Extracellular matrix educates an immunoregulatory tumor macrophage phenotype found in ovarian cancer metastasis.

Recent studies have shown that the tumor extracellular matrix (ECM) associates with immunosuppression, and that targeting the ECM can improve immune infiltration and responsiveness to immunotherapy. A question that remains unresolved is whether the ECM directly educates the immune phenotypes seen in tumors. Here, we identify a tumor-associated macrophage (TAM) population associated with poor prognosis, interruption of the cancer immunity cycle, and tumor ECM composition. To investigate whether the ECM was capable of generating this TAM phenotype, we developed a decellularized tissue model that retains the native ECM architecture and composition. Macrophages cultured on decellularized ovarian metastasis shared transcriptional profiles with the TAMs found in human tissue. ECM-educated macrophages have a tissue-remodeling and immunoregulatory phenotype, inducing altered T cell marker expression and proliferation. We conclude that the tumor ECM directly educates this macrophage population found in cancer tissues. Therefore, current and emerging cancer therapies that target the tumor ECM may be tailored to improve macrophage phenotype and their downstream regulation of immunity.

First steps to define murine amniotic fluid stem cell microenvironment.

Stem cell niche refers to the microenvironment where stem cells reside in living organisms. Several elements define the niche and regulate stem cell characteristics, such as stromal support cells, gap junctions, soluble factors, extracellular matrix proteins, blood vessels and neural inputs. In the last years, different studies demonstrated the presence of cKit+ cells in human and murine amniotic fluid, which have been defined as amniotic fluid stem (AFS) cells. Firstly, we characterized the murine cKit+ cells present both in the amniotic fluid and in the amnion. Secondly, to analyze the AFS cell microenvironment, we injected murine YFP+ embryonic stem cells (ESC) into the amniotic fluid of E13.5 wild type embryos. Four days after transplantation we found that YFP+ sorted cells maintained the expression of pluripotency markers and that ESC adherent to the amnion were more similar to original ESC in respect to those isolated from the amniotic fluid. Moreover, cytokines evaluation and oxygen concentration analysis revealed in this microenvironment the presence of factors that are considered key regulators in stem cell niches. This is the first indication that AFS cells reside in a microenvironment that possess specific characteristics able to maintain stemness of resident and exogenous stem cells.

Improvement of diaphragmatic performance through orthotopic application of decellularized extracellular matrix patch.

Muscle tissue engineering can provide support to large congenital skeletal muscle defects using scaffolds able to allow cell migration, proliferation and differentiation. Acellular extracellular matrix (ECM) scaffold can generate a positive inflammatory response through the activation of anti-inflammatory T-cell populations and M2 polarized macrophages that together lead to a local pro-regenerative environment. This immunoregulatory effect is maintained when acellular matrices are transplanted in a xenogeneic setting, but it remains unclear whether it can be therapeutic in a model of muscle diseases. We demonstrated here for the first time that orthotopic transplantation of a decellularized diaphragmatic muscle from wild animals promoted tissue functional recovery in an established atrophic mouse model. In particular, ECM supported a local immunoresponse activating a pro-regenerative environment and stimulating host muscle progenitor cell activation and migration. These results indicate that acellular scaffolds may represent a suitable regenerative medicine option for improving performance of diseased muscles.

Reprogramming of mouse amniotic fluid cells using a PiggyBac transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Amniotic fluid (AF) cells are easy to obtain from routinely scheduled procedures for prenatal diagnosis and iPS cells have been generated from human AF. Here, we generated iPS cells from mouse AF cells, using a non-viral-based approach constituted by the PiggyBac (PB) transposon system. All iPS cell lines obtained exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo.

A real-time 3D video tracking system for monitoring primate groups.

To date, assessing the solitary and social behaviors of laboratory primates' colonies relies on time-consuming manual scoring methods. Here, we describe a real-time multi-camera 3D tracking system developed to measure the behavior of socially-housed primates. Their positions are identified using non-invasive color markers such as plastic collars, thus allowing to also track colored objects and to measure their usage. Compared to traditional manual ethological scoring, we show that this system can reliably evaluate solitary behaviors (foraging, solitary resting, toy usage, locomotion) as well as spatial proximity with peers, which is considered as a good proxy of their social motivation. Compared to existing video-based commercial systems currently available to measure animal activity, this system offers many possibilities (real-time data, large volume coverage, multiple animal tracking) at a lower hardware cost. Quantitative behavioral data of animal groups can now be obtained automatically over very long periods of time, thus opening new perspectives in particular for studying the neuroethology of social behavior in primates.

Human bone marrow-derived CD133(+) cells delivered to a collagen patch on cryoinjured rat heart promote angiogenesis and arteriogenesis.

Transplanting hematopoietic and peripheral blood-derived stem/progenitor cells can have beneficial effects in slowing the effects of heart failure. We investigated whether human bone marrow CD133(+)-derived cells (BM-CD133(+) cells) might be used for cell therapy of heart injury in combination with tissue engineering. We examined these cells for: 1) their in vitro capacity to be converted into cardiomyocytes (CMs), and 2) their potential for in vivo differentiation when delivered to a tissue-engineered type I collagen patch placed on injured hearts (group II). To ensure a microvascular network ready for use by the transplanted cells, cardiac injury and patching were scheduled 2 weeks before cell injection. The cardiovascular potential of the BM-CD133(+) cells was compared with that of a direct injection (group I) of the same cells in heart tissue damaged according to the same schedule as for group II. While a small fraction (2 ± 0.5%) of BM-CD133(+)cells cocultured with rat CMs switched in vitro to a CM-like cell phenotype, in vivo-and in both groups of nude rats transplanted with BM-CD133(+)--there was no evidence of any CM differentiation (as detected by cardiac troponin I expression), but there were signs instead of new capillaries and small arterioles. While capillaries prevailed over arterioles in group II, the opposite occurred in group I. The transplanted cells further contributed to the formation of new microvessels induced by the patch (group II) but the number of vessels did not appear superior to the one developed after directly injecting the BM-CD133(+)cells into the injured heart. Although chimeric human-rat microvessels were consistently found in the hearts of both groups I and II, they represented a minority (1.5-2.3%) compared with those of rat origin. Smooth muscle myosin isoform expression suggested that the arterioles achieved complete differentiation irrespective of the presence or absence of the collagen patch. These findings suggest that: 1) BM-CD133(+) cells display a limited propensity for in vitro conversion to CMs; 2) the preliminarily vascularized bioscaffold did not confer a selective homing and differentiation advantage for the phenotypic conversion of BM-CD133(+) cells into CMs; and 3) combined patching and cell transplantation is suitable for angiogenesis and arteriogenesis, but it does not produce better results, in terms of endothelial and smooth muscle cell differentiation, than the "traditional" method of cell injection into the myocardium.

Unusual chromosome numbers in Paspalum L. (Poaceae: Paniceae) from Brazil.

Somatic chromosome numbers were determined for 20 new germplasm accessions of Paspalum, belonging to 17 species collected in Brazil. Chromosome number is reported for the first time for P. reduncum (2n = 18), P. cinerascens (2n = 20), P. cordatum (2n = 20), P. filgueirasii (2n = 24), P. ammodes (2n = 36), P. bicilium (2n = 40), P. heterotrichon (2n = 40), and P. burmanii (2n = 48). New cytotypes were confirmed for two germplasm accessions of P. carinatum (2n = 30) and P. trachycoleon (2n = 36), one of P. clavuliferum (2n = 40) and one of P. lanciflorum (2n = 40), indicating variability in these species. The remaining chromosome numbers reported here confirm previous counts. The unexpected chromosome numbers 2n = 18, 24, 36, and 48 in Paspalum species, which are usually shown to be multiples of 10, suggest that much more collection and cytogenetic characterization are necessary to assess the whole chromosomal and genomic multiplicity present in the genus, which seems to be much more diverse than currently thought to be.

Unusual chromosome numbers in Paspalum L. (Poaceae: Paniceae) from Brazil

Somatic chromosome numbers were determined for 20 new germplasm accessions of Paspalum, belonging to 17 species collected in Brazil. Chromosome number is reported for the first time for P. reduncum (2n = 18), P. cinerascens (2n = 20), P. cordatum (2n = 20), P. filgueirasii (2n = 24), P. ammodes (2n = 36), P. bicilium (2n = 40), P. heterotrichon (2n = 40), and P. burmanii (2n = 48). New cytotypes were confirmed for two germplasm accessions of P. carinatum (2n = 30) and P. trachycoleon (2n = 36), one of P. clavuliferum (2n = 40) and one of P. lanciflorum (2n = 40), indicating variability in these species. The remaining chromosome numbers reported here confirm previous counts. The unexpected chromosome numbers 2n = 18, 24, 36, and 48 in Paspalum species, which are usually shown to be multiples of 10, suggest that much more collection and cytogenetic characterization are necessary to assess the whole chromosomal and genomic multiplicity present in the genus, which seems to be much more diverse than currently thought to be.

Rosiglitazone modifies the adipogenic potential of human muscle satellite cells.

AIMS/HYPOTHESIS: Satellite cells are responsible for postnatal skeletal muscle regeneration. It has been demonstrated that mouse satellite cells behave as multipotent stem cells. We studied the differentiation capacities of human satellite cells and evaluated the effect of the insulin sensitiser rosiglitazone, a well known peroxisome proliferative activated receptor gamma (PPARG) agonist, on their adipogenic conversion. SUBJECTS, MATERIALS AND METHODS: We obtained human satellite cells from human muscle biopsies of healthy subjects by single-fibre isolation and cultured them under myogenic, osteogenic and adipogenic conditions. Moreover, we compared the morphological features and the adipose-specific gene expression profiling, as assessed by quantitative PCR, between adipocytes differentiated from human satellite cells and those obtained from the stromal vascular fraction of human visceral fat. RESULTS: We proved by morphological analysis, mRNA expression and immunohistochemistry that human satellite cells are able to differentiate into myotubes, adipocytes and osteocytes. The addition of rosiglitazone to the adipogenic medium strongly activated PPARG expression and enhanced adipogenesis in human satellite cells, but did not in itself trigger the complete adipogenic programme. Moreover, we observed a decrease in wingless-type MMTV integration site family member 10B and an upregulation of growth differentiation factor 8 expression, both being independent of PPARG activation. CONCLUSIONS/INTERPRETATION: Human satellite cells possess a clear adipogenic potential that could explain the presence of mature adipocytes within skeletal muscle in pathological conditions such as obesity, type 2 diabetes and ageing-related sarcopenia. Rosiglitazone treatment, while enhancing adipogenesis, induces a more favourable pattern of adipocytokine expression in satellite-derived fat cells. This could partially counteract the worsening effect of intermuscular adipose tissue depots on muscle insulin sensitivity.

Expression pattern of intracellular leukocyte-associated proteins in primary mediastinal B cell lymphoma.

Two microarray studies of mediastinal B cell lymphoma have shown that this disease has a distinct gene expression profile, and also that this is closest to the pattern seen in classical Hodgkin's disease. We reported previously an immunohistologic study in which the loss of intracellular B cell-associated signaling molecules in Reed-Sternberg cells was demonstrated, and in this study we have investigated the expression of the same components in more than 60 mediastinal B cell lymphomas. We report that these signaling molecules are frequently present, and in particular that Syk, BLNK and PLC-gamma2 (absent from Reed-Sternberg cells) are present in the majority of mediastinal B cell lymphomas. The overall pattern of B cell signaling molecules in this disease is therefore closer to that of diffuse large B cell lymphoma than to Hodgkin's disease, and is consistent with a common cell of origin as an explanation of the similar gene expression profiles.

Spontaneous superior mesenteric vein thrombosis (SMVT) in primary protein S deficiency. A case report and review of the literature.

Superior mesenteric vein thrombosis (SMVT) is an uncommon but important clinical entity that can induce ischemia or infarction of the small and large bowel. It is rare and accounts for 5-15% of mesenteric vascular occlusions. Bowel infarction due to SMVT can present as an acute abdominal disease, requiring urgent laparotomy with resection of the intestinal segment affected. However, the clinical diagnosis of this event remains difficult and invariably requires specific imaging investigations in order to be able to treat the condition as soon as possible. SMVT without bowel infarction can present as persistent, non-specific abdominal pain and nausea with minimal clinical signs, affecting young individuals without any known predisposing disorder, where laparotomy is not an urgent indication. We report a case of a young adult man with SMVT due to a hypercoagulable state (protein S deficiency), in whom an early diagnosis and appropriate anticoagulant treatment prevented any further extension of the thrombotic process and limited the hemorrhagic infarction of the ileum, which simply required a segmental resection.

Duplication of the chromosome number of diploid Brachiaria brizantha plants using colchicine.

Some species of Brachiaria, generally tetraploid apomictic varieties, have become important forage grasses in the tropics. Breeding of Brachiaria depends on compatibility with the available apomitic tretraploid cultivars. This paper describes a procedure for chromosome duplication of two Bracharia brizantha diploid sexual accessions, using colchicine treatment of basal segments of in-vitro-grown plants. Explants were cultured on a medium containing 1 mg/l naphthaleneacetic acid, 3 mg/l kinetin and 0.01% colchicine for 48 h and transferred to the same medium without colchicine until shoot regeneration occurred. Regenerated plants were screened by flow cytometry, and chromosome number duplication was confirmed by cytological analysis of root tips.

Gene transfer in skeletal muscle by systemic injection of DODAC lipopolyplexes.

Lipid-based vectors are a promising tool for gene therapy applications. Several studies have reported their use in vivo to transfect different organs. Few data, however, are available about lipid-mediated gene transfer in skeletal muscle. Here we report the initial results obtained after systemic administration of lipopolyplexes based on the DODAC cationic lipid in an animal model of muscle regeneration. In particular, we compared three routes of administration: intravenous (i.v.), intracardiac (IC) and intra-arterial (IA). Analysis of reporter gene expression (luciferase) showed that regenerating muscle is more efficiently transfected in all cases and that IA injection is by far the best approach.

Isolated vascular perfusion of human colon with adenocarcinoma.

Based on the premise that optimal drug delivery might improve the efficacy of locoregional treatment for solid tumors, the authors set up an experimental model for isolation perfusion in surgical specimens from patients resected for carcinoma of the colon. Ten surgical specimens were cannulated, washed internally and externally with saline solution, promptly cooled to 4 degreesC, connected to a circuit, and perfused with Krebs-Henselait modified solution, concentrated red blood cells, albumin, desamethasone, glucose, and heparin for 60 minutes at a target temperature of 37 degreesC. Organ temperature, flow rate, perfusion pressure, and metabolic and functional parameters were checked at 5, 20, and 60 minutes of perfusion. A paraphysiologic perfusion procedure was achieved. Mean values (and ranges) were as follows: temperature 37 degreesC (35. 1-39.6 degreesC); flow rate 10.2 (5.6-17.9) ml/min/100 g; arterial pressure 96 (42-154) mmHg; arterial pH 7.3 (7.1-7.5); arterial PO2 183 (78-304) mmHg; arterial PCO2 36 (31-46) mmHg. No important signs of tissue damage were found at histology. Autonomous or stimulated peristalsis (or both) was present throughout the experiment. Mean O2 extraction was 7.9 ml/min/100 g (range 3.1-11.0). Mean glucose consumption was 229 mg/100 g (range 174-252). The model worked well and appears promising, particularly for future use in various pharmacokinetic and pharmacodynamic studies of antiblastic agents.