, while surprisingly, those without recurrence of disease were characterized by an increased presence of the Gammaproteobacteria Shigella. This pilot study warrants further investigation into bacterial differences between rPSC and non-rPSC patients.
Assuntos
Colangite Esclerosante , Microbioma Gastrointestinal , Transplante de Fígado , Colangite Esclerosante/complicações , Humanos , Projetos Piloto , Fatores de RiscoRESUMO
OBJECTIVE: Pregnancy may affect the disease course of IBD. Both pregnancy and IBD are associated with altered immunology and intestinal microbiology. However, to what extent immunological and microbial profiles are affected by pregnancy in patients with IBD remains unclear. DESIGN: Faecal and serum samples were collected from 46 IBD patients (31 Crohn's disease (CD) and 15 UC) and 179 healthy controls during first, second and third trimester of pregnancy, and prepregnancy and postpartum for patients with IBD. Peripheral blood cytokine profiles were determined by ELISA, and microbiome analysis was performed by sequencing the V4 region of the bacterial 16S rRNA gene. RESULTS: Proinflammatory serum cytokine levels in patients with IBD decrease significantly on conception. Reduced interleukin (IL)-10 and IL-5 levels but increased IL-8 and interferon (IFN)γ levels compared with healthy controls were seen throughout pregnancy, but cytokine patterns remained stable during gestation. Microbial diversity in pregnant patients with IBD was reduced compared with that in healthy women, and significant differences existed between patients with UC and CD in early pregnancy. However, these microbial differences were no longer present during middle and late pregnancy. Dynamic modelling showed considerable interaction between cytokine and microbial composition. CONCLUSION: Serum proinflammatory cytokine levels markedly improve on conception in pregnant patients with IBD, and intestinal microbiome diversity of patients with IBD normalises during middle and late pregnancy. We thus conclude that pregnancy is safe and even potentially beneficial for patients with IBD.
Assuntos
Colite Ulcerativa/sangue , Colite Ulcerativa/microbiologia , Doença de Crohn/sangue , Doença de Crohn/microbiologia , Citocinas/sangue , Microbioma Gastrointestinal , Complicações na Gravidez/sangue , Complicações na Gravidez/microbiologia , Adulto , Estudos de Casos e Controles , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Fezes/microbiologia , Feminino , Humanos , Interferon gama/sangue , Interleucina-10/sangue , Interleucina-5/sangue , Interleucina-8/sangue , Gravidez , Complicações na Gravidez/imunologia , Trimestres da Gravidez/sangue , Trimestres da Gravidez/imunologiaRESUMO
OBJECTIVE: International guidelines recommend endoscopic surveillance of premalignant gastric lesions. However, the diagnostic yield and preventive effect require further study. We therefore aimed to assess the incidence of neoplastic progression and to assess the ability of various tests to identify patients most at risk for progression. DESIGN: Patients from the Netherlands and Norway with a previous diagnosis of atrophic gastritis (AG), intestinal metaplasia (IM) or dysplasia were offered endoscopic surveillance. All histological specimens were assessed according to the updated Sydney classification and the operative link on gastric intestinal metaplasia (OLGIM) system. In addition, we measured serum pepsinogens (PG) and gastrin-17. RESULTS: 279 (mean age 57.9 years, SD 11.4, male/female 137/142) patients were included and underwent at least one surveillance endoscopy during follow-up. The mean follow-up time was 57 months (SD 36). Four subjects (1.4%) were diagnosed with high-grade adenoma/dysplasia or invasive neoplasia (ie, gastric cancer) during follow-up. Two of these patients were successfully treated with endoscopic submucosal dissection, while the other two underwent a total gastrectomy. Compared with patients with extended AG/IM (PGI/II≤3 and/or OGLIM stage III-IV), patients with limited AG/IM (PG I/II>3 and OLGIM stage 0-II) did not develop high-grade adenoma/dysplasia or invasive neoplasia during follow-up (p=0.02). CONCLUSION: In a low gastric cancer incidence area, a surveillance programme can detect gastric cancer at an early curable stage with an overall risk of neoplastic progression of 0.3% per year. Use of serological markers in endoscopic surveillance programmes may improve risk stratification.
Assuntos
Gastroscopia , Vigilância da População , Lesões Pré-Cancerosas/epidemiologia , Neoplasias Gástricas/epidemiologia , Biomarcadores Tumorais/sangue , Progressão da Doença , Feminino , Gastrinas/sangue , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Países Baixos/epidemiologia , Noruega/epidemiologia , Pepsinogênio A/sangue , Medição de Risco , Fatores de RiscoRESUMO
Multiple myeloma (MM) is a neoplasm of plasma cell origin that is largely confined to the bone marrow (BM). Chromosomal translocations and other genetic events are known to contribute to deregulation of signaling pathways that lead to transformation of plasma cells and progression to malignancy. However, the tumor stroma may also provide trophic support and enhance resistance to therapy. Phosphorylation of proteins on tyrosine, serine and threonine residues plays a pivotal role in cell growth and survival. Therefore, knowing the status of phosphorylation-based signaling pathways in cells may provide key insights into how cell growth and survival is promoted in tumor cells. To provide a more comprehensive molecular analysis of signaling disruptions in MM, we conducted a kinome profile comparison of normal plasma cells and MM plasma cells as well as their surrounding cells from normal BM and diseased BM. Integrated pathway analysis of the profiles obtained reveals deregulation of multiple signaling pathways in MM cells but also in surrounding bone marrow blood cells compared to their normal counterparts. The deregulated kinase activities identified herein, which include the mTOR (mammalian target of rapamycin)/p70S6K and ERK1/2 (extracellular signal-regulated kinases 1 and 2) pathways, are potential novel molecular targets in this lethal disease.
Assuntos
Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/fisiopatologia , Fosfotransferases/metabolismo , Transdução de Sinais , Western Blotting , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mieloma Múltiplo/patologia , Fosfoproteínas/metabolismo , Fosforilação , Plasmócitos/metabolismo , Proteômica/métodos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Pre-osteoblast adhesion attracts increasing interest in both medicine and dentistry. However, how this physiological event alters osteoblast phenotype is poorly understood. We therefore attempted to address this question by investigating key biochemical mechanism that governs pre-osteoblast adhesion on polystyrene surface. Importantly, we found that cofilin activity was strongly modulated by PP2A (Ser/Thr phosphatase), while cell-cycle was arrested. Accordingly, we observed that the profile of cofilin phosphorylation (at Ser03) was similar to phospho-PP2A (at Tyr307). Also, it is plausible to suggest during pre-osteoblast adhesion that PP2A phosphorylation at Y307 was executed by phospho-Src (Y416). In addition, it was observed that MAPKp38, but not MAPK-erk, played a key role on pre-osteoblast adhesion by phosphorylating MAPKAPK-2 and ATF-2 (also called CRE-BP1). Also, the up-modulation of RhoA reported here suggests its involvement at the beginning of osteoblast attachment, while Akt remained active during all periods. Altogether, our results clearly showed that osteoblast adhesion is under an intricate network of signaling molecules, which are responsible to guide their interaction with substrate mainly via cytoskeleton rearrangement.