In vivo expression of innate immunity markers in patients with Mycobacterium tuberculosis infection.

BACKGROUND: Toll-like receptors (TLRs), Coronin-1 and Sp110 are essential factors for the containment of Mycobacterium tuberculosis infection. The purpose of this study was to investigate the in vivo expression of these molecules at different stages of the infection and uncover possible relationships between these markers and the state of the disease. METHODS: Twenty-two patients with active tuberculosis, 15 close contacts of subjects with latent disease, 17 close contacts of subjects negative for mycobacterium antigens and 10 healthy, unrelated to patients, subjects were studied. Quantitative mRNA expression of Coronin-1, Sp110, TLRs-1,-2,-4 and -6 was analysed in total blood cells vs an endogenous house-keeping gene. RESULTS: The mRNA expression of Coronin-1, Sp110 and TLR-2 was significantly higher in patients with active tuberculosis and subjects with latent disease compared to the uninfected ones. Positive linear correlation for the expression of those factors was only found in the infected populations. CONCLUSIONS: Our results suggest that the up-regulation of Coronin-1 and Sp110, through a pathway that also includes TLR-2 up-regulation may be involved in the process of tuberculous infection in humans. However, further studies are needed, in order to elucidate whether the selective upregulation of these factors in the infected patients could serve as a specific molecular marker of tuberculosis.

Flow cytometry analysis of CD4+IFN-γ+ T-cells for the diagnosis of mycobacterium tuberculosis infection.

BACKGROUND: CD4+ cells expressing Interferon-γ (IFN-γ), following stimulation with specific mycobacterial antigens, identified with flow cytometry (FCM-CD4+IFN-γ+), is a new method for the diagnosis of Mycobacterium tuberculosis (MTB) infection. The aim of this study is to investigate the performance of FCM-CD4+IFN-γ+ in comparison with tuberculin skin test (TST) and Quantiferon TB Gold In-Tube (QFT-G-IT) in the diagnosis of latent MTB infection (LTBI), in close contacts and in patients with rheumatic diseases under treatment with anti-TNFa and other biologic agents. METHODS: TST, QFT-G-IT, and FCM-CD4+IFN-γ+ were performed in 56 immunocompetent close contacts and in 65 medically immunosuppressed patients under biologic treatment. RESULTS: In close contacts, 63% were FCM-CD4+IFN-γ+ ESAT-6(+), 70% FCM-CD4+IFN-γ+ PPD(+), 41% QFT-G-IT(+) and 57% TST(+). FCM-CD4+IFN-γ+ ESAT-6 was the only test that was strongly correlated to the exposure time to infection. In the immunosuppressed group, 49% were FCM-CD4+IFN-γ+ ESAT-6(+), 62% FCM-CD4+IFN-γ+ PPD(+), 4.6% QFT-G-IT(+), and 18% TST(+). CONCLUSION: FCM-CD4+IFN-γ+ assays are more sensitive than QFT-G-IT and TST for the diagnosis of LTBI in close contacts and in immunosuppressed patients under anti-TNF-a treatment. FCM-CD4+IFN-γ+ is not affected by the chronic use of biologic agents. © 2015 International Clinical Cytometry Society.

Intracellular ESAT-6: A new biomarker for Mycobacterium tuberculosis infection.

BACKGROUND: Early secreted antigenic target 6 (ESAT-6) is a virulent factor of Mycobacterium tuberculosis (MTB). The identification of intracellular (i/c) ESAT-6 in host cells would be a direct marker of MTB infection. We developed a method to detect i/cESAT-6 by flow cytometry. The aim of this study is to investigate the expression of i/cESAT-6 in the host cells of individuals with MTB infection. METHODS: The expression of i/cESAT-6 was examined in the blood of 58 active TB patients, in 10 naïve to TB infection controls, in 17 patients who completed anti-TB treatment, and in 56 close contacts with an index TB case. Additionally, it was examined in the sputum of 12 active TB patients. RESULTS: The i/cESAT-6 was positively detected in the blood of 52 out of 58 (90%) active TB patients. All naïve to TB infection controls were negative. Three out of 17 (18%) patients at the end of anti-TB treatment were positive. Twenty-six out of 56 (46%) close contacts tested positive. The i/cESAT-6 was detected in all culture positive TB sputum specimens. CONCLUSIONS: The i/cESAT-6 is a promising biomarker of MTB infection that could be used in the evaluation of active TB patients and in the diagnosis of latent TB infection. Further studies are needed to validate its potential diagnostic role. © 2014 International Clinical Cytometry Society.

Inducing bioactivity of dental ceramic/bioactive glass composites by Nd:YAG laser.

OBJECTIVES: Aims of this study were to investigate the optimal conditions of laser irradiation of a novel Bioactive Glass/Dental Ceramic-BP67 composite for acceleration of hydroxyapatite-HA formation and to assess cellular responses on the precipitated HA region. METHODS: BP67 (Bioactive Glass: 33.3%, Dental Ceramic: 66.7%) was fabricated by the sol-gel method. A laser assisted biomimetic-LAB process was applied to BP67 sintered specimens immersed in 1.5-times concentrated simulated body fluid-1.5×-SBF. The effect of various energy densities of pulsed nanosecond Nd-YAG (1064nm) laser and irradiation exposure times (30min, 1 and 3h) were evaluated for HA precipitation. The HA film was characterized by FTIR, XRD, SEM and micro Raman techniques. ICP-AES was used for revealing changes in chemical composition of the 1.5×-SBF during irradiation. Cell viability and morphological characteristics of periodontal ligament fibroblasts-PDLFs, human gingival fibroblasts-HGFs and SAOS-2 osteoblasts on the HA surface were evaluated by MTT assays and SEM. RESULTS: At optimal energy fluence of 1.52J/cm2 and irradiation time for 3h followed by immersion in 1.5×-SBF at 60°C, a dense HA layer was formed on laser-irradiated BP67 within 7 days. The resulting HA film was tightly bonded to the underlying substrate and had mineral composition similar to cementum. MTT assay showed a consistent reduction of cell proliferation on the HA layer in comparison to conventional control ceramic and BP67 for all 3 cell lines studied. SIGNIFICANCE: These findings suggest LAB is an effective method for acceleration of HA formation on materials with low bioactivity, while cellular responses need further investigation.

MIRU-VNTR typing of drug-resistant tuberculosis isolates in Greece.

The increasing immigration rate in Greece from countries with a high prevalence of Mycobacterium tuberculosis (MTB) and multidrug-resistant tuberculosis (MDR-TB) may have an impact οn the number of MDR-TB cases in Greece. The aim of this study was to genotypically characterize the MTB isolates from patients with pulmonary drug-resistant tuberculosis (DR-TB) in Greece, and to determine whether there is any association between the prevalent genotypes and drug resistance. Fifty-three drug-resistant MTB strains isolated from culture specimens of clinical material from native Greeks and immigrant patients with pulmonary tuberculosis were genotyped using the mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) method. The phylogenetically distinct groups of isolates identified were: the Beijing (34%), the LAM (11%), the Haarlem (24.5%), the Uganda I (9.4%), the Ural (3.8%), the Delhi/CAS (9.4%) and the Cameroon (3.8%) families. Greek patients were more likely to have monoresistant and polyresistant TB with the most prevalent isolates belonging to the Haarlem family. Among foreign-born patients with MDR-TB, the most prevalent genotypes belonged to the Beijing family. MIRU-VNTR rapidly obtained clinically useful genotyping data, by characterizing clonal MTB heterogeneity in the isolated strains. Our results underline the need for more effective antituberculosis control programs in order to control the expansion of DR-TB in Greece.

Malakoplakia of the lung caused by Pasteurella multocida in a patient with AIDS.

We present a case of malakoplakia of the lung caused by Pasteurella multocida in a patient with AIDS. This is the first report to implicate P. multocida in the pathogenesis of malakoplakia.

Theophylline treatment may adversely affect the anoxia-induced erythropoietic response without suppressing erythropoietin production.

OBJECTIVE: To investigate the influence of theophylline on erythropoiesis in chronic obstructive pulmonary disease (COPD) and explore the potential underlying mechanisms. METHODS: We evaluated the haematological parameters and erythropoietin (EPO) values in 38 COPD patients, 18 of which had been treated with theophylline (8 mg/kg daily) for at least 1 year, and the other 20 had never received this drug; 38 sex- and age-matched healthy volunteers served as controls. We further studied the development of BFU-E (bursts forming units of erythrocyte precursors) -derived colonies in semisolid methylcellulose cultures in blood samples from 7 patients randomly selected from both groups. In addition, we studied the effects of theophylline on the erythroid cell development by adding this agent to erythroid cell cultures from 6 healthy volunteers at various concentrations. RESULTS: Haemoglobin values were found to be significantly lower in COPD patients treated with theophylline than in those untreated ( P<0.05). Both groups of patients exhibited significantly higher haemoglobin values than normal subjects ( P<0.01 and P<0.001 for treated and untreated patients, respectively). Serum EPO levels did not differ among the three studied groups. Unlike untreated patients and controls, the serum of the theophylline-treated patients produced a significant growth inhibition of erythroid bursts ( P<0.05); the in vitro use of theophylline showed a concentration-dependent inhibition ( P<0.001). CONCLUSION: Our findings confirm the decrease of red cell production, which occurs following administration of theophylline, exclude the possibility of decreased EPO synthesis and suggest a direct inhibitory action of theophylline on erythropoiesis.