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Monocyte HLA-DR is an increasingly recognized markers of sepsis-induced immunodepression, but its regulatory mechanisms remain poorly understood in sepsis. Several evidence for positive selection on the 5’ promoter region of HLA class II transactivator (CIITA) gene, the master regulator of MHC class II, have been gathered in the European population, and its role in sepsis has never been demonstrated, whilst suggested in autoimmune disease. We aim to describe the effect of rs3087456 polymorphism, localized on CIITA promoter III (pIII), on mortality of patients with septic shock, and investigate the mechanisms regulating HLA-DR expression. Genotyping of 203 patients with septic shock showed that, in A dominant model, GG genotype was associated with 28-day mortality (OR 2.29; 95%CI: 1.01 to 5.22; P = 0.043). Monocyte HLA-DR remained low in patients with GG genotype whereas it increases as early as at the end of the first week in intensive care in patients with AA or AG genotype. Using site-directed mutagenesis, in vitro reporter gene promoter activity of the pIII was decreased in GG genotype in monocyte cell line. Interferon-γ (IFN-γ) restored pIII activity in GG genotype as well as restore, in ex vivo experiment in healthy volunteers, CIITA pIII expression of GG genotype. Hereby, we demonstrated that rs3087456, a positively selected polymorphism of CIITA proximal promoter, significantly impact monocyte HLA-DR expression in patients with septic shock through CIITA promoter activity, that can be rescued using IFN-γ, offering a new perspective in genetic susceptibility to sepsis and targeted immunomodulatory therapy.KeypointsCIITA G-286A polymorphism reduces promotor activity and significantly impact monocyte HLA-DR expression and mortality in septic shockDownregulatory effects of CIITA G-286A polymorphism on monocyte HLA-DR expression can be reverse by IFN-γ in patients with septic shock

Objective. We investigated genetic polymorphisms of MBL2 gene, in a cohort of 90 italian HIV-1 pregnant seropositive women and their children in order to understand whether the MBL2 genotype of HIV-1 positive mothers might be related to their ability to transmit the virus to their children.Materials and methods. DNA was extracted from Iso Code Stix cards, and MBL2 genoptyping was performed by Melting Temperature Assay.Results. The frequency of the MBL2 0/0 homozygotes was higher in HIV-1 positive mothers than in healthy controls, the MBL2 0/0 genotype was more frequent in children born from HIV positive mothers than healthy subjects.Conclusions. We have confirmed the association of polymorphisms involving a gene of the innate immunity with an increased risk of being infected by HIV. These polymorphisms were also evidenced in children born from HIV + mothers, but the risk of infection was strongly reduced by cesarean delivery and by antiretroviral treatment.

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.

In familial hidradenitis suppurativa (HS), mutations in the genes encoding three subunits of the gamma secretase complex, presenilin-1 (PSEN1), presenilin enhancer (PSENEN) and nicastrin (NCSTN), have pointed to impaired Notch signalling as a pathogenic disease mechanism.

Background Diagnosis of pyoderma gangrenosum, acne and hidradenitis suppurativa (PASH) and pyogenic arthritis, pyoderma gangrenosum, acne, and hidradenitis suppurativa (PAPASH) patients, in spite of recently identified genetic variations, is just clinical, since most patients do not share the same mutations, and the mutations themselves are not informative of the biological pathways commonly disrupted in these patients. Objective To reveal genetic changes more closely related to PASH and PAPASH etiopathogenesis, identifying novel common pathways involved in these diseases. Methods Cohort study on PASH (n = 4) and PAPASH (n = 1) patients conducted using whole exome sequencing (WES) approach and a novel bioinformatic pipeline aimed at discovering potentially candidate genes selected from density mutations and involved in pathways relevant to the disease. Results WES results showed that patients presented 90 genes carrying mutations with deleterious and/or damage impact: 12 genes were in common among the 5 patients and bared 237 ns ExonVar (54 and 183 in homozygosis and heterozygosis, respectively). In the pathway enrichment analysis, only 10 genes were included, allowing us to retrieve 4 pathways shared by all patients: (1) Vitamin D metabolism, (2) keratinization, (3) formation of the cornified envelope and (4) steroid metabolism. Interestingly, all patients had vitamin D levels lower than normal, with a mean value of 10 ng/mL. Conclusion Our findings, through a novel strategy for analysing the genetic background of syndromic HS patients, suggested that vitamin D metabolism dysfunctions seem to be crucial in PASH and PAPASH pathogenesis. Based on low vitamin D serum levels, its supplementation is envisaged.

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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.

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.

Primary hyperoxaluria type 1 is an autosomal recessive disorder of glyoxylate metabolism, caused by a deficiency of alanine:glyoxylate aminotransferase, which is encoded by a single copy gene (AGXT). The aim of this research was to standardize denaturing high-performance liquid chromatography, a new, sensitive, relatively inexpensive, and automated technique, for the detection of AGXT mutation. Denaturing high-performance liquid chromatography was used to analyze in blind the AGXT gene in 20 unrelated Italian patients with primary hyperoxaluria type 1 previously studied by other standard methods (single-strand conformation polymorphism analysis and direct sequencing) and 50 controls. Denaturing high-performance liquid chromatography allowed us to identify 13 mutations and the polymorphism at position 154 in exon I of the AGXT gene. Hence the method is more sensitive and less time consuming than single-strand conformation polymorphism analysis for the detection of AGXT mutations, thus representing a useful and reliable tool for detecting the mutations responsible for primary hyperoxaluria type 1. The new technology could also be helpful in the search for healthy carriers of AGXT mutations amongst family members and their partners, and for screening of AGXT polymorphisms in patients with nephrolithiasis and healthy populations.

We investigated the polymorphisms of the promoter region of the MBL2 gene, which codifies for the Mannose-binding protein (MBP). The study population included 90 children with vertically acquired HIV-infection, further divided on the basis of the disease rate, 27 HIV exposed-uninfected children, and 74 healthy control subjects matched for ethnic origin to evaluate the MBP involvement in the risk of HIV-1 infection and to assess the role of the MBP promoter in AIDS progression. A region of 380 bp in the promoter of the MBL2 gene was analysed by PCR and direct sequencing of both DNA strands. We found that the polymorphism at position -550 influences the risk of HIV-infection and AIDS progression. Also a 6 bp deletion at position -328 was correlated with HIV-1 infection. This study indicates that the promoter of the MBL2 gene influences vertical transmission of HIV and the course of perinatal infection.

We developed a rapid, sensitive and robust high risk human papillomavirus (HR HPV) detection protocol based on direct in situ PCR technology and fluorochrome-modified nucleotides on cytologic specimens (cell smears) and on HPV infected tissues (CIN III). Reproducible results on both cytologic specimens and paraffin-embedded tissues were obtained, providing a powerful tool for clinical investigation on HR HPV infection. Quantitative PCR performed on the same tissue sections adjacent to those used for in situ techniques allowed us to establish the sensitivity of our methods, able to detect rare copies (about 15 in our paraffin-embedded tissues) of HPV.

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disorder that is caused by a deficiency of alanine: glyoxylate aminotransferase (AGT), which is encoded by a single copy gene (AGXT). Molecular diagnosis was used in conjunction with clinical, biochemical, and enzymological data to evaluate genotype-phenotype correlation. Twenty-three unrelated, Italian PH1 patients were studied, 20 of which were grouped according to severe form of PH1 (group A), adult form (group B), and mild to moderate decrease in renal function (group C). All 23 patients were analyzed by using the single-strand conformation polymorphism technique followed by the sequencing of the 11 AGXT exons. Relevant chemistries, including plasma, urine and dialyzate oxalate and glycolate assays, liver AGT activity, and pyridoxine responsiveness, were performed. Both mutant alleles were found in 21 out of 23 patients, and 13 different mutations were recognized in exons 1, 2, 4, and 10. Normalized AGT activity was lower in the severe form than in the adult form (P < 0.05). Double heterozygous patients presented a lower age at the onset of the disease (P = 0.025), and they were more frequent in group A (75%) than in the group B (14%; P = 0.0406). The T444C mutation was more frequent in the severe form (P < 0.05), and the opposite was observed for G630A (P < 0.05). G630A mutation homozygotes had a higher AGT residual activity (P = 0.00001). This study confirms the allelic heterogeneity of the AGXT, which could to some extent be responsible for the phenotypic heterogeneity in PH1.

Coeliac disease (CD) is an autoimmune enteropathy triggered by ingestion of wheat gluten or related protein from rye and barley, and is one of the most frequently occurring, treatable, lifelong disorders.1,2 Undetected or untreated CD may cause other, more severe, complications later, such as autoimmune diseases, osteoporosis, neurological disorders, and infertility.3,4 Several studies have shown CD clusters in families with a sibling relative risk of 20–60 and high concordance between monozygotic twins (75%),5–8 which indicates a strong genetic component to coeliac disease. As expected in complex autoimmune diseases, human leukocyte antigen (HLA) linked genes are the main genetic factor involved in CD. More than 90% of patients suffering from CD share the major histocompatibility complex II class HLA-DQ2 haplotype. Most of the remainder present HLA-DQ8. However, there are patients who present neither HLA-DQ8 nor HLA-DQ2 on their antigen-presenting cells. These genetic findings strongly indicate the involvement of other genes in the pathogenesis of coeliac disease. So far, various genome-wide linkage analyses have been performed in order to identify non-HLA linked genes responsible for CD. A recent study by Greco et al. 9 on Italian coeliac families demonstrated the existence of a genetic risk factor on chromosome 5 (5q31–33), which has been reported in other linkage studies in different populations.10,11 This genomic region contains several candidate genes for CD, including the interleukin ( IL12B ) gene, the polymorphisms of which do not seem to be associated with CD,12 and the gene encoding for CD14.13 CD14 is a multifunctional receptor involved in the innate immune response. In fact, it is thought to be one of the most important receptors for lipopolysaccharide and other bacterial wall-derived components.14–16 However it is well known that CD14 has several other functions,17,18 including the clearance …

Celiac disease (CD) is an autoimmune enteropathy triggered by ingestion of gluten in genetically susceptible subjects and represents one of the most frequently occurring, treatable, lifelong autoimmune disorders. Undetected or untreated CD may cause late more severe complications (Farrell and Kelly, 2002). So far, several factors have been identified as possible agents responsible for CD. There is a strong evidence that CD is associated with specific HLA haplotypes (HLADQA1* 0501, DQB1*0201 or DQA1*0301, DQB0302) (Sollid and Thorsby, 1993). Recently it has been demonstrated on Italian patients that polymorphisms of the first exon of MBL2 gene, which encodes for Mannose Binding Protein (MBP), could play a pathophysiological role in celiac disease (Boniotto et al., 2002). MBP is a serum protein involved in the natural or innate immune response. MBP acts as an ante-antibody and can enhance opsonisation, or can activate the classical pathway of the complement on bacteria, viruses and fungi (Sastry and Ezekowitz, 1993).

SUMMARYImmune dysregulation, polyendocrinopathy and enteropathy with X-linked inheritance (IPEX) is a serious disease arising from mutations in FOXP3. This gene codifies for a transcription factor whose dysfunction results in hyperactivation of T cells. It is not clear, however, why an intermediate phenotype is not seen in heterozygous females, who are completely healthy. In order to address this question, we investigated X-chromosome inactivation in peripheral blood lymphocytes from a heterozygous female with a child affected by IPEX. No preferential inactivation was shown in freshly sorted CD4+, CD8+, CD19+ cells or in IL-2 cultured CD4 and CD8 T cells, indicating that peripheral blood lymphocytes in these women are randomly selected. Moreover, only one single FOXP3 transcript was expressed by CD4 T cell clones analysed by RT-PCR, confirming that this gene is subject to X- inactivation. We hypothesize that hyper-activation of T cell in carriers of FOXP3 mutations is regulated by the presence of normal regulatory T cells.