Angiogenic Modification of Microfibrous Polycaprolactone by pCMV-VEGF165 Plasmid Promotes Local Vascular Growth after Implantation in Rats.

Insufficient vascular growth in the area of artificial-material implantation contributes to ischemia, fibrosis, the development of bacterial infections, and tissue necrosis around the graft. The purpose of this study was to evaluate angiogenesis after implantation of polycaprolactone microfiber scaffolds modified by a pCMV-VEGF165-plasmid in rats. Influence of vascularization on scaffold degradation was also examined. We investigated flat microfibrous scaffolds obtained by electrospinning polycaprolactone with incorporation of the pCMV-VEGF-165 plasmid into the microfibers at concentrations of 0.005 ng of plasmid per 1 mg of polycaprolactone (0.005 ng/mg) (LCGroup) and 0.05 ng/mg (HCGroup). The samples were subcutaneously implanted in the interscapular area of rats. On days 7, 16, 33, 46, and 64, the scaffolds were removed, and a histological study with a morphometric evaluation of the density and diameter of the vessels and microfiber diameter was performed. The number of vessels was increased in all groups, as well as the resorption of the scaffold. On day 33, the vascular density in the HCGroup was 42% higher compared to the control group (p = 0.0344). The dose-dependent effect of the pCMV-VEGF165-plasmid was confirmed by enhanced angiogenesis in the HCGroup compared to the LCGroup on day 33 (p-value = 0.0259). We did not find a statistically significant correlation between scaffold degradation rate and vessel growth (the Pearson correlation coefficient was ρ = 0.20, p-value = 0.6134). Functionalization of polycaprolactone by incorporation of the pCMV-VEGF165 plasmid provided improved vascularization within 33 days after implantation, however, vessel growth did not seem to correlate with scaffold degradation rate.

Misdiagnosis of diabetic foot ulcer in patients with undiagnosed skin malignancies.

A growing number of studies report dermal malignancies mimicking diabetic foot ulcers (DFUs). We reviewed clinical cases reporting malignant tumours misdiagnosed to be DFU aiming to identify factors contributing to misdiagnosis. We systematically searched in PubMed for clinical cases reporting on misdiagnosis of DFU in patients with cancer. A chi-square analysis was conducted to show the link between the incidence of initial DFU misdiagnosis and patient age, gender and wound duration. Lesions misdiagnosed to be DFU were subsequently diagnosed as melanoma (68% of the cases), Kaposi's sarcoma (14%), squamous cell carcinoma (11%), mantle cell lymphoma, and diffuse B-cell lymphoma (both by 4%). Older age (≥65 years) was associated with a significantly increased risk of malignancy masked as DFU (OR: 2.452; 95% CI: 1.132 to 5.312; P value = .019). The risk of such suspicion in older patients (age ≥ 65 years) was 145% higher than in younger patients (age < 65 years). Clinicians should maintain a high level of awareness towards potentially malignant foot lesions in elderly patients with diabetes (age ≥ 65).

Plasma Sarcosine Measured by Gas Chromatography-Mass Spectrometry Distinguishes Prostatic Intraepithelial Neoplasia and Prostate Cancer from Benign Prostate Hyperplasia.

OBJECTIVE: Sarcosine was postulated in 2009 as a biomarker for prostate cancer (PCa). Here, we assess plasma sarcosine as a biomarker that is complementary to prostate-specific antigen (PSA). METHODS: Plasma sarcosine was measured using gas chromatography-mass spectrometry (GC-MS) in adults classified as noncancerous controls (with benign prostate hyperplasia [BPH], n = 36), with prostatic intraepithelial neoplasia (PIN, n = 16), or with PCa (n = 27). Diagnostic accuracy was assessed using receiver operating characteristic curve analysis. RESULTS: Plasma sarcosine levels were higher in the PCa (2.0 µM [1.3-3.3 µM], P <.01) and the PIN (1.9 µM [1.2-6.5 µM], P <.001) groups than in the BPH (0.9 µM [0.6-1.4 µM]) group. Plasma sarcosine had "good" and "very good" discriminative capability to detect PIN (area under the curve [AUC], 0.734) and PCa (AUC, 0.833) versus BPH, respectively. The use of PSA and sarcosine together improved the overall diagnostic accuracy to detect PIN and PCa versus BPH. CONCLUSION: Plasma sarcosine measured by GC-MS had "good" and "very good" classification performance for distinguishing PIN and PCa, respectively, relative to noncancerous patients diagnosed with BPH.

Extension of Maximal Lifespan and High Bone Marrow Chimerism After Nonmyeloablative Syngeneic Transplantation of Bone Marrow From Young to Old Mice.

The goal of this work was to determine the effect of nonablative syngeneic transplantation of young bone marrow (BM) to laboratory animals (mice) of advanced age upon maximum duration of their lifespan. To do this, transplantation of 100 million nucleated cells from BM of young syngeneic donors to an old nonablated animal was performed at the time when half of the population had already died. As a result, the maximum lifespan (MLS) increased by 28 ± 5%, and the survival time from the beginning of the experiment increased 2.8 ± 0.3-fold. The chimerism of the BM 6 months after the transplantation was 28%.

A Novel Lipopeptaibol Emericellipsin A with Antimicrobial and Antitumor Activity Produced by the Extremophilic Fungus Emericellopsis alkalina.

Soil fungi are known to contain a rich variety of defense metabolites that allow them to compete with other organisms (fungi, bacteria, nematodes, and insects) and help them occupy more preferential areas at the expense of effective antagonism. These compounds possess antibiotic activity towards a wide range of other microbes, particularly fungi that belong to different taxonomical units. These compounds include peptaibols, which are non-ribosomal synthesized polypeptides containing non-standard amino acid residues (alpha-aminoisobutyric acid mandatory) and some posttranslational modifications. We isolated a novel antibiotic peptide from the culture medium of Emericellopsis alkalina, an alkalophilic strain. This peptide, called emericellipsin A, exhibited a strong antifungal effect against the yeast Candida albicans, the mold fungus Aspergillus niger, and human pathogen clinical isolates. It also exhibited antimicrobial activity against some Gram-positive and Gram-negative bacteria. Additionally, emericellipsin A showed a significant cytotoxic effect and was highly active against Hep G2 and HeLa tumor cell lines. We used NMR spectroscopy to reveal that this peptaibol is nine amino acid residues long and contains non-standard amino acids. The mode of molecular action of emericellipsin A is most likely associated with its effects on the membranes of cells. Emericellipsin A is rather short peptaibol and could be useful for the development of antifungal, antibacterial, or anti-tumor remedies.

Insulin receptor in the brain: Mechanisms of activation and the role in the CNS pathology and treatment.

While the insulin receptor (IR) was found in the CNS decades ago, the brain was long considered to be an insulin-insensitive organ. This view is currently revisited, given emerging evidence of critical roles of IR-mediated signaling in development, neuroprotection, metabolism, and plasticity in the brain. These diverse cellular and physiological IR activities are distinct from metabolic IR functions in peripheral tissues, thus highlighting region specificity of IR properties. This particularly concerns the fact that two IR isoforms, A and B, are predominantly expressed in either the brain or peripheral tissues, respectively, and neurons express exclusively IR-A. Intriguingly, in comparison with IR-B, IR-A displays high binding affinity and is also activated by low concentrations of insulin-like growth factor-2 (IGF-2), a regulator of neuronal plasticity, whose dysregulation is associated with neuropathologic processes. Deficiencies in IR activation, insulin availability, and downstream IR-related mechanisms may result in aberrant IR-mediated functions and, subsequently, a broad range of brain disorders, including neurodevelopmental syndromes, neoplasms, neurodegenerative conditions, and depression. Here, we discuss findings on the brain-specific features of IR-mediated signaling with focus on mechanisms of primary receptor activation and their roles in the neuropathology. We aimed to uncover the remaining gaps in current knowledge on IR physiology and highlight new therapies targeting IR, such as IR sensitizers.

Cyclic AMP Pathway Suppress Autoimmune Neuroinflammation by Inhibiting Functions of Encephalitogenic CD4 T Cells and Enhancing M2 Macrophage Polarization at the Site of Inflammation.

Although it has been demonstrated that cAMP pathway affect both adaptive and innate cell functions, the role of this pathway in the regulation of T-cell-mediated central nervous system (CNS) autoimmune inflammation, such as in experimental autoimmune encephalomyelitis (EAE), remains unclear. It is also unclear how cAMP pathway affects the function of CD4 T cells in vivo at the site of inflammation. We found that adenylyl cyclase activator Forskolin besides inhibition of functions autoimmune CD4 T cells also upregulated microRNA (miR)-124 in the CNS during EAE, which is associated with M2 phenotype of microglia/macrophages. Our study further established that in addition to direct influence of cAMP pathway on CD4 T cells, stimulation of this pathway promoted macrophage polarization toward M2 leading to indirect inhibition of function of T cells in the CNS. We demonstrated that Forskolin together with IL-4 or with Forskolin together with IL-4 and IFNγ effectively stimulated M2 phenotype of macrophages indicating high potency of this pathway in reprogramming of macrophage polarization in Th2- and even in Th1/Th2-mixed inflammatory conditions such as EAE. Mechanistically, Forskolin and/or IL-4 activated ERK pathway in macrophages resulting in the upregulation of M2-associated molecules miR-124, arginase (Arg)1, and Mannose receptor C-type 1 (Mrc1), which was reversed by ERK inhibitors. Administration of Forskolin after the onset of EAE substantially upregulated M2 markers Arg1, Mrc1, Fizz1, and Ym1 and inhibited M1 markers nitric oxide synthetase 2 and CD86 in the CNS during EAE resulting in decrease in macrophage/microglia activation, lymphocyte and CD4 T cell infiltration, and the recovery from the disease. Forskolin inhibited proliferation and IFNγ production by CD4 T cells in the CNS but had rather weak direct effect on proliferation of autoimmune T cells in the periphery and in vitro, suggesting prevalence of indirect effect of Forskolin on differentiation and functions of autoimmune CD4 T cells in vivo. Thus, our data indicate that Forskolin has potency to skew balance toward M2 affecting ERK pathway in macrophages and indirectly inhibit pathogenic CD4 T cells in the CNS leading to the suppression of autoimmune inflammation. These data may have also implications for future therapeutic approaches to inhibit autoimmune Th1 cells at the site of tissue inflammation.

Human endometrial stem cells: High-yield isolation and characterization.

BACKGROUND: Menstrual blood is only recently and still poorly studied, but it is an abundant and noninvasive source of highly proliferative mesenchymal stromal cells (MSCs). However, no appropriate isolation method has been reported due to its high viscosity and high content of clots and desquamated epithelium. METHODS: We studied three different isolation approaches and their combinations: ammonium-containing lysing buffer, distilled water and gradient-density centrifugation. We tested the proliferative capacity, morphology, surface markers and pluripotency of the resulting cells. RESULTS: Our isolation method yields up to four million nucleated cells per milliliter of initial blood, of which about 0.2-0.3% are colony-forming cells expressing standard mesenchymal markers CD90, CD105 and CD73, but not expressing CD45, CD34, CD117, CD133 or HLA-G. The cells have high proliferative potential (doubling in 26 h) and the ability to differentiate into adipocytes and osteocytes. Early endometrial MSCs (eMSCs) express epithelial marker cytokeratin 7 (CK7). CK7 is easily induced in later passages in a prohepatic environment. We show for the first time that a satisfactory and stable yield of eMSCs is observed throughout the whole menstrual period (5 consecutive days) of a healthy woman. DISCUSSION: The new cost/yield adequate method allows isolation from menstrual blood a relatively homogenous pool of highly proliferative MSCs, which seem to be the best candidates for internal organ therapy due to their proepithelial background (early expression of CK7 and its easy induction in later passages) and for mass cryobanking due to their high yield and availability.

The potential role of tissue-engineered urethral substitution: clinical and preclinical studies.

Urethral strictures and anomalies remain among the difficult problems in urology, with urethroplasty procedures being the most effective treatment options. The two major types of urethroplasty are anastomotic urethroplasty and widening the urethral lumen using flaps or grafts (i.e. substitution urethroplasty). However, no ideal material for the latter has been found so far. Designing and selecting such a material is a necessary and challenging endeavour, driving the need for further bioengineered urethral tissue research. This article reviews currently available studies on the potentialities of tissue engineering in urethral reconstruction, in particular those describing the use of both acellular and recellularized tissue-engineered constructs in animal and human models. Possible future developments in this field are also discussed. Copyright © 2015 John Wiley & Sons, Ltd.