Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Molecular investigation of cellular immunity in marsupials

"The T cell signalling cascade is an important biochemical event in the adaptive immune system. In this study, various molecules involved in the T cell biology of marsupials were characterized. Investigation of possible differences in the molecular sequence of these genes was undertaken to explain why marsupials appear to respond differently to other mammals in their reaction to challenge by pathogens. Prior to this study it has been generally accepted that the adaptive immune system in marsupials may not function in the same manner as in other mammals"--Abstract

Molecular investigation of cellular immunity in marsupials

"The T cell signalling cascade is an important biochemical event in the adaptive immune system. In this study, various molecules involved in the T cell biology of marsupials were characterized. Investigation of possible differences in the molecular sequence of these genes was undertaken to explain why marsupials appear to respond differently to other mammals in their reaction to challenge by pathogens. Prior to this study it has been generally accepted that the adaptive immune system in marsupials may not function in the same manner as in other mammals"--Abstract

Molecular investigation of cellular immunity in marsupials

"The T cell signalling cascade is an important biochemical event in the adaptive immune system. In this study, various molecules involved in the T cell biology of marsupials were characterized. Investigation of possible differences in the molecular sequence of these genes was undertaken to explain why marsupials appear to respond differently to other mammals in their reaction to challenge by pathogens. Prior to this study it has been generally accepted that the adaptive immune system in marsupials may not function in the same manner as in other mammals"--Abstract

Molecular characterisation of the signaling molecules TCR? and ZAP-70 in the marsupial Macropus eugenii (tammar wallaby)

The debate about the state of the marsupial immune system has entered a new era with the recent publication of the tammar wallaby (Macropus eugenii) genome. The aim of this study was to investigate two important components of the T-cell signalling cascade in M. eugenii to determine whether there are any significant differences between the genome and the expressed gene sequences and to elucidate the putative structures. Molecular methods, predominantly RACE PCRand RT–PCR, using cDNA obtained from mRNA isolated from M. eugenii lymph node tissue, were used to determine the sequence of functional motifs in the TCRz and ZAP-70 molecules. Structure prediction algorithms were used to determine their secondary and tertiary structures for comparison with the structures elucidated by X-ray crystallography in humans and other mammals. Differences between the genome and the expressed sequence were found in the ZAP-70 molecule. Homology modelling demonstrated that the predicted structure of the TCRz molecule was different from those of other mammals while the structure of the ZAP-70 molecule was very similar. It is concluded that the T-cell signalling cascade in the adaptive immune system of marsupials shows similar features to that of other mammals

Molecular characterisation of Interleukin-2 in two Australian marsupials (the tammar wallaby, Notamacropus eugenii, and the Tasmanian devil, Sarcophilus harrisii) facilitates the development of marsupial-specific immunological reagents

Interleukin-2 (IL-2) is an important regulator of cellular immunity in mammals. For many years, our inability to identify the expression of this cytokine in marsupials hindered our capacity to progress studies in metatherian immunology. Here, we report the use of molecular techniques to characterise the IL-2 gene for the tammar wallaby (Notamacropus eugenii) and the Tasmanian devil (Sarcophilus harrisii), which allowed the prediction of the structure and probable functions of the IL-2 proteins of these species. Deduced marsupial IL-2 proteins show considerable sequence identity to each other and to common brushtail possum (Trichosurus vulpecula) IL-2 (≥65%) but shared only 35% (tammar wallaby) and 32% (Tasmanian devil) identity with human IL-2. This difference means that reagents used to study IL-2 in human and other eutherians are unlikely to cross-react with marsupials. As a key step in furthering our ability to study cellular immune responses in marsupials and, more specifically, the susceptibility of macropodoid marsupials to intracellular pathogens, a polyclonal antibody was designed for the detection and future investigation of tammar wallaby IL-2 protein expression. The molecular data and polyclonal antibody described herein will support our development of gene probes and immunological reagents that will aid studies of infection and disease in marsupials

The Transcription Factor IRF4 Is Crucial for CLL Progression and Regulates Survival and Proliferation in a Microenvironment Related Manner

Abstract Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by an accumulation of CD19+ CD5+ CLL cells in the peripheral blood and the lymphoid compartments. The disease is still incurable despite recent advances in the development of novel therapy concepts (e.g. inhibitors of BCR signaling). Although it is not entirely clear how CLL develops, proliferation centers located in the lymph node and the bone marrow are thought to be the crucial niche that provides the surrounding in which CLL cells originate and re-establish the clone after therapy, leading to relapse in almost 100% of CLL patients. In analogous normal lymphoproliferative environments the transcription factor IRF4 plays an important role in controlling B cell maturation, differentiation, proliferation and survival. A SNP in the human IRF4 3’UTR was linked to CLL susceptibility (DiBernardo Nat.Gen. 2008) and in the mouse model germline deletion of IRF4 in all lineages contributed to CLL development in the New Zealand Black and in the VH11 mouse model (Shukla Blood 2013, Ma J.Biol.Chem. 2013). Methods: CLL samples were collected from the peripheral blood of CLL patients according to the Declaration of Helsinki and with written informed consent. IRF4 mRNA expression analyses were performed in purified CD19+ CLL cells using TaqMan Real Time PCR. In vitro studies were performed in soloculture without stimulation or in coculture systems mimicking the lymph node microenvironment, as previously described (Asslaber Br.J.Haematol. 2013). IRF4 knockdown was performed using lipofection and IRF4 specific siRNAs or non-targeting control siRNAs. IRF4 protein expression was measured by intracellular flow cytometry. Proliferation was detected by Ki-67 intracellular staining. All intracellular stainings were performed after gating on viable CD19+ CD5+ CLL cells. Cell death was assessed by Annexin V / 7AAD staining or using a fixable viability dye. Results: Overall, IRF4 mRNA expression was decreased in CLL patients (N=101) when compared to healthy donors (N=8, P=0.04). In the CLL cohort low IRF4 mRNA expression (cut-off determined by ROC analysis and Youden Index calculation) was significantly correlated with earlier time to treatment (median treatment-free survival: 40 month in the IRF4LOW and 69 month in the IRF4HIGH CLL patient group (P&lt;0.01)). This was proved to be independent of mutation state and other CLL risk factors in multivariate analysis. Using in vitro knockdown of IRF4 in solo- and coculture models we found that lowered expression of IRF4 was associated with increased CLL cell survival and abrogated spontaneous apoptosis (N=11, P&lt;0.01). Moreover, we detected a failure of CLL cells to respond to CD40L signals derived from the microenvironment after IRF4 knockdown, which was associated with abrogated proliferation (N=9, P&lt;0.01). Indeed microenvironmental stimuli (e.g. CD40L ligation or addition of activated T cells) were required to maintain IRF4 expression in vitro (N=17, P &lt; 0.001) which also corresponds to our observation that IRF4 positive CLL cells were highly overrepresented in the CXCR4dim CD5brightCLL population (N = 55, P &lt; 0.0001) which is considered to be the proliferative CLL cell fraction emigrated from the lymph node (Calissano Mol. Med. 2011). Conclusion: We demonstrate that low IRF4 expression is an independent negative prognostic marker for CLL progression. In the microenvironmental context IRF4 plays a dual role, with high expression limiting CLL survival on the one hand but being a crucial mediator of CD40L mediated proliferation on the other hand. While our data suggest that IRF4 is upregulated in response to microenvironmental stimuli the association of low IRF4 levels with bad prognosis suggests that IRF4 levels may serve to limit CLL growth by enhancing cell death responses. The exact mechanisms by which IRF4 impacts CLL pathophysiology is currently being investigated in vivo using B-cell specific IRF4-deficient CLL mouse models. Disclosures No relevant conflicts of interest to declare. </jats:sec

Molecular characterisation of Interleukin-2 in two Australian marsupials (the tammar wallaby, Notamacropus eugenii, and the Tasmanian devil, Sarcophilus harrisii) facilitates the development of marsupial-specific immunological reagents

Interleukin-2 (IL-2) is an important regulator of cellular immunity in mammals. For many years, our inability to identify the expression of this cytokine in marsupials hindered our capacity to progress studies in metatherian immunology. Here, we report the use of molecular techniques to characterise the IL-2 gene for the tammar wallaby (Notamacropus eugenii) and the Tasmanian devil (Sarcophilus harrisii), which allowed the prediction of the structure and probable functions of the IL-2 proteins of these species. Deduced marsupial IL-2 proteins show considerable sequence identity to each other and to common brushtail possum (Trichosurus vulpecula) IL-2 (≥65%) but shared only 35% (tammar wallaby) and 32% (Tasmanian devil) identity with human IL-2. This difference means that reagents used to study IL-2 in human and other eutherians are unlikely to cross-react with marsupials. As a key step in furthering our ability to study cellular immune responses in marsupials and, more specifically, the susceptibility of macropodoid marsupials to intracellular pathogens, a polyclonal antibody was designed for the detection and future investigation of tammar wallaby IL-2 protein expression. The molecular data and polyclonal antibody described herein will support our development of gene probes and immunological reagents that will aid studies of infection and disease in marsupials

Molecular identification of interleukin-2 in the lymphoid tissues of the common brushtail possum, Trichosurus vulpecula

The common brushtail possum (Trichosurus vulpecula) is an Australian marsupial. Here we describe the identification of possum interleukin-2 in mitogen-stimulated lymph node cells. We used a strategy of Rapid amplification of cDNA ends using probes designed from recently-sequenced marsupial genomes to identify the IL2 gene and then confirmed that IL-2 expression in possum immune tissue occurs in a similar manner to that in their eutherian counterparts. The predictive possum IL-2 peptide showed 28% and 35% amino acid sequence homology with the mouse and human IL-2 molecules, respectively, consistent with the divergence found within this cytokine family. Despite this low sequence identity, possum IL-2 still possessed the characteristic hallmarks of mammalian IL-2, such as a predicted signal peptide and conserved family motifs

Small babies, big risks : global estimates of prevalence and mortality for vulnerable newborns to accelerate change and improve counting

Small newborns are vulnerable to mortality and lifelong loss of human capital. Measures of vulnerability previously focused on liveborn low-birthweight (LBW) babies, yet LBW reduction targets are off-track. There are two pathways to LBW, preterm birth and fetal growth restriction (FGR), with the FGR pathway resulting in the baby being small for gestational age (SGA). Data on LBW babies are available from 158 (81%) of 194 WHO member states and the occupied Palestinian territory, including east Jerusalem, with 113 (58%) having national administrative data, whereas data on preterm births are available from 103 (53%) of 195 countries and areas, with only 64 (33%) providing national administrative data. National administrative data on SGA are available for only eight countries. Global estimates for 2020 suggest 13·4 million livebirths were preterm, with rates over the past decade remaining static, and 23·4 million were SGA. In this Series paper, we estimated prevalence in 2020 for three mutually exclusive types of small vulnerable newborns (SVNs; preterm non-SGA, term SGA, and preterm SGA) using individual-level data (2010–20) from 23 national datasets (∼110 million livebirths) and 31 studies in 18 countries (∼0·4 million livebirths). We found 11·9 million (50% credible interval [Crl] 9·1–12·2 million; 8·8%, 50% Crl 6·8–9·0%) of global livebirths were preterm non-SGA, 21·9 million (50% Crl 20·1–25·5 million; 16·3%, 14·9–18·9%) were term SGA, and 1·5 million (50% Crl 1·2–4·2 million; 1·1%, 50% Crl 0·9–3·1%) were preterm SGA. Over half (55·3%) of the 2·4 million neonatal deaths worldwide in 2020 were attributed to one of the SVN types, of which 73·4% were preterm and the remainder were term SGA. Analyses from 12 of the 23 countries with national data (0·6 million stillbirths at ≥22 weeks gestation) showed around 74% of stillbirths were preterm, including 16·0% preterm SGA and approximately one-fifth of term stillbirths were SGA. There are an estimated 1·9 million stillbirths per year associated with similar vulnerability pathways; hence integrating stillbirths to burden assessments and relevant indicators is crucial. Data can be improved by counting, weighing, and assessing the gestational age of every newborn, whether liveborn or stillborn, and classifying small newborns by the three vulnerability types. The use of these more specific types could accelerate prevention and help target care for the most vulnerable babies