Vaccination with streptococcal extracellular cysteine protease (interleukin-1β convertase) protects mice against challenge with heterologous group A streptococci

Virtually all clinical isolates of group A streptococci secrete a highly conserved extracellular cysteine protease that cleaves human fibronectin and vitronectin, and converts IL-1β precursor to biologically active IL-1β. Based on the high degree of gene conservation within the species and its role in host pathogenicity, it was postulated that antibodies to the cysteine protease would confer protective immunity against S. pyogenes infection. To test this hypothesis, Swiss CD1 mice were intraperitoneally administered either saline, rabbit IgG, or IgG from rabbits immunized with the protease, and challenged with a highly virulent (minimum lethal dose 10 cfu) clinical isolate of S. pyogenes expressing a heterologous cysteine protease. The results indicate that mice administered IgG from rabbits immunized with purified cysteine protease had significantly enhanced survival when compared with mice given either non-specific rabbit IgG (log rank test; χ²; p = 0.0195) or saline (log rank test; χ²; p = 0.0002). Moreover, mice actively immunized with the cysteine protease had a significantly longer time to death than the control group (log rank test; χ²; p = 0.0418). The results show that the cysteine protease elicits non-type-specific immunity to challenge with heterologous S. pyogenes.     

Functional evidence for a breast cancer growth suppressor gene on chromosome 17

Rearrangements or deletions of chromosome 17 are the most frequentlyobserved genetic changes identified in breast tumors. Molecularanalyses suggest that in addition to the p53 gene on 17p13.1there may be at least three other tumor suppressor genes onchromosome 17 involved in breast cancer. Regions of loss ofheterozygosity (LOH) identified on 17p13.3 and 17q12-qter occurfrequently in breast tumors, and the BRCA-1 gene has been mappedto 17q21 by genetic linkage analysis. Here we provide biologicalevidence for the presence of a growth suppressor gene(s) onchromosome 17 that results In the In vitro growth suppressionof the p53 wild-type MCF 7 breast cancer cell line. We haveIntroduced a normal chromosome 17 into MCF 7 cells by microcellmediatedchromosome transfer (MMCT), and demonstrate that cells growtharrest before 10 to 12 population doublings. In contrast, theintroduction of a normal chromosome 13 had no effect upon growthof these cells either In vitro or In vivo. These data providedirect functional evidence for the presence of a growth suppressorgene(s) on chromosome 17, which is not p53, and which may representone of several gene(s) that play a critical role in the developmentof breast cancer.     

Aberrant retinal projections to midbrain targets mediate spared visual orienting function in hamsters with neonatal lesions of superior colliculus

Summary Rodents, cats, and most nonmammalian vertebrates with bilateral tectal deafferentation or ablation in adulthood are extremely deficient at orienting to visual stimuli; yet animals with neonatal lesions of superficial layers of the superior colliculus (SC) show partial sparing of this response, particularly for targets in the central visual field. In this study, we sought to determine whether these spared orienting abilities are mediated by aberrant retinal projections to the remaining intermediate layers of the SC, or whether visual cortex (VC) mechanisms or alternative behavioral strategies are responsible. Neonatal golden hamsters received either bilateral heat lesions of the SC (rlSC), or a heat lesion of the right SC and enucleation of the right eye (rSCrE). This latter procedure causes axons from the left eye to recross the tectal midline and terminate in the wrong (left) SC (Schneider 1973). As adults, both groups of hamsters were extremely deficient in visually guided approach to stationary targets, although rlSC-lesioned hamsters showed some sparing for central field targets and rSCrE-lesioned hamsters often made wrong-direction turns for targets in the left peripheral field. We then subjected both groups of neonatally lesioned hamsters to bilateral aspiration lesions of the VC. Retesting showed no change in visual orienting behavior as a result of the cortical lesions. Labeling of the optic tract with horseradish peroxidase (HRP) revealed abundant aberrant retinal projections to remaining intermediate layers of the SC and thalamic nucleus lateralis posterior (LP), as well as supernormal innervation of pretectal nuclei, the dorsal terminal nucleus of the accessory optic tract, and the ventral nucleus of the lateral geniculate body (LGv). We conclude that the spared visual orienting capabilities of hamsters with rlSC and rSCrE lesions are mediated by the aberrant midbrain projections, and that cortical mechanisms are not involved in spared visual orienting functions following these neonatal lesions.     

Failure of norepinephrine to initiate procine malignant hyperthermia

Continuous intravenous infusion in pigs of norepinephrine, to blood concentrations of 140 ng.ml^–1, provided a test of the hypothesis that this sympathetic hormone can initiate malignant hyperthermia (MH). This study was performed during nitrous oxidepentobarbital anesthesia, and in part utilized sodium nitroprusside to maintain normal blood pressure and peripheral perfusion. Metabolic stimulation, or evidence of MH, did not occur in normal of susceptible pigs, as indicated by the lack of increase in both whole body O₂ consumption and arterial lactate concentration. Next, in contrast, susceptible pigs manifested MH when exposed to halothane and succinylcholine, while normal pigs did not. We conclude that norepinephrine does not mediate or initiate porcine whole body stress responses characteristic of MH.     

SHIP represses mast cell activation and reveals that IgE alone triggers signaling pathways which enhance normal mast cell survival

The hemopoietic specific, Src homology 2-containing inositol 5' phosphatase (SHIP) hydrolyzes the phosphatidylinositol (PI)-3-kinase generated second messenger, PI-3,4,5-trisphosphate (PIP(3)), to PI-3,4-bisphosphate (PI-3,4-P(2)) in normal bone marrow derived mast cells (BMMCs). As a consequence, SHIP negatively regulates IgE+antigen (Ag)-induced degranulation as well as leukotriene and inflammatory cytokine production. Interestingly, in the absence of SHIP, BMMCs degranulate extensively with IgE alone, i.e. without Ag, suggesting that IgE alone is capable of stimulating signaling in normal BMMCs and that SHIP prevents this signaling from progressing to degranulation. To test this, we compared signaling events triggered by monomeric IgE versus IgE+Ag in normal BMMCs and found that multiple pathways are triggered by monomeric IgE alone and, while they are in general weaker than those stimulated by IgE+Ag, they are more prolonged. Moreover, while SHIP prevents this IgE-induced signalling from progressing to degranulation or leukotriene production it allows sufficient production of autocrine acting cytokines, in part by activation of NFkappaB, to enhance BMMC survival. Interestingly, the activation of NFkappaB and the level of cytokines produced are far higher with IgE than with IgE+Ag. Moreover, IgE alone maintains Bcl-X(L) levels and enhances the adhesion of BMMCs to fibronectin and this likely enhances their survival still further.     

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.      

Genetic determinants: is there an "atherosclerosis gene"?

It is now clear that atherosclerotic disease is a chronic inflammatory disease triggered by a sequence of events initiated at sites with turbulent flow under normal conditions such as in the coronary arteries or at bifurcations or where normal laminar flow is replaced by turbulent flow because of vessel pathologies. Normally, laminar flow is protected by generation of NO by endothelial NO synthase (eNOS), which becomes activated via stretch activated channels. When the flow turns turbulent, such protective NO generation ceases, leading to endothelial cell activation and lipid deposition into the extra-cellular space. There, lipoproteins and specifically phospholipids become oxidized by cells of the monocytic-macrophage lineage. Only when the LDL-cholesterol level is high enough lipid peroxidation products are generated in sufficient amounts to perpetuate the disease by generating a feed forward loop of endothelial cell activation leading to an inflammatory response. That inflammatory response might also be added by bacterial or viral infections such as Chlamydia pneumoniae or viruses. The disease then progresses to a chronic inflammatory state, whereby the immune system seems to contribute significantly and markers of chronic inflammation such as fibrinogen, leukocytes, PAI-1 and CRP are found increased.