Purification and properties of a new ribosome-inactivating protein with RNA N-glycosidase activity suitable for immunotoxin preparation from the seeds of Momordica cochinchinensis.

A ribosome-inactivating protein similar to those already known (Stirpe and Barbieri (1986) FEBS Lett. 195, 1-8) was purified from the seeds of Momordica cochinchinensis. This protein, for which the name of momorcochin-S is proposed, is a glycoprotein, has an Mr of approx. 30,000, and an alkaline isoelectric point and can be considered as an iso-form of the previously purified momorcochin from the roots of M. cochinchinensis. Momorcochin-S inhibits protein synthesis by a rabbit-reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and alters rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Momorcochin-S was linked to a monoclonal antibody (8A) against human plasma cells, and the resulting immunotoxin was selectively toxic to target cells.

Ethiopian visceral leishmaniasis patients co-infected with human immunodeficiency virus.

This communication reports 7 Ethiopian visceral leishmaniasis (VL) patients co-infected with human immunodeficiency virus (HIV). The clinical and laboratory findings in 6 patients did not differ from classical VL. All patients had highly elevated anti-leishmanial antibody titres, determined by immunoglobulin G-based enzyme-linked immunosorbent assay; they most probably acquired the Leishmania infection before HIV. Amastigotes were identified in the splenic aspirates of 6 patients and in the lymph node aspirate of the 2 patients whose lymph nodes were examined. The CD4:CD8 lymphocyte ratio was depressed in those patients whose ratio was determined. Most patients showed some initial response to pentavalent antimonial therapy.

Induction of metal-binding proteins by zinc is accompanied by reduction of other proteins including antibodies.

Exposure of carp lymphoid cells to zinc causes formation of metal-binding proteins (metallothioneins). Metallothionein induction is accompanied by a suppression of the production of other proteins. Antibodies are among the proteins which are decreased by zinc exposure. Similar results from in vivo experiments indicate that zinc might affect even fish living in water still regarded as unpolluted.

Ribosome-inactivating proteins from plants inhibit ribosome activity of Trypanosoma and Leishmania.

Ribosomes from Trypanosoma brucei rhodesiense and from Leishmania infantum were isolated and optimal conditions for in vitro translation were established. The effect of ribosome-inactivating proteins extracted from several plants was then assessed in order to identify those suitable for the preparation of immunotoxins against these organisms. Ribosomes from both species were inactivated by some ribosome-inactivating proteins (dianthins, saporins, pokeweed antiviral proteins, and the ribosome-inactivating chain of abrin). The similarity of the effects on the ribosomes from the two species examined indicates that ribosome-inactivating proteins should also be effective in a similar way on ribosomes from other species of Trypanosoma and Leishmania.

Mononuclear cell subpopulations and cytokine levels in human visceral leishmaniasis before and after chemotherapy.

Serum cytokine levels and mononuclear cell subpopulations in the spleen and peripheral blood of patients with visceral leishmaniasis before and after antimony therapy were analyzed. The percentages of activated monocytes/macrophages, T cells, and possibly B cells; of gamma/delta T cell receptor (TCR)-bearing T cells; of CD4- CD8- alpha/beta TCR-bearing T cells; and serum levels of tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN-gamma), and interleukin-6 (IL-6) were high in patients with active visceral leishmaniasis. The proportion of both helper and suppressor CD4+ cells and of cells with NK and cytotoxic T phenotypes were depressed. Successful chemotherapy normalized these parameters with the exception of activated monocytes. Thus, the impaired cell-mediated immunity in human Leishmania donovani infection is primarily due to a decrease in the proportion and possibly the activity of helper CD4+ cells, while suppressor cells do not seem to play a relevant role. TNF alpha, IL-6, and IFN-gamma may prove to be useful markers for monitoring response to therapy.

Phenotype and cytokine expression of intralesional cells in borderline leprosy.

We studied the cytokine profiles and cellular compositions in the lesions of borderline lepromatous (BL) and borderline tuberculoid (BT) leprosy patients in order to ascertain the immunological distinctions between these two groups. Using a modified, reliable, noninvasive, suction-induced blister technique to sample lesions, we determined that CD4+ T cells predominated in BT lesions; whereas CD8+ T cells predominated in BL lesions. However, the numbers of CD8+ per mm2 surface area of the lesion did not differ significantly between the two patient groups. In BT lesions, the elevation in the number of CD4+ cells was paralleled by the levels of soluble interleukin-2 (IL-2) receptor and soluble CD4 in the lesions. The CD4+:CD8+ ratio was 16:1 in BT lesions and 0.36:1 in BL lesions, although this ratio in the peripheral blood was similar in both groups. In addition, cells expressing the CD8 molecule dominated in the TCR-gamma delta subpopulation. The cytokine profiles in the lesions were not as distinctly different between BL and BT patients as were the cellular compositions. However, trends observed included elevated concentrations of IL-6 in BL lesions and elevated TNF-alpha levels in BT lesions.

Effect of ribosome-inactivating proteins on ribosomes from Tetrahymena pyriformis and Acanthamoeba castellanii.

The effect of ribosome-inactivating proteins type 1 (single-chain) and type 2 (two-chain, toxins) on polyphenylalanine polymerization by Tetrahymena pyriformis and Acanthamoeba castellanii ribosomes has been studied. The reaction catalysed by tetrahymena ribosomes was inhibited by two ribosome-inactivating proteins type 1 (dianthin 32 and, less effectively, momordin) whereas the reaction catalysed by amoeba ribosomes was inhibited, in a decreasing order of activity, by three ribosome-inactivating proteins type 1 (dianthin 32, saporin 6 and bryodin) and by two toxins (abrin and volkensin).

Trichokirin, a ribosome-inactivating protein from the seeds of Trichosanthes kirilowii Maximowicz. Purification, partial characterization and use for preparation of immunotoxins.

A protein, here named trichokirin, was extracted from the seeds of Trichosanthes kirilowii and purified by ion-exchange and gel-filtration chromatography. Trichokirin is a basic glycoprotein of apparent relative molecular mass of 27,000 with a strong ribosome-inactivating activity. Alignment of the trichokirin, trichosanthin and momordin N-terminal sequences shows a substantial degree of homology. Trichokirin was conjugated to a monoclonal antibody directed against the Thy 1.2 antigen with the cleavable dimethyl 3,3'-dithiobispropionimidate cross-linking reagent. This immunotoxin selectively killed leukemia cells expressing the Thy 1.2 antigen. The addition of ammonium chloride, which increases the cytotoxicity of ricin A-chain immunotoxins, blocks that of the trichokirin immunotoxin, suggesting that they enter cells by different mechanisms. In vivo studies showed that the pharmacokinetic properties of the trichokirin immunotoxin could be more advantageous than those of the ricin A-chain immunotoxins for in vivo applications.

Ribosome-inactivating proteins from plant cells in culture.

1. Ribosome-inactivating proteins were found in high amounts in one line of cells of Phytolacca americana (pokeweed) cultured in vitro and, in less quantity, in lines of Saponaria officinalis (soapwort) and of Zea mays (corn) cells. 2. The main ribosome-inactivating protein from pokeweed cells was purified to homogeneity. It is a protein with Mr 29,000 and basic pI, similar to the 'pokeweed antiviral protein' (PAP), a ribosome-inactivating protein from pokeweed leaves. We propose to call the pokeweed antiviral protein isolated from pokeweed cells PAP-C. 3. PAP-C inactivates ribosomes in a less-than-equimolar ratio, thus inhibiting protein synthesis by a rabbit reticulocyte lysate with an IC50 (concentration causing 50% inhibition) of 0.067 nM (2 ng/ml), and modifies rRNA in a manner apparently identical to that of ricin and other ribosome-inactivating proteins. It inhibits protein synthesis by intact cells with an IC50 of 0.7-3.4 microM, and is toxic to mice with an LD50 of 0.95 mg/kg.