Cell Therapy of Pathological Gastrointestinal Lesions in Modeled NSAID-Induced Enterocolitis

Wistar rats with NSAID-induced (dexketoprofen) chronic enterocolitis of the gastrointestinal tract were treated with a cultured allogeneic cell suspension consisting of mononuclear cells (40 millions) and multipotent mesenchymal stromal cells (10 millions). The suspension was transplanted intraperitoneally, 2 times with an interval of 30 days. Cultured allogeneic fractions of bone marrow cells accelerate the regeneration of long-term non-healing gastrointestinal lesions by reducing the duration and severity of the inflammatory phase and activating the regenerative phase of the ulcerative process. It was found that the simultaneous administration of the studied cultured stem cells can be used for the treatment of chronic, long-term non-healing, poorly-scarring ulcerative necrotic gastrointestinal pathologies.

New bang-bang phase detectors for high-speed serial links

Bang-bang phase detector studies were carried out in this thesis. Based on the comparison of linear and non-linear phase detectors, a hybrid phase detector was proposed. It possesses the characteristics of two-XOR phase detectors and improved bang-bang phase detectors. PLLs with the proposed hybrid phase detector possess low timing jitter in lock states and a fast locking process. The effectiveness of the proposed hybrid phase detector was quantified by comparing the performance of three PLLs with identical loop components but different phase detectors. A new bang-bang phase detector with regenerative DFFs was also proposed. The regenerative bang-bang phase detector ensures a fast acquisition of incoming clocks. The effectiveness of the regenerative phase detector was assessed in a 2GHz PLL. A 1X bang-bang phase detector was proposed also. Compared to a 2X bang-bang phase detector, PLLs with a 1X bang-bang phase detector offer faster locking. A DFF frequency detector and a charge-pump frequency detector were also proposed. Both effectively detect the frequency difference.

New bang-bang phase detectors for high-speed serial links

Bang-bang phase detector studies were carried out in this thesis. Based on the comparison of linear and non-linear phase detectors, a hybrid phase detector was proposed. It possesses the characteristics of two-XOR phase detectors and improved bang-bang phase detectors. PLLs with the proposed hybrid phase detector possess low timing jitter in lock states and a fast locking process. The effectiveness of the proposed hybrid phase detector was quantified by comparing the performance of three PLLs with identical loop components but different phase detectors. A new bang-bang phase detector with regenerative DFFs was also proposed. The regenerative bang-bang phase detector ensures a fast acquisition of incoming clocks. The effectiveness of the regenerative phase detector was assessed in a 2GHz PLL. A 1X bang-bang phase detector was proposed also. Compared to a 2X bang-bang phase detector, PLLs with a 1X bang-bang phase detector offer faster locking. A DFF frequency detector and a charge-pump frequency detector were also proposed. Both effectively detect the frequency difference.

Stabilin-1+/SMA- macrophages in diagnostics of postinfarction tissue inflammation associated with adverse cardiac remodeling in patients with myocardial infarction

Introduction Molecular biomarkers of monocytes/macrophages identified to date have provided advanced diagnostic capabilities. We have accumulated a large amount of knowledge related to the role of innate immune response in the development of postinfarction cardiac remodeling. However, there is no significant advancement in clinical studies. Purpose The purpose was to assess prospects of macrophage biomarkers in diagnostics of postinfarction tissue inflammation associated with adverse cardiac remodeling in patients with myocardial infarction (MI). Methods The study included 41 patients with MI type 1, died in 2013–2014. We used a biobank of tissue samples for analysis. Group 1 (n=24) comprised patients who died within 72 hours of MI (the inflammatory phase), group 2 (n=17) comprised patients who died 4–28 days after MI (the regenerative phase). Macrophage infiltration in the heart was assessed by double immunofluorescence. We used stabilin-1 as a marker of M2 macrophages, while α-smooth muscle actin (α-SMA) was considered as a marker of cell transdifferentiation. Cells were counted in the infarct (IA) and non-infarct area (NIA). Morphological determination of adverse cardiac remodeling was based on the ratio of heart size, in particular, length/width ratio that was <1.1. Results We identified subpopulations of stabilin-1+/α-SMA− and stabilin-1+/α-SMA+ macrophages. In the IA the number of stabilin-1+/α-SMA− macrophages was lower during the inflammatory phase than during the regenerative phase (Table 1). The calculation of sensitivity and specificity of stabilin-1+/α-SMA− macrophages in the NIA for predicting of adverse cardiac remodeling has shown following: AUC=0.96, p<0.001. The cut-off value was 18 cells/mm2. The ROC curve is presented in Figure 1. Conclusions We identified that number of stabilin-1+/α-SMA− macrophages in the NIA ranged from 0 to 18 cells/mm2 was associated with adverse cardiac remodeling. The presence of stabilin-1+/α-SMA+ macrophages could indicate persistent tissue inflammation and possibility of macrophage transdifferentiation and plasticity. Our study supports prospects for implementation of macrophage biomarkers in clinical practice that might become a breakthrough in the development of new methods for management of MI and following complications. Figure 1 Funding Acknowledgement Type of funding source: None

P6390Macrophages as contributors to chronic inflammation and cardiac fibrosis in patients with myocardial infarction

Introduction The functional characteristics of tissue macrophages associated with the progression of cardiac fibrosis in clinic are still unknown. Purpose The purpose was to study cardiac macrophage phenotypes contributing to the development of chronic inflammation and fibrosis in patients with myocardial infarction (MI). Methods The study included 41 patients with fatal MI type 1. Group 1 (n=24) comprised patients who died within 72 hours of MI (the inflammatory phase) and group 2 (n=17) comprised patients who died 4–28 days after MI (the regenerative phase). Macrophage infiltration (number of cells) in the heart was assessed by double immunofluorescence in the non-infarct area. Each area was evaluated in 20 random fields. We used CD163, CD206, stabilin-1, α-smooth muscle actin (α-SMA), interleukin-10 (IL-10) as markers of M2-like macrophages. Results The number of CD163+/CD206− (p=0.087) and CD163+/206+ (p=0.072) macrophages was higher during the regenerative phase of MI. The number of CD163-/CD206+, stabilin-1+/α-SMA-, stabilin-1+/α-SMA+, stabilin-1+/IL-10−, stabilin-1+/IL-10+, stabilin-1-/IL-10+ did not significantly change throughout the entire period of MI. The comparison of various M2 macrophage subpopulations into groups revealed following. In group 1 the number of CD163+/CD206− and CD163+/CD206+ cells prevailed over CD163−/CD206+ (p=0.033 and p=0.003, respectively), stabilin1+/α-SMA− cells over stabilin-1+/α-SMA+ (p<0.001), and stabilin-1+/IL-10+ cells over stabilin-1+/IL-10− (p=0.018). In group 2 the quantity of stabilin-1+/α-SMA− macrophages prevailed over stabilin-1+/α-SMA+ (p=0.005) and stabilin-1+/IL-10+ over stabilin-1−/IL-10+ (p=0.028). In group 1 the number of CD163+/CD206+ cells correlated with the absolute and the relative number of peripheral blood monocytes prior to the onset of death (R=0.97), while the quantity of stablin-1+/α-SMA+ cells correlated with the absolute number of peripheral blood monocytes at admission (R=-0.53). In group 2 the quantity of CD206+/CD163− cells correlated with the absolute and the relative number of monocytes in the peripheral blood at admission (R=0.73 and R=0.59, respectively), the quantity of CD163+/CD206− macrophages with the incidence of recurrent MI (R= 0.54), and the number of stabilin-1+/α-SMA+ macrophages with the age of patients (R=-0.58). Conclusions We have suggested that the key cardiac macrophage phenotypes contributing to the development of chronic inflammation and cardiac fibrosis in the regenerative phase of MI were stabilin-1+/α-SMA− and stabilin-1+/IL-10+. We revealed subpopulation of stabilin-1+/α-SMA+ macrophages, that indicated the possibility of cellular transdifferentiation and macrophage plasticity. Thus, our results supports that understanding the role of macrophages in initiation, progression, and resolution of cardiac fibrosis is one of the most promising goal in the design of anti-fibrotic treatment strategies for patients with MI.

PHENOTYPIC HETEROGENEITY OF CARDIAC MACROPHAGES DURING WOUND HEALING FOLLOWING MYOCARDIAL INFARCTION: PERSPECTIVES IN CLINICAL RESEARCH

Purpose. Myocardial regeneration is one of the most ambitious goals in prevention of adverse cardiac remodeling. Macrophages play a key role in transition from inflammatory to regenerative phase during wound healing following myocardial infarction (MI). We have accumulated data on macrophage properties ex vivo and in cell culture. However, there is no clear information about phenotypic heterogeneity of cardiac macrophages in patients with MI. The purpose of the project was to assess cardiac macrophage infiltration during wound healing following myocardial infarction in clinical settings taking into consideration experimental knowledge.Material and Methods. The study included 41 patients with fatal MI type 1. In addition to routine analysis, macrophages infiltration was assessed by immunohistochemistry. We used CD68 as a marker for the cells of the macrophage lineage, while CD163, CD206, and stabilin-1 were considered as M2 macrophage biomarkers. Nine patients who died from noncardiovascular causes comprised the control group.Results. The intensity of cardiac macrophage infiltration was higher during the regenerative phase than during the inflammatory phase. Results of immunohistochemical analysis demonstrated the presence of phenotypic heterogeneity of cardiac macrophages in patients with MI. We noticed that numbers of CD68+, CD163+, CD206+, and stabilin-1+ macrophages depended on MI phase.Conclusion. Our study supports prospects for implementation of macrophage phenotyping in clinic practice. Improved understanding of phenotypic heterogeneity might become the basis of a method to predict adverse cardiac remodeling and the first step in developing myocardial regeneration target therapy.

BP-C3 POLYPHENOLIC COMPOSITION INFLUENCE ON DYNAMICS OF 5-FLUOROURACIL INDUCED DAMAGE IN SMALL INTESTINE EPITHELIUM IN MICE

Chemotherapeutic drugs negatively influence on normal tissues with high proliferative activity such as intestinal epithelium. The damage of chemotherapy to healthy tissues significantly reduces oncological patients’ quality of life. Drugs based on polyphenolic natural compounds are a perspective mean of adjuvant therapy due to their antioxidant and antiinflammatory properties. In this study we have showed that a single intragastric administration of BPC3 polyphenolic composition 2 hours before 5-fluorouracil treatment alleviates small intestine damage in C57Bl/6 mice after 3.5, 24 or 72 hours. BPC3 effect manifested in reduced apoptosis rate, increased mitotic activity of the epithelium in the regenerative phase and protection of LGR5-positive jejunum stem cells against 5-fluorouracil induced damage.

let-7 miRNA controls CED-7 homotypic adhesion and EFF-1–mediated axonal self-fusion to restore touch sensation following injury

Neuronal injury often leads to devastating consequences such as loss of senses or locomotion. Restoration of function after injury relies on whether the injured axons can find their target cells. Although fusion between injured proximal axon and distal fragment has been observed in many organisms, its functional significance is not clear. Here, using Caenorhabditis elegans mechanosensory neurons, we address this question. Using two femtosecond lasers simultaneously, we could scan and sever posterior lateral microtubule neurons [posterior lateral microtubules (PLMs)] on both sides of the worm. We showed that axotomy of both PLMs leads to a dramatic loss of posterior touch sensation. During the regenerative phase, only axons that fuse to their distal counterparts contribute to functional recovery. Loss of let-7 miRNA promotes functional restoration in both larval and adult stages. In the L4 stage, loss of let-7 increases fusion events by increasing the mRNA level of one of the cell-recognition molecules, CED-7. The ability to establish cytoplasmic continuity between the proximal and distal ends declines with age. Loss of let-7 overcomes this barrier by promoting axonal transport and enrichment of the EFF-1 fusogen at the growing tip of cut processes. Our data reveal the functional property of a regenerating neuron.