BackgroundIFN-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.MethodsWe 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.ResultsFluorescence-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.ConclusionsFluorescence-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.

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 waters.

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.

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 (IC(50) value 3.7 microg/ml), whilst only C. rupestris and U. lactuca had moderate trypanocidal activity against T. cruzi (IC(50) values 80.8 and 34.9 microg/ml). Again, all four extracts showed leishmanicidal activity with IC(50) values ranging between 12.0 and 20.2 microg/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 algae.

As part of our continuing research on seaweeds, we have screened the crude extracts of 23 red marine algae collected from England and Ireland. The clinically important blood-stage life forms of Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani and Mycobacterium tuberculosis were used as test organisms in the in vitro assays. The selectivity of the extracts was determined by using mammalian skeletal myoblast (L6) cells. All algal extracts showed activity against T. brucei rhodesiense, with Corallina officinalis and Ceramium virgatum being the most potent (IC(50) values 4.8 and 5.4 microg/ml), whilst none of the algal extracts inhibited the growth of T. cruzi. Except for Porphyra leucosticta, extracts from all seaweeds also showed leishmanicidal activity with IC(50) values ranging from 16.5 to 85.6 microg/ml. Only the crude extract of Calliblepharis jubata showed some weak activity against Mycobacterium tuberculosis (MIC value 256 microg/ml), while the others were inactive at this concentration. Corallina officinalis was the only seaweed that displayed some marginal cytotoxicity (IC(50) value 88.6 microg/ml), and all remaining extracts were non-toxic towards L6 cells at 90 microg/ml concentration. To our knowledge, this is the first study reporting antiprotozoal and antimycobacterial activity of British and Irish red algae.