Identification of Major Factors Influencing ELISpot-Based Monitoring of Cellular Responses to Antigens from Mycobacterium tuberculosis

A number of different interferon-γ ELISpot protocols are in use in laboratories studying antigen-specific immune responses. It is therefore unclear how results from different assays compare, and what factors most significantly influence assay outcome. One such difference is that some laboratories use a short in vitro stimulation period of cells before they are transferred to the ELISpot plate; this is commonly done in the case of frozen cells, in order to enhance assay sensitivity. Other differences that may be significant include antibody coating of plates, the use of media with or without serum, the serum source and the number of cells added to the wells. The aim of this paper was to identify which components of the different ELISpot protocols influenced assay sensitivity and inter-laboratory variation. Four laboratories provided protocols for quantifying numbers of interferon-γ spot forming cells in human peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis derived antigens. The differences in the protocols were compared directly. We found that several sources of variation in assay protocols can be eliminated, for example by avoiding serum supplementation and using AIM-V serum free medium. In addition, the number of cells added to ELISpot wells should also be standardised. Importantly, delays in peripheral blood mononuclear cell processing before stimulation had a marked effect on the number of detectable spot forming cells; processing delay thus should be minimised as well as standardised. Finally, a pre-stimulation culture period improved the sensitivity of the assay, however this effect may be both antigen and donor dependent. In conclusion, small differences in ELISpot protocols in routine use can affect the results obtained and care should be given to conditions selected for use in a given study. A pre-stimulation step may improve the sensitivity of the assay, particularly when cells have been previously frozen.     

Acceleration of human neutrophil apoptosis by TRAIL

Neutrophil granulocytes have a short lifespan, with their survival limited by a constitutive program of apoptosis. Acceleration of neutrophil apoptosis following ligation of the Fas death receptor is well-documented and TNF-alpha also has a transient proapoptotic effect. We have studied the role of the death receptor ligand TRAIL in human neutrophils. We identified the presence of mRNAs for TRAIL, TRAIL-R2, and TRAIL-R3, and cell surface expression of TRAIL-R2 and -R3 in neutrophil populations. Neutrophil apoptosis is specifically accelerated by exposure to a leucine zipper-tagged form of TRAIL, which mimics cell surface TRAIL. Using blocking Abs to TRAIL receptors, specifically TRAIL-R2, and a TRAIL-R1:FcR fusion protein, we have excluded a role for TRAIL in regulating constitutive neutrophil apoptosis. No additional proapoptotic effect of leucine zipper TRAIL was identified following TRAIL treatment of neutrophils in the presence of gliotoxin, an inhibitor of NF-kappaB, suggesting TRAIL does not activate NF-kappaB in human neutrophils. TRAIL treatment of human neutrophils did not induce a chemotactic response. The susceptibility of neutrophils to TRAIL-mediated apoptosis suggests a role for TRAIL in the regulation of inflammation and may provide a mechanism for clearance of neutrophils from sites of inflammation.     

Regulatory T cells in erythema nodosum leprosum maintain anti-inflammatory function

BackgroundThe numbers of circulating regulatory T cells (Tregs) are increased in lepromatous leprosy (LL) but reduced in erythema nodosum leprosum (ENL), the inflammatory complication of LL. It is unclear whether the suppressive function of Tregs is intact in both these conditions.MethodsA longitudinal study recruited participants at ALERT Hospital, Ethiopia. Peripheral blood samples were obtained before and after 24 weeks of prednisolone treatment for ENL and multidrug therapy (MDT) for participants with LL. We evaluated the suppressive function of Tregs in the peripheral blood mononuclear cells (PBMCs) of participants with LL and ENL by analysis of TNFα, IFNγ and IL-10 responses to Mycobacterium leprae (M. leprae) stimulation before and after depletion of CD25⁺ cells.Results30 LL participants with ENL and 30 LL participants without ENL were recruited. The depletion of CD25⁺ cells from PBMCs was associated with enhanced TNFα and IFNγ responses to M. leprae stimulation before and after 24 weeks treatment of LL with MDT and of ENL with prednisolone. The addition of autologous CD25⁺ cells to CD25⁺ depleted PBMCs abolished these responses. In both non-reactional LL and ENL groups mitogen (PHA)-induced TNFα and IFNγ responses were not affected by depletion of CD25⁺ cells either before or after treatment. Depleting CD25⁺ cells did not affect the IL-10 response to M. leprae before and after 24 weeks of MDT in participants with LL. However, depletion of CD25⁺ cells was associated with an enhanced IL-10 response on stimulation with M. leprae in untreated participants with ENL and reduced IL-10 responses in treated individuals with ENL. The enhanced IL-10 in untreated ENL and the reduced IL-10 response in prednisolone treated individuals with ENL was abolished by addition of autologous CD25⁺ cells.ConclusionThe findings support the hypothesis that the impaired cell-mediated immune response in individuals with LL is M. leprae antigen specific and the unresponsiveness can be reversed by depleting CD25⁺ cells. Our results suggest that the suppressive function of Tregs in ENL is intact despite ENL being associated with reduced numbers of Tregs. The lack of difference in IL-10 response in control PBMCs and CD25⁺ depleted PBMCs in individuals with LL and the increased IL-10 response following the depletion of CD25⁺ cells in individuals with untreated ENL suggest that the mechanism of immune regulation by Tregs in leprosy appears independent of IL-10 or that other cells may be responsible for IL-10 production in leprosy. The present findings highlight mechanisms of T cell regulation in LL and ENL and provide insights into the control of peripheral immune tolerance, identifying Tregs as a potential therapeutic target.     

Characterisation of a Tunisian coastal lagoon through hyperspectral underwater irradiance

North African coastal lagoons are unique ecosystems that often suffer degradation due to human activities. Therefore, monitoring methods are required to identify stressors and assist with the management of these valuable and often understudied ecosystems. A synthetic indicator of water ecological quality would be desirable for regular monitoring of these ecosystems under pressure. In 2008 an optical procedure was developed and applied in Ghar El Melh, a Tunisian lagoon which has been increasingly impacted by pollutant loading, especially from agriculture. In situ hyperspectral irradiance was measured at several stations, from which the apparent optical properties (AOPs), namely the irradiance attenuation coefficient K(λ) and the reflectance ratio R(λ), were obtained in order to relate them to water composition, in terms of light-attenuating substances (LASs). The significant relationships observed between R and LAS values enabled the application of a hyperspectral optical classification, which effectively highlighted threatened sectors of the lagoon. The pattern of differing water quality across the lagoon system that was derived from the hyperspectral classification agreed well with that obtained from a conventional optical classification that included AOPs and LASs. We suggest that hyperspectral analysis and classification is a useful monitoring tool for the assessment of change in coastal lagoons, and perhaps also in other shallow-water ecosystems.     

Oscillatory Transpiration and Water Uptake of Avena Plants IV. Transpiratory Response to Sine Shaped Light Cycles

The transpiration rate of individual 6-day-old oat plants was forced to oscillate by cyclic sine-shaped changes in the leaf irradiance (frequency 2 cycles h^?1, amplitude and average value 1.4 mW cm^?2, red light 620–800 nm). By means of a specially designed cuvette with three chambers the transpiration rate from three different segments of the leaf could be measured simultaneously. The leaf segments were illuminated individually and the illumination on each leaf segment could be modulated independently. The experiments showed that there was a strong correlation between the transpiration rates from the different leaf segments, dependent on a coupling mechanism in the plant. The coupling phenomenon disappeared when the root system was eliminated or when the water potential of the root medium was lowered. It was experimentally shown that CO₂ diffusion in the leaf could not be the primary cause for the coupling. Therefore the stomatal dependence on the leaf water potential was considered the most probable reason for the coupling. The frequency of the forcing light cycles could be linearly changed during an experiment and this swept-frequency technique was used to obtain a frequency response of one single oat plant. The technique made it also possible to study the strength of the coupling between different leaf segments.     

Evaluation of new chromogenic substrates for the detection of coliforms

Aims: To evaluate a new range of chromogenic substrates for the detection of β‐galactosidase activity in coliforms and to compare their performance in agar media and broths. Methods and Results: Sixteen novel galactoside substrates were prepared and incorporated into agar and broth. Their performance was compared using Escherichia coli (five strains), Salmonella (two strains), Enterobacter (two strains), Klebsiella, Pseudomonas, Listeria, Serratia, Shigella, Citrobacter, Proteus and Staphylococcus as well as pathological urine samples. The six substrates out of the initial 16 that showed the greatest sensitivity were VQE‐gal, VQM‐gal, VLPr‐gal, VLE‐gal, VLM‐gal and VBzTM‐gal, whose released chromophores were red, brown or purple. VQE‐gal and VLPr‐gal were studied in greater detail and were incorporated into agar medium. Coliform colonies appeared red and brown respectively, following incubation at 37°C for 24 h; however, positive results were obtained within a working day. The VQE‐gal medium was compared with some commercially available media. Conclusions: The range of substrates described can be used in broths as well as in agars. The VQE agar allows the detection of coliforms within a working day. VQE‐gal medium proved to be more sensitive when compared to other available chromogenic media and allows the unambiguous detection of coliforms.     

The Identification of Markers of Macrophage Differentiation in PMA-Stimulated THP-1 Cells and Monocyte-Derived Macrophages

Differentiated macrophages are the resident tissue phagocytes and sentinel cells of the innate immune response. The phenotype of mature tissue macrophages represents the composite of environmental and differentiation-dependent imprinting. Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD₃) are stimuli commonly used to induce macrophage differentiation in monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. We have compared the phenotype of the promonocytic THP-1 cell line after various protocols of differentiation utilising VD₃ and PMA in comparison to primary human monocytes or monocyte-derived macrophages (MDM). Both stimuli induced changes in cell morphology indicative of differentiation but neither showed differentiation comparable to MDM. In contrast, PMA treatment followed by 5 days resting in culture without PMA (PMAr) increased cytoplasmic to nuclear ratio, increased mitochondrial and lysosomal numbers and altered differentiation-dependent cell surface markers in a pattern similar to MDM. Moreover, PMAr cells showed relative resistance to apoptotic stimuli and maintained levels of the differentiation-dependent anti-apoptotic protein Mcl-1 similar to MDM. PMAr cells retained a high phagocytic capacity for latex beads, and expressed a cytokine profile that resembled MDM in response to TLR ligands, in particular with marked TLR2 responses. Moreover, both MDM and PMAr retained marked plasticity to stimulus-directed polarization. These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells.     

Streptococcus pneumoniae: the role of apoptosis in host defense and pathogenesis

Programmed cell death or apoptosis is a recognised feature of infection with Streptococcus pneumoniae, and is observed during pneumococcal meningitis and pneumonia. The cholesterol-dependent cytolysin, pneumolysin, is a major trigger of apoptosis in the brain in association with pneumococcal production of hydrogen peroxide. Pneumococcal cell wall is also an important stimulus for apoptosis. Microbial factors and host factors combine in causing apoptosis in the brain, with hippocampal neurons being particularly susceptible. In pulmonary infection epithelial cell apoptosis contributes to tissue injury but macrophage apoptosis may benefit the host, aiding microbial killing and downregulating the inflammatory response. During sepsis lymphocyte apoptosis may be harmful to the host while dendritic cell apoptosis may limit the generation of an adaptive immune response during infection. Apoptosis induction may be harmful or potentially beneficial during pneumococcal infection and understanding its function in each setting is essential to allow specific therapeutic intervention.