Mesenchymal stem cells transfer mitochondria to allogeneic Tregs in an HLA-dependent manner improving their immunosuppressive activity.

Cell-based immunotherapies can provide safe and effective treatments for various disorders including autoimmunity, cancer, and excessive proinflammatory events in sepsis or viral infections. However, to achieve this goal there is a need for deeper understanding of mechanisms of the intercellular interactions. Regulatory T cells (Tregs) are a lymphocyte subset that maintain peripheral tolerance, whilst mesenchymal stem cells (MSCs) are multipotent nonhematopoietic progenitor cells. Despite coming from different origins, Tregs and MSCs share immunoregulatory properties that have been tested in clinical trials. Here we demonstrate how direct and indirect contact with allogenic MSCs improves Tregs' potential for accumulation of immunosuppressive adenosine and suppression of conventional T cell proliferation, making them more potent therapeutic tools. Our results also demonstrate that direct communication between Tregs and MSCs is based on transfer of active mitochondria and fragments of plasma membrane from MSCs to Tregs, an event that is HLA-dependent and associates with HLA-C and HLA-DRB1 eplet mismatch load between Treg and MSC donors.

Determination of tocopherols and tocotrienols in human breast adipose tissue with the use of high performance liquid chromatography-fluorescence detection.

Tocopherols and tocotrienols have been extensively studied owing to their anticancer potential, especially against breast cancer. Therefore, the aim of this study was to quantitatively determine tocochromanols in human breast adipose tissue with the use of HPLC-FLD. The sample preparation procedure included homogenization and solvent extraction with isopropanol-ethanol-0.1% formic acid mixture prior to solid-phase extraction. After implementation of central composite design, satisfactory separation of all eight target compounds was achieved within 10.5 min. Chromatographic runs were carried out with the use of a naphthylethyl chromatographic column with methanol-water mixture (89:11, v/v) as the mobile phase. Fluorescence detection of tocochromanols was performed with excitation and emission wavelengths 298 and 330 nm, respectively. The method was validated in terms of linearity, carryover, recovery, precision, accuracy and stability. Extraction yield was also determined for accurate evaluation of vitamin E content in human breast adipose tissue samples. Finally, concentrations of particular tocochromanols compounds were assessed in human breast adipose tissue samples obtained from 99 patients, including women with breast cancer, healthy volunteers and deceased women who had died as a result of accidents. The raw data was transformed according to the newly developed equation for accurate estimation of the concentrations of tocochromanols in breast adipose tissue samples. Results obtained in the study indicated that the proposed analytical assay could be useful in breast cancer research.

Immunophenotyping and transcriptional profiling of in vitro cultured human adipose tissue derived stem cells.

Adipose-derived stem cells (ASCs) have become an important research model in regenerative medicine. However, there are controversies regarding the impact of prolonged cell culture on the ASCs phenotype and their differentiation potential. Hence, we studied 10 clinical ASCs replicates from plastic and oncological surgery patients, in six-passage FBS supplemented cultures. We quantified basic mesenchymal cell surface marker transcripts and the encoded proteins after each passage. In parallel, we investigated the differentiation potential of ASCs into chondrocytes, osteocytes and adipocytes. We further determined the effects of FBS supplementation and subsequent deprivation on the whole transcriptome by comprehensive mRNA and miRNA sequencing. Our results show that ASCs maintain differentiation potential and consistent profile of key mesenchymal markers, with apparent expression of distinct isoforms, in long-term cultures. No significant differences were observed between plastic and oncological surgery cohorts. ASCs in FBS supplemented primary cultures are almost committed to mesenchymal lineages as they express key epithelial-mesenchymal transition genes including early mesenchymal markers. Furthermore, combined mRNA/miRNA expression profiling strongly supports a modulatory role for the miR-30 family in the commitment process to mesenchymal lineages. Finally, we propose improvements to existing qPCR based assays that address alternative isoform expression of mesenchymal markers.

Transcriptional profile of in vitro expanded human epidermal progenitor cells for the treatment of non-healing wounds.

BACKGROUND: Epidermal progenitor cells (EPCs) have been under extensive investigation due to their increasing potential of application in medicine and biotechnology. Cultured human EPCs are used in the treatment of chronic wounds and have recently became a target for gene therapy and toxicological studies. One of the challenges in EPCs culture is to provide a high number of undifferentiated, progenitor cells displaying high viability and significant biological activity. OBJECTIVES: The goal of this study was to characterize the in vitro cultured progenitor cells and to assess whether the cells with the progenitor phenotype are able to enhance wound healing. Additionally, we aimed to establish the complete procedure of the culture, analysis and clinical application of epidermal progenitor cells. METHODS: In this study we present a method of cell isolation and culture followed by a technique of transplantation of the cultured cells onto the wound bed. The applied isolation technique involves two enzymatic steps (dispase, trypsin) and it is characterized by a high yield of cells. The obtained cells were cultured in vitro up to the second passage in serum-free and xeno-free keratinocytes-dedicated medium. Key stem cell markers were determined with means of flow cytometry and quantitative real-time PCR. RESULTS: The in vitro expanded cells displayed high proliferative activity without features of neither apoptosis nor necrosis. The flow cytometry and transcriptomic analyses showed enhanced expression of stem cell markers (i.e. proteins: ΔNp63, CD29, CD49f and BNC1, CDKN1A transcripts) in the expanded cells. In the presented compassionate use study, cultured autologous cells from an oncological patient were suspended in fibrin sealant and transplanted directly to a non-healing wound, resulting in wound closure within 2 months. CONCLUSION: The cells cultured in serum-free media display epidermal stem cells features and a potential to stimulate wound healing. This promising procedure of isolation, culture and application warrants further clinical trials in the treatment of chronic wounds.

TTF-1 and PAX5 Are Frequently Expressed in Combined Merkel Cell Carcinoma.

Thyroid transcription factor-1 (TTF-1) is a marker of tumors of pulmonary and thyroid origin, and its expression practically excludes diagnosis of Merkel cell carcinoma (MCC). However, TTF-1 expression in combined MCC was recently reported. PAX5 is a marker of B-cell origin that is also expressed in most classical MCC cases; however, its expression was not described in combined MCC. The authors decided to evaluate the expression of both these markers in a group of 5 combined MCCs (2 with invasive squamous cell carcinoma, 2 with squamous cell carcinoma in situ, and 1 with basal cell carcinoma). Expression of TTF-1 was found in 4 of 5 cases; in 3 cases, the marker was shown in the MCC component (weakly in 2 cases and strongly in 1 case), whereas the non-MCC component presented TTF-1 expression in 2 cases. A weak-to-moderate immunoreactivity for PAX5 was identified in all cases of the MCC component but in none of the non-MCC component. The results show that the expression of TTF-1 is a frequent finding in combined MCC and can be present in the neuroendocrine component, which differs from the conventional MCC. In contrast, PAX5 expression pattern is similar to that of the classical MCC.

Active dynamic infrared thermal imaging in burn depth evaluation.

The aim of this study was to find the relationship between active dynamic thermography (ADT) with cold excitation and burn depth. This new modality of evaluation of burns seems to be an attractive proposal for quantitative classification, allowing proper choice of burn wound treatment: conservative or surgical, especially compared with static thermography. The work was an in vivo experiment on domestic pigs, and a small number of patients were also diagnosed as part of the study. Statistical analysis showed a high correlation between the ADT synthetic parameter--thermal time constant, τ--and the classification of burn wounds that were predicted to heal within 3 weeks and so were treated conservatively and those that were predicted to not heal within 3 weeks and so were surgically treated. The results of the study show an accuracy of 60.7% for clinical evaluation, 69.6% for static thermography, 83.0% for ADT, and 84.0% for histopathologic assessment. The authors have concluded that the ADT method with cold excitation is suitable for the qualitative and quantitative assessment of burn depth.

Adipose tissue-derived stem cells in clinical applications.

INTRODUCTION: In the past decade human adipose tissue has been identified as a source of multipotent stem cells. Adipose tissue derived stem cells (ASCs) are characterised by immunosuppressive properties and low immunogenicity. Therefore, they can be used in regenerative medicine, as well as applied to induce graft tolerance or prevent autoimmunity. ASCs can be easily harvested with low morbidity, which is their main advantage over mesenchymal stem cells (MSCs) derived from other sources. AREAS COVERED: The review focuses on reported clinical applications of ASCs and discusses technical approaches of their isolation and processing. The differences in phenotype and differentiation preferences between ASCs and other MSCs that may affect the choice of a particular cell type for the future therapy are also described. EXPERT OPINION: ASCs seem to be the perfect tool for regenerative medicine and immunosuppressive cellular therapies. Nevertheless, there are some tasks that should be addressed by the future studies: i) ASCs require better characterisation; a set of markers determining ASCs should be clearly defined; ii) there is need for more studies on safety of reconstructive therapies with ASCs in cancer patients (e.g., after mastectomy); iii) release criteria should be determined for freshly isolated and ex vivo expanded ASCs designed for clinical applications.

Orthodontic characteristics of maxillary arch deficiency in 5-year-old patients undergoing unilateral cleft lip and palate repair with and without early gingivoplasty.

The purpose of this study was to compare the maxillary arch morphology in 5-year-old children treated for unilateral cleft lip and palate (UCLP) with early gingivoplasty (EGP) and without this procedure (non-EGP). Three-dimensional (3-D) imaging was used to verify which measurements and to what degree specific orthodontic parameters differ in both groups. The study included 120 non-syndromic 4.5-5.5-year-old children treated surgically at the age of 6-18 months. Fifty-six children underwent EGP utilising a Skoog-type of technique. The (non-EGP) group consisted of 64 patients. Patients in both groups were treated without nasoalveolar moulding prior to cleft lip repair. The maxillary arch models were obtained and subjected to the 3-D computer-aided imaging procedure and metric analysis. Ten selected orthodontic measurements were calculated based on imaging landmarks. The computed data derived from models in both (EGP and non-EGP) groups were compared. The differences between groups were statistically analysed using Student's test. Five orthodontic measurements: the angle of the lesser segment inclination, dental arch radius of this segment, anterior palatal depth, palatal surface and length of dental arch demonstrate more severe maxillary underdevelopment in patients with previous EGP. The results reaffirm the negative impact of EGP with wide undermining of periosteal flaps on maxillary development and suggest that these five parameters may be the most sensitive early indicators of growth alteration.

Thermal parametric imaging in the evaluation of skin burn depth.

The aim of this paper is to determine the extent to which infrared (IR) thermal imaging may be used for skin burn depth evaluation. The analysis can be made on the basis of the development of a thermal model of the burned skin. Different methods such as the traditional clinical visual approach and the IR imaging modalities of static IR thermal imaging, active IR thermal imaging and active-dynamic IR thermal imaging (ADT) are analyzed from the point of view of skin burn depth diagnostics. In ADT, a new approach is proposed on the basis of parametric image synthesis. Calculation software is implemented for single-node and distributed systems. The properties of all the methods are verified in experiments using phantoms and subsequently in vivo with animals with a reference histopathological examination. The results indicate that it is possible to distinguish objectively and quantitatively burns which will heal spontaneously within three weeks of infliction and which should be treated conservatively from those which need surgery because they will not heal within this period.

Burn depths evaluation based on active dynamic IR thermal imaging--a preliminary study.

Proper diagnostic assessment of burn wound depth is of the highest importance in selecting the mode of burn wound treatment. Several diagnostic methods--clinical and histopathological evaluation, as well as methods employing IR imaging--static thermography and active dynamic thermography (ADT)--are compared on the basis of in vivo experiments conducted on three domestic pigs (23 burn wounds). ADT is presented here as a new, reliable and quantitative method of assessing burn wound depth on the basis of discrimination of the thermal properties of burnt tissue. In the case of ADT registration of thermal images was performed following thermal pulse excitation. A series of captured infrared images was used as the basis for calculating the thermal time constant tau for each pixel. The parameter values were compared with histopathological and clinical assessments of burn depth. The mean value of tau was found for burns, which heal within 3 weeks (tau=12.08+/-1.94s) and for burns, which did not heal during this period (tau=9.07+/-0.68s), p<0.05. The accuracy, sensitivity and specificity of all the methods tested were compared, the best results coming from ADT. The ADT method is fast, non-invasive and relatively inexpensive, although it still requires further animal experimentation as well as clinical study to confirm the results.

Static thermography revisited--an adjunct method for determining the depth of the burn injury.

The aim of the study was to investigate the relationship between the static thermography figure of merit DeltaT (the difference in mean values of skin area temperature for the burn wound area and the unaffected reference skin area) and a means of burn classification which would be most suitable for the choice of treatment. The work was an in vivo animal experiment. Statistical analysis showed a high correlation between the DeltaT parameter and histopathological assessment. With regard to the choice of treatment, the most useful correlation was found to be that between DeltaT and the classification of burn wounds into those healed in 3 weeks and those unhealed. The results of this study have revealed a quantitative criterion DeltaT for burn classification. The study suggests that particular burn centres using static thermography use a DeltaT parameter based on their own values for burn classification so as to group burn wounds into those that healed in 3 weeks and those that did not heal. This criterion should be independent of and replace other classification systems. A criterion for the proper choice of burn treatment would then be made more readily available.

Early cleft lip repair in children with unilateral complete cleft lip and palate: a case against primary alveolar repair.

All children with complete unilateral cleft lip and palate will develop some degree of malocclusion regardless whether the alveolar cleft is repaired primarily or bone grafting is deferred. To evaluate the impact of early gingivoperiosteoplasty on occlusal relationships, dental models were obtained in 5-year-old patients who underwent early cleft lip and palate repair with primary boneless bone grafting (Skoog's method) (56 children) and without alveolar intervention (51 children). The Goslon's occlusion grading system was applied to evaluate occlusal relationships in both groups. Patients with early surgical intervention to repair alveolar cleft demonstrated poor occlusal relationship with the Goslon score 4 and 5, which will likely need an orthognathic corrective procedure (50% vs. 19.6% in patients without early primary dissection of the alveolar process). Results reaffirm that an inclusion of the alveolar process into the early primary lip repair adds to the severity of occlusal maldevelopment.