Characterization of a soluble suppressor of human B cell immunoglobulin biosynthesis produced by a continuous human suppressor T cell line.

A human suppressor T cell maintained in long-term culture with conditioned medium containing interleukin 2 elaborates a suppressor factor(s) that specifically inhibits human polyclonal B cell immunoglobulin biosynthesis. This soluble immune suppressor supernate of immunoglobulin production (CTC-SISS-B) shares a number of features with the previously described suppressive mediator elaborated by concanavalin A-activated human peripheral T cells (SISS-B) including: (a) the inhibition by a noncytotoxic mechanism, (b) the suppression of immunoglobulin biosynthesis either through direct action on the B cell or indirect action via the monocyte, (c) the loss of inhibition in the presence of the monosaccharide L-rhamnose, (d) the elaboration by cells irradiated with 500 ro 2,000 rad, and (e) molecular weights of 60,000--90,000. Furthermore, the suppression by this mediator appears to be specific for B cell immunoglobulin production in that CTC-SISS B has no effect on T cell proliferation to mitogens, antigens, an allogeneic cells or on T cell-mediated cytotoxicity. These data indicate that one possible mechanism of suppressor T cell inhibition of human immunoglobulin production is via the generation of a lectinlike suppressor lymphokine that interacts with defined saccharide determinants on the cell surface of either the B cell or monocyte.

Normal human B cells display ordered light chain gene rearrangements and deletions.

Human kappa-producing B cell lines and leukemias retain their excluded lambda light chain genes in the germ line configuration, whereas transformed lambda-producing B cells uniformly rearrange or delete their kappa genes (12). Whether the unexpected lambda gene recombinations within malignant lambda-producing B cells reflect a normal developmental process or are secondary to transformation and specific chromosomal translocations was uncertain. To resolve this issue, we purified circulating lambda-bearing B cells from a normal individual to 97% purity by using a series of negative selection steps and a final positive selection on a cell sorter. Over 95% of the collective kappa genes in these lambda B cells were no longer in their germ line form, with the majority (60%) deleted and the remainder present but in a rearranged state. The chromosomal loss of the germ line kappa genes included the joining (J kappa) segments as well as the constant (C kappa) region, yet the particular variable (V kappa) gene family studied was spared. In addition, the incidence of kappa gene deletions was higher in long-term than in freshly transformed lambda B cell lines. This implies that the deletion of aberrantly rearranged kappa genes may occur as a second event. Such a mechanism would serve to eliminate aberrant transcripts and light chain fragments that might interfere with the synthesis and assembly of effective immunoglobulin molecules. Thus, despite the nearly equal usage of kappa and lambda light chain genes in man, there appears to be a sequential order to their expression during normal B cell ontogeny in which kappa gene rearrangements precede those of lambda.

Expression of interleukin 2 receptors on activated human B cells.

Using anti-Tac, a monoclonal anti-interleukin 2 (IL-2) receptor antibody, we have explored the possibility that certain activated B cells display receptors for IL-2. Resting normal B cells and unselected B cell lines established from normal individuals were Tac antigen negative. In contrast, the cell surface Tac antigen expression was demonstrable on 6 of 10 B cell lines from patients with Burkitt's lymphoma, 5 of 6 B cell lines derived from patients with HTLV-I-associated adult T cell leukemia (including all four that had integrated HTLV-I into their genome), and on certain normal B cells activated with pokeweed mitogen. Furthermore, cloned Epstein-Barr virus-transformed B cell lines derived from Tac-positive normal B cells continued to express the Tac antigen in long-term cultures and manifested high affinity IL-2 receptors identified in binding studies with purified radiolabeled IL-2. The line 5B4 developed in the present study could be induced with purified JURKAT-derived or recombinant IL-2 to express a larger number of IL-2 receptors. Furthermore, the addition of IL-2 to the 5B4 B cell line augmented IgM synthesis, which could be blocked by the addition of anti-Tac. The size of the IL-2 receptors expressed on the cloned normal B cell lines was similar (53,000-57,000 daltons) to that of receptors on phytohemagglutinin-stimulated T cell lymphoblasts. Thus, certain malignant and activated normal B cells display the Tac antigen and manifest high affinity receptors for IL-2. These data suggest that IL-2 may play a role in the differentiation of activated B cells into immunoglobulin-synthesizing and -secreting cells.

Disorders of B cells and helper T cells in the pathogenesis of the immunoglobulin deficiency of patients with ataxia telangiectasia.

The pathogenesis of the immunoglobulin deficiency of 20 patients with ataxia telangiectasia was studied using an in vitro immunoglobulin biosynthesis system. 10 patients had no detectable IgA in their serum as assessed by radial diffusion in agar and 3 had a reduced serum IgA concentration. The peripheral blood mononuclear cells of 17 of the patients and 17 normal controls were cultured with pokeweed mitogen for 12 d and the immunoglobulin in the supernatants measured. The immunoglobulin synthesis was below the lower limit of the normal 95% confidence interval for IgM in 5 patients, for IgG in 8, and for IgA in 14. The mononuclear cells from 9 of the 10 patients with a serum IgA concentration less than 0.1 mg/ml failed to synthesize IgA in vitro. None of the patients manifested excessive suppressor cell activity. All patients had reduced but measurable helper T cell activity for immunoglobulin synthesis by co-cultured normal pokeweed mitogen-stimulated B cells (geometric mean 22% of normal). Furthermore, the addition of normal irradiated T cells to patient peripheral blood mononuclear cells led to an augmentation of IgM synthesis in 15 of 17 and to increased IgG synthesis in 9 of the 17 patients studied, including 9 of the 12 patients who had synthesized IgG before the addition of the irradiated T cells. In addition, IgA synthesis was increased in all eight patients examined that had serum IgA concentrations greater than 0.1 mg/ml. These studies suggest that a helper T cell defect contributes to the diminished immunoglobulin synthesis. However, a helper T cell defect does not appear to be the sole cause since there was no IgA synthesis by the peripheral blood mononuclear cells of 9 of the 10 patients with a profoundly reduced serum IgA even when co-cultured with normal T cells. Furthermore, the cells of the nine patients with profoundly reduced IgA levels examined also failed to produce IgA when stimulated with the relatively helper T cell-independent polyclonal activators, Nocardia water soluble mitogen or Epstein-Barr virus. Taken together these data support the view that the reduced immunoglobulin synthesis of these patients is due to defects of both B cells and helper T cells. Such a broad defect in lymphocyte maturation taken in conjunction with our demonstration of persistent alpha fetoprotein production by ataxia telangiectasia patients provides support for the proposal that these patients exhibit a generalized defect in tissue differentiation.

The Sézary syndrome: a malignant proliferation of helper T cells.

The Sézary syndrome is a frequently lethal disease characterized by circulating malignant cells of thymus-derived (T)-cell origin. The capacity of circulating malignant lymphocytes from patients with this syndrome to synthesize immunoglobulins and to function as helper or suppressor cells regulating immunoglobulin synthesis by bone marrow-derived (B) lymphocytes was determined. Peripheral blood lymphocytes from normal individuals had geometric mean immunoglobulin synthetic rates of 4,910 ng for IgM, 1,270 ng for IgA, and 1,625 ng for IgG per 2 X 10(6) cells in culture with pokeweed mitogen for 7 days. Purified normal B cells had geometric mean synthetic rates of 198 ng for IgM, 145 ng for IgA, and 102 ng for IgG. Leukemic cells from patients with the Sézary syndrome produced essentially no immunoglobulins. Adding normal T cells to normal B cells restored their immunoglobin producing capacity. Leukemic cells from four of five patients tested had a similar capacity to help immunoglobulin synthesis by purified normal B cells. Additionally, Sézary cells from one patient studied induced a nearly 10-fold increase in IgA synthesis by lymphocytes from a child with ataxia telangiectasia and selective IgA deficiency. Furthermore, these Sézary cells induced more than a 500-fold increase in IgG and IgA synthesis by lymphocytes from a child with Nezelof's syndrome. When Sézary cells were added to normal unfractionated lymphocytes, they did not suppress immunoglobulin biosynthesis. In addition, unlike the situation observed when large numbers of normal T cells were added to purified B cells, there was no depression of immunoglobulin synthesis at very high malignant T-cell to B-cell ratios. These data support the view that Sézary T cells do not express suppressor cell activity. The results presented in this paper suggest that neoplastic lymphocytes from the majority of patients with the Sézary syndrome originate from a subset of T cells programmed exclusively for helper-like interactions with B cells in their production of immunoglobulin molecules.

Functional and phenotypic comparison of human T cell leukemia/lymphoma virus positive adult T cell leukemia with human T cell leukemia/lymphoma virus negative Sézary leukemia, and their distinction using anti-Tac. Monoclonal antibody identifying the human receptor for T cell growth factor.

Adult T cell leukemia (ATL) and Sézary leukemia are malignant proliferations of T lymphocytes that share similar cell morphology and clinical features. ATL is associated with HTLV (human T cell leukemia/lymphoma virus), a unique human type C retrovirus, whereas most patients with the Sézary syndrome do not have antibodies to this virus. Leukemic cells of both groups were of the T3, T4-positive, T8-negative phenotype. Despite the similar phenotype, HTLV-negative Sézary leukemic cells frequently functioned as helper cells, whereas some HTLV-positive ATL and HTLV-positive Sézary cells appeared to function as suppressors of immunoglobulin synthesis. One can distinguish the HTLV-positive from the HTLV-negative leukemias using a monoclonal antibody (anti-Tac) that appears to identify the human receptor for T cell growth factor (TCGF). Resting normal T cells and most HTLV-negative Sézary cells were Tac-negative, whereas all ATL cell populations were Tac-positive. The observation that ATL cells manifest TCGF receptors suggests the possibility that an abnormality of the TCGF-TCGF receptor system may partially explain the uncontrolled growth of these cells.

In vitro and in vivo gene delivery mediated by a synthetic polycationic amino polymer.

A synthetic polyamino polymer with a glucose backbone was used for gene transfer in vitro and in vivo. Gene transfer in vitro to various human carcinoma cell lines was achieved with an efficiency superior to a commercially available cationic liposome preparation. The polymer was resistant to inhibition by serum, which allowed for efficient gene transfer in vivo. Direct Intracranial tumor injection using this reagent resulted in reporter gene expression levels comparable to those achieved by a recombinant adenoviral vector. Thus, this compound represents a new class of agent that may have broad utility for gene transfer and gene therapy applications.

Stable in vivo gene transduction via a novel adenoviral/retroviral chimeric vector.

Gene therapy to correct defective genes requires efficient gene delivery and long-term gene expression. The available vector systems have not allowed the simultaneous achievement of both goals. We have developed a chimeric viral vector system that incorporates favorable aspects of both adenoviral and retroviral vectors. Adenoviral vectors induce target cells to function as transient retroviral producer cells in vivo. The progeny retroviral vector particles are then able to stably transduce neighboring cells. In this system, the nonintegrative adenoviral vector is rendered functionally integrative via the intermediate generation of a retroviral producer cell. The chimeric vectors may allow realization of the requisite goals for specific gene-therapy applications.

Epidermal growth factor stimulates vascular endothelial growth factor production by human malignant glioma cells: a model of glioblastoma multiforme pathophysiology.

Hypervascularity, focal necrosis, persistent cerebral edema, and rapid cellular proliferation are key histopathologic features of glioblastoma multiforme (GBM), the most common and malignant of human brain tumors. By immunoperoxidase and immunofluorescence, we definitively have demonstrated the presence of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFr) in five out of five human glioma cell lines (U-251MG, U-105MG, D-65MG, D-54MG, and CH-235MG) and in eight human GBM tumor surgical specimens. In vitro experiments with glioma cell lines revealed a consistent and reliable relation between EGFr activation and VEGF production; namely, EGF (1-20 ng/ml) stimulation of glioma cells resulted in a 25-125% increase in secretion of bioactive VEGF. Conditioned media (CM) prepared from EGF-stimulated glioma cell lines produced significant increases in cytosolic free intracellular concentrations of Ca2+ ([Ca2+]i) in human umbilical vein endothelial cells (HUVECs). Neither EGF alone or CM from glioma cultures prepared in the absence of EGF induced [Ca2+]i increases in HUVECs. Preincubation of glioma CM with A4.6.1, a monoclonal antibody to VEGF, completely abolished VEGF-mediated [Ca2+]i transients in HUVECs. Likewise, induction by glioma-derived CM of von Willebrand factor release from HUVECs was completely blocked by A4.6.1 pretreatment. These observations provide a key link in understanding the basic cellular pathophysiology of GBM tumor angiogenesis, increased vascular permeability, and cellular proliferation. Specifically, EGF activation of EGFr expressed on glioma cells leads to enhanced secretion of VEGF by glioma cells. VEGF released by glioma cells in situ most likely accounts for pathognomonic histopathologic and clinical features of GBM tumors in patients, including striking tumor angiogenesis, increased cerebral edema and hypercoagulability manifesting as focal tumor necrosis, deep vein thrombosis, or pulmonary embolism.

IL-2Ralpha-Directed monoclonal antibodies provide effective therapy in a murine model of adult T-cell leukemia by a mechanism other than blockade of IL-2/IL-2Ralpha interaction.

Adult T-cell leukemia (ATL) develops in a small proportion of human T-cell lymphotrophic virus-I infected individuals. The leukemia consists of an overabundance of activated T cells, which are characterized by the expression of CD25, or IL-2Ralpha, on their cell surface. Presently, there is not an accepted curative therapy for ATL. We developed an in vivo model of ATL in non-obese diabetic/severe combined immunodeficient (NOD/ SCID) mice by introducing cells from an ATL patient (MET-1) into the mice. The leukemic cells proliferated in these mice that lack functional T, B, and natural killer (NK) cells. The MET-1 leukemic cells could be monitored by measurements of both serum soluble Tac (IL-2Ralpha) and soluble human beta2-microglobulin (beta2mu) by ELISA. The disease progressed to death in the mice after approximately 4-6 weeks. The mice developed grossly enlarged spleens and a leukemia involving ATL cells that retained the phenotype and the T-cell receptor rearrangement and human T-cell lymphotrophic virus-I integration pattern of the patient's ATL leukemia cells. This model is of value for testing the efficacy of novel therapeutic agents for ATL. The administration of humanized anti-Tac (HAT), murine anti-Tac (MAT), and 7G7/B6, all of which target IL-2Ralpha, significantly delayed the progression of the leukemia and prolonged the survival of the tumor-bearing mice. In particular, HAT induced complete remissions in 4 of 19 mice and partial remissions in the remainder. It appears that the antibodies act by a mechanism that had not been anticipated. The prevailing view is that antibodies to the IL-2Ralpha receptor have their effective action by blocking the interaction of IL-2 with its growth factor receptor, thereby inducing cytokine deprivation apoptosis. However, although both HAT and MAT block the binding of IL-2 to IL-2Ralpha of the high affinity receptor, the 7G7/B6 monoclonal antibody binds to a different epitope on the IL-2Ralpha receptor, one that is not involved in IL-2 binding. This suggested that the antibodies provide an effective therapy by a mechanism other than induction of cytokine deprivation. In accord with this view, the MET-1 cells obtained from the spleens of leukemic mice did not produce IL-2, nor did they express IL-2 mRNA as assessed by reverse transcription-PCR. Another possible conventional mechanism of action involves complement-mediated killing. However, although MAT and 7G7/B6 fix rabbit complement, HAT does not do so. Furthermore, in the presence of NOD/SCID mouse serum, there was no complement-mediated lysis of MET-1 cells. In addition, the antibodies did not manifest antibody-dependent cellular cytotoxicity with NOD/SCID splenocytes that virtually lack NK cells as the effector cells as assessed in an in vitro chromium-release assay. However, in contrast to the efficacy of intact HAT, the F(ab')2 version of this antibody was not effective in prolonging the survival of mice injected with MET-1 ATL cells. In conclusion, in our murine model of ATL, monoclonal antibodies, HAT, MAT, and 7G7/B6, appear to delay progression of the leukemia by a mechanism of action that is different from the accepted mechanism of IL-2 deprivation leading to cell death. We consider two alternatives: the first, antibody-dependent cellular cytotoxicity mediated by FcRI- or FcRIII-expressing cells other than NK cells, such as monocytes or polymorphonuclear leukocytes. The second alternative we consider involves direct induction of apoptosis by the anti-IL-2R antibodies in vivo. It has been shown that the IL-2R is a critical element in the peripheral self-tolerance T-cell suicide mechanism involved in the phenomenon of activation-induced cell death.

Targeted gene delivery to Kaposi's sarcoma cells via the fibroblast growth factor receptor.

Kaposi's sarcoma (KS) is a major AIDS-related malignancy associated with significant morbidity and mortality. Current chemotherapeutic regimens are associated with a dismal prognosis. In an effort to develop a new approach to KS treatment, we devised a gene therapy-based adenovirus retargeting schema that redirects the adenovirus to fibroblast growth factor receptors endogenously present on the cell surface of KS cells. By using a bifunctional conjugate consisting of a blocking antiadenoviral knob Fab linked to basic fibroblast growth factor, FGF2, the gene transduction of KS cells was enhanced 7.7-44 fold; recombinant adenoviruses encoding either the firefly luciferase reporter gene, or the herpes simplex thymidine kinase gene, demonstrated quantitative enhancement of expression in the KS cell lines. In this regard, two KS cell lines that were previously refractory to native adenovirus transduction could be successfully transduced by the addition of the conjugate. This study thus addresses the utility of adenoviral retargeting to the FGF receptor in KS cells that are ordinarily transduction refractory to standardized approaches and allows practical development of gene therapy approaches for the treatment of human KS.

Interleukin 2 receptor (Tac antigen) expression in HTLV-I-associated adult T-cell leukemia.

Interleukin-2 (IL-2) is a lymphokine synthesized by some T-cells following activation. Resting T-cells do not express IL-2 receptors, but receptors are rapidly expressed on T-cells following interaction of antigens, mitogens, or monoclonal antibodies with the antigen-specific T-cell receptor complex. Using anti-Tac, a monoclonal antibody that recognizes the IL-2 receptor, the receptor has been purified and shown to be a Mr 33,000 peptide that is posttranslationally glycosylated to a Mr 55,000 mature form. Normal resting T-cells and most leukemic T-cell populations do not express IL-2 receptors; however, the leukemic cells of the 11 patients examined who had human T-cell lymphotropic virus-associated adult T-cell leukemia expressed the Tac antigen. In human T-cell lymphotropic virus-I infected cells, the Mr 42,000 long open reading frame protein encoded in part by the pX region of this virus may act as a transacting transcriptional activator that induces IL-2 receptor gene transcription, thus providing an explanation for the constant association of IL-2 receptor expression with adult T-cell lymphotropic virus-I infection of lymphoid cells. The constant expression of large numbers of IL-2 receptors which may be aberrant may play a role in the uncontrolled growth of adult T-cell leukemia cells. Two patients with Tac-positive adult T-cell leukemia have been treated with the anti-Tac. One of the patients had 6- and 3-mo remissions of his leukemia following two courses of therapy with this monoclonal antibody directed toward this growth factor receptor.

Endothelial cell vehicles for delivery of cytotoxic genes as a gene therapy approach for carcinoma of the ovary.

Human umbilical vein endothelial cells (HUVECs) were evaluated for utility as a vector to achieve a bystander effect and killing of ovarian carcinoma cell lines. After demonstrating that HUVECs could be transduced with the reporter gene LacZ encoded by an adenoviral vector, appropriate cell killing of the AdCMVHSV-TK-transduced HUVECs was exhibited after treatment with 20 microM ganciclovir. Mixing experiments were then performed to determine whether the transduced HUVECs would demonstrate a bystander effect with the ovarian cancer cell lines. When 50% AdCMVHSV-TK-transduced HUVECs were mixed with untransduced ovarian cancer cells, > 70% of all cells were killed. Finally, s.c. and i.p. injections of herpes simplex-thymidine kinase-expressing HUVECs and SKOV3ip1 tumor cells were performed to evaluate the effects of HUVECs in in vivo models. These studies showed a decrease in tumor growth s.c. as well as a statistically significant survival prolongation (P < 0.05) in the i.p. model. These findings suggest that endothelial cells may be used as a vehicle for the delivery of cytotoxicity (bystander effect) in molecular chemotherapy.

Prolongation of graft survival in primate allograft transplantation by yttrium-90-labeled anti-Tac in conjunction with granulocyte colony-stimulating factor.

High-affinity IL-2 receptors are expressed by T cells activated in response to foreign histocompatibility antigens but not by normal resting T cells. To exploit this difference in IL-2R expression, anti-Tac, a murine monoclonal antibody specific for the IL-2R alpha subunit, was used to inhibit organ allograft rejection. To enhance its effector function, anti-Tac was armed by chelation with yttrium-90, a pure beta-emitting radionuclide. Animals received no immunosuppression (n = 5, group I, controls), unmodified anti-Tac (n = 5, 1 mg/kg q.o.d., group II), or 90Y-anti-Tac (n = 5, 1.6 mCi/kg divided into four doses, group III). The animals in group IV (n = 4) were treated identically to those in group III with the exception that 5 micrograms/kg/dose of granulocyte colony-stimulating factor was administered intramuscularly on the days when the yttrium-90 was given and on postoperative days 12 through 35 in order to reduce hematopoietic toxicity. Mean graft survival +/- S.E.M. for the control group was 8.2 +/- 0.5 days as compared with 13.8 +/- 2.1 days (P < 0.05) for those monkeys treated with unmodified anti-Tac. Graft survival was further prolonged in animals of group III that received 90Y-anti-Tac, with a mean graft survival of 45.0 +/- 11.8 days; however, three of the five monkeys retained viable grafts within this group but died secondary to bone marrow suppression. In comparison, the monkeys in group IV that were treated with G-CSF in conjunction with 90Y-anti-Tac had a mean graft survival of 49.2 +/- 2.9 days. In contrast to group III there were no deaths in the group (IV) receiving G-CSF. Furthermore, animals in group IV had a reduced magnitude and shortened duration of irradiation-induced neutropenia when compared with that observed in group III animals that did not receive G-CSF. Thus, treatment with 90Y-anti-Tac in conjunction with G-CSF may have potential applications in organ transplantation and the treatment of IL-2 receptor-expressing neoplastic diseases.

B cell, helper T cell, and suppressor T cell abnormalities contribute to disordered immunoglobulin synthesis in patients following bone marrow transplantation.

The pathogenic mechanisms responsible for the hypogammaglobulinemia that occurs in patients who have undergone bone marrow transplantation were studied using peripheral blood lymphocytes in an in vitro immunoglobulin biosynthesis assay. None of the nine marrow recipients between 2 and 15 months after transplantation produced normal amounts of IgG, IgA, or IgM in response to pokeweed mitogen (a T cell-dependent activator). However, three of these same patients responded to Epstein-Barr virus (a helper T cell- independent activator), suggesting that these three possessed responsive B cells. Cocultures of the marrow recipients' lymphocytes with lymphocytes from their respective donors or other normal subjects documented excessive suppressor cell activity in five cases. These suppressor cells frequently had profound activity (greater than 80% suppression of the immunoglobulin synthesis of cocultured normal cells), were predominantly radioresistant, and were T cells in the cases where analyzed. Helper T cell activity for immunoglobulin synthesis was not demonstrable in seven of the nine cases. In addition, four of the patients apparently possessed defective B cells. Although most patients had combined defects of several lymphocyte subpopulations, it appeared that either an isolated helper T cell or isolated B cell deficiency was sufficient to result in altered Ig synthesis. In this regard, two patients had demonstrable helper T cell defects with partially responsive B cells and no excessive suppressor activity. Interestingly, these two patients were experiencing a recurrence of their hypogammaglobulinemia following a period of normalized immunoglobulin levels. Thus, defects within all lymphocytic elements involved in the response and regulation of immunoglobulin synthesis were identified and may contribute to the humoral immunodeficiency which follows marrow transplantation.

Use of yttrium-90-labeled anti-Tac antibody in primate xenograft transplantation.

The high-affinity interleukin-2 receptor (IL-2R) is expressed by T cells activated in response to foreign histocompatibility antigens but not by normal resting cells. Thus, blockade of the interaction of IL-2 with its receptor could achieve selective immunosuppression. Accordingly, anti-Tac, a murine IgG2a class monoclonal antibody specific to the IL-2R, was used alone or in a chelated form with yttrium-90 (90Y), a pure beta emitter, to inhibit rejection of cardiac xenografts from Macaca fascicularis (cynomolgus) donors transplanted to the cervical or abdominal region of Macaca mulatta (rhesus) recipients (n = 20). Animals received no immunosuppression (n = 3, group I, controls), unmodified anti-Tac (n = 5, 2 mg/kg q.o.d., group II), or 90Y-anti-Tac (n = 5, 16 mCi, group III). To distinguish the nonspecific immunosuppressive effect of radiation, 90Y was administered bound to UPC-10 (n = 4, 16 mCi, group IV), another murine monoclonal antibody that does not specifically recognize activated immunoresponsive cells. All immunosuppression was administered in divided doses during the first 2 weeks posttransplant. Group I animals rejected their grafts at 6.7 +/- 1 days and demonstrated a rise in soluble IL-2R levels at the time of rejection, indicating the generation of Tac-expressing and -releasing cells. Graft survival in group II was not prolonged compared with controls (mean survival 6.2 +/- 1 days; P greater than 0.05). In contrast, graft survival in animals that received the designed dosage of 90Y-anti-Tac was significantly prolonged to an average of 38.4 +/- 5 days compared with groups I and II (P less than 0.005 and P les sthan 0.0005, respectively). Prolongation of graft survival occurred in animals that received 90Y-UPC-10 (mean survival 21.3 +/- 5 days, P less than 0.05 versus group I, P less than 0.01 versus group II). However, 90Y-UPC-10 was significantly less effective in prolonging graft survival than 90Y-anti-Tac, in which one-half the per-kilogram dosage of radioactivity was delivered in specific fashion via anti-Tac (P less than 0.025). Reversible nonlethal bone marrow suppression occurred without associated nephro- or hepatotoxicity, and virtually all animals developed antibodies to the murine monoclonal. Thus, the approach used in the present study, IL-2R-directed therapy with 90Y-anti-Tac, may have potential applications in organ transplantation and in the treatment of Tac-expressing neoplastic diseases.

Postsynaptic alpha 2-noradrenergic receptors mediate feeding induced by paraventricular nucleus injection of norepinephrine and clonidine.

This study examines the feeding response induced by hypothalamic noradrenergic stimulation, in terms of the type and synaptic position of its mediating receptor. Tests with norepinephrine or the alpha 2 receptor agonist clonidine, injected into the area of the paraventricular nucleus (PVN), revealed a potent feeding response in satiated animals. This response by either agonist was blocked, in a dose-dependent fashion, by local injection of the alpha 2-noradrenergic antagonists, rauwolscine and yohimbine. It was also blocked by the general antagonist, phentolamine. In contrast, it was unaffected by hypothalamic injection of the alpha 1-noradrenergic antagonists, prazosin and corynanthine. These results indicate that feeding elicited by noradrenergic stimulation in the region of the PVN is mediated through alpha 2-type receptors. These alpha 2 receptors appear to be located postsynaptically, since the effectiveness of clonidine in eliciting eating was undisturbed by prior injection of the catecholamine synthesis inhibitor, alpha-methyl-p-tyrosine.

Brain edema in meningiomas is associated with increased vascular endothelial growth factor expression.

OBJECTIVE: Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), an endothelial cell-specific cytokine, induces proliferation of endothelial cells and increases vascular permeability dramatically. All gliomas secrete significant amounts of VEGF, whereas meningiomas are variable in expression. Thus, we sought to determine whether the extent of VPF/VEGF expression in meningiomas correlated with differences in brain edema associated with these tumors. METHODS: Meningioma tissue samples from 37 patients (15 men, average age 65 +/- 13 yr; 22 women, average age 60 +/- 10 yr) who underwent surgery at or were referred to the University of Alabama Hospital were examined retrospectively for the extent of expression of immunoreactive VPF/VEGF. Additionally, peritumoral edema was assessed on a blinded basis radiographically from preoperative magnetic resonance imaging scans. Selected specimens were examined by in situ hybridization to document the source of VPF/VEGF. RESULTS: The predominant meningioma subclassifications were transitional (57%) or meningothelial (27%) subtypes. VPF/VEGF immunoreactivity ranged from 0 to 3.5, with a median value of 2 on a subjective 5-point scale; magnetic resonance imaging-assessed edema ranged in extent from 0 to 4 (subjective 5-point scale), with a median value of 2.5. The correlation of determination (R2) of magnetic resonance imaging-assessed tumor edema rating and VPF/VEGF staining intensity rating was 0.6087 (r = 0.78; P = 0.0001). In situ hybridization localized VPF/VEGF messenger ribonucleic acid in meningioma cells and not in normal parenchymal brain cells. CONCLUSION: These data suggest that meningioma-associated edema may be a result of the capacity of meningioma cells to produce VPF/VEGF locally, leading to increased tumor neovascularization and enhanced vascular permeability.

Vascular endothelial growth factor in human glioma cell lines: induced secretion by EGF, PDGF-BB, and bFGF.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is an endothelial cell-specific mitogen that is structurally related to platelet-derived growth factor (PDGF). Vascular endothelial growth factor/vascular permeability factor induces angiogenesis in vivo and may play a critical role in tumor angiogenesis. Using immunohistochemical analysis, the authors demonstrated the presence of VEGF/VPF protein in surgical specimens of glioblastoma multiforme and cultured glioma cells. By means of an enzyme-linked immunosorbent assay (ELISA) of cell supernatants, the authors showed that VEGF/VPF is variably secreted by all nine cultured human malignant glioma cell lines (CH-235MG, D-37MG, D-54MG, D-65MG, U-87MG, U-105MG, U-138MG, U-251MG, U-373MG) and by a single meningioma cell line (CH-157MN). An immunocytochemical survey of these cell lines revealed a cytoplasmic and cell-surface distribution of VEGF/VPF. In the U-105MG glioma cell line, VEGF/VPF secretion was induced with physiological concentrations of epidermal growth factor, PDGF-BB, or basic fibroblast growth factor, but not with PDGF-AA. Moreover, it was observed that activation of convergent growth factor signaling pathways led to increased glioma VEGF secretion. Similar results were obtained using these growth factor combinations in the D-54MG glioma cell line. The data obtained suggest a potential role for VEGF/VPF in tumor hypervascularity and peritumoral edema. These observations may lead to development of new therapeutic strategies.

Regulation of vascular endothelial growth factor secretion in human meningioma cells.

Previously, we induced vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) secretion in glioma cell lines by using physiologic concentrations of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), or platelet-derived growth factor-BB (PDGF-BB). We hypothesized that VEGF/VPF might enhance the blood supply required for the unregulated growth of tumors, and that it acts as the central mediator of tumor angiogenesis. The objective of this study was to determine whether the expression of VEGF/VPF by meningiomas is regulated by growth factors or sex hormones. By means of an enzyme-linked immunosorbent assay of CH-157MN meningioma cell supernatants, we demonstrated that EGF and bFGF similarly induce VEGF secretion by CH-157MN meningioma cells. At the maximum concentrations of EGF (50 ng/mL) and bFGF (50 ng/mL) used in this study, VEGF secretion was induced to 140% to 160% above baseline constitutive secretion. PDGF-BB homodimer did not enhance VEGF secretion significantly. Estradiol (up to 10(-7) mol/L), progesterone (up to 10(-5) mol/L), or testosterone (up to 10(-5) mol/L) did not stimulate or inhibit VEGF secretion in CH-157MN meningioma cells (p > 0.05). Furthermore, we demonstrated that dexamethasone decreased VEGF secretion to 32% of baseline constitutive secretion. This might explain the effect of corticosteroids in alleviating peritumoral brain edema in meningiomas. These results suggest that VEGF secretion in CH-157MN meningioma cells is mainly regulated by growth factors and corticosteroids, but not by sex hormones. Understanding the regulation of VEGF/VPF secretion in meningiomas might contribute to the development of a new therapeutic strategy.