Chemokine Homeostasis in Healthy Volunteers and during Pancreatic and Colorectal Tumor Growth in Murine Models.

Chemokines are involved in the humoral regulation of body homeostasis. Changes in the blood level of chemokines were found in cancer, atherosclerosis, diabetes, and other systemic diseases. It is essential to distinguish the effects of co-morbid pathologies and cancer on the level of chemokines in the blood. We aimed to analyze, by multiplex cytometry, the levels of chemokines in the blood of healthy young volunteers as well as of intact mice and mice with CT26 colon and Pan02 pancreatic tumors. Two types of chemokines were identified both in human and murine plasmas: homeostatic ones, which were found in high concentrations (>100 pg/mL), and inducible ones, which can be undetectable or determined at very low levels (0−100 pg/mL). There was a high variability in the chemokine levels, both in healthy humans and mice. To analyze chemokine levels during tumor growth, C57BL/6 and BALB/c were inoculated with Pan02 or CT26 tumor cells, accordingly. The tumors significantly differed in the growth and the mortality of mice. However, the blood chemokine levels did not change in tumor-bearing mice until the very late stages. Taken collectively, blood chemokine level is highly variable and reflects in situ homeostasis. Care should be taken when considering chemokines as prognostic parameters or therapeutic targets in cancer.

Asymmetric depth-filtration: A versatile and scalable method for high-yield isolation of extracellular vesicles with low contamination.

We developed a novel asymmetric depth filtration (DF) approach to isolate extracellular vesicles (EVs) from biological fluids that outperforms ultracentrifugation and size-exclusion chromatography in purity and yield of isolated EVs. By these metrics, a single-step DF matches or exceeds the performance of multistep protocols with dedicated purification procedures in the isolation of plasma EVs. We demonstrate the selective transit and capture of biological nanoparticles in asymmetric pores by size and elasticity, low surface binding to the filtration medium, and the ability to cleanse EVs held by the filter before their recovery with the reversed flow all contribute to the achieved purity and yield of preparations. We further demonstrate the method's versatility by applying it to isolate EVs from different biofluids (plasma, urine, and cell culture growth medium). The DF workflow is simple, fast, and inexpensive. Only standard laboratory equipment is required for its implementation, making DF suitable for low-resource and point-of-use locations. The method may be used for EV isolation from small biological samples in diagnostic and treatment guidance applications. It can also be scaled up to harvest therapeutic EVs from large volumes of cell culture medium.

Silicon-Gold Nanoparticles Affect Wharton's Jelly Phenotype and Secretome during Tri-Lineage Differentiation.

Multiple studies have demonstrated that various nanoparticles (NPs) stimulate osteogenic differentiation of mesenchymal stem cells (MSCs) and inhibit adipogenic ones. The mechanisms of these effects are not determined. The aim of this paper was to estimate Wharton's Jelly MSCs phenotype and humoral factor production during tri-lineage differentiation per se and in the presence of silicon-gold NPs. Silicon (SiNPs), gold (AuNPs), and 10% Au-doped Si nanoparticles (SiAuNPs) were synthesized by laser ablation, characterized, and studied in MSC cultures before and during differentiation. Humoral factor production (n = 41) was analyzed by Luminex technology. NPs were nontoxic, did not induce ROS production, and stimulated G-CSF, GM-CSF, VEGF, CXCL1 (GRO) production in four day MSC cultures. During MSC differentiation, all NPs stimulated CD13 and CD90 expression in osteogenic cultures. MSC differentiation resulted in a decrease in multiple humoral factor production to day 14 of incubation. NPs did not significantly affect the production in chondrogenic cultures and stimulated it in both osteogenic and adipogenic ones. The major difference in the protein production between osteogenic and adipogenic MSC cultures in the presence of NPs was VEGF level, which was unaffected in osteogenic cells and 4-9 times increased in adipogenic ones. The effects of NPs decreased in a row AuNPs > SiAuNPs > SiNPs. Taken collectively, high expression of CD13 and CD90 by MSCs and critical level of VEGF production can, at least, partially explain the stimulatory effect of NPs on MSC osteogenic differentiation.

Mesenchymal Stromal Cells Isolated from Ectopic but Not Eutopic Endometrium Display Pronounced Immunomodulatory Activity In Vitro.

A comparative analysis of the cell surface markers and immunological properties of cell cultures originating from normal endometrium and endometrioid heterotopias of women with extragenital endometriosis was carried out. Both types of cell cultures expressed surface molecules typical of mesenchymal stromal cells and did not express hematopoietic and epithelial markers. Despite similar phenotype, the mesenchymal stromal cells derived from the two sources had different immunomodulation capacities: the cells of endometrioid heterotopias but not eutopic endometrium could suppress dendritic cell differentiation from monocytes as well as lymphocyte proliferation in allogeneic co-cultures. A comparative multiplex analysis of the secretomes revealed a significant increase in the secretion of pro-inflammatory mediators, including IL6, IFN-γ, and several chemokines associated with inflammation by the stromal cells of ectopic lesions. The results demonstrate that the stromal cells of endometrioid heterotopias display enhanced pro-inflammatory and immunosuppressive activities, which most likely impact the pathogenesis and progression of the disease.

Ultrastructural and Immunohistochemical Features of Telocytes in Placental Villi in Preeclampsia.

A new cell type, interstitial Cajal-like cell (ICLC), was recently described in different organs. The name was recently changed to telocytes (TCs), and their typical thin, long processes have been named telopodes (Tp). TCs regulate the contractile activity of smooth muscle cells and play a role in regulating vessel contractions. Although the placenta is not an innervated organ, we believe that TCs are present in the placenta. We studied placenta samples from physiological pregnancies and in different variants of preeclampsia (PE). We examined these samples using light microscopy of semi-thin sections, transmission electron microscopy, and immunohistochemistry. Immunohistochemical examination was performed with primary antibodies to CD34, CD117, SMA, and vimentin, and TMEM16a (DOG-1), the latter was used for the diagnosis of gastrointestinal stromal tumours (GIST) consisting of TCs. We have identified a heterogenetic population of ТСs in term placentas, as these cell types differed in their localization, immunophenotype and ultrastructural characteristics. We assume TMEM16a could be used as the marker for identification of TCs. In PE we have revealed telocyte-like cells with ultrastructural signs of fibrocytes (significant process thickening and the granular endoplasmic reticulum content was increased) and a loss of TMEM16a immunohistochemical staining.

Isolation and characterization of trophoblasts from enzymatic explants of human term placenta.

In the present study, we describe a new method of isolation and culture of human villous and extravillous trophoblasts from term placenta. The cultivation of trypsinized placental villous tissue explants, followed by the isolation of cells from outgrowth islets allows for obtaining a cytotrophoblast subpopulation that is free from contamination by other cell types. Compared to other methods, our protocol is mild, simple and effective, does not request costly reagents and provides isolation of the mononuclear cytotrophoblast cell populations free from contamination by other types of placental cells. The isolated cells proliferated and formed a pleomorphic monolayer, where cells fused into a small number of binuclear or polynuclear syncytiotrophoblasts. Isolated cytotrophoblast cells expressed the specific epithelial intermediate filament cytokeratin 7 (CK7), the epithelium-specific cell-cell adhesion molecule E-cadherin and were CD9-, CD45- and vimentin-negative. Cyto- and syncytiotrophoblasts obtained by this method can be used as a model or tool for the fundamental research of differentiation and function of human placental cells, and can provide a new understanding of drug distribution in placenta. Their combination with other in vitro cell models can be useful for studying a variety of other aspects concerning placental functions, which will provide new knowledge for understanding immunology, endocrinology and development of placenta.

Lutein and its oxidized forms in eye structures throughout prenatal human development.

The presence of carotenoids in the vitreous body, retina, lens, retinal pigment epithelium together with choroid (hereinafter RPE), and ciliary body and iris together with choroidal stroma (hereinafter CBI) was studied throughout the second trimester of prenatal development of the human eye. It has been found that the vitreous body, retina, and RPE contain lutein and its oxidized forms. Zeaxanthin was not found in the tissues studied. The presence of lutein in the vitreous body is transient and no longer detected after 28 weeks of gestation. Lutein was not detected in the lens and CBI, but its oxidized forms were found. The presence of carotenoids in different tissues of the eye in the course of normal eye development and the antioxidant role of carotenoids are discussed.

Neural stem/progenitor cells maintained in vitro under different culture conditions alter differentiation capacity of monocytes to generate dendritic cells.

Cell therapy of the nervous system disorders using neural stem/progenitor cells (NSPCs) proved its efficacy in preclinical and pilot clinical studies. The mechanisms of the beneficial effects of NSPCs transplantation include replacement of damaged cells, paracrine activation of the regeneration, and immunomodulation. Detailed assessment of NSPCs-induced immunomodulation can contribute to better control of autoimmune reactions and inflammation in patients with neurodegenerative diseases. Interactions of NSPCs with dendritic cells (DCs), the key players in the induction of the immune system response to antigens are of particular interest. Here, we demonstrate that co-culturing of monocytes with NSPCs obtained and grown utilizing serum-containing medium instead of growth factor-containing serum-free medium, results in total suppression of monocyte differentiation into DCs. The effect is similar to the action of mesenchymal stem cells (MSCs). No significant effect on DCs maturation was observed. Cultures of NSPCs set up and maintained in serum-free medium have no influence on monocyte differentiation and DCs maturation. Therefore, the effects of NSPCs upon DC differentiation from monocytes strongly depend on culture conditions, whereas the molecular marker expression patterns are similar in both types of NSPCs cultures. In broader prospective, it means that cells with almost identical phenotypes can display opposite immunological properties depending upon culture conditions. It should be taken into account when developing NSPCs-based cell products for regenerative medicine.

Updates on the Production of Therapeutic Antibodies Using Human Hybridoma Technique.

Therapeutic antibodies are implicated into the very promising and fast growing area of pharmaceutics. Human hybridoma technology, allowing generation of natural human antibodies in a native form, seems to be the most direct way that require no additional modifications for production of therapeutic antibodies. However, technical difficulties in human hybridoma creation discovered in the 80s of the last century have switched the mainstream therapeutic antibody development into new directions like display and transgenic mice techniques. These approaches have provided remarkable achievements in antibody engineering within last 15 years, but also revealed other limitations. Thus, it is time to turn back to forgotten human hybridoma technology. In this review, we describe new advances in all components of human hybridoma technology and discuss challenges in generating novel therapeutic mABs based on hybridoma technologies.

Use of a cyanine dye as a probe for albumin and collagen in the extracellular matrix.

The aim of this work was to develop a quick method for analysis of macromolecules of the extracellular matrix. Of great interest are soluble components of the extracellular matrix, in particular, carrier proteins, whose variation dynamics can characterize the studied tissue in its development, adult stage, and aging. We suggest the method of analysis of the extracellular matrix to reveal the presence of albumin and collagen by using an anionic cyanine dye as a spectral and fluorescence probe. The method was applied for the analysis of the human vitreous body in the course of its development. Albumin was detected by the appearance of the trans monomer absorption and fluorescence bands in the dye spectra, and collagen was detected by the absorption and fluorescence bands of J aggregates. Hyaluronic acid present in the vitreous body does not interfere with the results of the analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis confirmed the presence of albumin in the vitreous body. We suppose that albumin as a protein carrying biologically active macromolecules plays an important role in the processes of differentiation and functional establishment of ocular tissues in the course of their prenatal development.