Streptolysin O enhances keratinocyte migration and proliferation and promotes skin organ culture wound healing in vitro

ML-05, a modified form of the hemolytic and cytotoxic bacterial toxin, streptolysin O, is currently being investigated as a treatment for collagen-related disorders such as scleroderma and fibrosis. Furthermore, ML-05 may be effective in promoting wound healing and alleviating the formation of hypertrophic scars and keloids. To investigate the effects of ML-05 on wound-healing processes, in vitro wound-healing scratch assays (using human primary epidermal keratinocytes and dermal fibroblasts) and a human skin organ culture wound model were utilized. ML-05 markedly enhanced keratinocyte migration and proliferation in wound scratch assays. ML-05 did not affect either proliferation or migration of dermal fibroblasts, indicating that ML-05's effects on cell migration/proliferation may be keratinocyte-specific. ML-05 was tested in a dose-dependent manner in a skin organ culture wound model using two different application methods: Through the culture media (dermal exposure) or direct topical treatment of the wound surface. ML-05 was found to accelerate wound healing as measured by reepithelialization, particularly after topical application. Therefore, ML-05 may have potential as a wound-healing agent that promotes reepithelialization through stimulation of keratinocyte migration and proliferation.     

Plasma and Urinary Amino Acid-Derived Catabolites as Potential Biomarkers of Protein and Amino Acid Deficiency in Rats

Objective: Dietary intakes must cover protein and essential amino acid (EAA) requirements. For this purpose, different methods have been developed such as the nitrogen balance method, factorial method, or AA tracer studies. However, these methods are either invasive or imprecise, and the Food and Agriculture Organization of the United Nations (FAO, 2013) recommends new methods and, in particular, metabolomics. The aim of this study is to determine total protein/EAA requirement in the plasma and urine of growing rats. Methods: 36 weanling rats were fed with diets containing 3, 5, 8, 12, 15, and 20% protein for 3 weeks. During experimentation, urine was collected using metabolic cages, and blood from the portal vein and vena was taken at the end of the experiment. Metabolomics analyses were performed using LC-MS, and the data were analyzed with a multivariate analysis model, partial least Squares (PLS) regression, and independent component-discriminant analysis (ICDA). Each discriminant metabolite identified by PLS or ICDA was tested by one-way ANOVA to evaluate the effect of diet. Results: PLS and ICDA allowed us to identify discriminating metabolites between different diet groups. Protein deficiency led to an increase in the AA catabolism enzyme systems inducing the production of breakdown metabolites in the plasma and urine. Conclusion: These results indicate that metabolites are specific for the state of EAA deficiency and sufficiency. Some types of biomarkers such as AA degradation metabolites appear to be specific candidates for protein/EAA requirement.     

The strength of combined cytogenetic and mate-pair sequencing techniques illustrated by a germline chromothripsis rearrangement involving FOXP2

Next-generation mate-pair sequencing (MPS) has revealed that many constitutional complex chromosomal rearrangements (CCRs) are associated with local shattering of chromosomal regions (chromothripsis). Although MPS promises to identify the molecular basis of the abnormal phenotypes associated with many CCRs, none of the reported mate-pair sequenced complex rearrangements have been simultaneously studied with state-of-the art molecular cytogenetic techniques. Here, we studied chromothripsis-associated CCR involving chromosomes 2, 5 and 7, associated with global developmental and psychomotor delay and severe speech disorder. We identified three truncated genes: CDH12, DGKB and FOXP2, confirming the role of FOXP2 in severe speech disorder, and suggestive roles of CDH12 and/or DGKB for the global developmental and psychomotor delay. Our study confirmes the power of MPS for detecting breakpoints and truncated genes at near nucleotide resolution in chromothripsis. However, only by combining MPS data with conventional G-banding and extensive fluorescence in situ hybridizations could we delineate the precise structure of the derivative chromosomes.     

A further case with a small supernumerary marker chromosome (sSMC) derived from chromosome 1--evidence for high variability in mosaicism in different tissues of sSMC carriers

A prenatally ascertained case with a de novo small supernumerary marker chromosome (sSMC) derived from chromosome 1 is reported. Due to a fetal heart defect the parents decided in favour of an induced abortion. Postmortem, a molecular cytogenetic study on eleven formalin fixed, paraffin-embedded tissues of the fetus was performed, to further characterize the levels of mosaicism of the sSMC(1). sSMC presence varied between 13 and 62% within different tissues of sSMC carriers. This finding is something common in sSMC carriers and could explain why up to the present no clinical correlations for sSMC mosaicism and clinical outcome in the corresponding carriers could be established.     

Non-T cell activation linker (NTAL): a transmembrane adaptor protein involved in immunoreceptor signaling

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non-T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcgamma- and Fc epsilon -receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non-T cells.     

Increased myeloid precursors in regenerating bone marrow; implications for detection of minimal residual disease in acute myeloid leukemia

Presented study is focused on exact immunophenotypic definition of myeloid precursors and their following stages in regenerating bone marrow during treatment of ALL/AML for correct interpretation of the immunophenotype results and proper distinction from minimal residual disease (MRD) by multiparameter flow cytometry. This study includes bone marrow samples from 36 controls, 27 patients with AML, 39 patients with B-ALL undergoing therapy who remained in complete remission after treatment and also 30 B-ALL patients one year after the end of therapy. We observed substantial expansion of immature bone marrow populations in the regenerating bone marrows, which were identified by expression of CD34 and/or CD117 markers by 4-color flow cytometry. Myeloid precursors were significantly increased after cessation of induction therapy cycle of B-ALL (1.27+/-2.04%, p=0.0064) and also AML patients (0.87+/-0.77%, p=0.001), but also during follow-up of B-ALL patients (1.42+/-2.36%, p=0.0001) when compared with non-treated controls (0.38+/-0.29%). Some cases where their frequencies achieved up to 12% reflect the massive regeneration of myeloid lineage in bone marrow after chemotherapy cycles. Especially in these cases accurate interpretation of such a high frequency of immature myeloid cells as myeloid precursors was very important to exclude incoming relapse or secondary leukemia. The myeloid precursors represented by CD34+ in regenerating bone marrow expressed CD45 (94.8+/-5.5%), CD117 (38.3+/-26.2%), CD38 (91.4+/-5.7%), HLA-DR (90.6+/-7.6%), CD13 (73.0+/-20.8%) and CD33 (85.2+/-15.6%), while CD90 (2.7+/-2.5%), CD133 (10.0+/-8.2%) and T or B lymphocyte markers were negative. Comparing immunophenotypes with control bone marrows, only difference in expression of CD33 marker was found (85.2+/-15.6% versus 63.0+/-17.4% p=0.024). In addition, according to expression of these markers three different subsets of myeloid precursor cells were identified in regenerating bone marrow samples: CD34+ CD117- HLA-DR+, CD34+ CD117+ HLA-DR+ and CD34- CD117+ HLA-DR-/+ without aberrant marker expression. In conclusion for the correct discrimination of MRD in acute leukemia it is indispensable to define the range of normality in myeloid differentiation by extensive studies of bone marrows not only from healthy donors but also from regenerating bone marrow of patients undergoing therapy.