[Show abstract][Hide abstract] ABSTRACT: Fecal microbiota transplants (FMT) are an effective treatment for patients with gut microbe dysbiosis suffering from recurrent C. difficile infections. To further understand how FMT reconstitutes the patient’s gut commensal microbiota, we have analyzed the colonization potential of the donor, recipient and recipient post transplant fecal samples using transplantation in gnotobiotic mice.
A total of nine samples from three human donors, recipient’s pre and post FMT were transplanted into gnotobiotic mice. Microbiome analysis of three donor fecal samples revealed the presence of a high relative abundance of commensal microbes from the family Bacteriodaceae and Lachnospiraceae that were almost absent in the three recipient pre FMT fecal samples (<0.01 %). The microbe composition in gnotobiotic mice transplanted with the donor fecal samples was similar to the human samples. The recipient samples contained Enterobacteriaceae, Lactobacillaceae, Enterococcaceae in relative abundance of 43, 11, 8 %, respectively. However, gnotobiotic mice transplanted with the recipient fecal samples had an average relative abundance of unclassified Clostridiales of 55 %, approximately 7000 times the abundance in the recipient fecal samples prior to transplant. Microbiome analysis of fecal samples from the three patients early (2–4 weeks) after FMT revealed a microbe composition with the relative abundance of both Bacteriodaceae and Lachnospiraceae that was approximately 7 % of that of the donor. In contrast, gnotobioitc mice transplanted with the fecal samples obtained from the three at early times post FMT revealed increases in the relative abundance of Bacteriodaceae and Lachnospiraceae microbe compositions to levels similar to the donor fecal samples. Furthermore, the unclassified Clostridiales in the recipient samples post FMT was reduced to an average of 10 %.
We have used transplantation into gnotobiotic mice to evaluate the colonization potential of microbiota in FMT patients early after transplant. The commensal microbes present at early times post FMT out competed non-commensal microbes (e.g. such as unclassified Clostridiales) for niche space. The selective advantage of these commensal microbes to occupy niches in the gastrointestinal tract helps to explain the success of FMT to reconstitute the gut microbe community of patients with recurrent C. difficile infections.
[Show abstract][Hide abstract] ABSTRACT: Background:
CD14, a co-receptor for several pattern recognition receptors and a widely used monocyte/macrophage marker, plays a key role in host responses to Gram-negative bacteria. Despite its central role in the inflammatory response to lipopolysaccharide and other microbial products, and in dissemination of bacteria in some infections, the signaling networks controlled by CD14 during urinary tract infection (UTI) are unknown.
We used uropathogenic Escherichia coli (UPEC) infection of wild-type (WT) C57BL/6 and Cd14-deficient mice and RNA-sequencing (RNA-seq) to define the CD14-dependent transcriptional signature, and role of CD14, in host defense against UTI in the bladder.
UPEC-induced the up-regulation of Cd14 and monocyte/macrophage related genes Emr1/F4/80 and Csf1r/c-fms, which was associated with lower UPEC burdens in WT compared to Cd14-deficient mice. Exacerbation of infection in Cd14-deficient mice was associated with the absence of a 491-gene transcriptional signature in the bladder that encompassed multiple host networks not previously associated with this receptor. CD14-dependent pathways included immune cell trafficking, differential cytokine production in macrophages, and IL-17 signaling. Depletion of monocytes/macrophages in the bladder by administration of liposomal clodronate led to higher UPEC burdens.
This study identifies new host protective and signaling roles for Cd14 in the bladder during UPEC UTI.
No preview · Article · Aug 2015 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Although most hypocalcemia with hypomagenesemia in the neonatal period is due to transient neonatal hypoparathyroidism, magnesium channel defects should also be considered.
We report a case of persistent hypomagnesemia in an 8-day-old Hispanic male who presented with generalized seizures. He was initially found to have hypomagnesemia, hypocalcemia, hyperphosphatemia and normal parathyroid hormone. Serum calcium normalized with administration of calcitriol and calcium carbonate. Serum magnesium improved with oral magnesium sulfate. However, 1 week after magnesium was discontinued, serum magnesium declined to 0.5 mg/dL. Magnesium supplementation was immediately restarted, and periodic seizure activity resolved after serum magnesium concentration was maintained above 0.9 mg/dL. The child was eventually weaned off oral calcium and calcitriol with persistent normocalemia. However, supraphysiologic oral magnesium doses were necessary to prevent seizures and maintain serum magnesium at the low limit of normal.
As his clinical presentation suggested primary renal magnesium wastage, TRPM6 gene mutations were suspected; subsequent genetic testing revealed the child to be compound heterozygous for TRPM6 mutations.
Two novel TRPM6 mutations are described with a new geographic and ethnic origin. This case highlights the importance of recognizing disorders of magnesium imbalance and describing new genetic mutations.
[Show abstract][Hide abstract] ABSTRACT: Systemic lupus erythematosus (SLE) is a severe autoimmune disease that is associated with increased circulating apoptotic cell autoantigens (AC-Ags) as well as increased type I IFN signaling. Here, we describe a pathogenic mechanism in which follicular translocation of marginal zone (MZ) B cells in the spleens of BXD2 lupus mice disrupts marginal zone macrophages (MZMs), which normally clear AC debris and prevent follicular entry of AC-Ags. Phagocytosis of ACs by splenic MZMs required the megakaryoblastic leukemia 1 (MKL1) transcriptional coactivator-mediated mechanosensing pathway, which was maintained by MZ B cells through expression of membrane lymphotoxin-alpha1beta2 (mLT). Specifically, type I IFN-induced follicular shuttling of mLT-expressing MZ B cells disengaged interactions between these MZ B cells and LTbeta receptor-expressing MZMs, thereby downregulating MKL1 in MZMs. Loss of MKL1 expression in MZMs led to defective F-actin polymerization, inability to clear ACs, and, eventually, MZM dissipation. Aggregation of plasmacytoid DCs in the splenic perifollicular region, follicular translocation of MZ B cells, and loss of MKL1 and MZMs were also observed in an additional murine lupus model and in the spleens of patients with SLE. Collectively, the results suggest that lupus might be interrupted by strategies that maintain or enhance mechanosensing signaling in the MZM barrier to prevent follicular entry of AC-Ags.
Full-text · Article · Jun 2015 · The Journal of clinical investigation
[Show abstract][Hide abstract] ABSTRACT: The apolipoprotein (apo)A-I mimetic peptide 4F favors the differentiation of human monocytes to an alternatively-activated M2 phenotype. The goal of the current study was to test whether the 4F-mediated differentiation of monocyte-derived macrophages (MDMs) requires the induction of an oxidative metabolic program. 4F treatment induced several genes in MDMs that play an important role in lipid metabolism, including peroxisome proliferator-activated receptor γ (PPARγ) and CD36. Addition of 4F was associated with a significant increase in fatty acid (FA) uptake and oxidation compared to vehicle treatment. Mitochondrial respiration was assessed by measurement of oxygen consumption rate (OCR). 4F increased basal and ATP-linked OCR as well as maximal uncoupled mitochondrial respiration. These changes were associated with a significant increase in mitochondrial membrane potential (ΔΨm). The increase in metabolic activity in 4F-treated MDMs was attenuated by etomoxir, an inhibitor of mitochondrial FA uptake. Finally, addition of the PPARγ antagonist T0070907 to 4F-treated MDMs reduced the expression of CD163 and CD36, cell surface markers for M2 macrophages, and reduced basal and ATP-linked OCR. These results support our hypothesis that the 4F-mediated differentiation of MDMs to an anti-inflammatory phenotype is due, in part, to an increase in FA acid uptake and mitochondrial oxidative metabolism.
This is the accepted version of the following article: The Biochemical journal, which has been published in final form at http://www.biochemj.org/
Full-text · Article · Mar 2015 · Biochemical Journal
[Show abstract][Hide abstract] ABSTRACT: Genetic changes occurring in different stages of pre-cancer lesions reflect causal events initiating and promoting the progression to cancer. Co-existing pre-cancerous lesions including low- and high-grade squamous intraepithelial lesion (LGSIL and HGSIL), and adjacent "normal" cervical epithelium from six formalin-fixed paraffin-embedded samples were selected. Tissues from these 18 samples were isolated using laser-capture microdissection, RNA was extracted and sequenced. RNA-sequencing generated 2.4 billion raw reads in 18 samples, of which ~50.1% mapped to known and annotated genes in the human genome. There were 40 genes up-regulated and 3 down-regulated (normal to LGSIL) in at least one-third of the sample pairs (same direction and FDR p < 0.05) including S100A7 and KLK6. Previous studies have shown that S110A7 and KLK7 are up-regulated in several other cancers, whereas CCL18, CFTR, and SLC6A14, also differentially expressed in two samples, are up-regulated specifically in cervical cancer. These differentially expressed genes in normal to LGSIL progression were enriched in pathways related to epithelial cell differentiation, keratinocyte differentiation, peptidase, and extracellular activities. In progression from LGSIL to HGSIL, two genes were up-regulated and five down-regulated in at least two samples. Further investigations using co-existing samples, which account for all internal confounders, will provide insights to better understand progression of cervical pre-cancer.
Full-text · Article · Nov 2014 · Frontiers in Oncology
[Show abstract][Hide abstract] ABSTRACT: Rationale: DNA methylation, a major epigenetic mechanism, may regulate
coordinated expression of multiple genes at specific time points during alveolar
septation in lung development. Objectives: To identify genes regulated by
methylation during normal septation in mice, and during disordered septation in
bronchopulmonary dysplasia. Methods: Mouse: Newborn lungs (pre-septation) and
adult lungs (post-septation) were evaluated by microarray analysis of gene
expression and immunoprecipitation of methylated DNA followed by sequencing
(MeDIP-Seq). Human: Microarray gene expression data were integrated with
genome-wide DNA methylation data from bronchopulmonary dysplasia versus preterm
and term lung. Genes with reciprocal changes in expression and methylation,
suggesting regulation by DNA methylation, were identified. Measurements and Main
Results: Mouse: 95 genes with inverse correlation between expression and
methylation during normal septation were identified. In addition to genes known
to be important in lung development (Wnt signaling, Angpt2, Sox9, etc.) and its
extracellular matrix (Tnc, Eln, etc), genes involved with immune and antioxidant
defense (Stat4, Sod3, Prdx6, etc.) were also observed. Human: 23 genes were
differentially methylated with reciprocal changes in expression in
bronchopulmonary dysplasia compared to preterm or term lung. Genes of interest
included those involved with detoxifying enzymes (Gstm3) and transforming growth
factor beta signaling (Bmp7). Overlap: 20 genes and three pathways methylated
during mouse lung development also demonstrated changes in methylation between
preterm and term human lung. Conclusions: Changes in methylation correspond to
altered expression of a number of genes associated with lung development,
suggesting that DNA methylation of these genes may regulate normal and abnormal
No preview · Article · Nov 2014 · American Journal of Respiratory Cell and Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Constitutional SMARCB1 mutations at 22q11.23 have been found in ∼50% of familial and <10% of sporadic schwannomatosis cases. We sequenced highly conserved regions along 22q from eight individuals with schwannomatosis whose schwannomas involved somatic loss of one copy of 22q, encompassing SMARCB1 and NF2, with a different somatic mutation of the other NF2 allele in every schwannoma but no mutation of the remaining SMARCB1 allele in blood and tumor samples. LZTR1 germline mutations were identified in seven of the eight cases. LZTR1 sequencing in 12 further cases with the same molecular signature identified 9 additional germline mutations. Loss of heterozygosity with retention of an LZTR1 mutation was present in all 25 schwannomas studied. Mutations segregated with disease in all available affected first-degree relatives, although four asymptomatic parents also carried an LZTR1 mutation. Our findings identify LZTR1 as a gene predisposing to an autosomal dominant inherited disorder of multiple schwannomas in ∼80% of 22q-related schwannomatosis cases lacking mutation in SMARCB1.
[Show abstract][Hide abstract] ABSTRACT: Aim:
Neoplastic transformation provides one of the few existing opportunities to analyze molecular changes in real time during the initiation and progression of breast cancer.
Materials & methods:
Human mammary epithelial cells underwent neoplastic reprogramming, generating one line of semitransformed, premalignant cells and two separate, temporal lines of fully transformed human mammary epithelial cells (THMECs). An Illumina Infinium HumanMethylation27 BeadChip was used to analyze DNA methylation alterations in 27,578 CpG loci at three consecutive time points over an 80-day (d) transformation period.
The mean β value for semitransformed human mammary epithelial cells CpG loci (0.245) was much greater than for either THMEC-40d (0.055) or THMEC-80d (0.066), indicating a large loss of methylation after neoplastic induction. In addition, 54% of CpG loci were hypermethylated during the THMEC-40d to THMEC-80d transition. We observed that the CpG loci exhibiting DNA methylation changes during early oncogenesis were enriched for biological functions like cellular movement; this was distinctly different than in the later, more progressive stages of the transformation process enriched for processes involving differentiation.
The timing of major methylomic changes may be important in directing the cell toward a more cancerous phenotype. In addition, gene-specific hypermethylation appears to silence developmentally related genes, leading to dedifferentiation.
[Show abstract][Hide abstract] ABSTRACT: Wnt5a is a non-canonical signaling Wnt. Low expression of WNT5A is correlated with poor prognosis in breast cancer patients. The highly invasive breast cancer cell lines, MDA-MB-231 and 4T1, express very low levels of WNT5A. To determine if enhanced expression of WNT5A would affect metastatic behavior, we generated WNT5A expressing cells from the 4T1 and MDA-MB-231 parental cell lines. WNT5A expressing cells demonstrated cobblestone morphology and reduced in vitro migration relative to controls. Cell growth was not altered. Metastasis to the lung via tail vein injection was reduced in the 4T1-WNT5A expressing cells relative to 4T1-vector controls. To determine the mechanism of WNT5A action on metastasis, we performed microarray and whole-transcriptome sequence analysis (RNA-seq) to compare gene expression in 4T1-WNT5A and 4T1-vector cells. Analysis indicated highly significant alterations in expression of genes associated with cellular movement. Down-regulation of a subset of these genes, Mmp13, Nos2, Il1a, Cxcl2, and Lamb3, in WNT5A expressing cells was verified by semi-quantitative RT-PCR. Significant differences in transcript splicing were also detected in cell movement associated genes including Cd44. Cd44 is an adhesion molecule with a complex genome structure. Variable exon usage is associated with metastatic phenotype. Alternative spicing of Cd44 in WNT5A expressing cells was confirmed using RT-PCR. We conclude that WNT5A inhibits metastasis through down-regulation of multiple cell movement pathways by regulating transcript levels and splicing of key genes like Cd44.
[Show abstract][Hide abstract] ABSTRACT: The apolipoprotein A-I (apoA-I) mimetic peptide 4F favors the differentiation of human monocytes to an anti-inflammatory phenotype and attenuates lipopolysaccharide (LPS)-induced inflammatory responses. We investigated the effects of LPS on the Toll-like receptor (TLR) signaling pathway in 4F-differentiated monocyte-derived macrophages.
Monocyte-derived macrophages were pretreated with 4F or vehicle for 7 days. 4F downregulated cell-surface TLRs (4, 5, and 6) as determined by flow cytometry. 4F attenuated the LPS-dependent upregulation of genes encoding TLR1, 2, and 6 and genes of the MyD88-dependent (CD14, MyD88, TRAF6, interleukin-1 receptor-associated kinase 4, and inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta) and MyD88-independent (interferon regulatory factor 3, TANK-binding kinase 1, and Toll-interleukin 1 receptor domain-containing adaptor-inducing interferon-β) pathways as determined by microarray analysis and quantitative reverse transcriptase polymerase chain reaction. Functional analyses of monocyte-derived macrophages showed that 4F reduced LPS-dependent TLR4 recycling, phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, activation and translocation of nuclear factor-κB and inhibited the secretion of tumor necrosis factor-α and interleukin-6 induced by LPS or lipoteichoic acid. These changes were associated with depletion of cellular cholesterol and caveolin, components of membrane lipid rafts.
These data suggest that disruption of rafts by 4F alters the assembly of TLR-ligand complexes in cell membranes and inhibits proinflammatory gene expression in monocyte-derived macrophages, thus attenuating the responsiveness of macrophages to LPS.
[Show abstract][Hide abstract] ABSTRACT: Rationale: DNA methylation is an important epigenetic mechanism, which often occurs in response to environmental stimuli and is crucial in regulating gene expression. It is likely that epigenetic alterations contribute to pathogenesis in idiopathic pulmonary fibrosis (IPF). Objectives: To determine the DNA methylation changes in IPF and their effects on gene expression. Methods: Total DNA methylation and DNA methyltransferase expression were compared in IPF and normal control lung tissues. IPF and normal tissues were subjected to comparative analysis of genome-wide DNA methylation and RNA expression using DNA hybridization to the Illumina HumanMethylation27 BeadChip and RNA hybridization to Illumina HumanHT-12 BeadChip. Functional analyses of differentially expressed and differentially methylated genes were done. Selected genes were validated at DNA, RNA, and protein levels. Measurements and Main Results: DNA methylation status was altered in IPF. IPF samples demonstrated higher DNA methyltransferase expression without observed alterations in global DNA methylation. Genome-wide differences in DNA methylation status and RNA expression were demonstrated by array hybridization. Among the genes whose DNA methylation status and RNA expression were both significantly altered, 16 genes were hypermethylated in DNA associated with decreased mRNA expression or vice versa. We validated CLDN5, ZNF467, TP53INP1, and DDAH1 genes at the level of DNA methylation status, RNA, and protein-level expression. Conclusions: Changes in DNA methylation correspond to altered mRNA expression of a number of genes, some with known and others with previously uncharacterized roles in IPF, suggesting that DNA methylation is important in the pathogenesis of IPF.
No preview · Article · Jun 2012 · American Journal of Respiratory and Critical Care Medicine
[Show abstract][Hide abstract] ABSTRACT: Renal injury induced by brain death is characterized by ischemia and inflammation, and limiting it is a therapeutic goal that could improve outcomes in kidney transplantation. Brain death resulted in decreased circulating nitrite levels and increased infiltrating inflammatory cell infiltration into the kidney. Since nitrite stimulates nitric oxide signaling in ischemic tissues, we tested whether nitrite therapy was beneficial in a rat model of brain death followed by kidney transplantation. Nitrite, administered over 2 h of brain death, blunted the increased inflammation without affecting brain death-induced alterations in hemodynamics. Kidneys were transplanted after 2 h of brain death and renal function followed over 7 days. Allografts collected from nitrite-treated brain-dead rats showed significant improvement in function over the first 2 to 4 days after transplantation compared with untreated brain-dead animals. Gene microarray analysis after 2 h of brain death without or with nitrite therapy showed that the latter significantly altered the expression of about 400 genes. Ingenuity Pathway Analysis indicated that multiple signaling pathways were affected by nitrite, including those related to hypoxia, transcription, and genes related to humoral immune responses. Thus, nitrite therapy attenuates brain death-induced renal injury by regulating responses to ischemia and inflammation, ultimately leading to better post-transplant kidney function.
Full-text · Article · Apr 2012 · Kidney International
[Show abstract][Hide abstract] ABSTRACT: To identify endoplasmic reticulum (ER) stress-induced microRNAs (miRNA) that govern ER protein influx during the adaptive phase of unfolded protein response, we performed miRNA microarray profiling and analysis in human airway epithelial cells following ER stress induction using proteasome inhibition or tunicamycin treatment. We identified miR-346 as the most significantly induced miRNA by both classic stressors. miR-346 is encoded within an intron of the glutamate receptor ionotropic delta-1 gene (GRID1), but its ER stress-associated expression is independent of GRID1. We demonstrated that the spliced X-box-binding protein-1 (sXBP1) is necessary and sufficient for ER stress-associated miR-346 induction, revealing a novel role for this unfolded protein response-activated transcription factor. In mRNA profiling arrays, we identified 21 mRNAs that were reduced by both ER stress and miR-346. The target genes of miR-346 regulate immune responses and include the major histocompatibility complex (MHC) class I gene products, interferon-induced genes, and the ER antigen peptide transporter 1 (TAP1). Although most of the repressed mRNAs appear to be indirect targets because they lack specific seeding sites for miR-346, we demonstrate that the human TAP1 mRNA is a direct target of miR-346. The human TAP1 mRNA 3'-UTR contains a 6-mer canonical seeding site for miR-346. Importantly, the ER stress-associated reduction in human TAP1 mRNA and protein levels could be reversed with an miR-346 antagomir. Because TAP function is necessary for proper MHC class I-associated antigen presentation, our results provide a novel mechanistic explanation for reduced MHC class I-associated antigen presentation that was observed during ER stress.
Full-text · Article · Dec 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Aberrant activation of the hedgehog (Hh) signaling pathway is implicated widely in both pediatric and adult malignancies. Inactivation of the Hh regulator PTCH is responsible for the Gorlin cancer predisposition syndrome. The spectrum of tumors found in Gorlin Syndrome includes basal cell carcinoma, medulloblastoma, and rarely, rhabdomyosarcoma (RMS). A previous report utilizing in situ hybridization has provided initial evidence for the expression of Hh targets GLI1 and PTCH in RMS tumors.
To investigate the role of Hh pathway signaling in pediatric RMS and undifferentiated sarcoma (US) tumors, the expression of Hh pathway targets GLI1 and PTCH was measured. RNA was extracted from archival human tumor specimens collected from pediatric patients enrolled on Intergroup Rhabdomyosarcoma Study III and IV, and subjected to quantitative reverse transcriptase-polymerase chain reaction.
Expression of GLI1 with or without PTCH was detected in substantial subsets of embryonal RMS (ERMS) and US tumors but only rarely in alveolar RMS tumors. Neither PTCH mutations nor activating SMO mutations were detected in ERMS tumors with high GLI1 expression. Microarray analysis demonstrated relative overexpression of downstream Hh targets in ERMS tumors with high or intermediate GLI1 expression. Unlike a recent report, Hh pathway activity in ERMS tumors did not correlate with a unique clinical phenotype.
Our findings support a role for Hh pathway activation in the genesis of a subset of ERMS and US tumors. Hh signaling may represent a novel therapeutic target in affected tumors.
Full-text · Article · Dec 2011 · Pediatric Blood & Cancer