Isolation of a novel LPS-induced component of the ML superfamily in Ciona intestinalis

ML superfamily represents a group of proteins playing important roles in lipid metabolism and innate immune response. In this study, we report the identification of the first component of the ML superfamily in the invertebrate Ciona intestinalis by means of a subtractive hybridization strategy. Sequence homology and phylogenetic analysis showed that this protein forms a specific clade with vertebrate components of the Niemann-Pick type C2 protein and, for this reason, it has been named Ci-NPC2. The putative Ci-NPC2 is a 150 amino acids long protein with a short signal peptide, seven cysteine residues, three putative lipid binding site and a three-dimensional model showing a characteristic b-strand structure. Gene expression analysis demonstrated that the Ci-NPC2 protein is positively upregulated after LPS inoculum with a peak of expression 1 h after challenge. Finally, in-situ hybridization demonstrated that the Ci-NPC2 protein is preferentially expressed in hemocytes inside the vessel lumen

Fine characterization of immunological mechanisms mediated by the major allergens of Parietaria judaica and hypoallergenic hybrid, rPjEDcys

Purpose: Allergy is a hypersensitivity disease IgE-mediated, affecting more than 25% of the population. The symptoms of IgE-mediated allergies reactions can be transiently ameliorated pharmacologically, but the only curative treatment of allergies is Allergen-Specific Immunotherapy (SIT). Recombinant hypoallergenic allergen derivatives with reduced allergenic activity have been engineered to reduce side effects during SIT. Parietaria judaica (Pj) pollen contains two major allergens belonging to the family of Lipid Tranfer Proteins (Par j 1 and Par j 2). By means of DNA recombinant technology, a hybrid hypoallergenic (PjEDcys), expressing disulphide bond variants of Par j 1 and Par j 2, was generated. The aim of this research project is to study the immunological mechanisms activated by the major allergens of Parietaria judaica, Par j 1 and Par j 2, and hypoallergenic hybrid rPjEDcys. Moreover, the project I am involved is trying to address the question whether this engineered hypoallergenic derivative can be a potential products for safer Allergen Specific Immunotherapy (SIT). Methods: Par j 1, Par j 2 and PjEDcys were produced as recombinant proteins. Human Peripheral Blood Mononuclear Cell (PBMC) from P. judaica allergic patients were stimulated in vitro with wild-type recombinant allergens and hybrid. PBMC proliferation assay, cytokine secretion assay, magnetic cell sorting of different subset of regulatory T cells, multiparametric flow cytometric analysis and molecular characterization using Real Time-PCR on sorted cells allow to study the biological properties of wild-type recombinant allergens and hybrid hypoallergenic derivate. Results: In vitro analysis suggested that PjEDcys have a reduced allergenity and maintained T cells reactivity. PBMC of P. judaica allergic patients stimulated in vitro with the hybrid and the wild-type recombinant allergens scored a percentage of proliferating CD4+ and CD56+ cell higher than unstimulated sample. Consistent with these data, cytokine secretion assay on CD4+ cells demonstrated that PBMC stimulation with rPjEDcys showed a percentage of IL-5 and IL-13 secreting T CD4+ cells lower than the wild-type allergens. Both rPjEDcys and wild-type stimulation promote the secretion of IFN- \u3b3 and IL-10 by T CD4+ cells. Finally whit the aim to study which subset of regulatory cells respond to wild-tipe allergens and hypoallergenic hybrid new experiment are setting. Discussion: In this experimental setting, the use of the major allergens of Pj and the hybrid polypeptides, rPjEDcys allows me to study the immunological mechanisms activated by the two different antigen stimulation and to investigate differences between the wild-type allergen and the hypoallergenic mutant rPjEDcys. Our data showed that CD4+ cells are clearly the predominant cell population proliferating in response to mixture of Par j 1 and Par j 2 allergens. The hypoallergenic derivate rPjEDcys retain the ability to stimulate CD4+ cells proliferation like the mixture of allergens (rPar j 1 and rPar j 2). Moreover these results highlighted a particular interesting datum; the mixture of allergens and the rPjEDcys hybrid showed the ability to stimulate an innate immune response, inducing CD56+ cells proliferative response. Cytokine secretion assay demonstrate that rPjEDcys reduce the secretion of IL-5 and IL-13, Th2 cytokines with a critical role in the development of allergy, compared to wild-type allergens. This may reflect the different biological function exerted by rPjEDcys. Conclusion: Collectivelly, our findings demonstrate that PjEDcys show a reduced allergenicity but maintained its immunogenicity and maybe it is also capable to regulate and redirect the immune response. These results suggest that PjEDcys represent a useful approach for immunotherapy of allergic disease

The major allergen of the Parietaria pollen contains an LPS-binding region with immuno-modulatory activity

Background: The major allergens in Parietaria pollen, Par j 1 and Par j 2, have been identified as lipid transfer proteins. The family of the Par j 1 allergens is composed of two isoforms, which differ by the presence of a 37 amino acid peptide (Par37) exclusive to the Par j 1.0101 isoform. The goal of this study was to elucidate the biological properties of the Par37 peptide. Methods: In silico analysis, spectrofluorimetric experiments and in vitro cell culture assays were used to identify the biological properties of Par37. In addition, a mouse model of sensitization was used to study the influence of Par37 in the murine immune response. Results: In silico analysis predicted that Par37 displays characteristics of a host defence peptide. Spectrofluorimetric analysis, real-time PCR and ELISA assays demonstrated that Par37 possesses an LPS-binding activity influencing cell signalling in vitro. In RAW264.7 cells, LPS-induced IL-6 and TNF-a transcription and translation were inhibited after preincubation with Par37. Consistent with these data, inhibition of IFN-c secretion was observed in murine spleen cells and in human PBMC. Finally, mice immunized with the two Par j 1 isoforms differing in the presence or absence of the Par37 peptide showed different immunological behaviours in vivo. Conclusions: This study demonstrates that the Par j 1.0101 allergen displays LPSbinding activity due to the presence of a 37 amino acid COOH-terminal region and that this region is capable of influencing cytokine and antibody responses in vitro and in vivo

An Analysis of the Neutralizing Antibodies against the Main SARS-CoV-2 Variants in Healthcare Workers (HCWs) Vaccinated against or Infected by SARS-CoV-2

: Although the anti-COVID-19 vaccination has proved to be an effective preventive tool, "breakthrough infections" have been documented in patients with complete primary vaccination courses. Most of the SARS-CoV-2 neutralizing antibodies produced after SARS-CoV-2 infection target the spike protein receptor-binding domain which has an important role in facilitating viral entry and the infection of the host cells. SARS-CoV-2 has demonstrated the ability to evolve by accumulating mutations in the spike protein to escape the humoral response of a host. The aim of this study was to compare the titers of neutralizing antibodies (NtAbs) against the variants of SARS-CoV-2 by analyzing the sera of recovered and vaccinated healthcare workers (HCWs). A total of 293 HCWs were enrolled and divided into three cohorts as follows: 91 who had recovered from SARS-CoV-2 infection (nVP); 102 that were vaccinated and became positive after the primary cycle (VP); and 100 that were vaccinated with complete primary cycles and concluded the follow-up period without becoming positive (VN). Higher neutralization titers were observed in the vaccinated subjects' arms compared to the nVP subjects' arms. Differences in neutralization titers between arms for single variants were statistically highly significant (p < 0.001), except for the differences between titers against the Alpha variant in the nVP and in VP groups, which were also statistically significant (p < 0.05). Within the nVP group, the number of subjects with an absence of neutralizing antibodies was high. The presence of higher titers in patients with a complete primary cycle compared to patients who had recovered from infection suggested the better efficacy of artificial immunization compared to natural immunization, and this further encourages the promotion of vaccination even in subjects with previous infections

Death, dying and informatics: misrepresenting religion on MedLine

BACKGROUND: The globalization of medical science carries for doctors worldwide a correlative duty to deepen their understanding of patients' cultural contexts and religious backgrounds, in order to satisfy each as a unique individual. To become better informed, practitioners may turn to MedLine, but it is unclear whether the information found there is an accurate representation of culture and religion. To test MedLine's representation of this field, we chose the topic of death and dying in the three major monotheistic religions. METHODS: We searched MedLine using PubMed in order to retrieve and thematically analyze full-length scholarly journal papers or case reports dealing with religious traditions and end-of-life care. Our search consisted of a string of words that included the most common denominations of the three religions, the standard heading terms used by the National Reference Center for Bioethics Literature (NRCBL), and the Medical Subject Headings (MeSH) used by the National Library of Medicine. Eligible articles were limited to English-language papers with an abstract. RESULTS: We found that while a bibliographic search in MedLine on this topic produced instant results and some valuable literature, the aggregate reflected a selection bias. American writers were over-represented given the global prevalence of these religious traditions. Denominationally affiliated authors predominated in representing the Christian traditions. The Islamic tradition was under-represented. CONCLUSION: MedLine's capability to identify the most current, reliable and accurate information about purely scientific topics should not be assumed to be the same case when considering the interface of religion, culture and end-of-life care

Epidemic spread of ST1-MRSA-IVa in a neonatal intensive care unit, Italy.

BACKGROUND: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has recently emerged as an important pathogen in neonatal intensive care units (NICUs). The purposes of this study were to characterize methicillin-resistant isolates from an outbreak in a NICU, to examine the genetic traits and clonality of CA-MRSA, and to review the characteristics and outcomes of the neonatal cases and investigate the routes of entry and transmission of the MRSA outbreak strain in the NICU under study. METHODS: The study NICU practiced an active surveillance program for multidrug-resistant organisms, including weekly cultures for detection of MRSA from nasal swabs among all the admitted neonates. All first isolates from surveillance cultures and all clinical isolates were submitted for susceptibility testing and genotyping. Data from each infant’s medical records were prospectively included in a database, and the clinical features and outcomes of the colonized/infected infants were assessed. RESULTS: A total of 14 infants were colonized or infected by a strain of ST1-MRSA-IVa between April and August 2011. The CA-MRSA strain appeared to have been introduced to the NICU by an infected infant transferred from another hospital. The outbreak was successfully contained by multifaceted infection control interventions. CONCLUSIONS: The results of this study confirm that NICU is a healthcare setting with a critical permeability to CA-MRSA. Active surveillance including molecular typing can help to detect and monitor the spread of antimicrobial drug-resistant organisms, and thus trigger timely control interventions

Panton-Valentine leukocidin positive sequence type 80 methicillin-resistant Staphylococcus aureus carrying a staphylococcal cassette chromosome mec type IVc is dominant in neonates and children in an Algiers hospital

Methicillin-resistant Staphylococcus aureus (MRSA) is a major antimicrobial drug-resistant pathogen causing serious infections. It was first detected in healthcare settings, but in recent years it has also become disseminated in the community. Children and young adults are most susceptible to infection by community-acquired (CA) MRSA strains. In this study 25 MRSA isolates implicated in infections of neonates and children admitted to an Algiers hospital during an 18 month period were characterized by molecular methods including staphylococcal cassette chromosome (SCC) mec typing, PCR amplification of pvl genes, pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Fifteen out of 25 isolates were from hospital-acquired infections. Twenty-four isolates carried SCCmec type IVc and belonged to the sequence type (ST) 80, one isolate carried SCCmec type II and was ST 39. Twenty-two out of 24 ST80-MRSA-IVc isolates carried pvl genes. Our results suggest that the Panton-Valentine leukocidin positive ST80- MRSA-IVc is the dominant MRSA clone causing disease in neonates and children in Algiers

Outbreak of colonizations by extended-spectrum β-lactamase-producing Escherichia coli sequence type 131 in a neonatal intensive care unit, Italy.

BACKGROUND: Extended spectrum β-lactamases (ESBLs) often associated with resistance to aminoglycosides and fluoroquinolones have recently emerged in community-associated Escherichia coli. The worldwide clonal dissemination of E. coli sequence type (ST)131 is playing a prominent role. We describe an outbreak of colonizations by ESBL-producing E. coli (ESBL-E. coli) in the neonatal intensive care unit (NICU) of the University Hospital, Palermo, Italy. METHODS: An epidemiological investigation was conducted with the support of molecular typing. All children admitted to the NICU and colonized by ESBL-E. coli between January and June 2012, were included in the study. Cases were defined as infants colonized by E. coli resistant to third generation cephalosporins and fluoroquinolones. A case–control study was also performed to identify possible risk factors. RESULTS: During the outbreak period, 15 infants were found to be colonized by ESBL-E. coli. The epidemic strain demonstrated continuous transmission throughout the outbreak period. Case–control study identified a lower birth weight as the only risk factor for colonization. The strain belonged to the sequence-type 131 community-associated clone. Transmission control interventions, including contact precautions and cohorting, restriction of the new admissions, sanitization of surfaces and equipment and targeted training sessions of the NICU staff, were successful in interrupting the outbreak. CONCLUSIONS: Although invasive infections did not develop in any of the 15 colonized neonates, our report highlights the need to strictly monitor the spill in the NICU setting of multidrug resistant community-associated organisms. Our findings confirm also the role of active surveillance in detecting the silent spread of ESBL-producing Gram negatives in a critical healthcare setting and trigging the implementation of infection control measures. As β-lactam and fluoroquinolone resistant E. coli strains are increasingly spreading in the community, this event could become a more serious challenge

Successful control of an outbreak of colonization by Klebsiella pneumoniae carbapenemase-producing K.\ua0pneumoniae sequence type 258 in a neonatal intensive care unit, Italy

This article reports an outbreak of colonization by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) sequence type (ST) 258 in a neonatal intensive care unit (NICU) in Palermo, Italy. KPC-Kp ST258 was detected by an active surveillance culture programme. Between 18th September and 14th November 2012, KPC-Kp was isolated from 10 out of 54 neonates admitted in the outbreak period. No cases of infection were recorded. Male sex was associated with colonization, whereas administration of ampicilline-sulbactam plus gentamicin was protective. Infection control interventions interrupted the spread of KPC-Kp without the need to close the NICU to new admissions. (C) 2013 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved