Laura Braida

A study of host defence peptide β-defensin 3 in primates

We have investigated the molecular evolution of the gene coding for beta-defensin 3 (DEFB103) in 17 primate species including humans. Unlike the DEFB4 genes (coding for beta-defensin 2) [Boniotto, Tossi, Del Pero, Sgubin, Antcheva, Santon and Masters (2003) Genes Immun. 4, 251-257], DEFB103 shows a marked degree of conservation in humans, Great Apes and New and Old World monkeys. Only the Hylobates concolor defensin hcBD3 showed an amino acid variation Arg17-->Trp17 that could have a functional implication, as it disrupts an intramolecular salt bridge with Glu27, which locally decreases the charge and may favour dimerization in the human congener hBD3. This is thought to involve the formation of an intermolecular salt bridge between Glu28 and Lys32 on another monomer [Schibli, Hunter, Aseyev, Starner, Wiencek, McCray, Tack and Vogel (2002) J. Biol. Chem. 277, 8279-8289]. To test the role of dimerization in mediating biological activity, we synthesized hBD3, hcBD3 and an artificial peptide in which the Lys26-Glu27-Glu28 stretch was replaced by the equivalent Phe-Thr-Lys stretch from human beta-defensin 1 and we characterized their structure and anti-microbial activity. Although the structuring and dimerization of these peptides were found to differ significantly, this did not appear to affect markedly the anti-microbial potency, the broad spectrum of activity or the insensitivity of the anti-microbial action to the salinity of the medium.

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 α1*05, β1*02 and DQ8 or α1*0301, β1*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.

Single-tube genotyping of MBL-2 polymorphisms using melting temperature analysis

Abstract Recently several authors correlated MBL-2 gene polymorphisms with different pathologies and there is a growing interest for MBL-2 genotyping in a large number of individuals. We have developed a single-tube, rapid, economic, and fully automated melting temperature analysis screening method, based on ABI 7700 Sequence Detection System technology and SYBR Green I chemistry, for the detection of three polymorphisms (exon 1, codons 52, 54, 57) in the MBL-2 gene. We also developed an electronic sheet for the automatic calling of different genotypes, based on the analysis of the first derivative of ABI 7700 raw data.

Detection of MBL-2 gene expression in intestinal biopsies of celiac patients by in situ reverse transcription polymerase chain reaction.

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).

Variant mannose-binding lectin alleles are associated with celiac disease

Abstract. 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.

Italian multicentric pilot study on MBL2 genetic polymorphisms in HIV positive pregnant women and their children

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.

Promoter polymorphisms of the CD14 gene in Italian patients with coeliac disease

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 …

Quantitative in situ detection of high-risk human papillomavirus in cytological specimens by SYBR Green I fluorescent labeling

Abstract In this study we developed an in situ protocol for quantitative detection of high-risk human papillomavirus (HPV), based on direct in situ polymerase chain reaction (PCR) with SYBR Green I labeling and GeneAmp 5700 Sequence Detection System technology. This protocol was applied on cytological specimens of patients with cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma (SCC). We performed direct in situ quantitative PCR on cell smears, uninfected human skin fibroblasts, Hela and Caski cells. After in situ amplification, slides were counterstained with propodium iodide and analyzed under a fluorescent microscope in order to localize high-risk HPV and verify preservation of morphology. After PCR optimization, we obtained the following results. The Hela cells showed values ranging from 15 to 33 copies of high-risk HPV per cell, the Caski cell line from 220 to 300 high-risk HPV copies per cell and the cell smear (both CIN and SCC) around 20–35 copies of high-risk HPV per cell. No high-risk HPV amplification was detected in uninfected human fibroblasts, healthy controls, non-amplification control, and non-specific primer control. A positive intranuclear high-risk HPV amplification was detected in cell smears from 20 patients with CIN and 10 with SCC. In conclusion, our in situ quantitative protocol for high-risk HPV detection on cell smears combines both quantitative data and in situ localization of the target, with preservation of morphology. For this reason it could be used as a rapid screening tool when both morphological and quantitative results are requested on the same slide.