Local injection of bone-marrow derived mesenchymal stromal cells alters a molecular expression profile of a contact frostbite injury wound and improves healing in a rat model.

INTRODUCTION: Frostbite is a traumatic injury of the tissues upon low temperature environment exposure, which is characterized by direct cell injury due to freezing-thawing followed by development of an acute inflammatory process. Severe frostbite can lead to necrosis of soft tissues and loss of a limb. Mesenchymal stromal cells (MSCs) have a unique ability to modulate pathogenic immune response by secretion of paracrine factors, which suppress inflammation and mediate more efficient tissue regeneration. It should be noted that potential of stem cell therapy for frostbite injury treatment has not been investigated so far. Here, we evaluated a healing capacity of bone-marrow derived MSCs for the treatment of contact frostbite injury wound in a rat model. METHODS: Cold-contact injury in a Wistar rat model was induced by 1-minute tight application of the cooled probe (-196 °C) to the skin surface of the left hip. Rat bone marrow MSCs were phenotypically characterized and used for local injections into non-damaged tissues surrounding the wound of animals from the experimental group. The second group of rats was treated in the same manner with 1 mL of isotonic sodium chloride solution. Analysis of cytokine and growth factor expression profile in сold-contact injury wounds was performed on days 5, 9, and 16 using immunoblotting and enzyme-linked immunosorbent assay. Animal recovery in MSC-treated and vehicle-treated groups was evaluated by several criteria including body weight recording, determination of eschar desquamation and re-epithelialization terms, assessment of wound closure kinetics, and histological scoring of the wounds on day 23. RESULTS: It turned out that a single subcutaneous administration of MSCs around the wound site resulted in elevated expression of pro-survival and pro-angiogenic VEGF-A and PDGF and 3-5-fold decrease in pro-inflammatory IL-1ß as compared with the frostbite wound treated with a vehicle. Moreover, treatment with MSCs caused accelerated wound re-epithelialization (p < 0.05) as well as a better histological score of the MSC-treated wounds. CONCLUSIONS: Thus, our data suggested that the use of MSCs is a promising therapeutic strategy for the treatment of cold-induced injury wounds.

The regenerative potential of skin and the immune system.

Skin has the natural ability to heal and replace dead cells regulated by a network of complex immune processes. This ability is conferred by the population of resident immune cells that act in coordination with other players to provide a homeostatic environment under constant challenge. Other than providing structure and integrity, the epidermis and dermis also house distinct immune properties. The dermal part is represented by fibroblasts and endothelial cells followed by an array of immune cells which includes dendritic cells (DCs), macrophages, mast cells, NK-cells, neutrophils, basophils, eosinophils, αß T lymphocytes, B-cells and platelets. On the other hand, the functionally active immune cells in the epidermis comprise keratinocytes, DCs, NKT-cells, γδ T cells and αß T cells (CD4+ and CD8+). Keratinocytes create a unique microenvironment for the cells of the immune system by promoting immune recognition and cellular differentiation. T lymphocytes exhibit tissue-specific tropism toward the epidermis and the lymphatic drainage system important for their function in immune regulation. This diversity in immune regulators makes the skin a unique organ to overcome pathogenic or foreign invasion. In addition, the highly coordinated molecular events make the skin an attractive model to understand and explore its regenerative potential.

Noninvasive label-free detection of circulating white and red blood clots in deep vessels with a focused photoacoustic probe.

Blood clotting is a serious clinical complication of many medical procedures and disorders including surgery, catheterization, transplantation, extracorporeal circuits, infections, and cancer. This complication leads to high patient morbidity and mortality due to clot-induced pulmonary embolism, stroke, and in some cases heart attack. Despite the clear medical significance, little progress has been made in developing the methods for detection of circulating blood clots (CBCs), also called emboli. We recently demonstrated the application of in vivo photoacoustic (PA) flow cytometry (PAFC) with unfocused ultrasound transducers for detection of CBCs in small vessels in a mouse model. In the current study, we extend applicability of PAFC for detection of CBCs in relatively large (1.5-2 mm) and deep (up to 5-6 mm) blood vessels in rat and rabbit models using a high pulse rate 1064 nm laser and focused ultrasound transducer with a central hole for an optic fiber. Employing phantoms and chemical activation of clotting, we demonstrated PA identification of white, red, and mixed CBCs producing negative, positive, and mixed PA contrast in blood background, respectively. We confirmed that PAFC can detect both red and white CBCs induced by microsurgical procedures, such as a needle or catheter insertion, as well as stroke modeled by injection of artificial clots. Our results show great potential for a PAFC diagnostic platform with a wearable PA fiber probe for diagnosis of thrombosis and embolism in vivo that is impossible with existing techniques.

Use of paracrine factors from stem cells to treat local radiation burns in rats.

BACKGROUND: Mesenchymal stem cells based paracrine bioactive factors that deploy their task as an essential mechanism, but their efficiency for skin regeneration still requires clarification. METHODS: The mesenchymal stem cell-based paracrine factors were administered by subcutaneous injection of 0.5 mL peptides (general protein 8 mg/mL). These were performed after radiation on different days like the first, third, sixth, eighth, and 10th. To determine the consequences, we performed photography, planimetry, and preclinical test each week after 15 days of radiation. MSC-based peptides were injected into a rat that had radiation burns, and its observation encouraged cell-free therapeutic remedies to regenerate skin. Both control and experimental groups were exposed to 110 Gy of X-rays, which resulted in the formation of localized radiation burns on the skin (S=6 cm2) 15 days later. Thirty days after radiation, the wound stabilized (surface of the wound was S=2.2±0.2 cm2) and fluctuated throughout the course of the pathological process. RESULTS: The wounded area on the skin from the 15th to the 29th day after radiation was practically the same in both groups. The wounded area gradually reduced by 6.1±0.4 cm2 (experimental group) and 5.9±0.6 cm2 (control group) 15 days after radiation up to 2.2±0.3 cm2 (in both control and experimental groups) on the 29th day after radiation. However, starting from the 36th day, there was a constant reduction in the burn area in the experimental group up to 0.2±0.1 cm2 till the 71st day after radiation. CONCLUSION: In the control group, the area of the lesion ranged from 1.4±0.6 cm2 on the 50th day to 1.9±0.8 cm2 on the 71st day. During the 57th to the 71st day, the difference between the affected area in the experimental and control groups was 1:8. The experimental group has a significantly higher level of skin regeneration and significant decrease in the level of leukocyte infiltration, thereby reducing necrosis.

A Nonsynonymous SNP Catalog of Mycobacterium tuberculosis Virulence Genes and Its Use for Detecting New Potentially Virulent Sublineages.

Mycobacterium tuberculosis is divided into several distinct lineages, and various genetic markers such as IS-elements, VNTR, and SNPs are used for lineage identification. We propose an M. tuberculosis classification approach based on functional polymorphisms in virulence genes. An M. tuberculosis virulence genes catalog has been established, including 319 genes from various protein groups, such as proteases, cell wall proteins, fatty acid and lipid metabolism proteins, sigma factors, toxin-antitoxin systems. Another catalog of 1,573 M. tuberculosis isolates of different lineages has been developed. The developed SNP-calling program has identified 3,563 nonsynonymous SNPs. The constructed SNP-based phylogeny reflected the evolutionary relationship between lineages and detected new sublineages. SNP analysis of sublineage F15/LAM4/KZN revealed four lineage-specific mutations in cyp125, mce3B, vapC25, and vapB34. The Ural lineage has been divided into two geographical clusters based on different SNPs in virulence genes. A new sublineage, B0/N-90, was detected inside the Beijing-B0/W-148 by SNPs in irtB, mce3F and vapC46. We have found 27 members of B0/N-90 among the 227 available genomes of the Beijing-B0/W-148 sublineage. Whole-genome sequencing of strain B9741, isolated from an HIV-positive patient, was demonstrated to belong to the new B0/N-90 group. A primer set for PCR detection of B0/N-90 lineage-specific mutations has been developed. The prospective use of mce3 mutant genes as genetically engineered vaccine is discussed.

In vivo acoustic and photoacoustic focusing of circulating cells.

In vivo flow cytometry using vessels as natural tubes with native cell flows has revolutionized the study of rare circulating tumor cells in a complex blood background. However, the presence of many blood cells in the detection volume makes it difficult to count each cell in this volume. We introduce method for manipulation of circulating cells in vivo with the use of gradient acoustic forces induced by ultrasound and photoacoustic waves. In a murine model, we demonstrated cell trapping, redirecting and focusing in blood and lymph flow into a tight stream, noninvasive wall-free transportation of blood, and the potential for photoacoustic detection of sickle cells without labeling and of leukocytes targeted by functionalized nanoparticles. Integration of cell focusing with intravital imaging methods may provide a versatile biological tool for single-cell analysis in circulation, with a focus on in vivo needleless blood tests, and preclinical studies of human diseases in animal models.

Real-Time Label-Free Embolus Detection Using In Vivo Photoacoustic Flow Cytometry.

Thromboembolic events are one of the world's leading causes of death among patients. Embolus or clot formations have several etiologies including paraneoplastic, post-surgery, cauterization, transplantation, or extracorporeal circuits. Despite its medical significance, little progress has been made in early embolus detection, screening and control. The aim of our study is to test the utility of the in vivo photoacoustic (PA) flow cytometry (PAFC) technique for non-invasive embolus detection in real-time. Using in vivo PAFC, emboli were non-invasively monitored in the bloodstream of two different mouse models. The tumor-free mouse model consisted of two groups, one in which the limbs were clamped to produce vessel stasis (7 procedures), and one where the mice underwent surgery (7 procedures). The melanoma-bearing mouse model also consisted of two groups, one in which the implanted tumor underwent compression (8 procedures), and one where a surgical excision of the implanted tumor was performed (8 procedures). We demonstrated that the PAFC can detect a single embolus, and has the ability to distinguish between erythrocyte-rich (red) and leukocyte/platelet-rich (white) emboli in small vessels. We show that, in tumor-bearing mice, the level of circulating emboli was increased compared to tumor-free mice (p = 0.0013). The number of circulating emboli temporarily increased in the tumor-free control mice during vessel stasis (p = 0.033) and after surgical excisions (signed-rank p = 0.031). Similar observations were noted during tumor compression (p = 0.013) and after tumor excisions (p = 0.012). For the first time, it was possible to detect unlabeled emboli in vivo non-invasively, and to confirm the presence of pigmented tumor cells within circulating emboli. The insight on embolus dynamics during cancer progression and medical procedures highlight the clinical potential of PAFC for early detection of cancer and surgery-induced emboli to prevent the fatal thromboembolic complications by well-timed therapy.

Biomaterials as carrier, barrier and reactor for cell-based regenerative medicine.

Cell therapy has achieved tremendous success in regenerative medicine in the past several decades. However, challenges such as cell loss, death and immune-rejection after transplantation still persist. Biomaterials have been designed as carriers to deliver cells to desirable region for local tissue regeneration; as barriers to protect transplanted cells from host immune attack; or as reactors to stimulate host cell recruitment, homing and differentiation. With the assistance of biomaterials, improvement in treatment efficiency has been demonstrated in numerous animal models of degenerative diseases compared with routine free cell-based therapy. Emerging clinical applications of biomaterial assisted cell therapies further highlight their great promise in regenerative therapy and even cure for complex diseases, which have been failed to realize by conventional therapeutic approaches.

In vivo photoswitchable flow cytometry for direct tracking of single circulating tumor cells.

Photoswitchable fluorescent proteins (PSFPs) that change their color in response to light have led to breakthroughs in studying static cells. However, using PSFPs to study cells in dynamic conditions is challenging. Here we introduce a method for in vivo ultrafast photoswitching of PSFPs that provides labeling and tracking of single circulating cells. Using in vivo multicolor flow cytometry, this method demonstrated the capability for studying recirculation, migration, and distribution of circulating tumor cells (CTCs) during metastasis progression. In tumor-bearing mice, it enabled monitoring of real-time dynamics of CTCs released from primary tumor, identifying dormant cells, and imaging of CTCs colonizing a primary tumor (self-seeding) or existing metastasis (reseeding). Integration of genetically encoded PSFPs, fast photoswitching, flow cytometry, and imaging makes in vivo single cell analysis in the circulation feasible to provide insights into the behavior of CTCs and potentially immune-related and bacterial cells in circulation.

Dynamic Fluctuation of Circulating Tumor Cells during Cancer Progression.

Circulating tumor cells (CTCs) are a promising diagnostic and prognostic biomarker for metastatic tumors. We demonstrate that CTCs' diagnostic value might be increased through real-time monitoring of CTC dynamics. Using preclinical animal models of breast cancer and melanoma and in vivo flow cytometry with photoacoustic and fluorescence detection schematics, we show that CTC count does not always correlate with the primary tumor size. Individual analysis elucidated many cases where the highest level of CTCs was detected before the primary tumor starts progressing. This phenomenon could be attributed to aggressive tumors developing from cancer stem cells. Furthermore, real-time continuous monitoring of CTCs reveals that they occur at highly variable rates in a detection point over a period of time (e.g., a range of 0-54 CTCs per 5 min). These same fluctuations in CTC numbers were observed in vivo in epithelial and non-epithelial metastatic tumors, in different stages of tumor progression, and in different vessels. These temporal CTC fluctuations can explain false negative results of a one-time snapshot test in humans. Indeed, we observed wide variations in the number of CTCs in subsequent blood samples taken from the same metastatic melanoma patient, with some samples being CTC-free. If these phenomena are confirmed in our ongoing in vivo clinical trials, this could support a personalized strategy of CTC monitoring for cancer patients.