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.The capsule of the pathogenic yeast Cryptococcus neoformans is a powerful activator of the alternative complement pathway (8, 16). Incubation of encapsulated cryptococci in normal human serum (NHS) leads to deposition of 10⁷ to 10⁸ molecules of C3 onto the typical yeast cell (18, 38); the capsule itself is the site for C3 binding (19, 21). Such activation and binding of C3 is due solely to the action of the alternative pathway (20, 21, 37). Binding of C3 via the alternative pathway in NHS is characterized by a delay of approximately 4 to 6 min before bound C3 is readily detectable (20).The major component of the cryptococcal capsule is the high-molecular-weight polysaccharide glucuronoxylomannan (GXM). Several anti-GXM monoclonal antibodies (MAbs) have been shown to provide a measure of protection in a murine model of cryptococcosis (9, 24, 30). In the accompanying report, we have examined the ability of anti-GXM MAbs to initiate the classical pathway, leading to accelerated deposition of C3 onto the yeast (17). These studies showed that most anti-GXM MAbs promote early deposition of C3 fragments into the capsule. However, depending on the epitope specificity of the MAb, some anti-GXM MAbs markedly reduced the apparent rate of amplification of bound C3, with the net result that fewer C3 molecules bound to the cell over a 20- to 30-min incubation period than would have bound in the absence of the antibody. When classical pathway initiation was blocked by the use of EGTA to chelate Ca²⁺ (12, 28), antibodies with the suppressive epitope specificity almost completely blocked the normal alternative pathway activation and binding of C3 that would have occurred in the absence of the MAbs.The ability of an antibody to block antibody-independent activation of the alternative pathway is without an obvious parallel in the literature. There are several potential mechanisms for antibody-mediated suppression. First, the antibody could bind to and occlude specific sites on the capsule that might be preferred acceptors for metastable C3b. Second, the capsule could contain specific domains that regulate the ability of the capsule to activate the alternative pathway. Antibodies specific for such regulatory domains could influence the ability of the cell to activate the alternative pathway. Finally, multivalent antibody could cross-link the capsule in a manner that prevents effective amplification. For example, binding of a multivalent antibody could reduce the ability of metastable C3b to diffuse from sites of C3 convertase activity. We have reasoned that the first two mechanisms for antibody-induced suppression of C3 binding would be mediated by intact antibody, F(ab)₂ fragments of the antibody, and Fab fragments. In contrast, inhibition that is dependent on cross-linking of the capsular matrix would be mediated by intact antibodies and F(ab)₂ fragments but not by Fab fragments.The objective of our study was to examine three anticapsular MAbs that suppress alternative pathway-dependent C3 binding. The suppressive activities of intact antibodies, F(ab)₂ fragments, and Fab fragments were compared. The results showed that intact antibodies and F(ab)₂ fragments of the antibodies suppressed accumulation of C3 fragments on the capsule. In contrast, Fab fragments of the suppressive antibodies showed markedly reduced or no ability to block alternative pathway activation by the capsule; indeed, Fab fragments derived from suppressive antibodies accelerated activation and binding of C3 via the alternative pathway.     

The Capsule of Cryptococcus neoformans Reduces T-Lymphocyte Proliferation by Reducing Phagocytosis, Which Can Be Restored with Anticapsular Antibody

Cell-mediated immunity is critical for the host defense to Cryptococcus neoformans, as demonstrated by numerous animal studies and the prevalence of the infection in AIDS patients. Previous studies have established that the polysaccharide capsule contributes to the virulence of C. neoformans by suppressing T-lymphocyte proliferation, which reflects the clonal expansion of T lymphocytes that is a hallmark of cell-mediated immunity. The present studies were performed to identify the major mechanism by which polysaccharide impairs lymphocyte proliferation, since capsular polysaccharide has the potential to affect the development of T-lymphocyte responses by stimulating production of interleukin-10 (IL-10), inhibiting phagocytosis, and inducing shedding of cell surface receptors. We demonstrate that polysaccharide inhibits lymphocyte proliferation predominantly by blocking uptake of C. neoformans, which is crucial for subsequent lymphocyte proliferation. In addition, we show that polysaccharide did not suppress lymphocyte proliferation via an IL-10-dependent mechanism, nor did it affect critical surface receptor interactions on the T cell or antigen-presenting cell. Having established that polysaccharide impairs phagocytosis, we performed studies to determine whether opsonization with human serum or with anticapsular antibody could reverse this effect. Impaired uptake and lymphocyte proliferation that were induced by polysaccharide can be enhanced through opsonization with monoclonal antibodies or human serum, suggesting that antipolysaccharide antibodies might enhance the host defense by restoring uptake of the organism and subsequent presentation to T lymphocytes. These studies support the therapeutic potential of stimulating cell-mediated immunity to C. neoformans with anticapsular antibody.     

An Opsonizing Monoclonal Antibody That Recognizes a Noncapsular Epitope Expressed on Cryptococcus neoformans

A mouse hybridoma secreting a monoclonal antibody (MAb) that bound a noncapsular epitope expressed on C. neoformans was developed by immunizing BALB/c mice with formalin-killed serotype A yeasts. The hybridoma, designated CSFi, secreted an immunoglobulin G2b MAb that reacted with all C. neoformans serotypes tested, including the acapsular mutant ATCC 52817 (Cap67). Postsectioned immune electron microscopy revealed extensive binding of the MAb to the cell walls of both encapsulated and acapsular yeasts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of secreted antigens recovered from concentrated culture supernatants from both encapsulated and acapsular strains was conducted. The results showed that this MAb bound predominantly to antigens with molecular masses of approximately 75 and 100 kDa. A competitive enzyme-linked immunosorbent assay was used to demonstrate that the MAb was not cross-reactive with purified glucuronoxylomannan derived from either serotypes A or D. Experiments conducted with mouse peritoneal phagocytes and the mouse phagocyte-like cell line, J774A.1, demonstrated that the CSFi MAb opsonized the yeasts and increased their adherence to both types of phagocytic cells. We conclude, therefore, that antibodies directed at noncapsular epitopes can serve as opsonins and may have a role in modulating cryptococcal infection.     

Monoclonal Antibodies Reactive with Immunorecessive Epitopes of Glucuronoxylomannan, the Major Capsular Polysaccharide of Cryptococcus neoformans

Cryptococcus neoformans is surrounded by an antiphagocytic capsule whose primary constituent is glucuronoxylomannan (GXM). An epitope shared by GXM serotypes A, B, C, and D is immunodominant when mice are immunized with serotype A GXM. In contrast, an epitope shared only by serotypes A and D is immunodominant when mice are immunized with serotype D. Hybridomas secreting antibodies reactive with subdominant epitopes were identified through a positive-negative screening procedure in which antibody-secreting colonies were characterized by reactivity with both the immunizing polysaccharide and GXMs from each of the four major serotypes. In this manner, a monoclonal antibody (MAb) that was reactive with an epitope shared only by serotypes A and B was identified and designated F10F5. Such an epitope has not been described previously. Immunization of mice with de-O-acetylated serotype A GXM generated a hybridoma that secreted an antibody, designated F12D2, that was reactive with all four serotypes. Unlike previously described monoclonal and polyclonal panspecific antibodies, the reactivity of MAb F12D2 was not altered by de-O-acetylation of GXM. These results indicate that there are at least two panspecific GXM epitopes; one epitope is dependent on O acetylation for antibody reactivity, and the other is independent of O acetylation. This study identifies strategies for production of MAbs that are reactive with subdominant or cryptic GXM epitopes and provides new information regarding the antigenic makeup and the humoral immune response to GXM, an essential virulence factor that is a target for active and passive immunization.     

In Vitro and In Vivo Stability a Cryptococcus neoformans Glucuronoxylomannan Epitope That Elicits Protective Antibodies

The monoclonal antibody (MAb) 2H1 defines an epitope in Cryptococcus neoformans capsular glucuronoxylomannan (GXM) that can elicit protective antibodies. In murine models of cryptococcosis, MAb 2H1 administration prolongs survival and reduces fungal burden but seldom clears the infection. The mechanism by which C. neoformans persists and escape antibody-mediated clearance is not understood. One possibility is that variants that do not bind MAb 2H1 emerge in the course of infection. Using an agglutination-sedimentation protocol, we recovered a variant of strain 24067 that did not agglutinate, could not be serotyped, and had marked reduction in GXM O-acetyl groups. Binding of MAb 2H1 to 24067 variant cells produced a different immunofluorescence pattern and lower fluorescence intensity relative to the parent 24067 cells. Addition of MAb 2H1 to 24067 variant cells had no effect on cell charge. Phagocytic assays demonstrated that MAb 2H1 was not an effective opsonin for the 24067 variant. The 24067 variant was less virulent than the 24067 parent strain in mice, and MAb 2H1 administration did not prolong survival in animals infected with the variant strain. To investigate whether variants which do not bind MAb 2H1 are selected in experimental infection, three C. neoformans strains were serially passaged in mice given either MAb 2H1 or no antibody. Analysis of passaged isolates by agglutination assay, flow cytometry, and indirect immunofluorescence revealed changes in MAb 2H1 epitope expression but no clear trend with regards to gain or loss of MAb 2H1 epitope. C. neoformans variants with reduced MAb 2H1 epitope content can be isolated in vitro, but persistence of infection in mice given MAb 2H1 does not appear to be a result of selection of escape variants that lack the MAb 2H1 epitope.     

Contribution of Epitope Specificity to the Binding of Monoclonal Antibodies to the Capsule of Cryptococcus neoformans and the Soluble Form of Its Major Polysaccharide, Glucuronoxylomannan

Incubation of encapsulated cryptococci with monoclonal antibodies (MAbs) specific for glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, produces two distinct capsular quellung-type reactions termed rim and puffy. The type of capsular reaction that occurs is determined by the epitope specificity of the MAb and the serotype of the yeast cell. Several biological activities, including opsonic activity, complement activation, and protective efficacy, are associated with the type of capsular reaction produced by a MAb. The goal of this study was to examine the reactivities of two families of anti-GXM MAbs with serotype A and D capsular polysaccharides in several immunochemical assays, including agglutination, immunofluorescence, quantitative precipitation, and enzyme-linked immunosorbent assay, in an effort to identify serological assays that are predictive of the capsular quellung reaction. The results showed that the type of capsular reaction (rim versus puffy) is a qualitative assessment of antibody-capsule interaction that cannot be predicted on the basis of a serological assay. The results further showed that antibody reactivity demonstrated in one serological assay is not necessarily predictive of results in another assay, particularly in cases where one assay examines antibody-capsule interactions, e.g., agglutination, and another assay examines interaction of antibody with soluble GXM. Taken together, the results suggest caution in interpretation of immunochemical assays for anti-GXM antibodies and recommend the use of multiple assays formats when studying anticryptococcal antibodies.     

抗胰蛋白酶原激活肽单克隆抗体的制备及研究

研究表明 ,血和尿中的胰蛋白酶原激活肽 (TAP)含量 ,能够特异地反映胰蛋白酶的肠外激活和胰腺坏死的程度。这对及时、准确地诊断急性胰腺炎有重要的临床意义。本研究用KLH连接的TAP作免疫原 ,共建立了 4株抗TAP的单克隆抗体杂交瘤细胞株 ,均属IgG1亚类。单克隆抗体效价达 1:5× 10 5至 1:1× 10 7。效价高 ,稳定性好 ,特异性强 ,与胰蛋白酶原无交叉反应。为建立更敏感、更特异的TAP免疫检测方法 ,并用于探讨胰蛋白酶异位激活的致病机制的研究 ,以及全身性炎症反应在局部损伤作用的研究创造了条件。     

Both Th1 and Th2 Cytokines Affect the Ability of Monoclonal Antibodies To Protect Mice against Cryptococcus neoformans

Variable-region-identical mouse immunoglobulin G1 (IgG1), IgG2b, and IgG2a monoclonal antibodies to the capsular polysaccharide of Cryptococcus neoformans prolong the lives of mice infected with this fungus, while IgG3 is either not protective or enhances infection. CD4+ T cells are required for IgG1-mediated protection, and CD8+ T cells are required for IgG3-mediated enhancement. Gamma interferon is required for both effects. These findings revealed that T cells and cytokines play a role in the modulation of cryptococcal infection by antibodies and suggested that it was important to more fully define the cytokine requirements of each of the antibody isotypes. We therefore investigated the efficacy of passively administered variable-region-identical IgG1, IgG2a, IgG2b, and IgG3 monoclonal antibodies against intravenous infection with C. neoformans in mice genetically deficient in interleukin-12 (IL-12), IL-6, IL-4, or IL-10, as well as in the parental C57BL/6J strain. The relative inherent susceptibilities of these mouse strains to C. neoformans were as follows: IL-12(-/-) > IL-6(-/-) > C57BL/6J approximately IL-4(-/-) > IL-10(-/-). This is consistent with the notion that a Th1 response is necessary for natural immunity against cryptococcal infection. However, none of the IgG isotypes prolonged survival in IL-12(-/-), IL-6(-/-), or IL-4(-/-) mice, and all isotypes significantly enhanced infection in IL-10(-/-) mice. These results indicate that passive antibody-mediated protection against C. neoformans requires both Th1- and Th2-associated cytokines and reveal the complexity of the mechanisms through which antibodies modulate infection with this organism.     

Measurement of Complement Activation in Rabbit Plasma or Serum Using Monoclonal Antibodies against C5a

Eight murine monoclonal antibodies (MoAb) raised against the major zymosan-induced chemotactic factor in rabbit serum were found to neutralize the chemotactic activity induced by lipopolysaccharides (LPS) and antigen-antibody complexes. A 15 kDa antigen was identified in plasma incubated with LPS by immunoblot analysis with MoAb. This is similar to the molecular weight of the major zymosan-induced chemotactic factor. Both the generation of this 15 kDa antigen and chemotactic activity were abrogated in a heat-inactivated plasma. A cross-reaction to human C5a was demonstrated for three MoAb (5H8B9, 4B1C11, and 2A5E3) in an indirect enzyme-linked immunosorbent assay (ELISA) of partially purified C5a and by the isolation of zymosan-induced chemotactic activity by affinity chromatography. MoAb 5H8B9 and 4B1C11 were able to neutralize the chemotactic activity in human zymosan-activated serum. MoAb 2A5E3 was able to bind 125I-labelled human C5a des Arg. We conclude that these MoAb are directed against rabbit C5a. MoAb 5B2C5 and 2B1A2, which are directed to different antigenic binding sites on C5a, may be applied in a sandwich ELISA for the detection and quantification of C5a des Arg in rabbit serum or plasma. The sandwich ELISA can be performed directly on serum or plasma samples without having to precipitate native C5. Complement activation is demonstrated by measuring the increased generation of C5a des Arg in rabbit plasma or serum activated with LPS, zymosan, antigen-antibody complexes, or cobra venom factor.     

Capsule structure changes associated with Cryptococcus neoformans crossing of the blood-brain barrier

Cryptococcus neoformans is a yeast responsible for disseminated meningoencephalitis in patients with cellular immune defects. The major virulence factor is the polysaccharide capsule. We took advantage of a relevant murine model of disseminated meningoencephalitis to study the early events associated with blood-brain barrier (BBB) crossing. Mice were sacrificed at 1, 6, 24, and 48 hours post-intravenous inoculation, and classical histology, electron microscopy, and double immunofluorescence were used to study tissues and yeasts. Crossing of the BBB occurred early after inoculation, did not involve the choroid plexus but instead occurred at the level of the cortical capillaries, and caused early and severe damage to the structure of the microvessels. Seeding of the leptomeninges was not the primary event but occurred secondary to leakage of cortical pseudocysts. Organ invasion was associated with changes in cryptococcal capsule structure and cell size, which differed in terms of magnitude and kinetics, depending on both the organs involved, and potentially, on the bed structure of the local capillary. The rapid changes in capsule structure could contribute to inability of the host immune response to control cryptococcal infection in extrapulmonary spaces.     

4种抗支原体单克隆抗体免疫学特性的研究

为了有效地控制细胞质量、我们从1987年开始、相继建立了抗口腔支原体(M.orale),精氨酸支原体(M.arginini),猪鼻支原体(M.hyorhinis).和莱氏无胆甾原体(A.laidlawii)的杂交瘤细胞系共20株。并用IFA法、BA法或APAAP法对单克隆抗体进行了检测和鉴定。单克隆抗体(McAb)亚类鉴定结果:8株为IgG1,2株为IgG2b,其余10株为IgM。抗体效价测定大多数在4000～8 000之间。利用支原体纯培养的菌落进行了IFA染色试验,封闭试验,生长抑制试验和免疫电镜检查,均证实了它们的特异性,并获得2株对同属支原体有抗原交叉的McAb(3D1和3A11)。利用上述McAb检测实验室常用细胞的支原体污染,较常规法和DNA荧光染色法简便,快速,特异性较高。     

Organ-Dependent Variation of Capsule Thickness in Cryptococcus neoformans during Experimental Murine Infection

In studies of murine infection, the capsule thickness of Cryptococcus neoformans varied depending on the organ. The relative order of thickness was as follows: lung > brain > in vitro isolates. The differences in capsule thickness suggest that there are organ-related differences in the expression of genes responsible for capsule thickness.     

Capsule size of Cryptococcus neoformans: control and relationship to virulence.

Capsule size of five isolates of Cryptococcus neoformans was controlled by cultivation in media containing varying amounts of sugar. High concentrations of sugar (e.g., 16%) suppressed encapsulation whereas low concentrations (e.g., 1%) allowed maximal encapsulation. Suppression of capsule size was attributed at least in part to the increased osmolarity of the medium because a medium with low sugar concentration but having high osmolarity (by virtue of added sodium chloride) also produced cells having small capsules. The extent of control was more marked with certain of the isolates than with others. Mice were intravenously inoculated with cells of a single isolate cultivated so as to have either small or large capsules, and virulence was measured by comparing death rates. Results indicate that virulence after such an inoculation is a constant characteristic of an isolate and is not affected by size of the capsule of the cells in the inoculum.     

Monoclonal Antibodies Specific for Sendai Virus I. Production and Characterization of Monoclonal Antibodies

Twelve monoclonal antibodies specific for Sendai virus were prepared by fusing immune LOU rat spleen cells with Y3 or FO myeloma cells. Six antibodies bound the viral glycoprotein HN, and six the viral protein F. Among the six HN-specific monoclonal antibodies, five reacted with the very same epitope and inhibited viral haemagglutination. Two antibodies against the F protein recognized the same epitope, but all the others reacted with different epitopes. All monoclonals were characterized with regard to specificity, biological function, epitope recognition, isotypes, and pI.     

Cryptococcus neoformans Variants Generated by Phenotypic Switching Differ in Virulence through Effects on Macrophage Activation

Macrophages have a central role in the pathogenesis of cryptococcosis since they are an important line of defense, serve as a site for fungal replication, and also can contribute to tissue damage. The objective of this study was to investigate the interaction of macrophages with cells from smooth-colony variants (SM) and mucoid-colony variants (MC) arising from phenotypic switching of Cryptococcus neoformans. Alveolar macrophages (AMs) isolated from SM- and MC-infected mice exhibited differences in gene and surface expression of PD-L1, PD-L2, and major histocompatibility class II (MHC-II). PD-L1 and PD-L2 are the ligands for PD1 and are differentially regulated in Th1- and Th2-type cells. In addition, macrophage activation in SM- and MC-infected mice was characterized as alternatively activated. Flow cytometric and cytokine analysis demonstrated that MC infection was associated with the emergence of Th17 cells and higher levels of interleukin-17 (IL-17) in lung tissue, which were reduced by AM depletion. In conclusion, our results indicate that macrophages play a significant role in maintaining damage-promoting inflammation in the lung during MC infection, which ultimately results in death.Cryptococcosis is an infection that occurs commonly in AIDS patients, as well as other immunocompromised patients, and is caused by Cryptococcus neoformans. Most affected patients present with subacute or chronic meningoencephalitis. C. neoformans is an encapsulated yeast that can undergo phenotypic switching in vivo, which affects the outcome of chronic cryptococcosis (15, 17). The model C. neoformans strain RC-2 switches from a smooth-parental-colony (SM) morphology to a hypervirulent mucoid-colony (MC) morphology. Phenotypic switching involves the emergence of morphological colony variants whose cells differ in the cell wall and polysaccharide capsule, including the biochemical composition of the major capsular polysaccharide, glucuronoxylomannan (GXM) (14, 15). Recent work in our laboratory demonstrated that the murine immune responses to infection with MC and SM cells are qualitatively different. Enhanced recruitment of CD8 cells, NK cells, and macrophages and an early polarized Th1-type cell response were observed in the lungs of MC-infected mice and were associated with altered cytokine production. The finding that interleukin-10 (IL-10) affected the survival of SM-infected mice but not MC-infected mice (20) further supported the conclusion that phenotypic switching alters cryptococcal virulence by changing the host-pathogen interaction in a way that is manifested through different immune responses. Histological analysis of MC-infected lungs demonstrated that there was enhanced macrophage recruitment and suggested that this recruitment resulted in damage to the alveolar tissue and decreased survival.The majority of individuals with chronic cryptococcosis have defects in cellular immunity (9, 11, 35, 46). The high incidence of cryptococcosis only in HIV-infected patients with low CD4 counts underscores the importance of T-cell-based defenses. Furthermore, the crucial role of T cells in host defense has been supported by findings with animal models (22, 32). Alveolar macrophages (AMs) also are important effector cells against C. neoformans (45). They are the primary phagocytic cells, and together with dendritic cells they facilitate antigen presentation (31, 39, 47, 50). In addition, C. neoformans is a facultative intracellular pathogen that can reside in a macrophage (12); hence, this type of cell is also a niche to which the pathogen adapts.The interaction of macrophages with a pathogen such as C. neoformans leads to activation, which can be classified as either classical or alternative activation (16, 18). Macrophages infected with C. neoformans are alternatively activated, but the role of macrophage activation during infection is unknown (3, 4, 37, 38). In African trypanosomiasis macrophages are activated classically early and alternatively late in infection, which leads to progression of the disease (4, 38). Given that there is a marked difference in virulence between the SM and MC variants and that persistent MC infection is associated with enhanced macrophage recruitment, the objective of this study was to further explore macrophage activation and function by examining infection with phenotypic switch variants.     

Involvement of Antilipoarabinomannan Antibodies in Classical Complement Activation in Tuberculosis

We examined alternative and classical complement activation induced by whole bacilli of Mycobacterium bovis BCG and Mycobacterium tuberculosis products. After exposure to BCG, there were higher levels of the terminal complement complex in sera from Indian tuberculosis patients than in sera from healthy controls. The addition of BCG with or without EGTA to these sera indicated that approximately 70 to 85% of the total levels of the terminal complement complex was formed by classical activation. Sera from Indian tuberculosis patients contained more antibody to lipoarabinomannan (LAM) than sera from healthy Indians. Levels of anti-LAM immunoglobulin G2 (IgG2), but not anti-LAM IgM, correlated positively with classical activation induced by BCG in the sera. By flow cytometry, deposition of C3 and terminal complement complex on bacilli incubated with normal human serum was demonstrated. The anticomplement staining was significantly reduced in the presence of EGTA and EDTA. Flow cytometry also revealed the binding of complement to BCG incubated with rabbit anti-LAM and then with factor B-depleted serum. This indicates that classical activation plays a major role in complement activation induced by mycobacteria and that anti-LAM IgG on the bacilli can mediate this response. Classical complement activation may be important for the extent of phagocytosis of M. tuberculosis by mononuclear phagocytes, which may influence the course after infection.Mycobacterium tuberculosis is a facultative intracellular parasite, and several studies have focused upon the mechanisms by which mycobacteria enter mononuclear phagocytes. The complement system plays a major role in opsonizing mycobacteria for cellular uptake. It has been shown that monocyte complement receptors (CR) mediate the phagocytosis of M. tuberculosis and Mycobacterium bovis BCG coated with C3 by alternative complement activation (17, 26). Phenolic glycolipid 1, which is found in abundance on Mycobacterium leprae, fixes C3 via serum antibody (Ab) binding and classical-pathway complement activation and mediates phagocytosis by monocytes (27). The authors have also shown that serum from healthy adults contains Ab to lipomannan, lipoarabinomannan (LAM), and arabinogalactan (28), and it is well established that anti-M. tuberculosis Ab occur in both tuberculous and nontuberculous individuals (1). Production of such Ab in the latter group may be influenced by BCG vaccination widely used against tuberculosis to induce cell-mediated protection against the disease or by exposure to epitopes shared by avirulent environmental mycobacteria and M. tuberculosis. Moreover, the presence of complement-activating Ab to avirulent mycobacteria that cross-react with M. tuberculosis may be decisive for the development of localized instead of disseminated tuberculosis (6).Complement activation culminates in the formation of the terminal complement complex (TCC). The presence of TCC containing C5b-9 with or without vitronectin (24) on the bacterial surface may explain the reported uptake of bacilli via monocyte vitronectin receptors (25). The soluble terminal complement activation product C5a is a potent chemotaxin and stimulator and may recruit activated host monocytes that can be invaded. Recently, the binding of M. tuberculosis to CR3 expressed in Chinese hamster ovary cells was reported to be predominantly nonopsonic (7). Previously, we have shown that antigen (Ag) 85C of M. bovis BCG and M. tuberculosis promotes monocyte CR3-mediated uptake of beads coated with mycobacterial products (13). Interestingly, 85C could be a ligand for the non-iC3b-binding epitope in CR3 found to bind M. tuberculosis to macrophages (29). In addition, several other ligands and receptors, unrelated to complement, are known to participate in the uptake of mycobacteria in mononuclear phagocytes (2, 12, 25, 30).We wanted to study complement activation induced by BCG and M. tuberculosis Ag in sera from nontuberculous and tuberculous subjects. Especially, we wished to investigate classical complement activation and its relationship to the specificity of anti-M. tuberculosis Ab. Therefore, sera from healthy subjects and tuberculosis patients were exposed to mycobacteria and differences in soluble complement activation products in the sera were examined by an enzyme-linked immunosorbent assay (ELISA) specific for neoepitopes in the activation products (11, 21). Ab to mycobacteria in the populations were identified, and the levels were determined by ELISA. In addition, deposition of complement on BCG exposed to different sera and Ab was studied by flow cytometry.(This work was presented at the Sixth European Meeting on Complement in Human Disease, Innsbruck, Austria 12 to 15 March, 1997.)     

Characterization of heteroresistance to fluconazole among clinical isolates of Cryptococcus neoformans

Strains of Cryptococcus neoformans expressing heteroresistance to fluconazole have been described previously. The present study was conducted to investigate the prevalence of heteroresistance among clinical isolates of C. neoformans and to characterize the heteroresistant phenotypes. A total of 107 clinical isolates of C. neoformans for which the MICs of fluconazole ranged from 0.25 to 32 microg/ml were selected. The isolates were chosen to represent a broad geographic distribution. Of the 107 C. neoformans isolates tested, 4 grew on medium containing fluconazole at concentrations that were four to eight times higher than the MICs for each strain. A fifth isolate, for which the fluconazole MIC was 32 microg/ml, grew on agar with 64 microg of fluconazole per ml. These five isolates (4.7% of the total number) were confirmed to exhibit heteroresistant compositions by population analysis. The degree and frequency of resistance varied among the isolates. Stepwise selection by exposure to fluconazole resulted in subclones of all five strains for which the fluconazole MIC was >64 microg/ml. Subclones of three strains demonstrated a homogeneous population of resistant cells on medium containing 64 microg of fluconazole/ml. The resistance was sensitive to incubation temperature, that is, heteroresistance was demonstrable only at 30 degrees C by agar-based tests, and was reversible through serial transfers on fluconazole-free medium over a period of 8 days. These results suggest that the fluconazole-heteroresistant phenotype of C. neoformans exists in a significant proportion of clinical isolates and that fluconazole resistance can be developed by selection from heteroresistant clones and induction by exposure to fluconazole.     

抗精脒单克隆抗体的制备及其特性鉴定

本文用鼠×鼠细胞杂交瘤技术首次建立了抗多胺（精脒）单克隆抗体的杂交瘤细胞系。以此杂交瘤经体外组织培养扩大，并注入同系小鼠腹腔，产生的腹水中抗体滴度达１：１０６。单抗的类别是ＩｇＧ１。单价抗体是由分子量５２ｋＤ和２７ｋＤ组成的复合体。这个单克隆抗体可用于癌症的快速诊断和预后随访。