The Alzheimer's disease-protective CD33 splice variant mediates adaptive loss of function via diversion to an intracellular pool.

The immunomodulatory receptor Siglec-3/CD33 influences risk for late-onset Alzheimer's disease (LOAD), an apparently human-specific post-reproductive disease. CD33 generates two splice variants: a full-length CD33M transcript produced primarily by the "LOAD-risk" allele and a shorter CD33m isoform lacking the sialic acid-binding domain produced primarily from the "LOAD-protective" allele. An SNP that modulates CD33 splicing to favor CD33m is associated with enhanced microglial activity. Individuals expressing more protective isoform accumulate less brain ß-amyloid and have a lower LOAD risk. How the CD33m isoform increases ß-amyloid clearance remains unknown. We report that the protection by the CD33m isoform may not be conferred by what it does but, rather, from what it cannot do. Analysis of blood neutrophils and monocytes and a microglial cell line revealed that unlike CD33M, the CD33m isoform does not localize to cell surfaces; instead, it accumulates in peroxisomes. Cell stimulation and activation did not mobilize CD33m to the surface. Thus, the CD33m isoform may neither interact directly with amyloid plaques nor engage in cell-surface signaling. Rather, production and localization of CD33m in peroxisomes is a way of diminishing the amount of CD33M and enhancing ß-amyloid clearance. We confirmed intracellular localization by generating a CD33m-specific monoclonal antibody. Of note, CD33 is the only Siglec with a peroxisome-targeting sequence, and this motif emerged by convergent evolution in toothed whales, the only other mammals with a prolonged post-reproductive lifespan. The CD33 allele that protects post-reproductive individuals from LOAD may have evolved by adaptive loss-of-function, an example of the less-is-more hypothesis.

Complement system activation contributes to the ependymal damage induced by microbial neuraminidase.

BACKGROUND: In the rat brain, a single intracerebroventricular injection of neuraminidase from Clostridium perfringens induces ependymal detachment and death. This injury occurs before the infiltration of inflammatory blood cells; some reports implicate the complement system as a cause of these injuries. Here, we set out to test the role of complement. METHODS: The assembly of the complement membrane attack complex on the ependymal epithelium of rats injected with neuraminidase was analyzed by immunohistochemistry. Complement activation, triggered by neuraminidase, and the participation of different activation pathways were analyzed by Western blot. In vitro studies used primary cultures of ependymal cells and explants of the septal ventricular wall. In these models, ependymal cells were exposed to neuraminidase in the presence or absence of complement, and their viability was assessed by observing beating of cilia or by trypan blue staining. The role of complement in ependymal damage induced by neuraminidase was analyzed in vivo in two rat models of complement blockade: systemic inhibition of C5 by using a function blocking antibody and testing in C6-deficient rats. RESULTS: The complement membrane attack complex immunolocalized on the ependymal surface in rats injected intracerebroventricularly with neuraminidase. C3 activation fragments were found in serum and cerebrospinal fluid of rats treated with neuraminidase, suggesting that neuraminidase itself activates complement. In ventricular wall explants and isolated ependymal cells, treatment with neuraminidase alone induced ependymal cell death; however, the addition of complement caused increased cell death and disorganization of the ependymal epithelium. In rats treated with anti-C5 and in C6-deficient rats, intracerebroventricular injection of neuraminidase provoked reduced ependymal alterations compared to non-treated or control rats. Immunohistochemistry confirmed the absence of membrane attack complex on the ependymal surfaces of neuraminidase-exposed rats treated with anti-C5 or deficient in C6. CONCLUSIONS: These results demonstrate that the complement system contributes to ependymal damage and death caused by neuraminidase. However, neuraminidase alone can induce moderate ependymal damage without the aid of complement.

The trans-sialidase, the major Trypanosoma cruzi virulence factor: Three decades of studies.

Chagas' disease is a potentially life-threatening disease caused by the protozoan parasite Trypanosoma cruzi. Since the description of Chagas'disease in 1909 extensive research has identified important events in the disease in order to understand the biochemical mechanism that modulates T. cruzi-host cell interactions and the ability of the parasite to ensure its survival in the infected host. Exactly 30 years ago, we presented evidence for the first time of a trans-sialidase activity in T. cruzi (T. cruzi-TS). This enzyme transfers sialic acid from the host glycoconjugates to the terminal ß-galactopyranosyl residues of mucin-like molecules on the parasite's cell surface. Thenceforth, many articles have provided convincing data showing that T. cruzi-TS is able to govern relevant mechanisms involved in the parasite's survival in the mammalian host, such as invasion, escape from the phagolysosomal vacuole, differentiation, down-modulation of host immune responses, among others. The aim of this review is to cover the history of the discovery of T. cruzi-TS, as well as some well-documented biological effects encompassed by this parasite's virulence factor, an enzyme with potential attributes to become a drug target against Chagas disease.

Trypanosoma cruzi trans-sialidase: enhancement of virulence in a murine model of Chagas' disease.

Trypanosoma cruzi, the etiological agent of Chagas' disease, expresses a trans-sialidase at highest levels in infective trypomastigotes, where it attaches to the plasma membrane by a glycophosphoinositol linkage. Bound enzyme sheds into the extracellular milieu in a soluble form. Experiments performed in vitro suggest that the trans-sialidase participates in several parameters of T. cruzi-host interactions, like cell adhesion and complement resistance. However, the role that membrane-bound and soluble trans-sialidase plays in the infection of mammals is not understood. To begin to study the role the enzyme may play in vivo, T. cruzi trypomastigotes were inoculated subcutaneously into mice that had been sensitized for various times with the purified protein. A single dose of either endogenous or recombinant trans-sialidase injected into the connective tissues of BALB/c mice greatly enhanced parasitemia and mortality. Maximum enhancement was achieved with 1-2-h priming. Injection of the enzyme after the parasites had been established in the inoculation site had little, if any, consequence in modifying virulence. The enhancement did not seem to be through a direct effect of the enzyme on trypomastigote-host cell interactions because it occurred when the sites of trans-sialidase sensitization and parasite inoculation were physically separate. Rather, virulence enhancement seemed to depend on inflammatory cells, since priming with trans-sialidase had no significant effect in severe combined immunodeficiency mice, which lack functional T and B lymphocytes. However, antibody response to T. cruzi in the trans-sialidase-primed BALB/c mice was the same as in the control animals. Virulence enhancement was specific for the trans-sialidase because it did not occur in mice primed with Newcastle virus sialidase, which has the same substrate specificity as the T. cruzi enzyme, or with the sialidase from the bacterium Vibrio cholerae, whose substrate specificity is broader than the trypanosome sialidase. Furthermore, no enhancement of virulence occurred after sensitization with another adhesion protein (penetrin) purified from T. cruzi trypomastigotes and engineered bacteria, nor with bacterial lipopolysaccharide. The virulence-promoting activity of soluble trans-sialidase in the mouse model may be physiologically relevant because it was achieved with tiny doses, approximately 1-2 microgram/kg, raising the possibility that neutralization of the enzyme with specific probes could impair the development of Chagas' disease. In fact, a monoclonal antibody specific for the tandem repeat in the trans-sialidase COOH terminus enhanced infection of BALB/c mice, in agreement with earlier experiments in vitro, whereas antibodies against an amino acid sequence in the Cys region had the opposite effect.

The Sialidase NanS Enhances Non-TcsL Mediated Cytotoxicity of Clostridium sordellii.

The clostridia produce an arsenal of toxins to facilitate their survival within the host environment. TcsL is one of two major toxins produced by Clostridium sordellii, a human and animal pathogen, and is essential for disease pathogenesis of this bacterium. C. sordellii produces many other toxins, but the role that they play in disease is not known, although previous work has suggested that the sialidase enzyme NanS may be involved in the characteristic leukemoid reaction that occurs during severe disease. In this study we investigated the role of NanS in C. sordellii disease pathogenesis. We constructed a nanS mutant and showed that NanS is the only sialidase produced from C. sordellii strain ATCC9714 since sialidase activity could not be detected from the nanS mutant. Complementation with the wild-type gene restored sialidase production to the nanS mutant strain. Cytotoxicity assays using sialidase-enriched culture supernatants applied to gut (Caco2), vaginal (VK2), and cervical cell lines (End1/E6E7 and Ect1/E6E7) showed that NanS was not cytotoxic to these cells. However, the cytotoxic capacity of a toxin-enriched supernatant to the vaginal and cervical cell lines was substantially enhanced in the presence of NanS. TcsL was not the mediator of the observed cytotoxicity since supernatants harvested from a TcsL-deficient strain displayed similar cytotoxicity levels to TcsL-containing supernatants. This study suggests that NanS works synergistically with an unknown toxin or toxins to exacerbate C. sordellii-mediated tissue damage in the host.

Dermatonecrotic lesions in rabbits caused by neuraminidase.

In an attempt to study the role of neuraminidase in cholera vibrio pathogenesis, the dermatonecrotic effect of a purified neuraminidase preparation was studied in rabbits. The experiments demonstrated that dermatonecrotic lesions resulted from intracutaneous injection of a purified neuraminidase preparation (0.5 specific human dose or 1 NU) as well as significant doses of cholera toxoid containing the enzyme (4,12,18 specific human doses).

Why was the 1918 influenza pandemic so lethal? The possible role of a neurovirulent neuraminidase.

Epidemiological, viral, behavioral and neuropathological evidence suggests that some influenza epidemics were neurovirulent. Re-examination of the data from the lethal 1918 pandemic armed with recent observations about the influenza virus implicates a neurovirulent influenza virus in manic-depressive disease, schizophrenia and Parkinson's disease. The neurovirulence seems to have been related to the species of neuraminidase.

[The induction by the influenza virus A/H1N1 (serovariant Hsw1N1) and its glycoproteins of congenital pathology in mice]. / Induktsiia virusom grippa A/H1N1 *serovariant Hsw1N1) i ego glikoproteidami vrozhdennoi patologii u myshei.

Influenza A/H1N1 (serovariant Hsw1N1) virus, a sum of isolated glycoproteins, separately neuraminidase "heads", inoculated into white random-bred female mice, induced in some of the offsprings the pathology clinically and pathomorphologically similar to previously described slow virus infection. At the same time, the pathology in the offsprings caused by the antigenic virus variant under study was characterized by complete absence of fur and more dynamic progress of the disease. It is quite obvious that glycoproteins, particularly neuraminidase, are the molecular biological basis of dystrophic and degenerative changes in the organs of baby mice due to desialization and increased fluidity of capillary endothelium membranes and, possibly, CNS and other organ cells.

[Role of enterotoxin and neuraminidase in the biological activity of E1 Tor vibrios]. / Rol' énterotoksina i neiraminidazy v biologicheskoi aktivnosti vibrionov E1 Tor.

The biological activity of the toxigenic strains of cholera vibrios in suckling rabbits used as a model is manifested irrespective of the amount of neuraminidase produced by these strains. Neuraminidase is important only in low-cholerogenic strains, which is confirmed by a significantly greater death rate among suckling rabbits infected with cultures producing 40-2560 ng/ml of the exoenzyme in comparison with that among suckling rabbits infected with strains producing less than 10 ng/ml of the exoenzyme, or not producing it at all. The relation between the amounts of enterotoxin and neuraminidase, produced in vitro, and the biological activity of strains in suckling rabbits has been established, which allows one to study the virulence of strains in the passive immune hemolysis and aggregate hemagglutination tests.

Investigating the role of pneumococcal neuraminidase A activity in isolates from pneumococcal haemolytic uraemic syndrome.

Streptococcus pneumoniae diseases are a rare but increasingly recognized trigger of atypical haemolytic uraemic syndrome (HUS) in young children and associated with a higher mortality rate than diarrhoea-associated HUS. This study aimed to determine the importance of neuraminidase A (NanA) and genomic diversity in the pathogenesis of pneumococcal HUS (pHUS). We investigated the nanA gene sequence, gene expression, neuraminidase activity and comparative genomic hybridization of invasive pneumococcal disease (IPD) isolates from patients with pHUS and control strains matched by serotype and sequence type (ST), isolated from patients with IPD but not pHUS. The nanA sequence of 33 isolates was determined and mutations at 142 aa positions were identified. High levels of diversity were observed within the NanA protein, with mosaic blocks, insertions and repeat regions present. When comparing nanA allelic diversity with ST and disease profile in the isolates tested, nanA alleles clustered mostly by ST. No particular nanA allele was associated with pHUS. There was no significant difference in overall neuraminidase activity between pHUS isolates and controls when induced/uninduced with N-acetylneuraminic acid. Comparative genomic hybridization showed little difference in genetic content between the pHUS isolates and the controls. Results of gene expression studies identified 12 genes differentially regulated in all pHUS isolates compared with the control. Although neuraminidase enzyme activity may be important in pHUS progression and contribute to pathogenesis, the lack of a distinction between pHUS isolates and controls suggests that host factors, such as acquired abnormalities of the alternative complement cascade in young children, may play a more significant role in the outcome of pHUS.

Haemolytic uraemic syndrome associated with Pseudomonas aeruginosa sepsis.

Haemolytic uraemic syndrome (HUS) is a recognized complication of infection with Shiga toxin-producing Escherichia coli (STEC) and Shigella dysenteriae type 1. Infections with other micro-organisms, especially Streptococcus pneumoniae, have been cited as causes of HUS. In addition, influenza virus and other viruses may rarely be associated with this syndrome. A 2-year-old girl presented with severe Pseudomonas aeruginosa sepsis with renal failure and ecthyma gangrenosum. Further investigations revealed features of HUS. She was managed with antibiotics and other supportive measures including peritoneal dialysis, and subsequently made a full recovery. A possible role of neuraminidase in the pathogenesis of P. aeruginosa-associated HUS was proposed. This is the first reported case of P. aeruginosa sepsis leading to HUS.

A safety evaluation of DAS181, a sialidase fusion protein, in rodents.

DAS181 is a novel inhaled drug candidate blocking influenza virus (IFV) and parainfluenza virus (PIV) infections through removal of sialic acid receptors from epithelial surface of the respiratory tract. To support clinical development, a 28-day Good Laboratory Practices inhalation toxicology study was conducted in Sprague-Dawley rats. In this study, achieved average daily doses based on exposure concentrations were 0.47, 0.90, 1.55, and 3.00 mg/kg/day of DAS181 in a dry powder formulation. DAS181 was well tolerated at all dose levels, and there were no significant toxicological findings. DAS181 administration did not affect animal body weight, food consumption, clinical signs, ophthalmology, respiratory parameters, or organ weight. Gross pathology evaluations were unremarkable. Histological examination of the lungs was devoid of pulmonary tissue damage, and findings were limited to mild and transient changes indicative of exposure and clearance of a foreign protein. DAS181 did not show any cytotoxic effects on human and animal primary cells, including hepatocytes, skeletal muscle cells, osteoblasts, or respiratory epithelial cells. DAS181 did not cause direct or indirect hemolysis. A laboratory abnormality observed in the 28-day toxicology study was mild and transient anemia in male rats at the 3.00 mg/kg dose, which is an expected outcome of enhanced clearance of desialylated red blood cells resulting from systemic exposure with DAS181. Another laboratory observation was a transient dose-dependent elevation in alkaline phosphatase (ALP), which can be attributed to reduced ALP clearance resulting from increased protein desialylation due to DAS181 systemic exposure. These laboratory parameters returned to normal at the end of the recovery period.

Desialylation decreases the resistance of apo B-containing lipoproteins to aggregation and increases their atherogenic potential.

Subfractions of apo B-containing lipoproteins (VLDL and intermediate-density lipoproteins) with reduced content of sialic acid were found in human blood. These lipoproteins are characterized by high capacity to spontaneous association (aggregation) and stimulated accumulation of cholesterol in smooth muscle cells of human aortic intima. In vitro treatment of apo B-containing lipoproteins with alpha-2,6-sialidase and alpha-2,3-sialidase stimulated aggregation and increased the ability of these particles to potentiate cholesterol accumulation in smooth muscle cells of the intact human aortic intima. Probably, desialylation of various apo B-containing lipoproteins can occur in the blood; this process decreases their resistance to aggregation, and increases the ability of these particles to stimulate accumulation of cholesterol in human aortic intima cells, i.e. increases their atherogenic potential.

A sexual dimorphism in intrathymic sialylation survey is revealed by the trans-sialidase from Trypanosoma cruzi.

Sialylation is emerging as an important issue in developing thymocytes and is considered among the most significant cell surface modifications, although its physiologic relevance is far from being completely understood. It is regulated by the concerted expression of sialyl transferases along thymocyte development. After in vivo administration of trans-sialidase, a virulence factor from the American trypanosomatid Trypanosoma cruzi that directly transfers the sialyl residue among macromolecules, we found that the alteration of the sialylation pattern induces thymocyte apoptosis inside the "nurse cell complex." This suggests a glycosylation survey in the development of the T cell compartment. In this study, we report that this thymocyte apoptosis mechanism requires the presence of androgens. No increment in apoptosis was recorded after trans-sialidase administration in females or in antiandrogen-treated, gonadectomized, or androgen receptor mutant male mice. The androgen receptor presence was required only in the thymic epithelial cells as determined by bone marrow chimeric mouse approaches. The presence of the CD43 surface mucin, a molecule with a still undefined function in thymocytes, was another absolute requirement. The trans-sialidase-induced apoptosis proceeds through the TNF-alpha receptor 1 deathly signaling leading to the activation of the caspase 3. Accordingly, the production of the cytokine was increased in thymocytes. The ability of males to delete thymocytes altered in their sialylation pattern reveals a sexual dimorphism in the glycosylation survey during the development of the T cell compartment that might be related to the known differences in the immune response among sexes.

Virulence, immunity, and vaccine related to Streptococcus pneumoniae.

The pathogenesis of bacterial infection involves a series of interactions between the virulence determinants of the microorganisms and the immunity of the host. Studies on the molecular structure and immunological properties of pneumococcal virulence factors have provided general knowledge for the chemical basis of immunogenicity and prevention of bacterial infection. Antibody responses to PS and protein antigens can be greatly affected by their physicochemical properties, e.g., molecular size, specific determinants, conformation, etc. Characterization of group 19 pneumolysins and cloning of their ply genes were studied to examine the relationship of ply to virulence. Group 19 pneumococci all contained ply; the disease-isolated types of 19F and 19A appeared to show a higher specific hemolytic activity and yield than the nonpathogenic types, 19B and 19C. Genomic DNA that contained the ply gene from group 19 strains were analyzed by the polymerase chain reaction (PCR). Type 2 oligonucleotide primers recognized and initiated synthesis of an identical 1.5 kb DNA fragment in types 2, 19F, 19A, 19B, and 19C. Their sizes of restriction DNA fragments were also found to be homologous. Thus, group 19 ply genes showed remarkably similar characteristics. A difficult problem in the development of vaccines against bacterial diseases is the poor immune response of young children to purified PSs. The efficacy of pneumococcal vaccine might be improved by supplementation with inactivated pneumolysin in the form of a PS-protein conjugate.

A role for pneumolysin but not neuraminidase in the hearing loss and cochlear damage induced by experimental pneumococcal meningitis in guinea pigs.

We investigated the roles of pneumolysin and neuraminidase in the pathogenesis of deafness and cochlear damage during experimental pneumococcal meningitis. Anesthetized guinea pigs were inoculated intracranially with 7.5 log10 CFU of either (i) wild-type Streptococcus pneumoniae D39 (n = 8), (ii) PLN-A, a defined isogenic derivative of D39 deficient in pneumolysin (n = 5), or (iii) deltaNA1, a new derivative of D39 deficient in neuraminidase constructed by insertion-duplication mutagenesis of the nanA gene (n = 5). To quantify hearing loss, the auditory nerve compound action potential evoked by a tone pulse was recorded from the round window membrane of the cochlea every 3 h for 12 h. The organ of Corti was intravitally fixed for subsequent examination by high-resolution scanning and transmission electron microscopy. All animals sustained similar meningeal inflammatory responses. PLN-A induced significantly less hearing loss than D39 over the frequency range of 3 to 10 kHz. Levels of mean hearing loss at 10 kHz 12 h postinoculation were as follows: D39, 50 dB; deltaNA1, 52 dB (P = 0.76 versus D39), and PLN-A, 12 dB (P < 0.0001 versus D39). The mean rates of hearing loss at 10 kHz were 4.4 dB/h for D39, 4.3 dB/h for deltaNA1, and just 1.0 dB/h for PLN-A (P < 0.0001 versus D39). Suppurative labyrinthitis was universal. PLN-A induced the accumulation of less protein in the cerebrospinal fluid (P = 0.04 versus D39). Infection with D39 and deltaNA1 induced significant damage to the reticular lamina, the sensory hair cells, and supporting cells of the organ of Corti. By contrast, after infection with PLN-A, the organ of Corti appeared virtually intact. Pneumolysin seems to be the principal cause of cochlear damage in this model of meningogenic deafness. No clear pathogenic role was demonstrated for neuraminidase.