Insect antimicrobial peptides: potential weapons to counteract the antibiotic resistance.

Misuse and overuse of antibiotics have contributed in the last decades to a phenomenon known as antibiotic resistance which is currently considered one of the principal threats to global public health by the World Health Organization. The aim to find alternative drugs has been demonstrated as a real challenge. Thanks to their biodiversity, insects represent the largest class of organisms in the animal kingdom. The humoral immune response includes the production of antimicrobial peptides (AMPs) that are released into the insect hemolymph after microbial infection. In this review, we have focused on insect immune responses, particularly on AMP characteristics, their mechanism of action and applications, especially in the biomedical field. Furthermore, we discuss the Toll, Imd, and JAK-STAT pathways that activate genes encoding for the expression of AMPs. Moreover, we focused on strategies to improve insect peptides stability against proteolytic susceptibility such as D-amino acid substitutions, N-terminus modification, cyclization and dimerization.

Evaluation of PD1 and PD-L1 expression in high-grade sarcomas of the limbs in the adults: possible implications of immunotherapy.

Sarcomas are a heterogeneous group of rare tumours. Improvements in immunotherapy and the important role of PD1 and PD-L1 expression in advancement and prognosis have opened new fields of research for the treatment of these neoplasia. We evaluated the immunohistochemistry of PD1 and PD-L1 expression in 60 adults' patients affected by high-grade sarcomas of the limbs. PD1 expression was 65% while PD-L1 was 68.3%. PD-L1 expression seems to correlate to Ki67 in liposarcomas, fibrosarcoma's and pleomorphic sarcomas, while it does not show any correlation to chondrosarcomas, while in rhabdomyosarcomas there is a correlation but, given the small sample size, it was not possible to perform a statistic analysis. Our study shows positivity among the different subgroups of positive PD1 lymphocytes infiltration and PD-L1 expression in high-grade sarcomas of the limbs.

The predictive value of CSF multiple assay in multiple sclerosis: A single center experience.

BACKGROUND: Multiple sclerosis (MS) is a chronic, immune-mediated, inflammatory, neurodegenerative disorder. Many studies are investigating the potential role of body fluid biomarkers as prognostic factors for early identification of patients presenting with clinical isolated syndrome (CIS) at high risk for conversion to MS or to recognize RRMS patients at high risk for progression. OBJECTIVES: To evaluate the correlation between levels of BAFF, chitinase 3-like 1 (CHI3L1), sCD163, Osteopontin (OPN), both on serum and cerebral spinal fluid (CSF), and the disease activity and progression. We also want to explore a possible relationship between serological and CSF biomarker's levels. PATIENTS AND METHODS: We enrolled 82 patients between June 2014 and June 2016. Seventy-one received a diagnosis of demyelinating disease of CNS (46 RRMS and 25 CIS), while 11 were affected by other neurological diseases. All patients underwent a neural axis MRI, lumbar puncture and blood samples. Levels of BAFF, CHI3L1, sCD163, OPN on serum and CSF were analyzed by Luminex xMAP system, with a kit 11-plex ad hoc. RESULTS: The CSF CHI3L1, sCD163 and OPN levels were significantly higher in MS patients than in controls. We did not find significant differences in serum CHI3L1, sCD163 and OPN levels, nor CSF or serum BAFF levels between patient and control groups. We found significantly higher CSF level of sCD163 and CHI3L1 in all patients' subgroups compared with controls, while OPN was higher in CIS and RR subgroups. We did not find significant differences for serum and CSF levels of all the markers between patients with or without clinical or radiological disease activity. CSF sCD163 and CHI3L1 levels was significant higher in CIS patients who converted to MS (p < 0.05). Using ROC curve analysis, CSF sCD163 resulted the best predictive factor. CSF CHI3L1 and OPN levels resulted useful independent predictors too. Combined ROCs of those three analytes demonstrated a better predictive value, with sCD163 and CHI3L1 resulting as the best combination. CONCLUSIONS: CSF sCD163 CHI3L1 and OPN levels were higher in MS patients whereas serum CHI3L1, sCD163 and OPN levels did not show differences compared with controls. This finding confirms the high CSF specificity with regards to the analysis of processes, inflammatory and non-inflammatory, that occur within the CNS.

IFN-γ and other serum cytokines in head and neck squamous cell carcinomas.

SUMMARY: Altered immune responses have been reported in head and neck cancer, and some of these responses have been associated with poor clinical outcomes. A multiple-array technology platform was used to simultaneously evaluate the levels of 25 cytokines. Pre-treatment serum levels were evaluated in 31 HNSCC patients and 6 healthy controls. The levels of 8 cytokines, specifically IL-1ra, IL-2, IL-5, IL-6, IL-8, IL-17, IFN-γ and IP-10, were significantly higher in patients than in controls. Among cancer patients we observed lower levels of IFN-γ and IL-7 in cases with nodal metastases compared to those with cN0 disease. We observed increases in the levels of some serum cytokines in HNSCC patients, as well as reductions in selected cytokines associated with regional progression. These findings provide an intriguing perspective on the development and validation of novel markers for follow-up evaluations and predictions of regional spreading in HNSCC patients.

Palladium nanoparticle effects on endocrine reproductive system of female rats.

The widespread industrial application of nanomaterials (NMs) has dramatically increased the likelihood of environmental and occupational exposure of humans to such xenobiotics. This issue, together with the increasing public health interest in understanding the effects of chemicals on endocrine system, encouraged to investigate the disruptive potential of NMs on the endocrine function. Therefore, the aim of this study was to evaluate the effects of palladium nanoparticles (Pd-NPs) on the female reproductive system of Wistar rats, intravenously exposed to different doses (0.12, 1.2, and 12 µg/kg), through the assessment of possible quantitative changes in the serum concentrations of several sex hormones. Our results demonstrated that the highest exposure doses significantly reduced the estradiol and testosterone concentrations, while increased the luteinizing hormone levels in treated animals compared to controls. Such alterations are indicative for an abnormal reproductive axis function. However, further investigations are needed to clarify the role of the different NP physicochemical properties in determining such effects, and possible underlining molecular mechanisms, as well as their relevance for the development of diseases in the female reproductive system. Overall, this may be helpful to define accurate risk assessment and management strategies to protect the health of the general and occupational populations exposed to Pd-NPs.

Subchronic exposure to palladium nanoparticles affects serum levels of cytokines in female Wistar rats.

Recently, palladium nanoparticles (PdNPs) have been increasingly used in many industrial sectors, and this has led to a significant release of nano-sized palladium particles into the environment. However, despite the increase in occupational and general population exposure, information on the potential adverse effects of these PdNPs is still limited and their impact on the immune system constitutes a major health concern. Therefore, the aim of this study was to investigate the potential adverse effects induced by subchronic intravenous administration of PdNPs on the immune system of female Wistar rats by evaluating alterations in Interleukin (IL)-1α, IL-2, IL-4, IL-6, IL-10, IL-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), Interferon (INF)-γ, and Tumor Necrosis Factor (TNF)-α serum levels. Exposed and control animals were randomly divided into five groups (0, 0.012, 0.12, 1.2, and 12 µg PdNPs per kg body weight) which were treated with repeated intravenous injections of vehicle or PdNPs (on day 1, 30, and 60). Subchronic exposure to PdNPs induced a decreasing trend in serum levels in most of the cytokines investigated, with the highest concentration (12 µg/kg) determining significant inhibitory effects. Overall, these results showed that PdNPs are able to alter cytokine serum levels in subchronically treated Wistar rats, suggesting a possible impact of these xenobiotics on the immune system after long-term exposures.

Soft tissue adhesion patterns over Trevira tube on modular endoprosthesis for malignant bone tumours: an in vitro study.

A reliable and effective technique in case of limb salvage surgery after resection of extensive bone tumors is represented by the implant of modular or custom-made megaprosthesis. Fixation of the residual surrounding soft tissue on the implant represent a challenge for the surgeon and the use of a polyethylene terephthalate (PET) tube over it, also known as Trevira, is currently a common choice for reattachment with good clinical outcomes. We compared fibroblastic cell culture potential over simple titanium coating vs titanium surrounded by Trevira and evaluated cell viability and replication at 24, 48 and 72 h using MTT cell growth assay and scanning electron microscopy to determine if there was any difference in the potential of cell growth associated to the material used. No significant difference was found at different timings in terms of total cell count for cultures over the two materials, but the absolute cell count was slightly higher in the Trevira group in the early time points, reversing the trend at 72 h of incubation. Ninety-four % of the cells analyzed were vital, regardless of the materials involved in the experiment, confirming the biocompatibility of titanium and PET. According to the results shown, we are able to confirm the in vitro safety and efficacy, in terms of newly formed cells extension and adhesion pattern, of using an attachment tube made from Trevira fibers surrounding an oncological megaprosthesis in order to achieve the most anatomical reinsertion of remaining soft tissue following resection.

In vitro evaluation of the potential toxic effects of palladium nanoparticles on fibroblasts and lung epithelial cells.

Palladium nanoparticles have been increasingly used in catalytic processes, wastewater treatment, electronics, and biomedicine. However, recent evidence proved that these nanoparticles are able to induce adverse effects both in in vitro and in vivo models. Nevertheless, molecular mechanisms underlying the toxic effects are still poorly understood. Therefore, this study aimed to investigate the potential toxicological mechanisms of palladium nanoparticles assessing their effects on normal diploid rat fibroblast and lung carcinoma human epithelial cell lines. Several endpoints such as cell growth, cell cycle progression, DNA damage, induction of apoptosis, reactive oxygen species production and expression of cell cycle regulatory proteins were evaluated. Results showed that palladium nanoparticles inhibited cell growth in a dose- and time-dependent manner in both cell lines, although with a more evident action on fibroblasts. Interestingly, inhibition of cell growth was not associated with the induction of apoptosis. Cell cycle progression was arrested in the G0/G1 phase and DNA damage was evident in both cell lines even if only a slight increase in the intracellular reactive oxygen species levels was detected. These findings provide valuable insight into understanding the molecular mechanisms responsible of palladium nanoparticles toxicity whose identification is essential to define an adequate risk assessment process.

Immunopathological characterization of cryptoglandular anal fistula: a pilot study investigating its pathogenesis.

AIM: The pathogenesis of cryptoglandular anal fistula (AF) is still under debate. Tissue inflammation could play a primary role. The pathological process of epithelial mesenchymal transition (EMT) might be involved but has never been investigated. METHOD: In a prospective pilot study, 12 patients with an AF had a fistulectomy. The excised track was divided into proximal (intrasphincteric) and distal (extrasphincteric) parts which were subjected to standard histopathological examination. The cytokines IL-8 and IL-1beta were analysed as markers of inflammation, while EMT was evaluated by expression of TGF-beta, Vimentin, Zeb-1, Snail and E-cadherin. The mRNA and protein expression of these molecules was investigated by real-time PCR (RT-PCR), Western blot analysis and immunohistochemistry and was compared with that of the normal adjacent tissue. RESULTS: Chronic inflammation and granulation tissue and a stratified epithelium were evident on standard histopathological examination. The cytokine IL-8 was more expressed in the proximal than the distal part of the track (fold increase 4.34 vs 3.60), while the reverse was found for IL-1beta (fold increase 1.33 vs 2.01); both were more intensely expressed compared with the normal anal mucosa. EMT was demonstrated, in both proximal and distal parts of the track, with an increase of TGF-beta, Vimentin, Zeb-1 and Snail and a mean decrease of E-cadherin. Western blot analysis and immunohistochemistry confirmed the protein expression. CONCLUSION: The study suggests that chronic inflammation is present in cryptoglandular fistulas. The inflammatory pattern might be different in the proximal than in the distal part of the fistula track. The cytokines IL-1beta and IL-8 could play a possible role in fistula formation. The study demonstrates for the first time the potential importance of EMT in the pathogenesis of cryptoglandular AF.

Mechanical and structural comparison between primary tumor and lymph node metastasis cells in colorectal cancer.

SW480 and SW620 colon carcinoma cell lines derive from primary tumour and lymph-node metastasis of the same patient, respectively. For this reason, these cells represent an ideal system to analyse phenotypic variations associated with the metastatic process. In this study we analysed SW480 and SW620 cytoskeleton remodelling by measuring the cells' mechanics and morphological properties using different microscopic techniques. We observed that different specialized functions of cells, i.e. the capacity to metastasize of elongated cells inside the primary tumour and the ability to intravasate and resist shear forces of the stream of cells derived from lymph node metastasis, are reflected in their mechanical properties. We demonstrated that, together with stiffness and adhesion between the AFM tip and the cell surface, cell shape, actin organization and surface roughness are strictly related and are finely modulated by colorectal cancer cells to better accomplish their specific tasks in cancer growth and invasion.

TNF-alpha gene polymorphisms can help to predict response to etanercept in psoriatic patients.

BACKGROUND: Genetic factors might have a role for lack of therapeutic response to anti-TNF-alpha agents, as previously suggested in patients with rheumatoid arthritis and inflammatory bowel disease. OBJECTIVES: We evaluated the role of the main TNF-alpha polymorphisms (-238G>A, -308G>A, -857C>T) in predicting the response to etanercept, an anti-TNF-alpha fusion protein. MATERIAL AND METHODS: Genomic DNA was extracted from buccal epithelial cells in a series of 97 psoriatic patients who received etanercept for at least 3 months. Patients were classified as responders, if they achieved a PASI improvement ≥ 75% after 12 weeks of etanercept treatment, and non-responders, if PASI improvement was <75%. Single-nucleotide polymorphisms (SNPs) in TNF-alpha gene (-238G>A, -308G>A, -857C>T) were genotyped by PCR restriction fragment length polymorphism (RFLP) assays. RESULTS: We found that patients heterozygous (GA) for the -238G>A polymorphism were more likely not responsive to therapy compared to the GG genotype. In fact, the GA genotype was found in 5/59 (8.5%) responders and in 14/38 (36.8%) non-responders (P = 0.001). A significant relationship with therapy was also observed for the -308G>A polymorphisms. In fact, the GG, GA and AA genotypes were detected in 48 (81.4%), 9 (15.3%) and 2 (3.4%) of the 59 responders and in 22 (57.9%), 11 (28.9%) and 5 (13.2%) of the 38 non-responder patients (P = 0.03). No association with therapy was observed for the -857C>T polymorphisms. CONCLUSION: Our study supports the role of TNF-alpha polymorphisms in predicting the response to anti-TNF-alpha agents. In particular, we found that the presence of -238G>A and -308G>A polymorphisms is associated with poor response to a 3-month therapy with etanercept. However, our data have yet to be validated in larger cohorts.

Circulating endothelial cell levels in psoriatic patients and their modification after an anti-TNF-alpha (Etanercept) treatment.

BACKGROUND: Endothelial function in psoriatic patients has been mainly evaluated through a high-resolution ultrasound measurement of flow-mediated vasodilation in the brachial artery, which is an operator-dependent and technically demanding technique: this characteristic, together with different patient selection criteria, could account for the conflicting results emerging from different studies. Recently, Circulating Endothelial Cells (CECs) level has been suggested as a novel biomarker of vascular injury. METHODS: The number of CECs was determined by a semi-automated immunomagnetic system (CellSearch system) in peripheral blood of psoriatic patients (n = 48) and healthy subjects (n = 50). In 15 patients, CEC level was also evaluated after 6 months of treatment with an anti-TNF-alpha agent, Etanercept. The plasma levels of high-sensitivity C-reactive Protein (CRP), E-selectin, VEGF and PAI-1 were measured by ELISA. The psoriasis severity was assessed by PASI score. RESULTS: A statistically significant difference (P = 0.001) was found between CEC level in psoriatic patients (10.6 ± 9.4 cells/mL) vs. the control group (3.9 ± 0.9 cells/mL). This count inversely correlated with sE-selectin levels (r(2) = 0.16; P = 0.03). After 6 months of therapy, patients experienced a significant (P < 0.05) decrease in CEC levels (3.4 ± 1.3 cells/mL) and in PASI score (from 11.7 ± 8.1 to 2.1 ± 4.0). CONCLUSIONS: The elevated CECs level that we found in a sample of high selected psoriatic patients could be expression of endothelial damage. Lowering of CECs count after treatment with Etanercept support the hypothesis that an effective systemic therapy of psoriasis may also improve the endothelial function.

Pre- and posttherapy assessment of intestinal soluble mediators in IBD: where we stand and future perspectives.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition characterized by an abnormal immune response against food or bacterial antigens in genetically predisposed individuals. Several factors of innate and adaptive immune system take part in the inflammatory process, probably actively contributing in endoscopic and histological healing at molecular level. Although it is difficult to discriminate whether they are primary factors in determining these events or they are secondarily involved, it would be interesting to have a clear map of those factors in order to have a restricted number of potentially "good candidates" for mucosal healing. The present review will present a class of these factors and their modulation in course of therapy, starting from pathogenic studies involving several treatments associated with good clinical outcomes. This approach is meant to help in the difficult task of identifying "good candidates" for healing signatures, which could also be possible new therapeutic targets for clinical management of IBD patients.

Intestinal gas production and gastrointestinal symptoms: from pathogenesis to clinical implication.

Intestinal gases are the expression of metabolic activity of gut microbiota in the gut, particularly carbohydrates in the case of H2, CH4. Alterations in composition of gases and air handling, directly or upon challenge with food are relevant for GI and extra-GI diseases. Assessing gas composition in breath can be a very useful tool for clinic, but technical issues are crucial (breath sampling, storing and analyzing). Aim of the present review is to summarize the understanding of the importance of intestinal gases in gastro-intestinal physiology and patho-physiology. Practical considerations on how to collect samples and instruments available for the clinic have also been provided.

The role of EGFR tyrosine kinase inhibitors in the first-line treatment of advanced non small cell lung cancer patients harboring EGFR mutation.

Lung cancer continues to be the leading cause of cancer death worldwide. Among lung cancers, 80% are classified as nonsmall- cell lung cancer (NSCLC) and are mostly diagnosed at an advanced stage (either locally advanced or metastatic disease). In the last years, the discovery of the pivotal role in tumorigenesis of the Epidermal Growth Factor Receptor (EGFR) has provided a new class of targeted therapeutic agents: the EGFR tyrosine kinase inhibitors (EGFR-TKIs). Since the first reports of an association between somatic mutations in EGFR exons 19 and 21 and response to EGFR-TKIs, treatment of advanced NSCLC has changed dramatically. Histologic profile, clinical characteristics, and mutational profile of lung carcinoma have all been reported as predictive factors of response to EGFR-TKIs and other targeted therapies. In advanced NSCLC patients harboring EGFR mutations, the use of EGFR TKIs in first-line treatment has provided an unusually large progression-free survival (PFS) benefit with a negligible toxicity when compared with cytotoxic chemotherapy in phase III randomized trials. Considering the findings regarding the excellent benefit and better safety profile of EGFR TKIs in EGFR mutation positive patients, these targeted therapeutic agents can be now considered as first-line treatment in this setting of patients. This review will discuss the new evidences in the role of EGFR-TKIs in the first-line treatment of advanced NSCLC and their implication in the current clinical decision-making.

Excised melanocytic lesions in children and adolescents - a 10-year survey.

BACKGROUND: Early recognition and prompt excision is to date the only available strategy for reducing mortality from melanoma. Little is known about the accuracy of melanoma detection in children and adolescents. OBJECTIVES: To assess the accuracy of melanoma detection in a paediatric population. METHODS: From the Department of Dermatology, Medical University of Graz, Austria, we reviewed the dermatopathology reports of naevi and melanomas excised in patients younger than 20 years over a 10-year period (1998-2007). Patients were subdivided into four age groups: 0-4, 5-9, 10-14 and 15-19 years. RESULTS: Accuracy in melanoma detection was tested using the number needed to excise (NNE) value that is obtained by dividing the total number of excised lesions by the number of melanomas. A total of 22564 lesions were reviewed, disclosing 22526 naevi and 38 melanomas, for an overall NNE value of 593.8. Five melanomas were excised in children aged 10-14 years (NNE 1141) and 33 in children aged 15-19 years (NNE 479.8), whereas no melanomas were found among 1026 lesions excised in children younger than 10 years. In children aged 0-4 years, congenital and Spitz/Reed naevi accounted for 34.5% and 20% of lesions, respectively. These percentages decreased progressively when moving to older age groups (P<0.0001). In contrast, the percentage of dermal and compound naevi rose in direct proportion with age, being 3.4% and 20.7%, respectively, in the youngest age group, and 36.7% and 31.9%, respectively, among the oldest patients (P<0.0001). CONCLUSIONS: The overall NNE value in paediatric patients over the 10-year study period was 593.8, meaning that about 594 lesions were excised to find one melanoma. This value is 20 times higher than the rates found in adult patients.

Low prevalence of human papillomavirus infection in the healthy oral mucosa of a Northern Italian population.

BACKGROUND: Oral cancer is the sixth most common malignancy in developed countries, representing almost 3% of malignant tumors. Tobacco use and alcohol consumption are well-established risk factors. However, the observation that most patients with oral cancer have not been exposed to these risk factors suggests that additional causes may promote oral carcinogenesis. A link has been suggested between human papillomavirus (HPV) and oral cavity cancer but the significance of HPV contribution to oral carcinogenesis as well as the prevalence of HPV infection in normal oral cavity mucosa remains debated. METHODS: In this study, the prevalence of oral HPV infection was evaluated in 81 randomly selected Northern Italian subjects with clinically normal oral mucosa using a nested PCR on DNA extracted by oral smears. RESULTS AND CONCLUSIONS: No HPV-related lesions were detectable in any of the smears analyzed by cytological approach. nPCR identified HPV DNA in only one (1.2%) of the specimens obtained from clinically healthy oral mucosa and subsequent characterization assigned the positive case to HPV type 90. These data suggest that the incidence of HPV infection in the healthy population might be very low and that other risk factors are likely responsible to promote oral carcinogenesis.

Zirconia toughened alumina (ZTA) powders: ultrastructural and histological analysis.

Ceramic materials, as Alumina and Zirconia, has made an improvement in the choice of new biomaterials for the load bearing application in dental and orthopaedic implants. These materials has shown mechanical resistance to high stress related to weight bearing and low debris in time. For this reason they are indicated on young patients implant, with high demanding activities and long life expectance. In literature however the risk of chronic inflammation due to chronic wear debris release and the possibility of carcinogenesis, is still to be definitively investigated. Another point to investigate is the acute reaction of the tissue in case of acute release of powders of these materials. The aim of this study was to investigate the possible local and systemic acute effects of ceramic precursors in form of powders of different size when released into articular joint. Powders of ZTA were implanted in the knee joint of twenty-four New Zealand white adult rabbits, that were sacrificed at 1,3,6, and 12 months. Radiographic, histological and immunoistochemestry analysis were conducted on periprosthetic tissue and peripheral organs, to verifying local host response and systemic toxic effects.