A loss-of-function NCSTN mutation associated with familial Dowling Degos disease and hidradenitis suppurativa.

Dowling Degos disease (DDD) is a rare autosomal dominant genodermatosis characterized by acquired, slowly progressive reticulated pigmented lesions primarily involving flexural skin areas. Mutations in KRT5, POGLUT-1 and POFUT-1 genes have been associated with DDD, and loss-of-function mutations in PSENEN, a subunit of the gamma-secretase complex, were found in patients presenting with DDD or DDD comorbid with hidradenitis suppurativa (HS). A nonsense mutation in NCSTN, another subunit of the gamma-secretase, was already described in a patient suffering from HS and DDD but whether NCSTN could be considered a novel gene for DDD is still debated. Here, we enrolled a four-generation family with HS and DDD. Through Whole Exome Sequencing (WES) we identified a novel nonsense mutation in the NCSTN gene in all the affected family members. To study the impact of this variant, we isolated outer root sheath cells from patients' hair follicles. We showed that this variant leads to a premature stop codon, activates a nonsense-mediated mRNA decay, and causes NCSTN haploinsufficiency in affected individuals. In fact, cells treated with gentamicin, a readthrough agent, had the NCSTN levels corrected. Moreover, we observed that this haploinsufficiency also affects other subunits of the gamma-secretase complex, possibly causing DDD. Our findings clearly support NCSTN as a novel DDD gene and suggest carefully investigating this co-occurrence in HS patients carrying a mutation in the NCSTN gene.

A rare loss-of-function genetic mutation suggest a role of dermcidin deficiency in hidradenitis suppurativa pathogenesis.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a multifactorial aetiology that involves a strict interplay between genetic factors, immune dysregulation and lifestyle. Familial forms represent around 40% of total HS cases and show an autosomal dominant mode of inheritance of the disease. In this study, we conducted a whole-exome sequence analysis on an Italian family of 4 members encompassing a vertical transmission of HS. Focusing on rare damaging variants, we identified a rare insertion of one nucleotide (c.225dupA:p.A76Sfs*21) in the DCD gene encoding for the antimicrobial peptide dermcidin (DCD) that was shared by the proband, his affected father and his 11-years old daughter. Since several transcriptome studies have shown a significantly decreased expression of DCD in HS skin, we hypothesised that the identified frameshift insertion was a loss-of-function mutation that might be associated with HS susceptibility in this family. We thus confirmed by mass spectrometry that DCD levels were diminished in the affected members and showed that the antimicrobial activity of a synthetic DCD peptide resulting from the frameshift mutation was impaired. In order to define the consequences related to a decrease in DCD activity, skin microbiome analyses of different body sites were performed by comparing DCD mutant and wild type samples, and results highlighted significant differences between the groins of mutated and wild type groups. Starting from genetic analysis conducted on an HS family, our findings showed, confirming previous transcriptome results, the potential role of the antimicrobial DCD peptide as an actor playing a crucial part in the etio-pathogenesis of HS and in the maintenance of the skin's physiological microbiome composition; so, we can hypothesise that DCD could be used as a novel target for personalised therapeutic approach.

Nucleolin Targeting by N6L Inhibits Wnt/ß-Catenin Pathway Activation in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and resistant cancer with no available effective therapy. We have previously demonstrated that nucleolin targeting by N6L impairs tumor growth and normalizes tumor vessels in PDAC mouse models. Here, we investigated new pathways that are regulated by nucleolin in PDAC. We found that N6L and nucleolin interact with ß-catenin. We found that the Wnt/ß-catenin pathway is activated in PDAC and is necessary for tumor-derived 3D growth. N6L and nucleolin loss of function induced by siRNA inhibited Wnt pathway activation by preventing ß-catenin stabilization in PDAC cells. N6L also inhibited the growth and the activation of the Wnt/ß-catenin pathway in vivo in mice and in 3D cultures derived from MIA PaCa2 tumors. On the other hand, nucleolin overexpression increased ß-catenin stabilization. In conclusion, in this study, we identified ß-catenin as a new nucleolin interactor and suggest that the Wnt/ß-catenin pathway could be a new target of the nucleolin antagonist N6L in PDAC.

Photobiomodulation as potential novel third line tool for non-invasive treatment of hidradenitis suppurativa.

Hidradenitis Suppurativa (HS) is a severe inflammatory pathology of the skin characterized by chronic recurrent inflamed lesions, nodules, sinus tracts and abscesses usually manifests after puberty, which involves scalp, neck, axillae, perineum and infra-mammary areas. Nowadays treatment options range from short or long courses of antibiotics, anti-inflammatory and biologic drugs, to surgery. Other suggested treatments consider the employment of laser devices, mainly microsurgical lasers (such as CO2 and intense pulsed lasers) and photodynamic therapy. This review explores the potential use of photobiomodulation (PBM), already used for the treatment of other skin conditions, such as acne, hypertrophic scars, wrinkles, and burns, as potential novel therapy for HS. PBM has been reported to have beneficial effects on promoting wound healing, angiogenesis, vasodilation, and relieving from pain and inflammation, as recently demonstrated in an in-vitro model mimicking HS disease. In addition, PBM, specifically set at the blue wavelength, has been recently reported as exerting an anti-bacterial activity. Therefore, considering all these PBM features especially its ability to decrease pain and inflammation and to lead to faster wound healing, thus improving patients' quality of life, we hypothesize its employment as adjuvant third line treatment for the management of HS both in young and adult patients.

Photobiomodulation therapy promotes in vitro wound healing in nicastrin KO HaCaT cells.

Mutations in NCSTN gene (encoding for nicastrin protein) are associated with hidradenitis suppurativa (HS), a chronic inflammatory disease involving hair follicles. HS is clinically handled with drugs but the most severe cases are treated with surgery. Photobiomodulation (PBM) therapy, already used in the treatment of skin diseases such as acne, herpes virus lesions, ultraviolet damage, vitiligo, hypertrophic scar, keloid, burn, psoriasis and diabetic chronic wounds, could be beneficial as an adjuvant supportive treatment to promote and foster the healing process after skin excision in HS. The effects of PBM therapy in promoting the wound closure are evaluated in a HaCaT cells NCSTN-/-, assessing cell metabolism, migration rate, proliferation and cell cycle progression. In our experimental model, PBM exerts a potent action on metabolism of mutated keratinocytes, incrementing adenosine triphosphate (ATP) production at 2 hours, while after 24 hours an increase of metabolism with a decrement of intracellular ATP levels were recorded. Moreover, PBM speeds up the wound closure, inducing cells' migration without affecting their proliferation.Based on our findings, we suggest the use of PBM in HS patients, who undergo major surgery with large skin excision.

Lactotransferrin gene functional polymorphisms do not influence susceptibility to human immunodeficiency virus-1 mother-to-child transmission in different ethnic groups

Lactotransferrin, also known as lactoferrin, is an iron binding glycoprotein that displays antiviral activity against many different infectious agents, including human immunodeficiency virus (HIV)-1. Lactotransferrin is present in the breast milk and in the female genitourinary mucosa and it has been hypothesised as a possible candidate to prevent mother-to-child HIV-1 transmission. To verify if two functional polymorphisms, Thr29Ala and Arg47Lys, in the lactotransferrin encoding gene (LTF) could affect HIV-1 infection and vertical transmission, a preliminary association study was performed in 238 HIV-1 positive and 99 HIV-1 negative children from Brazil, Italy, Africa and India. No statistically significant association for the Thr29Ala and Arg47Lys LTF polymorphisms and HIV-1 susceptibility in the studied populations was found. Additionally LTF polymorphisms frequencies were compared between the four different ethnic groups.

Human hematopoietic reconstitution and HLA-restricted responses in nonpermissive alymphoid mice.

We generated a new humanized mouse model to study HLA-restricted immune responses. For this purpose, we created unique murine hosts by enforcing the expression of human SIRPα by murine phagocytes in murine MHC-deficient HLA-transgenic alymphoid hosts, an approach that allowed the immune reconstitution of nonpermissive mice following injection of human hematopoietic stem cells. We showed that these mouse/human chimeras were able to generate HLA-restricted responses to immunization. These new humanized mice may offer attractive models to study immune responses to human diseases, such as HIV and EBV infections, as well as to assay new vaccine strategies.

The evolutionary landscape of cytosolic microbial sensors in humans.

Host-pathogen interactions are generally initiated by host recognition of microbial components or danger signals triggered by microbial invasion. This recognition involves germline-encoded microbial sensors or pattern-recognition receptors (PRRs). By studying the way in which natural selection has driven the evolution of these microbial sensors in humans, we can identify genes playing an essential role and distinguish them from other, more redundant genes. We characterized the sequence diversity of the NOD-like receptor family, including the NALP and NOD/IPAF subfamilies, in various populations worldwide and compared this diversity with that of other PRR families, such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs). We found that most NALPs had evolved under strong selective constraints, suggesting that their functions are essential and possibly much broader than previously thought. Conversely, most NOD/IPAF subfamily members were subject to more relaxed selective constraints, suggesting greater redundancy. Furthermore, some NALP genes, including NLRP1, NLRP14, and CIITA, were found to have evolved adaptively. We identified those variants conferring a selective advantage on some human populations as the most likely targets of positive selection. More generally, the strength of selection differed considerably between the major families of microbial sensors. Endosomal TLRs and most NALPs were found to evolve under stronger purifying selection than most NOD/IPAF subfamily members and cell-surface TLRs and RLRs, suggesting some degree of redundancy in the signaling pathways triggered by these molecules. This study provides novel perspectives and experimentally testable hypotheses concerning the relative biological relevance of the various families of microbial sensors in humans.

Inhibition of beta-defensin gene expression in airway epithelial cells by low doses of residual oil fly ash is mediated by vanadium.

Poor ambient air quality is associated with increased morbidity and mortality, including respiratory infections. However, its effects on various host-defense mechanisms are poorly understood. This study utilized an in vitro model to study the effect of particulate matter (PM(2.5)) on one antimicrobial mechanism of host defense in the airway, beta-defensin-2 and its bovine homologue, tracheal antimicrobial peptide (TAP) induction in response to lipopolysaccharide (LPS) and IL-1beta. Our model utilized cultured primary bovine tracheal epithelial (BTE) cells and the human alveolar type II epithelial cell line, A549, treated with 0-20 microg/cm(2) residual oil fly ash (ROFA) for 6 h. The cells were then washed and stimulated for 18 h with 100 ng/ml LPS or for 6 h with 100 ng/ml IL-1beta. ROFA inhibited the LPS-induced increase in TAP mRNA and protein without inducing significant cytotoxicity. As little as 2.5 microg/cm(2) of ROFA inhibited LPS-induced TAP gene expression by 30%. The inhibitory activity was associated with the soluble fraction and not the washed particle. The activity in the leachate was attributed to vanadium, but not nickel or iron. SiO(2) and TiO(2) were utilized as controls and did not inhibit LPS induction of TAP gene expression in BTE. ROFA also inhibited the increase of IL-1beta-induced human beta-defensin-2, a homologue of TAP, in A549 cells. The results show that ROFA, V(2)O(5), and VOSO(4) inhibit the ability of airway epithelial cells to respond to inflammatory stimuli at low, physiologically relevant doses and suggest that exposure to these agents could result in an impairment of defense against airborne pathogens.

Human beta-defensin 2 induces a vigorous cytokine response in peripheral blood mononuclear cells.

beta-Defensins are a family of small cationic peptides involved in the innate response to microbial infection. Although their role in microbial killing is well established, the mechanisms through which this occurs remain largely undefined. Here, using protein array technology, we describe a role for human beta-defensins in the induction of an inflammatory cytokine response by human peripheral blood mononuclear cells (PBMCs). Human beta-defensins 1, 2, and 3 were examined for induction of an array of cytokines and chemokines. Some cytokines, such as interleukin 8 (IL-8) and monocyte chemoattractant protein 1, were up-regulated by all three defensins, while others, such as IL-6 and IL-10, were induced more selectively. It was notable that each defensin induced a unique pattern of cytokines. This report documents, for the first time, an analysis of the composite cytokine response of human PBMCs to beta-defensins. The induction or up-regulation of a number of cytokines involved in the adaptive immune response suggests a possible role for these defensins in linking innate and acquired immunity.

MBL2 polymorphisms screening in a regional Italian CF Center.

We performed MBL2 genotyping in 47 CF patients-cared of at the regional CF Centre of Trieste-trying to establish a correlation within allelic variants of MBL2 and modification of patients' clinical outcome. FEV1 values were significantly lowered and a significantly earlier age at onset of Pseudomonas aeruginosa colonisation was found in CF patients with at least one MBL2 variant.

Evidence of a correlation between mannose binding lectin and celiac disease: a model for other autoimmune diseases.

Celiac disease is a multifactorial disorder caused, in genetically susceptible patients, by the ingestion of dietary gluten. Very little is known about the genetic factors, but there is a strong association of two HLA haplotypes (DQ2 or alpha1*05, beta1*02 and DQ8 or alpha1*0301, beta1*0302) with the disease. We investigated the relationship between polymorphisms in the first exon of the MBL2 gene, which encodes for mannose binding lectin (MBL) and celiac disease. Moreover we studied the MBL role by immunohistochemistry and TUNEL. Results were confirmed by clinical findings. We enrolled 149 Italian celiac patients; 116 were characterized by the presence of DQ2 or DQ8. The HLA haplotype was established by allelic specific PCR while the MBL2 genotype was resolved by melting temperature assay. Immunohistochemistry and TUNEL assays were performed on serial sections of biopsy specimens from celiac patients and healthy controls. MBL2 allele and genotype frequencies varied significantly between celiac patients and healthy controls. The frequencies of the 0 allele were 28% in DQ2 or DQ8 celiac patients, 36% in HLA atypical celiac patients, and 22% in healthy controls. Interestingly, the MBL2 0/0 genotype was present in 7 of 33 HLA atypical celiac patients (21%) and in 13 of 116 HLA typical celiac patients (13%) but in only 7 of 147 healthy controls (5%). Furthermore, we found that MBL2 genotype is strongly associated with the occurrence of secondary autoimmune diseases. Immunohistochemistry and TUNEL findings support a role of MBL2 in the clearance of apoptotic cells. In conclusion, MBL2 variants, responsible for lower MBL levels, are associated with celiac disease and higher risk of developing autoimmune diseases. Here we propose a role for MBL in the disease which could be easily applied to other autoimmune disorders.

Variant mannose-binding lectin alleles are associated with celiac disease.

In this study, we investigated the role of mannose-binding lectin (MBL) in celiac disease, by performing genotype analysis for the three point mutations in the first exon of the gene in 117 Italian celiac patients (characterized by flat biopsy and positive for anti-endomysium antibody and human transglutaminase antibodies) and 130 pan-ethnic healthy controls. The frequency of homozygous mutant 0/ 0 was significantly higher in the 117 Italian celiac patients (0.13) than in the 130 pan-ethnic healthy controls (0.05; P=0.0405). An increased frequency of homozygous 0/0 allele was found among patients with celiac disease compared with controls. These results suggest an involvement of MBL in the pathophysiology of celiac disease.