Immunological alterations in intracranial aneurysm: a prospective study on selected biomarker profiles in blood collected during endovascular neurointervention.

INTRODUCTION: Previous studies showed that the concentrations of selected chemokines are locally elevated in samples collected from the lumen of intracranial aneurysms (IA). Our objective was to determine whether the observed differences in analyte concentrations were influenced by the origin of the blood samples (i.e. cerebral versus peripheral), thus providing insight into the localised nature of these alterations and their significance in IA pathogenesis. MATERIAL AND METHODS: This prospective study included 24 patients with IA who underwent endovascular embolisation. Concentrations of selected analytes were analysed in blood samples from the IA lumen, feeding artery, and aorta. The analytes included MPO, Lipocalin-2/NGAL, sICAM-1, sVCAM-1, and serum amyloid A. RESULTS: Higher median plasma concentrations of MPO, lipocalin-2/NGAL, sVCAM-1, and SAA were found in samples obtained from the IA lumen and the feeding artery compared to the aorta. The concentration of sICAM-1 was significantly higher in the IA compared to the aorta, but did not differ between the proximal artery and the aorta. No significant differences in any analyte concentration were observed between the IA and the proximal artery. CONCLUSIONS: These findings suggest that the IA and the proximal vessel share similarities in the local immunological environment, which is different from that observed in the aorta. Further studies are needed to fully understand and elucidate these observations.

Higher platelet counts correlate to tumour progression and can be induced by intratumoural stroma in non-metastatic breast carcinomas.

BACKGROUND: Platelets support tumour progression. However, their prognostic significance and relation to circulating tumour cells (CTCs) in operable breast cancer (BrCa) are still scarcely known and, thus, merit further investigation. METHODS: Preoperative platelet counts (PCs) were compared with clinical data, CTCs, 65 serum cytokines and 770 immune-related transcripts obtained using the NanoString technology. RESULTS: High normal PC (hPC; defined by the 75th centile cut-off) correlated with an increased number of lymph node metastases and mesenchymal CTCs in the 70 operable BrCa patients. Patients with hPC and CTC presence revealed the shortest overall survival compared to those with no CTC/any PC or even CTC/normal PC. Adverse prognostic impact of hPC was observed only in the luminal subtype, when 247 BrCa patients were analysed. hPC correlated with high content of intratumoural stroma, specifically its phenotype related to CD8+ T and resting mast cells, and an increased concentration of cytokines related to platelet activation or even production in bone marrow (i.e. APRIL, ENA78/CXCL5, HGF, IL16, IL17a, MDC/CCL22, MCP3, MMP1 and SCF). CONCLUSIONS: Preoperative platelets evaluated alone and in combination with CTCs have prognostic potential in non-metastatic BrCa and define patients at the highest risk of disease progression, putatively benefiting from anti-platelet therapy.

Biodistribution PET/CT Study of Hemoglobin-DFO-89Zr Complex in Healthy and Lung Tumor-Bearing Mice.

Proteins, as a major component of organisms, are considered the preferred biomaterials for drug delivery vehicles. Hemoglobin (Hb) has been recently rediscovered as a potential drug carrier, but its use for biomedical applications still lacks extensive investigation. To further explore the possibility of utilizing Hb as a potential tumor targeting drug carrier, we examined and compared the biodistribution of Hb in healthy and lung tumor-bearing mice, using for the first time 89Zr labelled Hb in a positron emission tomography (PET) measurement. Hb displays a very high conjugation yield in its fast and selective reaction with the maleimide-deferoxamine (DFO) bifunctional chelator. The high-resolution X-ray structure of the Hb-DFO complex demonstrated that cysteine ß93 is the sole attachment moiety to the αß-protomer of Hb. The Hb-DFO complex shows quantitative uptake of 89Zr in solution as determined by radiochromatography. Injection of 0.03 mg of Hb-DFO-89Zr complex in healthy mice indicates very high radioactivity in liver, followed by spleen and lungs, whereas a threefold increased dosage results in intensification of PET signal in kidneys and decreased signal in liver and spleen. No difference in biodistribution pattern is observed between naïve and tumor-bearing mice. Interestingly, the liver Hb uptake did not decrease upon clodronate-mediated macrophage depletion, indicating that other immune cells contribute to Hb clearance. This finding is of particular interest for rapidly developing clinical immunology and projects aiming to target, label or specifically deliver agents to immune cells.

Human bone marrow mesenchymal stem cell-derived extracellular vesicles attenuate neuroinflammation evoked by focal brain injury in rats.

BACKGROUND: Ischemic stroke is the major cause of long-term severe disability and death in aged population. Cell death in the infarcted region of the brain induces immune reaction leading to further progression of tissue damage. Immunomodulatory function of mesenchymal stem cells (MSCs) has been shown in multiple preclinical studies; however, it has not been successfully translated to a routine clinical practice due to logistical, economical, regulatory, and intellectual property obstacles. It has been recently demonstrated that therapeutic effect of intravenously administered MSCs can be recapitulated by extracellular vesicles (EVs) derived from them. However, in contrast to MSCs, EVs were not capable to decrease stroke-induced neuroinflammation. Therefore, the aim of the study was to investigate if intra-arterial delivery of MSC-derived EVs will have stronger impact on focal brain injury-induced neuroinflammation, which mimics ischemic stroke, and how it compares to MSCs. METHODS: The studies were performed in adult male Wistar rats with focal brain injury induced by injection of 1 µl of 50 nmol ouabain into the right hemisphere. Two days after brain insult, 5 × 105 human bone marrow MSCs (hBM-MSCs) labeled with Molday ION or 1.3 × 109 EVs stained with PKH26 were intra-arterially injected into the right hemisphere under real-time MRI guidance. At days 1, 3, and 7 post-transplantation, the rats were decapitated, the brains were removed, and the presence of donor cells or EVs was analyzed. The cellular immune response in host brain was evaluated immunohistochemically, and humoral factors were measured by multiplex immunoassay. RESULTS: hBM-MSCs and EVs transplanted intra-arterially were observed in the rat ipsilateral hemisphere, near the ischemic region. Immunohistochemical analysis of brain tissue showed that injection of hBM-MSCs or EVs leads to the decrease of cell activation by ischemic injury, i.e., astrocytes, microglia, and infiltrating leucocytes, including T cytotoxic cells. Furthermore, we observed significant decrease of pro-inflammatory cytokines and chemokines after hBM-MSC or EV infusion comparing with non-treated rats with focal brain injury. CONCLUSIONS: Intra-arterially injected EVs attenuated neuroinflammation evoked by focal brain injury, which mimics ischemic stroke, and this effect was comparable to intra-arterial hBM-MSC transplantation. Thus, intra-arterial injection of EVs might be an attractive therapeutic approach, which obviates MSC-related obstacles.

Salivary interleukin 6, interleukin 8, interleukin 17A, and tumour necrosis factor α levels in patients with periodontitis and rheumatoid arthritis.

INTRODUCTION: Rheumatoid arthritis (RA) and periodontitis share risk factors and inflammatory pathways that could be related to cytokines, such as interleukin (IL)-6, IL-8, IL-17A, and tumour necrosis factor-α (TNF-α). The aim of this study was to compare periodontal status and salivary levels of selected cytokines between patients diagnosed with RA and periodontitis. RA patients were assessed for the potential influence of anti-rheumatic therapy. MATERIAL AND METHODS: One hundred and six patients were enrolled in a cross-sectional study. Medical assessment and periodontal examination were performed in 35 patients with chronic periodontitis, in 35 patients with RA and chronic periodontitis, and in 36 controls. Unstimulated whole saliva samples were analysed for IL-6, IL-8, IL-17A, and TNF-α. RESULTS: Significant differences in biomarkers and periodontal parameters were found among groups. Study groups exhibited higher mean pocket depth (PD), number of PD > 4 mm, and mean clinical attachment loss, when compared with controls. The RA group had lower bleeding on probing index and PD, but higher values of plaque indices than the periodontitis group. Concentration of evaluated cytokines were higher in the RA and periodontitis groups, compared with controls. The periodontitis group showed also higher levels of IL-6, IL-17A, and TNF-α in comparison to RA. RA patients were treated with disease-modifying anti-rheumatic drugs (DMARDs) and glucocorticosteroids. CONCLUSIONS: Salivary levels of IL-6, IL-8, IL-17A, and TNF-α can be affected by periodontitis, RA, and presumably DMARDs. DMARD therapy appears to reduce destructive and inflammatory processes in periodontal tissues because lower values of PD, BOP, and salivary levels of IL-6, IL-17A, and TNF-α were found in RA.

Survival rates of homozygotic Tp53 knockout rats as a tool for preclinical assessment of cancer prevention and treatment.

BACKGROUND: The gene that encodes tumor protein p53, Tp53, is mutated or silenced in most human cancers and is recognized as one of the most important cancer drivers. Homozygotic Tp53 knockout mice, which develop lethal cancers early in their lives, are already used in cancer prevention studies, and now Tp53 knockout rats have also been generated. This study assessed feasibility of using homozygous Tp53 knockout rats to evaluate the possible outcome of cancer chemoprevention. METHODS: A small colony of Tp53 knockout rats with a Wistar strain genetic background was initiated and maintained in the animal house at our institution. Tp53 heterozygotic females were bred with Tp53 homozygous knockout males to obtain a surplus of knockout homozygotes. To evaluate the reproducibility of their lifespan, 4 groups of Tp53 homozygous knockout male rats born during consecutive quarters of the year were kept behind a sanitary barrier in a controlled environment until they reached a moribund state. Their individual lifespan data were used to construct quarterly survival curves. RESULTS: The four consecutive quarterly survival curves were highly reproducible. They were combined into a single "master" curve for use as a reference in intervention studies. The average lifespan of untreated male Tp53 homozygous knockout rats was normally distributed, with a median of 133 days. Sample size vs. effect calculations revealed that confirming a 20% and 30% increase in the lifespan would respectively require a sample size of 18 and 9 animals (when assessed using the t-test with a power of 80% and alpha set at 0.05). As an example, the Tp53 homozygous knockout rat model was used to test the chemopreventive properties of carnosine, a dipeptide with suspected anticancer properties possibly involving modulation of the mTOR pathway. The result was negative. CONCLUSION: Further evaluation of the Tp53 homozygous knockout male rat colony is required before it can be confirmed as a viable tool for assessing new methods of cancer prevention or treatment.

Non-radioactive imaging strategies for in vivo immune cell tracking.

In vivo tracking of administered cells chosen for specific disease treatment may be conducted by diagnostic imaging techniques preceded by cell labeling with special contrast agents. The most commonly used agents are those with radioactive properties, however their use in research is often impossible. This review paper focuses on the essential aspect of cell tracking with the exclusion of radioisotope tracers, therefore we compare application of different types of non-radioactive contrast agents (cell tracers), methods of cell labeling and application of various techniques for cell tracking, which are commonly used in preclinical or clinical studies. We discuss diagnostic imaging methods belonging to three groups: (1) Contrast-enhanced X-ray imaging, (2) Magnetic resonance imaging, and (3) Optical imaging. In addition, we present some interesting data from our own research on tracking immune cell with the use of discussed methods. Finally, we introduce an algorithm which may be useful for researchers planning leukocyte targeting studies, which may help to choose the appropriate cell type, contrast agent and diagnostic technique for particular disease study.

Neuroinflammation evoked by brain injury in a rat model of lacunar infarct.

Stroke is the leading cause of long-term, severe disability worldwide. Immediately after the stroke, endogenous inflammatory processes are upregulated, leading to the local neuroinflammation and the potentiation of brain tissue destruction. The innate immune response is triggered as early as 24 h post-brain ischemia, followed by adaptive immunity activation. Together these immune cells produce many inflammatory mediators, i.e., cytokines, growth factors, and chemokines. Our study examines the immune response components in the early stage of deep brain lacunar infarct in the rat brain, highly relevant to the clinical scenario. The lesion was induced by stereotactic injection of ouabain into the adult rat striatum. Ouabain is a Na/K ATPase pump inhibitor that causes excitotoxicity and brings metabolic and structural changes in the cells leading to focal brain injury. We have shown a surge of neurodegenerative changes in the peri-infarct area in the first days after brain injury. Immunohistochemical analysis revealed early microglial activation and the gradual infiltration of immune cells with a significant increase of CD4+ and CD8+ T lymphocytes in the ipsilateral hemisphere. In our studies, we identified the higher level of pro-inflammatory cytokines, i.e., interleukin-1α, interleukin-1ß, tumor necrosis factor-α, and interferon-γ, but a lower level of anti-inflammatory cytokines, i.e., interleukin-10 and transforming growth factor-ß2 in the injured brain than in normal rats. Concomitantly focal brain injury showed a significant increase in the level of chemokines, i.e., monocyte chemoattractant protein-1 and CC motif chemokine ligand 5 compared to control. Our findings provide new insights into an early inflammatory reaction in our model of the deep-brain lacunar infarct. The results of this study may highlight future stroke immunotherapies for targeting the acute immune response accompanied by the insult.

Bone Defect Repair Using a Bone Substitute Supported by Mesenchymal Stem Cells Derived from the Umbilical Cord.

OBJECTIVE: Bone defects or atrophy may arise as a consequence of injury, inflammation of various etiologies, and neoplastic or traumatic processes or as a result of surgical procedures. Sometimes the regeneration process of bone loss is impaired, significantly slowed down, or does not occur, e.g., in congenital defects. For the bone defect reconstruction, a piece of the removed bone from ala of ilium or bone transplantation from a decedent is used. Replacement of the autologous or allogenic source of the bone-by-bone substitute could reduce the number of surgeries and time in the pharmacological coma during the reconstruction of the bone defect. Application of mesenchymal stem cells in the reconstruction surgery may have positive influence on tissue regeneration by secretion of angiogenic factors, recruitment of other MSCs, or differentiation into osteoblasts. Materials and Methods. Mesenchymal stem cells derived from the umbilical cord (Wharton's jelly (WJ-MSC)) were cultured in GMP-grade DMEM low glucose supplemented with heparin, 10% platelet lysate, glucose, and antibiotics. In vitro WJ-MSCs were seeded on the bone substitute Bio-Oss Collagen® and cultured in the StemPro® Osteogenesis Differentiation Kit. During the culture on the 1st, 7th, 14th, and 21st day (day in vitro (DIV)), we analyzed viability (confocal microscopy) and adhesion capability (electron microscopy) of WJ-MSC on Bio-Oss scaffolds, gene expression (qPCR), and secretion of proteins (Luminex). In vivo Bio-Oss® scaffolds with WJ-MSC were transplanted to trepanation holes in the cranium to obtain their overgrowth. The computed tomography was performed 7, 14, and 21 days after surgery to assess the regeneration. RESULTS: The Bio-Oss® scaffold provides a favourable environment for WJ-MSC survival. WJ-MSCs in osteodifferentiation medium are able to attach and proliferate on Bio-Oss® scaffolds. Results obtained from qPCR and Luminex® indicate that WJ-MSCs possess the ability to differentiate into osteoblast-like cells and may induce osteoclastogenesis, angiogenesis, and mobilization of host MSCs. In animal studies, WJ-MSCs seeded on Bio-Oss® increased the scaffold integration with host bone and changed their morphology to osteoblast-like cells. CONCLUSIONS: The presented construct consisted of Bio-Oss®, the scaffold with high flexibility and plasticity, approved for clinical use with seeded immunologically privileged WJ-MSC which may be considered reconstructive therapy in bone defects.

TP53-Deficient Angiosarcoma Expression Profiling in Rat Model.

Sarcomas are a heterogeneous group of malignant tumors, that develop from mesenchymal cells. Sarcomas are tumors associated with poor prognosis and expected short overall survival. Efforts to improve treatment efficacy and treatment outcomes of advanced and metastatic sarcoma patients have not led to significant improvements in the last decades. In the Tp53C273X/C273X rat model we therefore aimed to characterize specific gene expression pattern of angiosarcomas with a loss of TP53 function. The presence of metabolically active tumors in several locations including the brain, head and neck, extremities and abdomen was confirmed by magnetic resonance imaging (MRI) and positron emission tomography (PET) examinations. Limb angiosarcoma tumors were selected for microarray expression analysis. The most upregulated pathways in angiosarcoma vs all other tissues were related to cell cycle with mitosis and meiosis, chromosome, nucleosome and telomere maintenance as well as DNA replication and recombination. The downregulated genes were responsible for metabolism, including respiratory chain electron transport, tricarboxylic acid (TCA) cycle, fatty acid metabolism and amino-acid catabolism. Our findings demonstrated that the type of developing sarcoma depends on genetic background, underscoring the importance of developing more malignancy susceptibility models in various strains and species to simulate the study of the diverse genetics of human sarcomas.