Research interests

Development of Humanized SCID Mice and Studies of Single Gene Models for Human Hematological Diseases
Advances in our understanding of the development and regulation of the immune system in normal and pathologic states rely heavily on studies of mice with genetically determined immunodeficiency and autoimmunity. Research in our laboratory encompasses (1) basic studies focused on elucidating the mechanisms underlying immunodeficiency, autoimmunity, and hematological diseases; and (2) applied studies focused on the development of effective immunodeficient mouse models that support heightened engraftment with human hematopoietic stem cells. These "humanized SCID mice" will facilitate studies of human immunity, regenerative medicine, and neoplasia. Examples of the studies carried out over the last year are summarized below:


Development of Humanized Mice for Translational Biomedical Research
Complex biological processes often require in vivo analysis, and important research advances have been obtained using mice as a model system for the study of many biological systems. However, mice are not humans, and the study of human biology in vivo is severely limited by ethical and technical constraints. There is a growing need for animal models to carry out in vivo studies on human cells, tissues, and organs without putting individuals at risk. Humanized mice, or mouse–human chimeras, have been developed to overcome these limitations and have become an important research tool for the in vivo study of human cells and tissues. We have developed a NOD-Prkdc^scid (NOD/SCID) mouse strain harboring a null mutation of the common cytokine receptor γ chain (Il2rgtm1Wjl) (NOD/SCID/IL2rγ^null) (full name NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ). In collaboration with Dale Greiner and his colleagues at The University of Massachusetts Medical School along with Fumihiko Ishikawa and his colleagues at The Riken Research Center for Allergy and Immunology, we have shown that these mice can efficiently support development of all myelo-erythroid components and functional lymphoid cells following human hematopoietic stem cell (HSC) engraftment. Development of mice that are ‘humanized’ by engraftment of human tissues, HSCs, and peripheral-blood mononuclear cells (PBMCs) provides an opportunity to study human biological processes in vivo that would otherwise not be possible. The new generations of humanized mice are proving to be powerful tools in preclinical testing and in the investigation of many human biological processes. The culmination of decades of research on humanized mice is leading to advances in our understanding of human hematopoiesis, innate and adaptive immunity, autoimmunity, infectious diseases, cancer biology, and regenerative medicine. The potential for new advances in our understanding of human biological systems provided by studies in humanized mice remains promising. The development of humanized mice based on immunodeficient IL2rγ^null hosts has overcome many of the limitations and constraints of previously available models.


The Mouse Mutation "Thrombocytopenia and Cardiomyopathy" (trac) is within the Abcg5 Gene: A Single Gene Model for Human Mediterranean Macrothrombocytopenia
Homozygosity for a new spontaneous mouse mutation named "thrombocytopenia and cardiomyopathy" (trac) results in thrombocytopenia, dilated cardiomyopathy, and infertility. A/J-trac/trac mice show a precipitous drop in platelet numbers and increases in platelet volume by 4 weeks of age. By 2-3 months of age, trac/trac mice have a 20-fold decrease in platelet number, a 3-fold increase in platelet volume, and a greatly increased bleeding time. Blood smears showed abnormally large platelets and megakaryocytoid cells. The trac/trac mice also developed mild microcytic anemia accompanied by the presence of stomatocytes, a doubling of reticulocyte numbers, and a two-fold decrease in WBC counts. Increased numbers of megakaryocytes were present in bone marrow, spleen, and lungs. Ultrastructural studies of trac/trac megakaryocytes showed a poorly developed demarcation system and a failure to form platelet territories. The trac/trac platelets were enlarged, spherical, and contained numerous small alpha granules. The thrombocytopenia was not associated with defects intrinsic to bone marrow progenitor cells. Although thrombopoietin (TPO) levels were decreased, TPO treatment failed to reverse the thrombocytopenia. To identify the responsible gene, we produced a fine-structure genetic map of a 5-megabase interval containing the trac locus on mouse chromosome 17. The human syntenic region is Chr 2p21-p22. Analyses of 1100 F2 progeny from intercross matings of (A/J x C57BL/6J) F1 +/trac mice narrowed the interval to 0.3 Mb containing 4 genes. Sequencing of these genes revealed a G-to-A mutation at base 1435 (refseq nm031884) of Abcg5 (ATP-binding cassette sub-family G, member 5). This G>A base change results in a tryptophan codon (UGG) at amino acid position 463(uniprot) being changed to a premature stop codon (UAG). The transmembrane helices prediction program, TMHMM, predicts that the premature stop codon would truncate the last four of the six transmembrane domains of the ABCG5 protein. No DNA alterations were found in any of the other candidate genes. Genetic crosses of +/trac mice with mice doubly transgenic for the closely linked human ABCG5 and ABCG8 genes (stock B6SJL-Tg(ABCG5/ABCG8)14-2Hobb/J) showed that the transgenes normalized platelet counts and volumes in trac/trac mice. ABCG5 (sterolin-1) functions as part of a heterodimer, with ABCG8, that regulates plant sterol uptake. The trac/trac mutant mice have greatly elevated plasma levels of plant sterols. When placed on a phytosterol-free diet, the thrombocytopenia was reversed. Recent studies have shown that Mediterranean Macrothrombocytopenia is caused by mutations in ABCG5 or ABCG8. Identification of the molecular basis of the mouse Abcg5trac mutation provides a new model for studying the role of phytosterols in pathogenic changes in the hematopoietic, cardiovascular, and reproductive systems.

Publications

• Yamamoto T, Kaizu C, Kawasaki T, Hasegawa G, Umezu H, Ohashi R, Sakurada J, Jiang S, Shultz L, Naito M. 2008. Macrophage colony-stimulating factor is indispensable for repopulation and differentiation of Kupffer cells but not for splenic red pulp macrophages in osteopetrotic (op/op) mice after macrophage depletion. Cell Tissue Res 332:245-256.
• Ishikawa F, Saito Y, Yoshida S, Harada M, Shultz LD. 2008. The differentiative and regenerative properties of human hematopoietic stem/progenitor cells in NOD-SCID/IL2rgamma(null) mice. Curr Top Microbiol Immunol 324: 87-94.
• King M, Pearson T, Shultz LD, Leif J, Bottino R, Trucco M, Atkinson MA, Wasserfall C, Herold KC, Woodland RT, Schmidt MR, Woda BA, Thompson MJ, Rossini AA, Greiner DL. 2008. A new Hu-PBL model for the study of human islet alloreactivity based on NOD-scid mice bearing a targeted mutation in the IL-2 receptor gamma chain gene. Clin Immunol 126: 303-314.
• Kong Y, Yoshida S, Saito Y, Doi T, Nagatoshi Y, Fukata M, Saito N, Yang SM, Iwamoto C, Okamura J, Liu KY, Huang XJ, Lu DP, Shultz LD, Harada M, Ishikawa F. 2008. CD34+CD38+CD19+ as well as CD34+CD38-CD19+ cells are leukemia-initiating cells with self-renewal capacity in human B-precursor ALL. Leukemia 22:1207-1213.
• Pearson T, Greiner DL, Shultz LD. 2008. Creation of "humanized" mice to study human immunity. Curr Protoc Immunol Chapter 15: Unit 15 21.
• Pearson T, Greiner DL, Shultz LD. 2008. Humanized SCID mouse models for biomedical research. Curr Top Microbiol Immunol 324: 25-51.
• Simpson-Abelson MR, Sonnenberg GF, Takita H, Yokota SJ, Conway TF, Jr., Kelleher RJ, Jr., Shultz LD, Barcos M, Bankert RB. 2008. Long-term engraftment and expansion of tumor-derived memory T cells following the implantation of non-disrupted pieces of human lung tumor into NOD-scid IL2R?null Mice. J Immunol 180: 7009-7018.
• Gaines P, Tien CW, Olins AL, Olins DE, Shultz LD, Carney L, Berliner N. 2008. Mouse neutrophils lacking lamin B-receptor expression exhibit aberrant development and lack critical functional responses. Exp Hematol 36: 965-976.
• le Viseur C, Hotfilder M, Bomken S, Wilson K, Rottgers S, Schrauder A, Rosemann A, Irving J, Stam RW, Shultz LD, Harbott J, Jurgens H, Schrappe M, Pieters R, Vormoor J. 2008. In childhood acute lymphoblastic leukemia, blasts at different stages of immunophenotypic maturation have stem cell properties. Cancer Cell 14: 47-58.
• Pearson T, Shultz LD, Lief J, Burzenski L, Gott B, Chase T, Foreman O, Rossini AA, Bottino R, Trucco M, Greiner DL. 2008. A new immunodeficient hyperglycaemic mouse model based on the Ins2 Akita mutation for analyses of human islet and beta stem and progenitor cell function. Diabetologia 51:1449-1456.
• Giassi LJ, Pearson T, Shultz LD, Laning J, Biber K, Kraus M, Woda BA, Schmidt MR, Woodland RT, Rossini AA, Greiner DL. 2008. Expanded CD34+ Human umbilical cord blood cells generate multiple lymphohematopoietic lineages in NOD-scid IL2r?null Mice. Exp Biol Med (Maywood) 233:997-1012.
• Pearson T, Shultz LD, Miller D, King M, Laning J, Fodor W, Cuthbert A, Burzenski L, Gott B, Lyons B, Foreman O, Rossini AA, Greiner DL. 2008. Non-obese diabetic-recombination activating gene-1 (NOD-Rag 1null) interleukin (IL)-2 receptor common gamma chain (IL 2 r?null)) null mice: a radioresistant model for human lymphohaematopoietic engraftment. Clin Exp Immunol.
• Agliano A, Martin-Padura I, Mancuso P, Marighetti P, Rabascio C, Pruneri G, Shultz LD, Bertolini F. 2008. Human acute leukemia cells injected in NOD/LtSz-scid/IL-2R? null mice generate a faster and more efficient disease compared to other NOD/scid-related strains. Int J Cancer 123:2222-2227.
• Kumar P, Ban HS, Kim SS, Wu H, Pearson T, Greiner DL, Laouar A, Yao J, Haridas V, Habiro K, Yang YG, Jeong JH, Lee KY, Kim YH, Kim SW, Peipp M, Fey GH, Manjunath N, Shultz LD, Lee SK, Shankar P. 2008. T Cell-Specific siRNA Delivery Suppresses HIV-1 Infection in Humanized Mice. Cell 134:577-586.
• Croker BA, Lawson BR, Berger M, Eidenschenk C, Blasius AL, Moresco EM, Sovath S, Cengia L, Shultz LD, Theofilopoulos AN, Pettersson S, Beutler BA. 2008. Inflammation and autoimmunity caused by a SHP1 mutation depend on IL-1, MyD88, and a microbial trigger. Proc Natl Acad Sci U S A 105: 15028-15033.
• Schmidt MR, Appel MC, Giassi LJ, Greiner DL, Shultz LD, Woodland RT. 2008. Human BLyS Facilitates Engraftment of Human PBL Derived B Cells in Immunodeficient Mice. PLoS ONE 3: e3192.
• Dash Y, Ramesh M, Greiner D, Shultz LD, Klei TR, Rajan TV. 2007. Determinants of memory in experimental filarial infections in mice. Parasite Immunol 29: 567-574.
• Ishikawa F, Niiro H, Iino T, Yoshida S, Saito N, Onohara S, Miyamoto T, Minagawa H, Fujii S, Shultz LD, Harada M, Akashi K. 2007. The developmental program of human dendritic cells is operated independently of conventional myeloid and lymphoid pathways. Blood 110(10):3591-3660.
• Ishikawa F, Yoshida S, Saito Y, Hijikata A, Kitamura H, Tanaka S, Nakamura R, Tanaka T, Tomiyama H, Saito N, Fukata M, Miyamoto T, Lyons B, Ohshima K, Uchida N, Taniguchi S, Ohara O, Akashi K, Harada M, Shultz LD. 2007. Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region. Nat Biotechnol 25: 1315-1321.
• King M, Pearson T, Shultz LD, Leif J, Bottino R, Trucco M, Atkinson M, Wasserfall C, Herold K, Mordes JP, Rossini AA, Greiner DL. 2007. Development of new-generation HU-PBMC-NOD/SCID mice to study human islet alloreactivity. Ann N Y Acad Sci 1103: 90-93.
• Meyerrose TE, De Ugarte DA, Hofling AA, Herrbrich PE, Cordonnier TD, Shultz LD, Eagon JC, Wirthlin L, Sands MS, Hedrick MA, Nolta JA. 2007. In vivo distribution of human adipose-derived mesenchymal stem cells in novel xenotransplantation models. Stem Cells 25: 220-227.
• Ramesh M, Paciorkowski N, Dash Y, Shultz L, Rajan TV. 2007. Acute but not chronic macrophage recruitment in filarial infections in mice is dependent on C-C chemokine ligand 2. Parasite Immunol 29: 395-404.
• Seymour RE, Hasham MG, Cox GA, Shultz LD, Hogenesch H, Roopenian DC, Sundberg JP. 2007. Spontaneous mutations in the mouse Sharpin gene result in multiorgan inflammation, immune system dysregulation and dermatitis. Genes Immun 8: 416-421.
• Shultz LD, Ishikawa F, Greiner DL. 2007. Humanized mice in translational biomedical research. Nat Rev Immunol 7: 118-130.
• Shultz LD, Pearson T, King M, Giassi L, Carney L, Gott B, Lyons B, Rossini AA, Greiner DL. 2007. Humanized NOD/LtSz-scid IL2 receptor common gamma chain knockout mice in diabetes research. Ann N Y Acad Sci 1103: 77-89.
• Thornley TB, Phillips NE, Beaudette-Zlatanova BC, Markees TG, Bahl K, Brehm MA, Shultz LD, Kurt-Jones EA, Mordes JP, Welsh RM, Rossini AA, Greiner DL. 2007. Type 1 IFN mediates cross-talk between innate and adaptive immunity that abrogates transplantation tolerance. J Immunol 179: 6620-6629.
• Yamazaki M, Pearson T, Brehm MA, Miller DM, Mangada JA, Markees TG, Shultz LD, Mordes JP, Rossini AA, Greiner DL. 2007. Different mechanisms control peripheral and central tolerance in hematopoietic chimeric mice. Am J Transplant 7: 1710-1721.