Characterization of heterologously expressed recombinant retinoic acid receptors with natural or synthetic retinoids

The first step in retinoid action is binding to their nuclear receptors. Therefore, characterization of binding characteristics of retinoids is of major importance. Human retinoic acid receptors alpha (hRAR alpha), hRAR beta, and mouse RAR gamma (mRAR gamma) were expressed heterologously in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein. The expressed fusion proteins were functional and bound specifically to the all-trans-retinoic acid (RA). The dissociation constants (Kd) for RA were 1.4 nM for GST-hRAR alpha, 1.4 nM for GST-hRAR beta, and 3.3 nM for GST-mRAR gamma, respectively. The fusion proteins were further used for competitive displacement assays to determine the displacement constant (DC50) for other selected retinoids. All-trans-RA and 4-oxo-all-trans-RA have high affinity with all three receptors (DC50 = 0.8-55 nM). The 13-cis RA binds to hRAR alpha with low affinity, but not to other RARs evaluated here. All-trans-N-ethylretinamide, all-trans-retinylacetate, and an ethyl ester of tetrahydronaphthalene derivative had no affinity to any RARs. The hRAR alpha and mRAR gamma receptors did not bind a naphthalene carboxylic acid derivative of RA, but hRAR beta binds this chemical with high affinity. Results indicated that the three recombinant proteins were functional in binding various RA congeners. The affinity and binding data of these retinoids were compared to their observed teratogenic activity.     

Phagocytosis of nonopsonized Cryptococcus neoformans by swine microglia involves CD14 receptors

The interaction of the opportunistic fungus Cryptococcus neoformans with swine microglia was studied in vitro in the presence and absence of anti-CD14 monoclonal antibodies. In the absence of anti-CD14 antibodies, 36% of microglia had phagocytized nonopsonized, encapsulated cryptococci after 2 hr incubation (effector-to-target ratio, 1:50). Preincubation of microglia with anti-CD14 antibodies resulted in a 63% reduction of phagocytosis. These findings suggest that CD14 receptors facilitate uptake of nonopsonized C, neoformans by resident macrophages within the brain.     

Cryptococcus neoformans resides in an acidic phagolysosome of human macrophages

Recently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogen Cryptococcus neoformans. Both of these agents raise the lysosomal pH, which suggested that the increased antifungal activity was a function of alkalinizing the phagolysosome. Moreover, there was an inverse correlation between growth of C. neoformans in cell-free media and pH. These data suggested that C. neoformans was well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containing C. neoformans. Fungi were labeled with the isothiocyanate derivatives of two pH-sensitive probes: fluorescein and 2',7'-difluorofluorescein (Oregon Green). These probes have pKas of 6.4 and 4.7, respectively, allowing sensitive pH detection over a broad range. The phagosomal pH averaged approximately 5 after ingestion of either live or heat-killed fungi and remained relatively constant over time, which suggested that C. neoformans does not actively regulate the pH of its phagosome. The addition of 10 and 100 microM chloroquine resulted in increases in the phagosomal pH from a baseline of 5.1 up to 6.5 and 7.3, respectively. Finally, by immunofluorescence, colocalization of C. neoformans and the MDM lysosomal membrane protein LAMP-1 was demonstrated, establishing that fusion of C. neoformans-laden phagosomes with lysosomal compartments takes place. Thus, unlike many other intracellular pathogens, C. neoformans does not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.     

Comparison of heterologously expressed human cardiac and skeletal muscle sodium channels

In this study we have expressed and characterized recombinant cardiac and skeletal muscle sodium channel alpha subunits in tsA-201 cells under identical experimental conditions. Unlike the Xenopus oocyte expression system, in tsA-201 cells (transformed human embryonic kidney) both channels seem to gate rapidly, as in native tissue. In general, hSkM1 gating seemed faster than hH1 both in terms of rate of inactivation and rate of recovery from inactivation as well as time to peak current. The midpoint of the steady-state inactivation curve was approximately 25 mV more negative for hH1 compared with hSkM1. In both isoforms, the steady-state channel availability relationships ("inactivation curves") shifted toward more negative membrane potentials with time. The cardiac isoform showed a minimal shift in the activation curve as a function of time after whole-cell dialysis, whereas hSkM1 showed a continued and marked negative shift in the activation voltage dependence of channel gating. This observation suggests that the mechanism underlying the shift in inactivation voltage dependence may be similar to the one that is causing the shift in the activation voltage dependence in hSkM1 but that this is uncoupled in the cardiac isoform. These results demonstrate the utility and limitations of measuring cardiac and skeletal muscle recombinant Na+ channels in tsA-201 cells. This baseline characterization will be useful for future investigations on channel mutants and pharmacology.     

Virulence factors of Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast which causes cryptococcosis, a disease typified by an initial pulmonary infection which can disseminate to cause a life threatening meningoencephalitis. Although the disease may occur in individuals who show no evidence of immunosuppression it has had it most significant impact as an infection in patients with AIDS. Research into the potential virulence factors of this yeast has recently attracted particular attention. Capsule synthesis has been the focus of most interest and it is now established as a major virulence determinant. The mechanisms by which the capsule and capsular material effect the immune response have now largely been elucidated, and the genes underlying capsular synthesis are now under investigation. The isolation of mutants incapable of melanogenesis have implicated this process in the pathogenesis of C. neoformans infections, and evidence suggests that the production of melanin protects the yeast against oxidant induced damage. There is also some genetic evidence for the potential involvement of temperature tolerance and mating types in the virulence of this encapsulated yeast. The roles of other potential C. neoformans virulence determinants are more speculative; these include proteinase production, release of polyol metabolites, interaction with hormones, adherence and production of mannoproteins. The involvement of housekeeping enzyme systems in the maintenance of infection by C. neoformans is now also under active investigation.     

Functional expression of olfactory-adrenergic receptor chimeras and intracellular retention of heterologously expressed olfactory receptors

Replacing the G-protein-coupling domains of the beta 2-adrenergic receptor with homologous domains of putative olfactory receptors produced chimeric receptors which were able to stimulate pigment dispersion in Xenopus melanophores, a G-protein-mediated pathway. A multiple replacement chimera containing the second, third and C-terminal cytoplasmic domains of receptor OR5 elevated cyclic adenosine 3':5'-monophosphate (cAMP) and suppressed production of inositol phosphates. Co-expression of G alpha olf did not alter the strength of response of this chimera. A novel rat olfactory receptor cDNA (U131) was isolated and sequenced. Expression of U131 and OR5 constructs containing an N-terminal epitope-tag or C-terminal fusion to green fluorescent protein occurred in an intracellular network but not in the plasma membrane of heterologous cells. Similarly treated beta 2-adrenergic receptors were functional and were observed in the plasma membrane and the intracellular network. These results demonstrate that the putative cytoplasmic domains of olfactory receptors are capable of functional interaction with heterologous G-proteins of the G alpha s subtype. Instead, the absence of these receptors from the plasma membrane of heterologous cells appears to explain our inability to determine if odorants can activate the olfactory receptor clones. We hypothesize that the olfactory receptors have requirements for maturation and targeting to the plasma membrane that are different from most other G-protein-coupled receptors.     

Wortmannin-sensitive activation of p70s6k by endogenous and heterologously expressed Gi-coupled receptors

In order to study the regulation of the ribosomal protein S6 kinase, p70s6k, by G protein-coupled receptors, Rat-1 fibroblasts were stably transfected with two versions of the alpha2 adrenergic receptor. Stimulation of clone 1C cells, which express 3.5 pmol/mg of protein of the human alpha2C10 receptor, with the alpha2 agonist UK 14304 led to a transient increase in p70s6k activity. UK 14304 also activated p70s6k in a clone expressing the porcine alpha2A receptor (400 fmol/mg of protein). Lysophosphatidic acid (LPA), acting through endogenous G protein-coupled receptors, also activated p70s6k in alpha2 receptor-transfected and in nontransfected cells. Activation of p70s6k by both UK 14304 and LPA was accompanied by increased phosphorylation of the protein. Rapamycin completely blocked the activation of p70s6k by both agents. Activation of p70s6k by UK 14304 and by LPA, but not by platelet-derived growth factor (PDGF), was blocked by preincubation of cells with pertussis toxin. Wortmannin, a selective inhibitor of phosphoinositide (PI) 3-OH kinase, prevented activation of p70s6k by UK 14304, LPA, and PDGF. These data indicate that p70s6k is regulatable by Gi-coupled receptor agonists in a pertussis toxin-sensitive fashion in Rat-1 fibroblasts and that activation of p70s6k by such agents appears to involve an isoform of PI 3-kinase.     

Exposure to Cryptococcus neoformans var. gattii--a seroepidemiological study

The lateral cephalogram is a film that is routinely used in orthodontics. Orthodontists should therefore be familiar with the normal radiographic appearance of the skull as seen on the lateral cephalogram. We present a case of an enlarged sella turcica that was discovered during routine orthodontic workup. Referral and further investigation led to a diagnosis of a prolactinoma.     

Characterization of anticapsular monoclonal antibodies that regulate activation of the complement system by the Cryptococcus neoformans capsule

PURPOSE: To evaluate the effectiveness of adding interferon (IFN) alfa-2b to chemotherapy in the induction treatment of low-grade non-Hodgkin's lymphoma (NHL), and to assess the role of maintenance IFN. PATIENTS AND METHODS: A multicenter, two-phase controlled trial with double randomization was conducted in 155 patients with low-grade NHL. In the first randomization, 78 patients received cyclophosphamide, vincristine, and prednisone (CVP) and IFN, 3 MU/m2 three times a week for 3 months, and 77 patients received CVP alone. Responding patients were randomized to receive IFN for 1 year versus observation. RESULTS: Of 144 assessable patients, 73 received CVP + IFN and 71 received CVP. Responses were similar: CVP + IFN 79% versus CVP 76% (P = .62). The number of patients who did not complete the treatment was higher in the CVP + IFN group than in the CVP group (18% v 4%; P = .009), although the received dose-intensity of chemotherapy was comparable. Duration of response and progression-free survival (PFS) were significantly higher in the CVP + IFN group than in the CVP group (P = .0004). However, we observed no differences in overall survival (OS) (P = .30), with a median follow-up for the surviving patients of 3 years. Grade 3/4 granulocytopenia was the most frequent toxicity and was similar in both groups (33% v32%). Eighty-three (74%) of the 112 responding patients were randomized to maintenance IFN or observation. The duration of response was similar between 42 patients that received IFN compared with 41 control patients (P = .83), independently of treatment previously administered. CONCLUSION: Adding IFN alfa-2b to induction CVP in low-grade NHL did not induce a higher response rate, but it significantly increased the duration of the responses. We found significant differences in PFS that favored the patients who received CVP + IFN, but not in OS. To date, no additional benefit has been seen from the administration of IFN for maintenance.     

Cryptococcus neoformans and Candida albicans regulate CD4 expression on human monocytes

This study examined the capability of Candida albicans and Cryptococcus neoformans to modulate CD4 expression on human monocytes. C. albicans and an acapsular strain of C. neoformans induced higher levels of CD4 expression than encapsulated strains. Purified glucuronoxylomannan did not regulate CD4 expression on monocytes, but down-regulation of CD4 expression compared with stimulation by acapsular C. neoformans alone was observed when glucuronoxylomannan was used in combination with acapsular C. neoformans. The ability of opsonic factors to facilitate fungal-mediated CD4 overexpression suggests that binding or internalization (or both) of the yeast cells is a critical event. Protein synthesis was required, excluding redistribution of the intracellular pool of CD4 receptors to the cellular surface as the sole possible mechanism. Results demonstrate a new effect of fungi on professional phagocytic cells and raise the possibility that modulation of CD4 could influence gp120-mediated human immunodeficiency virus entry.     

Phenotypic and genotypic differentiation of several human and avian isolates of Cryptococcus neoformans

The Cryptococcus neoformans strains isolated from two human cases could be diagnosed as Cr. neoformans var. neoformans by differentiation on the basis of their characteristics determined by proline, canavanine and EDTA urease tests. The results of the serovar assignment were: for the isolate from the meningoencephalitis patient with lethal outcome, serovar A; for the strain isolated from the osteomyelitis patient with benign course, serovar D. Also, the PCR fingerprinting using primers (GACA)4, (CAC)5 and FM 1 resulted in a clear and reproducible assignment of the Cr. neoformans strains to the varieties neoformans and gattii, respectively, and, in addition, it confirmed the serovar assignment. No statistically confirmed differences in virulence between the osteomyelitis and the meningoencephalitis strain could be established by i.v. testing in mice, nor did the PCR with several primers provide any clues to a genetically determined higher virulence of the meningoencephalitis strain. The different classification as serovars A and D does not allow any conclusions concerning different virulence. It was not possible to retrospectively establish the sources of infection of the two Cr. neoformans infections, but pigeon faeces may well have played a role as a reservoir for one of the illnesses.     

Bivalency is required for anticapsular monoclonal antibodies to optimally suppress activation of the alternative complement pathway by the Cryptococcus neoformans capsule

Encapsulated cells of Cryptococcus neoformans are potent activators of the alternative complement pathway. Previous studies found that monoclonal antibodies (MAbs) specific for the major capsular polysaccharide, termed glucuronoxylomannan (GXM), can markedly suppress the ability of the capsule to accumulate C3 from normal human serum via the alternative pathway. The present study examined the abilities of F(ab)2 and Fab fragments of three MAbs (MAbs 439, 3C2, and 471) to mediate the suppressive effect. The results showed that F(ab)2 fragments of all three MAbs suppressed activation and binding of C3 via the alternative pathway in a manner similar to that of intact antibodies. In contrast, Fab fragments of MAb 439 and MAb 3C2 showed no suppressive activity, and Fab fragments of MAb 471 were markedly reduced in suppressive activity. Indeed, there was an earlier accumulation of C3 on encapsulated cryptococci in the presence of the Fab fragments. Study of subclass switch families of MAb 439 and MAb 471 found that MAbs of an immunoglobulin G (IgG) subclass with increased flexibility in the hinge region (IgG2b) had less suppressive activity than MAbs of IgG subclasses with less flexibility (IgG1 or IgG2a). Taken together, these results indicate that cross-linking of the capsular matrix is an essential component in suppression of the alternative complement pathway by anti-GXM MAbs.     

Direct activity of human T lymphocytes and natural killer cells against Cryptococcus neoformans

Lymphocytes constitute a critical component of host defenses against cryptococcosis. Previously, we demonstrated that human lymphocytes cultured with interleukin-2 formed conjugates with, and directly inhibited the growth of, Cryptococcus neoformans. Here, we explore the anticryptococcal activity of freshly isolated, highly purified populations of human peripheral blood lymphocytes. Lymphocytes were incubated with encapsulated C. neoformans for 24 h, after which the lymphocytes were lysed, dilutions and spread plates were made, and CFU were counted. Fungistasis was determined by comparing growth in wells with and without lymphocytes. Nylon wool-nonadherent peripheral blood mononuclear cells (NWNA PBMC) were highly fungistatic, even if either T cells or natural killer (NK) cells were depleted by panning. A mixed population of T cells and NK cells, obtained by rosetting NWNA PBMC with sheep erythrocytes, completely inhibited cryptococcal growth, whereas the nonrosetting cells had little fungistatic activity. CD4+, CD8+, and CD16/56+ lymphocytes, isolated by positive immunoselection, had potent growth-inhibitory activity. In contrast, purified B cells had no activity. Fungistasis was seen even in the absence of opsonins. Antifungal activity was markedly diminished when surface receptors on NWNA PBMC were cleaved by treatment with trypsin or bromelain. Supernatants from stimulated lymphocytes or concentrated lymphocyte sonicates were not active. Lymphocyte-mediated fungistasis was seen with two different strains of C. neoformans. CD4+, CD8+, and CD16/56+ lymphocytes formed conjugates with C. neoformans, as observed under Nomarski differential interference contrast microscopy and videomicroscopy. These data demonstrate that freshly isolated peripheral blood T cells and NK cells have the capacity to bind and directly inhibit the growth of C. neoformans.     

Isolation of specific DNA probes from Cryptococcus neoformans

OBJECTIVE: To construct plasmid library and screen specific DNA probes for Cryptococcus neoformans. METHODS: Serotype A Cryptococcus neoformans was used as the study strain, plasmid pUC18 as vector, and Escherichia coli JM103 as host cell. The plasmid library of cryptococcus neoformans was constructed (pCN). Other pathogenes causing affection diseases which should be distinguished from cryptococcusis clinically, and other fungi similar to Cryptococcus neoformans with physiological and biochemical characteristics were used as a distinguishing system, specific colonies were screened by hibridization in double steps. RESULTS: The inserts of the library were 280 to 1800 base pairs and 580 base pairs in average length. Repeated sequence was 32.43% and single copy sequence was 67.57% in genome of cryptococcus neoformans respectively. Three specific colonies were isolated from the library. Colony pCNII A6 was serotype A specific, pCNII B5 species specific and pCNIII G1-specific for var. neoformans. CONCLUSION: A rapid diagnosis of Cryptococcus neoformans infection at early stage can be made by using species-specific probe, and serotype and variaty of neoformans and gattii be distinguished in epidemic study.     

Effects of Aspergillus sulphureus mycotoxins on Cryptococcus neoformans

Many studies have evaluated the toxicity of mycotoxins to mammals, but there is little information on their action against fungal cells, even although mycotoxins are frequently active against fungi in nature. A crude extract of Aspergillus sulphureus was tested for its growth-inhibitory effect on Cryptococcus neoformans. The reduction in cell growth of Cr. neoformans caused by the extract was dose dependent. Using a liquid medium containing 2% A. sulphureus extract, the RNA content of Cryptococcus amounted to about 60% of that of non-treated cells. Capsule thickening, demonstrated biochemically and with cytological stains, occurred at doses that had minimal effect on cell growth and RNA content. Our results suggest that the virulence of Cr. neoformans may increase in cases of coenobiosis with A. sulphureus, which is theoretically possible in places where corn-fed pigeons are numerous.     

CD4 lymphocyte count in HIV-positive persons exposed to Cryptococcus neoformans

A report is presented on four HIV-positive homosexual men examined after several months of exposure during cleaning of a flat from masses of pigeon droppings heavily colonized by Cryptococcus neoformans. Only one out of the four persons, with a CD4 lymphocyte count of 50/microL, fell sick from systemic cryptococcosis, but not the others, with CD4 lymphocyte counts of 180, 250, and 630/microL, respectively; they remained clinically and mycologically inconspicuous and free from C. neoformans. Open questions in view of the epidemiology of opportunistic pathogens in AIDS are discussed with regard to the CD4 cell count as a parameter indicating a predisposition for cryptococcosis as an airborne AIDS-defining opportunistic infection. This has been confirmed by specific cultural diagnosis of the agent in both the environment and the patient. Already in 1987/88, the probable source of infection had been the subject of epidemiological studies on C. neoformans in Berlin.     

Chloroquine induces human mononuclear phagocytes to inhibit and kill Cryptococcus neoformans by a mechanism independent of iron deprivation

Infections due to Cryptococcus neoformans are common in AIDS patients. We investigated the effect of chloroquine, which raises the pH of phagolysosomes, on the anticryptococcal activity of mononuclear phagocytes. C. neoformans multiplied within monocyte-derived macrophages (MDM) in the absence of chloroquine but were killed with the addition of chloroquine. Ammonium chloride was also beneficial, suggesting that effects were mediated by alkalinizing the phagolysosome. Chloroquine inhibits growth of other intracellular pathogens by limiting iron availability. However, chloroquine-induced augmentation of MDM anticryptococcal activity was unaffected by iron nitriloacetate, demonstrating that chloroquine worked by a mechanism independent of iron deprivation. There was an inverse correlation between growth of C. neoformans in cell-free media and pH, suggesting that some of the effect of chloroquine on the anticryptococcal activity of MDM could be explained by relatively poor growth at higher pH. Chloroquine enhanced MDM anticryptococcal activity against all tested cryptococcal strains except for one large-capsule strain which was not phagocytosed. Positive effects of chloroquine were also seen in monocytes from both HIV-infected and -uninfected donors. Finally, chloroquine was therapeutic in experimental cryptococcosis in outbred and severe combined immunodeficient mice. Thus, chloroquine enhances the activity of mononuclear phagocytes against C. neoformans by iron-independent, pH-dependent mechanisms and is therapeutic in murine models of cryptococcosis. Chloroquine might have clinical utility for the prophylaxis and treatment of human cryptococcosis.     

Methylation of mercaptopurine, thioguanine, and their nucleotide metabolites by heterologously expressed human thiopurine S-methyltransferase

Thiopurine S-methyltransferase (TPMT), a cytosolic enzyme that exhibits genetic polymorphism, catalyzes S-methylation of mercaptopurine (MP) and thioguanine (TG), yielding S-methylated nucleobases that are inactive, whereas S-methylated nucleotides of these thiopurines are cytotoxic. A yeast-based heterologous expression system was therefore used to characterize human TPMT-catalyzed methylation of MP, TG, and their principal nucleotide metabolites [thioinosine monophosphate (TIMP) and thioguanosine monophosphate (TGMP), respectively]. MP, TG, TIMP, and TGMP were all substrates for human TPMT, exhibiting similar Michaelis-Menten kinetic parameters (Km, 10.6-27.1 microM; Vmax, 31-59 nmol/min/mg of TPMT). Consistent with these kinetic parameters, human leukemia cells (CEM) incubated for 24 hr with 10 microM MP or TG accumulated significantly higher (2.3-fold, p = 0.01) concentrations of methyl-TIMP after MP incubation than methyl-TGMP after TG incubation, due to the 2.7-fold higher concentration of TIMP after MP incubation, compared with TG nucleotides (TGN) after TG incubation. Moreover, intracellular accumulation of TGN was 2.5-fold greater after TG incubation than after MP incubation (p = 0.01). These data establish that MP, TG, and their principal nucleotide metabolites are comparable substrates for polymorphic TPMT, and they demonstrate significant differences in the accumulation of active TGN and methylated nucleotides when leukemia cells are treated with MP versus TG.     

Pathogenesis of pulmonary Cryptococcus neoformans infection in the rat

The pathogenesis of Cryptococcus neoformans pulmonary infection in the rat was studied after intratracheal inoculation. Lungs were examined at various times following infection for histopathology in conjunction with macrophage markers, proliferating cell nuclear antigen (PCNA), and capsular glucuronoxylomannan (GXM) antigen. Serum GXM, immunoglobulin M (IgM) and IgG titers and organ fungal burden were compared with pathological findings. C. neoformans organisms were in the lung parenchyma 2 h postinoculation, and GXM antigen was present in surrounding tissues shortly thereafter. Extrapulmonary dissemination occurred early in infection. Two phases of host cellular inflammatory response were discernible: early local macrophage recruitment at 2 to 4 days followed by granulomatous inflammation, which reached maximum intensity 14 days after infection. The granulomatous phase was preceded by lymphocyte influx with macrophage proliferation and maturation into epithelioid histiocytes; this was paralleled by a shift of yeasts from extracellular to intracellular spaces. Tissue IgG deposits, serum IgG to GXM, and localization of tissue GXM immunoreactivity to epithelioid cells were noted at 2 to 4 weeks. A 10-fold decrease in lung fungal burden occurred 25 days postinfection and was associated with resolving granulomas, fewer proliferating cells, and decreased tissue GXM. The present study demonstrates that (i) C. neoformans penetrates the lung parenchyma shortly after infection; (ii) immunocompetent rats control pulmonary cryptococcosis efficiently, with minimal extrapulmonary dissemination and low levels of serum GXM; and (iii) macrophage activation is likely to play a crucial role in limiting C. neoformans infection in the rat lung.     

The cell wall and membrane of Cryptococcus neoformans possess a mitogen for human T lymphocytes

The mechanism of human T-lymphocyte activation by the pathogenic yeast Cryptococcus neoformans has not been established. Previous investigations have suggested that C. neoformans contains a mitogen for T lymphocytes, while other investigators have attributed lymphocyte proliferation in vitro to a recall antigen. Because of the potential importance of the mechanism of T-cell activation for our understanding of the immune response to C. neoformans, the present studies were performed to determine whether C. neoformans contains a mitogen for T lymphocytes. C. neoformans stimulates fetal blood lymphocytes to proliferate and stimulates proliferation of CD45RA+ cells from adults, indicating that it stimulates naive T cells. The T-cell response to C. neoformans was dependent upon the presence of accessory cells. However, allogeneic cells were sufficient for accessory cell function, indicating that the response was not major histocompatibility complex restricted. The percentage of T cells in the cell cycle was higher than that with the recall antigen tetanus toxoid but lower than that with the mitogenic lectin phytohemagglutinin A or the superantigen Staphylococcus enterotoxin B. Precursor frequency analysis established that 1 in 7,750 +/- 2, 270 T cells proliferated in response to the cryptococcal cell wall and membrane. Compared to the case for most mitogens or superantigens, the proliferative response is late and the number of T cells that enter the cell cycle and the precursor frequency are low, indicating that the mitogenic effect is modest. However, the mitogenic effect of C. neoformans should be considered when interpreting the immune response to C. neoformans, since even weak mitogens can have profound effects on host defense.