Changes in the Sensitization Pattern to Alternaria alternata Allergens in Patients Treated with Alt a 1 Immunotherapy.

Alternaria alternata is the most important allergenic fungus, with up to 20% of allergic patients affected. The sensitization profile of patients sensitized to A. alternata and how it changes when treated with immunotherapy is not known. Our objective is to determine the allergen recognition pattern of allergic patients to A. alternata and to study its association to the parameters studied in a clinical trial recently published. Sera of 64 patients from the clinical trial of immunotherapy with native major allergen Alt a 1 were analyzed by immunoblotting; 98. 4% of the patients recognized Alt a 1. The percentage of recognition for Alt a 3, Alt a 4, and/or Alt a 6, Alt a 7, Alt a 8, Alt a 10 and/or Alt a 15 was 1.6%, 21.9%, 12.5%, 12.5%, and 12.5% respectively. Of the 64 patients, 45 (70.3%) only recognized Alt a 1 among the allergens present in the A. alternata extract. Immunotherapy with Alt a 1 desensitizes treated patients, reducing their symptoms and medication consumption through the elimination of Alt a 1 sensitization, which is no longer present in the immunoblotting of some patients. There may be gender differences in the pattern of sensitization to A. alternata allergens, among others.

Apoptosis of Candida albicans during the Interaction with Murine Macrophages: Proteomics and Cell-Death Marker Monitoring.

Macrophages may induce fungal apoptosis to fight against C. albicans, as previously hypothesized by our group. To confirm this hypothesis, we analyzed proteins from C. albicans cells after 3 h of interaction with macrophages using two quantitative proteomic approaches. A total of 51 and 97 proteins were identified as differentially expressed by DIGE and iTRAQ, respectively. The proteins identified and quantified were different, with only seven in common, but classified in the same functional categories. The analyses of their functions indicated that an increase in the metabolism of amino acids and purine nucleotides were taking place, while the glycolysis and translation levels dropped after 3 h of interaction. Also, the response to oxidative stress and protein translation were reduced. In addition, seven substrates of metacaspase (Mca1) were identified (Cdc48, Fba1, Gpm1, Pmm1, Rct1, Ssb1, and Tal1) as decreased in abundance, plus 12 proteins previously described as related to apoptosis. Besides, the monitoring of apoptotic markers along 24 h of interaction (caspase-like activity, TUNEL assay, and the measurement of ROS and cell examination by transmission electron microscopy) revealed that apoptotic processes took place for 30% of the fungal cells, thus supporting the proteomic results and the hypothesis of macrophages killing C. albicans by apoptosis.

Methodologies to generate, extract, purify and fractionate yeast ECM for analytical use in proteomics and glycomics.

BACKGROUND: In a multicellular organism, the extracellular matrix (ECM) provides a cell-supporting scaffold and helps maintaining the biophysical integrity of tissues and organs. At the same time it plays crucial roles in cellular communication and signalling, with implications in spatial organisation, motility and differentiation. Similarly, the presence of an ECM-like extracellular polymeric substance is known to support and protect bacterial and fungal multicellular aggregates, such as biofilms or colonies. However, the roles and composition of this microbial ECM are still poorly understood. RESULTS: This work presents a protocol to produce S. cerevisiae and C. albicans ECM in an equally highly reproducible manner. Additionally, methodologies for the extraction and fractionation into protein and glycosidic analytical pure fractions were improved. These were subjected to analytical procedures, respectively SDS-PAGE, 2-DE, MALDI-TOF-MS and LC-MS/MS, and DAE and FPLC. Additional chemical methods were also used to test for uronic acids and sulphation. CONCLUSIONS: The methodologies hereby presented were equally efficiently applied to extract high amounts of ECM material from S. cerevisiae and C. albicans mats, therefore showing their robustness and reproducibility for yECM molecular and structural characterization. yECM from S. cerevisiae and C. albicans displayed a different proteome and glycoside fractions. S. cerevisiae yECM presented two well-defined polysaccharides with different mass/charge, and C. albicans ECM presented a single different one. The chemical methods further suggested the presence of uronic acids, and chemical modification, possibly through sulphate substitution. All taken, the procedures herein described present the first sensible and concise approach to the molecular and chemical characterisation of the yeast ECM, opening the way to the in-depth study of the microbe multicellular aggregates structure and life-style.

Molecular stress responses to nano-sized zero-valent iron (nZVI) particles in the soil bacterium Pseudomonas stutzeri.

Nanotoxicological studies were performed in vitro using the common soil bacterium Pseudomonas stutzeri to assess the potentially toxic impact of commercial nano-sized zero-valent iron (nZVI) particles, which are currently used for environmental remediation projects. The phenotypic response of P. stutzeri to nZVI toxicity includes an initial insult to the cell wall, as evidenced by TEM micrographs. Transcriptional analyses using genes of particular relevance in cellular activity revealed that no significant changes occurred among the relative expression ratios of narG, nirS, pykA or gyrA following nZVI exposure; however, a significant increase in katB expression was indicative of nZVI-induced oxidative stress in P. stutzeri. A proteomic approach identified two major defence mechanisms that occurred in response to nZVI exposure: a downregulation of membrane proteins and an upregulation of proteins involved in reducing intracellular oxidative stress. These biomarkers served as early indicators of nZVI response in this soil bacterium, and may provide relevant information for environmental hazard assessment.

Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.

Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis--the paradigm of mesophilic hydrocarbonoclastic bacteria--O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.

Proteomic analysis of porcine mesenteric lymph-nodes after Salmonella typhimurium infection.

In this study we employed for the first time an in vivo approach coupled to DIGE-based proteomics to explore the response of porcine mesenteric lymph nodes (MLN) to Salmonella typhimurium infection. MLN samples were collected from four control and twelve infected pigs (at 1, 2 and 6 days post infection) for histological analysis, protein and RNA purification. Afterwards, expressed proteins were screened by differential in gel analysis and data were analyzed by bioinformatic tools to generate interaction networks, and identify enriched signaling pathways and biological annotations. S. typhimurium labeling in tissue and phagocyte infiltration were analyzed by immunohistochemistry and RNA was employed to determine the relative expression of immune-related genes by quantitative RNA analysis. The proteome response of porcine MLN to infection was associated to the induction of processes such as phagocyte infiltration, cytoskeleton remodeling and pyroptosis. Moreover, our results suggest that S. typhimurium antigens are cross-presented via MHC-I in a proteasome-dependent manner in porcine MLN. Since pathogen burden in tissue was noticeably reduced at the end of the time course, we infer that host innate and adaptive immunity act in association in MLN to control S. typhimurium dissemination in swine infections.

Sub-proteomic study on macrophage response to Candida albicans unravels new proteins involved in the host defense against the fungus.

In previous proteomic studies on the response of murine macrophages against Candida albicans, many differentially expressed proteins involved in processes like inflammation, cytoskeletal rearrangement, stress response and metabolism were identified. In order to look for proteins important for the macrophage response, but in a lower concentration in the cell, 3 sub-cellular extracts were analyzed: cytosol, organelle/membrane and nucleus enriched fractions from RAW 264.7 macrophages exposed or not to C. albicans SC5314 for 3 h. The samples were studied using DIGE technology, and 17 new differentially expressed proteins were identified. This sub-cellular fractionation permitted the identification of 2 mitochondrion proteins, a membrane receptor, Galectin-3, and some ER related proteins, that are not easily detected in total cell extracts. Besides, the study of different fractions allowed us to detect, not only total increase in Galectin-3 protein amount, but its distinct allocation along the interaction. The identified proteins are involved in the pro-inflammatory and oxidative responses, immune response, unfolded protein response and apoptosis. Some of these processes increase the host response and others could be the effect of C. albicans resistance to phagocytosis. Thus, the sub-proteomic approach has been a very useful tool to identify new proteins involved in macrophage-fungus interaction. This article is part of a Special Issue entitled: Translational Proteomics.

Identification of Candida albicans exposed surface proteins in vivo by a rapid proteomic approach.

We have set up a fast and easy methodology to identify cell-surface proteins in live yeasts. A non-gel proteomic approach was based on a short period of trypsin treatment followed by peptide separation and identification using nano-LC followed by off-line MS/MS. Candida albicans was used as a model organism and proteins involved in cell wall organization, cell rescue, defense, virulence, transport, protein fate and metabolism were identified. This strategy is a powerful tool to study host-pathogen interactions and to look for potential vaccine candidates and drug targets.

The Pseudomonas putida Crc global regulator controls the hierarchical assimilation of amino acids in a complete medium: evidence from proteomic and genomic analyses.

The Crc protein is a global translational regulator involved in catabolite repression of catabolic pathways for several non-preferred carbon sources in Pseudomonads when other preferred substrates are present. Using proteomic and transcriptomic approaches, we have analyzed the influence of Crc in cells growing in a complete medium, where amino acids are the main carbon source. Inactivation of the crc gene modified the expression of at least 134 genes. Most of them were involved in the transport and assimilation of amino acids or sugars. This allowed envisioning which amino acids are preferentially used. Crc did not inhibit the pathways for proline, alanine, glutamate, glutamine and histidine. These amino acids are good carbon sources for P. putida. In the case of arginine, lysine, aspartate and asparagine, which can be assimilated through several pathways, Crc favored one particular route, inhibiting other alternatives. Finally, Crc-inhibited genes needed to assimilate valine, isoleucine, leucine, tyrosine, phenylalanine, threonine, glycine and serine, amino acids that provide a less efficient growth. Crc has therefore a key role in coordinating metabolism, controlling the sequential assimilation of amino acids when cells grow in a complete medium. Inactivation of crc reduced growth rate, suggesting that Crc optimizes metabolism.

Proteomic analysis of cytoplasmic and surface proteins from yeast cells, hyphae, and biofilms of Candida albicans.

Candida albicans is a human commensal and opportunistic pathogen that participates in biofilm formation on host surfaces and on medical devices. We used DIGE analysis to assess the cytoplasmic and non-covalently attached cell-surface proteins in biofilm formed on polymethylmethacrylate and planktonic yeast cells and hyphae. Of the 1490 proteins spots from cytoplasmic and 580 protein spots from the surface extracts analyzed, 265 and 108 were differentially abundant respectively (>or=1.5-fold, p <0.05). Differences of both greater and lesser abundance were found between biofilms and both planktonic conditions as well as between yeast cells and hyphae. The identity of 114 cytoplasmic and 80 surface protein spots determined represented 73 and 25 unique proteins, respectively. Analyses showed that yeast cells differed most in cytoplasmic profiling while biofilms differed most in surface profiling. Several processes and functions were significantly affected by the differentially abundant cytoplasmic proteins. Particularly noted were many of the enzymes of respiratory and fermentative pentose and glucose metabolism, folate interconversions and proteins associated with oxidative and stress response functions, host response, and multi-organism interaction. The differential abundance of cytoplasmic and surface proteins demonstrated that sessile and planktonic organisms have a unique profile.

Mitochondrial dysregulation of osteoarthritic human articular chondrocytes analyzed by proteomics: a decrease in mitochondrial superoxide dismutase points to a redox imbalance.

Mitochondria are involved in many cellular processes; mitochondrial dysfunctions have been associated with apoptosis, aging, and a number of pathological conditions, including osteoarthritis (OA). Mitochondrial proteins are attractive targets for the study of metabolism of the chondrocyte, the unique cell type present in mature cartilage, and its role in tissue degradation. Using a proteomics approach based on two-dimensional DIGE and MALDI-TOF/TOF mass spectrometric identification of mitochondria- enriched protein fractions from human articular chondrocytes, we analyzed mitochondrial protein changes that are characteristic of OA chondrocytes. A total of 73 protein forms were unambiguously identified as significantly altered in OA; 23 of them have been previously described as mitochondrial. An extensive statistical and cluster analysis of the data revealed a mitochondrial protein profile characteristic for OA. This pattern includes alterations in energy production, maintenance of mitochondrial membrane integrity, and free radical detoxification. Real time PCR, Western blot, and immunohistofluorescence assays confirmed a significant decrease of the major mitochondrial antioxidant protein manganese-superoxide dismutase (SOD2) in the superficial layer of OA cartilage. As possible outputs for this antioxidant deficiency, we found an increase of intracellular reactive oxygen species generation in OA chondrocytes and also verified an OA-dependent increase in the mitochondrial tumor necrosis factor-alpha receptor-associated protein 1 (TRAP1), a chaperone with a reported reactive oxygen species antagonist role. Our results describe the differences between the mitochondrial protein profiles of normal and OA chondrocytes, demonstrating that mitochondrial dysregulation occurs in cartilage cells during OA and highlighting redox imbalance as a key factor in OA pathogenesis.

Molecular staging of prostatic cancer with RT-PCR assay for prostate-specific antigen in peripheral blood and lymph nodes. 5 Year follow-up

Objective: Thirty percent of patients with localized prostate cancer undergoing radical prostatectomy experience biochemical recurrence with rising serum prostate-specific antigen (PSA). More than 50% of these develop distant metastases. Methods: Presence of PSA mRNA in pathologically normal pelvic lymph nodes from 154 patients undergoing radical prostatectomy was investigated with non-quantitative PSA reverse transcriptase polymerase chain reaction (RT-PCR). In 135 of these patients preoperative serum PSA RT-PCR was also assessed. RT-PCR positivity was correlated with biochemical recurrence and compared with other clinical risk factors. Results: At a median follow-up of 58 months the biochemical failure-free survival of patients with positive versus negative lymph node RT-PCR was 68.4% and 76.7% respectively (p=0.2). Biochemical failure-free survival was not influenced by the serum PSA RT-PCR result either (72.3% versus 72.6%). Surgical margin status, preoperative serum PSA, pT category and Gleason score were independent prognostic risk factors for biochemical recurrence with a hazard ratio of 5.48, 2.56, 2.56 and 2.13 respectively. Conclusions: At 5 year follow-up after radical prostatectomy, both serum and lymph node RT-PCR are not correlated with biochemical failure-free survival. Established clinical risk factors have a much stronger impact on biochemical recurrence (AU)

Objetivo: El 30% de los pacientes con cáncer de próstata localizado sometidos a prostatectomía radical sufren recurrencia bioquímica con aumento del antígeno prostático específico (PSA). Más del 50% de ellos desarrollan metástasis a distancia. Métodos: Utilizando la prueba de reacción en cadena de polimerasa con transcriptasa inversa (RT-PCR) se investigó en 154 pacientes sometidos a prostatectomía radical la presencia de mRMA de PSA en ganglios linfáticos normales según el análisis anatomopatológico. En 135 pacientes también se evaluó la RT-PCR de PSA preoperatoria en sangre periférica. Se correlacionó un resultado positivo de RT-PCR con recurrencia bioquímica y se comparó con otros factores de riesgo clínicos. Resultados: La supervivencia libre de recurrencia bioquímica en los pacientes con RT-PCR positiva y negativa en ganglios linfáticos fue de 68,4% y 76,7% respectivamente (p = 0,2), con una mediana de seguimiento de 58 meses. El resultado de RT-PCR en suero no influyó en la supervivencia libre de recurrencia bioquímica en ninguno de los grupos (72,3% frente a 72,6%). El estado de los márgenes quirúrgicos, el PSA sérico preoperatorio, la categoría pT y el escore de Gleason fueron factores de riesgo independientes para el pronóstico de recurrencia bioquímica con un riesgo relativo de 5,48, 2,56, y 2,13 respectivamente. Conclusiones: Ni la RT-PCR de PSA sérica ni la de ganglios linfáticos se correlacionan con supervivencia libre de recidiva bioquímica a los cinco años de seguimiento después de prostatectomía radical. Factores de riesgo clínico establecidos tienen un impacto mucho más fuerte sobre la recurrencia bioquímica (AU)

Molecular staging of prostatic cancer with RT-PCR assay for prostate-specific antigen in peripheral blood and lymph nodes. 5 year follow-up.

OBJECTIVE: Thirty percent of patients with localized prostate cancer undergoing radical prostatectomy experience biochemical recurrence with rising serum prostate-specific antigen (PSA). More than 50% of these develop distant metastases. METHODS: Presence of PSA mRNA in pathologically normal pelvic lymph nodes from 154 patients undergoing radical prostatectomy was investigated with non-quantitative PSA reverse transcriptase polymerase chain reaction (RT-PCR). In 135 of these patients preoperative serum PSA RT-PCR was also assessed. RT-PCR positivity was correlated with biochemical recurrence and compared with other clinical risk factors. RESULTS: At a median follow-up of 58 months the biochemical failure-free survival of patients with positive versus negative lymph node RT-PCR was 68.4% and 76.7% respectively (p=0.2). Biochemical failure-free survival was not influenced by the serum PSA RT-PCR result either (72.3% versus 72.6%). Surgical margin status, preoperative serum PSA, pT category and Gleason score were independent prognostic risk factors for biochemical recurrence with a hazard ratio of 5.48, 2.56, 2.56 and 2.13 respectively. CONCLUSIONS: At 5 year follow-up after radical prostatectomy, both serum and lymph node RT-PCR are not correlated with biochemical failure-free survival. Established clinical risk factors have a much stronger impact on biochemical recurrence.

Molecular staging of prostatic cancer with RT-PCR assay for prostate-specific antigen in peripheral blood and lymph nodes: comparison with standard histological staging and immunohistochemical assessment of occult regional lymph node metastases.

BACKGROUND: About 30-40% of men with localized prostate cancer undergoing radical prostatectomy will have cancer recurrence. It is estimated that one third recur locally and two thirds develop distant metastases with or without local recurrence. METHODS: In the present study we investigate the detection of prostate-specific antigen (PSA) mRNA in peripheral blood samples (n=200 patients) and pelvic lymph nodes (n=154 patients) by PSA reverse transcriptase polymerase chain reaction (RT-PCR) and compare these results to standard histological and immunohistochemical staging. RESULTS: We have observed a statistically significant correlation of lymph node PSA RT-PCR with standard pathologic risk factors, such as Gleason score (p=0.011), the presence of Gleason patterns 4 or 5 (p=0.005), lymph node metastasis (p<0.001) and a nearly significant correlation with the pT category (p=0.087). 39.5% (57/145) of the pN0 patients had PSA mRNA detectable in their lymph nodes. Blood PSA RT-PCR showed no correlation with the aforementioned factors and was even inversely correlated with preoperative serum PSA and lymph node status. Immunohistochemistry did not detect unsuspected prostate micrometastases in any pN0 patient. CONCLUSIONS: Lymph node PSA RT-PCR correlates with the Gleason score and the presence of Gleason patterns 4 or 5. Further clinical follow-up and correlation of RT-PCR status with overall outcome is required to allow validation of lymph node RT-PCR as a predictor of distant disease recurrence.

Cell stress and MEKK1-mediated c-Jun activation modulate NFkappaB activity and cell viability.

Chemotherapeutic agents such as cisplatin induce persistent activation of N-terminal c-Jun Kinase, which in turn mediates induction of apoptosis. By using a common MAPK Kinase, MEKK1, cisplatin also activates the survival transcription factor NFkappaB. We have found a cross-talk between c-Jun expression and NFkappaB transcriptional activation in response to cisplatin. Fibroblast derived from c-jun knock out mice are more resistant to cisplatin-induced cell death, and this survival advantage is mediated by upregulation of NFkappaB-dependent transcription and expression of MIAP3. This process can be reverted by ectopic expression of c-Jun in c-jun(-/-) fibroblasts, which decreases p65 transcriptional activity back to normal levels. Negative regulation of NFkappaB-dependent transcription by c-jun contributes to cisplatin-induced cell death, which suggests that inhibition of NFkappaB may potentiate the antineoplastic effect of conventional chemotherapeutic agents.