Alessandra Pontillo

Evolution of the primate cathelicidin : Correlation between structural variations and antimicrobial activity

Cathelicidin genes homologous to the human CAMP gene, coding for the host defense peptide LL-37, have been sequenced and analyzed in 20 primate species, including Great Apes, hylobatidae, cercopithecidae, callithricidae, and cebidae. The region corresponding to the putative mature antimicrobial peptide is subject to a strong selective pressure for variation, with evidence for positive selection throughout the phylogenetic tree relating the peptides, which favors alterations in the charge while little affecting overall hydrophobicity or amphipathicity. Selected peptides were chemically synthesized and characterized, and two distinct types of behavior were observed. Macaque and leaf-eating monkey RL-37 peptides, like other helical antimicrobial peptides found in insect, frog, and mammalian species, were unstructured in bulk solution and had a potent, salt and medium independent antimicrobial activity in vitro, which may be the principal function also in vivo. Human LL-37 and the orangutan, hylobates, and callithrix homologues instead showed a salt-dependent structuring and likely aggregation in bulk solution that affected antimicrobial activity and its medium dependence. The two types of peptides differ also in their interaction with host cells. The evolution of these peptides has thus resulted in distinct mechanisms of action that affect the direct antimicrobial activity and may also modulate accessory antimicrobial functions due to interactions with host cells.

PIN1 promoter polymorphisms are associated with Alzheimer's disease.

In our study, we analyzed the coding and promoter regions of the PIN1 gene in a group of 111 Alzheimers disease (AD) patients looking for a possible genotype-phenotype correlation. The presence of SNPs - which could affect and modify the clinical phenotype of AD patients was also investigated. We identified two single nucleotide polymorphisms (SNPs) at positions -842 (G-->C) and -667 (C-->T) in the promoter region of the PIN1 gene. Our results evidenced a significantly higher percentage of -842C allele carriers in AD subjects with respect to healthy controls. We found that this allele significantly raised the risk of developing AD (OR 3.044, CI 1.42-6.52). The -842 and -667 SNPs were in linkage disequilibrium and combined to form haplotypes. The CC haplotype conferred a higher risk of developing AD (OR 2.95, confidence interval 1.31-6.82). Finally, protein expression analyses revealed that subjects carrying the -842 CC genotype or the CC haplotype showed reduced levels of the PIN1 protein in peripheral mononuclear cells.

The Missense Variation Q705K in CIAS1/NALP3/ NLRP3 Gene and an NLRP1 Haplotype Are Associated With Celiac Disease

OBJECTIVES:Celiac disease (CD) is a multifactorial common disorder with several susceptibility loci. Variations in the NALP1/NLRP1 and NALP3/NLRP3 genes have been reported to confer risk for several autoimmune conditions. We hypothesized that polymorphisms in these genes, due to their role in innate immunity and inflammatory processes, may affect susceptibility to CD.METHODS:Two single-nucleotide polymorphisms (SNPs) in NLRP1 (rs12150220, rs2670660) and two SNPs (rs10754558, rs35829419) in NLRP3 genes were genotyped in 504 CD Italian patients and 256 healthy controls.RESULTS:The minor A allele of NLRP3 rs35829419 (Q705K) polymorphism appeared to exert a protective role against the development of CD (P=0.029; odds ratio (OR)=0.56). Moreover, a particular NLRP1 haplotype was associated with predisposition to CD (P=0.003; OR=1.38), even more when present in combination with the rs35829419 major C allele (P=0.002; OR=1.42).CONCLUSIONS:We hypothesized that the deregulation of CIAS1/NALP3/NLRP3 and NALP1/NLRP1 inflammasomes could have a role in CD pathogenesis.

DEFB1 gene polymorphisms and increased risk of HIV-1 infection in Brazilian children

In our study we analysed three single nucleotide polymorphisms (SNPs) in the 5′ untranslated region (UTR) of the DEFB1 gene, namely −52(G/A) −44(C/G) and −20(G/A), in three groups of northeastern Brazilian children in order to assess their role in HIV-1 infection. Our results allowed us to hypothesize that the SNPs located in the 5′ UTR of the DEFB1 gene can be employed as a marker of risk for HIV-1 infection.

NALP1/NLRP1 genetic variants are associated with Alzheimer disease

Alzheimer disease (AD) is a complex neurodegenerative disease. Genetic and molecular studies have confirmed that in the human brain, amyloid-&bgr; fibrils can induce, through the activation of NALP1 inflammosome, inflammatory and apoptotic responses involved in the pathogenesis of AD. Considering that AD pathogenesis is multifactorial, we hypothesized that NALP1/NLRP1 could be a susceptibility gene involved in the devolvement of the disease. The possible association between 9 selected polymorphisms in the NALP1/NLRP1 gene and AD was evaluated by comparing their frequency distribution in an Italian cohort of AD patients (AD, n=276) and in a group of Italian sex-matched and age-matched healthy controls without dementia (HC, n=266). Our study, evidences the association of 4 nonsynonymous polymorphisms in the NLRP1 gene (rs2137722, rs34733791, rs11657747, rs11651595) with AD. The major alleles of all 4 single nucleotide polymorphisms and the corresponding homozygote genotypes were more frequent in AD patients than in healthy controls, suggesting an association of these variants in the predisposition versus the development of the disease. These findings seem to support the previously reported role of NALP1 in neuronal damage, and provide evidence of an association between single nucleotide variations in the NLRP1 gene and AD.

Natural isoprenoids are able to reduce inflammation in a mouse model of mevalonate kinase deficiency

Mevalonate kinase deficiency (MKD) is a rare disorder characterized by recurrent inflammatory episodes and, in most severe cases, by psychomotor delay. Defective synthesis of isoprenoids has been associated with the inflammatory phenotype in these patients, but the molecular mechanisms involved are still poorly understood, and, so far, no specific therapy is available for this disorder. Drugs like aminobisphosphonates, which inhibit the mevalonate pathway causing a relative defect in isoprenoids synthesis, have been also associated to an inflammatory phenotype. Recent data asserted that cell inflammation could be reversed by the addition of some isoprenoids, such as geranylgeraniol and farnesyl pyrophosphate. In this study, a mouse model for typical MKD inflammatory episode was obtained treating BALB/c mice with aminobisphosphonate alendronate and bacterial muramyldipeptide. The effect of exogenous isoprenoids—geraniol, farnesol, and geranylgeraniol—was therefore evaluated in this model. All these compounds were effective in preventing the inflammation induced by alendronate-muramyldipeptide, suggesting a possible role for these compounds in the treatment of MKD in humans.

Genetic predictors of glucocorticoid response in pediatric patients with inflammatory bowel diseases

Background Glucocorticoids (GCs) are used in moderate-to-severe inflammatory bowel diseases (IBD) but their effect is often unpredictable. Aim To determine the influence of 4 polymorphisms in the GC receptor [nuclear receptor subfamily 3, group C, member 1 (NR3C1)], interleukin-1&bgr; (IL-1&bgr;), and NACHT leucine-rich-repeat protein 1 (NALP1) genes, on the clinical response to steroids in pediatric patients with IBD. Methods One hundred fifty-four young IBD patients treated with GCs for at least 30 days and with a minimum follow-up of 1 year were genotyped. The polymorphisms considered are the BclI in the NR3C1 gene, C-511T in IL-1&bgr; gene, and Leu155His and rs2670660/C in NALP1 gene. Patients were grouped as responder, dependant, and resistant to GCs. The relation between GC response and the genetic polymorphisms considered was examined using univariate, multivariate, and Classification and Regression Tree (CART) analysis. Results Univariate analysis showed that BclI polymorphism was more frequent in responders compared with dependant patients (P=0.03) and with the combined dependant and resistant groups (P=0.02). Moreover, the NALP1 Leu155His polymorphism was less frequent in the GC responsive group compared with resistant (P=0.0059) and nonresponder (P=0.02) groups. Multivariate analysis comparing responders and nonresponders confirmed an association between BclI mutated genotype and steroid response (P=0.030), and between NALP1 Leu155His mutant variant and nonresponders (P=0.033). An association between steroid response and male sex was also observed (P=0.034). In addition, Leu155His mutated genotype was associated with steroid resistance (P=0.034). Two CART analyses supported these findings by showing that BclI and Leu155His polymorphisms had the greatest effect on steroid response (permutation P value=0.046). The second CART analysis also identified age of disease onset and male sex as important variables affecting response. Conclusions These results confirm that genetic and demographic factors may affect the response to GCs in young patients with IBD and strengthen the importance of studying high-order interactions for predicting response.

The inhibition of mevalonate pathway induces upregulation of NALP3 expression: new insight in the pathogenesis of mevalonate kinase deficiency.

Mevalonate kinase deficiency (MKD) is a rare hereditary auto-inflammatory syndrome due to mutations in mevalonate kinase, the second enzyme of mevalonate pathway of cholesterol, and nonsterol-isoprenoids biosynthesis. The shortage of mevalonate-derived intermediates, and in particular of geranylgeranyl pyrophosphate (GGPP), has been linked with the activation of caspase-1 and thereby with the production of IL-1β, but the true concatenation of these two events has not been clarified yet. We hypothesized that inflammasomes could mediate the activation of caspase-1 due to the shortage of GGPP. We monitored the expression of the principal proteins (NALP1, NALP3 and IPAF) of the three known inflammasomes, first in a cellular model of MKD and then in two MKD patients, after bacterial lipopolysaccharide (LPS) stimulation. In healthy subjects, alendronate alone induced the expression of NALP1 and NALP3, and then together with LPS it induced a dramatic increase in NALP3 expression. In MKD patients, NALP3 expression was higher than in untreated healthy controls. Our results, although preliminary, showed that the inhibition of the mevalonate pathway led to a hyper-expression of NALP3, suggesting a possible involvement of NALP3-inflammasome in the activation of caspase-1 consequent to GGPP decrement. This is the first time that the involvement of the inflammasome complexes was shown in MKD pathogenesis.

Diagnostics and Therapeutic Insights in a Severe Case of Mevalonate Kinase Deficiency

Mevalonate kinase deficiency is a rare inborn disorder of isoprenoid and sterol biosynthesis characterized by a recurrent autoinflammatory syndrome and, in most severe cases, psychomotor delay. Clinical manifestations can be very complex and, in some cases, mimic a chronic inflammatory disease. Diagnosis is also complex and often requires immunologic, genetic, and biochemical investigations. There is no standardized therapy, but biological agents could help to control inflammatory complaints in some cases. A severe case of mevalonate kinase deficiency that was associated with nephritis and successfully treated with anakinra (interleukin 1 receptor antagonist) is reported here, and new insights into diagnosis and therapy of this complex disorder are discussed.

Natural isoprenoids inhibit LPS-induced-production of cytokines and nitric oxide in aminobisphosphonate-treated monocytes.

The inhibition of mevalonate pathway through genetic defects (mevalonate kinase deficiency, MKD) or pharmacologic drugs (aminobisphosphonates) causes a shortage of intermediate compounds and, in particular, of geranylgeranyl-pyrophosphate (GGPP) associated to the activation of caspase-1 and IL-1beta release. Geraniol (GOH), farnesol (FOH), geranylgeraniol (GGOH) and menthol (MOH), due to their isoprenoid structure, are supposed to enter the mevalonate pathway and to by-pass the biochemical block, reconstituting the pathway. Considering the already known side effects of aminobisphosphonates, and the lack of a specific treatment for MKD, we evaluated the impact of these natural isoprenoids compounds in a RAW cell lines chemically treated with the aminobisphosphonate alendronate, and in monocytes isolated from 2 patients affected by MKD. GOH, FOH, GGOH and MOH were all capable to diminish inflammatory marker levels induced by LPS. These natural isoprenoids could be proposed as novel therapeutic approach for the still orphan drug MKD, but also considered for the evaluation of possible inflammatory side effects of aminobisphosphonates.