Expression profiling of lymph nodes in tuberculosis patients reveal inflammatory milieu at site of infection
ABSTRACT: Extrapulmonary manifestations constitute 15-20% of tuberculosis cases, with lymph node as the most common site. Understanding of disease etiology is limited due to the lack of understanding patients’ infected tissue milieu. This study was designed to perform global transcriptome analysis of lymph node tissues from healthy individuals and Mycobacterium tuberculosis infected lymph nodes of patients to decipher the local response of infected tissue. This study was designed to elucidate gene expression signatures in the event of Lymph Node Tuberculosis. Total RNA was extracted from lymph node tissue samples of LNTB patients in BSL3 facility and together with healthy lymph node RNA samples (commercially purchased) their global transcriptome profiling was performed using Illumina HumanHT-12 V4 expression beadchip.
Project description:RNA was extracted from formalin-fixed specimens of paired intratumoral and peritumoral tissues of patients with lymph node-positive (n=20) or negative (n=20) HCC. A cDNA-mediated annealing, selection, extension, and ligation assay was performed with an array of 502 known cancer-related genes to identify differentially expressed genes in 80 RNA samples. Total RNA was purified from the tissue specimens using the High Pure RNA Paraffin Kit according to the manufacturer’s protocol. DASL experiments were performed to identify genes that were differentially expressed between the lymph node metastasis (LNM) and non-lymph node metastasis (NLNM) groups of matched intratumoral and peritumoral tissues by using A cDNA-mediated annealing, selection, extension, and ligation assay.
Project description:Lymph node status is a crucial predictor for the overall survival of invasive breast cancer. However, lymph node involvement is only detected in about half of HER2 positive patients. Currently, there are no biomarkers available for distinguishing small size HER2-positive breast cancers with different lymph node statuses. Thus, in the present study, we applied label-free quantitative proteomic strategy to construct plasma proteomic profiles of ten patients with small size HER2-positive breast cancers (5 patients with lymph node metastasis versus 5 patients with lymph node metastasis).
Project description:Endobronchial ultrasound (EBUS) guided biopsy is becoming the mainstay for investigation of mediastinal lymphadenopathy to aid laboratory diagnosis of malignancy, sarcoidosis or tuberculosis. However, diagnostic uncertainty is still common in the distinction between culture negative tuberculosis and sarcoidosis or malignancy and reactive lymphadenitis. We sought to evaluate the role of genome-wide transcriptional profiling of EBUS guided lymph node samples to aid diagnostic processes in this setting.
Project description:Bovine tuberculosis (bTB), caused by Mycobacterium bovis (Mycobacterium tuberculosis complex), is a zoonotic disease that affects cattle and wildlife worldwide. In some regions of Spain, Iberian red deer (Cervus elaphus hispanicus) can serve as reservoir of infection, thus increasing the risk of human and cattle exposure and infection. Mesenteric lymph nodes are naturally infected with M. bovis in Iberian red deer, in which the digestive route of infection is particularly important in Mediterranean Spain. In this study we characterized the differential expression of inflammatory and immune response genes in mesenteric lymph nodes of Iberian red deer naturally infected with M. bovis using a Ruminant Immuno-inflammatory Gene Universal Array (RIGUA) and real-time RT-PCR. Of the 600 genes that were analyzed in the microarray, 157 showed ≥ 1.2 fold changes in expression in infected or uninfected deer and 17 genes displayed an expression fold change greater than 1.7 with a P-value ≤ 0.05 and were selected for further analysis. These genes included tight junction proteins (Z02 and occluding), IL-11R, bactenecin, CD62L, CD74, desmoglein, IgA and IgM that constitute new findings and suggest new mechanisms by which M. bovis may modulate host inflammatory and immune responses. Identification of genes differentially expressed in animals and tissues naturally infected with M. bovis contributes to our basic understanding of the mechanisms of pathogenesis and protective immunity to mycobacterial infections and may have important implications for future functional genomic and vaccine studies to aid in the control of bTB in deer and other wildlife reservoir species. Mesenteric lymph node RNA from four different uninfected Iberian red deer stags and two Iberian red deer stags infected with Mycobacterium bovis. Infected animals were naturally infected with M. bovis. All animals were hunter-harvested and the tissues retrieved 2-6 hrs after animal hunting.
Project description:The objective of this study was to identify porcine genes which expression was affected by experimental infection with A. pleuropneumoniae within 24 hours after experimental challenge. Microarray experiments were conducted to reveal genes being significantly differentially expressed in infected versus non-infected lung tissue and in liver and lung lymph node tissue from three challenged versus two non-challenged animals. Keywords: Host response Three comparisons were done: - infected versus non-infected lung tissue sampled from three challenged pigs - liver tissue from three challenged versus two non-challenged animals - lung lymph node tissue from three challenged versus two non-challenged animals All experiments were conducted as common reference design.
Project description:Targeted HIV cure strategies require definition of the mechanisms that maintain the virus. Here, we tracked HIV replication and the persistence of infected CD4 T cells in individuals with natural virologic control by sequencing viruses, T cell receptor genes, HIV integration sites and cellular transcriptomes. Our results revealed three mechanisms of HIV persistence operating within distinct anatomic and functional compartments. In lymph node, we detected viruses with genetic and transcriptional attributes of active replication in both T follicular helper (TFH) cells and non-TFH memory cells. In blood, we detected inducible proviruses of archival origin among highly differentiated, clonally expanded cells. Linking the lymph node and blood was a small population of circulating cells harboring inducible proviruses of recent origin. Thus, HIV replication in lymphoid tissue, clonal expansion of infected cells, and recirculation of recently infected cells act together to maintain the virus in HIV controllers despite effective antiviral immunity. Fluorescence-activated cell sorting (FACS) was used to sort subsets of CD4 T cells from blood (peripheral blood mononuclear cells; PBMC) and lymph node from a cohort of HIV-infected people with natural control of the virus (termed HIV controllers). Subsets of CD4 T cells that were sorted are as follows: from blood, (1) naïve (N), (2) central memory (CM), (3) transitional memory (TM), and (4) effector memory (EM); from lymph node, (1) naïve (N), (2) non-germinal center T-follicular helpers (nGC), (3) germinal center T-follicular helpers (Tfh), (4) effector memory (EM), and (5) other central memory-like subsets (CMPD1lo57lo, CMPD1lo57hi, and/or CMPD1lo). Total RNA and total DNA were extracted from these sorted subsets in separate fractions using RNAzol RT and DNAzol. Total cellular DNA was used for HIV quantification and sequence analysis as describe in the publication. Total RNA used for mRNA library construction by oligo-dT purification (Dynabeads), random fragmentation by heating in the presence of Magnesium (in form of 5x first-strand buffer, LifeTech), reverse transcription (SuperScript III), and then second-strand synthesis, end repair, a-tailing, and adaptor ligation using NEBNext enzyme master mixes and oligonucleotides. Libraries were sequenced on an Illumina HiSeq2000. Please note that each 'source name' value represent individual who havs HIV infection with natural control of the virus.
Project description:Mycobacterium tuberculosis (M. tb), the cause of tuberculosis (TB), utilizes the blood circulation to spread systemically and establish infection, and the risk of developing active TB (pulmonary and extrapulmonary) is significantly increased in individuals infected with human immunodeficiency virus (HIV). In this work, we have used DNA microarray analysis to investigate the transcriptome of M. tb replicating in human whole blood from both HIV-negative and HIV-positive donors compared to M. tb grown in Middlebrook 7H9 broth media in order to identify M. tb adaptations to this host environment as well as M. tb mechanisms/factors contributing to increased active and disseminated TB during M. tb/HIV co-infection. We compared the global gene expression of M. tb H37Rv replicating in whole blood from 6 HIV- and 6 HIV+ individulas at 96 hr to M. tb grown to log phase in Middlebrook 7H9 media.
Project description:Expression profiles of porcine liver and tracheobronchial lung lymph node tissues were studied to identify genes being significantly affected by bacterial infection of the lungs. Samples were taken from liver and tracheobronchial lung lymph node tissues in pigs experimentally infected with A. pleuropneumoniae, serotype 5b, and from healthy non-inoculated control pigs from the same herd. Samples were investigated for changes in gene expression by means of global cDNA microarrays. Expression profiling of samples of liver and lung lymph node tissues from ten pigs challenged with Actinobacillus pleuropneumoniae and from five healthy control pigs was conducted by hybridising all 2x15 samples against a tissue specific common reference consisting of a pool of equal amount of total-RNA from all samples of each tissue type and were balanced with respect to control and challenged animals.
Project description:We provide direct in vivo evidence for activation of the BCR and canonical NF-KB pathways in MCL that, in the absence of activating mutations, is dependent on the lymph node microenvironment. This finding provides a mechanistic explanation for the surprising efficacy of ibrutinib for the treatment of this type of lymphoma. Mutations in components of the BCR and NF-KB pathways are associated with cell-autonomous signaling and resistance to ibrutinib. Lymph node biopsies and peripheral blood samples were obtained from 55 patients with previously untreated MCL. The samples are distributed into 3 groups: peripheral blood (purified CD19, N=17), unpurified lymph node biopsy (mixed tissue biopsy, N=34), and purified lymph node (purified CD19 Tumor, N=4).
Project description:Objective: The purpose of this study was to investigate the molecular basis of tumorigenesis and regional lymph node metastasis in LSCC, and provide a set of genes that may be useful for the development of novel diagnostic markers and/or more effective therapeutic strategies. Methods: A total number of 10 patients who underwent surgery for primary laryngeal squamous cell carcinoma were recruited for microarray analysis. LSCC tissues compared with corresponding adjacent non-neoplastic tissues were analysed by Illumina mRNA microarrays,and LSCC tissues with regional lymph node metastasis and LSCC tissues without regional lymph node metastasis were analyzed in the same manner.The most frequently differently expressed genes screened by microarrays were also validated by qRT-PCR in another 42 patients diagnosed for LSCC . Results: Analysed by Illumina mRNA microarrays,there were 361 genes significantly related to tumorigenesis while 246 genes significantly related to regional lymph node metastasis in LSCC. We found that the six genes (CDK1,CDK2,CDK4,MCM2,MCM3,MCM4) were most frequently differently expressed functional genes related to tumorigenesis while eIF3a and RPN2 were most frequently differently expressed functional genes related to regional lymph node metastasis in LSCC. The expressions of these genes were also validated by qRT-PCR. Conclusions: The research revealed a gene expression signature of tumorigenesis and regional lymph node metastasis in laryngeal squamous cell carcinoma.Of the total, the deregulation of several genes (CDK1, CDK2, CDK4, MCM2, MCM3, MCM4, EIF3a and RPN2) were potentially associated with disease development and progression. The result will contribute to the understanding of the molecular basis of LSCC and help to improve diagnosis and treatment. A total number of 10 patients who underwent surgery for primary laryngeal squamous cell carcinoma were recruited for microarray analysis. LSCC tissues compared with corresponding adjacent non-neoplastic tissues were analysed by Illumina mRNA microarrays,and LSCC tissues with regional lymph node metastasis and LSCC tissues without regional lymph node metastasis were analyzed in the same manner.The most frequently differently expressed genes screened by microarrays were also validated by qRT-PCR in another 42 patients diagnosed for LSCC .