Maria Satra

TLR4 gene polymorphisms: evidence for protection against type 2 diabetes but not for diabetes-associated ischaemic heart disease

OBJECTIVE Several factors either predisposing or protecting from the onset of diabetes mellitus type 2 (DM2) have been proposed. Two specific polymorphisms of toll-like receptor 4 (TLR4; Asp299Gly and Thr399Ile) have recently been identified either as candidate protector genes against DM2 and associated neuropathy or risk alleles for the manifestation of diabetic retinopathy. The impact of these alleles on the risk for ischaemic heart disease (IHD) is controversial while their role in diabetes-associated IHD has never been studied. DESIGN AND METHODS In order to clarify the potential impact of TLR4 polymorphisms on the predisposition for DM2 as well as on diabetes-related IHD vulnerability, the distribution of the mutant TLR4 Asp299Gly and Thr399Ile alleles in 286 DM2 patients and 413 non-DM2 controls with or without IHD, was examined. RESULTS Mutant alleles were predominantly detected in 79/413 non-diabetic individuals versus 15/286 DM2 patients (P<0.0001). The rates of positivity for mutant alleles were similar among diabetic patients with or without IHD (7/142 vs 8/144, P>0.1), whereas they proved different among non-diabetic individuals with or without IHD (39/145 vs 40/268, P=0.004). Following multivariate analysis, the difference between diabetic and non-diabetic subjects, with regard to TLR4 mutations alone, remained significant (P=0.04). CONCLUSIONS Mutant TLR4 alleles confer protection against DM2. However, their presence does not seem to play any role, protective or aggravating, in the manifestation of IHD either in diabetic or in non-diabetic individuals.

Influence of Interleukin 1α (IL-1α), IL-4, and IL-6 Polymorphisms on Genetic Susceptibility to Chronic Osteomyelitis

ABSTRACT The association between cytokine gene polymorphisms and chronic osteomyelitis was investigated in order to determine whether genetic variability in cytokine genes predisposes to osteomyelitis susceptibility. Significant genotypic and allelic associations were observed between interleukin 1α (IL-1α) −889-C/T, IL-4 −1098-G/T and −590-C/T, and IL-6 −174-G/C polymorphisms and osteomyelitis in the Greek population, pointing towards their potential involvement in osteomyelitis pathogenesis.

Telomerase reverse transcriptase mRNA expression in peripheral lymphocytes of patients with chronic HBV and HCV infections

Summary.  Telomerase activity is present at low levels in peripheral lymphocytes (PL) and is upregulated upon activation, possibly protecting PL from telomere shortening. As decreased telomere length is considered a sign of cellular senescence, telomerase may, therefore, play an important role on immune function, organ regeneration and carcinogenesis. So far, quantification of human telomerase reverse transcriptase levels (hTERT) in PL, has not been reported. We determined hTERT mRNA levels in PL of hepatitis B virus (HBV) and hepatitis C virus (HCV) patients, in an attempt to address whether hTERT transcripts in PL are altered in these viral diseases, which are characterized by immune dysfunction and increased incidence of hepatocarcinogenesis. hTERT mRNA levels in PL of HBV (n = 17), HCV (n = 24) patients and healthy controls (n = 22) were quantified by real‐time polymerase chain reaction. We observed significantly lower hTERT mRNA levels in HBV and HCV patients compared with healthy individuals (P < 0.05). hTERT mRNA levels were not associated with the patients’ clinical status (inactive, hepatitis and cirrhosis). Also no correlation was observed between hTERT mRNA expression, and HBV and HCV replicative activity. In the inactive group (n = 18) we observed a negative correlation between hTERT mRNA expression and disease duration (rs = −0.52, P < 0.03). We performed for the first time an accurate quantification of hTERT mRNA expression in PL of HBV and HCV patients. The observed low levels of hTERT mRNA expression in the above patients may suggest its involvement in the immunopathogenesis of chronic viral hepatitis.

Association of KLOTHO Gene Polymorphisms with Knee Osteoarthritis in Greek Population

Based on the fact that klotho‐deficient mice exhibit multiple aging phenotypes, including osteopenia and subchondral sclerosis of joints and on the recent observation that KLOTHO gene plays an important role in calcium/phosphate homeostasis, we explored the possibility whether human KLOTHO gene polymorphisms are associated with osteoarthritis (OA). A total of 752 individuals participated in the study. The knee OA group consisted of 369 patients; 298 women (mean age 65.9 ± 8.2; range 40–92 years) and 71 men (mean age 65.7 ± 9.1; range 30–82 years). The control population consisted of 383 subjects; 231 women (mean age 65.8 ± 8.4; range 35–90 years) and 152 men (mean age 61.5 ± 9.3; range 28–87 years). Four SNPs—G395A in the promoter region, G1110C in exon 2, C1818T and C2298T in exon 4—were genotyped. A significant genotypic and allelic association was observed in SNP C2998T and knee OA, while genotype GA of SNP G395A was significantly associated (p = 0.039) with knee OA in females only. For the first time, an association was observed between SNPs G395A and C2998T of the KLOTHO gene and knee osteoarthritis implicating KLOTHO in OA pathogenesis.

Real‐time quantification of human telomerase reverse transcriptase mRNA in liver tissues from patients with hepatocellular cancer and chronic viral hepatitis

Summary.  We determined, for the first time, the human telomerase reverse transcriptase (hTERT) mRNA expression, using real‐time quantitative PCR, in liver tissues from patients with hepatocellular cancer (HCC; n = 13), chronic hepatitis B (n = 19) and C (n = 13). Liver tissues from the 45 patients and 17 patients without liver disease in whom liver biopsy was performed during cholecystectomy (control group), were investigated for telomerase activity (TA) and hTERT mRNA expression using the LightCycler technology. TA was detected in all HCC tissues compared with 15.6% of chronic hepatitis (P < 0.001) and none of controls (P < 0.001). TA levels and hTERT mRNA were higher in HCC compared with chronic hepatitis (P < 0.001) and normal livers (P < 0.001). hTERT mRNA expression was correlated with TA (P < 0.05). Chronic hepatitis patients who tested negative for TA and hTERT mRNA had significantly lower disease duration (58 ± 85 months) compared with those tested positive (144 ± 50 months; P < 0.05). Detection of TA and quantification of hTERT mRNA expression in liver tissues could be useful and additional markers for HCC diagnosis and may serve as prognostic markers for HCC development in chronic viral hepatitis patients. However, we were not able to draw general conclusions at this moment, as the number of chronic hepatitis patients positive for hTERT mRNA was relatively small. Real‐time quantification of hTERT mRNA expression as a diagnostic/prognostic marker in patients with chronic hepatitis B and C and its relationship with hepatocarcinogenesis needs further evaluation.

Correlation between radiation-induced telomerase activity and human telomerase reverse transcriptase mRNA expression in HeLa cells

Purpose: To quantify and correlate human telomerase reverse transcriptase (hTERT) mRNA expression with telomerase activity (TA) after ionizing irradiation of HeLa cells. Materials and methods: TA and hTERT mRNA expression were evaluated, at 24-h intervals, in HeLa cells cultured for up to 144 h, before and after treatment with increasing doses of 6 MV photon ionizing radiation (5 – 20 Gy), using the telomeric repeat amplification protocol (TRAP) assay and real-time reverse transcriptase polymerase chain reaction (RT-PCR), respectively. Cell viability was determined using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. A prototype phantom was constructed for accurate irradiation of HeLa cells. Results: Treated cells showed a decrease in viability with increasing radiation dose, and a correlation was observed with post-treatment period. TA and hTERT mRNA expression of HeLa cells increased for the first 24 h after irradiation. The maximal increases were approximately two times the un-irradiated cell levels at 24 h post-irradiation, followed by a decrease and a return to the control levels 72 h post-irradiation. The time-course of telomerase activation after 24 h, differed among radiation doses. A dose-dependent G2/M arrest was observed 24 h post-irradiation, along with an increase in polyploidy 48 h post-irradiation and afterwards. Conclusion: A correlation between TA and hTERT mRNA expression and a radiation induced cell cycle dependent modification of hTERT mRNA expression was established for the first 24 h post-irradiation.

Sequence-based genotyping HPV L1 DNA and RNA transcripts in clinical specimens

We developed a direct sequence-based genotyping method to detect single and multiple HPV L1 DNA and RNA types in genital and dermatological specimens. Our method couples PCR amplification of a highly conserved HPV L1 segment using a broad spectrum-generic primer cocktail mix with automated sequencing of amplified PCR products, followed by GenBank sorting of sequencing data. We genotyped 5 skin and 30 cervical HPV DNA-positive specimens using this method and established its first experimentally derived working cutoff value with the aid of commercial hybridization-based techniques. We suggest that sequence-based genotyping of appropriately amplified DNA and RNA products may serve as a primary HPV detection method in dermatological specimens. It can be applied as an all-purpose genotyping method for rare HPV types not detectable by commercial hybridization-based techniques and for sorting multiple HPV infections by order of prevalence.

Impact of TLR‐4 Polymorphisms on Circulating Levels of Antibodies Against Helicobacter pylori

Dear Editor, Over the years, Helicobacter pylori infection has been associated with a variety of intestinal, as well as extraintestinal disorders [1], thus underlining the need for the development and application of successful diagnostic and therapeutic strategies [2]. Among the procedures used for the diagnosis of infection from this Gram-negative bacterium, serology antibodies against H. pylori is one of the most commonly applied in research and clinical practice [2]. On the other hand, various factors capable of modifying immune responses against pathogens are currently known, including polymorphisms of toll-like receptors (TLR) [3,4]. These molecules and their polymorphic alleles have been implicated in the recognition of several bacterial components i.e. lipopolysaccharides of Gram-negative bacteria from TLR-4 [3–5]. To investigate whether the presence of polymorphic alleles could influence the production of antibodies against H. pylori, antibody titers were compared between H. pylori-infected individuals, with either wild-type or mutant TLR-4 genotypes. The TLR-4 Asp299Gly and Thr399Ile polymorphic alleles were genotyped using a polymerase chain reaction (PCR) and direct sequencing, in 246 individuals with documented, via both campylobacter-like organism (CLO) test and histology, H. pylori infection. For the purposes of the PCR amplification, a forward, 5¢-TCTAGAGGGCCTGTGCAATT-3¢, and a reverse primer, 5¢-TGAAACTCACTCATTTGTTTCAA-3¢, were used. Using an enzyme-linked immunosorbent assay (ELISA, Enzygnost; Dade Behring Marburg GmbH, Marburg, Germany), IgG and IgA antibodies against H. pylori were determined. All study participants lacked any disorder that could alter the immune response and were not under immuno-modulatory or -suppressive medication. A total of 40 individuals were tested positive for both mutant alleles, heterozygotes, while the rest 206 were homozygotes for the wild-type allele. The simultaneous presence –cosegregationof TLR-4 Asp299Gly and Thr399Ile alleles, in our study was 100%. The mean ± SEM of anti-H. pylori IgG titers were 51.90 ± 4.927 U ⁄ mL, in individuals with the wild-type genotype, and 48.94 ± 9.878 U ⁄ mL, in the group carrying the mutant alleles (p > .05). On the contrary, a statistically significant difference was recorded when antiH. pylori IgA titers were compared between carriers of wild-type (11.61 ± 1.330 U ⁄ mL) and mutant polymorphic alleles (5.887 ± 0.6506 U ⁄ mL) (p < .001). Evidently, lower IgA levels were found in the group of TLR-4 Asp299Gly and Thr399Ile gene carriers. This observation does not come entirely as a surprise, because interactions between TLR-4 and Gram-negative bacteria have already been described [3–5]. The impact of the TLR-4 Asp299Gly and Thr399Ile polymorphic alleles, however, on immune responses and especially those triggered by pathogens, has been debated [3]. One of the major reasons for the lack of unanimity regarding this matter was that the simultaneous presence of the two alleles did not seem to increase susceptibility or alter the cytokine response to microorganisms [3]. The results from this study seem to contradict the notion that the cosegregated mutant alleles ‘‘behave’’ pretty much like the wild-type allele. As for the rather selective downregulation of IgA response against H. pylori, it could be hypothesized that it reflects a defect or a dysregulation in the mucosal defense against H. pylori. Besides, the production of IgAs, circulating and mucosal, which represent the most common immunoglobulin subclass in the mucosa, can be triggered by TLR-facilitated recognition of microorganisms, in the intestinal epithelium [4,5]. In addition, a similar blunt antibody production has been reported for anti-chitobioside (ACCA) and anti-outer membrane porin (Omp) IgA, in patients with inflammatory bowel disease carrying the TLR-4 Asp299Gly allele [6]. This, however, is the first attempt to record a potential interaction between widely studied polymorphisms, tightly linked to the host’s immune responses toward Gram-negative bacteria, and H. pylori. In view of the notion that the presence of these specific TLR-4 polymorphic alleles could reduce the diagnostic utility of H. pylori serology, our findings are not consistent with this remark, because the IgG response against H. pylori remained intact. All in all, it seems that the idea that the host’s genetic background can ‘‘carve’’ the reaction toward environmental challenges is once more verified this time via the suppression of anti-H. pylori Helicobacter ISSN 1523-5378