In vivo study of pro-inflammatory cytokine changes in serum and synovial fluid during treatment with celecoxib and etoricoxib and correlation with VAS pain change and synovial membrane penetration index in patients with inflammatory arthritis.

OBJECTIVES: To determine the impact of celecoxib and etoricoxib therapy on serum and synovial fluid levels of IL-1ß, IL-6, TNF-α, sTNFR1, sTNFR2 and IL-1Ra in patients with inflammatory arthritis. To determine the correlation between cytokine changes and synovial membrane penetration index of the study drugs, and pain VAS change. METHODS: Fifty-one patients with inflammatory synovial fluid accumulation in a knee joint (33 women), randomized on 3 groups of 17 each: 100 mg b.i.d. celecoxib treated group, 90 mg o.d. etoricoxib treated group, and the control group with no NSAID treatment. Cytokines serum and synovial fluid levels as well as membrane penetration index were assessed prior and after treatment. RESULTS: Celecoxib led to decrease of both synovial fluid and serum levels of IL-6 (p=0.017 and p=0.003, respectively). In the etoricoxib treated group synovial fluid IL-6 concentration was significantly decreased after treatment (p=0.019). Correlating the study drugs penetration index with the change of cytokines and their receptors levels, positive correlation was found with the reduction of synovial fluid IL-1ß for the celecoxib (p=0.032) and with the increase of synovial fluid sTNFR1 for the etoricoxib group (p=0.028). Pain VAS reduction was positively correlated with decrease of synovial fluid IL-1ß (p=0.041) and IL-6 levels (p<0.005) and negative with synovial fluid sTNFR1 changes (p=0.045) in celecoxib group, and negative with serum TNF-α decrease (p=0.044) in the etoricoxib group. CONCLUSION: Our results suggest that celecoxib and etoricoxib inhibit the inflammatory cytokines, mostly in synovial fluid but also in serum, causing through this mechanism, decrease of inflammation, irrespective to COX-2 inhibition.

Detection of the tetM resistance determinant among phenotypically sensitive Ureaplasma species by a novel real-time PCR method.

The study aimed to identify the proportion of tetM-positive Ureaplasma spp. isolates phenotypically susceptible to tetracycline by real-time PCR. Ureaplasma spp. strains of urogenital origin were isolated from 100 female or male adults on A7 agar plates. The presence of Ureaplasma was confirmed by the presence of urease gene by a novel real-time PCR method. Genotyping and sensitivity to tetracyclines were examined using commercial methods. The tetM gene was detected by a novel real-time PCR method especially designed for this study. Ureaplasma parvum was isolated from 87 of the specimens; Ureaplasma urealyticum, from 12; and both species were isolated from a single specimen. All isolates were phenotypically susceptible to tetracyclines. Thirty-five strains were tetM carriers; 29 (82.9%), U. parvum; 5 (14.3%), U. urealyticum; and 1 (2.9%), U. parvum/U. urealyticum. No statistically significant difference was observed between the 3 groups. Four (40%) tetM carriers were isolated from 10 symptomatic men; 11 (32.4%), from 34 symptomatic women; and 20 (35.7%), from 56 asymptomatic women. No statistically significant difference was observed between the 3 groups. The tetM determinant is detected in 35% of phenotypically susceptible to tetracycline Ureaplasma spp. Greek isolates. The use of a real-time PCR technique is particularly helpful, as it makes its detection easy; cost-effective; rapid; and, therefore, more convenient for the surveillance of the dissemination of the tetM resistance gene.

Comparison of interleukin-6 and matrix metalloproteinase expression in the subretinal fluid and the vitreous during proliferative vitreoretinopathy: correlations with extent, duration of RRD and PVR grade.

INTRODUCTION: The full extent of IL-6 involvement in PVR pathophysiology has not yet been comprehensively investigated. The aim of this study was the comparison of the IL-6 effect on MMP expression between SRF and the vitreous in the context of RRD complicated by PVR. MATERIALS AND METHODS: Thirty-one SRF samples from 31 eyes of 31 consecutive patients suffering from RRD with PVR were collected during treatment by scleral buckling. Twenty-eight vitreous samples from 28 eyes of 28 RRD patients with PVR were collected during surgical management with pars plana vitrectomy (PPV). Enzyme Linked Immunosorbent Assay was employed for the measurement of MMP-1, -3, -8 and TIMP-1 concentrations (in ng/ml). MMP gelatinolytic activity was determined with the use of gelatin zymography analysis using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: Correlation analysis in the SRF revealed a significant correlation between MMP-1/IL6 and RRD duration. Regression analysis in the SRF revealed a significant correlation between the MMP-9/IL-6 and RRD extent. In the same fluid, with respect to PVR grade, ANOVA revealed a significant relationship with the proMMP-2/IL-6, MMP-2/IL6 and TIMP-1/IL-6 ratios. Graphical representation of the results revealed that, between the SRF and vitreous groups, significant peak values were observed for all MMP/IL-6 and TIMP-1/IL-6 ratios included in this study with the exception of the MMP-2/IL-6 ratio. CONCLUSIONS: It appears that there is a significant correlation between the presence of IL-6 and MMP/TIMP ratio in the SRF, indicating that IL-6 may contribute to the increased MMP/TIMP ratio during PVR.

Chemokine CXCL-1 expression in the subretinal fluid during rhegmatogenous retinal detachment.

PURPOSE: To investigate the expression of chemokine CXCL-1 in the subretinal fluid (SRF) during rhegmatogenous retinal detachment (RRD) and identify potential correlations with number of quadrants involved and duration of the detachment. METHODS AND RESULTS: Inclusion criteria were patients aged 18 years or older and primary RRD possibly complicated by proliferative vitreoretinopathy (PVR). CXCL-1 levels were measured in 36 SRF samples from 36 RRD patients. Mean CXCL-1 levels (102 ± 37 pg/mL) were significantly higher (p = 0.050) compared to controls. CXCL-1 levels correlated significantly with age (p = 0.001) and RRD duration (p = 0.002). Maximum CXCL-1 levels coincided with total RRD, 29- to 60-day duration and PVR grade C. CONCLUSIONS: The findings of this study may contribute to increased understanding regarding the role of CXCL-1 during the onset and progression of the wound healing process in the context of RRD and PVR.

Salivary concentration of free LL-37 in edentulism, chronic periodontitis and healthy periodontium.

OBJECTIVE: The antimicrobial peptide LL-37, a component of innate immunity, has an important role in maintaining oral health. This study aimed to investigate the concentration of free LL-37 in whole saliva of periodontally healthy, edentulous and chronic periodontitis subjects. DESIGN: Unstimulated whole saliva was sampled from 154 subjects (76 periodontally healthy, 20 edentulous, and 58 subjects with chronic periodontitis). All participants were in good general health. The salivary LL-37 was determined by ELISA. RESULTS: The median salivary concentrations of free LL-37 were 30.5, 22.5, and 1.8ng/ml for the healthy, the chronic periodontitis and the edentulous group, respectively. The differences in concentration between the edentulous and the others were statistically significant (Mann-Whitney test, p<0.001). In the healthy subjects, women displayed significantly higher peptide concentrations compared to men (Mann-Whitney test, p<0.05). The intra-subject variation in LL-37 concentration was wider for the healthy (range 0.75-285ng/ml) and chronic periodontitis patients (range 1-207ng/ml) than for the edentulous subjects (range 0.15-4.4ng/ml). CONCLUSIONS: The present findings show that edentulism correlates with a substantial decrease in salivary levels of free LL-37, thus indicating the considerable contribution of the gingival tissues in the secretion of the peptide in the oral environment.

Interleukin-6 and matrix metalloproteinase expression in the subretinal fluid during proliferative vitreoretinopathy: correlation with extent, duration of RRD and PVR grade.

PURPOSE: To investigate interleukin (IL)-6 protein levels in the subretinal fluid (SRF) of patients with rhegmatogenous retinal detachment (RRD) complicated by proliferative vitreoretinopathy (PVR); to correlate the IL-6 levels with matrix metalloproteinases (MMP)-1, -2, -3, -8, -9 and tissue inhibitor of metalloproteinases (TIMP)-1 with respect to RRD extent, duration and PVR grade. METHODS: Thirty-one SRF samples from 31 eyes of 31 patients with RRD complicated with PVR and five SRF samples from five eyes of five patients suffering from RRD not complicated with PVR were collected during treatment by scleral buckling. Enzyme-Linked Immunosorbent Assay was employed for the measurement of IL-6, MMP-1, -3, -8 and TIMP-1 levels while the enzymatic activity of MMP-2 and MMP-9 was assessed by gelatin zymography. RESULTS: Protein levels of IL-6 (p=0.050), MMP-1 (p=0.001), MMP-3 (p=0.005), MMP-8 (p=0.003), TIMP-1 (p=0.001) as well as enzymatic activity of proMMP-2 (p=0.001), MMP-2 (p=0.023) and MMP-9 (p=0.015), were significantly higher in the SRF of PVR patients compared to controls. IL-6 levels correlated significantly with TIMP-1 (r=0.528, p=0.035). Regarding clinical parameters of the detachment, IL-6 levels correlated with RRD extent (r=0.592, p=0.016), but not with RRD duration (p=0.857) and PVR grade (p=0.594). Regression analysis revealed positive correlations between IL-6 and MMP-2. CONCLUSIONS: There was a significant correlation between IL-6 and TIMP-1 levels in the SRF of PVR patients. The findings of this study are in agreement with relevant studies concerning IL-6 involvement in the modulation of MMP expression and are indicative of IL-6 and MMP activity during PVR, mainly that of MMP-2 and TIMP-1.

Salivary concentration of the antimicrobial peptide LL-37 in children.

OBJECTIVE: Antimicrobial peptides are important components of innate immunity, especially in the unique environment of the oral cavity. Lack of the human cathelicidin LL-37 has been implicated in severe periodontitis, whilst high salivary levels of LL-37 seem to increase caries resistance. Limited data exists about the concentration of LL-37 in saliva of young children. In this study, the salivary concentration of LL-37 was examined in relation to age, gender, type of dentition (primary, mixed or permanent) and caries experience of children. DESIGN: Unstimulated whole saliva was collected from 49 systemically healthy and gingivitis free children aged 2-18 years old. Their caries activity was recorded. The salivary LL-37 concentration was determined by enzyme linked immunosorbent assay (ELISA). RESULTS: LL-37 was detected in all saliva samples. Its concentration varied widely, with girls exhibiting higher peptide levels than boys. A positive correlation of the LL-37 concentration was observed with age. Children with primary dentition had significantly lower peptide concentration than those with mixed or permanent dentition. Significantly lower concentrations of LL-37 were also found in children with high caries activity, compared to caries free children or to children with low to moderate caries activity. CONCLUSIONS: Our results reinforce the belief that LL-37 is an important molecule of immunity in the oral environment and it seems to play a protective role against caries.

Post‒cataract surgery endophthalmitis outbreak caused by multidrug-resistant Pseudomonas aeruginosa.

In June 2010, a severe outbreak of 13 cases of post-cataract surgery endophthalmitis caused by multidrug-resistant Pseudomonas aeruginosa occurred. Pulse-field gel electrophoresis in eye isolates found 95% genetic similarity; however, extensive environmental and carriage investigation revealed no source of infection.

Interleukin-6 and the matrix metalloproteinase response in the vitreous during proliferative vitreoretinopathy.

PURPOSE: To investigate the levels of IL-6 in the vitreous of patients with RRD complicated with PVR and correlate the IL-6 levels with matrix metalloproteinase (MMP)-1,-2,-3,-8,-9 and tissue inhibitor of metalloproteinases (TIMP)-1 with respect to RRD extent, duration and PVR grade. DESIGN: Cohort study. PARTICIPANTS: Twenty-eight vitreous samples from 28 eyes of 28 patients with RRD complicated with PVR. METHODS: Institutional study. Twenty-eight vitreous samples from 28 eyes of 28 patients with RRD complicated with PVR were collected during pars plana vitrectomy (PPV) and were compared to vitreous control samples. IL-6, MMP-1,-3,-8 and TIMP-1 levels were measured using ELISA while enzymatic activity of MMP-2, and -9 was determined employing gelatin zymography. RESULTS: Protein IL-6 (p=0.030), MMP-1 (p=0.003), MMP-3 (p=0.003), TIMP-1 (p=0.001) levels as well as enzymatic activity of proMMP-9 (p=0.013), MMP-9 (p=0.017) and proMMP-2 (p=0.010), were significantly increased in PVR patients as compared to controls. IL-6 levels correlated with MMP-1 (p=0.002), proMMP-2 (p=0.006), MMP-3 (p=0.001) and TIMP-1 (p=0.006). Regression analysis revealed positive correlations between IL-6 and all MMPs and TIMP-1. CONCLUSIONS: Taking into account the previously established effect of interleukins in MMP activity, the findings of this study suggest a role of IL-6 in MMP stimulation during PVR development.

Correlation of matrix metalloproteinase levels with the grade of proliferative vitreoretinopathy in the subretinal fluid and vitreous during rhegmatogenous retinal detachment.

PURPOSE: We investigated the activity of matrix metalloproteinase (MMP)-2 and -9 and their latent pro-forms (proMMP-2, -9), and protein levels of MMP-1, -3, -8 and tissue inhibitor of MMPs (TIMP)-1 in the subretinal fluid (SRF) and vitreous of patients with rhegmatogenous retinal detachment (RRD). Potential correlations with proliferative vitreoretinopathy (PVR) grade were determined. METHODS: Thirty-seven SRF and 32 vitreous samples from RRD patients and nine vitreous samples from human organ donors (controls), were collected and assayed for MMP-1, -3, -8/TIMP-1 levels using enzyme-linked immunosorbent assay (ELISA), and for proMMP-2, -9, MMP-2, -9 activity employing gelatine zymography. RESULTS: ProMMP-2, -9, MMP-1, -3, -9, TIMP-1 were significantly higher in the SRF and vitreous of RRD patients compared to the vitreous of organ donors. MMP-8 levels were higher in RRD patients' SRF. Regarding PVR grade, MMPs and TIMP-1 were differentially present in SRF and vitreous. PVR grade correlated significantly with the levels of MMP-2 in SRF, while proMMP-2, MMP-1, -2, -3, -8, -9 and TIMP-1 levels correlated with PVR grade in the vitreous. CONCLUSION: MMP/TIMP-1 levels are elevated in SRF and vitreous during RRD. Significant correlations between PVR grade and MMP-2 in SRF and proMMP-2, MMP-1, -2, -3, -8, -9 and TIMP-1 levels in vitreous were revealed. Investigation of MMP activity in vitreous may provide more valid conclusions compared to SRF pertaining to the role of the MMPs during RRD. The observations of the present study suggest a possible role for MMPs and TIMP-1 in PVR pathophysiology.

Molecular and phylogenetic analysis of the haemagglutinin gene of pandemic influenza H1N1 2009 viruses associated with severe and fatal infections.

The objectives of this research is molecular and phylogenetic analysis of pandemic influenza A(H1N1) 2009 strains that circulated in northern Greece, focusing on severe or fatal infections, identification of sequence variations in relation with the severity of the illness and comparison of circulating viruses with the vaccine strain. A total of 1598 infections were attributed to the novel influenza A(H1N1) virus. Molecular analysis revealed a number of variations at the HA1 sequences of northern Greek circulating strains, some of which were more frequent in viruses that caused severe or fatal infections. Such mutations, the most common being D222G, demand close monitoring to continuously assess associated risks. Phylogenetic analysis confirmed the close match of the majority of circulating strains with A/California/7/09. However it also reveals a trend of 2010 strains to accumulate amino acid variations and form new plylogenetic clades. Constant molecular surveillance is important to monitor the pathogenicity of circulating strains and evaluate the vaccine efficacy.

Determination of two COX-2 inhibitors in serum and synovial fluid of patients with inflammatory arthritis by ultra performance liquid chromatography-inductively coupled plasma mass spectroscopy and quadrupole time-of-flight mass spectrometry.

The determination of two sulphur-containing drugs, the COX-2 inhibitors celecoxib and etoricoxib, in the serum and synovial fluid of inflammatory arthritis patients, is described using a sensitive ultra performance liquid chromatography-inductively coupled plasma mass spectroscopy (UPLC/ICPMS) method. Confirmation of the identity of the analytes in the samples was also performed by electrospray quadruple time-of-flight mass spectrometry in positive electrospray ionisation mode. The two COX-2 inhibitors were extracted from serum and synovial fluid following dilution with acetate buffer (pH 5) and liquid-liquid extraction (LLE) into ethyl acetate. Extracted samples were then analysed using UPLC/ICPMS with sulphur-specific detection. The limit of detection by UPLC/ICPMS was 0.45 ng/ml of sulphur in both serum and synovial fluid. The UPLC/ICPMS method was applied to the analysis of samples from patients receiving either 200 mg/day of celecoxib (2x 100 mg), 90 mg/day etoricoxib or placebo. The range of concentrations detected in the samples for the two drugs was from 0.3 to 3.3 microg/ml.

Genetic study of the distribution of Greek goat encephalitis virus in Greece.

Tick-borne encephalitis virus (TBEV) and Louping ill virus (LIV) are viruses in the Mammalian tick-borne virus group/genus Flavivirus, causing central nervous system disease. Greek goat encephalitis virus (GGEV), which was isolated from the brain of a newborn goat with neurological symptoms, is currently classified in the TBEV group. The vector of GGEV has not yet been specifically identified but is considered likely to be Ixodes ricinus. A total of 3,144 Ixodidae ticks collected during 2003-2006 from goats and sheep in rural areas of Northern Greece were tested by reverse transcription-polymerase chain reaction (RT-PCR) for the presence of TBEV-specific RNA. Two pools of I. ricinus ticks were shown to be RNA-positive. Sequence analysis showed that the virus was GGEV. The RNA-positive ticks were detected in regions where high prevalence of TBE antibodies in humans was present. Prevalence in ticks varied according to year, season, and geographic region. TBEV is not endemic in Greece, and most probably the seroprevalence of TBE antibodies in humans is due to cross-reactivity to GGEV.

Correlation of the extent and duration of rhegmatogenous retinal detachment with the expression of matrix metalloproteinases in the vitreous.

BACKGROUND: Investigation of the activity of matrix metalloproteinase (MMP)-2 and -9 and protein levels of MMP-1, -3, -8, and the tissue inhibitor of MMPs (TIMP)-1 in the vitreous of patients with rhegmatogenous retinal detachment (RRD) and establishment of potential correlations of MMPs with clinical parameters. METHODS: Thirty-two vitreous samples from patients with RRD and 9 vitreous samples from human organ donors (controls) were assayed for MMP-1,-3, -8, and TIMP-1 levels using enzyme-linked immunosorbent assay and MMP-2 and -9 activity employing gelatin zymography. RESULTS: MMP-1, MMP-3, proMMP-2, proMMP-9, MMP-9, and TIMP-1 were higher in vitreous from patients with RRD as compared to organ donors. Overall, MMPs and TIMPs were differentially expressed in vitreous from RRD with respect to the duration and extent of RRD. Regression analysis for all data indicated that a model consisting of MMP-2 and TIMP-1 could estimate the extent of RRD. CONCLUSION: Levels of MMPs and TIMP-1 studied are elevated in vitreous during RRD. MMP-2 and TIMP-1 may have a more prominent and persistent role than other MMPs in the wound healing process of the retina during RRD. A regression model consisting of MMP-2 and TIMP-1 may prove to be of potential use in providing information for the evaluation of the extent of RRD.

Evaluation of different PCR assays for early detection of acute and relapsing brucellosis in humans in comparison with conventional methods.

Human brucellosis is a significant public health problem in many Mediterranean countries including Greece. The conventional serological methods, as well as blood cultures, have serious limitations, especially in chronic, relapsing, and focal forms of the disease. Four different PCR assays were applied in 4,926 buffy coat, whole-blood, and serum samples received from 200 patients admitted with brucellosis to the Infectious Diseases Hospital, Thessaloniki, Greece, for the rapid diagnosis of acute infection and relapses and compared to blood culture and serological tests (i.e., Wright's seroagglutination test, Coombs' antibrucella test, and the complement fixation test). The four PCR assays had excellent sensitivity and specificity and were able to detect all of the cases of acute disease. Buffy coat and whole blood were the optimal specimens. All four PCR assays were negative in all follow-up samples from 183 patients who had completed a successful treatment and were positive in all follow-up samples from 17 patients who had relapses in the first year after therapy, including the times of the relapses. In conclusion, PCR is a very useful tool for the rapid diagnosis of acute brucellosis and a good marker for the posttreatment follow-up and the early detection of relapses.

Expression of matrix metalloproteinases in the subretinal fluid correlates with the extent of rhegmatogenous retinal detachment.

BACKGROUND: We investigated the activity of matrix metalloproteinase (MMP)-2 and -9 and the protein levels of MMP-1, -3, -8 and the tissue inhibitor of MMPs (TIMP)-1 in the subretinal fluid (SRF) of patients with rhegmatogenous retinal detachment (RRD) and establishment of potential correlations with clinical parameters. METHODS: Thirty-seven SRF from RRD patients and nine vitreous samples from the human eye of organ donors (controls) were collected and assayed for MMP-1,-3,-8 and TIMP-1 levels using ELISA and for MMP-2 and -9 activity employing gelatin zymography. RESULTS: MMP-1, MMP-3, MMP-8, proMMP-2, proMMP-9, MMP-9 and TIMP-1 levels were higher in SRF compared with vitreous fluid. Overall, MMPs and TIMPs were differentially expressed in SRF with respect to duration and extent of RRD, as well as to stage of proliferative vitreoretinopathy. Regression analysis for all data indicated that a model consisting of MMP-3, MMP-8 and proMMP-9 could estimate the extent of RRD. CONCLUSIONS: MMPs and TIMP-1 levels are elevated in SRF during RRD. A regression model consisting of MMP-3, MMP-8 and proMMP-9 may be proved to be of potential use in providing information for evaluation of the extent of RRD.

Epidemiological study of tick-borne encephalitis virus in northern Greece.

Serum samples from 921 apparently healthy individuals living in different prefectures of Northern Greece were investigated for the presence of antibodies against tick-borne encephalitis (TBE) virus. In addition, serum and cerebrospinal fluid samples of 302 patients with central nervous system (CNS) infection were tested for the presence of specific IgG and IgM antibodies and TBE virus RNA. Two percent of the general population was found to have antibodies to the virus, with no significant differences among the age groups. Most of the seropositive individuals were male farmers, while seroprevalence varied among different prefectures (0%-5.8%). TBE was not confirmed by laboratory findings in any of the patients with CNS infection. Results of this study revealed that a flavivirus of the TBE serocomplex is circulating in Greece, yet is not a major public health problem.

Enterovirus meningitis in Greece from 2003-2005: diagnosis, CSF laboratory findings, and clinical manifestations.

Enteroviruses (EVs) are the most commonly identified cause of aseptic meningitis. Rapid detection and characterization of EV meningitis is essential in making decisions for patient management and treatment. A total of 52 cases of acute aseptic meningitis that occurred from March 2003 to April 2005 were investigated for EVs using viral culture and/or molecular methods directly in the cerebrospinal fluid (CSF). EVs were detected in 21 out of 52 (40.4%) patients using reverse transcription-PCR (RT-PCR) and/or tissue culture. EVs were isolated from six out of 37 (16.2%) cultured specimens, while 20 out of 52 (38.4%) specimens yielded positive results when 5'non-coding region (5'NCR) RT-PCR assay was used. One specimen that was culture-positive was RT-PCR-negative. Using the VP1-2A RT-PCR and sequence analysis, 14 of the 21 positive EVs were identified as: four strains of Coxsackie virus B5, five echovirus 11, two echovirus 9, one echovirus 5, one echovirus 14, and one Coxsackie virus A9. Fever, headache, vomiting, and stiff neck were the most pronounced symptoms. Pleocytosis with the predominance of lymphocytes and mild elevated protein levels characterized the CSF specimens. Coxsackie virus B5 and echovirus 11 were the predominant serotypes during the study period. Although there was seasonal enteroviral activity (April-November), cases also occurred in the cold months. The 5'NCR and VP1-2A RT-PCR with sequence analysis were found to be superior to conventional methods for direct diagnosis and the typing of EVs.