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

Teachers observing classroom communication: An application of the Communicating Supporting Classroom Observation Tool for children aged 4-7 years

The nature of ‘Teacher talk’ is likely to have a considerable bearing on the child’s learning but measuring the communication environment in the classroom can present challenges. One tool which does this is the Communication Supporting Classroom Observation Tool (CSCOT). Initial use suggested that it was valid and reliably used by specialists (psychologists and speech and language therapists) and special educational needs coordinators (SENCOs). A key question is whether it can be used routinely by classroom teachers and whether results coincide with those in earlier studies. CSCOT observations were carried out by teachers in 33 schools (32 Reception classrooms, 25 in Year 1 and 25 in Year 2) in two local authorities in the North East of England and teachers were asked afterwards to reflect on their experiences using the tool. Scores were in line with those in earlier studies and were consistently higher on all dimensions for reception compared to Year 2, but there was no difference between other year groups. Results were mostly consistent with the original studies. Language learning environment was higher relative to both language learning opportunities and interactions across all years (which again did not differ). There was a moderate interaction between language learning environment where scores were significantly higher in the Reception group and lower in the Year 2 group. Teachers supported the use of the CSCOT in their feedback, suggesting that CSCOT was easy to use and useful in informing practice. The CSCOT clearly has utility as a starting point in auditing classroom communication. It allows teachers to compare between classrooms and year groups and potentially fosters collaboration between teachers and specialist practitioners who focus on communication such as speech and language therapists. Further work could link the observation tool into an intervention program co-constructed with teachers

Identification of major factors influencing ELISpot-based monitoring of cellular responses to antigens from mycobacterium tuberculosis

A number of different interferon-c 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-c 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

Screening vaccine formulations for biological activity using fresh human whole blood.

Understanding the relevant biological activity of any pharmaceutical formulation destined for human use is crucial. For vaccine-based formulations, activity must reflect the expected immune response, while for non-vaccine therapeutic agents, such as monoclonal antibodies, a lack of immune response to the formulation is desired. During early formulation development, various biochemical and biophysical characteristics can be monitored in a high-throughput screening (HTS) format. However, it remains impractical and arguably unethical to screen samples in this way for immunological functionality in animal models. Furthermore, data for immunological functionality lag formulation design by months, making it cumbersome to relate back to formulations in real-time. It is also likely that animal testing may not accurately reflect the response in humans. For a more effective formulation screen, a human whole blood (hWB) approach can be used to assess immunological functionality. The functional activity relates directly to the human immune response to a complete formulation (adjuvant/antigen) and includes adjuvant response, antigen response, adjuvant-modulated antigen response, stability, and potentially safety. The following commentary discusses the hWB approach as a valuable new tool to de-risk manufacture, formulation design, and clinical progression

Youth Use of E-Liquid Flavours – A systematic review exploring patterns of use of e liquid flavours and associations with continued vaping, tobacco smoking uptake, or cessation

Background and Aims: There is concern that young people may be attracted to e-liquid flavours, prompting long-term vaping in naive users and potentially subsequent tobacco smoking. We aimed to review the use of e-liquid flavours by young people and describe associations with uptake or cessation of both regular vaping and tobacco smoking, adverse effects and subjective experiences. Design: Systematic review, including interventional, observational and qualitative studies reporting on the use of e-cigarette flavours by young people (aged < 18 years). Setting: Studies published in English language from any country or cultural setting. Participants: Young people and their carers (aged < 18 years). Measurements: A meta-analysis was not possible due to substantial heterogeneity, inconsistency in reporting of flavour categorizations and non-interventional study designs; thus, we narratively report findings. Findings: In total, 58 studies were included. The quality of the evidence was extremely low. Most (n = 39) studies were cross-sectional survey designs. In total, 11 longitudinal cohort studies assessed trajectories; eight qualitative studies reported on user experiences. Studies reported views and experiences of a total of 512 874 young people. Both cross-sectional and longitudinal studies suggested that flavours are important for initiation and continuation of vaping. Qualitative evidence shows interest and enjoyment in flavours. There was judged to be insufficient evidence that use of e-liquid flavours specifically is associated with uptake of smoking. No studies found clear associations between flavours and cessation in this population. We found no included reports of adverse effects of flavours. Conclusions: Flavours may be an important motivator for e-cigarette uptake, but the role of flavours in tobacco smoking uptake or cessation is unclear. The quality of the evidence on use of e-cigarette flavours by young people is low overall

Narrative skills in adolescents with a history of SLI in relation to non-verbal IQ scores

There is a debate about whether the language of children with primary language disorders and normal cognitive levels is qualitatively different from those with language impairments who have low or borderline non-verbal IQ (NVIQ). As children reach adolescence, this distinction may be even harder to ascertain, especially in naturalistic settings. Narrative may provide a useful, ecologically valid way in which to assess the language ability of adolescents with specific language impairment (SLI) who have intact or lowered NVIQ and to determine whether there is any discernable difference in every day language. Nineteen adolescents with a history of SLI completed two narrative tasks: a story telling condition and a conversational condition. Just under half the group (n = 8) had non-verbal IQs of 85. The remaining 11 had NVIQs in the normal range or above. Four areas of narrative (productivity, syntax, cohesion and performance) were assessed. There were no differences between the groups on standardized tests of language. However, the group with low NVIQ were poorer on most aspects of narrative, suggesting that cognitive level is important, even when language is the primary disorder. The groups showed similar patterns of differences between story telling and conversational narrative. It was concluded that adolescents with a history of SLI and poor cognitive levels have poorer narrative skills than those with normal range NVIQ even though these may not be detected by standardized assessment. Their difficulties present as qualitatively similar to those with normal range NVIQ and narratives appear impoverished rather than inaccurate

A mathematical model shows macrophages delay staphylococcus aureus replication, but limitations in microbicidal capacity restrict bacterial clearance

S. aureus is a leading cause of bacterial infection. Macrophages, the first line of defence in the human immune response, phagocytose and kill S. aureus but the pathogen can evade these responses. Therefore, the exact role of macrophages is incompletely defined. We develop a mathematical model of macrophage - S. aureus dynamics, built on recent experimental data. We demonstrate that, while macrophages may not clear infection, they significantly delay its growth and potentially buy time for recruitment of further cells. We find that macrophage killing is a major obstacle to controlling infection and ingestion capacity also limits the response. We find bistability such that the infection can be limited at low doses. Our combination of experimental data, mathematical analysis and model fitting provide important insights in to the early stages of S. aureus infections, showing macrophages play an important role limiting bacterial replication but can be overwhelmed with large inocula

Monocytes regulate the mechanism of T-cell death by inducing Fas-mediated apoptosis during bacterial infection.

Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed 'classic' features of apoptosis following exposure to pneumococci. Conversely, purified CD3(+) T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3(+) T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3(+) T-cells in PBMC cultures required 'classical' CD14(+) monocytes, which enhanced T-cell activation. CD3(+) T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3(+) T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease

Circulating levels of GDF15 in patients with myalgic encephalomyelitis/chronic fatigue syndrome

Abstract: Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating condition characterised by fatigue and post-exertional malaise. Its pathogenesis is poorly understood. GDF15 is a circulating protein secreted by cells in response to a variety of stressors. The receptor for GDF15 is expressed in the brain, where its activation results in a range of responses. Among the conditions in which circulating GDF15 levels are highly elevated are mitochondrial disorders, where early skeletal muscle fatigue is a key symptom. We hypothesised that GDF15 may represent a marker of cellular stress in ME/CFS. Methods: GDF15 was measured in serum from patients with ME/CFS (n = 150; 100 with mild/moderate and 50 with severe symptoms), “healthy volunteers” (n = 150) and a cohort of patients with multiple sclerosis (n = 50). Results: Circulating GDF15 remained stable in a subset of ME/CFS patients when sampled on two occasions ~ 7 months (IQR 6.7–8.8) apart, 720 pg/ml (95% CI 625–816) vs 670 pg/ml (95% CI 598–796), P = 0.5. GDF15 levels were 491 pg/ml in controls (95% CI 429–553), 546 pg/ml (95% CI 478–614) in MS patients, 560 pg/ml (95% CI 502–617) in mild/moderate ME/CFS patients and 602 pg/ml (95% CI 531–674) in severely affected ME/CFS patients. Accounting for potential confounders, severely affected ME/CFS patients had GDF15 concentrations that were significantly increased compared to healthy controls (P = 0.01). GDF15 levels were positively correlated (P = 0.026) with fatigue scores in ME/CFS. Conclusions: Severe ME/CFS is associated with increased levels of GDF15, a circulating biomarker of cellular stress that appears which stable over several months