Relation of the Types of DNA Damage to Replication Stress and the Induction of Premature Chromosome Condensation

Rozdział książki New Research Directions in DNA Repair Edited by Clark ChenThe work was funded by “POMOST” fellowship from the Foundation for Polish Science (the contract no. POMOST/2011-4/8)

Monocyte response receptors in BCG driven delayed type hypersensitivity to tuberculin.

Tuberculosis (TB) still remains the leading cause of mortality due to bacterial pathogen. The only currently available vaccine against TB, Bacille Calmette-GuĂŠrin (BCG) is at best credited with a 50% overall protective efficacy. Skin testing with purified protein derivative (PPD) from Mycobacterium tuberculosis is the method of detecting BCG-induced cell mediated immunity, in vivo. In the previous study we found that approximately 60% young volunteers with no history of TB, who had been subjected to neonatal BCG vaccination and revaccination(s) at school age, developed delayed type hypersensitivity (DTH) to tuberculin. The remaining volunteers were persistently tuberculin negative. Moreover, we found a significant association between BCG driven development of DTH to PPD and the polymorphism within the CD14 C/T(-159) gene for macrophage receptor recognising mycobacterial compounds. It has suggested that the CD14 gene variants may play a role in the appearance and persistence of DTH to PPD in BCG vaccinated subjects. In order to extend our study on a possible role of CD14 in BCG driven DTH response to PPD, we measured the expression of mCD14 on macrophages, stimulated or not stimulated with mycobacterial antigens, and the serum levels of sCD14. Considering the importance of CD14 - TLR2/TLR4 interactions in macrophage signalling, we determined the polymorphism of TLR2 and TLR4 genes as well as macrophage expression of TLR2 for the volunteers with and without skin reactivity to PPD. We observed a subtle but significant decrease in CD14 density on adherent monocytes from tuberculin positive versus tuberculin negative volunteers. However, we found no difference in CD14 density on monocytes enriched in CD14+ cells using anti-CD14 mAb coupled to magnetic beads. A significant increase in CD14 density was observed on macrophages stimulated with PPD and LPS but not with live BCG bacilli. However, this increase as well as serum levels of soluble sCD14 were similar in the volunteers with and without skin reactions to PPD. Thus, our suggestion on the role of CD14 in the generation of DTH to tuberculin in BCG vaccinated subjects should be further explored. The most important CD14 co-receptors are Toll-like receptors (TLRs) which activate nuclear factors for the production of inflammatory cytokines. However, we could see no association between the polymorphisms of TLR4 (Asp299Gly and Thr399Ile) and TLR2 genes (Arg753Gln and Arg677Trp) and skin responses to PPD. Also, the TLR2 density was similar on monocytes from tuberculin negative and tuberculin positive volunteers

Interferon alpha and rapamycin inhibit the growth of pheochromocytoma PC12 line in vitro

Wstęp: Guzy chromochłonne (pheochromocytoma/paraganglioma) należą do łagodnych lub złośliwych nowotworów neuroendokrynnych.Wobec niezadowalającej skuteczności standardowych sposobów leczenia pacjentów z rozsianą postacią choroby, wciąż poszukuje się nowych metod terapii, w tym możliwości skutecznego leczenia celowanego. W związku ze wzmożonym unaczynieniem tych nowotworów, preparaty o działaniu antyangiogennym mogą potencjalnie stanowić nową grupę leków stosowanych w leczeniu pheochromocytoma/paraganglioma.Materiał i metody: W badaniu oceniano wpływ różnych angiomodulatorów: VEGF (naczyniowo-środbłonkowy czynnik wzrostu) orazpięciu endo- i egzogennych czynników antyangiogennych (endostatyna; IFN-alfa [interferon alfa]; rapamycyna — inhibitor szlaku mTOR[mammalian target of rapamycin]; JV1-36 and SU5416 [semaxinib]) na wzrost szczurzej linii pheochromocytoma PC12.Wyniki: IFN-alfa (105 U/mL) silnie hamował wzrost komórek PC12 w hodowli 72 h, nasilając apoptozę i hamując cykl komórkowy. Rapamycynaw szerokim zakresie stężeń (10-5 to 10-8 M) nieznacznie zmniejszała żywotność komórek PC12, a w stężeniu 10-5 M także hamowałaich proliferację. VEGF, endostatyna oraz JV1-36 nie wpływały na wzrost lini PC12.Wnioski: W badaniu po raz pierwszy wykazano, że IFN-alfa hamuje wzrost linii komórkowej pheochromocytoma PC12, a także potwierdzonohamujący wpływ rapamycyny wobec tej linii komórkowej. Uzyskane wyniki sugerują zatem, że IFN-alfa oraz inhibitory szlakumTOR mogą być potencjalnie skuteczne w leczeniu złośliwych postaci guzów chromochłonnych i zachęcają do dalszych badań w tymkierunku.(Endokrynol Pol 2013; 64 (5): 368–374)Introduction: Pheochromocytomas are benign or malignant neuroendocrine tumours. The unsatisfactory efficacy of the traditionaltherapeutic methods for patients with metastatic disease results in a continuing search for more effective and targeted agents. Due to theincreased vascularisation of these tumours, inhibitors of angiogenesis could be potentially a new group of drugs in pheochromocytoma/paraganglioma therapy.Material and methods: The aim of this study was to evaluate the influence of angiomodulators: VEGF (vascular endothelial growth factor)and five endogenous and exogenous antiangiogenic compounds (endostatin; IFN-alpha [interferon alpha]; rapamycin — mTOR [mammaliantarget of rapamycin] inhibitor; JV1-36 and SU5416 (semaxinib]) on the growth of rat pheochromocytoma PC12 cell line.Results: IFN-alpha (105 U/mL) strongly inhibited PC12 growth in a 72 h culture, increasing apoptosis and arresting the cell cycle. Rapamycinin a wide range of concentrations (10-5 to 10-8 M) induced a slight inhibitory effect on PC12 viability and decreased cell proliferation at theconcentration of 10-5 M. VEGF, endostatin and JV1-36 did not influence the growth of PC12.onclusions: The study has shown for the first time that IFN-a inhibited the growth of pheochromocytoma PC12 line and confirmed theinhibitory action of rapamycin on these cells. The results suggest that IFN-alpha and mTOR inhibitors could be potentially effective in thetherapy of malignant pheochromocytoma, and encourage further study in this field.(Endokrynol Pol 2013; 64 (5): 368–374

Interaction of Helicobacter pylori with C-Type Lectin Dendritic Cell-Specific ICAM Grabbing Nonintegrin

In this study we asked whether Helicobacter pylori whole cells and lipopolysaccharide (LPS) utilize sugar moieties of Lewis (Le) antigenic determinants to interact with DC-SIGN (dendritic cell specific ICAM grabbing nonintegrin) receptor on dendritic cells (DCs). For this purpose the soluble DC-SIGN/Fc adhesion assay and the THP-1 leukemia cells with induced expression of DC-SIGN were used. We showed that the binding specificity of DC-SIGN with H. pylori LeX/Y positive whole cells and H. pylori LPS of LeX/Y type was fucose dependent, whereas in LeXY negative H. pylori strains and LPS preparations without Lewis determinants, this binding was galactose dependent. The binding of soluble synthetic LeX and LeY to the DC-SIGN-like receptor on THP-1 cells was also observed. In conclusion, the LeXY dependent as well as independent binding of H. pylori whole cells and H. pylori LPS to DC-SIGN was described. Moreover, we demonstrated that THP-1 cells may serve as an in vitro model for the assessment of H. pylori-DC-SIGN interactions mediated by LeX and LeY determinants

Recombinant BCG to Enhance Its Immunomodulatory Activities

The bacillus Calmette–Guérin (BCG) is an attenuated Mycobacterium bovis derivative that has been widely used as a live vaccine against tuberculosis for a century. In addition to its use as a tuberculosis vaccine, BCG has also been found to have utility in the prevention or treatment of unrelated diseases, including cancer. However, the protective and therapeutic efficacy of BCG against tuberculosis and other diseases is not perfect. For three decades, it has been possible to genetically modify BCG in an attempt to improve its efficacy. Various immune-modulatory molecules have been produced in recombinant BCG strains and tested for protection against tuberculosis or treatment of several cancers or inflammatory diseases. These molecules include cytokines, bacterial toxins or toxin fragments, as well as other protein and non-protein immune-modulatory molecules. The deletion of genes responsible for the immune-suppressive properties of BCG has also been explored for their effect on BCG-induced innate and adaptive immune responses. Most studies limited their investigations to the description of T cell immune responses that were modified by the genetic modifications of BCG. Some studies also reported improved protection by recombinant BCG against tuberculosis or enhanced therapeutic efficacy against various cancer forms or allergies. However, so far, these investigations have been limited to mouse models, and the prophylactic or therapeutic potential of recombinant BCG strains has not yet been illustrated in other species, including humans, with the exception of a genetically modified BCG strain that is now in late-stage clinical development as a vaccine against tuberculosis. In this review, we provide an overview of the different molecular engineering strategies adopted over the last three decades in order to enhance the immune-modulatory potential of BCG

BCG and protection against inflammatory and auto-immune diseases

Introduction: Bacillus Calmette-Guérin (BCG) is the only available vaccine against tuberculosis. Although its protective efficacy against pulmonary tuberculosis is still under debate, it provides protection against other mycobacterial diseases. BCG is also an effective therapy against superficial bladder cancer and potentially decreases overall childhood mortality. Areas covered: The purpose of this paper is to provide a state-of-the-art summary of the beneficial effects of BCG in inflammatory and auto-immune diseases. As a strong inducer of Th1 type immunity, BCG has been reported to protect against atopic conditions, such as allergic asthma, a Th2-driven disorder. Its protective effect has been well documented in mice, but still awaits definitive evidence in humans. Similarly, murine studies have shown a protective effect of BCG against auto-immune diseases, such as multiple sclerosis and insulin-dependent diabetes, but studies in humans have come to conflicting conclusions. Expert commentary: Studies in mice have shown a beneficial effect of the BCG vaccine against allergic asthma, multiple sclerosis and diabetes. However, the understanding of its mechanism is still fragmentary and requires further in depth research. Some observational or intervention studies in humans have also suggested a beneficial effect, but definitive evidence for this requires confirmation in carefully conducted prospective studies

Interleukin 18 (IL-18) as a target for immune intervention

Interleukin 18 (IL-18) is a pleiotropic cytokine involved in the regulation of innate and acquired immune response. In the milieu of IL-12 or IL-15, IL-18 is a potent inducer of IFN-gamma in natural killer (NK) cells and CD4 T helper (Th) 1 lymphocytes. However, IL-18 also modulates Th2 and Th17 cell responses, as well as the activity of CD8 cytotoxic cells and neutrophils, in a host microenvironment-dependent manner. It is produced by various hematopoietic and nonhematopoietic cells, including dendritic cells and macrophages. In an organism, bioactivity of the cytokine depends on the intensity of IL-18 production, the level of its natural inhibitory protein - IL-18BP (IL-18 binding protein) and the surface expression of IL-18 receptors (IL-18R) on the responding cells. This review summarizes the biology of the IL-18/IL-18BP/IL-18R system and its role in the host defense against infections. The prospects for IL-18 application in immunotherapeutic or prophylactic interventions in infectious and non-infectious diseases are discussed

The role of IL-18 in dendritic cell interactions with <em>M. bovis</em> Bacille Calmette-Guérin (BCG)

International audienceINTRODUCTION. The initiation and maintenance of specific responses to infective agents depend on dendritic cells (DC) playing the role of antigen presenting cells. IFN-γ has been demonstrated to be crucial to antimycobacterial immunity. The production of this cytokine by Th1 cells is effectively enhanced by IL-18, however, the role of IL-18 in human responsiveness to M. bovis BCG vaccine remains poorly understood. AIM. We asked a question whether human IL-18 released by the recombinant BCG strain (rBCGhIL-18) influences DC driven response to mycobacterial antigens in healthy humans who were vaccinated with BCG in childhood. MATERIALS AND METHODS. Immature DC were obtained from CD14+ monocytes, separated by magnetic sorting and stimulated for 6-days with IL-4 and GM-CSF. Dendritic cell response to live rBCGhIL-18 and BCG strains was estimated on the level of DC receptors (flow cytometry) and cytokines (ELISA) in 24-h cultures. RESULTS. Both mycobacterial strains caused a significant decrease in specific dendritic cell DC-SIGN receptor and an increase in CD86 co-receptor of the immune synapse. Neither rBCGhIL-18 nor BCG changed the expression of CD40 and HLA-DR on DC. Recombinant rBCGhIL-18 but not nonrecombinant BCG enhanced the DC expression of CD80 co-receptor and production of IP-10 chemokine, which promotes T cell migration to lymph nodes. Recombinant rBCGhIL-18 stimulated the production of T cell activating IL-23 and regulatory IL-10 by DC more intensively than nonrecombinant BCG. CONCLUSIONS. The obtained results suggest that IL-18 can positively modulate adaptive immune response to BCG bacilli by the intensification of antigen presentation and regulatory functions of dendritic cells

Interferon-gamma assay in combination with tuberculin skin test are insufficient for the diagnosis of culture-negative pulmonary tuberculosis.

OBJECTIVE:Early diagnosis of infectious cases and treatment of tuberculosis (TB) are important strategies for reducing the incidence of this disease. Unfortunately, traditional TB diagnostic methods are time-consuming and often unreliable. This study compared the accuracy and reliability of the tuberculin skin test (TST) and interferon (IFN)-γ-based assay (IGRA) for the diagnosis of active pulmonary TB Polish cases that could or could not be confirmed by M. tuberculosis (M.tb) culture. METHODS:In total, 126 adult patients with clinically active TB or non-mycobacterial, community-acquired lung diseases (NMLD) hospitalised at the Regional Specialised Hospital of Tuberculosis, Lung Diseases and Rehabilitation in Tuszyn, Poland were enrolled in the present study. Sensitivity, specificity, positive predicted value (PPV), negative predicted value (NPV), and analytic accuracy (Acc) of TST and IGRA testing for the diagnosis of culture-positive and culture-negative TB patients were calculated. The quantities of IFN-γ produced in the response to M.tb specific antigens (TB Ag - Nil) in the cultures of blood from patients with active TB and NMLD patients were also analysed. RESULTS:The IGRA sensitivity in culture-positive and culture-negative TB patients was similar, measuring 65.1% and 55.6%, respectively. The sensitivity of TST did not differ from the parameters designated for IGRA, measuring 55.8% in culture-positive and 64.9% in culture-negative TB. The sensitivity of TST and IGRA was age-dependent and decreased significantly with the age of the patients. No differences in the frequency or intensity of M.tb-stimulated IFN-γ production, as assessed by IGRA testing between culture-positive and culture-negative TB were noticed. Significantly lower concentrations of IFN-γ were observed in patients with advanced TB forms compared with those with mild or moderate TB pathologies. CONCLUSIONS:Our results do not show that a combination of IGRA and TST might be a step forward in the diagnosis of culture-negative TB cases. However, M. tuberculosis-stimulated IFN-γ levels might help to assess the extent of pulmonary TB lesions