Antonio Cassone

Agglutination of blastospores of Candida albicans by concanavalin A and its relationship with the distribution of mannan polymers and the ultrastructure of the cell wall.

Blastospores of Candida albicans were readily agglutinated by Concanavalin A (Con A) owing to the specific binding of this lectin to the mannan receptors of the cell surface. When mannan was extracted from the cell wall by neutral buffers, alkali and acid, the agglutination was decreased or lost depending on the degree of extraction. A relatively mild alkali treatment was sufficient to derange the multilayered wall organization and transform it into a uniform, medium-density structure having about the same thickness as the untreated wall. After a more drastic extraction, all the electron-dense components of the wall were lost, the residual, alkali-insoluble wall fabric being completely electron-transparent and of about the same thickness as the inner wall region of untreated cells. Thiol-reducing agents like mercaptoethanol or dithiothreitol also extracted wall materials, an effect which was enhanced by pronase. After dithiothreitol-pronase treatment, the outer wall layers were removed but the inner wall region was not apparently damaged and some electron-dense components remained. None of these treatments significantly affected blastospore agglutination by Con A--this was reduced (but not abolished) only by the sequential action of pronase and helicase, which led to sphaeroplast formation. These sphaeroplasts showed a varied amount of residual wall consisting of evenly distributed, fibrogranular components. Two main conclusions were drawn from these results: (i) mannan polymers extend throughout the wall of the blastospore of C. albicans; (ii) the layering of the wall, as seen by ordinary fixation and staining for electron microscopy, essentially reflects the distribution of the various alkali-soluble complexes, at different levels, both over and in the rigid, glucan-chitin matrix.

Immunoadjuvant effects of Candida albicans and its cell wall fractions in a mouse lymphoma model

SummaryChemical, ultrastructural, and immunoadjuvant properties of Candida albicans (CA) and of a number of its fractions have been characterized through the analysis of the antitumor activity of soluble and insoluble cell wall components.CD2F1 mice were inoculated IP with Moloney virus-induced lymphoma LSTRA and treated with bis-1-chloroethyl-nitrosurea (BCNU) on day +5 after tumor challenge. A significant increase of the antitumor efficacy of BCNU treatment was found in mice inoculated with CA as immunoadjuvant on days −14 and +1 (−14/+1 schedule) with respect to tumor challenge.However, no significant difference in survival time was found between mice treated with BCNU alone and those treated with BCNU plus either soluble mannan or glucan-protein fractions extracted from CA and administered according to −14/+1 or −7/+1 schedules. On the other hand, mice treated with BCNU plus the insoluble glucan fraction (wall ‘ghosts’) given on days −14/+1 or even on day −7 only (i.e., without boosting after tumor challenge) survived longer than animals treated with BCNU alone.The immunoadjuvant effect of CA and of other ‘classic’ immunoadjuvants, such as BCG and Corynebacterium parvum, was completely abolished by total-body irradiation (400 R) given 5 h before the first administration of the agent on day −7 prior to tumor challenge.These results indicate that: (a) the minimal structure required for the expression of the immunoadjuvant effect of CA is the insoluble, β-glucan component of the cell wall; (b) the soluble components of CA cell wall (i.e., mannan and glucan-protein) per se do not show any detectable immunoadjuvant effect in the present animal-tumor system; they may, however, ‘modulate’ this effect, as shown by the fact that whole CA, but not the insoluble β-glucan, needs a boosting injection for the expression of its immunoadjuvant properties; (c) the immunoadjuvanticity of CA is radiosensitive.

Cell Wall of Candida Albicans and Host Response

Modulation in chemistry and organization of cell wall macromolecules play decisive roles in the morphogenic processes and virulence of Candida albicans. Cell wall components also have a diversified range of effects on the hosts immune system, including immunopotentiating or immunodepressing activities. Mannan, mannan-protein, and glucan fractions have been especially studied in this context. In in vitro cultured human peripheral blood mononuclear cells, a mannan-protein fraction (GMP) from the cell wall of the yeast form acted as a strong antigenic activator by stimulating lymphokine production and lymphocyte proliferation. Cytolytic effectors active against several tumor targets were also generated. In the mouse, GMP was a strong inducer of natural killer lymphocytes. Other cell wall components, mostly the insoluble beta-glucan, modulated the activity of macrophages and monocyte precursors. Some of the immunomodulating properties of artificially extracted components were shared, even with greater potency, by antigens which were released from C. albicans during its growth and hyphal morphogenesis. Altogether, the range of the immunoresponses elicited and the intensity of the observed effects are such as to individuate in this human indigenous fungus a microorganism capable of profoundly affecting the hosts immune system.

Inhibition of uridine transport in cultured mammalian cells by theophylline.

Theophylline (theobromine, caffeine) reversibly inhibits the incorporation of labeled RNA precursors both in confluent 37 RC and in exponentially growing HeLa cells. As measured in 37 RC after 2 h labeling, 20 mM theophylline reduces the incorporation of [3H]UTP and [14C]uridine into acid-precipitable material to 5% and 9% of the control, respectively. This reduction is paralleled by a comparably lowered incorporation of the same precursors into the acid-soluble pool. The initial rate of incorporation into total cell material is similarly affected by theophylline, the inhibition being of a simple competitive type. Theophylline does not alter the turnover rate of pulse labeled RNA during actinomycin D chase nor does it preclude the utilization of the endogenous pool of nucleoside phosphates. Upto a concentration of 10 mM, it does not inhibit uridine kinase neither in 37 RC nor in HeLa cells. The mentioned inhibitory effects of theophylline cannot be mimicked by exogenously added cyclic AMP. All the data support the conclusion that theophylline inhibits the transport of uridine into the cell.

Long-Term Evaluation of Cellular Immunity during Antiretroviral Therapy and Immunization with Human Immunodeficiency Virus Type 1 (HIV-1) Env Glycoprotein in HIV-1-Infected Persons

Cellular immune responses to human immunodeficiency virus type 1 (HIV-1) antigens, microbial recall antigens, and CD3 monoclonal antibody were studied in HIV-1-infected asymptomatic patients in a phase II, double-blind trial of immunization with recombinant HIV-1 gp160 in or not in association with zidovudine. A vigorous and persistent lymphoproliferative response (LPR) to HIV-1 Env antigens was observed in vaccinated patients. Neither Env-specific lymphocyte cytotoxicity nor LPR to recall antigens was significantly influenced by gp160 administration. The induction of LPRs to HIV-1 envelope proteins did not show positive effects on the course of HIV-1 infection. Patients treated with zidovudine alone or in combination with the immunogen showed improvement of T lymphocyte responses and transient reduction of viremia. These results suggest that antiretroviral therapy is more beneficial than immunization with gp160 and should always be considered in association with future vaccination and immunotherapeutic interventions in HIV-1-infected subjects.

Enhanced uptake of actinomycin d in cultured mammalian cells by the anti-inflammatory, non-steroid drug benzydamine

Abstract The anti-inflammatory, non-steroid drug benzydamine, used at non-toxic concentrations ranging from 10 to 50 μg ml −1 , markedly enhanced the uptake of the antiblastic drug actinomycin D in several lines of cultured mammalian cells including tumor cells. In particular, the synthesis of RNA of cells derived from African Green Monkey Kidney, which showed a degree of resistance to actinomycin D, was rendered sensitive to this drug by benzydamine. The nucleocytoplasmic distribution of actinomycin D at equilibrium was not altered by benzydamine, nor did this drug increase the amount of actinomycin D taken up by isolated nuclei. Experiments performed with radiolabeled benzydamine showed that the binding of the drug to the cell is rapid but promptly reversible, almost all bound material sedimenting at 100,000g in the ultracentrifuge. The experimental evidence favours the idea that benzydamine exerts the described effects by interacting with plasmamembrane and allowing for a more effective penetration of actinomycin D into the cell.

Antitumor Adjuvants from Candida Albicans : Effects on Human Allogenic T-Cell Responses “In Vitro”

Candida albicans (CA) and other yeasts have recently been shown to act as strong, non-specific immunoadjuvants in combined chemoimmunotherapy in a mouse lymphoma model (1,2). Several cell wall fractions of distinct antigenicity and chemical composition have been used in “in vivo” experiments and showed a differential capacity of stimulating both anticancer effects and immunoresponsiveness (2). Other pieces of experimental evidence strongly suggest that the overall immunostimulating activity of yeast adjuvants depend on the potentiation of classical T-cell responses against products of minor histocompatibility loci or tumor-associated transplantation antigens of experimental, virus-induced tumors (2,3).