Intracellular Cytokine Staining and Flow Cytometry: Considerations for Application in Clinical Trials of Novel Tuberculosis Vaccines

Intracellular cytokine staining combined with flow cytometry is one of a number of assays designed to assess T-cell immune responses. It has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation and functional parameters pertaining to responding T-cells, most notably, the expression of multiple effector cytokines. These attributes make the technique particularly suitable for the assessment of T-cell immune responses induced by novel tuberculosis vaccines in clinical trials. However, depending upon the particular nature of a given vaccine and trial setting, there are approaches that may be taken at different stages of the assay that are more suitable than other alternatives. In this paper, the Tuberculosis Vaccine Initiative (TBVI) TB Biomarker Working group reports on efforts to assess the conditions that will determine when particular assay approaches should be employed. We have found that choices relating to the use of fresh whole blood or peripheral blood mononuclear cells (PBMC) and frozen PBMC; use of serum-containing or serum-free medium; length of stimulation period and use of co-stimulatory antibodies can all affect the sensitivity of intracellular cytokine assays. In the case of sample material, frozen PBMC, despite some loss of sensitivity, may be more advantageous for batch analysis. We also recommend that for multi-site studies, common antibody panels, gating strategies and analysis approaches should be employed for better comparability.     

Pneumolysin activates the NLRP3 inflammasome and promotes proinflammatory cytokines independently of TLR4

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system.     

Selective and orally bioavailable phenylglycine tissue factor/factor VIIa inhibitors

We describe the structure-based design and synthesis of highly potent, orally bioavailable tissue factor/factor VIIa inhibitors which interfere with the coagulation cascade by selective inhibition of the extrinsic pathway.     

Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival

Many pathogenic organisms produce pore-forming toxins as virulence factors. Target cells however mount a response to such membrane damage. Here we show that toxin-induced membrane permeabilization leads to a decrease in cytoplasmic potassium, which promotes the formation of a multiprotein oligomeric innate immune complex, called the inflammasome, and the activation of caspase-1. Further, we find that when rendered proteolytic in this context caspase-1 induces the activation of the central regulators of membrane biogenesis, the Sterol Regulatory Element Binding Proteins (SREBPs), which in turn promote cell survival upon toxin challenge possibly by facilitating membrane repair. This study highlights that, in addition to its well-established role in triggering inflammation via the processing of the precursor forms of interleukins, caspase-1 has a broader role, in particular linking the intracellular ion composition to lipid metabolic pathways, membrane biogenesis, and survival.     

cFLIP regulation of lymphocyte activation and development

Cellular caspase-8 (FLICE)-like inhibitory protein (cFLIP) was originally identified as an inhibitor of death-receptor signalling through competition with caspase-8 for recruitment to FAS-associated via death domain (FADD). More recently, it has been determined that both cFLIP and caspase-8 are required for the survival and proliferation of T cells following T-cell-receptor stimulation. This paradoxical finding launched new investigations of how these molecules might connect with signalling pathways that link to cell survival and growth following antigen-receptor activation. As discussed in this Review, insight gained from these studies indicates that cFLIP and caspase-8 form a heterodimer that ultimately links T-cell-receptor signalling to activation of nuclear factor-kappaB through a complex that includes B-cell lymphoma 10 (BCL-10), mucosa-associated-lymphoid-tissue lymphoma-translocation gene 1 (MALT1) and receptor-interacting protein 1 (RIP1).     

High Prevalence of Bovine Tuberculosis in Dairy Cattle in Central Ethiopia: Implications for the Dairy Industry and Public Health

Background

Ethiopia has the largest cattle population in Africa. The vast majority of the national herd is of indigenous zebu cattle maintained in rural areas under extensive husbandry systems. However, in response to the increasing demand for milk products and the Ethiopian government''s efforts to improve productivity in the livestock sector, recent years have seen increased intensive husbandry settings holding exotic and cross breeds. This drive for increased productivity is however threatened by animal diseases that thrive under intensive settings, such as bovine tuberculosis (BTB), a disease that is already endemic in Ethiopia.

Methodology/Principal Findings

An extensive study was conducted to: estimate the prevalence of BTB in intensive dairy farms in central Ethiopia; identify associated risk factors; and characterize circulating strains of the causative agent, Mycobacterium bovis. The comparative intradermal tuberculin test (CIDT), questionnaire survey, post-mortem examination, bacteriology, and molecular typing were used to get a better understanding of the BTB prevalence among dairy farms in the study area. Based on the CIDT, our findings showed that around 30% of 2956 tested dairy cattle from 88 herds were positive for BTB while the herd prevalence was over 50%. Post-mortem examination revealed gross tuberculous lesions in 34/36 CIDT positive cattle and acid-fast bacilli were recovered from 31 animals. Molecular typing identified all isolates as M. bovis and further characterization by spoligotyping and MIRU-VNTR typing indicated low strain diversity within the study area.

Conclusions/Significance

This study showed an overall BTB herd prevalence of 50% in intensive dairy farms in Addis Ababa and surroundings, signalling an urgent need for intervention to control the disease and prevent zoonotic transmission of M. bovis to human populations consuming dairy products coming from these farms. It is suggested that government and policy makers should work together with stakeholders to design methods for the control of BTB in intensive farms in Ethiopia.     

Danger signaling through the inflammasome acts as a master switch between tolerance and sensitization

Efficient priming of adaptive immunity depends on danger signals provided by innate immune pathways. As an example, inflammasome-mediated activation of caspase-1 and IL-1beta is crucial for the development of reactive T cells targeting sensitizers like dinitrofluorobenzene (DNFB). Surprisingly, DNFB and dinitrothiocyanobenzene provide cross-reactive Ags yet drive opposing, sensitizing vs tolerizing, T cell responses. In this study, we show that, in mice, inflammasome-signaling levels can be modulated to turn dinitrothiocyanobenzene into a sensitizer and DNFB into a tolerizer, and that it correlates with the IL-6 and IL-12 secretion levels, affecting Th1, Th17, and regulatory T cell development. Hence, our data provide the first evidence that the inflammasome can define the type of adaptive immune response elicited by an Ag, and hint at new strategies to modulate T cell responses in vivo.     

Identification of granzyme A isolated from cytotoxic T-lymphocyte-granules as one of the proteases encoded by CTL-specific genes

A serine esterase called granzyme A, which is specifically expressed in cytolytic lymphocytes has been characterized. It is a disulfide-linked dimer and exhibits a trypsin-like specificity cleaving best after Arg. N-terminal sequence analysis revealed that granzyme A is identical to a protease recently predicted from a cloned CTL-specific gene.