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

Upper respiratory tract mucosal immunity for SARS-CoV-2 vaccines

SARS-CoV-2 vaccination significantly reduces morbidity and mortality, but has less impact on viral transmission rates, thus aiding viral evolution; and the longevity of vaccine-induced immunity rapidly declines. Immune responses in respiratory tract mucosal tissues are crucial for early control of infection, and can generate long-term antigen-specific protection with prompt recall responses. However, currently approved SARS-CoV-2 vaccines are not amenable to adequate respiratory mucosal delivery, particularly in the upper airways, which could account for the high vaccine breakthrough infection rates and limited duration of vaccine-mediated protection. In view of these drawbacks, we outline a strategy that has the potential to enhance both the efficacy and durability of existing SARS-CoV-2 vaccines, by inducing robust memory responses in the upper respiratory tract mucosa

Does autonomous macrophage-driven inflammation promote alveolar damage in COVID-19?

SARS-CoV-2 has caused devastating effects with over 550 million infections by July 2022 and approximately 6.4 million deaths [1]. Societal and economic impacts will reverberate for years, with continuous evolution of SARS-CoV-2 as it persistently spreads through the human population as exemplified by reduced activity of vaccines and monoclonals against Omicron BA.4 or BA.5 subvariants [2]. A greater understanding of pathogenesis and more tailored therapeutic approaches are therefore essential

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

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

Evidence based pathways to intervention for children with language disorders

Background: Paediatric SLT roles often involve planning individualised intervention for specific children (provided directly by SLTs or indirectly through non-SLTs), working collaboratively with families and education staff and providing advice and training. A tiered approach to service delivery is currently recommended, whereby services become increasingly specialised and individualised for children with greater needs.  Aims: To examine 1) evidence of intervention effectiveness for children with language disorders at different tiers and 2) evidence regarding SLT roles; and to propose an evidence-based model of SLT service delivery.  Methods: Controlled, peer-reviewed studies, meta-analyses and systematic reviews of interventions for children with language disorders are reviewed and their outcomes discussed, alongside the differing roles SLTs play in these interventions. We indicate where gaps in the evidence base exist and present a possible model of service delivery consistent with current evidence, and a flowchart to aid clinical decision making.  Main Contribution: The service delivery model presented resembles the tiered model commonly used in education services, but divides individualised (Tier 3) services into Tier3A: indirect intervention delivered by non-SLTs, and Tier 3B: direct intervention by an SLT. We report the evidence for intervention effectiveness and which children might best be served by each tier, the role SLTs could take within each, and the evidence of effectiveness of these roles. Regarding universal interventions provided to all children (Tier 1) and those targeted at children with language weaknesses (Tier 2), there is growing evidence that approaches led by education services can be effective when staff are highly trained and well-supported. There is currently limited evidence regarding additional benefit of SLT-specific roles at Tiers 1 and 2. With regard to individualised intervention (Tier 3): children with complex or pervasive language disorders progress significantly following direct individualised intervention (Tier 3B), whereas children with milder or less pervasive difficulties can make progress when intervention is managed by an SLT, but delivered indirectly by others (Tier 3A), provided they are well-trained, -supported and -monitored.  Conclusions: SLTs have a contribution to make at all tiers, but where prioritisation for clinical services is a necessity, we need to establish the benefits and cost-effectiveness of each contribution. Good evidence exists for SLTs delivering direct individualised intervention, and we should ensure that this is available to those children with pervasive and/or complex language impairments. In cases where service models are being provided which lack evidence, we strongly recommend that SLTs investigate the effectiveness of their approaches

Evidence-based pathways to intervention for children with language disorders

Background  Paediatric speech and language therapist (SLT) roles often involve planning individualized intervention for specific children, working collaboratively with families and education staff, providing advice, training and coaching and raising awareness. A tiered approach to service delivery is currently recommended whereby services become increasingly specialized and individualized for children with greater needs.  Aims  To stimulate discussion regarding delivery of SLT services by examining evidence regarding the effectiveness of (1) intervention for children with language disorders at different tiers and (2) SLT roles within these tiers; and to propose an evidence‐based model of SLT service delivery and a flowchart to aid clinical decision‐making.  Methods & Procedures  Meta‐analyses and systematic reviews, together with controlled, peer‐reviewed group studies where recent systematic reviews were not available, of interventions for children with language disorders are discussed, alongside the differing roles SLTs play in these interventions. Gaps in the evidence base are highlighted.  Main Contribution  The service‐delivery model presented resembles the tiered model commonly used in education services, but divides individualized (Tier 3) services into Tier 3A: indirect intervention delivered by non‐SLTs, and Tier 3B: direct intervention by an SLT. We report evidence for intervention effectiveness, which children might best be served by each tier, the role SLTs could take within each tier and the effectiveness of these roles. Regarding universal interventions provided to all children (Tier 1) and those targeted at children with language weaknesses or vulnerabilities (Tier 2), there is growing evidence that approaches led by education services can be effective when staff are highly trained and well supported. There is currently limited evidence regarding additional benefit of SLT‐specific roles at Tiers 1 and 2. With regard to individualized intervention (Tier 3), children with complex or pervasive language disorders can progress following direct individualized intervention (Tier 3B), whereas children with milder or less pervasive difficulties can make progress when intervention is managed by an SLT, but delivered indirectly by others (Tier 3A), provided they are well trained and supported, and closely monitored.  Conclusions & Implications  SLTs have a contribution to make at all tiers, but where prioritization for clinical services is a necessity, we need to establish the relative benefits and cost‐effectiveness at each tier. Good evidence exists for SLTs delivering direct individualized intervention and we should ensure that this is available to children with pervasive and/or complex language disorders. In cases where service models are being provided which lack evidence, we strongly recommend that SLTs investigate the effectiveness of their approaches