A potent and specific immunotoxin for tumor cells expressing disialoganglioside GD2

Summary Monoclonal antibody 14G2a (anti-GD₂) reacts with cell lines and tumor tissues of neuroectodermal origin that express disialoganglioside GD₂. mAb 14G2a was coupled to the ribosome-inactivating plant toxin gelonin with the heterobifunctional cross-linking reagentN-succinimidyl-3(2-pyridyldithio)propionate. The activity of the immunotoxin was assessed by a cell-free translation assay that confirmed the presence of active gelonin coupled to 14G2a. Data from an enzyme-linked immunosorbent assay demonstrated the specificity and immunoreactivity of the 14G2a-gelonin immunotoxin, which was identical to that of native 14G2a. Assays for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) revealed that these functional properties of the native 14G2a antibody were also preserved in the 14G2a-gelonin immunotoxin. The gelonin-14G2a immunotoxin was directly cytotoxic to human melanoma (A375-M and AAB-527) cells and was 1000-fold more active than native gelonin in inhibiting the growth of human melanoma cells in vitro. The augmentation of tumor cell killing of 14G2a-gelonin immunotoxin was examined with several lysosomotropic compounds. Chloroquine and monensin, when combined with 14G2a-gelonin immunotoxin, augmented its cytotoxicity more than 10-fold. Biological response modifiers such as tumor necrosis factor and interferon and chemotherapeutic agents such as cisplatinum andN,N-bis(2-chloroethyl)-N-nitrosourea (carmustine) augmented the cytotoxicity of 14G2a-gelonin 4- to 5-fold. The results of these studies suggest that 14G2a-gelonin may operate directly by both cytotoxic efforts and indirectly by mediating both ADCC and CDC activity against tumor cells; thus it may prove useful in the future for therapy of human neuroectodermal tumors.Research conducted, in part, by the Clayton Foundation for Research     

Seasonal cycles of reserves in relation to reproduction inSebastes

Synopsis Seasonal cycles of reserve deposition and utilization in many fishes, amphibians and reptiles alleviate temporal mismatches of energy supply and demand. Utilization of reserves can be related to maintenance during periods of reduced food supply, to reproduction, particularly during periods of poor food availability, and to migration. Published data on the seasonal cycles of reserves and reproduction inSebastes suggest that reserves are important for maintenance during wintertime periods of low food availability. Unlike many other ectothermic vertebrates, some species ofSebastes deposit fat reserves at the same time as gametogenesis, a pattern consistent with the longevity and iteroparity evident in the genus. Other species ofSebastes have similar seasonal timing of fat cycles, but since reproduction takes place later in the year, the decline in reserves during winter coincides with the main period of reproductive activity. The significance of this is not clear. Interspecific differences in amounts of reserves may be related to geographical differences in the seasonality or abundance of food. Intraspecific variation in reserves, marked most strongly by allometry of reserves with regard to fish legth, bears further study, since it may link the proces of sexual maturation and the responses of repeat spawners to variability in food supply and other environmental factors.     

Relationship between sensitivity to 4'-(9-acridinylamino)methanesulfon-m-anisidide and DNA topoisomerase II in a cold-sensitive cell-cycle mutant of a murine mastocytoma cell line

The cold-sensitive (proliferating at 39.5 degrees C, reversibly arrested in GI-phase at 33 degrees C) cell-cycle mutant 21-Fb of the murine mastocytoma cell line P815 was used to study the effect of amsacrine on non-cycling cells. The sensitivity of arrested 21-Fb cells decreased less than 2-fold in cell survival experiments when compared to proliferating cells. In contrast, DNA breakage and stimulation of protein-DNA complex formation in intact or lysed cells was reduced approx. 10-fold in arrested cells and DNA topoisomerase II activity in arrested cells was only 5% of the activity in proliferating cells. Thus, there was no correlation between cell survival and DNA damage or DNA topoisomerase II activity in drug-treated cells.     

Influence of human T lymphocytes identified by antibodies to dipeptidyl peptidase IV on differentiation of human B lymphocytes stimulated with Staphylococcus aureus Cowan I and pokeweed mitogen

The influence of human T lymphocytes expressing the enzyme dipeptidyl peptidase IV (DPP IV) was investigated with respect to human peripheral B-lymphocyte differentiation. B cells stimulated with pokeweed mitogen in the presence of DPP IV-positive T cells produced high amounts of immunoglobulin. Moderate amounts of immunoglobulin could be measured when B cells were cultured in the presence of DPP IV-negative T cells. DPP IV defines a T-cell subset partially overlapping the subsets characterized by the differentiation antigens Leu 3a (helper/inducer) and Leu 2a (suppressor/cytotoxic). DPP IV-positive T cells exert, in contrast to DPP IV-negative T cells, high interleukin-2 activity after stimulation with phytohemagglutinin and pokeweed mitogen. To further functionally characterize DPP IV-positive and DPP IV-negative T cells, the helper effects of Leu 3a-positive T-cell subsets, differing in DPP IV expression, were investigated in pokeweed mitogen- and Staphylococcus aureus-driven B-cell differentiation systems. After pokeweed mitogen stimulation, immunoglobulin production was markedly reduced when B cells were cultured in the presence of Leu 3a-positive T cells expressing DPP IV (DPP IV+/Leu 3a+). In contrast, high amounts of immunoglobulin were produced in cultures with Leu 3a-positive but DPP IV-negative T cells (DPP IV-/Leu 3a+). This difference in immunoglobulin production of B cells cultured with DPP IV+/Leu 3a+ and DPP IV-/Leu 3a+ T cells could not be observed in Staphylococcus aureus-stimulated cultures. Here, both T-cell subsets supported terminal differentiation of B cells. We conclude that in the pokeweed mitogen-driven culture systems, DPP IV+/Leu 3a+ and DPP IV-/Leu 3a+ T cells may differ in the production of growth and/or differentiation factors distinct from interleukin-2.     

Rapid and reversible modulation of T4 (CD4) on monocytoid cells by phorbol myristate acetate: effect on HIV susceptibility

The effect of phorbol myristate acetate (PMA) on T4 (CD4) expression by monocytoid cells was studied. Greater than 99% of untreated U937 and HL-60 cells expressed surface T4 as measured with a fluorescence-activated cell sorter. The percentage of T4 positive cells decreased to less than 20% after incubation with PMA (10(-8) M). A decrease was observed within 15 min of PMA exposure, was maximal within 1 hr, and persisted for at least 3 days in the continuous presence of PMA. The susceptibility of untreated and PMA-treated U937 cells to human immunodeficiency virus (HIV) was also studied. Pretreatment of cells with PMA for 18 hr decreased the production of viral RNA and p24 antigen 24 hr after infection. The dose of PMA resulted in a parallel reduction of both T4 expression and infection by HIV. When PMA was washed from cultures and replaced with fresh medium for 48 hr, then T4 expression and the production viral RNA and p24 antigen following infection were restored. These data suggest that pharmacologic manipulation of surface T4 expression may have a potential role in the prevention or treatment of HIV infection.     

Stimulation of macrophage antibody-dependent killing of tumor targets by recombinant lymphokine factors and M-CSF

Macrophage colony-stimulating factor (M-CSF) was investigated as a stimulator of ADCC to the murine R1.1 thymoma target by murine peritoneal exudate macrophages which were elicited by proteose peptone. Both an 125IUdR release and a viable cell count assay were used. The latter assay avoids radiation damage, and the fate of the targets can be determined over a long period. Pretreatment of macrophages for several days in culture with lymphokine (LK) from concanavalin A-induced mouse spleen cells moderately stimulated ADCC. Preincubation of macrophages with conventional or recombinant human M-CSF or immunoaffinity-purified mouse M-CSF alone had little effect. However, M-CSF greatly enhanced ADCC to the tumor target when used as a costimulant with LK, IFN-gamma, IFN-alpha, IFN-beta, or IL-2 to pretreat macrophages. Incubation of macrophages with LK or LK plus M-CSF for 2 days generated stronger ADCC than 1- or 3-day incubations. Enhancement of LK-stimulated ADCC by M-CSF appeared to plateau at about 1000 U/ml. The enhancement of macrophage cytotoxicity when stimulated with IFNs or IL-2 was most effective at the lowest active concentration of these LKs. At 1 U/ml IFN-gamma or IL-2, or 5 U/ml IFN-alpha or IFN-beta, M-CSF boosted ADCC activity to that using 10-fold of the LK alone. IL-1, IL-4, and TNF had little or no stimulating activity for ADCC alone or with M-CSF, and the other hemopoietic growth factors IL-3 and GM-CSF did not promote this effector function alone or with IFN-gamma. We previously showed that M-CSF boosted macrophage antibody-independent killing of TU5 sarcoma targets with or without LK (Cell. Immunol. 105, 270, 1987). These studies thus show that M-CSF is a positive regulator of both macrophage-nonspecific tumor lysis and ADCC.     

Seed morphology under SEM and light microscopy in Kansas Juncus (Juncaceae)

Seed morphology was studied in 15 species of four subgenera ofJuncus occurring in Kansas, to determine if seeds provide traits useful in assessing systematic relations within the genus. In this study seed size and shape were of limited value, while surface ornamentation of the hard inner seed coat provided encouraging results. SubgenusPoiophylli showed little variation in surface ornamentation among taxa; similar ornamentation was observed in subgenusGenuini. SubgeneraGraminifolii andSeptati were separately distinct with the taxa in theSeptati forming a continuum of variation.     

Improved Agar Bottle Plate for Isolation of Methanogens or Other Anaerobes in a Defined Gas Atmosphere

A bottle plate for the cultivation of methanogens or other organisms in a defined pressurized-gas atmosphere was developed. The bottle provides the convenience of an agar streak plate, solves the problem of the water exudate from agar medium, and provides a convenient way of adding or sampling a defined gas atmosphere.     

Life Cycles in the Methanogenic Archaebacterium Methanosarcina mazei

Methanosarcina mazei S6 and LYC were used to study the structure and differentiation of the aggregating methanogens. Cultures harvested under various conditions are described at the ultrastructural level. Cells of strain S6 are enclosed by a layer 12 nm thick in contact with the plasma membrane. In sarcinal colonies, cells are held in close association by a fibrous matrix up to 60 nm thick. Colony maturation was examined in strain S6 over a period of 1 year. Changes occurred in the shape and staining of individual cells. Also, various inclusion bodies were observed that either persist throughout colony maturation or are only found at certain growth stages. Two types of cores that are composed of double membranes in M. mazei S6 are described. One has a 90-nm diameter and contains electron-dense granules similar to those found in the cytoplasm. The other core type does not contain granules, is more numerous, and is found in older cultures. Two life cycles are described for M. mazei based on electron microscope examinations. A complex life cycle involving the release of single cells is described with two variations for strains S6 and LYC. When released cells of strain S6 are placed in fresh medium they can repeat the cycle. In addition, a limited cycle is described for both strains of M. mazei. This limited cycle contains the only sarcinal morphotypes observed in M. barkeri. When M. mazei S6 remains in the limited cycle and does not disaggregate in stationary phase, several types of possible resting forms are found.     

Macrophage growth factor CSF-1 stimulates human monocyte production of interferon, tumor necrosis factor, and colony stimulating activity

CSF-1, a macrophage colony stimulating factor that causes proliferation and differentiation of progenitor cells, may also have effects on mature cells. Human peripheral blood monocytes were used to examine this possibility. Monocytes, separated from normal blood by density centrifugation and adherence, were incubated for 3 days with or without CSF-1 (1,000 U/ml, purified from the MIA PaCa pancreatic carcinoma line). The two groups of cells were then washed and tested for the ability, when induced, to produce several factors. When induced for 2 days with LPS and PMA, the monocytes produced a factor that was cytotoxic to L929 cells, and this factor was completely neutralized by polyclonal antibody to tumor necrosis factor. The cells preincubated with CSF-1 consistently produced an average of 12 times more of this factor than cells not exposed to CSF-1. Monocytes induced with LPS and PMA also produced a colony stimulating activity, as measured by colony formation when using mouse bone marrow. Cells preincubated with CSF-1, washed, and induced with LPS and PMA produced more than three times as much activity compared with control monocytes. When monocytes were induced with poly-I.C, 22-fold higher levels of interferon were produced by the cells exposed to CSF-1. These results show that CSF-1 has direct stimulating effects on mature human monocytes, and suggest that the macrophage growth factor may have clinical application in the treatment of infectious diseases and cancer.