Effect of Poly(I:C) and melanoma B16-F10 on the immunophenotype of murine spleen cells

Introduction. It is known that the agonist of TLR-3 Poly(I:C), used as an adjuvant in a number of models of antitumor vaccines, causes inhibition of melanoma B16 growth, but the immunological aspects involved in this process have not been fully studied.The aim of the study was to evaluate changes of the immunophenotype of the spleen cells of C57BL / 6 mice caused by the tumor load and / or Poly(I:C), which is necessary for better understanding of the processes occurring during Poly(I:C) inhibition of melanoma B16-F10.Materials and methods. The immunophenotype of splenocytes of C57Bl / 6 mice was studied by flow cytometry asfollowing: the group 1 was a control (intact animals), the group 2 was mice with subcutaneously transplanted melanoma B16-F10, the group 3 was mice without a tumor treated with Poly(I:C) and the group 4 – mice with subcutaneously transplanted melanoma B16-F10 treated with Poly(I:C).Results. Median values of parameters such as the CD4 / CD8 immunoregulatory index, the percentage of CD69+ CD4+ and CD8+ T cells, the number of B and NK cells for the group of mice with melanoma treated with Poly(I:C) were between the values in the control group and in the group of mice with B16-F10. when comparing the results, the number of B and NK cells, the percentage of CD69+ on CD4+ and CD8+ T cells, their median in the group of mice with melanoma treated with Poly(I:C) was closer to the control than to the values obtained in the B16-F10 group and in the group of healthy mice receiving Poly(I:C). At the same time, we found that the total number of CD3+ cells, the number of naive CD4+ and CD8+ T cells was higher in the group of mice with melanoma treated with Poly(I:C) compared to all other groups.Conclusion. The analysis revealed the changes of the immunophenotype of murine spleen cells (CD4 / CD8, the percentage of CD69+ CD4+ and CD8+ T cells, the number of B and NK cells), which were affected by the tumor load and / or the administration of Poly adjuvant (I:C). Changes in the immunophenotype of murine splenocytes were associated with the tumor load and its size. It was also found that the splenocyte immunophenotype was affected by the repeated administration of Poly(I:C) during the tumor growth.

Maca against Echinococcosis?—A Reverse Approach from Patient to In Vitro Testing

Drug-based treatment of alveolar echinococcosis (AE) with benzimidazoles is in most cases non-curative, thus has to be taken lifelong. Here, we report on a 56-year-old male AE patient who received standard benzimidazole treatment and biliary plastic stents, and additionally self-medicated himself with the Peruvian plant extract Maca (Lepidium meyenii). After 42 months, viable parasite tissue had disappeared. Based on this striking observation, the anti-echinococcal activity of Maca was investigated in vitro and in mice experimentally infected with Echinococcus multilocularis metacestodes. Albendazole (ABZ)-treated mice and mice treated with an ABZ+Maca combination exhibited a significantly reduced parasite burden compared to untreated or Maca-treated mice. As shown by a newly established UHPLC-MS/MS-based measurement of ABZ-metabolites, the presence of Maca during the treatment did not alter ABZ plasma levels. In vitro assays corroborated these findings, as exposure to Maca had no notable effect on E. multilocularis metacestodes, and in cultures of germinal layer cells, possibly unspecific, cytotoxic effects of Maca were observed. However, in the combined treatments, Maca inhibited the activity of ABZ in vitro. While Maca had no direct anti-parasitic activity, it induced in vitro proliferation of murine spleen cells, suggesting that immunomodulatory properties could have contributed to the curative effect seen in the patient.

Tissue Sampling and Homogenization in the Sub-Microliter Scale with a Nanosecond Infrared Laser (NIRL) for Mass Spectrometric Proteomics

It was recently shown that ultrashort pulse infrared (IR) lasers, operating at the wavelength of the OH vibration stretching band of water, are highly efficient for sampling and homogenizing biological tissue. In this study we utilized a tunable nanosecond infrared laser (NIRL) for tissue sampling and homogenization with subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for mass spectrometric proteomics. For the first time, laser sampling was performed with murine spleen and colon tissue. An ablation volume of 1.1 × 1.1 × 0.4 mm³ (approximately 0.5 µL) was determined with optical coherence tomography (OCT). The results of bottom-up proteomics revealed proteins with significant abundance differences for both tissue types, which are in accordance with the corresponding data of the Human Protein Atlas. The results demonstrate that tissue sampling and homogenization of small tissue volumes less than 1 µL for subsequent mass spectrometric proteomics is feasible with a NIRL.

Single-cell transcriptomic analysis of mIHC images via antigen mapping

Highly multiplexed immunohistochemistry (mIHC) enables the staining and quantification of dozens of antigens in a tissue section with single-cell resolution. However, annotating cell populations that differ little in the profiled antigens or for which the antibody panel does not include specific markers is challenging. To overcome this obstacle, we have developed an approach for enriching mIHC images with single-cell RNA sequencing data, building upon recent experimental procedures for augmenting single-cell transcriptomes with concurrent antigen measurements. Spatially-resolved Transcriptomics via Epitope Anchoring (STvEA) performs transcriptome-guided annotation of highly multiplexed cytometry datasets. It increases the level of detail in histological analyses by enabling the systematic annotation of nuanced cell populations, spatial patterns of transcription, and interactions between cell types. We demonstrate the utility of STvEA by uncovering the architecture of poorly characterized cell types in the murine spleen using published cytometry and mIHC data of this organ.

Joint probabilistic modeling of paired transcriptome and proteome measurements in single cells

The paired measurement of RNA and surface protein abundance in single cells with CITE-seq is a promising approach to connect transcriptional variation with cell phenotypes and functions. However, each data modality exhibits unique technical biases, making it challenging to conduct a joint analysis and combine these two views into a unified representation of cell state. Here we present Total Variational Inference (totalVI), a framework for the joint probabilistic analysis of paired RNA and protein data from single cells. totalVI probabilistically represents the data as a composite of biological and technical factors such as limited sensitivity of the RNA data, background in the protein data, and batch effects. To evaluate totalVI, we performed CITE-seq on immune cells from murine spleen and lymph nodes with biological replicates and with different antibody panels measuring over 100 surface proteins. With this dataset, we demonstrate that totalVI provides a cohesive solution for common analysis tasks like the integration of datasets with matched or unmatched protein panels, dimensionality reduction, clustering, evaluation of correlations between molecules, and differential expression testing. totalVI enables scalable, end-to-end analysis of paired RNA and protein data from single cells and is available as open-source software.

Human and mouse transcriptome profiling identifies cross-species homology in pulmonary and lymph node mononuclear phagocytes

SummaryThe mononuclear phagocyte (MP) system consists of macrophages, monocytes, and dendritic cells (DCs). MP subtypes play distinct functional roles in steady state and inflammatory conditions. Though murine MPs are well characterized, their pulmonary and lymph node (LN) human homologs remain poorly understood. To address this gap, we created a gene expression compendium across 15 distinct human and 9 distinct murine MPs from lung, LN, blood, and spleen. Human blood MPs and murine spleen MPs served as validation datasets, as the human-mouse MP homologs are relatively well-defined in these tissues. In-depth RNA sequencing identified corresponding human-mouse MP subtypes and determined marker genes shared and divergent across between species counterparts. Unexpectedly, at the gene expression level, only 13-23% of the top 1000 marker genes (i.e., genes not shared across species-specific MP subtypes) overlapped in corresponding human-mouse MP counterparts, indicating a need for caution when translating mouse studies to human gene targets and functions. Lastly, CD88 was useful in both species to distinguish macrophage and tissue monocytes from DCs. Our cross-species gene expression compendium serves as a resource for future translational studies to investigate beforehand whether pursuing specific MP subtypes, or genes will prove fruitful.