Methods for studying the nematophagous fungus Verticillium chlamydosporium in the root environment

In order to exploit fully the biocontrol potential of the nematophagous fungus Verticillium chlamydosporium, it is important to understand the ecology of the fungus in soil, and interactions with both plant and nematode hosts. Several approaches for studying the fungus in soil and the root environment are compared. These include a semi-selective medium for V. chlamydosporium, PCR primers specific for the fungal -tubulin gene, and monoclonal antibodies. In addition to providing a target for species-specific primers, the -tubulin gene is implicated in resistance to the fungicides used in the semi-selective medium, and the genetic basis for this is investigated. Culture and PCR-based methods were used to screen for the presence of the fungus in field soils known to have been suppressive to cereal cyst nematode and that contained V. chlamydosporium populations. Monoclonal antibodies specific for either hyphae or conidia of the fungus were obtained, and their application as a tool for visualising the infection process on the root was explored.     

Toxocara canis: Some characteristics of larval precipitating antibodies in rat serum

1972. Toxocara canis: Some characteristics of larval precipitating antibodies in rat serum. International Journal for Parasitology 2: 471–479. Serum from rats infected with 1000 T. canis eggs had larval precipitating antibodies present at least between 21 and 183 days post infection. The precipitating antibodies in whole serum were stable to heating at 60°C for 1 h and excretory pore precipitates developed in serum treated with 2-mercaptoethanol.

Fractionation of serum on G-200 and DEAE-cellulose and analysis of the fractions by immunoelectrophoresis indicated the fraction containing fast 7S globulin was primarily responsible for precipitating activity. Purified precipitating antibodies were stable to heating at 70°C for 15 min.

No skin fixing antibodies were detected in immune sera employing both a homologous and heterologous PCA text.     

Identification of protein C epitopes altered during its nanoencapsulation

Protein C is a plasmatic inhibitor which regulates the blood coagulation mechanism by modulating the anticoagulant response. The improvement of its bioavailability would be beneficial for the treatment of the disorders caused by its homozygous deficiency or by an other plasmatic inhibitor deficiency. In this context, the protein C encapsulation into biodegradable nanoparticles could be used to approach the problem. However, the method used to prepare the nanoparticles requires the use of ultrasonication and of an organic solvent such as methylene chloride which interferes with protein activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that neither ultrasonication nor methylene chloride, singly or in combination, led to protein C aggregation or cleavage. Thus, a binding study using an ELISA assay with four characterized monoclonal antibodies was carried out to identify the epitopes damaged by these experimental constraints. The correlation between the immunological assay and a functional one i.e. by the means of the assay of its anticoagulant activity (activated partial thromboplastin time) made it possible to show that protein C amino acids 166–169 of the activation peptide were probably altered after ultrasonication and methylene chloride treatment. Indeed, it is likely that these residues were no longer surface-exposed but had moved inside the protein core.     

Antibody response to Candida albicans cell wall antigens

The cell wall of Candida albicans is not only the structure where many essential biological functions reside but is also a significant source of candidal antigens. The major cell wall components that elicit a response from the host immune system are proteins and glycoproteins, the latter being predominantly mannoproteins. Both carbohydrate and protein moieties are able to trigger immune responses. Proteins and glycoproteins exposed at the most external layers of the wall structure are involved in several types of interactions of fungal cells with the exocellular environment. Thus, coating of fungal cells with host antibodies has the potential to profoundly influence the host-parasite interaction by affecting antibody-mediated functions such as opsonin-enhanced phagocytosis and blocking the binding activity of fungal adhesins to host ligands. In this review we examine various members of the protein and glycoprotein fraction of the C. albicans cell wall that elicit an antibody response in vivo. Some of the studies demonstrate that certain cell wall antigens and anti-cell wall antibodies may be the basis for developing specific and sensitive serologic tests for the diagnosis of candidiasis, particularly the disseminated form. In addition, recent studies have focused on the potential of antibodies against the cell wall protein determinants in protecting the host against infection. Hence, a better understanding of the humoral response triggered by the cell wall antigens of C. albicans may provide the basis for the development of (i) effective procedures for the serodiagnosis of disseminated candidiasis, and (ii) novel prophylactic (vaccination) and therapeutic strategies to control this type of infections.     

Possible apoptotic mechanism in epidermal cell acantholysis induced by pemphigus vulgaris autoimmunoglobulins

Through a still unclear mechanism, pemphigus vulgaris autoantibodies (PV-IgG) induce intra-epidermal acantholytic lesions responsible for severe to fatal skin wounding. We present evidence that PV lesions contain apoptotic keratinocytes, and that cell death is induced in the lesional tissue apparently before cell separation. These data suggest that apoptosis could be the cause of the acantholytic phenomenon. We show that PV-IgG and an antibody against Fas receptor (anti-FasR) induce lesions in vitro in a similar way, causing: (1) secretion of soluble FasL; (2) elevated cellular amounts of FasR, FasL (soluble and membranal), Bax and p53 proteins; (3) reduction in levels of cellular Bcl-2; (4) enrichment in caspase 8, and activation of caspases 1 and 3; (5) co-aggregation of FasL and FasR with caspase 8 in membranal death-inducing signaling complex (DISC). Hence, the Fas-mediated death signaling pathway seems to be involved in lesion formation. Moreover, we have shown that in skin organ cultures and in keratinocyte cultures, PV-IgG can induce caspase activation and DNA fragmentation, and caspase inhibitors can prevent the formation of PV-IgG-induced epidermal lesions. Altogether, these results suggest that PV-IgG-induced acantholysis may proceed through the death-signaling pathway. They highlight new perspectives on mechanisms of tissue damage in autoimmune diseases.     

Therapeutic potential of yeast killer toxin-like antibodies and mimotopes

This review focuses on the potential of yeast killer toxin (KT)-like antibodies (KTAbs), that mimic a wide-spectrum KT through interaction with specific cell wall receptors (KTR) and their molecular derivatives (killer mimotopes), as putative new tools for transdisease anti-infective therapy. KTAbs are produced during the course of experimental and natural infections caused by KTR-bearing micro-organisms. They have been produced by idiotypic vaccination with a KT-neutralizing mAb, also in their monoclonal and recombinant formats. KTAbs and KTAbs-derived mimotopes may exert a strong therapeutic activity against mucosal and systemic infections caused by eukaryotic and prokaryotic pathogenic agents, thus representing new potential wide-spectrum antibiotics.     

Mitochondria and autoimmunity in primary biliary cirrhosis

Primary biliary cirrhosis is an enigmatic autoimmune liver disease that predominantly affects women and is characterized by antimitochondrial antibodies and specific destruction of small bile ducts. Interestingly, patients with this disease not only have high titer antibodies to mitochondria, but also highly directed, liver-specific CD4 and CD8 cells directed at the same mitochondrial autoantigens. These mitochondrial autoantigens are all members of the 2-oxo dehydrogenase complex family and include the E2 component of pyruvate dehydrogenase as the major autoantigen. Moreover, the epitopes recognized by CD4, CD8 T cells and autoantibody, are all directed within the same region, namely the lipoyl domain of pyruvate dehydrogenase complex-E2. All cells in the body have mitochondria but there appear to be specific destruction of biliary cells. We believe that this specific destruction is secondary to a highly directed mucosal response that focuses on biliary cells because of the involvement of a polymeric immunoglobulin receptor, the presence of immunoglobulin A in mucosal secretions, and the unique apoptotic properties of biliary epithelium.     

Prey selection by linyphiid spiders: molecular tracking of the effects of alternative prey on rates of aphid consumption in the field

A molecular approach, using aphid-specific monoclonal antibodies, was used to test the hypothesis that alternative prey can affect predation on aphids by linyphiid spiders. These spiders locate their webs in cereal crops within microsites where prey density is high. Previous work demonstrated that of two subfamilies of Linyphiidae, one, the Linyphiinae, is web-dependent and makes its webs at sites where they were more likely to intercept flying insects plus those (principally aphids) falling from the crop above. The other, the Erigoninae, is less web-dependent, making its webs at ground level at sites with higher densities of ground-living detritivores, especially Collembola. The guts of the spiders were analysed to detect aphid proteins using enzyme-linked immunosorbent assay (ELISA). Female spiders were consuming more aphid than males of both subfamilies and female Linyphiinae were, as predicted, eating more aphid than female Erigoninae. Rates of predation on aphids by Linyphiinae were related to aphid density and were not affected by the availability of alternative prey. However, predation by the Erigoninae on aphids was significantly affected by Collembola density. Itinerant Linyphiinae, caught away from their webs, contained the same concentration of aphid in their guts as web-owners. However, nonweb-owning Erigoninae, living away from Collembola aggregations at web-sites, contained significantly higher concentrations of aphid. For both subfamilies there was evidence of a disproportionate increase in predation on aphids once Collembola populations had declined. It was concluded that nonaphid prey, by helping to maintain spiders in the field, can significantly affect predation on aphids.     

Amyloid-Beta Immunization in Alzheimer's Disease Transgenic Mouse Models and Wildtype Mice

Alzheimer's disease is the most prevalent form of dementia worldwide. Therapies are desperately needed to prevent and cure the disease. Mouse models of amyloid- deposition [APP and PSAPP transgenic (tg) mice] have been useful in determining the role of amyloid- (A) in both the pathogenesis and cognitive changes in AD. In addition, they have allowed scientists to investigate potential AD therapies in living animals. Active and passive A immunizations have been employed successfully in APP and PSAPP tg mice to lower cerebral A levels and improve cognition. Optimization of immunization protocols and characterization of immune responses in wildtype mice have been reported. Based on the promising results of A immunization studies in mice, a clinical trial was initiated for A vaccination in humans with AD. Although no adverse effects were reported in the Phase I safety trials, about 5% of AD patients in the phase II clinical trial developed meningoencephalitis, ending the trial prematurely in March 2002. Studies in AD mouse models and wildtype mice may help elucidate the mechanism for these unwanted side effects and will be useful for testing newer, safer vaccines for future use in human clinical trials.     

Special Features of the DNA-Hydrolyzing Activity of the Antibodies in Systemic Lupus Erythematosus

Two types of IgG anti-DNA antibodies exhibiting DNA-hydrolyzing activity have been isolated from blood serum of patients with systemic lupus erythematosus. This DNase activity of antibodies differs from serum DNases by the non-processive mode, temperature resistance, pH optimum, and the rate of DNA hydrolysis. It is suggested that the anti-DNA antibody molecule possessing DNase activity contains two sites: one site determines specificity of antibody-DNA interaction, whereas the other is responsible for manifestation of the catalytic activity.