Enumeration of Functional T-Cell Subsets by Fluorescence-Immunospot Defines Signatures of Pathogen Burden in Tuberculosis

IFN-γ and IL-2 cytokine-profiles define three functional T-cell subsets which may correlate with pathogen load in chronic intracellular infections. We therefore investigated the feasibility of the immunospot platform to rapidly enumerate T-cell subsets by single-cell IFN-γ/IL-2 cytokine-profiling and establish whether immunospot-based T-cell signatures distinguish different clinical stages of human tuberculosis infection.We used fluorophore-labelled anti-IFN-γ and anti-IL-2 antibodies with digital overlay of spatially-mapped colour-filtered images to enumerate dual and single cytokine-secreting M. tuberculosis antigen-specific T-cells in tuberculosis patients and in latent tuberculosis infection (LTBI). We validated results against established measures of cytokine-secreting T-cells.Fluorescence-immunospot correlated closely with single-cytokine enzyme-linked-immunospot for IFN-γ-secreting T-cells and IL-2-secreting T-cells and flow-cytometry-based detection of dual IFN-γ/IL-2-secreting T-cells. The untreated tuberculosis signature was dominated by IFN-γ-only-secreting T-cells which shifted consistently in longitudinally-followed patients during treatment to a signature dominated by dual IFN-γ/IL-2-secreting T-cells in treated patients. The LTBI signature differed from active tuberculosis, with higher proportions of IL-2-only and IFN-γ/IL-2-secreting T-cells and lower proportions of IFN-γ-only-secreting T-cells.Fluorescence-immunospot is a quantitative, accurate measure of functional T-cell subsets; identification of cytokine-signatures of pathogen burden, distinct clinical stages of M. tuberculosis infection and long-term immune containment suggests application for treatment monitoring and vaccine evaluation

The role of C-reactive protein in immune responses to Neisseria meningitidis.

Immunity to Neisseria meningitidis (N. meningitidis) involves both innate and antibody-mediated mechanisms. The acute-phase serum protein C-reactive protein (CRP) helps to protect the host from several bacterial pathogens, often by binding to surface phosphorylcholine (PC). Pathogenic Neisseria species can exhibit phase-variable PC modification on type 4 pili of class 1 and 2. This investigation demonstrates that CRP can bind to piliated meningococci in a calcium-dependent manner that is concentration dependent, of low affinity and specific for PC. Separate experiments confirmed that CRP can bind to both live and paraformaldehyde-fixed meningococci, suggesting that CRP has a role in the immune response to meningococci. CRP binding to PC on commensal species of Neisseria is known to increase serum bactericidal activity by initiating the classical complement cascade. In these investigations, CRP-opsonisation did not increase serum killing of N. meningitidis. CRP binding did, however, significantly increase phagocytic uptake of fixed meningococci by macrophages and neutrophils (and increased uptake by dendritic cells was also indicated). This CRP-mediated uptake was attributed to recruitment of the cellular receptor for CRP, FcyR, as blocking of these receptors with human IgG abrogated the increased uptake by macrophages in a concentration-dependent manner. In contrast, macrophage uptake of live meningococci was not increased by CRP-opsonisation and preliminary investigations detected no increase in intracellular killing of CRP-opsonised meningococci, indicating that live meningococci may evade CRP-mediated phagocyte detection. The absence of a direct killing effect of CRP led to the hypothesis that CRP provides an indirect advantage to the host by modulation of inflammation and antigen presentation by phagocytes. CRP-opsonised N. meningitidis induced greater macrophage HLA-DR expression, IL-10 and IL-1beta secretion compared to non-opsonised N. meningitidis in the majority of human donors. These studies indicate that CRP may increase antigen presentation and therefore accelerate the development of acquired immunity to meningococcal infection, whilst limiting the inflammatory damage to the host via increased IL-10 production

The Acute-Phase Reactant C-Reactive Protein Binds to Phosphorylcholine-Expressing Neisseria meningitidis and Increases Uptake by Human Phagocytes▿

Neisseria meningitidis is a global cause of meningitis and septicemia. Immunity to N. meningitidis involves both innate and specific mechanisms with killing by serum bactericidal activity and phagocytic cells. C-reactive protein (CRP) is an acute-phase serum protein that has been shown to help protect the host from several bacterial pathogens, which it recognizes by binding to phosphorylcholine (PC) on their surfaces. Pathogenic Neisseria species can exhibit phase-variable PC modification on type 1 and 2 pili. We have shown that CRP can bind to piliated meningococci in a classical calcium-dependent manner. The binding of CRP to the meningococcus was concentration dependent, of low affinity, and specific for PC. CRP appears to act as an opsonin for N. meningitidis, as CRP-opsonized bacteria showed increased uptake by human macrophages and neutrophils. Further investigation into the downstream effects of CRP-bound N. meningitidis may lead us to a better understanding of meningococcal infection and help direct more effective therapeutic interventions

Infect and Inject: How Mycobacterium tuberculosis Exploits Its Major Virulence-Associated Type VII Secretion System, ESX-1.

Mycobacterium tuberculosis is an ancient master of the art of causing human disease. One important weapon within its fully loaded arsenal is the type VII secretion system. M. tuberculosis has five of them: ESAT-6 secretion systems (ESX) 1 to 5. ESX-1 has long been recognized as a major cause of attenuation of the FDA-licensed vaccine Mycobacterium bovis BCG, but its importance in disease progression and transmission has recently been elucidated in more detail. This review summarizes the recent advances in (i) the understanding of the ESX-1 structure and components, (ii) our knowledge of ESX-1's role in hijacking macrophage function to set a path for infection and dissemination, and (iii) the development of interventions that utilize ESX-1 for diagnosis, drug interventions, host-directed therapies, and vaccines

Antiprotozoal, Antimycobacterial and Cytotoxic Potential of Some British Green Algae

In the continuation of our search for natural sources for antiprotozoal and antitubercular molecules, we have screened the crude extracts of four green marine algae (Cladophora rupestris, Codium fragile ssp. tomentosoides, Ulva intestinalis and Ulva lactuca) collected from the Dorset area of England. Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Mycobacterium tuberculosis were used as test organisms in the in vitro assays. The selective toxicity of the extracts was also determined toward mammalian skeletal myoblast (L6) cells. The crude seaweed extracts had no activity against M. tuberculosis, but showed antiprotozoal activity against at least two protozoan species. All algal extracts were active against T. brucei rhodesiense, with C. rupestris being the most potent one (IC50 value 3.7 μg/ml), whilst only C. rupestris and U. lactuca had moderate trypanocidal activity against T. cruzi (IC50 values 80.8 and 34.9 μg/ml). Again, all four extracts showed leishmanicidal activity with IC50 values ranging between 12.0 and 20.2 μg/ml. None of the extracts showed cytotoxicity toward L6 cells, indicating that their antiprotozoal activity is specific. This is the first study reporting antiprotozoal and antimycobacterial activity of British marine alga

Evaluation of Turkish Seaweeds for Antiprotozoal, Antimycobacterial and Cytotoxic Activities

As part of our continuing research on seaweeds, crude MeOH extracts of two green, three brown and six red algae collected from Marmara, Black, Aegean and Mediterranean Seas were screened. Four parasitic protozoa, i.e. Plasmodium falciparum, Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani and the tubercle bacillus Mycobacterium tuberculosis were used as test organisms for the in vitro assays. The selective toxicity of the extracts was also determined against mammalian L6 cells. All seaweed extracts were active against T. brucei rhodesiense; the Dasya pedicellata extract was the most potent (IC50 value 0.37 µg/mL). The same extract also weakly inhibited the growth of T. cruzi (IC50 62.02 µg/mL). All seaweed extracts also showed leishmanicidal activity (IC50 values 16.76–69.98 µg/mL). The majority of the extracts also exhibited antiplasmodial potential and the most potent extracts were those from D. pedicellata (IC50 0.38 µg/mL), Codium bursa (IC50 1.38 µg/mL) and Caulerpa rasemosa (IC50 3.12 µg/mL). One brown and two red algal extracts showed some weak activity against Mycobacterium tuberculosis (MIC values 125–256 µg/mL). Except for the extract of Dasya pedicellata, none of the extracts displayed any cytotoxicity. This is the second study investigating the antiprotozoal activities of Turkish marine algae and identifies Dasya pedicellata, an understudied algal species, as a candidate for further studies

Antimycobacterial, Antiprotozoal and Cytotoxic Potential of Twenty-one Brown Algae (Phaeophyceae) from British and Irish Waters

In the continuation of our research on seaweeds, crude extracts of 21 brown algae collected from the south coast of England and the west coast of Ireland were screened for in vitro trypanocidal, leishmanicidal and antimycobacterial activities. Mammalian stages of a small set of parasitic protozoa; i.e. Trypanosoma brucei rhodesiense, T. cruzi and Leishmania donovani, and the tubercle bacillus Mycobacterium tuberculosis were used as test organisms. The extracts were also evaluated for selectivity by testing on a mammalian cell line (L6 cells). Only four extracts were moderately active against T. cruzi, whereas all algal extracts showed significant activity against T. brucei rhodesiense, with Halidrys siliquosa and Bifurcaria bifurcata (Sargassaceae) being the most potent (IC50 values 1.2 and 1.9 μg/mL). All algal extracts also displayed leishmanicidal activity, with H. siliquosa and B. bifurcata again being the most active (IC50s 6.4 and 8.6 μg/mL). When tested against M. tuberculosis, only the B. bifurcata extract was found to have some antitubercular potential (MIC value 64.0 μg/mL). Only three seaweed extracts, i.e. H. siliquosa, B. bifurcata and Cystoseira tamariscifolia showed some cytotoxicity. To our knowledge, this is the first study on the antiprotozoal and antimycobacterial activity of brown algae from British and Irish water

Data from: Recurrent insect outbreaks caused by temperature-driven changes in system stability

Insect species often undergo regular outbreaks in population density, but identifying the causal mechanism for such outbreaks in any particular species has proven difficult. Here we show that outbreak cycles in the tea tortrix Adoxophyes honmai can be explained by temperature-driven changes in system stability. Wavelet analysis of a 51yr time series spanning over 200 outbreaks reveals a threshold in outbreak amplitude each spring when temperature exceeds 15°C, and a secession of outbreaks each fall as temperature decreases. This is in close agreement with our independently parameterized mathematical model that predicts the system crosses a Hopf bifurcation from stability to sustained cycles as temperature increases. These results suggest that temperature can alter system stability and provide an explanation for generation cycles in multivoltine insects

Undetected multidrug-resistant tuberculosis amplified by first-line therapy in mixed infection

Infections with >1 Mycobacterium tuberculosis strain(s) are underrecognized. We show, in vitro and in vivo, how first-line treatment conferred a competitive growth advantage to amplify a multidrug-resistant M. tuberculosis strain in a patient with mixed infection. Diagnostic techniques that identify mixed tubercle bacilli populations are needed to curb the spread of multidrug resistance