Proteome profiling of triple negative breast cancer cells overexpressing NOD1 and NOD2 receptors unveils molecular signatures of malignant cell proliferation.

BACKGROUND: Triple negative breast cancer (TNBC) is a malignancy with very poor prognosis, due to its aggressive clinical characteristics and lack of response to receptor-targeted drug therapy. In TNBC, immune-related pathways are typically upregulated and may be associated with a better prognosis of the disease, encouraging the pursuit for immunotherapeutic options. A number of immune-related molecules have already been associated to the onset and progression of breast cancer, including NOD1 and NOD2, innate immune receptors of bacterial-derived components which activate pro-inflammatory and survival pathways. In the context of TNBC, overexpression of either NOD1or NOD2 is shown to reduce cell proliferation and increase clonogenic potential in vitro. To further investigate the pathways linking NOD1 and NOD2 signaling to tumorigenesis in TNBC, we undertook a global proteome profiling of TNBC-derived cells ectopically expressing each one of these NOD receptors. RESULTS: We have identified a total of 95 and 58 differentially regulated proteins in NOD1- and NOD2-overexpressing cells, respectively. We used bioinformatics analyses to identify enriched molecular signatures aiming to integrate the differentially regulated proteins into functional networks. These analyses suggest that overexpression of both NOD1 and NOD2 may disrupt immune-related pathways, particularly NF-κB and MAPK signaling cascades. Moreover, overexpression of either of these receptors may affect several stress response and protein degradation systems, such as autophagy and the ubiquitin-proteasome complex. Interestingly, the levels of several proteins associated to cellular adhesion and migration were also affected in these NOD-overexpressing cells. CONCLUSIONS: Our proteomic analyses shed new light on the molecular pathways that may be modulating tumorigenesis via NOD1 and NOD2 signaling in TNBC. Up- and downregulation of several proteins associated to inflammation and stress response pathways may promote activation of protein degradation systems, as well as modulate cell-cycle and cellular adhesion proteins. Altogether, these signals seem to be modulating cellular proliferation and migration via NF-κB, PI3K/Akt/mTOR and MAPK signaling pathways. Further investigation of altered proteins in these pathways may provide more insights on relevant targets, possibly enabling the immunomodulation of tumorigenesis in the aggressive TNBC phenotype.

Expression and in vitro assessment of tumorigenicity for NOD1 and NOD2 receptors in breast cancer cell lines.

OBJECTIVE: Immune-related pathways have been frequently associated to tumorigenesis. NOD1 and NOD2 are innate immune receptors responsible for sensing a subset of bacterial-derived components, and to further translate these pathogenic signals through pro-inflammatory and survival pathways. NOD1 and NOD2 have been further associated with tumorigenesis, particularly in gastrointestinal cancers. NOD1 has also been suggested to be a tumor suppressor gene in a model of estrogen receptor-dependent breast cancer. Contrarily, NOD2 polymorphisms are associated with higher risk of breast cancer, with no tumor suppressor role being reported. To better delineate this issue, we investigated NOD1 and NOD2 expression in a panel of breast cancer cell lines, as well as their potential impact in breast tumorigenesis based on in vitro assays. RESULTS: The highly invasive Hs578T breast cell line presented the second highest NOD1 expression and the lowest NOD2 expression in our panel. Therefore, we investigated whether NOD1 and/or NOD2 might act as a tumor suppressor in this cell model. Our studies indicate that overexpression of either NOD1 or NOD2 reduces cell proliferation and increases clonogenic potential in vitro. Elucidation of NOD1 and NOD2 effects on tumor cell viability and proliferation may unveil potential targets for future therapeutic intervention.

NOD1 in the modulation of host-microbe interactions and inflammatory bone resorption in the periodontal disease model.

Periodontitis is a chronic inflammatory condition characterized by destruction of non-mineralized and mineralized connective tissues. It is initiated and maintained by a dysbiosis of the bacterial biofilm adjacent to teeth with increased prevalence of Gram-negative microorganisms. Nucleotide-binding oligomerization domain containing 1 (NOD1) is a member of the Nod-like receptors (NLRs) family of proteins that participate in the activation of the innate immune system, in response to invading bacteria or to bacterial antigens present in the cytoplasm. The specific activating ligand for NOD1 is a bacterial peptidoglycan derived primarily from Gram-negative bacteria. This study assessed the role of NOD1 in inflammation-mediated tissue destruction in the context of host-microbe interactions. We used mice with whole-genome deletion of the NOD1 gene in a microbe-induced periodontitis model using direct injections of heat-killed Gram-negative or Gram-negative/Gram-positive bacteria on the gingival tissues. In vitro experiments using primary bone-marrow-derived macrophages from wild-type and NOD1 knockout mice provide insight into the role of NOD1 on the macrophage response to Gram-negative and Gram-negative/Gram-positive bacteria. Microcomputed tomography analysis indicated that deletion of NOD1 significantly aggravated bone resorption induced by Gram-negative bacteria, accompanied by an increase in the numbers of osteoclasts. This effect was significantly attenuated by the association with Gram-positive bacteria. In vitro, quantitative PCR arrays indicated that stimulation of macrophages with heat-killed Gram-negative bacteria induced the same biological processes in wild-type and NOD1-deficient cells; however, expression of pro-inflammatory mediators was increased in NOD1-deficient cells. These results suggest a bone-sparing role for NOD1 in this model.

Manual de monitoramento da qualidade dos registros de enfermagem na assistência domiciliar / Manual de monitoreo de la calidad de los registros de enfermería en la atención domiciliaria / Manual for monitoring the quality of nursing home care records

RESUMO Objetivo: construir e validar um instrumento para monitorar a qualidade dos registros de enfermagem no Programa de Assistência Domiciliar (PAD) em um hospital universitário. Método: estudo metodológico envolvendo a elaboração de um manual e submetido à validação de conteúdo por seis juízes sob consenso ≥ 80%. A coleta ocorreu em 2012 por meio de questionário contendo: evolução de enfermagem, diagnóstico e prescrição de enfermagem e normas para os registros da equipe de enfermagem preconizadas pelo Conselho Regional de Enfermagem-SP e pela instituição. Os itens do manual foram julgados de acordo com as variáveis - relevância, pertinência, clareza e simplicidade. Resultados: das 39 proposições 100% atingiram consenso ≥ 80% em relevância, pertinência e clareza; 92,3% em simplicidade. Os itens sono/repouso, mobilidade e checagem nas atividades prescritas não atingiram consenso mínimo favorável, sendo aprimorados pelas sugestões dos juízes. Conclusão: acreditamos que o instrumento possibilitará a melhoria dos processos de trabalho no PAD. .

RESUMEN Objetivo: construir y validar un instrumento para monitorear la calidad del registros de enfermería en Programa de Atención Domiciliaria (PAD) de un Hospital Universitario. Metodo: estudio metodológico. Fue construido un manual y sometió a validación de contenido por seis jueces bajo el consenso ≥80%. La recogida currió en 2012, con un cuestionario que contiene: evolución de enfermería, diagnóstico y prescripción de enfermería y normas para los registros del personal de enfermaria estabelecidas por Consejo Regional de Enfermería-SP y por la institución. Los artículos del manual fueran juzgadso conforme las variables relevancia, pertinencia, claridad y sencillez. Resultados: de las 39 proposiciones 100% alcanzó consenso ≥ 80% en la relevancia, pertinencia y claridad; 92,3% en la simplicidad. Los itens sueño/resto, movilidad y verificar las actividades prescritas no alcanzó consenso favorable, siendo mejoradas por las sugerencias de los jueces. Conclusión: creemos que el instrumento permitirá la mejora de los procesos de trabajo en PAD. .

ABSTRACT Objective: to build and validate an instrument aimed at monitoring the quality of nursing records in the Home Care Program (HCP) of a university hospital. Method: methodological study involving the elaboration of a manual, whose content was later submitted to six experts for validation, reaching a ≥ 80% consensus. The data collection process was carried out in 2012 by means of a questionnaire comprised of the following issues: nursing evolution, nursing diagnosis, and nursing prescription, and standards for the nursing team recommended by the Regional Nursing Council of São Paulo and by the assessed institution. Manual items were judged according to the following variables: relevance, pertinence, clarity and simplicity. Results: of the 39 propositions, 100% achieved ≥ 80% agreement in the relevance, pertinence and clarity variables; 92.3% in the simplicity variable. Sleep/rest, Mobility and Check-out variables did not reach a favorable minimum consensus in the prescribed activities and were improved following suggestions from the experts. Conclusion: we believe that the instrument will enable the improvement of the HCP’s work process. .

MyD88-, but not Nod1- and/or Nod2-deficient mice, show increased susceptibility to polymicrobial sepsis due to impaired local inflammatory response.

Pathogen recognition and triggering of the inflammatory response following infection in mammals depend mainly on Toll-like and Nod-like receptors. Here, we evaluated the role of Nod1, Nod2 and MyD88-dependent signaling in the chemokine production and neutrophil recruitment to the infectious site during sepsis induced by cecal ligation and puncture (CLP) in C57Bl/6 mice. We demonstrate that Nod1 and Nod2 are not involved in the release of chemokines and recruitment of neutrophils to the infectious site during CLP-induced septic peritonitis because these events were similar in wild-type, Nod1-, Nod2-, Nod1/Nod2- and Rip2-deficient mice. Consequently, the local and systemic bacterial loads were not altered. Accordingly, neither Nod1 nor Nod2 was involved in the production of the circulating cytokines and in the accumulation of leukocytes in the lungs. By contrast, we showed that MyD88-dependent signaling is crucial for the establishment of the local inflammatory response during CLP-induced sepsis. MyD88-deficient mice were susceptible to sepsis because of an impaired local production of chemokines and defective neutrophil recruitment to the infection site. Altogether, these data show that Nod1, Nod2 and Rip2 are not required for local chemokine production and neutrophil recruitment during CLP-induced sepsis, and they reinforce the importance of MyD88-dependent signaling for initiation of a protective host response.

Transcription of pattern recognition receptors and abortive agents induced chemokines in the bovine pregnant uterus.

Pattern recognition receptors (PRRs) are important components of the innate immune system whose ligands are specific pathogen associated molecular patterns (PAMPs). Considering the scarcity of studies on transcription of PRRs in the pregnant uterus of cows, and its response to PAMPs and microorganisms that cause abortion in cattle, this study aimed to characterize the transcription of TLR1-10, NOD1, NOD2 and MD2 in bovine uterus throughout gestation and to investigate the sensitivity of different uterine tissues at third trimester of pregnancy to purified TLR ligands or heat-killed Brucella abortus, Salmonella enterica serotype Dublin (S. Dublin), Listeria monocytogenes, and Aspergillus fumigatus, by assessing chemokine transcription. RNA extracted from endometrium, placentome and intercotiledonary region of cows at the first (n=6), second (n=6), and third (n=6) trimesters of pregnancy were subjected to real time RT-PCR. After stimulation of endometrium and intercotiledonary regions with purified TLR ligands or heat-killed microorganisms, gene transcription was assessed by real time RT-PCR. In the placentome, there was no significant variation in TLRs transcription throughout the three trimesters of pregnancy. In the endometrium, there was significant variation in TLR4 and TLR5 transcription during the three stages of gestation; i.e. TLR4 transcription was higher during the third trimester, whereas TLR5 transcription was higher during the last two trimesters. In the intercotiledonary region, there was significant variation in transcription of TLR1/6, TLR7, and TLR8, which were more strongly expressed during the first trimester of pregnancy. At the third trimester of gestation, significant transcription of CXCL6 and CXCL8 was detected mostly in endometrial tissues in response to purified TLR4 and TLR2 ligands. Transcription of these chemokines was induced in the endometrium and intercotiledonary region at the third trimester of pregnancy when stimulated with heat-killed B. abortus or S. Dublin. Therefore, this study demonstrates that some PRRs are expressed in the uterus during pregnancy, which coincides with its ability to respond to stimulation with TLRs ligands as well as heat-killed organisms known to cause abortion in cattle.

Nucleotide-binding oligomerization domain-1 and -2 play no role in controlling Brucella abortus infection in mice.

Nucleotide-binding oligomerization domain proteins (NODs) are modular cytoplasmic proteins implicated in the recognition of peptidoglycan-derived molecules. Further, several in vivo studies have demonstrated a role for Nod1 and Nod2 in host defense against bacterial pathogens. Here, we demonstrated that macrophages from NOD1-, NOD2-, and Rip2-deficient mice produced lower levels of TNF-α following infection with live Brucella abortus compared to wild-type mice. Similar reduction on cytokine synthesis was not observed for IL-12 and IL-6. However, NOD1, NOD2, and Rip2 knockout mice were no more susceptible to infection with virulent B. abortus than wild-type mice. Additionally, spleen cells from NOD1-, NOD2-, and Rip2-deficient mice showed unaltered production of IFN-γ compared to C57BL/6 mice. Taken together, this study demonstrates that NOD1, NOD2 and Rip2 are dispensable for the control of B. abortus during in vivo infection.