Characterization of Anticapsular Monoclonal Antibodies That Regulate Activation of the Complement System by the Cryptococcus neoformans Capsule

Incubation of the encapsulated yeast Cryptococcus neoformans in human serum leads to alternative pathway-mediated deposition of C3 fragments in the capsule. We examined the ability of monoclonal antibodies (MAbs) specific for different epitopes of the major capsular polysaccharide to alter the kinetics for classical and alternative pathway-mediated deposition of C3 onto a serotype A strain. We studied MAbs reactive with capsular serotypes A, B, C, and D (MAb group II); serotypes A, B, and D (MAb group III); and serotypes A and D (MAb group IV). The MAb groupings are based on antibody variable region usage which determines the antibody molecular structure. When both the classical and alternative pathways were operative, group II MAbs induced early classical pathway-mediated binding of C3 but reduced the overall rate of C3 accumulation and the amount of bound C3. Group III MAbs closely mimicked the effects of group II MAbs but exhibited reduced support of early classical pathway-facilitated accumulation of C3. Depending on the antibody isotype, group IV MAbs slightly or markedly enhanced early binding of C3 but had no effect on either the rate of C3 accumulation or the amount of bound C3. When the classical pathway was blocked, group II and III MAbs markedly suppressed C3 binding that normally would have occurred via the alternative pathway. In contrast, MAbs of group IV had no effect on alternative pathway-mediated C3 binding. These results indicate that anticapsular antibodies with different epitope specificities may have distinct regulatory effects on activation and binding of C3.Cryptococcus neoformans is the etiological agent of cryptococcal meningitis, a life-threatening infection of particular importance in patients with deficiencies in cellular immunity, most notably patients with the AIDS. The yeast is surrounded by a polysaccharide capsule that is composed primarily of glucuronoxylomannan (GXM), which has a linear (1→3)-α-d-mannopyranan backbone bearing β-d-xylopyranosyl, β-d-glucopyranosyluronic acid, and O-acetyl substituents (3, 9, 54). The cryptococcal capsule occurs as four major serotypes (A, B, C, and D) and is an essential virulence factor for the yeast.One of the most striking features of the cryptococcal capsule is its ability to activate the alternative complement pathway. Incubation of encapsulated cryptococci in normal human serum (NHS) leads to the deposition of 10⁷ to 10⁸ C3 fragments on the yeast (28, 56). The C3 is deposited at the surface and throughout the capsule (30). Available evidence indicates that the amount of anti-GXM antibodies found in NHS is not sufficient to initiate the classical pathway (24); consequently, activation and binding of C3 to the cryptococcal capsule are mediated entirely by the alternative complement pathway (29, 30, 55). One of the hallmark features of alternative pathway deposition of C3 onto encapsulated cryptococci is a delay of 5 to 8 min before readily detectable amounts of C3 are found on yeast cells incubated in NHS (29, 55). Once past the initial lag, C3 fragments rapidly accumulate on the yeast cells as incubation proceeds for an additional 10 min.Recently, there has been interest in antibody-mediated resistance to cryptococcosis. Monoclonal antibodies (MAbs) have been proposed for treatment of cryptococcosis (7), and immunization with GXM-protein conjugates has been suggested for prevention of cryptococcosis (6, 12, 13). However, it is becoming increasingly clear that anti-GXM MAbs may have distinct specificities and biological activities. Anti-GXM MAbs which differ in (i) reactivities with GXM of the four major serotypes (2), (ii) apparent binding sites in the cryptococcal capsule (32, 37), and (iii) abilities to provide protection in a murine model of cryptococcosis (32, 37) have been described. Some differences in biological activity are related to differences in the epitope specificities of the various MAbs (32, 37).One means by which antibodies could enhance resistance to cryptococcosis is through accelerated deposition of opsonic C3 fragments via the action of the classical pathway. Such an acceleration would reduce or eliminate the 5- to 8-min lag that occurs during alternative pathway-mediated deposition of C3 fragments. The objectives of our study were to evaluate the effects of anti-GXM MAbs on the kinetics and sites for deposition of C3 fragments into the cryptococcal capsule. We examined several well-characterized antibodies that differed in the epitope specificity of the MAbs. The results showed that MAbs with different isotypes and epitope specificities had distinctly different effects on activation and binding of C3 via the classical and alternative pathways; many antibodies markedly suppressed C3 binding, some antibodies accelerated C3 binding, and other antibodies had little or no effect.     

Heminested inverse PCR for IS6110 fingerprinting of Mycobacterium tuberculosis strains.

A heminested inverse PCR (HIP) for the amplification of sequences flanking the Mycobacterium tuberculosis insertion sequence IS6110 has been developed. The method depends upon primers that anneal to IS6110 at sites between its 5' end and the closest BsrFI site. The accuracy of HIP was demonstrated by the amplification of sequences within plasmid constructs carrying one or two copies of the insertion sequence IS986 in different orientations. The identities of the amplicons produced from strains carrying a single copy of IS6110 were verified by nucleotide sequencing. Analyses of 204 M. tuberculosis strains including those involved in outbreaks showed that IS6110 HIP is highly discriminatory and reproducible. HIP fingerprinting of these 204 strains generated 136 distinct types, and its discriminatory power was equivalent to that of standard restriction fragment length polymorphism analysis. The method is therefore of value for the rapid fingerprinting of M. tuberculosis strains for epidemiological purposes.     

Bryostatin 1-induced modulation of nucleoside transporters and 2-chlorodeoxyadenosine influx in WSU-CLL cells

WSU-CLL cells, a fludarabine resistant B-cell chronic lymphocytic leukemia cell line, has been shown to exhibit enhanced sensitivity to 2-chlorodeoxyadenosine (2-CdA) following 48-72 h exposure to bryostatin 1. For 2-CdA to manifest its chemotherapeutic activity, it must first enter the cell through one of several specific nucleoside transporter systems. We present data to show that bryostatin 1-induced enhanced influx of 2-CdA is in part the result of bryostatin 1-induced modulation of nucleoside transporters in WSU-CLL cells. The bi-directional equilibrative NBMPR sensitive transporters in WSU-CLL cells were significantly down-regulated 90 min post-exposure to 1-200 nM bryostatin 1. This down-regulation was evident up to 144 h. In contrast, WSU-CLL cells exhibited a transient increase in Na+-dependent concentrative 2-CdA influx from 48 to 96 h after bryostatin 1 exposure which was evident for a longer duration than that accounted for by the increase in deocycytidine kinase activity. These data may, in part, explain the enhanced efficacy of 2-CdA seen in WSU-CLL cells following 48-72 h exposure to bryostatin 1. It may raise questions as to the importance of the bi-directional transporters in determining the resistance or sensitivity of CLL cells to 2-CdA or other nucleoside analogues.     

Isolation of a new clathrin heavy chain gene with muscle-specific expression from the region commonly deleted in velo-cardio-facial syndrome

Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are developmental disorders characterized by a spectrum of phenotypes including velopharyngeal insufficiency, conotruncal heart defects and facial dysmorphology among others. Eighty to eighty-five percent of VCFS/DGS patients are hemizygous for a portion of chromosome 22. It is likely that the genes encoded by this region play a role in the etiology of the phenotypes associated with the disorders. Using a cDNA selection protocol, we isolated a novel clathrin heavy chain cDNA (CLTD) from the VCFS/DGS minimally deleted interval. The cDNA encodes a protein of 1638 amino acids. CLTD shares significant homology, but is not identical to the ubiquitously expressed clathrin heavy chain gene. The CLTD gene also shows a unique pattern of expression, having its maximal level of expression in skeletal muscle. Velopharyngeal insufficiency and muscle weakness are common features of VCFS patients. Based on the location and expression pattern of CLTD, we suggest hemizygosity at this locus may play a role in the etiology of one of the VCFS-associated phenotypes.      

METHYLATION STATUS OF BCL6 DISTINGUISHES DNA SAMPLES FROM BENIGN AND MALIGNANT LYMPHOPROLIFERATIVE DISORDERS

INTRODUCTION: Core biopsy of the breast has become the method of choice for tissue diagnosis of screen detected microcalcifications and some mass lesions in many breast assessment centres. Biopsy results are not available until the following day. Imprint cytology of fresh breast core samples allows same-day reporting and patient counselling.
AIM: To determine the accuracy of core imprint cytology when compared with core biopsy diagnosis when used in a breast assessment centre setting.
METHODS: Core imprints (CI) were prepared and reported on all fresh core biopsies (CB) performed at the Sir Charles Gairdner Hospital Breast Centre from May to December 2000. Fresh core samples were placed on a glass microscope slide. Core radiographs were taken for microcalcification lesions (MC). A laboratory technician gently and quickly rolled the cores on the slide with fine forceps. The cores were fixed in formalin, processed and reported next day. The imprint slide was air dried and stained with DiffQuik. CI were reported using four categories: Insufficient, Benign, Indeterminate and Malignant. Counselling and planning for management were possible on the same day in women with malignant diagnoses. Clinicians were advised not to discuss negative or indeterminate CI results with women and to defer to the final CB report.
RESULTS: Cores were performed on 381 lesions. There were 83 carcinomas (38 in MC and 45 in masses) and 56 were called malignant on CI (absolute sensitivity 67.5%; 78.9% for MC and 57.8% for masses). 3 malignancies on CB were negative on CI giving a false negative rate of 3.6%. There were no false positive diagnoses. The predictive value of a benign diagnosis was 95.3%. There were no adverse effects in the histology of CB.
CONCLUSION: CI was an accurate method of providing an immediate diagnosis of malignancy in two thirds of malignancies confirmed on CB.