Addition of interleukin-2in vitro augments detection of lymphokine-activated killer activity generatedin vivo

Summary Thein vivo administration of repetitive weekly cycles of interleukin-2 (IL-2) to patients with cancer enhances the ability of freshly obtained peripheral blood lymphocytes (PBL) to lyse both the natural-killer(NK)-susceptible K562 and the NK-resistant Daudi targets. Lysis of both targets is significantly augmented by inclusion of IL-2 in the medium during the cytotoxicity assay. This boost is much greater for cells obtained following thein vivo IL-2 therapy than for cells obtained prior to the initiation of therapy or for cells from healthy control donors. In addition to direct lytic activity, the PBL obtained followingin vivo IL-2 show a rapid increase in lymphokine-activated killer (LAK) activity with more prolongedin vitro IL-2 exposure, indicating that LAK effectors primedin vivo respond with secondary-like kinetics to subsequent IL-2in vitro. Lymphocytes from healthy control individuals, cultured in IL-2 under conditions attempting to simulate thein vivo IL-2 exposure, function similarly to PBL obtained from patients following IL-2, in that low-level LAK activity was significantly boosted by inclusion of IL-2 during the cytotoxic assay and the cells also responded with secondary-like kinetics to subsequent IL-2in vitro. The augmentation of the LAK effect was also dependent on the dose of IL-2 added during the 4-h⁵¹Cr-release cytotoxicity assay, with higher doses of IL-2 having a more pronounced effect. While continuous infusion of IL-2 induces a greater cytotoxic potential per milliliter of blood obtained from patients, the peak serum IL-2 levels attained are greater with bolus IL-2 infusions. These pharmacokinetic results, together with the IL-2 dose dependence of LAK activity generatedin vivo shown in this report, suggest that a combination of treatment with bolus IL-2 infusions superimposed on continuous IL-2 infusion may transiently expose IL-2 dependent LAK cells, activatedin vivo, to higher concentrations of IL-2, facilitating theirin vivo cytotoxic potential.This work was supported by NIH contract NO1 CM-47669-02, NIH grants CA-32685, RR-031086, NO1 CM-47669-03, and American Cancer Society grant CH-237     

Three classes of signalling molecules on B-cell membranes

The question of whether surface immunoglobulin and Ia molecules have a signalling function in helper T cell-dependent activation of B cells has been evaluated. Two sources of B cells have been used, one a purified population of hapten-binding B cells, the other a B-cell lymphoma, CH12, with known antigen specificity. Evidence is presented that both immunoglobulin and Ia molecules are receptors actively involved in the initial activation of resting B cells. Nevertheless, the requirements for ligand binding to either receptor can be bypassed under appropriate conditions, and the implications of this result for the function of these molecules is discussed. With respect to B-cell Ia, the authors present data that demonstrate two distinct functions of this molecule, one as a restricting element for T-cell activation, the second as a signalling receptor for B-cell excitation. On the CH12 surface, the I-A molecule fulfills the former function, but T-cell interactions with I-A fail to result in B-cell stimulation, suggesting that B-cell Ia may limit helper T cell-B cell interactions. We suggest that the binding of antigen surface immunoglobulin and binding of helper T-cell receptors to the appropriate Ia molecule(s) results in the activation of genes that encode for a third class of membrane B-cell receptors, those that bind B-cell stimulating factors.     

Infection of DBA/2 or C3H/HeJ mice by intraperitoneal injection of vaccinia virus elicits activated macrophages,cytolytic and cytostatic for S91-melanoma tumor cells

Summary Murine peritoneal macrophages harvested 3–4 days after IP injection of vaccinia virus lysed S91-melanoma tumor cells in vitro; enhanced tumoricidal activity was measured with effector macrophages prepared 5–6 days after vaccinia virus infection. Treatment of virus-elicited macrophages prepared from DBA/2 mice with anti-asialo-GM1 antiserum, anti-Thy 1.2 antiserum or anti-Ia^d antiserum in the presence of complement so that cells sensitized with antibodies were lysed, did not reduce the measured level of tumoricidal activity indicating that macrophages [Ia(–); asialo GM1(–)] and not natural killer cells [asialo GM1(+); Thy 1.2(±)] or T-cells [Thy 1.2(+)] were responsible for mediating the lysis of S91-melanoma tumor cells. When incubated with virus-elicited macrophages but not thioglycollate-elicited macrophages, the ability of S91-melanoma tumor cells, to synthesize DNA was completely blocked. The results of these experiments support the view that one aspect of antitumor immunity enhanced during immunotherapy with vaccinia virus is the activation of macrophages which have cytolytic as well as cytostatic effects on melanoma tumor cells.     

Diversity in the available repertoire of murine antibodies reactive with bromelain-treated isologous erythrocytes

Antibodies specific for bromelain-treated mouse RBC (BrMRBC) are of interest as models of "natural autoantibodies" and because of their primary source is Ly-1+ (CD5+) B cells. In earlier work by others, anti-BrMRBC hybridomas prepared by using CBA or NZB "spontaneously activating" peritoneal B cells were all found to produce mAb with a single common H chain V region sequence, by using a novel gene (VH11p), and a single common L chain V region sequence, a member of the Vk9 group (VkBrMp). We prepared anti-BrMRBC hybridomas by using LPS-activated B10.A splenic B cells in order to reveal the maximum available diversity in this repertoire. Data based on binding studies, Northern blot analyses with V region-specific probes, and mRNA nucleotide sequence analysis indicated that there is combining-site diversity in the repertoire of anti-BrMRBC hybridomas. There was considerable variation in trimethylammonium (a constituent of phosphatidyl choline) binding efficiency, and one of the anti-BrMRBC mAb showed no detectable binding. Northern blot analyses indicated 6 of 11 mAb to be of the VH11p/VkBrMp type, including one dual reactive anti-[BrMRBC + SRBC] mAb. Sequence analyses of the H chain V regions of four of the non-VH11p mAb revealed utilization of four distinct VH, three of which are very similar to the VH expressed by Ly-1+ B cell clones or lymphomas, as reported by others. However, because the VH11p/VkBrMp-type mAb were all relatively efficient at lysing BrMRBC and binding trimethylammonium, we suggest that affinity considerations may determine the selective predominance of B cells with this V region configuration from an available repertoire of considerable diversity.     

Conjugal transfer replication of R64drd11 plasmid DNA in the donor cells of Escherichia coli K-12

Summary Conjugation, the process of genetic transfer requiring cell-to-cell contact, has been the focus of many investigations. In recent years, the molecular aspect of conjugation has been questioned. Since it has been shown that during exponential growth plasmid DNA forms a complex with the folded chromosomal complex (FCC), the relationship of R64drd11 plasmid DNA to the FCC (chromosome plus membrane) during conjugal replication was examined. A cell system was used which allowed specific observation of conjugal events as they occurred in the donor cell. Evidence is presented to show that conjugally replicating R64drd11 covalently closed circular molecules co-sediment with the FCC in neutral sucrose gradients. The use of density gradients to separate DNA from membrane-bound DNA from free membrane, indicate that the membrane is the preferential structure for conjugally replicating plasmid DNA association.     

A revision of Erythrochiton sensu lato (Cuspariinae,Rutaceae)

In the rutaceous subtribe Cuspariinae, species with relatively large, valvate, colored calyces have been assigned to Erythrochiton, but differences in arrangement of leaves, type of inflorescence, union of petals, of filaments, and of carpels, indument of corolla and testa, appendages of anthers, height of the intrastaminal disc, and exine of the pollen argue for the recognition of three genera. Erythrochiton s. str., characterized by often perennating inflorescences, connate, usually glabrous petals, free carpels, tomentulose seeds, and spinulose exine, consists of seven species of which four are new: E. fallax from the eastern flanks of the Andes from Colombia to Bolivia, E. odontoglossus from western Ecuador and adjacent Peru, E. trichanthus from eastern Peru, and E. gymnanthus from Costa Rica. The assignment to Toxosiphon of four species with woolly, coherent petals, connate carpels, glabrous seeds, and reticulate exine necessitates three new combinations: T. carinatus, T. macropodus, and T. trifoliatus. Recognition of a third unispecific genus with opposite simple leaves, sparsely pubescent, coherent, clawed petals, and spinulose exine requires a new genus name, Desmotes, and a new combination, D. incomparabilis.