J. Graham Sharp

Does the number or quality of pluripotent bone marrow stem cells decrease with age

In the next 25 years, as the 37 million baby boomers age, we can expect a 400% increase in total joint arthroplasties that will challenge surgeons, the healthcare system, and the scientific community. The burden may be eased if we are able to manipulate side population stem cells and enhance peri-prosthetic bone remodeling thereby reducing the incidence of revisions. Therefore, as a preliminary question, we asked if the number and quality of side population stem cells, with the ability to proliferate into multiple cell lineages for long periods, correlates with age and can be evaluated in peripheral blood. Using flow cytometry we analyzed the quantity and quality of side population stem cells from bone marrow and peripheral blood in 54 patients (20 under 60 years of age, 34 over) undergoing THA. The total side population of stem cells decreased with age, but their long-term repopulating ability (quality) remained constant. The total count of side population stem cells in marrow correlated with the number found in peripheral blood. If these populations can be manipulated, periprosthetic remodeling may be beneficially enhanced.

A murine model of bone marrow micrometastasis in breast cancer

Bone marrow (BM) is one of the most common sites and often the first clinical indication of metastatic progression of breast cancer. Multivariate analyses have shown that the presence of cytokeratin positive tumor cells in the marrow of women with newly diagnosed stage I, II or III breast cancer is an independent predictor of survival. The objective of this study was to develop an orthotopic model of spontaneous BM metastasis to facilitate studies of this process. A murine mammary adenocarcinoma cell line, Clone 66, was transduced with the neomycin resistance gene (Cl66neo) and injected orthotopically into female Balb/c mice. Polymerase chain reaction (PCR) for the neo gene performed on BM cells harvested from tumor bearing mice demonstrated as few as 102 injected tumor cells produced BM micrometastases at 4 weeks post-injection. Small foci of tumor cells were identified in the mammary fatpad (mfp) without gross evidence of primary tumors. Higher doses of tumor cells produced BM micrometastases, detectable by PCR, at one week post-injection. Constructs containing green fluorescent protein (GFP) and the neomycin resistance gene (neo) were also transduced into Clone 66 cells (Cl66-GFPneo) and injected into the mfp. GFP transduced tumor cells were identified in multiple tissues in addition to BM by flow cytometric analysis (FACS) but less 13% of the animals developed gross metastases. This model is a clinically relevant tool for the analysis of organ specificity of metastasis.

VLA-4 Mediated Adhesion to Bone Marrow Stromal Cells Confers Chemoresistance to Adherent Lymphoma Cells

The mechanisms of maintenance of residual lymphoma in bone marrow during chemotherapy are currently not well understood. Previous studies have shown that primary lymphoma cells obtained from histologically negative bone marrow of non-Hodgkins lymphoma (NHL) patients grew in long-term bone marrow cultures primarily in association with bone marrow stromal cells. Furthermore, the interaction of NHL patient cells with bone marrow stromal cells inhibited their spontaneous apoptosis. The current studies were designed to characterize the components of the heterotypic interaction between lymphoma cells and bone marrow stromal cells as well as to probe the consequences of this interaction as it pertains to the potential survival of minimal numbers of lymphoma cells during chemotherapy. Cellular adhesion assays performed in the presence of either neutralizing antibodies to VCAM-or the α and β subunit of VLA-4 resulted in >95%, 82% and 35% inhibition of lymphoma cell line adhesion to the bone marrow stromal line MS-5, respectively. Modulation of VLA-4 affinity by the 8A2 antibody resulted in enhanced secondary adhesion at 24 and 72 hours to either cellular fibronectin (65% and 65%) or MS-5 cells (60% and 55%), superceding levels obtained using untreated lymphoma cells (< 20%). The bone marrow stromal cells induced a chemoprotective effect for adherent lymphoma cells over a 3-log dose range of vincristine, resulting in a 2-log increase in the ED50 at day 6 of culture. The failure of glutaraldehyde fixed stromal cells to induce a chemoprotective effect demonstrated that viable bone marrow stromal cells were necessary. Similarly, lymphoma/stromal cell conditioned medium also failed to provide a survival advantage. These data demonstrated that viable bone marrow stromal cells possessed the ability to actively inhibit the apoptotic pathways of intimately adherent lymphoma cells and this potentially contributes to their survival during chemotherapy.

The effect of neonatal thymectomy on the induction of autoimmune orchitis in rats.

The objective of these experiments was to determine the effects of neonatal thymectomy on the induction of experimental autoimmune orchitis in inbred rats of the Fischer 344 and Lewis strains. It was found that thymectomy alone in Lewis rats, and thymectomy followed by immunization with testicular extract in both Lewis and Fischer 344 rats, led to the development of autoimmune orchitis, as indicated by decreased testes weights, increased serum spermagglutinating antibody titers and histopathological changes in the testes. These data indicate that rats of the Lewis strain are genetically predisposed to the development of autoimmune orchitis, and thymectomy alone leads to active manifestations of the disease, which are further enhanced by subsequent immunization with testicular extract. In Fischer 344 rats, thymectomy followed by immunization leads to indications of early signs of experimental autoimmune orchitis. This is in contrast to experimentally induced autoimmune diseases in other model systems, where previous investigators have reported that thymectomy lessens or prevents induction of autoimmune disease. It is suggested that these differences may be related to the timing of thymectomy with regard to differences in the time of appearance of sperm antigens (at puberty) as compared to pre-natal and early neonatal appearances of other autoantigens.

Circulating factors may be responsible for murine strain-specific responses to mobilizing cytokines

OBJECTIVE To determine if circulating factors influence strain-specific responses to administration of hematopoietic stem-cell mobilizing cytokines, a murine model was employed. METHODS Plasma aliquots from intact DBA2, Balb/c, and C57Bl/6 mice were injected into intact Balb/c mice prior to delivery of mobilizing cytokines. Control Balb/c mice were injected with mobilizing cytokines alone. Plasma from hemi-body irradiated Balb/c mice, known to inhibit mobilization, was also injected into Balb/c mice. Twenty-four hours later, spleen cells were harvested and assayed for granulocyte-macrophage colony-forming cells (GM-CFC) and high-proliferative-potential colony-forming cells (HPP-CFC). Simultaneously harvested blood aliquots were assayed for CD45(+)/CD34(+) cells using flow cytometric techniques. RESULTS Mice receiving plasma from any source demonstrated significant inhibition of mobilization of HPP-CFC and GM-CFC to the spleen as compared to mobilized controls; for HPP-CFC, plasma from C57Bl/6 mice was more inhibitory than plasma from Balb/c (p = 0.001) or from DBA2 mice (p = 0.01), while for GM-CFC, plasma from C57Bl/6 mice was more inhibitory than Balb/c plasma but not more inhibitory than DBA2 plasma. Mice injected with plasma from previously irradiated Balb/c mice exhibited the expected HPP-CFC and GM-CFC mobilization inhibition, which was not statistically different from the inhibition seen in animals that received C57Bl/6 plasma. Mobilization of CD34(+)/CD45(+) cells to the blood also appeared to be inhibited by pretreatment with C57Bl/6 plasma, but not DBA2 plasma. CONCLUSION These data suggest that strain-specific patterns of mobilization may be influenced by a circulating mobilization inhibitor(s).

Novel therapy for therapy‐resistant mantle cell lymphoma: Multipronged approach with targeting of hedgehog signaling

Mantle cell lymphoma (MCL) is one of the most aggressive B‐cell lymphomas with a median patient survival of only 5–7 years. The failure of existing therapies is mainly due to disease relapse when therapy‐resistant tumor cells remain after chemotherapy. Therefore, development and testing of novel therapeutic strategies to target these therapy‐resistant MCL are needed. Here, we developed an in vivo model of therapy‐resistant MCL by transplanting a patient‐derived MCL cell line (Granta 519) into NOD/SCID mice followed by treatment with combination chemotherapy. Cytomorphologic, immunophenotypic, in vitro and in vivo growth analyses of these therapy‐resistant MCL cells confirm their MCL origin and resistance to chemotherapy. Moreover, quantitative real‐time PCR revealed the upregulation of GLI transcription factors, which are mediators of the hedgehog signaling pathway, in these therapy‐resistant MCL cells. Therefore, we developed an effective therapeutic strategy for resistant MCL by treating the NOD/SCID mice bearing Granta 519 MCL with CHOP chemotherapy to reduce tumor burden combined with GLI‐antisense oligonucleotides or bortezomib, a proteosome inhibitor, to target therapy‐resistant MCL cells that remained after chemotherapy. This regimen was followed by treatment with MCL‐specific cytotoxic T lymphocytes to eliminate all detectable leftover minimal residual disease. Mice treated with this strategy showed a significantly increased survival and decreased tumor burden compared to the mice in all other groups. Such therapeutic strategies that combine chemotherapy with targeted therapy followed by tumor‐specific immunotherapy are effective and have excellent potential for clinical application to provide long‐term, disease‐free survival in MCL patients.

The Leukemic Myeloid Cell Line OMA-AML-1: an In Vitro Model of Hematopoietic Cell Differentiation

The OMA-AML-1, acute myelogenous leukemia cell line is unique in that it spontaneously maintains both a CD34+ precursor cell compartment and a CD15+ differentiating cell compartment in vitro. A third transitional cell type with co-expression of CD34 and CD15 can also be identified in in vitro cultures. The cell line shows dynamic fluctuations in the relative sizes of these three cell compartments in suspension culture. In contrast, OMA-AML-1 fails to show phenotypic or morphologic evidence of differentiation when grown subcutaneously in immunodeficient mice. OMA-AML-1 responds to a number of hematopoietic cytokines. Delineation of cytokine responses on FACS isolated populations of CD34+ versus CD15+ cells demonstrated that proliferative responses occurred primarily at the level of the precursor cell (CD34+) while the production of endstage eosinophils occurred within the CD15+ compartment. OMA-AML-1 mimics a number of features of normal hematopoiesis and is proving to be a useful in vitro model for the study of hematopoietic differentiation.

Characterization of a human herpes virus-6 (HHV-6) and epstein-barr virus (EBV) associated leukemic cell line, J6-1

This report characterizes the J6-1 cell line derived from a Chinese acute myelomonocytic leukemia patient and previoulsy reported to be associated with EBV. These studies showed that J6-1 cells were also infected with HHV-6 as demonstrate at the DNA level by PCR and Southern blot hybridization and by expression of HHV-6 early membrane antigen on the J6-1 cell surface. Further characterization showed J6-1 was co-infected with EBV type 2. Generally, cells infected with EBV type 2 do not grow wellin vitro, However, J6-1, although difficult to maintainin vitro, has been growth for 15 years. Possibly, co-infection with HHV-6 confers this property. In this regard, J6-1 cells exhibited density dependent growth which could be inhibited with an anti-HHV-6-MA monoclonal antibody (MAb). In contrast, anti-HHV-6-VCA MAb stimulate the J6-1 cell proliferation. Electron microscopic analysis showed that, morphologically, there were two types of J6-1 cell, one with lymphoblastoid features and one with a monocytoid appearance. Accordingly, the flow profile of the J6-1 cell line showed heterogeneity with two populations comprised of CD15-, CD19+cells with low light scatter (small cells) and a population with greater light scatter (larger cells) which was CD15+, CD19+, The population was negative for progenitor cell markers (CD33, 34), and T cell markers. Southern analysis showed no T cell receptor rearrangement, however there was a clonal JH and kappa light chain expressing population. Glycocytochemical analysis showed several endogenous lectin receptors on the J6-1 cell surface: BSA-Xylose, BSA-Rhamnose, BSA-Gal, BSA-Lac. This cell line shares many characteristics with other monocytic/lymphoblastoid cell lines isolated elsewhere and provides circumstantial evidence linking Herpes viruses, as least as co-factors, to leukemia cell growth.

Thymic non-lymphoid cells

SummaryIn formulating this summary of our simon-pure knowledge of the structure/function relationships in the thymus, we decided that the time may have come to introduce a suitable dose of cynicism to balance the sometimes hopeless optimism of the past.Are the non-lymphoid cells of the thymus necessary for thymic function? Probably, but not to the extent or uniqueness that some authors including ourselves have previously claimed; T cells can probably differentiate in other tissues but may acquire their preference for MHC class II in the thymus.Mouse thymic lymphoid cell traffic and surface phenotype has recently been summarized pictorally byScollay and Shortman [95]. Briefly stated, within the thymus, cells are hatched, matched and then dispatched. Minimally, the non-lymphoid cells act either as scenically varied obstacles along the way, nurseries for newborn T cells, or as tombstones for life’s disenfranchized, effete and autoaggressive thymocytes. Hassall’s corpuscles are morphological structures unique to the thymus, which are most useful to medical students for identification of this tissue. Their function remains one of life’s great mysteries. Morphologically, they are suitable companions to the more recently described strange multicellular complexes of lymphocytes and epithelial cells which might be functionally important. The thymus of the much studied inbred, environmentally mollycoddled, laboratory mouse has been often and majestically described. It is probably typical for that of man and most mammals. It may, however, be unrepresentative of the thymus of stressed and parasitized wild animals. Diseases of the thymus generally can be categorized as not having enough thymus, having a neoplastic thymus or having a thymus which does not work properly. The bottom line in our knowledge of thymic non-lymphoid cells is that if you are born without them, you get sick and die; unless, of course, you are a nude mouse in Omaha, in which case you just freeze to death.

Effect of 6-hydroxydopamine on murine hematopoietic stem cells: Enhanced cytotoxicity on megakaryocyte colony forming units

We examined the effect of catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) on murine committed megakaryocyte progenitor cells, the megakaryocyte-colony forming unit (CFU-Meg). More mature cells of the megakaryocyte series have the capacity for active uptake of catecholamines, and we speculated that the CFU-Meg would also take up 6-OHDA and be selectively killed. CFU-Meg were much more sensitive to the effects of this agent than were granulocyte-macrophage colony forming units (CFU-GM) or spleen-colony forming units. Co-incubation with catalase, which would destroy hydrogen peroxide generated extracellularly by the autoxidation of 6-OHDA, ablated 6-OHDA toxicity towards CFU-GM, but also significantly reduced the effect on CFU-Meg. Mazindol, a selective dopamine uptake inhibitor did not alter 6-OHDA effect on either CFU-Meg or CFU-GM. Finally, CFU-Meg were no more sensitive to incubation with hydrogen peroxide than were CFU-GM. These data suggest that CFU-Meg, unlike their more mature progeny, do not actively concentrate 6-OHDA, and the excess toxicity of this agent towards CFU-Meg is probably due to increased sensitivity to autoxidation products of 6-OHDA, other than hydrogen peroxide, generated extracellularly.