Modulation of Stat-1 in human macrophages infected with different species of intracellular pathogenic bacteria.

none6The infection of human macrophages by pathogenic bacteria induces different signaling pathways depending on the type of cellular receptors involved in the microorganism entry and on their mechanism(s) of survival and replication in the host cell. It was reported that Stat proteins play an important role in this process. In the present study, we investigate the changes in Stat-1 activation (phosphorylation in p-tyr(701)) after uptake of twoGram-positive (Listeria monocytogenes and Staphylococcus aureus) and two Gram-negative bacteria (Salmonella typhimurium and Legionella pneumophila) characterized by their varying abilities to enter, survive, and replicate in human macrophages. Comparing the results obtained with Gram-negative and Gram-positive bacteria, Stat-1 activation in macrophages does not seem to be related to LPS content. The p-tyr(701) Stat-1 expression levels were found to be independent of the internalized bacterial number and IFN-gamma release. On the contrary, Jak/Stat-1 pathway activation only occurs when an active infection has been established in the host macrophage, and it is plausible that the differences in the expression levels of p-tyr(701) Stat-1 could be due to different survival mechanisms or to differences in bacteria life cycles within macrophages.openSchiavano, Giuditta Fiorella; Dominici, Sabrina; Rinaldi, Laura; Cangiano, Alfonsina Mariarosaria; Brandi, Giorgio; Magnani, MauroSchiavano, GIUDITTA FIORELLA; Dominici, Sabrina; Rinaldi, Laura; Cangiano, ALFONSINA MARIAROSARIA; Brandi, Giorgio; Magnani, Maur

Transcriptional activation of the miR-17-92 cluster is involved in the growth-promoting effects of MYB in human Ph-positive leukemia cells.

MicroRNAs, non-coding regulators of gene expression, are likely to function as important downstream effectors of many transcription factors including MYB. Optimal levels of MYB are required for transformation/maintenance of BCR-ABL-expressing cells. We investigated whether MYB silencing modulates microRNA expression in Philadelphia-positive (Ph+) leukemia cells and if MYB-regulated microRNAs are important for the MYB addiction of these cells. Thirty-five microRNAs were modulated by MYB silencing in lymphoid and erythromyeloid chronic myeloid leukemia-blast crisis BV173 and K562 cells; 15 of these were concordantly modulated in both lines. We focused on the miR-17-92 cluster because of its oncogenic role in tumors and found that: i) it is a direct MYB target; ii) it partially rescued the impaired proliferation and enhanced apoptosis of MYB-silenced BV173 cells. Moreover, we identified FRZB, a Wnt/β-catenin pathway inhibitor, as a novel target of the miR-17-92 cluster. High expression of MYB in blast cells from 2 Ph+leukemia patients correlated positively with the miR-17-92 cluster and inversely with FRZB. This expression pattern was also observed in a microarray dataset of 122 Ph+acute lymphoblastic leukemias. In vivo experiments in NOD scid gamma mice injected with BV173 cells confirmed that FRZB functions as a Wnt/β-catenin inhibitor even as they failed to demonstrate that this pathway is important for BV173-dependent leukemogenesis. These studies illustrate the global effects of MYB expression on the microRNAs profile of Ph+cells and supports the concept that the MYB addiction of these cells is, in part, caused by modulation of microRNA-regulated pathways affecting cell proliferation and survival. Copyright© 2019 Ferrata Storti Foundation

Palytoxin and an Ostreopsis Toxin Extract Increase the Levels of mRNAs Encoding Inflammation-Related Proteins in Human Macrophages via p38 MAPK and NF-κB

BACKGROUND: Palytoxin and, likely, its analogues produced by the dinoflagellate genus Ostreopsis, represent a class of non-proteinaceous compounds displaying high toxicity in animals. Owing to the wide distribution and the poisonous effects of these toxins in humans, their chemistry and mechanism of action have generated a growing scientific interest. Depending on the exposure route, palytoxin and its Ostreopsis analogues may cause several adverse effects on human health, including acute inflammatory reactions which seem more typical of cutaneous and inhalation contact. These observations have led us to hypothesize that these toxins may activate pro-inflammatory signalling cascades. METHODOLOGY AND PRINCIPAL FINDINGS: Here we demonstrate that palytoxin and a semi-purified Ostreopsis cf. ovata toxin extract obtained from a cultured strain isolated in the NW Adriatic Sea and containing a putative palytoxin and all the ovatoxins so far known--including the recently identified ovatoxin-f--significantly increase the levels of mRNAs encoding inflammation-related proteins in immune cells, i.e. monocyte-derived human macrophages, as assessed by Real-Time PCR analysis. Western immunoblot and electrophoretic mobility shift assays revealed that nuclear transcription factor -κB (NF-κB) is activated in cells exposed to toxins in coincidence with reduced levels of the inhibitory protein IκB-α. Moreover, Mitogen-Activated Protein Kinases (MAPK) were phosphorylated in response to palytoxin, as also reported by others, and to the Ostreopsis toxin extract, as shown here for the first time. By using specific chemical inhibitors, the involvement of NF-κB and p38 MAPK in the toxin-induced transcription and accumulation of Cycloxigenase-2, Tumor Necrosis Factor-α, and Interleukin-8 transcripts has been demonstrated. CONCLUSIONS AND SIGNIFICANCE: The identification of specific molecular targets of palytoxin and its Ostreopsis analogues, besides contributing to expand the still limited knowledge of the intracellular signalling cascades affected by these toxins, may have important implications in setting up focused pharmacological interventions, replacing currently used symptomatic treatments

Immunization with HIV protease peptides linked to syngeneic erythrocytes

New potent vaccine adjuvants are desirable for increasing the efficacy of novel vaccine modalities such as DNA and peptides. We therefore tested if syngeneic erythrocytes could serve as delivery vectors for selected HIV peptides and compared the potency of these constructs to immunization with peptides in phosphate buffered saline or in incomplete Freunds adjuvant. Immunization of mice with peptides in a low dose (5 ng) coupled to erythrocytes induced a weak immune response in mice. These peptides alone (5 μg) gave no immune responses, while formulating the peptides (50 μg) in IFA induced strong homologous immunity as well as prominent cross reactivity to a related mutant epitope. Thus, vaccine delivery using syngeneic erythrocytes, although attractive for clinical use, might be of limited value due to the low amount of antigen that can be loaded per erythrocyte

The human antibody fragment DIATHIS1 specific for CEACAM1 enhances natural killer cell cytotoxicity against melanoma cell lines in vitro

Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1 malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1 melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1 melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro-expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell-mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin

Representações sociais dos sentimentos vivenciados pelo paciente portador de neoplasia

The aim was to identify the social representations about cancer of patients undergoing cancer treatment. This is an exploratory-descriptive study with a qualitative approach with data that used the research grounds following assumptions of the proposed Theory of Social Representations. The research had a total of 92 patients of various cancer diagnoses during chemotherapy treatment. Data were collected through individual interviews, using a semi-structured questionnaire where the patient had to enumerate the first 5 words that came to their mind from the most important to the least important. Among the words evoked, the ones that were repeated the most were fear, death and treatment, sadness and healing. Evidencing, therefore, the fear that the patient feels in the face of cancer. In the fifth line of words, the most evoked terms were healing, faith, God, hope. At the beginning of the treatment, the patient is afraid of a disease that still has an important psychosocial impact on his daily life. Oncological therapy performed with quality and efficiency, by qualified professionals who assist the patient in all its dimensions, who know parts of their feelings during treatment, can help to reduce suffering and reduce the physical and psychological impacts on the patient.Objetivou-se identificar as representações sociais sobre o câncer dos pacientes em tratamento oncológico. Trata-se de um estudo exploratório-descritivo com abordagem qualitativa com dados que utilizaram fundamentação da pesquisa seguindo pressupostos da Teoria das Representações Sociais proposta. A pesquisa teve o total de 92 pacientes de diversos diagnósticos de câncer durante o tratamento quimioterápico. Os dados foram coletados por meio de entrevistas individuais, utilizando um questionário semiestruturado onde o paciente precisou enumerar as 5 primeiras palavras que viessem a sua cabeça da de maior importância para a de menor importância. Entre as palavras evocadas as que mais se repetiram foram medo, morte e tratamento, tristeza e cura. Evidenciando, portanto, o temor que o paciente sente frente o câncer. Na quinta linha de palavras os termos mais evocados foram cura, fé, Deus, esperança. No início do tratamento, o paciente encontra-se temeroso diante de uma doença que ainda tem uma repercussão psicossocial importante no seu dia-a-dia. A terapêutica oncológica realizada com qualidade e eficiência, por profissionais qualificados que assistem o paciente em todas as suas dimensões que conhecem partes de seus sentimentos durante o tratamento, pode auxiliar na redução do sofrimento e diminuir os impactos físicos e psíquicos no paciente

Intraperitoneal drain placement and outcomes after elective colorectal surgery: international matched, prospective, cohort study

Despite current guidelines, intraperitoneal drain placement after elective colorectal surgery remains widespread. Drains were not associated with earlier detection of intraperitoneal collections, but were associated with prolonged hospital stay and increased risk of surgical-site infections.Background Many surgeons routinely place intraperitoneal drains after elective colorectal surgery. However, enhanced recovery after surgery guidelines recommend against their routine use owing to a lack of clear clinical benefit. This study aimed to describe international variation in intraperitoneal drain placement and the safety of this practice. Methods COMPASS (COMPlicAted intra-abdominal collectionS after colorectal Surgery) was a prospective, international, cohort study which enrolled consecutive adults undergoing elective colorectal surgery (February to March 2020). The primary outcome was the rate of intraperitoneal drain placement. Secondary outcomes included: rate and time to diagnosis of postoperative intraperitoneal collections; rate of surgical site infections (SSIs); time to discharge; and 30-day major postoperative complications (Clavien-Dindo grade at least III). After propensity score matching, multivariable logistic regression and Cox proportional hazards regression were used to estimate the independent association of the secondary outcomes with drain placement. Results Overall, 1805 patients from 22 countries were included (798 women, 44.2 per cent; median age 67.0 years). The drain insertion rate was 51.9 per cent (937 patients). After matching, drains were not associated with reduced rates (odds ratio (OR) 1.33, 95 per cent c.i. 0.79 to 2.23; P = 0.287) or earlier detection (hazard ratio (HR) 0.87, 0.33 to 2.31; P = 0.780) of collections. Although not associated with worse major postoperative complications (OR 1.09, 0.68 to 1.75; P = 0.709), drains were associated with delayed hospital discharge (HR 0.58, 0.52 to 0.66; P < 0.001) and an increased risk of SSIs (OR 2.47, 1.50 to 4.05; P < 0.001). Conclusion Intraperitoneal drain placement after elective colorectal surgery is not associated with earlier detection of postoperative collections, but prolongs hospital stay and increases SSI risk

An enzyme-linked immunosorbent assay for the measurement of plasma flavonoids in mice fed apigenin-C-glycoside

In the Chenopodiaceae family, the apigenin flavonoids vitexin-2-O-xyloside (VOX) and vitexin-2-O-rhamnoside (VOR) are important chemopreventive components. To investigate their bioavailability in in vivo animal studies an enzyme-linked immunosorbent assay (ELISA) method has been developed

Salmonella Abortusovis: An Epidemiologically Relevant Pathogen

The ovine pathogen Salmonella enterica serovar Abortusovis (SAO), a pathogen strictly adapted to ovine hosts, is endemic in several European and Asian countries, where it causes significant economic losses due to the high rates of abortion in infected flocks. In some countries (i.e. Switzerland and Croatia), re-emergence of infection by SAO occurred after decades during which the disease has not been reported. The introduction of (SAO) epidemic strains in new areas is difficult to control due to the asymptomatic behaviors in infected adult lambs, rams, and nonpregnant ewes. Culture-based diagnosis may provide false-negative results. Moreover, the retrospective identification of Salmonella infection in ewes is challenging as excretion of the causative agent is transient and the serum antibodies fall to low titres soon after the abortion. Therefore, regular monitoring of pathogen exposure, mainly through seroconversion assessment, is advisable to prevent disease introduction and spread in SAO-free areas, especially in case of animal export, and to reduce abortion risk