Complex Serology and Immune Response of Mice to Variant High-Molecular-Weight O Polysaccharides Isolated from Pseudomonas aeruginosa Serogroup O2 Strains

The O antigen of the Pseudomonas aeruginosa lipopolysaccharide is the optimal target for protective antibodies, but the unusual and complex nature of their sugar substituents has made it difficult to define the range of these structures needed in an effective vaccine. Most clinical isolates of P. aeruginosa can be classified into 10 O-antigen serogroups, but slight chemical differences among O polysaccharides within a serogroup give rise to subtype epitopes. These epitopes could impact the reactivity of O-antigen-specific antibodies, as well as the susceptibility of a target strain to protective, opsonic antibodies. To define parameters of serogroup and subtype-epitope immunogenicity, antigenicity, and surface expression on P. aeruginosa cells, we prepared high-molecular-weight O-polysaccharide vaccines from strains of P. aeruginosa serogroup O2, for which eight structurally variant O antigens expressing six defined subtype epitopes (O2a to O2f) have been identified. A complex pattern of immune responses to these antigens was observed following vaccination of mice. The high-molecular-weight O polysaccharides were generally more immunogenic at low doses (1 and 10 μg) than at a high dose (50 μg) and usually elicited antibodies that opsonized the homologous strain for phagocytic killing. Some of the individual polysaccharides elicited cross-opsonic antibodies to a variable number of strains that express all of the defined serogroup O2 subtype epitopes. Combination into one vaccine of two antigens that individually elicited cross-reactive opsonic antibodies to most members of the O2 serogroup inhibited, instead of enhanced, the production of antibodies broadly reactive with most serogroup O2 subtype strains. Thus, immune responses to P. aeruginosa O antigens may be restricted to a limited range of epitopes on structurally complex O antigens, and combining multiple related antigens into a single vaccine formulation may inhibit the production of those antibodies best able to protect against most P. aeruginosa strains within a given O-antigen serogroup.It has been established through animal and human experimentation that the lipopolysaccharide (LPS) O antigen of Pseudomonas aeruginosa is a target for protective antibodies (3, 36, 38). The studies of Knirel and colleagues (17, 19) on the chemical composition and structure of the major O-side-chain polysaccharides have provided important insights into the immunochemical properties of these antigens, but our understanding of their antigenic and immunogenic properties is incomplete. This point is highlighted by the inability to date to develop effective, LPS-specific immunotherapies for human P. aeruginosa infection (7).Results obtained with animals by using immunogens and antibodies specific to the O polysaccharides have indicated that slight chemical differences among bacterial strains with otherwise closely related O-side-chain structures can produce a complex pattern of reactions between antibodies and related antigens (13). With standard serologic methods using whole-cell agglutinations, strains of P. aeruginosa can be classified as members of one serogroup (serotype); members of each serogroup share a group-specific antigen. Further subdivision into subtypes, which correlate with structural variants determined by Knirel and colleagues (17), can be accomplished with appropriate antisera (22).To develop safe and effective O-antigen-specific P. aeruginosa vaccines, we have utilized the high-molecular-mass (>100,000-Da) fraction of O polysaccharides. These antigens are safe and immunogenic in humans and animals (13, 27, 37) and elicit protective antibodies to the strains from which they are isolated. However, in recent studies of animals immunized with a heptavalent high-molecular-weight O-polysaccharide vaccine whose individual components were isolated from single strains representative of the major serogroups causing P. aeruginosa infection, opsonic antibody responses to the group-specific antigens were not commonly elicited (13). Thus, in spite of chemical and serologic relatedness among subtype strains within a P. aeruginosa serogroup, single antigens isolated from one subtype strain do not always elicit opsonic antibodies to all of the strains within the serogroup (13). Previous results showed that a particular O antigen from a given serogroup may elicit group-specific immunity, while an O antigen from another serogroup may elicit only immunity specific to the subtype epitopes expressed on that particular O antigen.To explore this situation further and gain additional insight into the serologic diversity among P. aeruginosa LPS O antigens, we prepared high-molecular-weight O-polysaccharide immunogens from five strains of P. aeruginosa serogroup O2 that, together, express all six of the identified subtype antigens (Table ​(Table1).1). These polysaccharides were used to immunize mice, and the resultant sera were assessed by enzyme-linked immunosorbent assay (ELISA) and for opsonic killing activity. The results showed a complex interaction among the strains with regard to high-molecular-weight O-polysaccharide immunogenicity, antigenicity, serogroup and subtype epitope density, and susceptibility to opsonic killing. These findings indicate that the current serogroup classifications of P. aeruginosa are probably inadequate to define the full range of LPS antigens needed to elicit comprehensive immunity to a wide range of clinical isolates.

TABLE 1

Strains used for immunogen production, their serologic classification by subtype epitope, and chemical structures of the associated O antigens Open in a separate window^aBoldface type indicates a feature of a structure that distinguishes it from a related structure of the same serogroup. Abbreviations: FucNAc, 2-acetamido-2,6-dideoxygalactose (N-acetylfucosamine); Man(NAc)₂A, 2,3-diacetamido-2,3-dideoxymannuronic acid; Man(2NAc3N)A, 2-acetamido-3-acetamidino-2,3-dideoxymannuronic acid; Gul(NAc)₂A, 2,3-diacetamido-2,3-dideoxyguluronic acid. ^bThe lower structure is also part of the O antigen of strain 170007; there is about a 2:1 ratio of the upper and lower structures.      

Clinical,immunological, and molecular analysis in a large cohort of patients with X-linked agammaglobulinemia: an Italian multicenter study

A questionnaire-based retrospective clinical and immunological survey was conducted in 73 males with a definite diagnosis of X-linked agammaglobulinemia based on BTK sequence analysis. Forty-four were sporadic and 29 familial cases. At December 2000, the patients' ages ranged from 2 to 33 years; mean age at diagnosis and mean duration of follow-up were 3.5 and 10 years respectively. After the mid-1980s all but 2 were on intravenous immunoglobulin (IVIG) substitution therapy, with residual IgG >500 mg/dl in 94% of the patients at the time of enrollment. Respiratory infections were the most frequent manifestation both prior to diagnosis and over follow-up. Chronic lung disease (CLD) was present in 24 patients, in 15 already at diagnosis and in 9 more by 2000. The cumulative risk to present at diagnosis with CLD increased from 0.17 to 0.40 and 0.78 when the diagnosis was made at the ages of 5, 10, and 15 years respectively. For the 9 patients who developed CLD during follow-up, the duration of follow-up, rather than age at diagnosis; previous administration of intramuscular immunoglobulin; and residual IgG levels had a significant effect on the development of CLD. Chronic sinusitis was present in 35 patients (48%), in 15 already at diagnosis and in 20 by 2000. Sistemic infections such as sepsis and meningitis/meningoencephalitis decreased over follow-up, probably due to optimal protection provided by high circulating IgG levels reached with IVIG.     

Age and sex influence on oxidative damage and functional status in human skeletal muscle

A reduction in muscle mass, with consequent decrease in strength and resistance, is commonly observed with advancing age. In this study we measured markers of oxidative damage to DNA, lipids and proteins, some antioxidant enzyme activities as well Ca²⁺ transport in sarcoplasmic reticulum membranes in muscle biopsies from vastus lateralis of young and elderly healthy subjects of both sexes in order to evaluate the presence of age- and sex- related differences. We found a significant increase in oxidation of DNA and lipids in the elderly group, more evident in males, and a reduction in catalase and glutathione transferase activities. The experiments on Ca²⁺ transport showed an abnormal functional response of aged muscle after exposure to caffeine, which increases the opening of Ca²⁺ channels, as well a reduced activity of the Ca²⁺ pump in elderly males. From these results we conclude that oxidative stress play an important role in muscle aging and that oxidative damage is much more evident in elderly males, suggesting a gender difference maybe related to hormonal factors.This revised version was published online in September 2005 with corrections to the Cover Date.     

Using linked markers to infer the age of a mutation

Advances in sequencing and genotyping technologies over the last decade have enabled geneticists to easily characterize genetic variation at the nucleotide level. Hundreds of genes harboring mutations associated with genetic disease have now been identified by positional cloning. Using variation at closely linked genetic markers, it is possible to predict the times in the past at which particular mutations arose. Such studies suggest that many of the rare mutations underlying human genetic disorders are relatively young. Studies of variation at genetic markers linked to particular mutations can provide insights into human geographic history, and historical patterns of natural selection and disease, that are not available from other sources. We review two approaches for estimating allele age using variation at linked genetic markers. A phylogenetic approach aims to reconstruct the gene tree underlying a sample of chromosomes carrying a particular mutation, obtaining a “direct” estimate of allele age from the age of the root of this tree. A population genetic approach relies on models of demography, mutation, and/or recombination to estimate allele age without explicitly reconstructing the gene tree. Phylogenetic methods are best suited for studies of ancient mutations, while population genetic methods are better suited for studies of recent mutations. Methods that rely on recombination to infer the ages of alleles can be fine‐tuned by choosing linked markers at optimal map distances to maximize the information available about allele age. A limitation of methods that rely on recombination is the frequent lack of a fine‐scale linkage map. Maximum likelihood and Bayesian methods for estimating allele age that rely on intensive numerical computation are described, as well as “composite” likelihood and moment‐based methods that lead to simple estimators. The former provide more accurate estimates (particularly for large samples of chromosomes) and should be employed if computationally practical. Hum Mutat 18:87–100, 2001. © 2001 Wiley‐Liss, Inc.     

Ultrastructural alterations induced in quail ciliary neurons by postganglionic nerve crush and by Ricinus toxin administration, separately and in combination

The response to postganglionic nerve crush and Ricinus toxin administration by the ciliary neurons of the quail ciliary ganglion was investigated at the ultrastructural level. The toxin was either applied at the crush site on the postganglionic nerves or injected into the anterior eye chamber without any other operative intervention. Crush of postganglionic nerves without toxin administration and saline injection into the anterior eye chamber served as controls for the two toxin administration procedures. Postganglionic nerve crush caused a distinct chromatolytic reaction, accompanied by massive detachment of the preganglionic axon terminals from the ciliary neurons and loss of most of the synapses, both chemical and electrical. This process does not induce cell death and is reversible. Saline injection in the anterior eye chamber caused a moderate retrograde reaction in some of the ciliary neurons, presumably as a consequence of paracentesis. The changes consisted mainly of an increase of perikaryal neurofilaments with, at most, a minor detachment of the preganglionic boutons from a small portion of the cell body at the nuclear pole. Ricinus toxin administration induced neuronal degeneration following a pattern common to both delivery modes. The degenerative process consisted of disruption and detachment of polyribosomes from the rough endoplasmic reticulum, an increase of smooth cisterns and tubules, a dramatic increase of neurofilament bundles, compartmentalization of the cytoplasmic organelles and, finally, karyorrhexis and cell lysis. The final stages of Ricinus toxin degeneration involve a progressive accumulation of extracellular flocculo-filamentous material and cell lysis. After administration of Ricinus toxin to the crush site, ricin-affected neurons showed withdrawal of the preganglionic boutons from a portion of the ciliary neuron, especially at the nuclear pole. After Ricinus toxin injection into the anterior eye chamber, however, the bouton shell surrounding the affected ciliary neurons remained intact in the early stages of degeneration. Detachment of the preganglionic terminals and disruption of the cell junctions, therefore, is the consequence of nerve crush and not of the toxin itself.

This study demonstrates that quail ciliary neurons are a suitable model for experimental neuropathology and neurotoxicology.     

Mitochondrial import and enzymatic activity of PINK1 mutants associated to recessive parkinsonism

Parkinson's disease (PD) is a progressive neurodegenerative illness associated with a selective loss of dopaminergic neurons in the nigrostriatal pathway of the brain. Despite the overall rarity of the familial forms of PD, the identification of single genes linked to the disease has yielded crucial insights into possible mechanisms of neurodegeneration. Recently, a putative mitochondrial kinase, PINK1, has been found mutated in an inherited form of parkinsonism. Here, we describe that PINK1 mutations confer different autophosphorylation activity, which is regulated by the C-terminal portion of the protein. We also demonstrate the mitochondrial localization of both wild-type and mutant PINK1 proteins unequivocally and prove that a short N-terminal part of PINK1 is sufficient for its mitochondrial targeting.