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.

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.

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.

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.

Post-translational modulation of CD133 expression during sodium butyrate-induced differentiation of HT29 human colon cancer cells: implications for its detection.

The CD133 molecule has been proposed as a surface marker of cancer stem cells in several human malignancies, including colon cancers. The function and the mechanisms regulating CD133 expression remain unknown. The HT29 human colon cancer cells undergo differentiation following treatment with various agents and represent a useful in vitro model of colon differentiation. This study evaluated the behavior of CD133 during sodium butyrate-induced differentiation of HT29 cells. Treatment with sodium butyrate induced a progressive decrease of CD133 expression, as assessed by flow cytometry using the AC133 monoclonal antibody. Indeed, expression of CD133, which was about 47% in untreated control cells, gradually decreased down to about 3% after 72 h in a time- and dose-dependent manner. No relationship was observed between CD133 protein evaluated by flow cytometry and mRNA expression level, and no changes were detected in the methylation status of the CD133 gene promoter during HT29 differentiation. Moreover, the expression of the CD133 protein, evaluated by Western blot analysis using a specific anti-CD133 antibody directed against the C-terminal intracytoplasmic region of human CD133 protein, did not correlate with flow cytometry results. Different results were also obtained using the two antibodies to analyze the expression of the CD133 molecule in human colon cancers. These findings demonstrate that membrane expression of the CD133 stem cell marker might undergo a complex regulation during differentiation of colon cells and suggest that HT29 cells are a useful in vitro model to study the mechanisms involved in this regulation which likely occurs at a post-transcriptional level.

In vivo characterization of Zirconia Toughened Alumina material: a comparative animal study.

The development of a new chromia-doped Zirconia Toughened Alumina (ZTA) material was previously reported as displaying mechanical properties suitable for implants with load bearing applications, such as orthopaedic and dental implants. This type of biomaterial is expected to be in contact with living tissues for a long period of time and its long-term toxicity must be carefully evaluated. In this study the suitability of this ZTA material as a candidate biomaterial for orthopaedic implants and dental devices was further investigated in vivo in comparison to alumina and zirconia, which are currently used in orthopaedic and dental surgery. Cylinders of the materials were implanted in vivo in white rabbits, and local and systemic tissue reactions were analyzed at different time intervals after surgery. Radiologic examinations displayed the absence of radiolucence around cylinders and no signs of implant loosening up to twelve months. No tumours developed in the animals either locally (at the site of implantation), or systemically in the peripheral organs. The results obtained suggest that this new ZTA material does not display any long term pathogenic effect in vivo. These findings extend our previous observations on the biocompatibility and the absence of any long-term carcinogenic effect in vitro of this material which displays interesting properties for biomedical applications. In conclusion, we report the in vivo characterization of a new chromia-doped ZTA material and confirm its suitability as a candidate biomaterial for orthopaedic implants and dental devices since it does not give any local nor systemic toxicity even after a long period of time after implantation.

Inflammation and cancer: a multifaceted link.

STATE OF THE ART: Mounting evidence indicates a link between inflammation and cancer. However, the molecular mechanism(s) remains unclear. Indeed, although preclinical and clinical studies suggest that chronic inflammation can promote cancer development, the role(s) of inflammation in the process is likely very complex and far to be completely understood. Inflammation can promote all stages of tumor development through multiple mechanisms which include enhanced proliferation and resistance to apoptosis of initiated cells, induction of DNA mutations, promotion of angiogenesis, invasion and metastasis. On the other hand, components of tumor microenviroment, including tumor cells themselves, may promote an inflammatory state by producing inflammatory mediators. Moreover, while chronic inflammation might promote tumor formation, acute inflammation might well hamper the process and is indeed used therapeutically to inhibit tumor formation. CONCLUSIONS: The present review briefly highlights the relationship between inflammation and tumorigenesis and discusses the possibility to develop chemoprevention and/or therapeutical approaches targeting components of the inflammatory responses.

Development of a new zirconia-toughened alumina: promising mechanical properties and absence of in vitro carcinogenicity.

High purity alumina as well as zirconia ceramics have been widely used as orthopaedic implant biomaterials and dental devices displaying optimal, but sometimes exclusive, mechanical properties. In order to combine the advantages of alumina and zirconia ceramic materials different types of composites have been developed in which either zirconia is dispersed in an alumina matrix or vice versa. Orthopaedic and dental implant biomaterials are expected to be in contact with living tissues for a long period of time and their long term toxicity must be carefully evaluated. In this study we report the development of a high performance chromia-doped zirconia toughened alumina (ZTA) material which displays promising mechanical properties in terms of hardness, strength and fracture toughness that make it suitable for prosthesis even for small joints. The long-term biocompatibility of this material was also evaluated, mainly in terms of DNA damage, mutagenicity and cancerogenetic potential in mammalian cells. The results obtained suggest that this new ZTA material does not display any longterm carcinogenic effect and it is suitable for biomedical applications from a cancerogenetic point of view. In conclusion, we report the development of a new chromia-doped ZTA material with interesting properties, both from a mechanical and a biocompatibility point of view which warrant further studies on its suitability as a candidate biomaterial for orthopaedic implants and dental devices.

Isolation and characterization of CD133+ cell population within human primary and metastatic colon cancer.

BACKGROUND: "Cancer stem cells" (CSC) have been identified as a minority of cancer cells responsible for tumor initiation, maintenance and spreading. Although a universal marker for CSC has not yet been identified, CD133 has been proposed as the hallmark of CSC in colon cancer. The aim of our study was to assess the presence of a CD133+ cell fraction in samples of colon cancer and liver metastasis from colon cancer and evaluate their potential as tumor-initiating cells. METHODS: Tissue samples from 17 colon cancers and 8 liver metastasis were fragmented and digested using collagenase. Cell suspensions were characterized by flow cytometry using anti-CD133, CD45 and CD31 antibodies. CD133+ cells were also isolated by magnetic cell sorting and their tumor-initiating potential was assessed versus the remaining CD133- fraction by soft-agar assay. RESULTS: Our results confirmed the existence of a subset of CD133+ tumor cells within human colon cancers. Interestingly, CD133+ cells were detectable in liver metastasis at a higher percentage when compared to primary tumors. Soft-agar assay showed that CD133+ cell fraction was able to induce larger and more numerous colonies than CD133-cells. CONCLUSION: Our findings data that the CD133+ colon cancer cells might play an important role in both primary tumors as well as in metastatic lesions thus warranting further studies on the role(s) of this subset of cells in the metastatic process.

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.

Dermoscopic monitoring of melanocytic skin lesions: clinical outcome and patient compliance vary according to follow-up protocols.

BACKGROUND: Dermoscopic monitoring of melanocytic lesions increases the likelihood that featureless melanomas are not overlooked and minimizes the excision of benign lesions. Objective To examine clinical outcome and patient compliance using different follow-up protocols. METHODS: A retrospective analysis of 600 lesions from 405 patients (aged 6-79 years) was performed to examine patient compliance and clinical outcome in patients with multiple atypical melanocytic lesions undergoing sequential dermoscopy imaging during short-, medium- or long-term follow-up. Based on the degree of dermoscopic atypical features, patients were scheduled for short-term monitoring with follow-up after 3 months, medium-term monitoring with follow-up after 6 months or long-term monitoring with annual follow-up. Criteria leading to excision of monitored lesions differed according to the follow-up protocol. RESULTS: In a median follow-up period of 23 months, 54 (9%) lesions were excised, revealing 12 early melanomas (occurring in 3% of monitored patients), one basal cell carcinoma and 41 melanocytic naevi. The melanoma/benign ratio of excised lesions was 1 : 3.4. Seven of 12 melanomas showed changes after two to four visits, corresponding to 8-54 months of follow-up. Patient compliance was 84% for short-term monitoring, 63% for medium-term monitoring and 30% for long-term monitoring. CONCLUSIONS: In patients with multiple naevi sequential dermoscopy imaging is a useful strategy to avoid missing melanomas while minimizing unnecessary excision of benign lesions. For better compliance, the first re-examination should be scheduled at 3 months after the baseline visit. Regular annual follow-up monitoring is also needed to detect slow-growing melanomas in which subtle changes may become apparent only over time.