Identification of enolase as a laminin-binding protein on the surface of Staphylococcus aureus

We have previously demonstrated that Staphylococcus aureus, a highly invasive bacteria, presents a 52-kDa surface protein that mediates its binding to laminin. in order to better characterize this receptor, we excised this putative laminin receptor from two-dimensional (2-D) PAGE and used it as antigen for raising a mouse hyperimmune serum which was for screening an S. aureus expression library. A single clone of 0.3 kb was obtained, and its sequence revealed 100% homology with S. aureus a-enolase. Moreover, amino acid sequencing of the 52-kDa protein eluted from the 2-D gel indicated its molecular homology with a-enolase, an enzyme that presents a high evolutionary conservation among species. in parallel, monoclonal antibodies raised against the S. aureus 52-kDa band also recognized yeast a-enolase in western blot analysis. These monoclonal antibodies were also able to promote capture of iodine-labeled bacteria when adsorbed to a solid phase, and this capture was inhibited by the addition of excess rabbit muscle a-enolase. Finally, the cell surface localization of S. aureus a-enolase was further confirmed by flow cytometry. Hence, a-enolase might play a critical role in the pathogenesis of S. aureus by allowing its adherence to laminin-containing extracellular matrix. (C) 2004 Elsevier SAS. All rights reserved.Hosp Canc AC Camargo, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Discipline Immunol, São Paulo, BrazilUniv São Paulo, Sch Med, Inst Heart, Immunol Lab, São Paulo, BrazilLudwig Inst Canc Res, Sau Paulo Branch, BR-01509010 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Discipline Immunol, São Paulo, BrazilWeb of Scienc

Differential gene expression assessed by cDNA microarray analysis in breast cancer tissue under tamoxifen treatment

Our purpose was to identify tamoxifen (TAM) responsive genes after 30 days of TAM treatment in tumor tissues obtained from women with breast cancer using microarray expression analysis. In our study, we identified 12 candidates to be considered as tamoxifen-modulated genes. Among them, we selected two candidates the TEGT BI-1 (testis enhanced gene transcript Bax Inhibitor-1) and the CD63 gene in order to further confirm their differential expression under tamoxifen effects. We observed that both were down-regulated in tumor tissues of patients during TAM treatment. TEGT is able to inhibit the expression of Bax, which is known to promote apoptosis. On the other hand, CD63 encodes a cell membrane protein and it seems to be involved in mechanisms of platelet activation. cell adhesion and cell motility. We therefore hypothesize that TAM would be able to modulate tumor growth by down-regulating genes involved in mechanisms such as cell cycle control, tumor invasion and metastasis.Univ Fed Sao Paulo, Dept Gynecol, Mol Gynecol Lab, Paulista Sch Med, Sao Paulo, SP, BrazilLudwig Inst Canc Res, Sao Paulo Branch, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Gynecol, Mol Gynecol Lab, Paulista Sch Med, Sao Paulo, SP, BrazilWeb of Scienc

Correlation of Body Mass Index (BMI), initial neutralizing antibodies (nAb), ABO group and kinetics of nAb and anti-nucleocapsid (NP) SARS-CoV-2 antibodies in convalescent plasma (CCP) donors – A longitudinal study with proposals for better quality of CCP collections

AbstractIntroductionA cohort of COVID-19 convalescent volunteers allowed the study of neutralizing (nAb) and ligand antibodies kinetics by providing sequential samples during a median of 100 days after onset of disease.Material and MethodsA cohort of previously RT-PCR+ve (detected by nasopharyngeal swab during the acute phase), male convalescent patients, all with mild symptoms, were enrolled on serial blood sample collection for evaluation of longitudinal nAb titers and anti-nucleocapsid (NP) antibodies (IgM, IgG and IgA). Nabs were detected by a cytopathic effect-based virus neutralization test (CPE-based VNT), carried out with SARS-CoV-2 (GenBank: MT350282)ResultsA total of 78 male volunteers provided 316 samples, spanning a total of 4820 days of study. Although only 25% of donors kept nAb titers ≥160, after a median of 100 days after the onset of disease, there was a high probability of sustaining nAB titers ≥160 in volunteers whose initial nAb titer was ≥1280, weight ≥ 90kg or BMI classified as overweight or obese, evidenced by Kaplan-Meier estimates and Cox hazard regression. There was no correlation between ABO group, ABO antibody titers and persistent high nAb titers. High IgG anti-NP (S/CO ≥5.0) is a good surrogate for detecting nAB ≥160, defined by ROC curve (sensitivity = 90.5%; CI95% 84.5-94.7%)ConclusionSelection of CCP donors for multiple collections based on initial high nAb titers (≥1280) or overweight/obese (BMI) provides a simple strategy to achieve higher quality in CCP programs. High IgG anti-NP levels can also be used as surrogate markers for high nAb screening.</jats:sec

Mutational analysis of genes coding for cell surface proteins in colorectal cancer cell lines reveal novel altered pathways, druggable mutations and mutated epitopes for targeted therapy

We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of "general" immune checkpoint drugs in this subset of patients

Role of Endogenous IFN-γ in Macrophage Programming Induced by IL-12 and IL-18

Besides the established role of interleukin-12 (IL-12) and IL-18 on interferon-γ (IFN-γ) production by natural killer (NK), T, and B cells, the effects of these cytokines on macrophages are largely unknown. Here, we investigated the role of IL-12/IL-18 on nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production by CD11b+ adherent peritoneal cells, focusing on the involvement of endogenously produced IFN-γ. C57BL/6 cells released substantial amounts of NO when stimulated with IFN-γ or lipopolysaccharide (LPS), but failed to respond to IL-12 or IL-18 or both. However, IL-12/IL-18 pretreatment was able to program these cells to release 6–8-fold more NO and TNF-α in response to LPS or Trypanosoma cruzi stimulation, with NO levels directly correlating with macrophage resistance to intracellular parasite growth. Analysis of IL-12/IL-18-primed cells from mice deficient in IFN-γ, IFNGR, and IFN regulatory factor-1 (IRF-1) revealed that these molecules were essential for LPS-induced NO release, but TNF-α production was IFN-γ independent. Conversely, the myeloid differentiation factor 88 (MyD88)-dependent pathway was indispensable for IL-12/IL-18-programmed LPS-induced TNF-α production, but not for NO release. Contaminant T and NK cells largely modulated the IL-12/IL-18 programming of LPS-induced NO response through IFN-γ secretion. Nevertheless, a small population of IFN-γ+ cells with a macrophage phenotype was also identified, particularly in the peritoneum of chronically T. cruzi-infected mice, reinforcing the notion that macrophages can be an alternative source of IFN-γ. Taken together, our data contribute to elucidate the molecular basis of the IL-12/IL-18 autocrine pathway of macrophage activation, showing that endogenous IFN-γ plays an important role in programming the NO response, whereas the TNF-α response occurs through an IFN-γ-independent pathway

Shotgun sequencing of the human transcriptome with ORF expressed sequence tags

Theoretical considerations predict that amplification of expressed gene transcripts by reverse transcription-PCR using arbitrarily chosen primers will result in the preferential amplification of the central portion of the transcript. systematic, high-throughput sequencing of such products would result in an expressed sequence tag (EST) database consisting of central, generally coding regions of expressed genes. Such a database would add significant value to existing public EST databases, which consist mostly of sequences derived from the extremities of cDNAs, and facilitate the construction of contigs of transcript sequences. We tested our predictions, creating a database of 10,000 sequences from human breast tumors. The data confirmed the central distribution of the sequences, the significant normalization of the sequence population, the frequent extension of contigs composed of existing human ESTs, and the identification of a series of potentially important homologues of known genes. This approach should make a significant contribution to the early identification of important human genes, the deciphering of the draft human genome sequence currently being compiled, and the shotgun sequencing of the human transcriptome.9773491349

Avaliação dos danos do DNA na mucosa esofágica e sangue periférico de portadores da doença do refluxo gastroesofágico Evaluation of DNA damage in the esophageal mucosa and peripheral blood of patients with gastroesophageal reflux disease

RACIONAL: A doença do refluxo gastroesofágico é a afecção digestiva de maior prevalência. Os portadores podem apresentar na evolução algumas complicações, sendo o esôfago de Barrett a de maior importância, tendo em vista seu potencial de malignidade. Todavia os processos inflamatórios do trato gastrointestinal podem apresentar degeneração maligna. OBJETIVOS: Avaliar os possíveis danos do DNA em portadores de esofagite de refluxo gastroesofágico de vários graus e verificar a aplicação do ensaio Cometa na detecção dos mesmos. MÉTODOS: Foram estudados 25 pacientes distribuídos em quatro grupos: controle (n=5), esofagite leve (n=8), esofagite severa (n=5) e câncer (n=7). O ensaio Cometa foi realizado no sangue periférico (linfócitos) e biópsia do terço distal do esôfago. RESULTADOS: O ensaio Cometa detectou danos no DNA nos pacientes com esofagite leve e severa (sangue periférico e biópsia), sendo que na esofagite severa a intensidade dos danos foi maior (p<0,05). Os danos do DNA dos pacientes com esofagite severa e câncer não mostraram diferença significativa e a intensidade dos mesmos corresponde ao ensaio Cometa classe 4 (maior que 95% de danos). CONCLUSÕES: 1) As frequências de quebras do DNA da mucosa esofágica e linfócitos estão diretamente relacionadas ao grau de inflamação; 2) a esofagite severa apresenta praticamente a mesma frequência de danos no DNA do câncer esofágico; 3) o ensaio Cometa mostrou-se muito sensível para a detecção dos danos do DNA.<br>BACKGROUND: The gastroesophageal reflux disease is the most prevalent digestive disorder. Patients with it may present some complications during its development, and Barrett's esophagus is the most important in view of its potential malignancy. However, the inflammatory processes of the gastrointestinal tract may show malignant degeneration. AIM: To assess possible DNA damage in patients with gastroesophageal reflux esophagitis of various degrees and to evaluate the application of the Comet assay in its detection. METHODS: Twenty-five patients were studied. They were divided into four groups: control (n=5), mild esophagitis (n=8), severe esophagitis (n=5) and cancer (n=7). The Comet assay was performed on peripheral blood cells (lymphocytes) and biopsy of the distal esophagus. RESULTS: The Comet assay detected DNA damage in patients with mild and severe esophagitis (peripheral blood and biopsy), and damage intensity was greater in severe esophagitis (p<0,05). DNA damage in patients with severe esophagitis and cancer did not show significant difference, and its intensity corresponds to class-4 Comet assay (greater than 95% of damage). CONCLUSIONS: 1) The frequencies of DNA breakage in the esophageal mucosa and lymphocytes are directly related to inflammation level; 2) severe esophagitis shows virtually the same DNA damage frequency as that of esophageal cancer; 3) the Comet assay showed to be very sensitive for DNA damage detection