Clinical characteristics of patients in a case control study of sarcoidosis.

Sarcoidosis may be affected by sex, race, and age. A Case Control Etiologic Study of Sarcoidosis (ACCESS) enrolled 736 patients with sarcoidosis within 6 mo of diagnosis from 10 clinical centers in the United States. Using the ACCESS sarcoidosis assessment system, we determined organ involvement for the whole group and for subgroups differentiated by sex, race, and age (less than 40 yr or 40 yr and older). The study population was heterogeneous in terms of race (53% white, 44% black), sex (64% female, 36% male), and age (46% < 40 yr old, 54% > or = 40 yr old). Women were more likely to have eye and neurologic involvement (chi(2) = 4.74, p < 0.05 and chi(2) = 4.60, p < 0.05 respectively), have erythema nodosum (chi(2) = 7.28, p < 0.01), and to be age 40 yr or over (chi(2) = 6.07, p < 0.02) whereas men were more likely to be hypercalcemic (chi(2) = 7.38, p < 0.01). Black subjects were more likely to have skin involvement other than erythema nodosum (chi(2) = 5.47, p < 0.05), and eye (chi(2) = 13.8, p < 0.0001), liver (chi(2) = 23.3, p < 0.0001), bone marrow (chi(2) = 18.8, p < 0.001), and extrathoracic lymph node involvement (chi(2) = 7.21, p < 0.01). We conclude that the initial presentation of sarcoidosis is related to sex, race, and age.

Potent inhibition of neutrophil migration by cryptococcal mannoprotein-4-induced desensitization.

Cryptococcal capsular Ags induce the production of proinflammatory cytokines in patients with cryptococcal meningitis. Despite this, their cerebrospinal fluid typically contains few neutrophils. Capsular glucuronoxylomannan is generally considered to mediate the inhibition of neutrophil extravasation. In the current study, culture supernatant harvested from the nonglucuronoxylomannan-producing strain CAP67 was found to be as potent as supernatant from wild-type strains in preventing migration. We identified capsular mannoprotein (MP)-4 as the causative agent. Purified MP-4 inhibited migration of neutrophils toward platelet-activating factor, IL-8, and fMLP, probably via a mechanism involving chemoattractant receptor cross-desensitization, as suggested by its direct chemotactic activity. Supporting this hypothesis, MP-4 elicited Ca(2+) transients that were inhibited by preincubation with either fMLP, IL-8, or C5a, but not platelet-activating factor, and vice versa. Moreover, MP-4 strongly decreased the neutrophil surface expression of L-selectin and induced shedding of TNF receptors p55/p75, whereas CD11b/18 increased. Finally, MP-4 was clearly detectable in both serum and cerebrospinal fluid of patients suffering from cryptococcal meningitis. These findings identify MP-4 as a novel capsular Ag prematurely activating neutrophils and desensitizing them toward a chemoattractant challenge.

Role of mannoprotein in induction and regulation of immunity to Cryptococcus neoformans.

Our previous observations showed that mannoprotein (MP) induces early and massive production of interleukin-12 (IL-12) in vitro. This study was designed to investigate whether this phenomenon could be applied in vivo and to determine the biological significance of MP in Cryptococcus neoformans infection. The results reported here show that MP treatment induces IL-12 secretion by splenic macrophages and IL-12 p40 mRNA in the brain. During C. neoformans infection, MP reinforced IL-12 and IFN-gamma secretion that coincided with enhanced antifungal activity of natural effector cells, early resolution of the inflammatory process, and clearance of fungal load from the brain. These studies show that MP is a key inflammatory mediator that induces a protective immune response against C. neoformans infection. This information can be used to facilitate the design of a rational approach to manipulate the immune response to C. neoformans.

Cryptococcal glucuronoxylomannan delays translocation of leukocytes across the blood-brain barrier in an animal model of acute bacterial meningitis.

In bacterial meningitis, neurological damage is associated with a high influx of polymorphonuclear leukocytes (PMN) into the brain. Previous data suggest that the capsular component of the fungus C. neoformans, glucuronoxylomannan (GXM), interferes with PMN-migration into the cerebrospinal fluid (CSF). Therefore, a rabbit model of bacterial meningitis was treated intravenously with GXM. This resulted in (1) a reduction of PMN in the CSF at 6 h (P=0.05), (2) reduced peak TNF-alpha concentrations in the CSF, and (3) diminished tissue inflammation and intravascular margination of PMN in GXM-treated animals. Thus, GXM may represent a novel adjuvant anti-inflammatory agent in bacterial meningitis.

Early induction of interleukin-12 by human monocytes exposed to Cryptococcus neoformans mannoproteins.

Interleukin-12 (IL-12) production by human monocytes stimulated with mannoproteins (MPs) of Cryptococcus neoformans was investigated. The results reported show that secreted or cell-associated MPs induce an early and significant production of IL-12. MPs show different capabilities to quantitatively affect IL-12 production; MP2, an 8. 2-kDa MP purified from the culture supernatant of C. neoformans, appears to be the most potent stimulator. Cytochalasin B inhibits both internalization and IL-12 induction by MP. In addition, a drastic reduction of IL-12 was observed when monocytes were cultured in the absence of normal human serum or treated with soluble mannan. Early production of IL-12 promotes early secretion of gamma interferon by T cells but does not influence the magnitude of the MP-induced lymphoproliferative response. Overall our results identify cryptococcal antigens responsible for rapid and potent induction of IL-12 in monocytes. MPs appear to regulate IL-12 secretion by internalization via the endocytic pathway and by interaction with monocyte receptors or serum factors.

Cryptococcus neoformans and its cell wall components induce similar cytokine profiles in human peripheral blood mononuclear cells despite differences in structure.

We studied the cytokine profile of peripheral blood mononuclear cells after stimulation with various cryptococcal strains or its purified cell wall components. After 3 h of stimulation, tumor necrosis factor (TNF) alpha levels were strongly increased, whereas interferon (IFN) gamma and interleukin (IL) 10 levels were increased only slightly, or not at all (respectively). In contrast, after 18 h, TNF-alpha and IFN-gamma levels were (strongly) decreased, whereas the IL-10 levels were increased. The IL-1beta, IL-6 and IL-8 levels were equally high throughout the experiment. In order to establish which of the cryptococcal envelope components contributed most to the observed cytokine profile induced by whole cryptococci, glucuronoxylomannan, galactoxylomannan and mannoproteins were purified and partially characterized biochemically. All cryptococcal components elicited a similar cytokine pattern despite the differences in structure.

Phenotypic switching in Cryptococcus neoformans results in changes in cellular morphology and glucuronoxylomannan structure.

Cryptococcus neoformans strains exhibit variability in their capsular polysaccharide, cell morphology, karyotype, and virulence, but the relationship between these variables is poorly understood. A hypovirulent C. neoformans 24067A isolate, which usually produces smooth (SM) colony types, was found to undergo phenotypic switching and to produce wrinkled (WR) and pseudohyphal (PH) colony types at frequencies of approximately 10(-4) to 10(-5) when plated on Sabouraud agar. Cells from these colony types had large polysaccharide capsules and PH morphology, respectively. Scanning electron microscopy showed that different colony types were the result of altered cellular packing in the colony. Phenotypic switching was associated with quantitative and qualitative changes in capsular polysaccharide. Specifically, the glucuronoxylomannan (GXM) of the WR polysaccharide differed in the proportion of structural reporter groups and in increased xylose residue content linked at the 4 to 0 position. The relative virulence of the colony types was WR > PH > SM, as measured by CFU in rat lungs after intratracheal infection. Karyotype instability was observed in strain 24067A and involved primarily two chromosomes. Colonies with an alternative colony type exhibited more karyotype changes, which did not revert to the original karyotype in reverted colonies. In summary, this study revealed that phenotypic switching in C. neoformans (i) can produce WR colonies consisting of cells with either large capsule or PH morphology, (ii) is associated with production of structurally different GXM, (iii) is commonly associated with karyotype changes, (iv) can produce cells of PH morphology, and (v) can increase the virulence of a strain. Hence, phenotypic switching is an adaptive mechanism linked to virulence that can generate cell types with very different biological characteristics.

In vitro and in vivo stability of a Cryptococcus neoformans [corrected] 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.

Pathogenesis of Cryptococcus neoformans is associated with quantitative differences in multiple virulence factors.

Two isolates of Cryptococcus neoformans were previously described as being highly divergent in their level of capsule synthesis in vivo and in their virulence for mice. The highly virulent isolate (NU-2) produced more capsule than a weakly virulent isolate (184A) in vitro under tissue culture conditions and in vivo. This investigation was done to determine if there were differences between the two isolates in other factors that might also contribute to virulence. Growth rate was not a factor as NU-2 grew more slowly than 184A. Based on PCR fingerprinting the two isolates were genetically different providing an opportunity to examine differences in multiple virulence traits. Quantitative analysis revealed that NU-2 expressed significantly more melanin and mannitol than did 184A. Although the isolates expressed the same capsular chemotype, NU-2 produced an additional structure reporter group (SRG) under tissue culture conditions that was not present when grown in glucose salts/urea/basal medium (GSU). Capsular polysaccharide SRGs of 184A were unaffected by shifting the growth conditions from GSU to tissue culture conditions. Our results suggest that pathogenesis of a C. neoformans strain is dictated by the quantitative expression of the strain's combined virulence traits. Regulators of the expression of these genes may be playing key roles in virulence.

Structural characterization of the galactoxylomannan of Cryptococcus neoformans Cap67.

The galactoxylomannan (GalXM) obtained from the culture supernatant of an acapsular mutant of Cryptococcus neoformans Cap67 was purified by Concanavalin A affinity, ion-exchange, and gel-filtration chromatographies. The structure of GalXM was determined by methylation analysis and by 1D and 2D NMR spectroscopic studies of the intact polysaccharide and of the oligosaccharide fragments generated by Smith degradation and by acetolysis. GalXM is a complex polysaccharide with an alpha-(1-->6) -galactan backbone. The polysaccharide is branched at c-3 of alternate Gal units of the backbone. C-3 is the point of attachment of the oligosaccharide side chains comprised of alpha-D-Man- (1-->3)-alpha-D-Man-(1-->4)- beta-D-Gal-substituted with zero to three terminal beta-Xyl residues as shown in the following structure: [formula: see text].

Assimilation of xylose, mannose, and mannitol for synthesis of glucuronoxylomannan of Cryptococcus neoformans determined by 13C nuclear magnetic resonance spectroscopy.

Cryptococcus neoformans NIH 409 was cultured in a defined medium containing D-[1-13C]xylose (Xyl), D-[1-13C]mannose (Man), or D-[1-13C]mannitol as the sole carbon source. The distribution of 13C in the Man, Xyl, glucuronic acid (GlcA), and O-acetyl constituents of native and de-O-acetylated glucuronoxylomannan (GXM) was determined by one-dimensional 13C nuclear magnetic resonance spectroscopy. The carbon chain of Man was incorporated intact into GXM since 13C was observed only in carbon 1 of Man, GlcA, and Xyl. The carbon chain of mannitol was incorporated intact into GXM since 13C was observed only in carbons 1 and 6. This was expected since mannitol has an axis of symmetry. The carbon chain of Xyl was identified only in carbons 1 and 3 of Man, GlcA, and Xyl. This pattern of labeling is consistent with the assimilation of Xyl through the pentophosphate pathway.

Cryptococcus neoformans chemotyping by quantitative analysis of 1H nuclear magnetic resonance spectra of glucuronoxylomannans with a computer-simulated artificial neural network.

The complete assignment of the proton chemical shifts obtained by nuclear magnetic resonance (NMR) spectroscopy of de-O-acetylated glucuronoxylomannans (GXMs) from Cryptococcus neoformans permitted the high-resolution determination of the total structure of any GXM. Six structural motifs based on an alpha-(1-->3)-mannotriose substituted with variable quantities of 2-O-beta- and 4-O-beta-xylopyranosyl and 2-O-beta-glucopyranosyluronic acid were identified. The chemical shifts of only the anomeric protons of the mannosyl residues served as structure reporter groups (SRG) for the identification and quantitation of the six triads present in any GXM. The assigned protons for the mannosyl residues resonated at clearly distinguishable positions in the spectrum and supplied all the information essential for the assignment of the complete GXM structure. This technique for assigning structure is referred to as the SRG concept. The SRG concept was used to analyze the distribution of the six mannosyl triads of GXMs obtained from 106 isolates of C. neoformans. The six mannosyl triads occurred singularly or in combination with one or more of the other triads. The identification and quantitation of the SRG were simplified by using a computer-simulated artificial neural network (ANN) to automatically analyze the SRG region of the one-dimensional proton NMR spectra. The occurrence and relative distribution of the six mannosyl triads were used to chemotype C. neoformans on the basis of subtle variations in GXM structure determined by analysis of the SRG region of the proton NMR spectrum by the ANN. The data for the distribution of the six SRGs from GXMs of 106 isolates of C. neoformans yielded eight chemotypes, Chem1 through Chem8.

A 1H NMR spectroscopic approach to the unambiguous determination of glycosyl linkage positions in oligosaccharides.

A sensitive (< 1 mg) and nondestructive method was devised for the unambiguous determination of the glycosyl linkage positions in oligosaccharides using 1H NMR spectroscopy. The technique is based on the absence of a hydroxyl group on the carbon atom that participates in the glycosidic linkage. The "missing" hydroxyl group is identified by recording 1H NMR spectra of the oligosaccharide in H2O and D2O. The method is illustrated by determining the glycosyl linkage positions of maltose, Glc alpha 1-4Glc, and a branched mannopentaose, Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man.

Microevolution of a standard strain of Cryptococcus neoformans resulting in differences in virulence and other phenotypes.

Cryptococcus neoformans is a major fungal pathogen for patients with debilitated immune systems. However, no information is available on the stability of virulence or of phenotypes associated with virulence for C. neoformans laboratory strains. A serendipitous observation in our laboratory that one isolate of C. neoformans ATCC 24067 (strain 52D) became attenuated after continuous in vitro culture prompted us to perform a comparative study of nine strain 24067 isolates obtained from six different research laboratories. Each isolate was characterized by DNA typing, virulence for mice, proteinase production, extracellular protein synthesis, melanin synthesis, carbon assimilation pattern, antifungal drug susceptibility, colony morphology, growth rate, agglutination titers, phagocytosis by murine macrophages, capsule size, and capsular polysaccharide structure. All isolates had similar DNA typing patterns consistent with their assignment to the same strain, although minor chromosome size polymorphisms were observed in the electrophoretic karyotypes of two isolates. Several isolates had major differences in phenotypes that may be associated with virulence, including growth rate, capsule size, proteinase production, and melanization. These findings imply that C. neoformans is able to undergo rapid changes in vitro, probably as a result of adaptation to laboratory conditions, and suggest the need for careful attention to storage and maintenance conditions. In summary, our results indicate that C. neoformans (i) can become attenuated by in vitro culture and (ii) is capable of microevolution in vitro with the emergence of variants exhibiting new genotypic and phenotypic characteristics.

Biochemical characterization of Candida albicans epitopes that can elicit protective and nonprotective antibodies.

We previously reported that the immunoglobulin M (IgM) monoclonal antibody (MAb) B6.1 protects mice against disseminated candidiasis, whereas the IgM MAb B6 does not. Both MAbs are specific for an adhesin fraction isolated from the cell surface of Candida albicans, but their epitope specificities differ. In the present study, we examined the surface locations of both epitopes and obtained structural information regarding the B6.1 epitope. Immunofluorescence confocal microscopic analysis of C. albicans yeast forms showed that epitope B6.1 is displayed rather homogeneously over the entire cell surface, whereas epitope B6 appears to have a patchy distribution. Both antibodies were essentially nonreactive with the surfaces of mycelial forms of the fungus, indicating that neither epitope is expressed on the surfaces of these forms. For isolation of the B6.1 epitope, the adhesin fraction consisting of cell surface phosphomannan was subjected to mildly acidic (10 mM HCl) hydrolysis and was fractionated into acid-labile and acid-stable portions by size exclusion chromatography. Antibody blocking experiments showed that the B6.1 epitope is an acid-labile moiety of the phosphomannan and that the B6 epitope is located in the acid-stable fraction. The B6 epitope appeared to be mannan because it was stable to heat (boiling) and protease treatments but was destroyed by alpha-mannosidase digestion. The B6.1 epitope eluted from the size exclusion column in two fractions. Mass spectroscopic analyses showed that one fraction contained material with the size of a mannotriose and that the other was a mixture of mannotriose- and mannotetraose-size substances. Dose response inhibition tests of the fractions indicated that the B6.1 epitope is associated with the mannotriose. Nuclear magnetic resonance (NMR) spectroscopic analysis of the epitope yielded data consistent with a beta-(1-->2)-linked mannotriose. The fine structure of the B6 epitope is under investigation. Information derived from these investigations will be useful both in understanding protective versus nonprotective antibody responses to C. albicans and in improving anti-Candida vaccine formulations.

Induction of TNF-alpha in human peripheral blood mononuclear cells by the mannoprotein of Cryptococcus neoformans involves human mannose binding protein.

We have shown previously that specific receptors on PBMCs and a serum factor other than Ab and complement are involved in the TNF-alpha response to cryptococcal mannoprotein (MP2). To characterize the mechanism of MP2 recognition by PBMCs, 10(6) PBMCs were incubated with 25 microg of FITC-labeled MP2 in 10% normal human serum (1 h). The cells were analyzed by flow cytometry. FITC-MP2 binding was CaCl2 and temperature dependent and was enhanced by prestimulating PBMCs with unlabeled MP2. Binding to PBMCs was specific, since unlabeled MP and mannan produced dose-dependent inhibition. Beta-Glucan laminarin produced background inhibition. mAbs against CD14, CD11b, and CD18 did not prevent FITC-MP2 binding to PBMCs, implying that these receptors are not involved in MP2 recognition by PBMCs. mAb against CD14 blocked (>90%) MP2-induced TNF-alpha release by PBMCs, while mAbs against CD11b/CD18 caused no inhibition. Removal of human mannose binding protein (hMBP) by preincubation of serum with a specific mAb abrogated TNF-alpha induction by MP2 and strongly inhibited its binding to PBMCs. Recombinant hMBP enhanced TNF-alpha induction by MP2 as well as binding of FITC-MP2 to PBMCs. In addition, incubation of serum with MP2-coated beads and analysis by SDS-PAGE resulted in the detection of a protein of approximately 33/34 kDa that could be partially removed by preincubating the serum with hMBP mAb. We conclude that hMBP is involved in the binding of MP2 to PBMCs and the release of TNF-alpha.

Structure of the 13C-enriched O-deacetylated glucuronoxylomannan of Cryptococcus neoformans serotype A determined by NMR spectroscopy.

The complete assignment of 1H and 13C chemical shifts for 99% uniformly 13C-labeled O-deacetylated glucuronoxylomannan (GXM) of Cryptococcus neoformans serotype A isolate 9759-Mu-1 was accomplished by the analysis of HCCH-TOCSY and HCCH-COSY spectra. The sequence of the glycosyl residues was determined by a GHMBC experiment using 20% uniformly 13C-labeled GXM; GXM was prepared by a novel procedure that insured the virtual exclusion of adjacent 13C-labeled carbon atoms. For each residue in the GXM of 9759-Mu-1 we determined its linkage position, its anomeric configuration, and its position in the repeating sequence as follows: [sequence: see text]

Structure and biological activities of acapsular Cryptococcus neoformans 602 complemented with the CAP64 gene.

The extracellular polysaccharide capsule of Cryptococcus neoformans is a well-recognized virulence factor. Strain 602 is an acapsular clinical isolate of unknown serotype which has been widely used in studies of virulence and host-parasite interactions. In previous studies, strain 602 was compared with genetically unrelated strains of various serotypes because the wild-type equivalent of strain 602 was not available. We created an encapsulated strain, TYCC38-602, by transforming strain 602 with the CAP64 gene which was isolated from a serotype D strain. Serological tests and chemical analysis of the major polysaccharide capsule of TYCC38-602 indicated that strain 602 was originally derived from a serotype A strain. Restoration of the ability to produce a capsule enabled strain 602 to cause fatal infection in mice, whereas the acapsular strain 602 remained avirulent. Capsule-restored yeast cells of strain 602 activated the human complement system and bound C3 fragments in a manner that is characteristic of encapsulated cryptococci. In addition, the capsule in TYCC38-602 masked the ability of the organism to induce tumor necrosis factor alpha and subsequent nitric oxide synthase production in primed macrophage-like cells. These results indicate that the lack of capsule in strain 602 is the reason for its inability to cause fatal infection. Moreover, the acapsular phenotype accounts for differences in various biological activities of strain 602 compared to encapsulated strains. The results also indicate that the gene product of CAP64 does not contribute to serotype specificity of capsules in C. neoformans.

Structure of the capsular polysaccharide of Clostridium perfringens Hobbs 5 as determined by NMR spectroscopy.

The complete primary structure of the capsular polysaccharide of Clostridium perfringens Hobbs 5, an anaerobic bacterium implicated in food poisoning, was determined. The polysaccharide was isolated from C. perfringens Hobbs 5 cells, after deproteination, by ethanol precipitation and by ion-exchange chromatography. The polysaccharide was comprised of glucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, and glucuronic acid, in equimolar ratios. Sequence and linkage assignments of the glycosyl residues were obtained by NMR spectroscopy, specifically by the combination of two-dimensional homonuclear TOCSY and NOESY experiments and heteronuclear (1H, 13C) multiple-quantum coherence (HMQC, HMQC-COSY, HMQC-TOCSY and HMBC) experiments. Thus, the envelope polysaccharide of C. perfringens Hobbs 5 was found to be a polymer composed of a hexasaccharide repeating unit with the following structure: [formula: see text] This structure is novel among bacterial cell-surface polysaccharides, and it is the first of many serotypically distinct capsular polysaccharides of C. perfringens to be described.

Reactivity patterns and epitope specificities of anti-Cryptococcus neoformans monoclonal antibodies by enzyme-linked immunosorbent assay and dot enzyme assay.

Cryptococcus neoformans glucuronoxylomannans (GXM) are capsular polysaccharides important for virulence in cryptococcosis. This study used dot enzyme assays (DEA) and enzyme-linked immunosorbent assays (ELISA) to determine the reactivity patterns of 21 murine monoclonal antibodies (MAbs) with structurally defined GXMs from five serotypes. The MAbs were categorized into eight groups on the basis of DEA and five groups on the basis of ELISA. MAbs 302, 339, and 439 were studied extensively for their binding to various native and chemically modified GXMs. Quantitative variation in the inhibitory effects of GXMs on the binding of MAbs 302, 339, and 439 were observed by competitive ELISA. O-Deacetylation of serotype A, B, and D GXM resulted in the complete loss of their inhibitory properties. Carboxyl group reduction of GXMs from serotypes A and D resulted in a significant decrease of inhibitory activity for MAb. Xylomannans and methyl glycosides exhibited no detectable inhibitory activity on MAb binding to GXM. The results indicate (i) the existence of five to eight MAb-defined distinct epitopes in C. neoformans GXM that can elicit antibody responses, (ii) MAb detection of antigenic variation within GXMs assigned to a particular serotype, (iii) good correspondence between the patterns of MAb reactivities and polyclonal rabbit factor sera, (iv) good agreement between MAb molecular structure and serotype reactivity, and (v) a dependence of the serotype reactivity profile for a given MAb on the technique used to measure binding.