Late Treatment With Autologous Expanded Regulatory T-cell Therapy After Alemtuzumab Induction Is Safe and Facilitates Immunosuppression Minimization in Living Donor Renal Transplantation.

BACKGROUND: The TWO Study (Transplantation Without Overimmunosuppression) aimed to investigate a novel approach to regulatory T-cell (Treg) therapy in renal transplant patients, using a delayed infusion protocol at 6 mo posttransplant to promote a Treg-skewed lymphocyte repopulation after alemtuzumab induction. We hypothesized that this would allow safe weaning of immunosuppression to tacrolimus alone. The COVID-19 pandemic led to the suspension of alemtuzumab use, and therefore, we report the unique cohort of 7 patients who underwent the original randomized controlled trial protocol. This study presents a unique insight into Treg therapy combined with alemtuzumab and is therefore an important proof of concept for studies in other diseases that are considering lymphodepletion. METHODS: Living donor kidney transplant recipients were randomized to receive autologous polyclonal Treg at week 26 posttransplantation, coupled with weaning doses of tacrolimus, (Treg therapy arm) or standard immunosuppression alone (tacrolimus and mycophenolate mofetil). Primary outcomes were patient survival and rejection-free survival. RESULTS: Successful cell manufacturing and cryopreservation until the 6-mo infusion were achieved. Patient and transplant survival was 100%. Acute rejection-free survival was 100% in the Treg-treated group at 18 mo after transplantation. Although alemtuzumab caused a profound depletion of all lymphocytes, including Treg, after cell therapy infusion, there was a transient increase in peripheral Treg numbers. CONCLUSIONS: The study establishes that delayed autologous Treg therapy is both feasible and safe, even 12 mo after cell production. The findings present a new treatment protocol for Treg therapy, potentially expanding its applications to other indications.

Percutaneous Coronary Intervention vs. Coronary Artery Bypass Grafting in Left Main Coronary Artery Disease: An Updated Systematic Review and Meta-Analysis.

Recently, both US and European guidelines have predominantly recommended coronary artery bypass grafting (CABG) as the preferred revascularisation method. However, emerging data have raised the possibility of percutaneous coronary intervention (PCI) being a viable and effective alternative. This meta-analysis sought to evaluate the latest insights from major clinical trials to ascertain whether PCI could be as effective as CABG in treating left main coronary artery (LMCA) disease. To achieve this, a comprehensive systematic search was conducted across databases, including Medline (via PubMed), Embase, Cochrane, and clinicaltrials.gov. The search spanned from the inception of these databases to August 20, 2022, and exclusively focused on randomized controlled trials (RCTs). Employing the random effects model, selected studies underwent rigorous analysis. The study outcomes encompassed a spectrum of factors such as all-cause mortality, major adverse cerebrovascular and cardiovascular events (MACCE), myocardial infarction (MI), stroke, and revascularisation procedures. The observation periods of interest included the 30-day mark, 1 year, 5 years, and 10 years. The analysis integrated six RCTs, revealing noteworthy patterns. In terms of all-cause mortality, PCI demonstrated non-inferiority to CABG across all observed time frames: 30 days (OR 0.6), 1 year (OR 0.77), 5 years (OR 1.41), and 10 years (OR 1.08). Analysis of MACCE outcomes favored PCI at 30 days and CABG at 5 years. The utilisation of the original five-year EXCEL (Evaluation of XIENCE versus Coronary Artery Bypass Surgery for Effectiveness of Left Main Revascularisation) trial definition for MI highlighted higher MI rates for PCI compared to CABG (OR 1.66, P < 0.05). Intriguingly, when the subsequently released EXCEL data, aligned with the third universal MI definition, was incorporated, the five-year data consistently leaned towards CABG. Specifically, the PCI group exhibited 7.5% MI rates in contrast to the 3.6% in the CABG cohort (OR 2.19, P < 0.001). Concerning stroke, PCI proved advantageous at 30 days and 1 year while exhibiting no significant disparity at 5 and 10 years. Revascularisation procedures favoured CABG at one and five years, with comparability at the remaining time points. In summation, the outcomes of this comprehensive meta-analysis suggest that PCI could serve as a feasible alternative to CABG in the context of uncomplicated LMCA disease. It's worth noting that CABG might still hold an advantage for complex lesions.

Barriers to Treg therapy in Europe: From production to regulation.

There has been an increased interest in cell based therapies for a range of medical conditions in the last decade. This explosion in novel therapeutics research has led to the development of legislation specifically focused on cell and gene based therapies. In Europe, the European medicines agency (EMA) designates any medicines for human use which are based on genes, tissues, or cells as advanced therapy medicinal products or advanced therapy medicinal products (ATMPs). In this article we discuss the hurdles to widespread adoption of ATMPs in Europe, with a focus on regulatory T cells (Tregs). There are numerous barriers which must be overcome before mainstream adoption of Treg therapy becomes a reality. The source of the cells, whether to use autologous or allogenic cells, and the methods through which they are isolated and expanded, must all meet strict good manufacturing practice (GMP) standards to allow use of the products in humans. GMP compliance is costly, with the equipment and reagents providing a significant cost barrier and requiring specialized facilities and personnel. Conforming to the regulations set centrally by the EMA is difficult, and the different interpretations of the regulations across the various member states further complicates the regulatory approval process. The end products then require a complex and robust distribution network to ensure timely delivery of potentially life saving treatments to patients. In a European market whose logistics networks have been hammered by COVID and Brexit, ensuring rapid and reliable delivery systems is a more complex task than ever. In this article we will examine the impact of these barriers on the development and adoption of Tregs in Europe, and potential approaches which could facilitate more widespread use of Tregs, instead of its current concentration in a few very specialized centers.

Riding the waves: the ongoing impact of COVID-19 on a national surgical training cohort.

BACKGROUND: The World Health Organisation declared a global pandemic on the 11 March 2020 resulting in implementation of methods to contain viral spread, including curtailment of all elective and non-emergent interventions. Many institutions have experienced changes in rostering practices and redeployment of trainees to non-surgical services. Examinations, study days, courses, and conferences have been cancelled. These changes have the potential to significantly impact the education and training of surgical trainees. AIM: To investigate the impact of the COVID-19 pandemic on training, educational, and operative experiences of Irish surgical trainees. METHODS: Surgical trainees were surveyed anonymously regarding changes in working and educational practices since the declaration of the COVID-19 pandemic on 11 March 2020. The survey was circulated in May 2020 to both core and higher RCSI surgical trainees, when restrictions were at level five. Questions included previous and current access to operative sessions as well as operative cases, previous and current educational activities, access to senior-led training, and access to simulation-/practical-based training methods. A repeat survey was carried out in October 2020 when restrictions were at level two. RESULTS: Overall, primary and secondary survey response rates were 29% (n = 98/340) and 19.1% (n = 65/340), respectively. At the time of circulation of the second survey, the number of operative sessions attended and cases performed had significantly improved to numbers experienced pre-pandemic (p < 0.0001). Exposure to formal teaching and education sessions returned to pre-COVID levels (p < 0.0001). Initially, 23% of trainees had an examination cancelled; 53% of these trainees have subsequently sat these examinations. Of note 27.7% had courses cancelled, and 97% of these had not been rescheduled. CONCLUSION: Surgical training and education have been significantly impacted in light of COVID-19. This is likely to continue to fluctuate in line with subsequent waves. Significant efforts have to be made to enable trainees to meet educational and operative targets.

Anti-Viral Pattern Recognition Receptors as Therapeutic Targets.

Pattern recognition receptors (PRRs) play a central role in the inflammation that ensues following microbial infection by their recognition of molecular patterns present in invading microorganisms but also following tissue damage by recognising molecules released during disease states. Such receptors are expressed in a variety of cells and in various compartments of these cells. PRR binding of molecular patterns results in an intracellular signalling cascade and the eventual activation of transcription factors and the release of cytokines, chemokines, and vasoactive molecules. PRRs and their accessory molecules are subject to tight regulation in these cells so as to not overreact or react in unnecessary circumstances. They are also key to reacting to infection and in stimulating the immune system when needed. Therefore, targeting PRRs offers a potential therapeutic approach for chronic inflammatory disease, infections and as vaccine adjuvants. In this review, the current knowledge on anti-viral PRRs and their signalling pathways is reviewed. Finally, compounds that target PRRs and that have been tested in clinical trials for chronic infections and as adjuvants in vaccine trials are discussed.

Recent advances in our understanding of the allograft response.

Organ transplantation is a life-saving treatment for end-stage organ failure. However, despite advances in immunosuppression, donor matching, tissue typing, and organ preservation, many organs are still lost each year to rejection. Ultimately, tolerance in the absence of immunosuppression is the goal, and although this seldom occurs spontaneously, a deeper understanding of alloimmunity may provide avenues for future therapies which aid in its establishment. Here, we highlight the recent key advances in our understanding of the allograft response. On the innate side, recent work has highlighted the previously unrecognised role of innate lymphoid cells as well as natural killer cells in promoting the alloresponse. The two major routes of allorecognition have recently been joined by a third newly identified pathway, semi-direct allorecognition, which is proving to be a key active pathway in transplantation. Through this review, we detail these newly defined areas in the allograft response and highlight areas for potential future therapeutic intervention.

Inhibition of anti-viral responses in intestinal epithelial cells by epigenetic modifying drugs is mediated by a reduction in viral pattern recognition receptor expression and activity.

Background: Pattern recognition receptors form an essential part of the host defenses against pathogens, in particular in the intestinal epithelium. However, despite their importance relatively little is understood about the regulation of their expression. Increasing evidence suggesting that epigenetic mechanisms such as DNA methylation and histone acetylation have substantial effects on gene expression and regulation. Epigenetic modifying drugs are now used to treat certain cancers but not a lot is known about their effects on the innate immune system. Thus, we set out to examine the role of such drugs in the expression and function of Toll-like receptors. Methods: Using the HCT116 epithelial cell line, we determined the effects of genetic knockout of the DNA methyltransferases enzymes (DNMTs), as well as pharmacological inhibition of the DNMTs and histone deacetylase complexes (HDACs) on TLR responses to their ligands. Results: Our initial results showed that anti-viral responses were affected by changes in the epigenome, with TLR3 responses showing the most dramatic differences. We determined that inhibition of methylation and acetylation inhibited poly I:C induced increases in signaling protein phosphorylation, as well as increases in cytokine mRNA expression and release. We also observed that treatment with epigenetic modifying drugs were leading to large increases in IRF8 expression, a protein that is a known negative regulator of TLR3. When we overexpressed IRF8 in our WT cells we noticed inhibition of poly I:C responses. Conclusion: This research highlighted the potential immunoregulatory role of epigenetic modifying drugs specifically in response to viral stimulation.

Epigenetics and innate immunity: the 'unTolld' story.

Toll-like receptors (TLRs) are a family of 13 receptors known as pattern-recognition receptors (PRRs) and have a key role in the innate immune response. The TLRs are activated by pathogen-associated molecular patterns (PAMPs) that are structurally conserved molecules present on the surfaces of bacteria and viruses. The activation of these TLRs by pathogens results in the downstream activation of genes involved in the production of proinflammatory factors. There is a lack of understanding on the mechanisms by which TLR gene expression is regulated. Epigenetics could be one such mechanism, which is concerned with changes in gene expression/products that arise without a change in the nucleotide sequence. These changes are brought about by two main mechanisms, DNA methylation and histone modifications. This review seeks to examine the current knowledge regarding the epigenetic regulation of this family of receptors and their signalling pathways.

A dynamical model reveals gene co-localizations in nucleus.

Co-localization of networks of genes in the nucleus is thought to play an important role in determining gene expression patterns. Based upon experimental data, we built a dynamical model to test whether pure diffusion could account for the observed co-localization of genes within a defined subnuclear region. A simple standard Brownian motion model in two and three dimensions shows that preferential co-localization is possible for co-regulated genes without any direct interaction, and suggests the occurrence may be due to a limitation in the number of available transcription factors. Experimental data of chromatin movements demonstrates that fractional rather than standard Brownian motion is more appropriate to model gene mobilizations, and we tested our dynamical model against recent static experimental data, using a sub-diffusion process by which the genes tend to colocalize more easily. Moreover, in order to compare our model with recently obtained experimental data, we studied the association level between genes and factors, and presented data supporting the validation of this dynamic model. As further applications of our model, we applied it to test against more biological observations. We found that increasing transcription factor number, rather than factory number and nucleus size, might be the reason for decreasing gene co-localization. In the scenario of frequency- or amplitude-modulation of transcription factors, our model predicted that frequency-modulation may increase the co-localization between its targeted genes.

Compartmentalisation of the sperm plasma membrane: a FRAP, FLIP and SPFI analysis of putative diffusion barriers on the sperm head.

Spermatozoa are highly polarised cells with a compartmentalised distribution of lipids and proteins in their plasma membrane. It is not known how these compartments are stably maintained in what is essentially a fluid environment. In this investigation we have examined the hypothesis that intramembranous diffusion barriers selectively retain some components within compartments, while allowing free passage of others. A fluorescence loss in photobleaching analysis of the behaviour of the lipid reporter probe 1,1'-dihexadecyl-3,3,3'3'-tetramethyindocarbocyanine (DiIC16) on the head of boar spermatozoa revealed that it was freely diffusing between all three compartments (anterior acrosome, equatorial segment and postacrosome). Spermatozoa also contained rapidly diffusing particles of DiIC16 over the anterior acrosome and equatorial segment. These particles, approximately 200 nm in diameter, were tracked in real time and their trajectories analysed by mean square displacement. Particle diffusion was essentially random over the anterior acrosome and equatorial segment but showed a periodicity in jump sizes and diffusion coefficients suggestive of microheterogeneities. Particles did not exchange between the equatorial segment and postacrosome, indicating a barrier at the junction between these two compartments. No barrier was detected between the equatorial segment and anterior acrosome. A model is proposed in which a molecular 'filter' is present at the equatorial segment-postacrosomal boundary that allows free passage of single molecules but not molecular complexes. Passage of heterogeneous complexes, such as lipid rafts, requires disassembly and reassembly on either side of the filter.