Investigating the candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: conjugates based on core oligosaccharides.

In this study we have prepared glycoconjugates with core oligosaccharides (OS) from the lipopolysaccharide (LPS) of Neisseria meningitidis, thus avoiding the neo-epitopes of the deacylated lipid A region of the derived LPS molecule identified in our previous studies. A comprehensive investigation was performed with glycoconjugates prepared from the most extended to the most truncated core OS still maintaining the conserved inner core epitope. As previously, we have established reproducible bactericidal killing of the homologous antigen elaborating strain, but a failure to kill wild-type strains. In these studies it was evident that the linker molecules used in the conjugation methodologies were dominating the immune response. However, when galE core OS based conjugates were prepared without utilizing linkers, via direct reductive amination, we failed to generate an immune response to even the homologous antigen. We also identified that immunisation with the galE antigen via linker methodologies provoked an immune response that was dependent upon key residues of the conserved inner core OS structure, whereas the immune responses to lgtB and lgtA antigens did not involve the inner core OS. This comprehensive study has, despite our best efforts, cast significant doubt as to the utility of the conserved inner core region of the meningococcal LPS as a potential vaccine antigen.

Adjunctive use of intravitreal dexamethasone in presumed bacterial endophthalmitis: a randomised trial.

AIM: To evaluate the use of intravitreal dexamethasone as adjunctive therapy in the treatment of presumed bacterial endophthalmitis. Design Prospective, double masked, randomised placebo-controlled clinical trial. METHODS: Patients with 'post cataract surgery', 'bleb-related' and 'other' endophthalmitis were grouped and randomised to receive intravitreal ceftazidime (2.225 mg/0.1 ml), vancomycin (1 mg/0.1 ml), and either dexamethasone (0.4 mg/0.1) or placebo. All underwent vitreous and aqueous sampling for microbiological analysis. Injections were repeated after 48 h if necessary. The primary outcome measure was Snellen visual acuity on presentation, within the first 14 days post injection, and at 2-4 months. RESULTS: 62 patients completed the protocol from 2001 to 2005. Thirty patients received intravitreal dexamethasone and 32 received intravitreal placebo. There was no statistically significant difference in the visual outcomes of either group with a mean 2.79 Snellen lines improvement of the intravitreal dexamethasone group versus 1.8 lines in the placebo group. Subgroup analysis suggested a clinical trend to better visual acuity in the post cataract steroid subgroup with mean 4.1 lines improvement versus 2.7 in the placebo group (p=0.33). No adverse events attributable to the dexamethasone were reported. CONCLUSIONS: Intravitreal dexamethasone appears safe and may be of benefit in post cataract surgery bacterial endophthalmitis.

Course and complications of varicella zoster ophthalmicus in a high HIV seroprevalence population (Cape Town, South Africa).

AIM: To describe the course and complications of varicella zoster ophthalmicus (VZO) in patients attending an eye clinic in a community with a high HIV seroprevalence. STUDY DESIGN: Prospective cohort study of consecutive patients presenting to a tertiary hospital eye clinic with VZO. METHOD: Patients recruited in 2001 and 2002 received standardized initial topical and systemic management, which was then modified according to complications. Information on the course and complications of the disease was entered in a database prior to statistical analysis. RESULTS: Information on 102 patients who had 250 visits to the eye clinic was collected. HIV serology was positive, negative, and unknown in 66, 22, and 14 patients, respectively. The most common complication was uveitis (40/102). Median delay from onset of rash to starting acyclovir was 5 days. Complications were present in 33 patients at the first visit. Complications were commoner in patients with positive Hutchinson's sign and were less common at CD4 counts <200. At CD4 counts, > or =200 HIV infection had little effect on the course and complications of VZO. Timing of commencement of Acyclovir therapy within or after 72 h had no demonstrable effect on the incidence of new complications. CONCLUSION: In a resource-limited setting, patients with the following characteristics should have immediate ophthalmic assessment: symptoms suggesting ocular complications or the presence of Hutchinson's sign. All VZO patients should receive antiviral therapy at the first doctor's visit even if they present >72 h after onset of the rash.

The last 11 years of Molteno implantation at the University of Cape Town. Refining our indications and surgical technique.

AIMS: To analyse outcomes, factors influencing surgical success, and surgical technique of Molteno implantation over the past 11 years in order to identify ways of improving long-term control. METHODS: Retrospective interventional review of case records of all consecutive patients undergoing Molteno implantation at Groote Schuur Hospital between 1/1/1991 and 31/12/2002. Data were recorded on an MSAccess database and processed using Kaplan-Meier survival curves and life table analysis. RESULTS: We analysed 162 consecutive single-phase Molteno tube implantation procedures on 157 eyes of 148 patients with mean follow-up of 2.9 years. Intraocular pressure (IOP) dropped from a mean of 43.3 at booking to 19.1 at final follow-up. Overall 'complete success' was achieved in 30% and 'partial success' in 16%. A high preoperative IOP was a significant predictor of a high postoperative pressure. Pseudophakic patients had significantly better postoperative pressure control. Neovascular glaucoma was a risk factor for poor pressure control. Race, gender, previous surgery, uveitis, and trauma did not influence surgical outcome. Follow-up adjusted incidence of 2.4 cases of endophthalmitis per patient year was unexpectedly high. Tubes that migrated had been secured with absorbable sutures in 4/5 cases. CONCLUSIONS: In this study, high preoperative IOPs were probably a significant contributing factor to relatively poor postoperative pressure control. Addressing this issue may aid in improving outcomes in future surgery. The high postoperative pressure outcomes suggest that single plate Molteno implantation is not an ideal way of achieving low target pressure in third world glaucoma patients.

Corneal graft rejection precipitated by uveitis secondary to alendronate sodium therapy.

PURPOSE: To report a case of corneal graft rejection precipitated by severe uveitis secondary to alendronate therapy and to review the literature of relevance to this case. METHODS: A 77-year-old woman with a hypopyon and corneal graft rejection was studied for possible precipitants, including herpes viral and bacterial infection. Results were negative. She was treated unsuccessfully with systemic and topical steroids, systemic antivirals, and intraocular antibiotic therapy. RESULTS: Withdrawal of alendronate resulted in rapid resolution of intraocular inflammation and corneal edema. CONCLUSION: We recommend vigilance in corneal transplant patients on simultaneous bisphosphonate therapy. Caution is advised in the extension to human trials of animal studies investigating the use of bisphosphonates in corneal transplantation.

Candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: developmental chemistry and investigation of immunological responses following immunization of mice and rabbits.

Glycoconjugates were prepared by covalently linking the immunogenic protein carrier CRM(197) to O-deacylated lipopolysaccharide (LPS) derived from Neisseria meningitidis (strain H44/76), immunotype L3 galE LPS. This mutant strain elaborates a truncated LPS structure that displays immunological epitopes characteristic of 76% of Group B meningococcal (NmB) strains. CRM(197) was covalently linked either to the reducing glucosamine residue of the lipid A region of the O-deacylated LPS or to a 2-keto-3-deoxy-octulosonic acid (Kdo) residue in the inner core region of the O-deacylated LPS. In both rabbits and mice a much stronger IgG response to the immunising antigen was generated in those animals that received conjugates linked via the lipid A region. Sera from mice that were immunized with these conjugates were assayed for their reactivity with LPS, both mutant and wild-type, of several homologous and heterologous NmB strains. Sera obtained from mice immunized with conjugates in which the carrier protein was linked via the Kdo moiety were only able to react with O-deacylated, but not fully acylated (native), LPS from the homologous strain. However, sera obtained from mice that were immunized with conjugates, in which the carrier protein was coupled to the lipid A region, reacted predominately with inner core epitopes that contained phosphoethanolamine at the same 3-position of the distal heptose residue (HepII) of the inner core LPS as was present on the immunising antigen. Additionally it was observed that sera from rabbits immunised with lipid A linked conjugates, unlike the mice responses, were generally not as specific for LPS antigens that contained phosphoethanolamine at the same 3-position as was present on the immunising antigen, but showed a broader inner core recognition, whereas those rabbits that received the Kdo-linked conjugates gave only a very weak non-specific response to all immunotypes. Finally, the sera from two out of six mice that had received lipid A linked conjugates had bactericidal activity against L3 wild-type NmB strain 8047 and one of these was able to passively protect against meningococcal infection in an infant rat model. This study demonstrates evidence towards the proof-in-principle that by using Nm inner core LPS conjugates coupled via the lipid A region with an intact phosphoethanolamine at the O-3 position of the HepII of the inner core LPS, it is possible to elicit functional and protective antibodies against meningococcal infection.

Mapping bacterial glycolipid complexity using capillary electrophoresis and electrospray mass spectrometry.

This chapter presents the application of capillary electrophoresis coupled to electrospray mass spectrometry (CE-ES-MS) for the analysis of complex bacterial lipopolysaccharides (LPS) from pathogenic strains of Haemophilus influenzae and Neisseria meningitidis. A discussion is included of the development of electrophoretic conditions conducive to trace-level enrichment and separation of closely related glycoforms and isoforms, which provided sensitive detection of glycolipids from as little as five bacterial colonies. The chapter also describes the use of mixed MS scanning functions to aid the identification of specific functionalities and immunodeterminants of LPS, such as pyrophosphoethanolamine, phosphocholine, and N-acetyl neuraminic acid (Neu5Ac), which represent less than 2% of the overall LPS population. The combination of high-resolution capillary electrophoresis with sensitive tandem mass spectrometry (MS/MS) provides a unique analytical tool to probe the subtle structural changes resulting from oligosaccharide branching and location of substituted LPS isoforms. The ability to detect a diverse LPS population over a wide dynamic range of expression using CE-MS enables the correlation of structural changes between bacterial strains and isogenic mutants to assign functional gene relationship.

Functional genomics of pathogenic bacteria.

Microbial diseases remain the commonest cause of global mortality and morbidity. Automated-DNA sequencing has revolutionized the investigation of pathogenic microbes by making the immense fund of information contained in their genomes available at reasonable cost. The challenge is how this information can be used to increase current understanding of the biology of commensal and virulence behaviour of pathogens with particular emphasis on in vivo function and novel approaches to prevention. One example of the application of whole-genome-sequence information is afforded by investigations of the pathogenic role of Haemophilus influenzae lipopolysaccharide and its candidacy as a vaccine.

Genetic basis for expression of the major globotetraose-containing lipopolysaccharide from H. influenzae strain Rd (RM118).

A genetic basis for the biosynthetic assembly of the globotetraose containing lipopolysaccharide (LPS) of Haemophilus influenzae strain RM118 (Rd) was determined by structural analysis of LPS derived from mutant strains. We have previously shown that the parent strain RM118 elaborates a population of LPS molecules made up of a series of related glycoforms differing in the degree of oligosaccharide chain extension from the distal heptose residue of a conserved phosphorylated inner-core element, L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)-]-L-alpha-D-Hepp-(1-->5)-alpha-Kdo. The fully extended LPS glycoform expresses the globotetraose structure, beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp. A fingerprinting strategy was employed to establish the structure of LPS from strains mutated in putative glycosyltransferase genes compared to the parent strain. This involved glycose and linkage analysis on intact LPS samples and analysis of O-deacylated LPS samples by electrospray ionization mass spectrometry and 1D (1)H-nuclear magnetic resonance spectroscopy. Four genes, lpsA, lic2A, lgtC, and lgtD, were required for sequential addition of the glycoses to the terminal inner-core heptose to give the globotetraose structure. lgtC and lgtD were shown to encode glycosyltransferases by enzymatic assays with synthetic acceptor molecules. This is the first genetic blueprint determined for H. influenzae LPS oligosaccharide biosynthesis, identifying genes involved in the addition of each glycose residue.

The catastrophic misinterpretation model of panic disorder.

In the catastrophic misinterpretation model of panic Clark [Behav. Res. Ther. 24(1986)1461] proposes that panic attacks result from the misinterpretation of autonomic arousal stimuli as precursors to a physical or psychological emergency. The model has been widely examined, with many researchers suggesting that this specific cognitive bias is implicated in both the phenomenon of panic, and the aetiology and maintenance of panic disorder. Various research methodologies have provided only partial or inconclusive support for the model as being uniquely associated with panic, and as a cognitive process underpinning the experience of panic. This paper reviews the body of existing evidence and its implications for the model and proposes future research directions. The influence of implicit operational definitions of key terms in the catastrophic misinterpretation literature (e.g. 'catastrophe', 'threat', 'anxiety-related') are examined, and clarifications proposed. Inconsistencies and limitations in the measurement of catastrophic misinterpretation are highlighted, and subsequently developments to measurement instruments are proposed.

Deposition and pharmacokinetics of an HFA formulation of triamcinolone acetonide delivered by pressurized metered dose inhaler.

Novel formulations of asthma drugs contained in pressurized metered dose inhalers (pMDIs) are being developed containing hydrofluoroalkane (HFA) propellants. The objectives of this study were to assess the deposition in the lungs and oropharynx of triamcinolone acetonide (TAA; Azmacort, Aventis Pharma, Collegeville, PA) delivered by pMDI formulated with HFA-134a, together with the pharmacokinetic profile of TAA, and to determine the extent to which the Azmacort spacer improves targeting of TAA to the lungs. The deposition of TAA, labelled with 99mTc, was assessed by gamma scintigraphy in 10 patients with mild to moderate asthma (mean forced expiratory volume in one second [FEV1] 76% predicted), who received in randomized order three delivered (ex-device) doses of 75 microg TAA via pMDI coupled to an Azmacort spacer (TAA-spacer), and three delivered doses of 230 microg TAA via the same device, but with the spacer removed (TAA-no spacer). Mean lung deposition expressed as mass of drug was similar for each regimen (TAA-no spacer 175 microg; TAA-spacer 188 microg), but when expressed as percentage delivered dose, lung deposition was higher for TAA-spacer (53.8%) versus TAA-no spacer (26.0%), indicating superior drug targeting for TAA-spacer. The spacer reduced oropharyngeal deposition. The pharmacokinetic data showed higher plasma levels of drug for TAA-no spacer, resulting from higher oropharyngeal deposition. "Pharmacoscintigraphic" data showed proof of concept for a novel HFA delivery system for an inhaled corticosteroid based on pulmonary targeting of drug.

A rapid and sensitive procedure for determination of 5-N-acetyl neuraminic acid in lipopolysaccharides of Haemophilus influenzae: a survey of 24 non-typeable H. influenzae strains.

In view of the importance of 5-N-acetyl neuraminic acid in bacterial pathogenesis, a sensitive, reproducible and reliable method for the determination of 5-N-acetyl neuraminic acid levels in lipopolysaccharide (LPS) is described and applied to 24 different non-typeable Haemophilus influenzae (NTHi) strains. The method involves analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of terminal 5-N-acetyl neuraminic acid residues released by neuraminidase treatment of O-deacylated LPS. The procedure is relatively fast and the instrumental effort is moderate. The results of the procedure were compared with data obtained by 1H NMR and electrospray ionisation-mass spectrometry (ESI-MS). The analysis of LPS from 24 NTHi strains showed that 5-N-acetyl neuraminic acid was found to be a common constituent of LPS in NTHi. Only one strain (NTHi 432) did not show any sialylation. Molar ratios (LPS/5-N-acetyl neuraminic acid) ranged between 5/1 and 500/1. Several strains in which no 5-N-acetyl neuraminic acid could be determined by other methods including 1H NMR and ESI-MS were shown to contain 5-N-acetyl neuraminic acid by this HPAEC-PAD procedure. The method was applied to determine levels of terminal 5-N-acetyl neuraminic acid in LPS from NTHi strains grown under different conditions and mutant strains containing inactive LPS biosynthetic genes.

Structural analysis of the lipopolysaccharide from the nontypable Haemophilus influenzae strain SB 33.

The structure of the core region of the lipopolysaccharide (LPS) from the nontypable Haemophilus influenzae strain SB 33 was elucidated. The LPS was subjected to a variety of degradative procedures. The structures of the derived oligosaccharide products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. These analyses revealed a series of related phosphocholine (PCho) containing structures differing in the number of hexose residues. The results pointed to each species containing a conserved phosphoethanolamine (PEtn) substituted heptose-containing trisaccharide inner-core moiety. The major LPS glycoforms were identified as 2-Hex, 3-Hex and 4-Hex species according to the number of hexose residues present.

Structural variability and originality of the Bordetella endotoxins.

Structural studies of Bordetella endotoxins (LPSs) have revealed remarkable differences: (i) between their LPSs and those of other bacterial pathogens; (ii) among the LPSs of the seven identified Bordetella species; and (iii) among the LPSs of some Bordetella strains. The lipid As have the "classical" bisphosphorylated diglucosamine backbone but tend to have fewer and species-specific fatty acid components compared to those of other genera. Nevertheless, three strains of B. bronchiseptica have at least three different fatty acid distributions; however, the recently identified B. hinzii and B. trematum LPSs had identical lipid A structures. The B. pertussis core is a dodecasaccharide multi-branched structure bearing amino and carboxylic groups. Another unusual feature is the presence of free amino sugars in the central core region and a complex distal trisaccharide unit containing five amino groups of which four are acetylated and one is methylated. The B. pertussis LPS does not have O-chains and that of B. trematum had only a single O-unit, unlike the LPSs of all the other species of the smooth-type. The O-chain-free cores of non-B. pertussis LPSs were always built on the B. pertussis core model but most were species-specifically incomplete. The LPS structures of three B. bronchiseptica strains were found to be different from each other. The O-chains of B. bronchiseptica and B. parapertussis were almost identical and had some features in common with B. hinzii O-chain. Serological analyses are consistent with the determined LPS structures.

Functional opsonic activity of human serum antibodies to inner core lipopolysaccharide (galE) of serogroup B meningococci measured by flow cytometry.

A recently described flow cytometric opsonophagocytic assay (OPA) was adapted to quantify the functional activity of serum antibodies specifically directed against serogroup B inner core lipopolysaccharide (LPS) of Neisseria meningitidis. The percentage of human peripheral polymorphonuclear leukocytes and monocytes (PMNms) ingesting fluorescently labeled, ethanol-fixed N. meningitidis organisms (phagocytic activity) in the presence of human sera was measured to reflect the serum opsonic activity against the bacterium. The contribution to opsonophagocytic activity of antibodies to inner core LPS was estimated by comparing the opsonic activities of adult and infant sera before and after adsorbing anti-LPS antibodies from the sera using purified LPS extracted from an LPS mutant (galE) of N. meningitidis strain MC58 (B:15:P1.7,16:L3). The specificity of the assay was further investigated using monoclonal antibody (MAb) B5, which binds to an inner core LPS epitope of N. meningitidis. A dose-dependent decrease in phagocytic activity was observed when MAb B5 was incubated with LPS from an inner core LPS (galE) mutant. Similarly, the number of PMNms ingesting fluorescently labeled polystyrene beads coated with inner core (galE) LPS decreased in a dose-dependent fashion when MAb B5 was incubated with various concentrations of the homologous inner core LPS. Strong correlations were found between the concentration of serum antibodies to inner core LPS (galE) versus the phagocytic activity using healthy adult sera (r(2) = 0.89). There was a correlation between phagocytic ingestion and initiation of intracellular oxidative burst (r(2) = 0.99) using polystyrene beads coated with inner core LPS and opsonized with the same sera using the oxidative burst indicator system dihydrorhodamine123/rhodamine 123. OPA results were also found to correlate closely with the results of the serum bactericidal assay using MAb B5 against the N. meningitidis MC58 galE mutant in the presence of human complement (r(2) = 0.994, P = 0.003, two-tailed test). These studies demonstrate that functional antibodies are produced in humans against meningococcal inner core LPS and that the OPA is a useful approach to study the opsonic activity of antibodies to inner core LPS in health and disease.

A new structural type for Haemophilus influenzae lipopolysaccharide. Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 486.

Structural elucidation of the sialylated lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae (NTHi) strain 486 has been achieved by the application of high-field NMR techniques and ESI-MS along with composition and linkage analyses on O-deacylated LPS and oligosaccharide samples. It was found that the LPS contains the common element of H. influenzae, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A, but instead of glycosyl substitution of the terminal heptose residue (HepIII) at the O2 position observed in other H. influenzae strains, HepIII is chain elongated at the O3 position by either lactose or sialyllactose (i.e. alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp). The LPS is substituted by an O-acetyl group linked to the O2 position of HepIII and phosphocholine (PCho) which was located at the O6 position of a terminal alpha-D-Glcp residue attached to the central heptose, a molecular environment different from what has been reported earlier for PCho. In addition, minor substitution by O-linked glycine to the LPS was observed. By investigation of LPS from a lpsA mutant of NTHi strain 486, it was demonstrated that the lpsA gene product also is responsible for chain extension from HepIII in this strain. The involvement of lic1 in expression of PCho was established by investigation of a lic1 mutant of NTHi strain 486.

Identification of a lipopolysaccharide alpha-2,3-sialyltransferase from Haemophilus influenzae.

We have identified a gene for the addition of N-acetylneuraminic acid (Neu5Ac) in an alpha-2,3-linkage to a lactosyl acceptor moiety of the lipopolysaccharide (LPS) of the human pathogen Haemophilus influenzae. The gene is one that was identified previously as a phase-variable gene known as lic3A. Extracts of H. influenzae, as well as recombinant Escherichia coli strains producing Lic3A, demonstrate sialyltransferase activity in assays using synthetic fluorescent acceptors with a terminal galactosyl, lactosyl or N-acetyl-lactosaminyl moiety. In the RM118 strain of H. influenzae, Lic3A activity is modulated by the action of another phase-variable glycosyltransferase, LgtC, which competes for the same lactosyl acceptor moiety. Structural analysis of LPS from a RM118:lgtC mutant and the non-typeable strain 486 using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed that the major sialylated species has a sialyl-alpha-(2-3)-lactosyl extension off the distal heptose. This sialylated glycoform was absent in strains containing a lic3A gene disruption. Low amounts of sialylated higher molecular mass glycoforms were present in RM118:lgtC lic3A, indicating the presence of a second sialyltransferase. Lic3A mutants of H. influenzae strains show reduced resistance to the killing effects of normal human serum. Lic3A, encoding an alpha-2,3-sialyltransferase activity, is the first reported phase-variable sialyltransferase gene.

Glycine is a common substituent of the inner core in Haemophilus influenzae lipopolysaccharide.

A survey of both typeable and nontypeable strains of Haemophilus influenzae indicated that they contain glycine (Gly) in their lipopolysaccharide (LPS). Significant amounts (30-250 pmol Gly/microg LPS) were determined by high-performance anion-exchange chromatography using pulsed amperometric detection after treatment of the LPS with mild alkali. Oligosaccharides obtained from LPS after mild acid hydrolysis and gel filtration chromatography were investigated by electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis (CE) ESI-MS. In all cases, molecular ions corresponding to the major glycoforms were identified and were accompanied by ions differing by 57 Da, thus indicating the presence of glycine. The position of glycine in these glycoforms was determined by CE-ESI-MS/MS analyses. It was found that, depending on strain, glycine can substitute each of the heptoses of the inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-L-alpha-D-Hepp-(1-->5)-alpha-Kdo of H. influenzae LPS as well as Kdo. In some strains, mixtures of monosubstituted Gly-containing glycoforms having different substitution patterns were identified.

Structure and functional genomics of lipopolysaccharide expression in Haemophilus influenzae.

The involvement of genes in the lic loci in H. influenzae LPS expression has been known for some time. However, it was not until recently that it was shown that the lic1 locus contains genes required for phase variable expression of phosphocholine substituents, while genes in the lic2 locus and lgtC are required for expression of the globoside trisaccharide, alpha-D-Galp-(1 --> 4)-beta-D-Galp-(1 --> 4)-beta-D-Glcp (i.e., the pK blood group epitope). The availability of the complete sequence of the H. influenzae strain Rd genome has facilitated significant progress in understanding the role of these and other genes in the expression and biosynthesis of LPS. We have employed a comparative structural fingerprinting strategy to establish the structural relationships among LPS from H. influenzae mutant strains in which putative biosynthesis genes were inactivated. Using this functional genomics approach, we have gained considerable insight into the genetic basis for intra-strain and strain-to-strain variation in epitope expression.