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Item ANALYSIS OF CO-OCCURRING PHENOTYPES IN INFANTS WITH DOWN SYNDROME WITH CARDIAC DEFECTS(Office of the Vice Chancellor for Research, 2011-04-08) Shepherd, Nicole; Duvall, Nichole; Stone, Sandra B.; Davis, Charlene; Stanley, Maria; Roper, Randall J.Down syndrome (DS), caused by a trisomy 21, is the most common chromosomal aneuploidy occurring in approximately 1 of 750 live births. Individuals with DS exhibit craniofacial dysmorphology, cardiac defects, gastrointestinal problems, and cognitive impairment, although these phenotypes vary in incidence and severity. Common cardiac defects are usually recognized in young infants with DS and include atrial septal anomalies, ventricular septal abnormalities, atrioventricular canal defects, and patent ductus arteriosus. Additional abnormalities may also affect infants with DS, but not be identified until later in life. Since multiple phenotypes are found in these individuals, we hypothesize that children with a severe congenital heart defect may be at increased risk for additional medical issues. To investigate this hypothesis, we performed a retrospective chart review of 170 infants with DS between birth and 6 months of age who were referred to the Down Syndrome Program at Riley Hospital for Children from August 2005 to July 2010. We analyzed comorbidity in infants with upper airway obstruction (UAO) or a feeding problem with and without a severe congenital heart defect. Our data show that 33% of infants without a cardiac defect have identified UAO while 44% with a severe cardiac defect have identified UAO. Additionally, 59% of infants without a cardiac defect compared to 49% with a severe cardiac defect have a feeding problem. With the knowledge of these comorbid clinical features in DS, healthcare providers may be able to identify potential complications affecting infants with DS earlier in life.Item Antiretroviral Therapy Normalizes Autoantibody Profile of HIV Patients by Decreasing CD33⁺CD11b⁺HLA-DR⁺ Cells: A Cross-Sectional Study(Ovid Technologies (Wolters Kluwer) - Lippincott Williams & Wilkins, 2016-04) Meng, Zhefeng; Du, Ling; Hu, Ningjie; Byrd, Daniel; Amet, Tohti; Desai, Mona; Shepherd, Nicole; Lan, Jie; Han, Renzhi; Yu, Qigui; Department of Microbiology & Immunology, IU School of MedicineAutoimmune manifestations are common in human immunodeficiency virus (HIV) patients. However, the autoantibody spectrum associated with HIV infection and the impact of antiretroviral therapy (ART) remains to be determined. The plasma autoantibody spectrum for HIV patients was characterized by protein microarrays containing 83 autoantigens and confirmed by enzyme-linked immunosorbent assay (ELISA). Regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) were analyzed by flow cytometry and their effects on autoantibodies production were determined by B cell ELISpot. Higher levels of autoantibody and higher prevalence of elevated autoantibodies were observed in ART-naive HIV patients compared to healthy subjects and HIV patients on ART. The highest frequency of CD33(+)CD11b(+)HLA-DR(+) cells was observed in ART-naive HIV patients and was associated with the quantity of elevated autoantibodies. In addition, CD33(+)CD11b(+)HLA-DR(+) cells other than Tregs or MDSCs boost the B cell response in a dose-dependent manner by in vitro assay. In summary, HIV infection leads to elevation of autoantibodies while ART suppresses the autoimmune manifestation by decreasing CD33(+)CD11b(+)HLA-DR(+) cells in vivo.The roles of CD33(+)CD11b(+)HLA-DR(+) cells on disease progression in HIV patients needs further assessment.Item Clinical identification of feeding and swallowing disorders in 0-6 month old infants with Down syndrome(Wiley, 2019-02) Stanley, Maria A.; Shepherd, Nicole; Duvall, Nichole; Jenkinson, Sandra B.; Jalou, Hasnaa E.; Givan, Deborah C.; Steele, Gregory H.; Davis, Charlene; Bull, Marilyn J.; Watkins, Donna U.; Roper, Randall J.; Pediatrics, School of MedicineFeeding and swallowing disorders have been described in children with a variety of neurodevelopmental disabilities, including Down syndrome (DS). Abnormal feeding and swallowing can be associated with serious sequelae such as failure to thrive and respiratory complications, including aspiration pneumonia. Incidence of dysphagia in young infants with DS has not previously been reported. To assess the identification and incidence of feeding and swallowing problems in young infants with DS, a retrospective chart review of 174 infants, ages 0-6 months was conducted at a single specialty clinic. Fifty-seven percent (100/174) of infants had clinical concerns for feeding and swallowing disorders that warranted referral for Videofluroscopic Swallow Study (VFSS); 96/174 (55%) had some degree of oral and/or pharyngeal phase dysphagia and 69/174 (39%) had dysphagia severe enough to warrant recommendation for alteration of breast milk/formula consistency or nonoral feeds. Infants with certain comorbidities had significant risk for significant dysphagia, including those with functional airway/respiratory abnormalities (OR = 7.2). Infants with desaturation with feeds were at dramatically increased risk (OR = 15.8). All young infants with DS should be screened clinically for feeding and swallowing concerns. If concerns are identified, consideration should be given to further evaluation with VFSS for identification of dysphagia and additional feeding modifications.Item Glycosylphosphatidylinositol Anchor Deficiency Attenuates the Production of Infectious HIV-1 and Renders Virions Sensitive to Complement Attack(Mary Ann Liebert, 2016-11-01) Amet, Tohti; Lan, Jie; Shepherd, Nicole; Yang, Kai; Byrd, Daniel; Xing, Yanyan; Yu, Qigui; Microbiology and Immunology, School of MedicineHuman immunodeficiency virus type 1 (HIV-1) escapes complement-mediated lysis (CML) by incorporating host regulators of complement activation (RCA) into its envelope. CD59, a key member of RCA, is incorporated into HIV-1 virions at levels that protect against CML. Since CD59 is a glycosylphosphatidylinositol-anchored protein (GPI-AP), we used GPI anchor–deficient Jurkat cells (Jurkat-7) that express intracellular CD59, but not surface CD59, to study the molecular mechanisms underlying CD59 incorporation into HIV-1 virions and the role of host proteins in virus replication. Compared to Jurkat cells, Jurkat-7 cells were less supportive to HIV-1 replication and more sensitive to CML. Jurkat-7 cells exhibited similar capacities of HIV-1 binding and entry to Jurkat cells, but were less supportive to viral RNA and DNA biosynthesis as infected Jurkat-7 cells produced reduced amounts of HIV-1 RNA and DNA. HIV-1 virions produced from Jurkat-7 cells were CD59 negative, suggesting that viral particles acquire CD59, and probably other host proteins, from the cell membrane rather than intracellular compartments. As a result, CD59-negative virions were sensitive to CML. Strikingly, these virions exhibited reduced activity of virus binding and were less infectious, implicating that GPI-APs may be also important in ensuring the integrity of HIV-1 particles. Transient expression of the PIG-A gene restored CD59 expression on the surface of Jurkat-7 cells. After HIV-1 infection, the restored CD59 was colocalized with viral envelope glycoprotein gp120/gp41 within lipid rafts, which is identical to that on infected Jurkat cells. Thus, HIV-1 virions acquire RCA from the cell surface, likely lipid rafts, to escape CML and ensure viral infectivity.Item IDENTIFICATION OF TRANSCRIPTION FACTORS ASSOCIATED WITH DOWN SYNDROME SKELETAL ABNORMALITIES(Office of the Vice Chancellor for Research, 2012-04-13) Shepherd, Nicole; Deitz, Samantha; Roper, Randall J.Individuals with Down syndrome (DS) exhibit a variety of phenotypes, including craniofacial and skeletal dysmorphologies. It is believed that trisomic genes initiate phenotypes associated with Down syndrome, though specific gene-phenotype relationships for DS are largely unknown. We hypothesize that the altered expression of genes in three copies will also affect the expression of downstream genes, including non-trisomic genes and play an important role in DS phenotypes. Transcription factors, which encode proteins that bind to specific DNA sequences controlling the flow of transcription, are among the genes that may be affected by trisomy. We have identified genetic and phenotypic alterations in craniofacial precursors as early as embryonic dayE9.5of the Ts65Dn mouse model of human DS. This mouse model is trisomic for orthologs of approximately half of the genes on human chromosome 21. Previous microarray data from the developing mandible have shown dysregulation of multiple non-trisomic genes. We will test the expression of the Six2, Gata3, Gata6, Pth, Hoxb4, Runx2, Ets2, and Osterix transcription factors at two developmental time points, E9.5 and E13.5, to determine which are dysregulated in the Ts65Dn DS mouse model. Understanding the effect of trisomy on non-trisomic transcription factors will help identify links between trisomy and specific DS phenotypes.Item MMPs/TIMPs imbalances in the peripheral blood and cerebrospinal fluid are associated with the pathogenesis of HIV-1-associated neurocognitive disorders: A pilot study(Elsevier, 2017) Xing, Yanyan; Shepherd, Nicole; Lan, Jie; Rane, Sushmita; Gupta, Samir K.; Zhang, Shanxiang; Dong, Jun; Yu, Qigui; Department of Microbiology and Immunology, IU School of MedicineHIV-1-associated neurocognitive disorders (HAND) continue to be a major concern in the infected population, despite the widespread use of combined antiretroviral therapy (cART). Growing evidence suggests that an imbalance between matrix metalloproteinases (MMPs) and endogenous tissue inhibitors of MMPs (TIMPs) contributes to the pathogenesis of HAND. In our present study, we examined protein levels and enzymatic activities of MMPs and TIMPs in both plasma and cerebrospinal fluid (CSF) samples from HIV-1 patients with or without HAND and HIV-1-negative controls. Imbalances between MMPs and TIMPs with distinct patterns were revealed in both the peripheral blood and CSF of HIV-1 patients, especially those with HAND. In the peripheral blood, the protein levels of MMP-2, MMP-9, TIMP-1, TIMP-2, and the enzymatic activities of MMP-2 and MMP-9 were increased in HIV-1 patients with or without HAND when compared with HIV-1-negative controls. The enzymatic activity of MMP-2, but not MMP-9, was further increased in plasma samples of HAND patients than that of HIV-1 patients without HAND. Notably, the ratio of MMP-2/TIMP-2 in plasma was significantly increased in HAND patients, not in patients without HAND. In the CSF, MMP-2 activity was increased, but the ratio of MMP-2/TIMP-2 was not altered. De novo induction and activation of MMP-9 in the CSF of HAND patients was particularly prominent. The imbalances between MMPs and TIMPs in the blood and CSF were related to the altered profiles of inflammatory cytokines/chemokines and monocyte activation in these individuals. In addition, plasma from HIV-1 patients directly induced integrity disruption of an in vitro blood-brain barrier (BBB) model, leading to increased BBB permeability and robust transmigration of monocytes/macrophages. These results indicate that imbalances between MMPs and TIMPs are involved in BBB disruption and are implicated in the pathogenesis of neurological disorders such as HAND in HIV-1 patients.Item Obstructive Sleep Apnea in Young Infants with Down Syndrome Evaluated in a Down Syndrome Specialty Clinic(Wiley, 2015) Goffinski, Alida; Stanley, Maria A.; Shepherd, Nicole; Duvall, Nichole; Jenkinson, Sandra B.; Davis, Charlene; Bull, Marilyn J.; Roper, Randall J.; Biology, School of ScienceChildren with Down syndrome (DS) experience congenital and functional medical issues that predispose them to obstructive sleep apnea (OSA). Research utilizing stringent age criteria among samples of infants with DS and OSA is limited. This study examines clinical correlates of OSA among infants with DS. A retrospective chart review was conducted of infants ≤6 months of age referred to a DS clinic at a tertiary children's hospital over five-years (n = 177). Chi-square tests and binary logistic regression models were utilized to analyze the data. Fifty-nine infants underwent polysomnography, based on clinical concerns. Of these, 95% (56/59) had studies consistent with OSA. Among infants with OSA, 71% were identified as having severe OSA (40/56). The minimum overall prevalence of OSA among the larger group of infants was 31% (56/177). Significant relationships were found between OSA and dysphagia, congenital heart disease (CHD), prematurity, gastroesophageal reflux disease (GERD), and other functional and anatomic gastrointestinal (GI) conditions. Results indicate that odds of OSA in this group are higher among infants with GI conditions in comparison to those without. Co-occurring dysphagia and CHD predicted the occurrence of OSA in 36% of cases with an overall predictive accuracy rate of 71%. Obstructive sleep apnea is relatively common in young infants with DS and often severe. Medical factors including GI conditions, dysphagia and CHD may help to identify infants who are at greater risk and may warrant evaluation. Further studies are needed to assess the impact of OSA in infants with DS.Item Primary Human B Cells at Different Differentiation and Maturation Stages Exhibit Distinct Susceptibilities to Vaccinia Virus Binding and Infection(American Society for Microbiology, 2019-09-12) Shepherd, Nicole; Lan, Jie; Rane, Sushmita; Yu, Qigui; Microbiology and Immunology, School of MedicineVaccinia virus (VACV), the prototypical member of the poxvirus family, was used as a live-virus vaccine to eradicate smallpox worldwide and has recently received considerable attention because of its potential as a prominent vector for the development of vaccines against infectious diseases and as an oncolytic virus for cancer therapy. Studies have demonstrated that VACV exhibits an extremely strong bias for binding to and infection of primary human antigen-presenting cells (APCs), including monocytes, macrophages, and dendritic cells. However, very few studies have assessed the interactions of VACV with primary human B cells, a main type of professional APCs. In this study, we evaluated the susceptibility of primary human peripheral B cells at various differentiation and maturation stages to VACV binding, infection, and replication. We found that plasmablasts were resistant to VACV binding, while other B subsets, including transitional, mature naive, memory, and plasma cells, were highly susceptible to VACV binding. VACV binding preference was likely associated with differential expression of chemokine receptors, particularly CXCR5. Infection studies showed that plasmablast, plasma, transitional, and mature naive B cells were resistant to VACV infection, while memory B cells were preferentially infected. VACV infection in ex vivo B cells was abortive, which occurred at the stage of late viral gene expression. In contrast, activated B cells were permissive to productive VACV infection. Thus, primary human B cells at different differentiation stages exhibit distinct susceptibilities to VACV binding and infection, and the infections are abortive and productive in ex vivo and activated B cells, respectively. IMPORTANCE Our results provide critical information to the field of poxvirus binding and infection tropism. We demonstrate that VACV preferentially infects memory B cells that play an important role in a rapid and vigorous antibody-mediated immune response upon reinfection by a pathogen. Additionally, this work highlights the potential of B cells as natural cellular models to identify VACV receptors or dissect the molecular mechanisms underlying key steps of the VACV life cycle, such as binding, penetration, entry, and replication in primary human cells. The understanding of VACV biology in human primary cells is essential for the development of a safe and effective live-virus vector for oncolytic virus therapy and vaccines against smallpox, other pathogens, and cancer.Item Primary Human Macrophages Serve as Vehicles for Vaccinia Virus Replication and Dissemination(American Society for Microbiology (ASM), 2014-06) Byrd, Daniel; Shepherd, Nicole; Lan, Jie; Hu, Ningjie; Amet, Tohti; Yang, Kai; Desai, Mona; Yu, Qigui; Department of Microbiology & Immunology, IU School of MedicineHuman monocytic and professional antigen-presenting cells have been reported only to exhibit abortive infections with vaccinia virus (VACV). We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, but not human AB serum-derived cells, were permissive to VACV replication. The titers of infectious virions in both cell-free supernatants and cellular lysates of infected M1 and M2 markedly increased in a time-dependent manner. The majority of virions produced in permissive MDMs were extracellular enveloped virions (EEV), a secreted form of VACV associated with long-range virus dissemination, and were mainly found in the culture supernatant. Infected MDMs formed VACV factories, actin tails, virion-associated branching structures, and cell linkages, indicating that MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. VACV replication was sensitive to inhibitors against the Akt and Erk1/2 pathways that can be activated by VACV infection and M-CSF stimulation. Classical activation of MDMs by lipopolysaccharide (LPS) plus gamma interferon (IFN-γ) stimulation caused no effect on VACV replication, while alternative activation of MDMs by interleukin-10 (IL-10) or LPS-plus-IL-1β treatment significantly decreased VACV production. The IL-10-mediated suppression of VACV replication was largely due to Stat3 activation, as a Stat3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data demonstrate that primary human macrophages are permissive to VACV replication. After infection, these cells produce EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread. IMPORTANCE Our results provide critical information to the burgeoning fields of cancer-killing (oncolytic) virus therapy with vaccinia virus (VACV). One type of macrophage (M2) is considered a common presence in tumors and is associated with poor prognosis. Our results demonstrate a preference for VACV replication in M2 macrophages and could assist in designing treatments and engineering poxviruses with special considerations for their effect on M2 macrophage-containing tumors. Additionally, this work highlights the importance of macrophages in the field of vaccine development using poxviruses as vectors. The understanding of the dynamics of poxvirus-infected foci is central in understanding the effectiveness of the immune response to poxvirus-mediated vaccine vectors. Monocytic cells have been found to be an important part of VACV skin lesions in mice in controlling the infection as well as mediating virus transport out of infected foci.Item Provirus activation plus CD59 blockage triggers antibody-dependent complement-mediated lysis of latently HIV-1-infected cells(The American Association of Immunologists, 2014-10-01) Lan, Jie; Yang, Kai; Byrd, Daniel; Hu, Ningjie; Amet, Tohti; Shepherd, Nicole; Desai, Mona; Gao, Jimin; Gupta, Samir; Sun, Yongtao; Yu, Qigui; Department of Microbiology & Immunology, IU School of MedicineLatently HIV-1-infected cells are recognized as the last barrier toward viral eradication and cure. To purge these cells, we combined a provirus stimulant with a blocker of human CD59, a key member of the regulators of complement activation, to trigger Ab-dependent complement-mediated lysis. Provirus stimulants including prostratin and histone deacetylase inhibitors such as romidepsin and suberoylanilide hydroxamic acid activated proviruses in the latently HIV-1-infected T cell line ACH-2 as virion production and viral protein expression on the cell surface were induced. Romidepsin was the most attractive provirus stimulant as it effectively activated proviruses at nanomolar concentrations that can be achieved clinically. Antiretroviral drugs including two protease inhibitors (atazanavir and darunavir) and an RT inhibitor (emtricitabine) did not affect the activity of provirus stimulants in the activation of proviruses. However, saquinavir (a protease inhibitor) markedly suppressed virus production, although it did not affect the percentage of cells expressing viral Env on the cell surface. Provirus-activated ACH-2 cells expressed HIV-1 Env that colocalized with CD59 in lipid rafts on the cell surface, facilitating direct interaction between them. Blockage of CD59 rendered provirus-activated ACH-2 cells and primary human CD4(+) T cells that were latently infected with HIV-1 sensitive to Ab-dependent complement-mediated lysis by anti-HIV-1 polyclonal Abs or plasma from HIV-1-infected patients. Therefore, a combination of provirus stimulants with regulators of complement activation blockers represents a novel approach to eliminate HIV-1.