Upside-Down Preference in the Forskolin-Induced In Vitro Differentiation of 50B11 Sensory Neurons: A Morphological Investigation by Label-Free Non-Linear Microscopy.

In this study, we revealed a peculiar morphological feature of 50B11 nociceptive sensory neurons in in vitro culture related to the forskolin-induced differentiation of these cells growing upside-down on cover glass supports. Multi-photon non-linear microscopy was applied to monitor increased neurite arborization and elongation. Under live and unstained conditions, second harmonic generation (SHG) microscopy could monitor microtubule organization inside the cells while also correlating with the detection of cellular multi-photon autofluorescence, probably derived from mitochondria metabolites. Although the differentiated cells of each compartment did not differ significantly in tubulin or multi-photon autofluorescence contents, the upturned neurons were more elongated, presenting a higher length/width cellular ratio and longer neurites, indicative of differentiated cells. SHG originating from the axons' microtubules represented a proper tool to study neurons' inverted culture in live conditions without exogenous staining. This work represents the first instance of examining neuronal cell lines growing and differentiated in an upside-down orientation, allowing a possible improvement of 50B11 as a model in physiology studies of sensory neurons in peripheric nervous system disease (e.g., Fabry disease, Friedreich ataxia, Charcot-Marie-Tooth, porphyria, type 1 diabetes, Guillain-Barré syndrome in children) and analgesic drug screening.

Association of LTA gene haploblock with periodontal disease in Italian adults.

BACKGROUND: Periodontitis is a common oral disease caused by host inflammatory response towards bacteria biofilm. The chronic activation of immune response leads to destruction of teeth supporting tissue, bone loss and tooth detachment. Different factors could be involved in the development and severity of the disease; among them the host genetic background should be considered. OBJECTIVES: In our study, we analysed haploblocks in a genomic region within major histocompatibility complex (MHC) locus aimed at disclosing a possible correlation with the risk of periodontal disease in 602 adult subjects from North-East Italy. RESULTS: The CTTAC haploblock (formed by LTA-rs2857709, LTA-rs2844484, LTA- rs2229094, LTA-rs2229092 and LTA-rs1041981 polymorphisms) correlated with protection towards periodontitis condition, after regression analysis including age and smoking status as covariates (P-value = 0.015). CONCLUSION: Our results suggest that a haplotype within LTA gene (encoding for lymphotoxin alpha) is involved in the susceptibility towards chronic periodontitis.

Mannose-Binding Lectin 2 (MBL2) combined genotypes deficiency is associated with susceptibility for Oral Lichen Planus.

Oral Lichen Planus (OLP) is an oral inflammatory condition, mediated by host immune system reaction, presenting basal membrane damages with inflammatory lesions in the mouth and/or skin. In this study, the role of functional polymorphisms in the MBL2 gene, encoding for Mannose-Binding Protein C (MBP-C), a member of the innate immune response and an acute-phase protein able to activate the complement cascade, was investigated to assess a possible association with OLP susceptibility in Italian patients. Two variations at the promoter region (called H/L and X/Y) and three at the first exon (at codon 52, 54, and 57) of the MBL2 gene were analyzed in 69 OLP patients and 244 healthy controls from northeastern Italy. Considering the polymorphisms singularly, the MBL2 X allele and C/T genotype of the D allele (correlated with low MBP-C expression) were associated with susceptibility to develop OLP. Moreover, when taking into account MBL2 combined genotypes, more OLP patients were deficient MBP-C producers than not deficient, who were more represented among healthy controls. MBL2 combined genotypes, responsible for deficient MBP-C production, are associated with an increased risk of developing OLP.

Antiviral properties of blue laser in an in vitro model of HSV-1 infection.

Herpes simplex virus type-1 (HSV-1) is known to cause lifelong infections in humans. First infection is characterized by gingiva-stomatitis and pharyngitis, while virus reactivation causes recurrent herpes labialis with ulcerations on intraoral mucosa, mouth or external facial skin [1]. Laser therapy (LT), set at red and infrared wavelengths, has been reported as able to reduce HSV-1 recurrence and duration of herpetic sores [2]. Despite the blue wavelength already showed its efficacy in killing different strains of bacteria, it has never been tested on viruses [3].

Caries and Innate Immunity: DEFB1 Gene Polymorphisms and Caries Susceptibility in Genetic Isolates from North-Eastern Italy.

BACKGROUND: The DEFB1 gene, encoding for the constitutively expressed human ß-defensin 1 (hBD1) antimicrobial peptide is a potential candidate when studying genetic susceptibility to caries. DEFB1 genetic variations have been reported as contributing to hBD1 production impairment, leading to a greater susceptibility to be infected by oral pathogens, also leading to periodontitis. METHODS: We analysed 5 DEFB1 polymorphisms, namely 3 functional single-nucleotide polymorphisms (SNPs) at the 5'-untranslated region (UTR), -52G>A (rs1799946), -44C>G (rs1800972), and -20G>A (rs11362), 2 SNPs at the 3'-UTR, c*5G>A (rs1047031) and c*87A>G (rs1800971) SNP located in potential miRNA binding sites, looking for possible correlations with the risk to develop caries in 654 adult subjects from isolated populations of north-eastern Italy. Dental caries prevalence was evaluated with the DMFT (decayed, missing, filled teeth) index, calculated after an accurate oral examination. DEFB1 SNP genotyping was performed with an Illumina 370k high-density SNP array. RESULTS: Two DEFB1 SNPs were significantly associated with the DMFT index: the strongest association emerged from rs11362 SNP (p = 0.008). In particular G/G homozygous individuals showed a higher DMFT index compared to both G/A heterozygous and A/A homozygous individuals; rs1799946 SNP was also significantly associated with DMFT (p = 0.030), and individuals homozygous for the T allele had a higher DMFT value compared to heterozygous C/T and homozygous C/C individuals. CONCLUSIONS: Our study replicated, on a larger number of individuals, previous findings showing the association between two 5'-UTR SNPs in the DEFB1 gene and DMFT, suggesting that these polymorphisms could be considered as potential markers for assessing the risk to develop caries.

Polymorphisms of the vitamin D receptor gene (FOKI, CDX2, and GATA) and susceptibility to chronic periodontitis in diabetic and non-diabetic individuals: A case-control study.

AIM: The aim of the present study was to evaluate the distribution of single-nucleotide polymorphisms (SNP) (variants FOKI [rs2228570], CDX2 [rs47908762], and GATA [rs4516035]) in the vitamin D receptor in individuals with type 2 diabetes mellitus and chronic periodontitis (DM2 + CP), CP alone, and healthy individuals, and to investigate the relationship with susceptibility to CP. METHODS: In total, 280 individuals (116 with DM2 + CP, 95 with CP alone, and 69 healthy individuals) were genotyped using real-time polymerase chain reaction with allele-specific probes. Significant differences (P < .05) were found among the groups with regard to socio-epidemiological variables (sex, marital status, income, smoking habit, and schooling) and clinical-epidemiological variables (age, number of teeth, probing depth, clinical attachment loss, gingival bleeding index, and visible plaque index). RESULTS: The C allele was significantly more frequent among the healthy individuals (34.8%) than those with DM2 + CP (23.5%) (odds ratio [OR] = .58, 95% confidence interval [CI]: . 35-.94, P = .022). Likewise, the CC allele was significantly more frequent among healthy individuals (11.6%) than those with DM2 + CP (2.6%) (OR = .17, 95% CI: .03-.79, P = .015). CONCLUSION: The results suggest that the presence of these variants could lead to a lower susceptibility to DM2 and CP. No other significant differences among groups were found for the other SNP investigated.

Human beta defensin-1 is involved in the susceptibility to adeno-tonsillar hypertrophy.

INTRODUCTION: Innate immunity molecules are known to play a pivotal role in the homeostasis of the oral mucosa, permitting the presence of commensal microflora and, at the same time, providing a first line of defense against pathogens attempting to invade the oral cavity. Tonsils represent the local immune tissue in oral cavity, being able to provide a non-specific response to pathogens; however, in the presence of microbes or foreign materials present in the mouth tonsils could became infected and develop chronic inflammation, thus leading to hypertrophy. The etiology of the disease is multifactorial depending upon environmental and host factors, the latter including molecules of mucosal innate immunity. METHODS: Ninety-five children with adeno-tonsillar hypertrophy subjected to adeno-tonsillectomy were recruited at the pediatric otorhinolaryngology service of the Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste (Italy). The specimen discarded from the surgery were used for genomic DNA extraction and genotyping, for mRNA extraction and gene expression analysis, finally the samples were cut and used to prepare slides to perform immunohistochemistry. RESULTS: Functional polymorphisms within DEFB1 gene, encoding the human beta defensin-1 (hBD-1), were analyzed finding association between DEFB1 rare haplotypes and susceptibility to adeno-tonsillar hypertrophy. DEFB1 mRNA expression was detected in the tonsils and the hBD-1 protein was localized at the epithelia of tonsils mainly in the proximity of the basal lamina. CONCLUSION: Our findings lead us to hypothesize an involvement of hBD-1 mediated innate immunity in the modulation of the susceptibility towards adeno-tonsillar hypertrophy development.

Evaluation of DEFB1 polymorphisms in individuals with chronic periodontitis and diabetes mellitus type 2 in a population of northeastern Brazil.

AIMS: The role of genetic variations in genes related to innate response, as ß-defensin-1 (DEFB1), in the context of chronic periodontitis (CP) and diabetes mellitus type 2 (DM2), is still not clear. The present study evaluates the distribution of DEFB1 single nucleotide polymorphisms (SNPs) 5'-untranslated (5'UTR) region and its relation with the CP in DM2 individuals in northeastern Brazilians. METHODS: Two hundred and eighty individuals participated in the study, being 116 DM2+CP, 95 CP, and 69 healthy individuals. Three known DEFB1 functional SNPs [-52 G > A (rs1799946), -44 C > G (rs1800972), -20 G > A (rs11362)] were genotyped with allele-specific assays. RESULTS: Association was found for the DEFB1 -20 G > A SNP. The G allele, the GA and GG genotypes were significantly (P < 0.05) more frequent in the DM2+CP (59.5%, 50%, and 34.5%, respectively) and CP (61%, 44.2%, and 38.9%, respectively) than in healthy individuals (26.8%, 36.2%, and 8.7%, respectively). The GCG and ACG combinations (-52, -44, -20) were significantly more frequent among DM2+CP and CP than in the healthy individuals. CONCLUSION: The results indicate that genetic variations of DEFB1 gene (SNP-20: G allele and GA and GG genotypes) and the DEFB1 5'UTR haplotypes (GCG and ACG) may be associated with a susceptibility to CP in DM2 individuals as well as CP individuals without DM2.

NLRC5 polymorphism is associated with susceptibility to chronic periodontitis.

Periodontitis is a chronic oral pathology caused by impaired immune response against oral bacteria resulting in tissue inflammation and damage. Among the members of innate immune response, the first line of defence against pathogens, inflammasomes are macro-molecular protein complexes that can be activated by different stimuli, comprised bacteria infections. Different proteins are involved in inflammasoma formation; the most important are molecules belonging from the family of nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs). In this study, polymorphisms within 20 NLRs related genes were analysed in order to investigate their possible association with periodontitis susceptibility in a population from North-East Italy. One polymorphism, namely rs289723, in NLRC5 gene resulted associated with chronic slight and chronic localized periodontitis susceptibility, specifically A/A genotype was correlated with increased risk of disease development. Our study, for the first time, identified the possible involvement of a polymorphism within NLRC5 gene as a possible biomarker for periodontitis condition susceptibility among Italian individuals from genetic isolates.

DEFB1 polymorphisms and salivary hBD-1 concentration in Oral Lichen Planus patients and healthy subjects.

OBJECTIVES: The aetiology of Oral Lichen Planus (OLP), a chronic inflammatory disease of oral mucosa, is not yet well understood. Since innate immunity may be hypothesized as involved in the susceptibility to OLP, we studied human beta defensin 1 (hBD-1) an antimicrobial peptide constitutively expressed in the saliva, looking at functional genetic variants possibly able to diminish hBD-1 production an consequently conferring major susceptibility to OLP. DESIGN: We analysed three DEFB1 polymorphisms at 5' UTR, -52G>A (rs1799946), -44C>G (rs1800972), -20G>A (rs11362) and two DEFB1 polymorphisms at 3'UTR, c*5G>A (rs1047031), c*87A>G (rs1800971), with the aim of correlating these genetic variants and hBD-1 salivary level in a group of OLP patients and in healthy subjects. We also evaluated hBD-1 salivary concentrations, using ELISA, in OLP and healthy controls. RESULTS: We compared hBD-1 concentrations in OLP and healthy subjects: hBD-1 concentration was significantly higher in OLP patients respect to control. When considering the correlation between DEFB1 polymorphisms genotypes and hBD-1 expression levels, significant results were obtained for SNPs -52G>A (p=0.03 both in OLP patients and healthy individuals) and -44C>G (p=0.02 in OLP patients). CONCLUSIONS: hBD-1 production was different between OLP and healthy subjects (not age-matched with OLP). DEFB1 gene polymorphisms, -52G>A and -44C>G, correlated with hBD-1 salivary concentrations.

Lactoferrin gene polymorphisms in Italian patients with recurrent tonsillitis.

INTRODUCTION: Recurrent tonsillitis is an oral pathology characterized by inflammation of tonsils. The disease susceptibility depends upon environmental and host factors, specifically the innate immune response, the first line of host defence could play an important role. Among innate immunity members, lactoferrin, known for its antimicrobial properties, was previously correlated with the risk of oral pathology as periodontitis and dental caries. METHODS: 89 Italian children presenting recurrent tonsillitis and 95 healthy children were genotyped for two LTF non-synonymous polymorphisms, called Thr29Ala and Arg47Lys, in order to investigate their potential role in recurrent tonsillitis susceptibility. RESULTS: no different allele, genotype and haplotype frequency distributions were detected comparing patients and controls. CONCLUSION: data from the current study indicate that LTF polymorphisms might not be involved in recurrent tonsillitis development in our Italian population. However, since the importance of lactoferrin in oral immunity has been previously assessed, further studies should be necessary to unravel the potential role of LTF genetic variants in oral cavity.

CD209 promoter polymorphisms associate with HCV infection and pegylated-interferon plus ribavirin treatment response.

Hepatitis C is a severe liver disease caused by hepatitis C virus that could persist in the host causing progression towards chronic disease in about 80% of the cases. Pegylated-interferon plus ribavirin was the gold standard therapy, however treatment's response was quite variable among individuals and different host/viral factors may play a role in disease outcome. The cluster of differentiation 209 (CD209 antigen) is a component of the innate immune system able to recognize HCV and consequently activating the immune response. We enrolled 203 Italian HCV infected patients and 220 healthy controls investigating if five promoter polymorphisms within CD209 gene (encoding for CD209 antigen) correlated with HCV infection susceptibility, spontaneous viral clearance and interferon treatment response. CD209 -939G>A and -871A>G polymorphisms associated with HCV infection susceptibility, while, CD209 -871A>G and -336A>G polymorphisms associated with response to treatment. In conclusion, CD209 polymorphisms could play a role in the susceptibility to HCV infection as well as interferon treatment response in our study population from North-East of Italy.

Dentifrice Containing Extract of Rosmarinus officinalis Linn.: An Antimicrobial Evaluation.

This study aimed to evaluate the antimicrobial activity of a dentifrice containing an alcoholic extract of rosemary on oral bacteria, compared to a commercially available herbal dentifrice. Standard strains of Streptococcus mutans (ATCC 25175), Streptococcus oralis (ATCC 9811) and Lactobacillus rhamnosus (ATCC 7469) were used, as well as different toothpastes based on rosemary (TR), on propolis (TH), triclosan (positive control) (TPC) and non-fluoridated dentifrice (negative control) (TNC). Bacteria were seeded in Petri dishes and paper discs soaked with dilutions of dentifrice placed on the plates. The inhibition halos were analyzed. It was observed that TR did not show statistical difference in relation to the TH to inhibit S. mutans and S. oralis, while TH was more active against L. rhamnosus. The toothpaste containing rosemary extract had the ability to inhibit the growth of S. mutans, S. oralis and L. rhamnosus, revealing an antimicrobial activity similar to commercially available toothpastes for inhibition of S. mutans and S. oralis.

Impact of DEFB1 gene regulatory polymorphisms on hBD-1 salivary concentration.

OBJECTIVES: Human ß-defensin 1 (hBD-1) is an antimicrobial peptide involved in epithelial defence of various tissues, also present in the saliva. Individual genetic variations within the DEFB1 gene, encoding for hBD-1, could influence gene expression and protein production. DESIGN: Three DEFB1 polymorphisms at 5' untranslated region (UTR), -52G > A (rs1799946), -44C > G (rs1800972) and -20G > A (rs11362), and two polymorphisms at DEFB1 3' UTR, c*5G > A (rs1047031) and c*87A > G (rs1800971), were analysed by direct sequencing and correlated with hDB-1 salivary concentration (tested with enzyme-linked immunosorbent assay (ELISA)) in 40 healthy subjects. RESULTS: Significant associations were found between individuals presenting different DEFB1 polymorphisms at positions -52 and -44 of the gene and hBD-1 salivary concentrations: -52 G/G carriers had higher levels of protein than G/A and A/A; -44C/G subjects showed a higher protein concentration than homozygous wild-type C/C. For the -20G > A, c*5G > A and c*87A > G polymorphisms, no statistically significant differences were found. Combined haplotype analysis confirmed the results obtained considering the single-nucleotide polymorphisms (SNPs) singularly. CONCLUSION: Polymorphisms in the DEFB1 gene influence hBD-1 production and, therefore, could modify the innate immune system responses and, consequently, the oral health.

Ear nose throat manifestations in hypoidrotic ectodermal dysplasia.

The ectodermal dysplasias (EDs) are a large and complex group of inherited disorders. In various combinations, they all share anomalies in ectodermal derived structures: hair, teeth, nails and sweat gland function. Clinical overlap is present among EDs. Few causative genes have been identified, to date. Altered gene expression is not limited to the ectoderm but a concomitant effect on developing mesenchymal structures, with modification of ectodermal-mesenchymal signaling, takes place. The two major categories of ED include the hidrotic and hypohidrotic form, the latter more frequent; they differentiate each other for the presence or absence of sweat glands. We report Ear Nose Throat manifestations of ED, linked to the reduction of mucous glands in the nasal fossae with reduced ciliar function, and decrease salivary glands function. Often patients report an increased rate of infections of the upper respiratory tract and of the ear. Nasal obstruction due to the presence of nasal crusting, hearing loss and throat hoarseness are the most represented symptoms. Environmental measures, including a correct air temperature and humidification, is mandatory above all in subjects affected by hypohidrotic form.

Dentifrice Containing Extract of Rosmarinus officinalis Linn: An Antimicrobial Evaluation

Abstract This study aimed to evaluate the antimicrobial activity of a dentifrice containing an alcoholic extract of rosemary on oral bacteria, compared to a commercially available herbal dentifrice. Standard strains of Streptococcus mutans (ATCC 25175), Streptococcus oralis (ATCC 9811) and Lactobacillus rhamnosus (ATCC 7469) were used, as well as different toothpastes based on rosemary (TR), on propolis (TH), triclosan (positive control) (TPC) and non-fluoridated dentifrice (negative control) (TNC). Bacteria were seeded in Petri dishes and paper discs soaked with dilutions of dentifrice placed on the plates. The inhibition halos were analyzed. It was observed that TR did not show statistical difference in relation to the TH to inhibit S. mutans and S. oralis, while TH was more active against L. rhamnosus. The toothpaste containing rosemary extract had the ability to inhibit the growth of S. mutans, S. oralis and L. rhamnosus, revealing an antimicrobial activity similar to commercially available toothpastes for inhibition of S. mutans and S. oralis.

Resumo O estudo teve como objetivo avaliar a atividade antimicrobiana de um dentifrício extrato alcoólico de alecrim sobre bactérias orais, comparando-o a um dentifrício herbal disponível no mercado. Cepas padrão de Streptococcus mutans (ATCC 25175), Streptococcus oralis (ATCC 9811) e Lactobacillus rhamnosus (ATCC 7469) foram utilizadas, bem como diferentes dentifrícios à base de alecrim (TR), própolis (TH), triclosan (controle positivo) (TPC) e sem flúor (controle negativo) (TNC). Placas de Petri foram inoculadas com as bactérias e discos de papel embebidos com diluições de cada dentifrício foram colocados nas placas. Em seguida, foram analisados os halos de inibição. Observou-se que o TR não mostrou diferença estatística em relação ao TH para inibição dos S. mutans e S. oralis, enquanto TH foi mais ativo contra L. rhamnosus. O dentifrício contendo extrato de alecrim foi capaz de inibir o crescimento de S. mutans, S. oralis e L. rhamnosus, revelando uma atividade antimicrobiana semelhante ao dentifrício disponível comercialmente na inibição de S. mutans e S. oralis.

Association of polymorphisms in the first exon of mannose binding lectin gene (MBL2) in Brazilian patients with HCV infection.

In our study we investigated the role of the polymorphisms in the first exon of MBL2 gene in the susceptibility to HCV infection and disease progression in a Northeastern Brazilian population. One hundred and eleven patients seen at the Gastroenterology Service of the Oswaldo Cruz Hospital of the University of Pernambuco were included in this study. A total of 165 unexposed, uninfected individuals matched for place of origin were employed as healthy controls. MBL2 genotyping was performed by using a melting temperature assay. The 0 allele was significantly more frequent in the HCV positive group than the healthy controls (34% vs. 20%, p<0.01, respectively) and was associated to an increased risk of HCV-1 infection (O.R.=2.1; C.I. 1.41-3.19). Also genotypes frequencies were significantly different in HCV positive subjects when compared to healthy controls with the 00 and A0 genotypes being significantly overrepresented in HCV infected subject (15% and 37%, respectively) as compared to healthy subjects (6% and 27%, respectively, p<0.01 ) Allele and genotypes frequencies were also evaluated in HCV infected subjects according to their response to pegylated-INFalpha/riboviron therapy. There was a trend for HCV positive responders vs. non-responders to be 0 allele positive and a similar trend was observed for the MBL2 A0 and 00 genotypes, but neither of these reached statistical significance. Our findings indicate that MBL might represent an important antiviral molecule having a protective role in the first stages of HCV infection, as shown by the increased frequency of wild-type alleles in control population as compared to the infected group.

Mannose binding lectin genes (MBL2) polymorpshisms and the periodontal disease in diabetic patients / Polimorfimos da lectina de ligação da manose (MBL2) e doença periodontal em pacientes diabéticos

PURPOSE: To assess the association between the polymorphism in exon-1 of the MBL2 gene and the periodontal disease in type 2 diabetic patients. METHODS: The sample comprised of 100 patients, who were submitted to a clinical periodontal examination that evaluated in six sites per tooth the probing depth (PD), bleeding on probing (BOP), clinical attachment loss (CAL), plaque index (PI) and the number of teeth present. Periodontal disease was defined as at least four sites with loss of attachment of >5 mm, with one or more of those sites having a pocket of > 4 mm. The collection of scaling cells from the oral mucosa was carried out and the detection of MBL2 polymorphism was made by real time PCR and melting temperature curve analysis. RESULTS: In a type 2 diabetic population, no significant statistical differences in MBL2 polymorphisms genotype or allele frequencies were observed among subjects with periodontal disease. CONCLUSION: This study indicates that the polymorphisms in exon-1 of the MBL2 gene are not related to periodontal disease in a type 2 diabetic population.

OBJETIVO: Avaliar a associação entre o polimorfismo no exon-1 do gene MBL2 e a doença periodontal em pacientes diabéticos tipo 2. MÉTODO: A amostra foi composta por 100 pacientes que foram submetidos a um exame clínico periodontal que avaliou seis sítios por dente a profundidade de sondagem (PS), sangramento à sondagem (SS), perda de inserção clínica (PIC), índice de placa (IP) e o número de dentes presente. A doença periodontal foi definida como pelo menos quatro sítios com perda de inserção de >5mm, com um ou mais destes sítios tendo uma bolsa de >4mm. Foram coletadas células de descamação da mucosa oral e a detecção do polimorfismo foi feita através da PCR em tempo real e análise da temperatura de melting. RESULTADOS: Em uma população de diabéticos tipo 2, não houve diferenças estatisticamente significantes nos genótipos do polimorfismo da MBL2 ou freqüência alélica observadas entre os indivíduos com doença periodontal. CONCLUSÃO: Este estudo indicou que o polimorfimos no exon-1 do gene da MBL2 não foi relacionado à doença periodontal em uma população de diabéticos tipo 2.