Reiner Class

Propagation and senescence of human marrow stromal cells in culture: a simple colony‐forming assay identifies samples with the greatest potential to propagate and differentiate

Marrow stromal cells (MSCs) were isolated from bone marrow obtained by aspirates of the iliac crest of normal volunteers. The cells were isolated by their adherence to plastic and then passed in culture. Some of the samples expanded through over 15 cell doublings from the time frozen stocks were prepared. Others ceased replicating after about four cell doublings. The replicative potential of the cells in culture was best predicted by a simple colony‐forming assay in which samples from early passages were plated at low densities of about 10 cells per cm2. Samples with high colony‐forming efficiency exhibited the greatest replicative potential. The colonies obtained by plating early passage cells at low density varied in size and morphology. The large colonies readily differentiated into osteoblasts and adipocytes when incubated in the appropriate medium. As samples were expanded in culture and approached senescence, they retained their ability to differentiate into osteoblasts. However, the cells failed to differentiate into adipocytes. The loss of multipotentiality following serial passage in culture may have important implications for the use of expanded MSCs for cell and gene therapy.

Radioiodinated (I-125) monoclonal antibody 425 in the treatment of high grade glioma patients: ten-year synopsis of a novel treatment.

The present report is the follow-up of patients enrolled in a phase II clinical trial using 125I-MAb 425 as an adjuvant treatment for high grade gliomas. Patient median survivals support published data from an earlier preliminary report. From January 29, 1987 to January 25, 1997, 180 patients diagnosed with astrocytoma with anaplastic foci (AAF) and glioblastoma multiforme (GBM) were treated as outpatients with an average of three weekly intravenous or intraarterial injections of radiolabeled MAb 425. The mean dose was 140 mCi (5.2 GBq). Only one patient who received a single dose of more than 60 mCi (2.2 GBq) experienced acute toxicity. Patients received prior surgery and radiation therapy, with and without chemotherapy. Overall median survival for patients with GBM and AAF was 13.4 and 50.9 months, respectively, with Karnofsky Performance Status (KPS) ranging from 40 to 100 and age ranging from 11 to 75 years. Prognostic factors (KPS and age) correlated positively with increased survival, with KPS the most important determinant of median survival. Data analysis was performed on patients followed 5 years or longer. We conclude that the administration of 125I-MAb 425 with intensive medical management demonstrates a significant increase in median survival and should be considered a therapeutic regimen for the management of patients with high grade gliomas.

Rat marrow stromal cells rapidly transduced with a self-inactivating retrovirus synthesize L-DOPA in vitro.

Autologous bone marrow stromal cells engineered to produce 3,4,-dihydroxyphenylalanine (L-DOPA) can potentially be used as donor cells for neural transplantation in Parkinsons disease. Here, we examined the possibility of using several different promoters and either a self-inactivating retrovirus (pSIR) or standard retroviruses to introduce into marrow stromal cells (MSCs), the two genes necessary for the cells to synthesize L-DOPA. pSIR vectors were constructed using the mouse phosphoglycerate kinase-1 (PGK) promoter or the cytomegalovirus (CMV) promoter to drive expression of either a GFP reporter gene or a bicistronic sequence containing the genes for human tyrosine hydroxylase type I (TH) and rat GTP cyclohydrolase I (GC) separated by an internal ribosome entry site (IRES). rMSCs were successfully transduced with both standard retroviral vectors and pSIR containing the PGK promoter. Transduced rMSCs expressed GFP (90.4–94.4% of cells) or were able to synthesize and secrete L-DOPA (89.0–283 pmols/106 cells/h). After transduced rMSCs were plated at low density (3–6 cells/cm2), the cells expanded over 1000-fold in 3–4 weeks, and the rMSCs continued to either express GFP or produce L-DOPA. Furthermore, two high-expressing clones were isolated and expanded at low-density from rMSCs transduced with pSIR driven by the PGK promoter (97.0% GFP+ or 1096.0 pmols L-DOPA/106 cells/h).

Histone H1 suppresses tumor growth of leukemia cells in vitro, ex vivo and in an animal model suggesting extracellular functions of histones.

Purified histone H1 exerts growth inhibition of leukemia cells independent of lineage, stage, and maturation. At 200 micrograms/ml, H1 proved cytotoxic in 19 of 21 of the tested leukemia-derived cell lines and for 11 of 16 of the fresh tumor samples from leukemia patients. In all cases, normal peripheral blood mononuclear cells and bone marrow cells remained unaffected. Multicellular spheroids from the Burkitts lymphoma cell line IM-9 were growth arrested at 500 micrograms H1/ml. The clonogenic growth of the Burkitts lymphoma cell line Daudi was arrested at 160 micrograms H1/ml. Synthetic H1-peptides as well as peptides and proteins with biochemical properties similar to H1 had no inhibitory growth effect at equimolar concentrations. Furthermore, 250 micrograms H1 injected into a Burkitts lymphoma (Daudi), xenotransplanted into nude mice, arrested tumor growth. As shown by electron microscopy and flow cytometry, incubation of leukemia cells with H1 resulted in severe plasma membrane damage and ultimately cytolysis. This report characterizes a 33-kd protein that binds H1 and is responsible for the cell death via destruction of the cell membrane integrity. New extranuclear functions of histones are presented.

Radiation enhancement by gemcitabine-mediated cell cycle modulations.

The purpose of this study was to investigate the exact dose dependency and time dependency of the radiation-enhancing effect of gemcitabine (2′,2′difluoro desoxycytidine [dFdC]) in in vitro experiments (HeLa cells: cancer of the uterine cervix, #4197 cells: oropharyngeal squamous cell carcinoma), and to correlate this effect with the underlying changes in cell cycle distribution. Cell viability was determined fluorometrically after exposure to dFdC (0–20.0 &mgr;mol/l), irradiation (0–37.5 Gy), and both modalities. Combining both therapies, cells were exposed to dFdC (0–10.0 &mgr;mol/l) for 24 hours before further treatment and irradiated (0–30 Gy) immediately afterwards with or without removal of dFdC. For cell cycle analysis by flow cytometry, cells were irradiated (0–40 Gy) or treated with dFdC (0.012–1.0 &mgr;mol/l, 24–48 hours). Additionally, cells were exposed to dFdC (2.0 &mgr;mol/l, 0–4 hours). Cell cycle kinetics were evaluated using bromodeoxyuridine (BrdU) (10 &mgr;mol/l) S-phase labeling, given either 30 minutes before or in the last hour of dFdC treatment (2.0 &mgr;mol/l, 0–6 hours). The fluorometric assay revealed that dFdC enhances radiation-induced cytotoxicity at marginally toxic or nontoxic concentrations (<37 nmol/l). Radiation resulted in the anticipated G2/M arrest already at 2 Gy. DFdC induced concentration and exposure time-dependent cell cycle changes that were better resolved using BrdU, demonstrating a pronounced S-phase arrest already at 12 nmol/l. BrdU-pulse labeling revealed that the cell cycle block occurred at the G1/S boundary. Our data reconfirm the already known radiation enhancement, the S-phase specific activities of dFdC, and the relevance of the synchronized progression of cells through the S-phase with regard to the radiosensitizing properties of low-dose dFdC. However, we could demonstrate that before progressing in the S-phase, cells were blocked and partially synchronized at the more radiosensitive G1/S boundary. Furthermore, cells progressing past the block might accumulate proapoptotic signals caused by both radiation and dFdC, which will also results in cell death.

ICOS-B7 Homologous Protein Interactions Are Necessary for Mercury-Induced Autoimmunity

After exposure to subtoxic doses of heavy metals such as mercury, H-2s mice develop an autoimmune syndrome consisting of the rapid production of IgG autoantibodies that are highly specific for nucleolar autoantigens and a polyclonal increase in serum IgG1 and IgE. In this study, we explore the role of one of the members of the CD28-B7 costimulation families, ICOS-B7 homologous protein (B7h), in the regulation of mercury-induced autoimmunity. The expression of ICOS on T cells was more enhanced in susceptible A.SW mice than in non-responsive C57BL/6 and DBA/2 mice after HgCl2 treatment. Furthermore, in A.SW mice treated with HgCl2, administration of a blocking anti-ICOS Ab effectively inhibited anti-nucleolar autoantibodies and total serum IgE production. Taken together, these results indicate that the ICOS-B7h costimulation pathway is required for this autoimmune syndrome and suggest that targeting this pathway might have therapeutic benefits for human autoimmune diseases.

Differential activation and inhibition of human platelet thrombin receptors by structurally distinct α-, β- and γ-thrombin

The development of drugs to neutralize the action of thrombin has to date focused on the α form of the protease. It is generally agreed that inactive prothrombin is proteolytically converted to active α-thrombin which may be further hydrolyzed to β- and γ-thrombin. While all three forms of the enzyme retain catalytic activities, only α-thrombin is presumed to be physiologically important. The β- and γ-thrombin are presumed to be degradation products of no physiological significance. Our demonstration that β- and γ-thrombin selectively activate PAR-4 in this and a previous report (J. Biol. Chem. 276, 21173–21183, 2001) necessitates a reevaluation of how we view their physiological roles and how we approach the pharmacological regulation of their actions. β-Thrombin, like γ-thrombin, at nM levels selectively activates PAR-4. This was demonstrated by full retention of aggregatory activity with platelets whose PAR-1 and GP Ib receptors were inactivated. Furthermore, the β-thrombin response was abrogated by desensitizing platelets with suboptimal levels of the thrombin receptor activating peptide for PAR-4 (TRAP-4). For β-thrombin and γ-thrombin to have a physiological role, it is necessary to show they can be generated under physiological conditions. We demonstrate, for the first time, that α-thrombin is hydrolyzed in less than 1 min by activated factor X at physiological pH, in vitro. This implies that α-thrombin may be rapidly converted to β-thrombin and/or γ-thrombin in vivo in the proper microenvironment. The differential activation of the three platelet thrombin receptors by α-, β- and γ-thrombin implies selective structural variations between these thrombin species. Structural differences are likely to account for the marked differential responses observed with the antithrombotic, hirudin, which inhibits α-thrombin, is a slightly weaker inhibitor of β- thrombin and a very weak inhibitor of γ-thrombin-induced platelet aggregations. The converse order of inhibition is observed with the physiological protease inhibitor, α1-antitrypsin. Finally, a non-traditional inhibitor, histone-1, selectively inhibits only β- and γ-thrombin, primarily at the receptor level of PAR-4 rather than on the thrombin molecule. Trypsin, like β- and γ-thrombin, activates PAR-4 and is also inactive with TRAP-4 desensitized platelets. Therefore, it was reasoned that trypsin would be more structurally similar to γ-thrombin than to α-thrombin. The analysis of the crystalline structures of α-, γ-thrombin and trypsin from the databases confirm that this is the case. These findings should help to elucidate structure-function relationships of the different thrombins and may aid in the development of new anti-thrombotic drugs.

The Use of Epidermal Growth Factor Receptor-425 Monoclonal Antibodies Radiolabeled with Iodine-125 in the Adjuvant Treatment of Patients with High Grade Gliomas of the Brain

Astrocytomas with anaplastic foci and glioblastoma multiforme have a poor prognostic outlook despite multiple efforts of aggressive combined integrated multi-modality treatment. In 1994, the American Cancer Society estimates that 17 500 new cases of brain and central nervous system tumors will be diagnosed with a projected death rate of 12600 (Boring et al. 1994). Of these new cases, approximately 7000 or 40% will be malignant astrocytomas and the majority of these will be high grade gliomas of the brain- 4200 to 5560 (Mahaley et al. 1989). Standard treatment is generally surgical resection followed by conventionally fractionated external beam radiotherapy to approximately 60–65 Gy in 6–7 weeks of treatment. However, survival for these patients remains poor, with median survival of 42 weeks in large cooperative trial studies with conventional treatment (Walker et al. 1978). The addition of chemotherapy to the management of high grade gliomas has been of little benefit in altering this bleak outlook for management.

In vitro evaluation of iodine-125-labeled monoclonal antibody (MAb 425) in human high-grade glioma cells.

Human high-grade glioma cell lines (A1207, U-87MG, U-373MG, and F39) with high levels of epidermal growth factor receptor (EGF-R) expression were incubated for 2-48 h with 1 microCi/ml of the EGF-R-specific 125I-MAb 425 and measured for surface-bound, cytoplasmic, and nuclear radioactivity. The A1207 and U-373MG cell lines showed the highest surface-bound radioactivity with 215.9 +/- 8.7 nCi (30 h) and 287.8 +/- 23.2 nCi (24 h)/10(6) cells, respectively, whereas the U-87MG and the F39 cell lines bound significantly less antibody (48.8 +/- 5.4 nCi [48 h] and 31.1 +/- 0.7 nCi [24 h]). Surface-bound antibody was efficiently internalized into the cytoplasm. The U-373MG, U-87MG, and A1207 cell lines achieved 19.8% +/- 2.1 internalization of the surface-bound antibody in contrast to > 40% for the F39 cell line. Only the A1207 cell line showed significant nuclear radioactivity. There was no correlation between the reported EGF-R number and amount of antibody bound or internalized. We conclude that binding and uptake of the 125I-MAb 425 is specific for human glioma cells and shows saturation kinetics independent of receptor density.

Cytokine mediated proliferation of cultured sea turtle blood cells: morphologic and functional comparison to human blood cells.

Blood cells from three different sea turtle species were cultured for approximately 3 weeks in nutrient medium supplemented with recombinant human cytokines known to induce terminal maturation of human hematological stem cells. Cultured turtle erythrocytes were translucent, approximately 10x larger than human erythrocytes, contained a single fluorescent inclusion body, contained nuclear epsilon (embryonic) globin proteins, and, absent of organelles while fresh cells contained few, but well defined mitochondria. Cells with basophilic cytoplasm and in all stages of proliferation were observed in cytokine-supplemented cultures and appeared to possess active protein synthesis. Cultured thrombocytes aggregated in response to agonists for at least 8 days, post-collection, contained P-selectin in the nucleus of 6 day cultured cells which appeared to be released after activation with collagen, and after 6 days had no organelles or open canalicular-like system (OCS) while freshly isolated cells demonstrated few, if any organelles but had a well developed OCS. The response of turtle cells to apparently homologous but unnatural human cytokines and the sustained biological properties of thrombocytes identify this suspension culture system as a powerful tool to explore the evolution of cell types and molecular components of hematopoiesis and hemostasis.