Myeloid Cells in Intact Human Cervical Explants Capture HIV and Can Transmit It to CD4 T Cells

The importance of myeloid cells in HIV transmission in the female genital tract is uncertain. Because it is difficult to study the early events in HIV transmission in humans, most of our knowledge is based on animal models of SIV infection in Rhesus macaques and more recently HIV infection in humanized mice. However, these models may not accurately recapitulate transmission in the human genital tract. CD14+ myeloid cells are the most abundant hematopoietic cells in the human cervical mucosa, comprising 40–50% of CD45+ mononuclear cells. Most CD14+ cells are CD14+CD11c– macrophages and about a third are CD14+CD11c+ tissue dendritic cells, which express the HIV-binding receptors, DC-SIGN and CX3CR1. To examine the role of mucosal myeloid cells in HIV transmission, we infected intact healthy human cervical explants with CCR5–tropic HIV-1 ex vivo and then sorted populations of cervical immune cells 20 h later to determine whether they took up virus and could transmit it to activated CD4 T cells. Viral RNA was detected in CD14+ myeloid cells in all but one of 10 donor tissue samples, even when HIV RNA was not detected in CD4+ T cells. HIV RNA was detected predominantly in CD14+CD11c+ dendritic cells rather than in CD14+CD11c– macrophages. The reverse transcriptase inhibitor, nevirapine, reduced HIV RNA in CD4+ T cells, but not in CD14+ cells. Moreover, integrated HIV DNA were not detected above background in myeloid cells but was detected in T cells. These data suggest that although HIV replicates in T cells, myeloid cells in the female genital mucosa capture viral particles, but do not replicate the virus at early timepoints. However, sorted CD14+ myeloid cells isolated 20 h post-infection from 5 HIV-infected cervical explants tested all transmitted HIV to activated CD4+ T cells, while only 1 sample of sorted CD4+ T cells did. Thus, myeloid cells in human cervical tissue capture HIV and are an important early cellular storage site of infectious virus

Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

Background: Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP)-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP) labelling is complicated by autofluorescence (AF) of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. Methods: Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. Results: A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP) and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP), AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. Discussion Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis of parasite-infected samples with in the intention of gene-expression profiling and analysis. The approach outlined here results in substantially improved yield of GFP-expressing parasites, and requires decreased sorting time in comparison to standard methods. It is anticipated that this protocol will be useful for a wide range of applications involving rare events

Microguards and micromessengers of the genome

The regulation of gene expression is of fundamental importance to maintain organismal function and integrity and requires a multifaceted and highly ordered sequence of events. The cyclic nature of gene expression is known as ‘transcription dynamics’. Disruption or perturbation of these dynamics can result in significant fitness costs arising from genome instability, accelerated ageing and disease. We review recent research that supports the idea that an important new role for small RNAs, particularly microRNAs (miRNAs), is in protecting the genome against short-term transcriptional fluctuations, in a process we term ‘microguarding’. An additional emerging role for miRNAs is as ‘micromessengers’—through alteration of gene expression in target cells to which they are trafficked within microvesicles. We describe the scant but emerging evidence that miRNAs can be moved between different cells, individuals and even species, to exert biologically significant responses. With these two new roles, miRNAs have the potential to protect against deleterious gene expression variation from perturbation and to themselves perturb the expression of genes in target cells. These interactions between cells will frequently be subject to conflicts of interest when they occur between unrelated cells that lack a coincidence of fitness interests. Hence, there is the potential for miRNAs to represent both a means to resolve conflicts of interest, as well as instigate them. We conclude by exploring this conflict hypothesis, by describing some of the initial evidence consistent with it and proposing new ideas for future research into this exciting topic

A flow cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells.

Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, to make it easier to quantify and to allow a streamlined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (takes no more than a working day from sample collection to quantification), requires less starting material compared with standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy.Research in our laboratory is funded by Cancer Research UK (CRUK; programme grant C6/A11224), the European Research Council and the European Community Seventh Framework Programme (grant agreement no. HEALTH¬‐F2¬‐2010¬‐259893 (DDResponse)). Core funding is provided by Cancer Research UK (C6946/A14492) and the Wellcome Trust (WT092096). J.V.F. is funded by Cancer Research UK programme grant C6/A11224 and the Ataxia Telangiectasia Society. S.P.J. receives his salary from the University of Cambridge, supplemented by CRUK.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/nprot.2015.06

Circulating microparticles: square the circle

Background: The present review summarizes current knowledge about microparticles (MPs) and provides a systematic overview of last 20 years of research on circulating MPs, with particular focus on their clinical relevance. Results: MPs are a heterogeneous population of cell-derived vesicles, with sizes ranging between 50 and 1000 nm. MPs are capable of transferring peptides, proteins, lipid components, microRNA, mRNA, and DNA from one cell to another without direct cell-to-cell contact. Growing evidence suggests that MPs present in peripheral blood and body fluids contribute to the development and progression of cancer, and are of pathophysiological relevance for autoimmune, inflammatory, infectious, cardiovascular, hematological, and other diseases. MPs have large diagnostic potential as biomarkers; however, due to current technological limitations in purification of MPs and an absence of standardized methods of MP detection, challenges remain in validating the potential of MPs as a non-invasive and early diagnostic platform. Conclusions: Improvements in the effective deciphering of MP molecular signatures will be critical not only for diagnostics, but also for the evaluation of treatment regimens and predicting disease outcomes

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

A60

It has previously been shown that additional local electromagnetic impact on the tumor en-hances the systemic antitumor effect of low-intensity electromagnetic radiation (EMR) used in the activation therapy regimes designed by Garkavi L.H. et al. (1990–2008). At the same time the question of the influence of weak EMR, applied to the peritumoral area, on the tumor development has not been studied. The aim of the study was to investigate the changes in the tumor caused by low-intensity microwave electromagnetic radiation of bioeffective frequency that acted on the head and peritumoral zone. Materials and methods: The effects of resonance radiation (RR) with frequency corresponding to that of one of the water-containing medium radiations – 1 GHz (“SPE-effect”) – were studied on 53 adult male outbred white rats with transplantable sarcoma 45. Special anticancer agents were not used. The power flux density of RR was less than 1 μW/cm2, surface area of the emitter – 4 cm2, 3–10 min. exposure depending on algorithms of the activation therapy. RR exposure in different groups of animals was localized to the head only, or to the peri-tumoral area only, or successively to the head and peritumoral area (“double” exposure). The course lasted for 4 weeks. The exposure effect was assessed according to the dynamics of the tumor size and results of the light and electron microscopy analysis of tumor changes (JEOL JEM-1011, Japan). Results: The effect of RR in different groups of animals depended on the exposure localization. Central systemic exposure was decisive. The group receiving RR localized to the head showed regression of the tumor or inhibition of its growth in 60% of animals – almost complete tumor regression in 10% and tumor growth inhibition by 70% in the rest cases. RR to the peritumoral area did not show significant influence on the tumor development, while it increased antitumor effect of the central exposure (p < 0.01–0.05). Antitumor effect was registered in 77% of the animals receiving the “double” RR exposure: 30% – morphologically verified complete regression, 24% – partial regression (tumor shrinkage by 2–2.5 times) and tumor growth inhibition by 40% was detected in 23% of animals. Regressing tumor, unlike sarcoma 45 with an active growth, was characterized by significant thickening of the capsule (by 7 times, p < 0.01) and increased intensity of lymphoplasmacytic infiltration (p < 0.01). Immune system cells were present in the capsule, subcapsular zone and as leukocytic barrier in peritumoral area of the conjunctive tissue of up to 170 μm width. Different numbers of lymphocytes and plasma cells were noted in tumor cells. Macrophages were found. Migrating lymphocytes were often noted in the vessels among tumor cells. Electron microscopy showed multiple contacts of lymphocytes with the surface of tumor cells through cytoplasmic excrescences. Such lymphocytes had distinct signs of activation. Simultaneous contacts of lymphocytes and macrophages among themselves and with tumor cells were found. Analysis of ultrastructural characteristics of cells in regressing tumors and detection of collagen in intercellular spaces during histochemical examination of tumor tissues showed the increase in the degree of differentiation of some sarcoma 45 cells. Conclusion: Possibility to increase the systemic antitumor effect of the low-intensity microwave radiation with an additional weak exposure on the peritumoral area was demonstrated for the first time. Damaging effect of RR on tumor was mediated by the changes in composition and activity of elements of the tumor’s immune microenvironment and by the increase in the degree of differentiation of some tumor cells, probably under the influence of bioactive factors of leukocytic origin

A66

In previous experiments the self-dependent antitumor effect of magnetite nanoparticles (NPs) injected in peritumoral area in form of magnetic fluid (MF) was shown. Elucidation of the mechanisms of this phenomenon is of theoretical and practical interest. The aim of the study was to investigate the cellular and ultrastructural changes in the tissue tumors regressed under the influence of magnetite NPs, as well as the composition of the cells of the immune system in the peritumoral area. Materials and methods: The study was carried on white male rats, 180–200 g, with transplanted sarcoma 45 (59) or Pliss lymphosarcoma (50). Special antitumor agents were not used. Magnetite NPs (10 ± 2 nm) were applied in the form of the water-based MF. Original MF (20 kA/m) was diluted with saline in different degree and was injected into peritumoral zone along the tumor borders at a distance of 1.5 cm twice a week in a volume of 0.4–0.9 ml (depending on the animal weight) within 3 weeks. Special anti-tumor agents were not used. At the end of the experiments fragments of the tumor and surrounding tissue were taken for research. The study of changes in the tumor and peritumoral area were performed by the methods of cytology, histology, histochemistry, electron microscopy (microscope JEOL JEM-1011, Japan), flow cytometry, X-ray fluorescence spectroscopy (spectrometer M4 Tornado Bruker). Results: At a dilution of the original MF in 30 times the treatment was effective in 75% of animals. Complete and partial (more than 2-fold) tumor regression was observed in 2/3 cases. In rats with Pliss lymphosarcoma tumor regression on 70–100% has been reported in 20–40% cases. The results of microscopic examination of sarcoma 45 with partial regression showed significant changes in their immune microenvironment compared with the cases of progressive tumor growth (p < 0.05–0.01). This was expressed in the increase in the number of lymphocytes and plasmacytes (respectively, in 12 and 2.5 fold), and in the appearance of macrophages and basophils that were missing in tumors with progressive growth. The results of flow cytometry of tissue from the tumor as well as from peritumoral zone indicate the predominance of plasmacytes in the case of inhibition of tumor growth and increasing the proportion of mature T lymphocytes in the cases of tumor regression (more than 1.5 times, p < 0.05). By electron microscopy, it was shown that in the cases of efficiency of the MF in addition to necrosis, observed also in growing tumors, such types of cell death were marked as apoptosis and autophagy. Numerous signs of activation of cell–cell interactions involved cells of the immune system were revealed. Different groups of 2–4 contacting cells (macrophages, lymphocytes, plasmacytes, mast cells, tumor-associated fibroblasts and neutrophils in various combinations) with signs of metabolic activity were marked. X-ray fluorescence spectroscopy preliminary results in rats with Pliss lymphosarcoma regression indicated dense arrangement of magnetite NPs and their aggregates in the peritumoral area and their practical absence in tumor. Conclusion: The study results reflect the changes in the immune microenvironment of experimental tumors under effective action of magnetite NPs and indicate significant activation of local immune processes caused by these factors