Department of Microbiology and Immunology

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Introduction of human erythropoietin receptor complementary DNA by retrovirus-mediated gene transfer into murine embryonic stem cells enhances erythropoiesis in developing embryoid bodies
(Elsevier, 2000-07-01) Dai, Mu-Shui; Ge, Yue; Xia, Zhen-Biao; Broxmeyer, Hal E.; Lu, Li; Microbiology and Immunology, School of Medicine
To evaluate the role of the erythropoietin (Epo) receptor (R) in erythropoiesis in more primitive stem cells, we assessed the influence of retrovirus-mediated gene transfer of human (h) EpoR complementary DNA (cDNA) into murine embryonic stem (ES) cells on erythroid differentiation of these cells. The hEpoR cDNA was efficiently transduced into ES cells, forming hEpoR that stably expressed ES (ES-hEpoR) cells. Expression of hEpoR cDNA was confirmed in ES-hEpoR cells by reverse transcriptase-polymerase chain reaction and Northern blot analysis. Colony assays demonstrated that definitive erythroid and primitive erythroid colonies were significantly increased from ES-hEpoR cells, when compared with mock virus-transduced ES (ES-Neo) cells, during the time course of differentiation induced by withdrawal of leukemia inhibitory factor, in either the presence or the absence of Epo. Multipotential colony-forming units (CFU-Mix) were also increased in ES-hEpoR cells at different stages of differentiation, but no changes were detected for CFU-granulocyte-macrophage colonies (CFU-GM). Time course studies by Northern blot analysis demonstrated elevated levels of expression of beta-H1 and beta-Major globin genes in embryoid bodies derived from ES-hEpoR cells stimulated with Epo, when compared with similar expression from ES-Neo cells. Expression of the GATA-1 gene was enhanced in ES-hEpoR cells, when compared with ES-Neo cells, beginning immediately after initiation of the cultures until 8 days of differentiation. These data indicate that primitive and definitive erythropoiesis in differentiating embryoid bodies can be enhanced by retrovirus-mediated gene transfer of an hEpoR gene. Biol Blood Marrow Transplant 2000;6(4):395-407.
Counterpoint: Cord blood stem cell therapy for acquired immune deficiency syndrome
(Mary Ann Liebert, 2001-01) Alkhatib, Ghalib; Microbiology and Immunology, School of Medicine
Comment on Cord blood stem cell therapy for acquired immune deficiency syndrome. [Stem Cells Dev. 2009]
Adeno-associated Virus 2-Mediated Transduction and Erythroid Lineage-Restricted Long-Term Expression of the Human β-Globin Gene in Hematopoietic Cells from Homozygous β-Thalassemic Mice
(Elsevier, 2001-06) Tan, Mengqun; Qing, Keyun; Zhou, Shangzhen; Yoder, Mervin C.; Srivastava, Arun; Microbiology and Immunology, School of Medicine
Adeno-associated virus 2 (AAV), a nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. Here, we report successful AAV-mediated stable transduction and high-efficiency, long-term, erythroid lineage-restricted expression of a human β-globin gene in primary murine hematopoietic stem cells in vivo. Bone marrow-derived primitive Sca-1+, lin− hematopoietic stem cells from homozygous β-thalassemic mice were transduced ex vivo with a recombinant AAV vector containing a normal human β-globin gene followed by transplantation into low-dose-irradiated B6.c-kitW41/41 anemic recipient mice. Six months posttransplantation, tail-vein blood samples were analyzed by PCR amplification to document the presence of the transduced human β-globin gene sequences in the peripheral blood cells. Semiquantitative PCR analyses revealed that the transduced human β-globin gene sequences were present at ∼1 copy per cell. The efficiency of the human β-globin gene expression was determined to be up to 35% compared with the murine endogenous β-globin gene by semiquantitative RT-PCR analyses. Peripheral blood samples from several positive recipient mice obtained 10 months posttransplantation were fractionated to obtain enriched populations of granulocytes, lymphocytes, and erythroid cells. PCR analyses revealed the presence of the human β-globin gene sequences in granulocytes and lymphocytes, indicating multilineage reconstitution. However, only the erythroid population was positive following RT-PCR analyses, suggesting lineage-restricted expression of the transduced human β-globin gene. Southern blot analyses of total genomic DNA samples isolated from bone marrow cells from transplanted mice also documented proviral integration. These results provide further support for the potential use of recombinant AAV vectors in gene therapy of β-thalassemia and sickle-cell disease.
Infection of Purified Nuclei by Adeno-associated Virus 2
(Elsevier, 2001-10) Hansen, Jonathan; Qing, Keyun; Srivastava, Arun; Microbiology and Immunology, School of Medicine
Adeno-associated virus 2 (AAV), a nonpathogenic human parvovirus, requires co-infection with a helper virus for its optimal replication. Although AAV possesses a broad host range, certain cell types lack the machinery necessary for efficient entry into the cell and intracellular trafficking of AAV into the nucleus, where the viral second-strand DNA synthesis must occur before gene expression. We have demonstrated that in less-permissive mouse fibroblasts, the virus fails to transport to the nucleus due to altered endocytic processing. However, relatively little is known about the intracellular site of viral uncoating and transport of the virion across the nuclear envelope. Here, we provide evidence that AAV can efficiently enter intact nuclei purified from both permissive and less-permissive cell types. Furthermore, entry into the nucleus is time- and temperature-dependent, but is not saturable and seems to occur independently of the nuclear pore complex. We also demonstrate that purified nuclei contain all of the machinery necessary for uncoating and viral second-strand DNA synthesis even in the absence of a helper virus. These studies provide new insights into the basic biology of AAV and may also have implications for the optimal use of AAV vectors in human gene therapy.
Current Status of Cord Blood Banking and Transplantation in the United States and Europe
(Elsevier, 2001-12-01) Ballen, Karen; Broxmeyer, Hal E.; McCullough, Jeffrey; Piaciabello, Wanda; Rebulla, Paolo; Verfaillie, Catherine M.; Wagner, John E.; Microbiology and Immunology, School of Medicine
Cord blood (CB) transplantation has expanded the ability of the transplantation community to meet the growing needs of their patients. Clinical data over the last decade show promising results in CB transplantation using blood from related as well as unrelated donors. Basic science continues to look for ways to expand the quality and quantity of CB. CB banks are now established around the world, with major efforts to standardize banking to facilitate regulation, collection, processing, and distribution as a way of providing the highest-quality CB for patient use. This review article discusses the current status of CB transplantation and banking in the United States and Europe.
Recognition of bacterial peptidoglycan by the innate immune system
(Springer, 2003) Dziarski, Roman; Microbiology and Immunology, School of Medicine
The innate immune system recognizes microorganisms through a series of pattern recognition receptors that are highly conserved in evolution. Peptidoglycan (PGN) is a unique and essential component of the cell wall of virtually all bacteria and is not present in eukaryotes, and thus is an excellent target for the innate immune system. Indeed, higher eukaryotes, including mammals, have several PGN recognition molecules, including CD14, Toll-like receptor 2, a family of peptidoglycan recognition proteins, Nod1 and Nod2, and PGN-lytic enzymes (lysozyme and amidases). These molecules induce host responses to microorganisms or have direct antimicrobial effects.
Certification assays for HIV-1-based vectors: frequent passage of gag sequences without evidence of replication-competent viruses
(Elsevier, 2003-11-01) Sastry, Lakshmi; Xu, Yi; Johnson, Terry; Desai, Kunal; Rissing, David; Marsh, Jonathan; Cornetta, Kenneth; Microbiology and Immunology, School of Medicine
A principal concern regarding the safety of HIV-1-based vectors is replication-competent lentivirus (RCL). We have developed two PCR assays for detecting RCL; the first detects recombination between gag regions in the transfer vector and the packaging construct (sensitivity of detection ∼10–100 copies of target sequence). The second assay uses real-time PCR to detect vesicular stomatitis virus glycoprotein (VSVG) envelope DNA (sensitivity ∼5–50 VSVG sequences). In an attempt to amplify any RCL, test vectors were used to transduce C8166 and 293 cells, which were then screened weekly for 3 weeks. Psi–gag recombinants were routinely detected (20 of 21 analyses) in four transductions using the RRL-CMV-GFP vector. In contrast, VSVG sequences were detected only once in 21 analyses. Interestingly, p24 levels (as measured by ELISA) were occasionally detectable after 3 weeks of culture. To determine if a true RCL was present, 21-day cell-free medium was used to transduce naïve cells. No evidence of psi–gag or VSVG transfer was detected, indicating that the recombination events were insufficient to reconstitute a true RCL. These findings have important implications for the design and safety of HIV-1-based vectors intended for clinical applications.
High levels of soluble herpes virus entry mediator in sera of patients with allergic and autoimmune diseases
(Springer Nature, 2003-12-01) Jun, Hyo Won; La, Su Jin; Kim, Ji Young; Heo, Suk Kyeung; Kim, Ju Yang; Wang, Sa; Kim, Kack Kyun; Lee, Ki Man; Cho, Hong Rae; Lee, Hyeon Woo; Kwon, Byungsuk; Kim, Byung Sam; Kwon, Byoung Se; Microbiology and Immunology, School of Medicine
Herpes virus entry mediator (HVEM) is a newly discovered member of the tumor necrosis factor receptor (TNFR) superfamily that has a role in herpes simplex virus entry, in T cell activation and in tumor immunity. We generated mAb against HVEM and detected soluble HVEM (SHVEM) in the sera of patients with various autoimmune diseases. HVEM was constitutively expressed on CD4+ and CD8+ T cells, CD19+ B cells, CD14+ monocytes, neutrophils and dendritic cells. In three-way MLR, mAb 122 and 139 were agonists and mAb 108 had blocking activity. An ELISA was developed to detect sHVEM in patient sera. sHVEM levels were elevated in sera of patients with allergic asthma, atopic dermatitis and rheumatoid arthritis. The mAbs discussed here may be useful for studies of the role of HVEM in immune responses. Detection of soluble HVEM might have diagnostic and prognostic value in certain immunological disorders.
Growth inhibitory effect of Hcc-1/CIP29 is associated with induction of apoptosis, not just with G2/M arrest
(Springer, 2005) Fukuda, S.; Pelus, L. M.; Microbiology and Immunology, School of Medicine
hSef potentiates EGF-mediated MAPK signaling through affecting EGFR trafficking and degradation
(Elsevier B.V., 2008-03) Ren, Yongming; Cheng, Long; Rong, Zhili; Li, Zhiyong; Li, Yinghua; Zhang, Xinjun; Xiong, Shiqin; Hu, Jim; Fu, Xin-Yuan; Chang, Zhijie; Department of Microbiology & Immunology, IU School of Medicine
Sef (similar expression to fgf genes) was identified as an effective antagonist of fibroblast growth factor (FGF) in vertebrates. Previous reports have demonstrated that Sef interacts with FGF receptors (FGFRs) and inhibits FGF signaling, however, its role in regulating epidermal growth factor receptor (EGFR) signaling remains unclear. In this report, we found that hSef localizes to the plasma membrane (PM) and is subjected to rapid internalization and well localizes in early/recycling endosomes while poorly in late endosomes/lysosomes. We observed that hSef interacts and functionally colocalizes with EGFR in early endosomes in response to EGF stimulation. Importantly, we demonstrated that overexpression of hSef attenuates EGFR degradation and potentiates EGF-mediated mitogen-activated protein kinase (MAPK) signaling by interfering EGFR trafficking. Finally, our data showed that, with overexpression of hSef, elevated levels of Erk phosphorylation and differentiation of rat pheochromocytoma (PC12) cells occur in response to EGF stimulation. Taken together, these data suggest that hSef plays a positive role in the EGFR-mediated MAPK signaling pathway. This report, for the first time, reveals opposite roles for Sef in EGF and FGF signalings.

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