Cells derived from the coelomic epithelium contribute to multiple gastrointestinal tissues in mouse embryos.

Gut mesodermal tissues originate from the splanchnopleural mesenchyme. However, the embryonic gastrointestinal coelomic epithelium gives rise to mesenchymal cells, whose significance and fate are little known. Our aim was to investigate the contribution of coelomic epithelium-derived cells to the intestinal development. We have used the transgenic mouse model mWt1/IRES/GFP-Cre (Wt1(cre)) crossed with the Rosa26R-EYFP reporter mouse. In the gastrointestinal duct Wt1, the Wilms' tumor suppressor gene, is specific and dynamically expressed in the coelomic epithelium. In the embryos obtained from the crossbreeding, the Wt1-expressing cell lineage produces the yellow fluorescent protein (YFP) allowing for colocalization with differentiation markers through confocal microscopy and flow cytometry. Wt1(cre-YFP) cells were very abundant throughout the intestine during midgestation, declining in neonates. Wt1(cre-YFP) cells were also transiently observed within the mucosa, being apparently released into the intestinal lumen. YFP was detected in cells contributing to intestinal vascularization (endothelium, pericytes and smooth muscle), visceral musculature (circular, longitudinal and submucosal) as well as in Cajal and Cajal-like interstitial cells. Wt1(cre-YFP) mesenchymal cells expressed FGF9, a critical growth factor for intestinal development, as well as PDGFRα, mainly within developing villi. Thus, a cell population derived from the coelomic epithelium incorporates to the gut mesenchyme and contribute to a variety of intestinal tissues, probably playing also a signaling role. Our results support the origin of interstitial cells of Cajal and visceral circular muscle from a common progenitor expressing anoctamin-1 and SMCα-actin. Coelomic-derived cells contribute to the differentiation of at least a part of the interstitial cells of Cajal.

Expression of PROKR1 and PROKR2 in Human Enteric Neural Precursor Cells and Identification of Sequence Variants Suggest a Role in HSCR

Background. The enteric nervous system (ENS) is entirely derived from neural crest and its normal development is regulated by specific molecular pathways. Failure in complete ENS formation results in aganglionic gut conditions such as Hirschsprung's disease (HSCR). Recently, PROKR1 expression has been demonstrated in mouse enteric neural crest derived cells and Prok-1 was shown to work coordinately with GDNF in the development of the ENS. Principal Findings. In the present report, ENS progenitors were isolated and characterized from the ganglionic gut from children diagnosed with and without HSCR, and the expression of prokineticin receptors was examined. Immunocytochemical analysis of neurosphere-forming cells demonstrated that both PROKR1 and PROKR2 were present in human enteric neural crest cells. In addition, we also performed a mutational analysis of PROKR1, PROKR2, PROK1 and PROK2 genes in a cohort of HSCR patients, evaluating them for the first time as susceptibility genes for the disease. Several missense variants were detected, most of them affecting highly conserved amino acid residues of the protein and located in functional domains of both receptors, which suggests a possible deleterious effect in their biological function. Conclusions. Our results suggest that not only PROKR1, but also PROKR2 might mediate a complementary signalling to the RET/GFRα1/GDNF pathway supporting proliferation/survival and differentiation of precursor cells during ENS development. These findings, together with the detection of sequence variants in PROKR1, PROK1 and PROKR2 genes associated to HSCR and, in some cases in combination with RET or GDNF mutations, provide the first evidence to consider them as susceptibility genes for HSCR.

Role of Leptin in the Activation of Immune Cells

Adipose tissue is an active endocrine organ that secretes various humoral factors (adipokines), and its shift to production of proinflammatory cytokines in obesity likely contributes to the low-level systemic inflammation that may be present in metabolic syndrome-associated chronic pathologies such as atherosclerosis. Leptin is one of the most important hormones secreted by adipocytes, with a variety of physiological roles related to the control of metabolism and energy homeostasis. One of these functions is the connection between nutritional status and immune competence. The adipocyte-derived hormone leptin has been shown to regulate the immune response, innate and adaptive response, both in normal and pathological conditions. The role of leptin in regulating immune response has been assessed in vitro as well as in clinical studies. It has been shown that conditions of reduced leptin production are associated with increased infection susceptibility. Conversely, immune-mediated disorders such as autoimmune diseases are associated with increased secretion of leptin and production of proinflammatory pathogenic cytokines. Thus, leptin is a mediator of the inflammatory response

Modulation of MDR1 and MRP3 Gene Expression in Lung Cancer Cells after Paclitaxel and Carboplatin Exposure

Carboplatin-paclitaxel is a reference regimen in the treatment of locally advanced or disseminated non-small cell lung cancer (NSCLC). This paper discusses the multidrug resistance developed with this drug combination, which is one of the major obstacles to successful treatment. In order to understand and overcome the drug resistance pattern of NSCLC after carboplatin plus paclitaxel exposure, levels of mRNA expression of multidrug resistance 1 (MDR1) and multidrug resistance-associated protein 3 (MRP3) were investigated in primary NSCLC cell lines (A-549 and A-427) and a metastasis-derived NSCLC cell line (NODO). Our results showed that exposure of the three NSCLC lines to plasma concentrations of paclitaxel (5 μM) produced an increase in MDR1 expression, while MRP3 showed no alteration in expression. By contrast, the same cells exposed to carboplatin plasma concentrations (30 μM) showed overexpression of MRP3. In these cells, MDR1 showed no expression changes. Interestingly, the combination of both paclitaxel and carboplatin caused increased expression of the MDR1 drug resistance gene rather than the individual treatments. These results suggest that carboplatin and paclitaxel may induce drug resistance mediated by MDR1 and MRP3, which may be enhanced by the simultaneous use of both drugs.

Synergistic Effect between Amoxicillin and TLR Ligands on Dendritic Cells from Amoxicillin-Delayed Allergic Patients.

Amoxicillin, a low-molecular-weight compound, is able to interact with dendritic cells inducing semi-maturation in vitro. Specific antigens and TLR ligands can synergistically interact with dendritic cells (DC), leading to complete maturation and more efficient T-cell stimulation. The aim of the study was to evaluate the synergistic effect of amoxicillin and the TLR2, 4 and 7/8 agonists (PAM, LPS and R848, respectively) in TLR expression, DC maturation and specific T-cell response in patients with delayed-type hypersensitivity (DTH) reactions to amoxicillin. Monocyte-derived DC from 15 patients with DTH to amoxicillin and 15 controls were cultured with amoxicillin in the presence or absence of TLR2, 4 and 7/8 agonists (PAM, LPS and R848, respectively). We studied TLR1-9 gene expression by RT-qPCR, and DC maturation, lymphocyte proliferation and cytokine production by flow cytometry. DC from both patients and controls expressed all TLRs except TLR9. The amoxicillin plus TLR2/4 or TLR7/8 ligands showed significant differences, mainly in patients: AX+PAM+LPS induced a decrease in TLR2 and AX+R848 in TLR2, 4, 7 and 8 mRNA levels. AX+PAM+LPS significantly increased the percentage of maturation in patients (75%) vs. controls (40%) (p=0.036) and T-cell proliferation (80.7% vs. 27.3% of cases; p=0.001). Moreover, the combinations AX+PAM+LPS and AX+R848 produced a significant increase in IL-12p70 during both DC maturation and T-cell proliferation. These results indicate that in amoxicillin-induced maculopapular exanthema, the presence of different TLR agonists could be critical for the induction of the innate and adaptive immune responses and this should be taken into account when evaluating allergic reactions to these drugs.

Angiographic demonstration of neoangiogenesis after intra-arterial infusion of autologous bone marrow mononuclear cells in diabetic patients with critical limb ischemia.

Critical limb ischemia in diabetic patients is associated with high rates of morbidity and mortality. Suboptimal responses to the available medical and surgical treatments are common in these patients, who also demonstrate limited vascular homeostasis. Neovasculogenesis induced by stem cell therapy could be a useful approach for these patients. Neovasculogenesis and clinical improvement were compared at baseline and at 3 and 12 months after autologous bone marrow-derived mononuclear cell (BMMNC) transplantation in diabetic patients with peripheral artery disease. We conducted a prospective study to evaluate the safety and efficacy of intra-arterial administration of autologous BMMNCs (100-400 × 10(6) cells) in 20 diabetic patients with severe below-the-knee arterial ischemia. Although the time course of clinical effects differed among patients, after 12 months of follow-up all patients presented a notable improvement in the Rutherford-Becker classification, the University of Texas diabetic wound scales, and the Ankle-Brachial Index in the target limb. The clinical outcome was consistent with neovasculogenesis, which was assessed at 3 months by digital subtraction angiography and quantified by MetaMorph software. Unfortunately, local cell therapy in the target limb had no beneficial effect on the high mortality rate in these patients. In diabetic patients with critical limb ischemia, intra-arterial perfusion of BMMNCs is a safe procedure that generates a significant increase in the vascular network in ischemic areas and promotes remarkable clinical improvement.

Angiographic demonstration of neoangiogenesis after intra-arterial infusion of autologous bone marrow mononuclear cells in diabetic patients with critical limb ischemia.

Critical limb ischemia in diabetic patients is associated with high rates of morbidity and mortality. Suboptimal responses to the available medical and surgical treatments are common in these patients, who also demonstrate limited vascular homeostasis. Neovasculogenesis induced by stem cell therapy could be a useful approach for these patients. Neovasculogenesis and clinical improvement were compared at baseline and at 3 and 12 months after autologous bone marrow-derived mononuclear cell (BMMNC) transplantation in diabetic patients with peripheral artery disease. We conducted a prospective study to evaluate the safety and efficacy of intra-arterial administration of autologous BMMNCs (100-400 × 10(6) cells) in 20 diabetic patients with severe below-the-knee arterial ischemia. Although the time course of clinical effects differed among patients, after 12 months of follow-up all patients presented a notable improvement in the Rutherford-Becker classification, the University of Texas diabetic wound scales, and the Ankle-Brachial Index in the target limb. The clinical outcome was consistent with neovasculogenesis, which was assessed at 3 months by digital subtraction angiography and quantified by MetaMorph software. Unfortunately, local cell therapy in the target limb had no beneficial effect on the high mortality rate in these patients. In diabetic patients with critical limb ischemia, intra-arterial perfusion of BMMNCs is a safe procedure that generates a significant increase in the vascular network in ischemic areas and promotes remarkable clinical improvement.