Suzie Hingley-Wilson

A mycolic acid–specific CD1-restricted T cell population contributes to acute and memory immune responses in human tuberculosis infection

Current tuberculosis (TB) vaccine strategies are largely aimed at activating conventional T cell responses to mycobacterial protein antigens. However, the lipid-rich cell wall of Mycobacterium tuberculosis (M. tuberculosis) is essential for pathogenicity and provides targets for unconventional T cell recognition. Group 1 CD1-restricted T cells recognize mycobacterial lipids, but their function in human TB is unclear and their ability to establish memory is unknown. Here, we characterized T cells specific for mycolic acid (MA), the predominant mycobacterial cell wall lipid and key virulence factor, in patients with active TB infection. MA-specific T cells were predominant in TB patients at diagnosis, but were absent in uninfected bacillus Calmette-Guérin-vaccinated (BCG-vaccinated) controls. These T cells were CD1b restricted, detectable in blood and disease sites, produced both IFN-γ and IL-2, and exhibited effector and central memory phenotypes. MA-specific responses contracted markedly with declining pathogen burden and, in patients followed longitudinally, exhibited recall expansion upon antigen reencounter in vitro long after successful treatment, indicative of lipid-specific immunological memory. T cell recognition of MA is therefore a significant component of the acute adaptive and memory immune response in TB, suggesting that mycobacterial lipids may be promising targets for improved TB vaccines.

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 waters. Copyright

Ethnicity and mycobacterial lineage as determinants of tuberculosis disease phenotype

Background Emerging evidence suggests that Mycobacterium tuberculosis (Mtb) lineage and host ethnicity can determine tuberculosis (TB) clinical disease patterns but their relative importance and interaction are unknown. Methods We evaluated prospectively collected TB surveillance and Mtb strain typing data in an ethnically heterogeneous UK population. Lineage assignment was denoted using 15-loci mycobacterial interspersed repetitive units containing variable numbers of tandem repeats (MIRU-VNTR) and MIRU-VNTRplus. Geographical and ethnic associations of the six global Mtb lineages were identified and the influence of lineage and demographic factors on clinical phenotype were assessed using multivariate logistic regression. Results Data were available for 1070 individuals with active TB which was pulmonary only, extrapulmonary only and concurrent pulmonary–extrapulmonary in 52.1%, 36.9% and 11.0% respectively. The most prevalent lineages were Euro-American (43.7%), East African Indian (30.2%), Indo-Oceanic (13.6%) and East Asian (12.2%) and were geo-ethnically restricted with, for example, Indian subcontinent ethnicity inversely associated with Euro-American lineage (OR 0.23; 95% CI 0.14 to 0.39) and positively associated with the East African-Indian lineage (OR 4.04; 95% CI 2.19 to 7.45). Disease phenotype was most strongly associated with ethnicity (OR for extrathoracic disease 21.14 (95% CI 6.08 to 73.48) for Indian subcontinent and 14.05 (3.97 to 49.65) for Afro-Caribbean), after adjusting for lineage. With East Asian lineage as the reference category, the Euro-American (OR 0.54; 95% CI 0.32 to 0.91) and East-African Indian (OR 0.50; 95% CI 0.29 to 0.86) lineages were negatively associated with extrathoracic disease, compared with pulmonary disease, after adjusting for ethnicity. Conclusions Ethnicity is a powerful determinant of clinical TB phenotype independently of mycobacterial lineage and the role of ethnicity-associated factors in pathogenesis warrants investigation.

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 algae. Copyright

Individual Mycobacterium tuberculosis universal stress protein homologues are dispensable in vitro

Summary Mycobacterium tuberculosis has 10 universal stress proteins, whose function is unknown. However, proteomic and transcriptomic analyses have shown that a number of usp genes are significantly upregulated under hypoxic conditions and in response to nitric oxide and carbon monoxide, as well as during M. tuberculosis infection of macrophage cell lines. Six of these USPs are part of the DosR regulon and this, along with their expression pattern and the phenotypes of usp mutants in other bacterial species, suggests a potential role in the persistence and/or intracellular survival of Mtb. Knock-out mutants of individual usp genes encoding the USPs Rv1996, Rv2005c, Rv2026c and Rv2028c were generated and their growth and survival under hypoxic and other stress conditions examined. Although the majority of usp genes are highly induced in hypoxic conditions, mutation did not affect the long term survival of Mtb under these conditions, or in response to a range of stress conditions chosen to represent the environmental onslaughts experienced by the bacillus during an infection, nor during infection of mouse and human – derived macrophage cell lines. The possibility remains that these USPs are functionally redundant in Mtb.

Antiprotozoal, Antimycobacterial and Cytotoxic Potential of Twenty-Three British and Irish Red 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 (IC50 values 4.8 and 5.4 μg/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 IC50 values ranging from 16.5 to 85.6 μg/ml. Only the crude extract of Calliblepharis jubata showed some weak activity against Mycobacterium tuberculosis (MIC value 256 μg/ml), while the others were inactive at this concentration. Corallina officinalis was the only seaweed that displayed some marginal cytotoxicity (IC50 value 88.6 μg/ml), and all remaining extracts were non‐toxic towards L6 cells at 90 μg/ml concentration. To our knowledge, this is the first study reporting antiprotozoal and antimycobacterial activity of British and Irish red algae. Copyright

Sarcoidosis and tuberculosis cytokine profiles: indistinguishable in bronchoalveolar lavage but different in blood.

Background The clinical, radiological and pathological similarities between sarcoidosis and tuberculosis can make disease differentiation challenging. A complicating factor is that some cases of sarcoidosis may be initiated by mycobacteria. We hypothesised that immunological profiling might provide insight into a possible relationship between the diseases or allow us to distinguish between them. Methods We analysed bronchoalveolar lavage (BAL) fluid in sarcoidosis (n = 18), tuberculosis (n = 12) and healthy volunteers (n = 16). We further investigated serum samples in the same groups; sarcoidosis (n = 40), tuberculosis (n = 15) and healthy volunteers (n = 40). A cross-sectional analysis of multiple cytokine profiles was performed and data used to discriminate between samples. Results We found that BAL profiles were indistinguishable between both diseases and significantly different from healthy volunteers. In sera, tuberculosis patients had significantly lower levels of the Th2 cytokine interleukin-4 (IL-4) than those with sarcoidosis (p = 0.004). Additional serum differences allowed us to create a linear regression model for disease differentiation (within-sample accuracy 91%, cross-validation accuracy 73%). Conclusions These data warrant replication in independent cohorts to further develop and validate a serum cytokine signature that may be able to distinguish sarcoidosis from tuberculosis. Systemic Th2 cytokine differences between sarcoidosis and tuberculosis may also underly different disease outcomes to similar respiratory stimuli.

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. Copyright

An exit strategy for the tubercle bacillus

Most studies on the host–pathogen interactions that occur between the residing infecting agent and its niche cell focus on the pathogenic manipulation of host cell entrance and intracellular propagation, but the timely and in-depth review by Kevin Hybiske and Richard S. Stephens (Exit strategies of intracellular pathogens. Nature Rev. Microbiol. 6, 99–110 (2008))1 focuses on exit of the pathogen from its host cell. The timing and mode of action of the cellular egress by the bacteria may, as the authors denote, determine the efficacy of secondary infection and the immune response. In addition, it may affect the dissemination of the disease within the host and also the transmission between hosts. We understand that it was impossible for the reviewers to cover all intracellular infections that depend on extracellular egress. However, we also think it should be noted that cytolysis-dependent cellular egress has recently been described for the causative agent of tuberculosis, Mycobacterium tuberculosis, which remains the leading cause of bacterial mortality worldwide. Indeed, the key mechanism involved in host cell egress by the tubercle bacilli forms the basis of the primary attenuating mutation (region of difference 1; RD1) of the vaccine strain Mycobacterium bovis bacille Calmette–Guérin (BCG). Mycobacterial RD1 mutants exhibit a host cell lysis defect and exhibit reduced egress from host macrophages2–4. In the murine model of infection, RD1 was necessary to gain access to the deeper interstitial tissue of the lung and an M. tuberculosis RD1 mutant displayed both reduced disease progression and virulence4,5. Thus, in tuberculosis, RD1-mediated host cell exit is crucial both to cellular egress and to virulence. Interestingly, RD1 has recently been found to encode a novel type of secretion system and is responsible for both the production and secretion of a highly immunodominant virulence factor called early secreted antigenic target 6 (ESAT6)4,6–8. We found that ESAT6 is itself capable of lysing an artificial membrane4, and it has since been reported that RD1mediated whole-cell lysis is induced through the depletion of intracellular ATP owing to lysis of the mitochondrial membrane3. To conclude, we note that M. tuberculosis elicits cellular egress though RD1-mediated cytolysis, in a similar manner to many of the pathogens noted in the review, and that this is a requisite for disease progression and virulence. The mycobacterial mediators of cellular egress could be potential drug targets with which to halt dissemination and transmission of this global health threat. In addition, the benefits of understanding this potentially fundamental mechanism of disease progression include the development of much-needed improved strategies for the treatment and prevention of tuberculosis.

Rhinovirus induction of fractalkine (CX3CL1) in airway and peripheral blood mononuclear cells in asthma

Rhinovirus infection is associated with the majority of asthma exacerbations. The role of fractalkine in anti-viral (type 1) and pathogenic (type 2) responses to rhinovirus infection in allergic asthma is unknown. To determine whether (1) fractalkine is produced in airway cells and in peripheral blood leucocytes, (2) rhinovirus infection increases production of fractalkine and (3) levels of fractalkine differ in asthmatic compared to non-asthmatic subjects. Fractalkine protein and mRNA levels were measured in bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMCs) from non-asthmatic controls (n = 15) and mild allergic asthmatic (n = 15) subjects. Protein levels of fractalkine were also measured in macrophages polarised ex vivo to give M1 (type 1) and M2 (type 2) macrophages and in BAL fluid obtained from mild (n = 11) and moderate (n = 14) allergic asthmatic and non-asthmatic control (n = 10) subjects pre and post in vivo rhinovirus infection. BAL cells produced significantly greater levels of fractalkine than PBMCs. Rhinovirus infection increased production of fractalkine by BAL cells from non-asthmatic controls (P<0.01) and in M1-polarised macrophages (P<0.05), but not in BAL cells from mild asthmatics or in M2 polarised macrophages. Rhinovirus induced fractalkine in PBMCs from asthmatic (P<0.001) and healthy control subjects (P<0.05). Trends towards induction of fractalkine in moderate asthmatic subjects during in vivo rhinovirus infection failed to reach statistical significance. Fractalkine may be involved in both immunopathological and anti-viral immune responses to rhinovirus infection. Further investigation into how fractalkine is regulated across different cell types and into the effect of stimulation including rhinovirus infection is warranted to better understand the precise role of this unique dual adhesion factor and chemokine in immune cell recruitment.