Childrens Memorial Research Center

Chicago, IL, United States

Childrens Memorial Research Center

Chicago, IL, United States
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Barry K.A.,Feinberg Cardiovascular Research Institute | Schultz K.M.,Feinberg Cardiovascular Research Institute | Payne C.J.,Childrens Memorial Research Center | McGarry T.J.,Feinberg Cardiovascular Research Institute
Developmental Biology | Year: 2012

Spermatogonial stem cells divide throughout life, maintaining their own population and giving rise to differentiated gametes. The unstable regulatory protein Geminin is thought to be one of the factors that determine whether stem cells continue to divide or terminally differentiate. Geminin regulates the extent of DNA replication and is thought to maintain cells in an undifferentiated state by inhibiting various transcription factors and chromatin remodeling proteins. To examine how Geminin might regulate spermatogenesis, we developed two conditional mouse models in which the Geminin gene (Gmnn) is deleted from either spermatogonia or meiotic spermatocytes. Deleting Geminin from spermatogonia causes complete sterility in male mice. Gmnn(-/-) spermatogonia disappear during the initial wave of mitotic proliferation that occurs during the first week of life. Gmnn(-/-) spermatogonia exhibit more double-stranded DNA breaks than control cells, consistent with a defect in DNA replication. They maintain expression of genes associated with the undifferentiated state and do not prematurely express genes characteristic of more differentiated spermatogonia. In contrast, deleting Geminin from spermatocytes does not disrupt meiosis or the differentiation of spermatids into mature sperm. In females, Geminin is not required for meiosis, oocyte differentiation, or fertility after the embryonic period of mitotic proliferation has ceased. We conclude that Geminin is absolutely required for mitotic proliferation of spermatogonia but does not regulate their differentiation. Our results suggest that Geminin maintains replication fidelity during the mitotic phase of spermatogenesis, ensuring the precise duplication of genetic information for transmission to the next generation. © 2012 Elsevier Inc.


Rouster-Stevens K.A.,Wake forest University | Morgan G.A.,Childrens Memorial Research Center | Wang D.,Childrens Memorial Research Center | Pachman L.M.,Northwestern University
Arthritis Care and Research | Year: 2010

Objective. To determine if mycophenolate mofetil (MMF) diminishes skin and muscle disease activity in children with juvenile dermatomyositis (DM), thereby permitting a decrease in corticosteroid dose. Methods. A retrospective data review for 50 children with juvenile DM (mean ± SD age 12.2 ± 5.0 years) who had received MMF for 12 months identified the following characteristics: 38 (76%) were girls, 39 (78%) were white, 10 (20%) were Hispanic, and 1 (2%) was African American. The MMF dose and frequency, type of infection, white blood cell (WBC) count, corticosteroid dose, and the validated disease activity score (DAS) subscores for skin (DAS-S) and muscle (DAS-M) were obtained. Results. Twelve months after the start of MMF, the mean ± SD DAS-S decreased from 5.24 ± 0.29 to 3.72 ± 0.29 (P = 0.001), and the mean ± SD DAS-M decreased from 2.44 ± 0.39 to 1.17 ± 0.28 (P = 0.002). The mean ± SD prednisone dosage decreased from 0.39 ± 0.06 to 0.23 ± 0.02 mg/kg/day (P = 0.0001), with resumption of linear growth (P = 0.008). The WBC/lymphocyte count was unchanged over the 12 months on MMF. The infection rate was assessed in a subset of 26 children with juvenile DM who were observed for 12 months before the start of MMF and then compared with the ensuing 12 months of MMF therapy. There was no significant difference between the pretreatment period and the first 6 months of MMF therapy (P = 0.44), but the infection rate decreased in months 7-12 (P = 0.001). Conclusion. MMF appears to be worthy of consideration as an additional therapeutic modality for treatment of children with juvenile DM. These data suggest that the use of MMF decreases skin and muscle disease activity and is steroid sparing. MMF appears to be well tolerated, but patients should be monitored for infection. © 2010, American College of Rheumatology.


Shrestha S.,Childrens Memorial Research Center | Wershil B.,Northwestern University | Sarwark J.F.,Northwestern University | Niewold T.B.,University of Chicago | And 2 more authors.
Arthritis and Rheumatism | Year: 2010

Objective. To investigate the distribution of mast cells and dendritic cell (DC) subsets in paired muscle and skin (lesional/nonlesional) from untreated children with juvenile dermatomyositis (DM). Methods. Muscle and skin biopsy samples (4 skin biopsy samples with active rash) from 7 patients with probable/definite juvenile DM were compared with muscle and skin samples from 10 healthy pediatric controls. Mast cell distribution and number were assessed by toluidine blue staining and analyzed by Student's t-test. Immunohistochemical analysis was performed to identify mature DCs, myeloid DCs (MDCs), and plasmacytoid DCs (PDCs) by using antibodies against DC-LAMP, blood dendritic cell antigen 1 (BDCA-1), and BDCA-2, respectively. Myxovirus resistance protein A (MxA) staining indicated active type I interferon (IFN) signaling; positive staining was scored semiquantitatively and analyzed using the Mann-Whitney U test. Results. Both inflamed and nonlesional skin from patients with juvenile DM contained more mast cells than did skin from pediatric controls (P = 0.029), and comparable numbers of mast cells were present in lesional and nonlesional skin. Interestingly, mast cell numbers were greater in skin than in paired muscle tissue from patients with juvenile DM (P = 0.014) and were not increased in muscle from patients with juvenile DM compared with control muscle. Both muscle and skin from patients with juvenile DM showed more mature PDCs and MxA staining than did their corresponding control tissues (P < 0.05). In both muscle and skin from patients with juvenile DM and in pediatric control muscle, there were fewer MDCs than PDCs, and the distributions of MDCs and PDCs were similar in pediatric control skin samples. Conclusion. The identification of mast cells in skin (irrespective of rash) from patients with juvenile DM, but not in paired muscle tissue, suggests that they have a specific role in juvenile DM skin pathophysiology. In skin from patients with juvenile DM, increased numbers of PDCs and increased expression of type I IFN-induced protein suggest a selective influence on T cell differentiation and subsequent effector function. © 2010, American College of Rheumatology.


Leir S.-H.,Childrens Memorial Research Center | Leir S.-H.,Northwestern University | Harris A.,Childrens Memorial Research Center | Harris A.,Northwestern University
Experimental Cell Research | Year: 2011

The MUC6 mucin has a critical protective function in the normal stomach, pancreas and duodenum and is aberrantly expressed during the progression of some gastrointestinal cancers. Our aim was to determine whether MUC6 contributes to the etiology or progression of pancreatic cancer and elucidate the molecular basis of its involvement. Expression of MUC6 glycoprotein was examined in pancreatic cancer tissues by immunofluorescence and loss of MUC6 was observed. Next, to determine whether MUC6 inhibits tumor growth and metastasis by altering cell adhesion and invasion, recombinant MUC6 cDNA and separate MUC6 N-terminal and C-terminal domains were transfected into pancreatic, colorectal and breast cancer cell lines. The recombinant N- and C-terminal proteins were each seen to oligomerize under non-reducing conditions. Overexpression of both domains of the MUC6 glycoprotein significantly inhibited cell adhesion to matrix proteins (collagen I, collagen IV, fibronectin and laminin) in LS 180 but not in PANC-1 cells. Moreover, the N- and C-terminal domains of MUC6 inhibited invasion of both LS 180 and PANC-1 cells by 40% and 70%, respectively, in comparison with controls. These results suggest that MUC6 may inhibit invasion of tumor cells through the basement membrane of the pancreatic duct and slow the development of infiltrating carcinoma. © 2011 Elsevier Inc.


Kofman A.E.,Childrens Memorial Research Center | McGraw M.R.,Childrens Memorial Research Center | Payne C.J.,Childrens Memorial Research Center | Payne C.J.,Northwestern University
Aging | Year: 2012

Balancing quiescence with proliferation is of paramount importance for adult stem cells in order to avoid hyperproliferation and cell depletion. In some models, stem cell exhaustion may be reversed with the drug rapamycin, which was shown can suppress cellular senescence in vitro and extend lifespan in animals. We hypothesized that rapamycin increases the expression of oxidative stress response genes in adult stem cells, and that these gene activities diminish with age. To test our hypothesis, we exposed mice to rapamycin and then examined the transcriptome of their spermatogonial stem cells (SSCs). Gene expression microarray analysis revealed that numerous oxidative stress response genes were upregulated upon rapamycin treatment, including superoxide dismutase 1, glutathione reductase, and deltaaminolevulinate dehydratase. When we examined the expression of these genes in 55-week-old wild type SSCs, their levels were significantly reduced compared to 3-week-old SSCs, suggesting that their downregulation is coincident with the aging process in adult stem cells. We conclude that rapamycin-induced stimulation of oxidative stress response genes may promote cellular longevity in SSCs, while a decline in gene expression in aged stem cells could reflect the SSCs' diminished potential to alleviate oxidative stress, a hallmark of aging. © Kofman et al.


Kerschner J.L.,Childrens Memorial Research Center | Kerschner J.L.,Northwestern University | Harris A.,Childrens Memorial Research Center | Harris A.,Northwestern University
Biochemical Journal | Year: 2012

A critical cis-regulatory element for the CFTR (cystic fibrosis transmembrane conductance regulator) gene is located in intron 11, 100 kb distal to the promoter, with which it interacts. This sequence contains an intestine-selective enhancer and associates with enhancer signature proteins, such as p300, in addition to tissue-specific TFs (transcription factors). In the present study we identify critical TFs that are recruited to this element and demonstrate their importance in regulating CFTR expression. In vitro DNase I footprinting and EMSAs (electrophoretic mobility-shift assays) identified four cell-type-selective regions that bound TFs in vitro. ChIP (chromatin immunoprecipitation) identified FOXA1/A2 (forkhead box A1/A2), HNF1 (hepatocyte nuclear factor 1) and CDX2 (caudal-type homeobox 2) as in vivo trans-interacting factors. Mutation of their binding sites in the intron 11 core compromised its enhancer activity when measured by reporter gene assay. Moreover, siRNA (small interfering RNA)-mediated knockdown of CDX2 caused a significant reduction in endogenous CFTR transcription in intestinal cells, suggesting that this factor is critical for the maintenance of high levels of CFTR expression in these cells. The ChIP data also demonstrate that these TFs interact with multiple cis-regulatory elements across the CFTR locus, implicating a more global role in intestinal expression of the gene. © The Authors Journal compilation © 2012 Biochemical Society.


Kluppel M.,Northwestern University | Kluppel M.,Childrens Memorial Research Center
Molecular and Cellular Biochemistry | Year: 2011

Proteoglycans carrying chondroitin sulfate side chains have been shown to fulfill important biological functions in development, disease, and signaling. One area of considerable interest is the functional importance of chondroitin sulfates as inhibitors of the regeneration of axonal projections in the mammalian central nervous system. In animal models of spinal cord injury, injections of the enzyme Chondroitinase ABC from the bacterium Proteus vulgaris into the lesion site leads to degradation of chondroitin sulfates, and promotes axonal regeneration and significant functional recovery. Here, a mammalian expression system of an epitope-tagged Chondroitinase ABC protein is described. It is demonstrated that the addition of a eukaryotic secretion signal sequence to the N-terminus of the bacterial Chondroitinase ABC sequence allowed secretion, but interfered with function of the secreted enzyme. In contrast, expression of the Chondroitinase ABC gene without N-terminal eukaryotic secretion sequence or bacterial hydrophobic leader sequence led to efficient secretion of a biologically active Chondroitinase ABC protein from both immortalized and primary cells. Moreover, the C-terminal epitope tag could be utilized to follow expression of this protein. This novel Chondroitinase ABC gene is a valuable tool for a better understanding of the in vivo roles of chondroitin sulfates in mammalian development and disease, as well as in gene therapy approaches, including the treatment of spinal chord injuries. © 2011 Springer Science+Business Media, LLC.


Ostrowski R.A.,Loyola University Chicago | Sullivan C.L.,Childrens Memorial Research Center | Morgan G.A.,Childrens Memorial Research Center | Pachman L.M.,Northwestern University
Arthritis and Rheumatism | Year: 2010

Objective. To determine the association of normal numbers of end row loops (ERLs) in nailfold capillaries at the time of diagnosis of juvenile dermatomyositis (DM) with clinical findings in untreated children with the disease and to identify predictors of the development of decreased numbers of ERLs. Methods. Clinical and laboratory data from 80 untreated children with juvenile DM were collected. ERL numbers were recorded at the time of diagnosis and at 24 months and 36 months thereafter. The 12 children who had normal ERLs at diagnosis were compared with the remaining 68 children. Outcomes included the duration of untreated disease, the duration of treatment with immunosuppressive medications, family medical history, Disease Activity Score (DAS) for juvenile DM, creatinine phosphokinase level, aldolase level, absolute number of CD3-CD56+/16+ natural killer cells, and von Willebrand factor antigen level. Crosssectional and longitudinal analyses were performed. Results. At diagnosis, children with normal ERLs had a shorter duration of untreated disease (P = 0.03) and a lower skin DAS (P = 0.045). Over time, an increased likelihood of having decreased numbers of ERLs was associated with a longer duration of untreated disease and with a higher skin DAS. Conclusion. The presence of a normal number of ERLs in juvenile DM appears to be associated with a shorter duration of symptoms and may be a useful indicator of disease chronicity in the newly diagnosed child. Normal ERLs is also associated with a lower skin DAS. The lack of association between normal ERLs and other variables indicates that normal findings on nailfold capillaroscopy should not be used as justification to delay immunosuppressive therapy in children with typical symptoms of juvenile DM. © 2010, American College of Rheumatology.


Heier C.R.,Childrens Memorial Research Center | Heier C.R.,Northwestern University | Satta R.,Childrens Memorial Research Center | Satta R.,Northwestern University | And 3 more authors.
Human Molecular Genetics | Year: 2010

Proximal spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. Traditionally, SMA has been described as a motor neuron disease; however, there is a growing body of evidence that arrhythmia and/or cardiomyopathy may present in SMA patients at an increased frequency. Here, we ask whether SMA model mice possess such phenotypes. We find SMA mice suffer from severe bradyarrhythmia characterized by progressive heart block and impaired ventricular depolarization. Echocardiography further confirms functional cardiac deficits in SMA mice. Additional investigations show evidence of both sympathetic innervation defects and dilated cardiomyopathy at late stages of disease. Based upon these data, we propose a model in which decreased sympathetic innervation causes autonomic imbalance. Such imbalance would be characterized by a relative increase in the level of vagal tone controlling heart rate, which is consistent with bradyarrhythmia and progressive heart block. Finally, treatment with the histone deacetylase inhibitor trichostatin A, a drug known to benefit phenotypes of SMA model mice, produces prolonged maturation of the SMA heartbeat and an increase in cardiac size. Treated mice maintain measures of motor function throughout extended survival though they ultimately reach death endpoints in association with a progression of bradyarrhythmia. These data represent the novel identification of cardiac arrhythmia as an early and progressive feature of murine SMA while providing several new, quantitative indices of mouse health. Together with clinical cases that report similar symptoms, this reveals a new area of investigation that will be important to address as we move SMA therapeutics towards clinical success. © The Author 2010. Published by Oxford University Press. All rights reserved.


Hendrickson P.G.,Childrens Memorial Research Center | Silliker M.E.,DePaul University
Current Genetics | Year: 2010

Similarity searches with Didymium iridis mitochondrial genomic DNA identified six possible ribosomal protein-coding regions, however, each region contained stop codons that would need to be removed by RNA editing to produce functional transcripts. RT-PCR was used to amplify these regions from total RNA for cloning and sequencing. Six functional transcripts were verified for the following ribosomal protein genes: rpS12, rpS7, rpL2, rpS19, rpS3, and rpL16. The editing events observed, such as single C and U nucleotide insertions and a dinucleotide insertion, were consistent with previously observed editing patterns seen in D. iridis. Additionally, a new form of insertional editing, a single A insertion, was observed in a conserved region of the rpL16 gene. While the majority of codons created by editing specify hydrophobic amino acids, a greater proportion of the codons created in these hydrophilic ribosomal proteins called for positively charged amino acids in comparison to the previously characterized hydrophobic respiratory protein genes. This first report of edited soluble mitochondrial ribosomal proteins in myxomycetes expands upon the RNA editing patterns previously seen; there was: a greater proportion of created codons specifying positively charged amino acids, a shift in the codon position edited, and the insertion of single A nucleotides. © 2010 Springer-Verlag.

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