Wide cross-species aminoacyl-tRNA synthetase replacement in vivo: yeast cytoplasmic alanine enzyme replaced by human polymyositis serum antigen.

Because of variations in tRNA sequences in evolution, tRNA synthetases either do not acylate their cognate tRNAs from other organisms or execute misacylations which can be deleterious in vivo. We report here the cloning and primary sequence of a 958-aa Saccharomyces cerevisiae alanyl-tRNA synthetase. The enzyme is a close homologue of the human and Escherichia coli enzymes, particularly in the region of the primary structure needed for aminoacylation of RNA duplex substrates based on alanine tRNA acceptor stems with a G3.U70 base pair. An ala1 disrupted allele demonstrated that the gene is essential and that, therefore, ALA1 encodes an enzyme required for cytoplasmic protein synthesis. Growth of cells harboring the ala1 disrupted allele was restored by a cDNA clone encoding human alanyl-tRNA synthetase, which is a serum antigen for many polymyositis-afflicted individuals. The human enzyme in extracts from rescued yeast was detected with autoimmune antibodies from a polymyositis patient. We conclude that, in spite of substantial differences between human and yeast tRNA sequences in evolution, strong conservation of the G3.U70 system of recognition is sufficient to yield accurate aminoacylation in vivo across wide species distances.

Regulation of the polarization of T cells toward antigen-presenting cells by Ras-related GTPase CDC42.

The mechanisms by which cells rapidly polarize in the direction of external signals are not understood. Helper T cells, when contacted by an antigen-presenting cell, polarize their cytoskeletons toward the antigen-presenting cell within minutes. Here we show that, in T cells, the mammalian Ras-related GTPase CDC42 (the homologue of yeast CDC42, a protein involved in budding polarity) can regulate the polarization of both actin and microtubules toward antigen-presenting cells but is not involved in other T-cell signaling processes such as those which culminate in interleukin 2 production. Although T-cell polarization appears dispensable for signaling leading to interleukin 2 production, polarization may direct lymphokine secretion towards the correct antigen-presenting cell in a crowded cellular environment. Inhibitor experiments suggest that phosphatidylinositol 3-kinase is required for cytoskeletal polarization but that calcineurin activity, known to be important for other aspects of signaling, is not. Apparent conservation of CDC42 function between yeast and T cells suggests that this GTPase is a general regulator of cytoskeletal polarity in many cell types.

Analysis of the interaction of ZAP-70 and syk protein-tyrosine kinases with the T-cell antigen receptor by plasmon resonance.

Tyrosine phosphorylation of a 17-amino acid immunoreceptor tyrosine-based activation motif (ITAM), conserved in each of the signaling subunits of the T-cell antigen receptor (TCR), mediates the recruitment of ZAP-70 and syk protein-tyrosine kinases (PTKs) to the activated receptor. The interaction between the two tandemly arranged Src-homology 2 (SH2) domains of this family of PTKs and each of the phosphotyrosine-containing ITAMs was examined by real-time measurements of kinetic parameters. The association rate and equilibrium binding constants for the ZAP-70 and syk SH2 domains were determined for the CD3 epsilon ITAM. Both PTKs bound with ka and Kd values of 5 x 10(6) M-1.sec-1 and approximately 25 nM, respectively. Bindings to the other TCR ITAMs (zeta 1, zeta 2, gamma, and delta ITAMs) were comparable, although the zeta 3 ITAM bound approximately 2.5-fold less well. Studies of the affinity of a single functional SH2 domain of ZAP-70 provided evidence for the cooperative nature of binding of the dual SH2 domains. Mutation of either single SH2 domain decreased the Kd by > 100-fold. Finally, the critical features of the ITAM for syk binding were found to be similar to those required for ZAP-70 binding. These data provide insight into the mechanism by which the multisubunit TCR interacts with downstream effector molecules.

Regulation of T-cell antigen receptor (TCR) alpha-chain expression by TCR beta-chain transcripts.

The TCR is an alpha beta heterodimer, a part of the multimeric structure through which physiological T-cell activation occurs. The expression of TCR alpha chain is greatly diminished in a beta-chain-deficient mutant Jurkat cell line (J.RT3-T3.5). The relationship between the expression of the TCR alpha and beta chains has been examined by stable transfection of a series of TCR beta-chain mutant constructs into this mutant cell line. The level of alpha-chain transcript was dramatically upregulated by the expression of the beta chain and specifically by a transcript of the beta-chain variable region alone, including a transcript in which the ATG start codon was mutated. The downregulation of the endogenous alpha-chain transcripts in mutants cells lacking complete beta-chain transcripts occurred primarily at the posttranscriptional level. This evidence for a regulatory function of the TCR beta-chain gene represents an unusual regulatory pathway in which the transcript of one gene is required for the optimal expression of another gene.

A serine proteinase inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene.

The squamous cell carcinoma antigen (SCCA) is a member of the ovalbumin family of serine proteinase inhibitors (serpins). A neutral form of the protein is found in normal and some malignant squamous cells, whereas an acidic form is detected exclusively in tumor cells and in the circulation of patients with squamous cell tumors. In this report, we describe the cloning of the SCCA gene from normal genomic DNA. Surprisingly, two genes were found. They were tandemly arrayed and flanked by two other closely related serpins, plasminogen activator inhibitor type 2 (PAI2) and maspin at 18q21.3. The genomic structure of the two genes, SCCA1 and SCCA2, was highly conserved. The predicted amino acid sequences were 92% identical and suggested that the neutral form of the protein was encoded by SCCA1 and the acidic form was encoded by SCCA2. Further characterization of the region should determine whether the differential expression of the SCCA genes plays a causal role in development of more aggressive squamous cell carcinomas.

Immunogenicity of transgenic plant-derived hepatitis B surface antigen.

The focus of the Children's Vaccine Initiative is to encourage the discovery of technology that will make vaccines more readily available to developing countries. Our strategy has been to genetically engineer plants so that they can be used as inexpensive alternatives to fermentation systems for production of subunit antigens. In this paper we report on the immunological response elicited in vivo by using recombinant hepatitis B surface antigen (rHBsAg) purified from transgenic tobacco leaves. The anti-hepatitis B response to the tobacco-derived rHBsAg was qualitatively similar to that obtained by immunizing mice with yeast-derived rHBsAg (commercial vaccine). Additionally, T cells obtained from mice primed with the tobacco-derived rHBsAg could be stimulated in vitro by the tobacco-derived rHBsAg, yeast-derived rHBsAg, and by a synthetic peptide that represents part of the a determinant located in the S region (139-147) of HBsAg. Further support for the integrity of the T-cell epitope of the tobacco-derived rHBsAg was obtained by testing the ability of the primed T cells to proliferate in vitro after stimulation with a monoclonal anti-idiotype and an anti-idiotype-derived peptide, both of which mimic the group-specific a determinant of HBsAg. In total, we have conclusively demonstrated that both B- and T-cell epitopes of HBsAg are preserved when the antigen is expressed in a transgenic plant.

Autoimmunity in Chagas disease cardiopathy: biological relevance of a cardiac myosin-specific epitope crossreactive to an immunodominant Trypanosoma cruzi antigen.

Heart tissue destruction in chronic Chagas disease cardiopathy (CCC) may be caused by autoimmune recognition of heart tissue by a mononuclear cell infiltrate decades after Trypanosoma cruzi infection. Indirect evidence suggests that there is antigenic crossreactivity between T. cruzi and heart tissue. As there is evidence for immune recognition of cardiac myosin in CCC, we searched for a putative myosin-crossreactive T. cruzi antigen. T. cruzi lysate immunoblots were probed with anti-cardiac myosin heavy chain IgG antibodies (AMA) affinity-purified from CCC or asymptomatic Chagas disease patient-seropositive sera. A 140/116-kDa doublet was predominantly recognized by AMA from CCC sera. Further, recombinant T. cruzi protein B13--whose native protein is also a 140- and 116-kDa double band--was identified by crossreactive AMA. Among 28 sera tested in a dot-blot assay, AMA from 100% of CCC sera but only 14% of the asymptomatic Chagas disease sera recognized B13 protein (P = 2.3 x 10(-6)). Sequence homology to B13 protein was found at positions 8-13 and 1442-1447 of human cardiac myosin heavy chain. Competitive ELISA assays that used the correspondent myosin synthetic peptides to inhibit serum antibody binding to B13 protein identified the heart-specific AAALDK (1442-1447) sequence of human cardiac myosin heavy chain and the homologous AAAGDK B13 sequence as the respective crossreactive epitopes. The recognition of a heart-specific T. cruzi crossreactive epitope, in strong association with the presence of chronic heart lesions, suggests the involvement of crossreactivity between cardiac myosin and B13 in the pathogenesis of CCC.

Retinal microglia are activated by systemic fungal infection

Purpose: To determine whether systemic fungal infection could cause activation of retinal microglia and therefore could be potentially harmful for patients with retinal degenerative diseases. Methods: Activation of retinal microglia was measured in a model of sublethal invasive candidiasis in C57BL/6J mice by (i) confocal immunofluorescence and (ii) flow cytometry analysis, using anti-CD11b, anti-Iba1, anti-MHCII and anti-CD45 antibodies. Results: Systemic fungal infection causes activation of retinal microglia, with phenotypic changes in morphology, surface markers expression, and microglial re-location in retinal layers. Conclusions: As an excessive or prolonged microglial activation may lead to chronic inflammation with severe pathological side effects, causing or worsening the course of retinal dystrophies, a systemic infection may represent a risk factor to be considered in patients with ocular neurodegenerative diseases, such as diabetic retinopathy, glaucoma, age-related macular degeneration or retinitis pigmentosa.

Carbon and nitrogen limitation increase chitosan antifungal activity in Neurospora crassa and fungal human pathogens

Chitosan permeabilizes plasma membrane and kills sensitive filamentous fungi and yeast. Membrane fluidity and cell energy determine chitosan sensitivity in fungi. A five-fold reduction of both glucose (main carbon (C) source) and nitrogen (N) increased 2-fold Neurospora crassa sensitivity to chitosan. We linked this increase with production of intracellular reactive oxygen species (ROS) and plasma membrane permeabilization. Releasing N. crassa from nutrient limitation reduced chitosan antifungal activity in spite of high ROS intracellular levels. With lactate instead of glucose, C and N limitation increased N. crassa sensitivity to chitosan further (4-fold) than what glucose did. Nutrient limitation also increased sensitivity of filamentous fungi and yeast human pathogens to chitosan. For Fusarium proliferatum, lowering 100-fold C and N content in the growth medium, increased 16-fold chitosan sensitivity. Similar results were found for Candida spp. (including fluconazole resistant strains) and Cryptococcus spp. Severe C and N limitation increased chitosan antifungal activity for all pathogens tested. Chitosan at 100 μg ml-1 was lethal for most fungal human pathogens tested but non-toxic to HEK293 and COS7 mammalian cell lines. Besides, chitosan increased 90% survival of Galleria mellonella larvae infected with C. albicans. These results are of paramount for developing chitosan as antifungal.

Fungal Contamination of Sandpits from Recreational Parks and Schools: a Potential Risk for Human Health

Sandpits used by children are frequently visited by wild life which constitutes a source of fungal pathogens and allergenic fungi. This study aimed to take an unannounced snapshot of the urban levels of fungal contaminants in sands, using for this purpose two public recreational parks, three elementary schools and two kindergartens. All samples were from Lisbon and neighboring municipalities and were tested for fungi of clinical interest. Potentially pathogenic fungi were isolated from all samples besides one. Fusarium dimerum (32.4%) was found to be the dominant species in one park and Chrysonilia spp. in the other (46.6%). Fourteen different species and genera were detected and no dermatophytes were found. Of a total of 14 species and genera, the fungi most isolated from the samples of the elementary schools were Penicillium spp. (74%), Cladophialophora spp. (38%) and Cladosporium spp. (90%). Five dominant species and genera were isolated from the kindergartens. Penicillium spp. was the only genus isolated in one, though with remarkably high counts (32500 colony forming units per gram). In the other kindergarten Penicillium spp. were also the most abundant species, occupying 69% of all the fungi found. All of the samples exceeded the Maximum Recommended Value (MRV) for beach sand defined by Brandão et al. 2011, which are currently the only quantitative guidelines available for the same matrix. The fungi found confirm the potential risk of exposure of children to keratinophilic fungi and demonstrates that regular cleaning or replacing of sand needs to be implemented in order to minimize contamination.

Oral fungal microflora associated with implant-supported VS. mucous-supported dentures

Poster presented at the 1st International Congress of CiiEM - From Basic Sciences to Clinical Research, 27-28 November 2015, Egas Moniz, Caparica, Portugal.

N-terminal fusion of a toll-like receptor 2-ligand to a Neospora caninum chimeric antigen efficiently modifies the properties of the specific immune response

Immunoprophylactic products against neosporosis during pregnancy should induce an appropriately balanced immune response. In this respect, OprI, a bacterial lipoprotein targeting toll like receptor (TLR)2, provides promising adjuvant properties. We report on the manipulation of the innate and the T-cell immune response through the fusion of OprI with the Neospora caninum chimeric protein Mic3-1-R. In contrast to Mic3-1-R, OprI-MIC3-1-R significantly activated bone-marrow dendritic cells from naïve mice. Mice immunized with OprI-Mic3-1-R induced an immune response with mixed T helper (Th)1 and Th2 properties (high levels of both immunoglobulin (Ig)G1 and IgG2a and of interleukin (IL)-10, IL-12(p70) and interferon-γ responses) whereas Mic3-1-R+saponin induced a clear Th2-biased response (low IgG2a and high IL-4 and IL-10). After mating and challenge with N. caninum, increased expression of interferon-γ was only found in placentas from OprI-Mic3-1-R immunized dams. However, no protection against vertical transmission and neonatal mortality was observed in either of the two groups. These results indicated that more exhaustive studies must be done to elucidate the immune mechanisms associated with transplacental transmission. Antigen linkage to TLR2-ligands, such as OprI, is a useful tool to investigate this enigma by reorienting the innate and adaptive immune responses against other candidate antigens in future studies.

Immunization with a circumsporozoite epitope fused to Bordetella pertussis adenylate cyclase in conjunction with cytotoxic T-lymphocyte-associated antigen 4 blockade confers protection against Plasmodium berghei liver-stage malaria

The adenylate cyclase toxoid (ACT) of Bordetella pertussis is capable of delivering its N-terminal catalytic domain into the cytosol of CD11b-expressing professional antigen-presenting cells such as myeloid dendritic cells. This allows delivery of CD8+ T-cell epitopes to the major histocompatibility complex (MHC) class I presentation pathway. Recombinant detoxified ACT containing an epitope of the Plasmodium berghei circumsporozoite protein (CSP), indeed, induced a specific CD8+ T-cell response in immunized mice after a single application, as detected by MHC multimer staining and gamma interferon (IFN-gamma) ELISPOT assay. This CSP-specific response could be significantly enhanced by prime-boost immunization with recombinant ACT in combination with anti-CTLA-4 during the boost immunization. This increased response was accompanied by complete protection in a number of mice after a challenge with P. berghei sporozoites. Transient blockade of CTLA-4 may overcome negative regulation and hence provide a strategy to enhance the efficacy of a vaccine by amplifying the number of responding T cells.

The fungal diseases of the common larch,

Bibliography: p. [193]-199.

The Antigenic response to a booster injection of a multiple antigen preparation containing diphtheria, pertussis, and tetanus antigens given seven years or more after primary immunization /

Mode of access: Internet.