Sexual and apomictic plant reproduction in the genomics era: exploring the mechanisms potentially useful in crop plants

Arabidopsis, Mimulus and tomato have emerged as model plants in researching genetic and molecular basis of differences in mating systems. Variations in floral traits and loss of self-incompatibility have been associated with mating system differences in crops. Genomics research has advanced considerably, both in model and crop plants, which may provide opportunities to modify breeding systems as evidenced in Arabidopsis and tomato. Mating system, however, not recombination per se, has greater effect on the level of polymorphism. Generating targeted recombination remains one of the most important factors for crop genetic enhancement. Asexual reproduction through seeds or apomixis, by producing maternal clones, presents a tremendous potential for agriculture. Although believed to be under simple genetic control, recent research has revealed that apomixis results as a consequence of the deregulation of the timing of sexual events rather than being the product of specific apomixis genes. Further, forward genetic studies in Arabidopsis have permitted the isolation of novel genes reported to control meiosis I and II entry. Mutations in these genes trigger the production of unreduced or apomeiotic megagametes and are an important step toward understanding and engineering apomixis.     

A Case of Cunninghamella bertholettiae Rhino-cerebral Infection in a Leukaemic Patient and Review of Recent Published Studies

Cunninghamella bertholletiae infection occurs most frequently in neutropenic patients affected by haematological malignancies, is associated with an unfavourable outcome. We report a case of rhino-mastoidal fungal infection in a leukaemic patient. Bioptical tissue cultures yield the isolation of a mould with typical properties of Cunninghamella species. Liposomal amphotericin B (L-Amb) therapy combined with surgical intervention brought the lesion to recovery. Nevertheless, the patient died 14 days after bone marrow transplantation (BMT) from bacterial sepsis. Mastoiditis was documented at CT-scan. The conditioning regimen probably caused the reactivation of the Cunninghamella infection that led to the patient's fatal outcome; fungal hyphae were detected after autopsy of brain and lung tissue.     

Identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis C virus E2 protein

Identification of anti-hepatitis C virus (anti-HCV) human antibody clones with broad neutralizing activity is important for a better understanding of the interplay between the virus and host and for the design of an effective passive immunotherapy and an effective vaccine. We report the identification of a human monoclonal Fab (e137) able to bind the HCV E2 glycoprotein of all HCV genotypes but genotype 5. The results of antibody competition assays and testing the reactivity to alanine mutant E2 proteins confirmed that the e137 epitope includes residues (T416, W420, W529, G530, and D535) highly conserved across all HCV genotypes. Fab e137 neutralized HCV pseudoparticles bearing genotype 1a, 1b, and 4 E1-E2 proteins and to a lesser extent, genotype 2b. Fab e137 was also able to inhibit cell culture-grown HCV (genotype 2a). These data indicate that broadly cross-reacting and cross-neutralizing antibodies are generated during HCV infection.     

Denitrification activity of Bradyrhizobium sp. isolated from Argentine soybean cultivated soils

Two hundred and fifty strains, all of them representatives of native Bradyrhizobium sp., isolated from soils cultivated with soybean have been characterized by their denitrification activity. In addition, the denitrification potential of those soils was also measured by evaluating the most-probable-number (MPN) of denitrifying bacteria and the denitrification enzyme assay (DEA). Of the 250 isolates tested, 73 were scored as probable denitrifiers by a preliminary screening method. Only 41 were considered denitrifiers because they produced gas bubbles in Durham tubes, cultures reached an absorbance of more than 0.1 and NO³⁻ and NO²⁻ were not present. Ten of these 41 were selected to confirm denitrification and to study denitrification genes. According to N₂O production and cell protein concentration with NO³⁻, the isolates could be differentiated in three categories of denitrifiers. The presence of the napA, nirK, norC and nosZ genes was detected by production of a diagnostic PCR product using specific primers. RFLP from the 16S-23S rDNA spacer region (IGS) revealed that denitrifiers strains could be characterized as Bradyrhizobium japonicum and strains which were non-respiratory denitrifiers as B. elkanii.     

Rationally designed Human Cytomegalovirus gB nanoparticle vaccine with improved immunogenicity

Human cytomegalovirus (HCMV) is the primary viral cause of congenital birth defects and causes significant morbidity and mortality in immune-suppressed transplant recipients. Despite considerable efforts in vaccine development, HCMV infection still represents an unmet clinical need. In recent phase II trials, a MF59-adjuvanted gB vaccine showed only modest efficacy in preventing infection. These findings might be attributed to low level of antibodies (Abs) with a neutralizing activity induced by this vaccine. Here, we analyzed the immunogenicity of each gB antigenic domain (AD) and demonstrated that domain I of gB (AD5) is the main target of HCMV neutralizing antibodies. Furthermore, we designed, characterized and evaluated immunogenic responses to two different nanoparticles displaying a trimeric AD5 antigen. We showed that mice immunization with nanoparticles induces sera neutralization titers up to 100-fold higher compared to those obtained with the gB extracellular domain (gB_ECD). Collectively, these results illustrate with a medically relevant example the advantages of using a general approach combining antigen discovery, protein engineering and scaffold presentation for modern development of subunit vaccines against complex pathogens.     

Potential distribution of <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> in Argentina: implications in amphibian conservation

Within the last two decades Batrachochytrium dendrobatidis (B.d.), the causative agent of chytridiomycosis, seems to have become a pandemic parasite, and is proposed as one of the more important causes of amphibian declines worldwide. In Argentina, the first report was in 2002 in Leptodactylus ocellatus. Since then, the fungus has expanded through different environments and species. In this study, we predict B.d. distribution in Argentina applying niche modelling based on reports of infected amphibians and environmental variables. The distribution hypothesis showed regions with highest suitability for B.d. including habitat types with (1) the most diverse amphibian fauna in Argentina such as the Paraná River Basin, (2) endangered species, such as north Patagonia and northwest Andean highlands, and (3) wide range of optimum precipitation and temperatures allowing development of B.d.     

Eukaryotic ectosymbionts of Acari

The Acari is the most numerous and diverse group of the subphylum Chelicerata. With approximately 55 000 described species (and estimates of up to 1 million extant species), their adaptations for parasitism, phytophagy, mycophagy, saprophagy and predation rival other arthropods and challenge us with a wide variety of biological interactions. While a few studies have unravelled the nature of some endosymbiotic associations between mites or ticks and prokaryotes, almost nothing has been done yet regarding acarine eukaryotic ectosymbionts. Microbial ectosymbionts can benefit their hosts by providing nutrients, by aiding digestion, by enhancing communication, by assisting in mating and/or fertilization, by protecting their host against pathogenic microorganisms, against predation and so on. In this sketch, we introduce a number of described cases of fungal and protist ectosymbionts and discuss the role they might play in the life of their acarine hosts.     

GMP production of anti-tumor cytotoxic T-cell lines for adoptive T-cell therapy in patients with solid neoplasia

BACKGROUND: The adoptive transfer of ex vivo-induced tumor-specific T-cell lines provides a promising approach for cancer immunotherapy. We have demonstrated previously the feasibility of inducing in vitro long-term anti-tumor cytotoxic T-cell (CTL) lines directed against different types of solid tumors derived from both autologous and allogeneic PBMC. We have now investigated the possibility of producing large amounts of autologous anti-tumor CTL, in compliance with good manufacturing practices, for in vivo use. METHODS: Four patients with advanced solid tumors (two sarcoma, one renal cell cancer and one ovarian cancer), who had received several lines of anticancer therapy, were enrolled. For anti-tumor CTL induction, patient-derived CD8-enriched PBMC were stimulated with DC pulsed with apoptotic autologous tumor cells (TC) as the source of tumor Ag. CTL were then restimulated in the presence of TC and expanded in an Ag-independent way. RESULTS: Large amounts of anti-tumor CTL (range 14-20 x 10(9)), which displayed high levels of cytotoxic activity against autologous TC, were obtained in all patients by means of two-three rounds of tumor-specific stimulation and two rounds of Ag-independent expansion, even when a very low number of viable TC was available. More than 90% of effector cells were CD3(+) CD8(+) T cells, while CD4(+) T lymphocytes and/or NK cells were less than 10%. DISCUSSION: Our results demonstrate the feasibility of obtaining large quantities of anti-tumor specific CTL suitable for adoptive immunotherapy approaches.     

Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute

There is great interest in mesenchymal stromal cells (MSCs) for cell-therapy and tissue engineering approaches. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns when used in clinical grade preparations. The aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL), already shown to promote MSC growth, are endowed with biological properties appropriate for cell-therapy approaches. We confirm previously published data showing that MSCs expanded in either FCS or PL display comparable morphology, phenotype, and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. We further extended these data by investigating the immune-regulatory effect of MSCs on the alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to: (i) decrease alloantigen-induced cytotoxic activity; (ii) favor differentiation of CD4+ T-cell subsets expressing a Treg phenotype; (iii) increase early secretion of IL-10 in MLC supernatant, as well as induce a striking augmentation of IL-6 production. As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and reducing early IFNgamma-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by molecular karyotyping and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the immunological functional plasticity of MSCs and suggest that MSCs-PL can be used as an alternative to MSCs-FCS, although these latter cells might be more suitable for preventing/treating alloreactivity-related immune complications.