B7-1 and B7-2 co-stimulatory molecules are required for mercury-induced autoimmunity.

B7-1 (CD80) and B7-2 (CD86) molecules on antigen presenting cells play important roles in providing co-stimulatory signals required for activation and expansion of autoreactive T cells. Moreover, some reports have suggested that these molecules may have distinct functions in the differentiation of Th1 and Th2 cells. Mercury-induced autoimmunity in H-2s mice is characterized by lymphoproliferation of T and B cells, serum increases in IgG1 and IgE and production of antinucleolar antibodies (ANoA). The mechanisms responsible for the various manifestations of this syndrome have yet to be elucidated. To examine the contributions of B7 co-stimulatory molecules to this model, susceptible mice were treated with antibodies to B7-1, B7-2, or both during the development of mercury-induced autoimmunity. The combination of anti-B7-1 and anti-B7-2 antibodies prevented Hg-induced disease in H-2s mice. Additionally, single anti-B7-1 antibody treatment was sufficient to prevent Hg-induced ANoA production, but not IgG1 and IgE hypergammaglobulinaemia. Further, single antibody treatment with anti-B7-2 resulted in a partial reduction of ANoA titres but had no significant effect on total serum IgG1 and IgE levels. Taken together, these results indicate that B7-1 and B7-2 molecules are critical for the development of Hg-induced autoimmunity and suggest that the different manifestations of the syndrome are regulated by independent mechanisms.

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 Parkinson's 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/10(6) cells/h). After transduced rMSCs were plated at low density (3--6 cells/cm(2)), 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/10(6) cells/h).

Unique pathway of thrombin-induced platelet aggregation mediated by glycoprotein Ib.

Thrombin plays a central role in normal and abnormal hemostatic processes. It is assumed that alpha-thrombin activates platelets by hydrolyzing the protease-activated receptor (PAR)-1, thereby exposing a new N-terminal sequence, a tethered ligand, which initiates a cascade of molecular reactions leading to thrombus formation. This process involves cross-linking of adjacent platelets mediated by the interaction of activated glycoprotein (GP) IIb/IIIa with distinct amino acid sequences, LGGAKQAGDV and/or RGD, at each end of dimeric fibrinogen molecules. We demonstrate here the existence of a second alpha-thrombin-induced platelet-activating pathway, dependent on GP Ib, which does not require hydrolysis of a substrate receptor, utilizes polymerizing fibrin instead of fibrinogen, and can be inhibited by the Fab fragment of the monoclonal antibody LJIb-10 bound to the GP Ib thrombin-binding site or by the cobra venom metalloproteinase, mocarhagin, that hydrolyzes the extracellular portion of GP Ib. This alternative alpha-thrombin pathway is observed when PAR-1 or GP IIb/IIIa is inhibited. The recognition sites involved in the cross-linking of polymerizing fibrin and surface integrins via the GP Ib pathway are different from those associated with fibrinogen. This pathway is insensitive to RGDS and anti-GP IIb/IIIa antibodies but reactive with a mutant fibrinogen, gamma407, with a deletion of the gamma-chain sequence, AGDV. The reaction is not due to simple trapping of platelets by the fibrin clot, since ligand binding, signal transduction, and second messenger formation are required. The GP Ib pathway is accompanied by mobilization of internal calcium and the platelet release reaction. This latter aspect is not observed with ristocetin-induced GP Ib-von Willebrand factor agglutination nor with GP Ib-von Willebrand factor-polymerizing fibrin trapping of platelets. Human platelets also respond to gamma-thrombin, an autoproteolytic product of alpha-thrombin, through PAR-4. Co-activation of the GP Ib, PAR-1, and PAR-4 pathways elicit synergistic responses. The presence of the GP Ib pathway may explain why anti-alpha-thrombin/anti-platelet regimens fail to completely abrogate thrombosis/restenosis in the cardiac patient.

Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow.

Cultures of plastic-adherent cells from bone marrow have attracted interest because of their ability to support growth of hematopoietic stem cells, their multipotentiality for differentiation, and their possible use for cell and gene therapy. Here we found that the cells grew most rapidly when they were initially plated at low densities (1.5 or 3.0 cells/cm(2)) to generate single-cell derived colonies. The cultures displayed a lag phase of about 5 days, a log phase of rapid growth of about 5 days, and then a stationary phase. FACS analysis demonstrated that stationary cultures contained a major population of large and moderately granular cells and a minor population of small and agranular cells here referred to as recycling stem cells or RS-1 cells. During the lag phase, the RS-1 cells gave rise to a new population of small and densely granular cells (RS-2 cells). During the late log phase, the RS-2 cells decreased in number and regenerated the pool of RS-1 cells found in stationary cultures. In repeated passages in which the cells were plated at low density, they were amplified about 10(9)-fold in 6 wk. The cells retained their ability to generate single-cell derived colonies and therefore apparently retained their multipotentiality for differentiation.

Influence of vortex speed on fresh versus stored platelet aggregation in the absence and presence of extracellular ATP.

Platelets are subjected to vastly differing shear forces under laminar and nonlaminar flow patterns throughout the tortuous cardiovascular system. Different activation pathways appear to be associated with platelet adhesion and aggregation under high shear rates vs. low shear rates. We found that platelets continue to aggregate at very low stirring rates (100 RPM) and low shear forces although significantly less than at high stirring rates (1000 RPM). These conditions may model vortices encountered in vivo, such as downstream of partially occluded blood vessels. The extent of agonist-induced platelet aggregation, at varying stir rates, remained essentially unchanged between 1200 and 600 RPM. This was true for both freshly prepared and stored platelets even though the extent of aggregation was significantly reduced with stored platelets. Agonists used were thrombin, thrombin receptor activating peptide (TRAP), SFLLRNP, the thromboxane A2 mimetic, U46619, plus epinephrine and ADP+epinephrine. At lower stir rates (100-400 RPM), little or no difference in aggregation levels was observed between fresh and stored platelets, depending upon agonist used. This may indicate that old and young platelets, in vivo, would be equally active at vessel walls exposed to blood flowing through a slow vortex at low shear rates. ATP, released from activated platelets, may act as a potent regulator of platelet aggregation within a vortex where the resident time of platelets and bioactive molecules is greater than in laminar flow regions. High levels of extracellular ATP (100 microM) inhibited agonist-induced aggregation of fresh platelets to a greater extent than stored platelets, except with ADP+epinephrine where the converse was observed. Inhibition, in general, appeared to be inversely related to stir rates. Low levels of extracellular ATP (10 nM, 1 microM) generally stimulated agonist-induced aggregations independent of stir rates and to a greater extent with stored platelets than fresh platelets. Unraveling how hemostasis functions within microenvironments may facilitate ways to further regulate this process.

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.

A central role of Bcl-X(L) in the regulation of keratinocyte survival by autocrine EGFR ligands.

The epidermal growth factor receptor has multiple roles in epidermal biology relating to growth, migration, and, as shown recently, survival of keratinocytes. In cultured keratinocytes activation of the epidermal growth factor receptor upregulates expression of Bcl-x(L), an anti-apoptotic Bcl-2 homolog. The functional contribution of epidermal growth factor receptor-dependent Bcl-x(L) expression to keratinocyte survival is poorly understood. Here we demonstrate that inhibition of the epidermal growth factor receptor tyrosine kinase activity with either an epidermal growth factor receptor antagonistic monoclonal antibody (MoAb 425) or an epidermal growth factor receptor-selective tyrosine kinase inhibitor (AG 1478) downregulated Bcl-x(L) expression in normal human keratinocytes but had no effect on expression of the pro-apoptotic Bcl-2 homologs Bad, Bak, and Bax. Bovine pituitary extract and insulin partially alleviated both, downregulation of Bcl-x(L) expression and cell death upon epidermal growth factor receptor inhibition. Forced expression of Bcl-x(L) attenuated cell death of immortalized keratinocytes (HaCaT) induced by either forced suspension (anoikis) or by epidermal growth factor receptor blockade. These results demonstrate that epidermal growth factor receptor-dependent signaling pathways control the balance of pro-apoptotic and anti-apoptotic Bcl-2 family members expressed in normal keratinocytes. Inappropriate survival supported by aberrant signaling through the epidermal growth factor receptor may contribute to the pathogenesis of psoriasis and of squamous cell carcinomas.

Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta.

Marrow stromal cells from wild-type mice were infused into transgenic mice that had a phenotype of fragile bones resembling osteogenesis imperfecta because they expressed a human minigene for type I collagen. In mice that were irradiated with potentially lethal levels (700 cGy) or sublethal levels (350 cGy), DNA from the donor marrow stromal cells was detected consistently in marrow, bone, cartilage, and lung either 1 or 2.5 mo after the infusions. The DNA also was detected but less frequently in the spleen, brain, and skin. There was a small but statistically significant increase in both collagen content and mineral content of bone 1 mo after the infusion. Similar results were obtained with infusion of relatively large amounts of wild-type whole marrow cells into the transgenic mice. In experiments in which male marrow stromal cells were infused into a female osteogenesis imperfecta-transgenic mouse, fluorescense in situ hybridization assays for the Y chromosome indicated that, after 2.5 mo, donor male cells accounted for 4-19% of the fibroblasts or fibroblast-like cells obtained in primary cultures of the lung, calvaria, cartilage, long bone, tail, and skin. In a parallel experiment in which whole marrow cells from a male mouse were infused into a female immunodeficient rag-2 mouse, donor male cells accounted for 4-6% of the fibroblasts or fibroblast-like cells in primary cultures. The results support previous suggestions that marrow stromal cells or related cells in marrow serve as a source for continual renewal of cells in a number of nonhematopoietic tissues.

Cytokine-mediated erythroid maturation in megakaryoblastic human cell line HU-3.

HU-3 is a bipotential cell line derived from the bone marrow of a patient with megakaryoblastic leukemia. Continuously proliferating cells evolved from cultures supplemented with nutrient medium containing human serum and granulocyte-macrophage (GM) colony-stimulating factor (CSF). Growth and viability of the HU-3 cell line was strictly dependent on the presence of GM-CSF, interleukin-3, or thrombopoietin (Tpo). Independent of the cytokine, the cells constitutively expressed a well-defined megakaryocyte phenotype, with 70-95% of the cells positive for CD4, CD34, and platelet glycoproteins Ib, IIb, and IIIa. Fewer than 10% of the cells had detectable erythroid glycophorin A. Erythropoiesis was induced in HU-3 parental cells and five clones harvested from culture medium containing GM-CSF by replacement of the growth-promoting cytokine with stem cell factor (SCF) and erythropoietin (Epo). During the first week of induction, the proliferating cells slowly acquired erythroid markers. Concomitant with a maturational growth arrest during the second week, there was a rapid accumulation of gamma and beta globin chains and benzidine reactive hemoglobin, as well as a distinct erythroid morphology. The culture declined after 12 days because of the transient effect of SCF in maintaining viability. Parental and cloned cells cultured for 7 days in Tpo-supplemented medium responded to the synergistic growth effect of SCF and Epo but were markedly suppressed in their yield of hemoglobinized cells. Recycling of the cells in GM-CSF for 4 days did not reverse the suppressive effect of Tpo. These results suggest a role for Tpo in the lineage commitment of erythromegakaryocytic progenitors by suppressing the erythroid potential. With its constitutive megakaryocyte phenotype and inducible erythroid potential, the self-renewing bipotential HU-3 cell line may represent one of the earliest stages in megakaryocytopoiesis before irreversible lineage commitment. The suppressive effect of Tpo on the erythroid potential of cloned HU-3 cells enhances the value of this cell line for deciphering the molecular and cellular events during lineage commitment of progenitor cells.

Biodistribution of 125I-MAb 425 in a human glioma xenograft model: effect of chloroquine.

Chloroquine has been shown to increase the cellular retention and nuclear incorporation of 125I-labeled monoclonal antibody (MAb) 425, a murine anti-epidermal growth factor receptor monoclonal antibody, in human high-grade glioma cells in vitro. The objective of this study was to examine the effect of chloroquine on the biodistribution of 125I-MAb 425 in an intracerebral xenogeneic transplant of glioma cells. Nude rats were stereotaxically implanted in the right hemisphere with A1207 human high-grade glioma cells. After 14 days, animals were injected i.v. with chloroquine (40 mg/kg) followed 2 h later by an 125I-MAb 425 (9 MBq) infusion. Tissue distributions were performed up to 168 h post 125I-MAb 425 injection. From 24 to 168 h, tumor-to-contralateral left brain ratios increased from 9 to 15 for 125I-MAb 425 alone, and 7 to 13 for the 125I-MAb 425/chloroquine combination, respectively. A single administration of chloroquine did not result in any significant difference in radiolabeled MAb accumulation in either the tumor site or other tissues. We conclude that chloroquine did not increase the amount of 125I-MAb 425 into the tumor; however, it is safe to administer i.v. at the 40 mg/kg dose. Under these experimental conditions, the increased radioactive accumulation observed for in vitro data did not translate into similar in vivo results.

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.

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 Burkitt's lymphoma cell line IM-9 were growth arrested at 500 micrograms H1/ml. The clonogenic growth of the Burkitt's 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 Burkitt's 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.

Limitations of the severe combined immunodeficiency (SCID) mouse model for study of human B-cell responses.

Mice lacking functional T and B lymphocytes offer an in vivo animal model for the study of human immune functions. We have attempted to optimize the reconstitution of severe combined immunodeficiency (SCID) mice with human peripheral blood lymphocytes (PBL) using radiation, anti-asialo GM1 antibody or cyclophosphamide (Cy) treatment of the mice and in vitro stimulation of human PBL with interleukin (IL)-2 prior to their transfer to the mice. Total human IgG and tetanus-toxoid (TT)-specific human IgG responses of the mice were used as parameters of successful reconstitution. Treatment of the mice with anti-asialo GM1 antibody significantly enhanced total human IgG levels, but not TT-specific antibody responses, whereas irradiation or Cy treatment of the mice had no effect on human antibody production. In vitro treatment of human PBL with IL-2 prior to engraftment significantly decreased total human IgG responses of human PBL-grafted SCID mice. The immune responses of individual mice within a group were highly variable, which constitutes a major disadvantage of this model.

Epidermal growth factor receptor 425 monoclonal antibodies radiolabeled with iodine-125 in the adjuvant treatment of high-grade astrocytomas.

Fifty-nine patients with primary presentation of high-grade gliomas of the brain, 13 with astrocytomas with anaplastic foci and 46 with glioblastoma multiforme, were treated with surgical intervention and definitive postoperative radiation therapy followed by multiple intravenous administration of iodine-125-labeled monoclonal antibody-425, which binds specifically to human epidermal growth factor receptor. The total cumulative labeled antibody doses ranged from 40 to 296 mCi. The administration of the radiolabeled antibody was performed in most instances within 3 months following completion of the primary surgery and radiation therapy. No significant life-threatening toxicities were observed during the trial. At one year, 34 (58%) of the 59 patients in the trial were alive. The median overall survival for both groups was 13.5 months.

Effects of hyperkalemia on the electrocardiogram of patients receiving digitalis.

In a prospective and a retrospective study, the effects of hyperkalemia on the electrocardiogram (ECG) of patients treated with customary maintenance doses of digoxin were examined and the results were compared with the effects of hyperkalemia in patients not receiving digitalis. The prospective study included 11 patients treated and 11 not treated with digitalis, and the retrospective study 27 patients treated and 61 not treated with digitalis. In all patients serum potassium concentrations (Ks) were determined within 1 hour of the recorded electrocardiogram. Serum digoxin concentrations, measured in 11 patients in the prospective and in 4 in the retrospective study, ranged from 0.7 to 5.0 ng/ml, and exceeded 2.0 ng/ml in 10 of 15 patients. Since the results of the prospective and of the retrospective study were similar, they were combined. In patients treated with digitalis, Ks ranged from 5.5 to 6.6 mEq/liter in 21 patients, from 6.7 to 7.5 mEq/liter in 17 and from 7.6 to 8.5 mEq/liter in 6; the Ks was 9.1 mEq/liter in 1 patient. The ventricular rate in patients treated with digitalis ranged from 48 to 140 beats/min, and was not significantly different from that in untreated patients within each range of Ks. Atrioventricular (AV) junctional rhythm occurred more frequently in the electrocardiograms of digitalis-treated patients (15 of 45 vs 2 of 76, p less than 0.001). The average PR intervals were longer in patients treated with digitalis who had Ks greater than 6.6 mEq/liter, but no patient in the study had greater than first-degree AV block, and no patient required a pacemaker.(ABSTRACT TRUNCATED AT 250 WORDS)

Unusual echocardiographic findings in primary pulmonary hypertension.

A patient with primary pulmonary hypertension showed unusual echocardiographic findings. These included echoes arising from an intracavitary right ventricular muscle band mimicking a divided interventricular septum. The hemodynamic study excluded subvalvular right ventricular obstruction. Severe right ventricular hypertrophy was also present. Both findings were probably related and may possibly be found in similar cases with severe right ventricular hypertrophy, especially due to primary pulmonary hypertension. The right ventricular muscle band possibly represented hypertrophied moderator band.

Value of 67Gallium scintigraphy in the diagnosis of localized renal and perirenal inflammation.

Eight patients with renal or perirenal abscesses were evaluated by 67gallium scintigraphy. The correlation of localized 67gallium uptake to the inflammatory lesion was proved by tissue diagnoses in 7 patients and by response to treatment and repeated studies in the remaining case. 67Gallium scanning was found to be an accurate, non-invastive technique. It should be applied early in diagnosing inflammatory lesions of the retroperitoneum and for examining fever of unknown origin.