PURIFICATION, CHARACTERIZATION AND CULTURE OF ENSHEATHING CELLS FROM HUMAN OLFACTORY MUCOSA BIOPSIES

Among all the possible sources of mesenchymal stem cells, adipose tissue and olfactory mucosa have raised great interest and have become some of the most investigated sources. Adipose tissue-derived mesenchymal stem cells and the fat itself as a source of human adipose derived stem cells, represent one of the major fields of research in regenerative medicine. A great advantage is represented by the minimal invasive and high accessibility to adipose tissue and its ready availability. In the present study, hADSCs were isolated from the adipose tissue donated by several patient and have been investigated and characterized through different technical approaches, such as flow cytometry and immunocytochemistry. These hADSCs reproducibly fulfill the general definition of MSCs by both phenotypic and differentiation capabilities criteria, showing also the expression of neural markers, as observed by confocal microscope analysis. Lipoaspirated adipose tissue showed positivity to f-tubulin III that was also maintained in lipoaspirate-derived hADSCs. A population of stem cells retaining typical characteristics of surface markers of classical adipose tissue stem cells and MSC was obtained when adipose tissue was subjected to culture in vitro, either by processing through centrifugation or by direct plating without enzymatic digestion with collagenase. Flow cytometry analyses showed that hADSCs expressed classical mesenchymal markers such as CD44, CD73, CD90, CD105 and CD166, while endothelial (CD31, CD34, CD144, CD146) and hematopoietic (CD45, CD133) markers were much less represented. Also the ability to give rise to tissue of mesenchymal origins, such as osteoblastic and adipogenic lineages, were present in hADSCs. In addition, the immunofluorescence staining indicated the expression of neural stem markers in hADSCs which consequently co-expressed nestin, \u3b2-tubulin III and glial GFAP. We have also characterized human olfactory ensheathing stem cells. Olfactory mucosa is specialized tissue inside the nasal cavity involved in olfactory perception and capable of lifelong regeneration throughout adulthood. Multipotent stem cells obtained from it offer the possibility of promoting regeneration and reconstruction in regenerative medicine, being readily accessible with minimal invasive techniques, capable of expansion in vitro and retaining broadly potent differentiative capacity as stem cell progenitors. Among the several members of the olfactory mucosa, Olfactory Ensheathing Cells (OECs) are well known to be useful in repairing the nervous system. By following our method, cells can be easily isolated and maintained in TCM, and their cultivation in large flasks allowed obtaining rich cultures of OECs in 2 weeks. Cell cycle analysis showed that the majority of cells are in G0/G1 phase, while just a lesser part is in S/G2 phase. In our growth conditions, no chromosomal abnormalities were observed also at high culture passage (p14). Live morphology of obtained cells showed a fibroblast-like phenotype and the immunohistochemical analyses showed the expression of beta-Tubulin III, Vimentin, Nestin, Glial Fibrillary Acidic Protein and Microtubule-Associated Protein 2. By FACS analysis we demonstrated that OECs are positive to typical surface mesenchymal markers (CD44, CD73, CD90, CD105, CD146 and CD166). As expected, some endothelial (CD31, CD34) and hematopoietic (CD45) markers were very few represented, while some others (CD56, CD144, CD146, CD133) are partially found. These cells also express genes that constitute the core circuitry of self-renewal such as SOX2, NANOG and OCT4 and the stemness marker CD133. OECs incubated with serum-free medium, normally used for the formation of neurospheres, spontaneously formed large spheroids reaching a mean diameter of 100 \u3bcm in 10 days of culture. Immunofluorescence of specific proteins showed that spheres were positive to markers such as Nestin, Vimentin, TUJ-1, MAP2 and GFAP. In conclusion, our method allows the quickly and easily hADSCs and hOESCs isolation from human adipose tissue and nasal biopsies. The obtained cells can be cultured without altering their mesenchymal properties, suggesting the pluripotency nature of these cells and that they are a reliable source for regenerative medicine

Tumor biopsy and patient enrollment in clinical trials for advanced hepatocellular carcinoma

Tumor biopsies may help to reliably distinguish hepatocellular carcinoma (HCC) from other tumors, mostly cholangiocarcinoma as well as to identify the patient populations who most benefit from target-driven HCC treatments, in order to improve the success rate of experimental therapies. Clarifying tumor biology may also lead to identify biomarkers with prognostic role and/or enabling to predict response or resistance to therapies. Recently, clinical trials have more efficiently included biomarker endpoints and increasingly collected tumor tissue from enrolled patients. Due to their frail status and sometimes fast-progressing disease, the performance status of patients with HCC progressing on first-line therapy can deteriorate quickly, preventing their enrollment in clinical trials. However, the challenge of identifying the proper patient at the proper time can be overcome by periodic inter-department meetings involving the key specialists taking care of HCC patients, and solid networks between research centers and referring institutions. An early planned biopsy would also facilitate timely inclusion of patients in biology-driven clinical trials. Ultimately, institution of multidisciplinary teams can optimize treatment choice, biopsy timing, and quick enrollment of patients in clinical trials, before their performance status deteriorates

EFFECT: a randomized phase II study of efficacy and impact on function of two doses of nab-paclitaxel as first-line treatment in older women with advanced breast cancer

Background: Limited data are available regarding the use of nab-paclitaxel in older patients with breast cancer. A weekly schedule is recommended, but there is a paucity of evidence regarding the optimal dose. We evaluated the efficacy of two different doses of weekly nab-paclitaxel, with a specific focus on their corresponding impact on patient function, in order to address the lack of data specifically relating to the older population. Methods: EFFECT is an open-label, phase II trial wherein 160 women with advanced breast cancer aged 65 65 years were enrolled from 15 institutions within Italy. Patients were randomly assigned 1:1 to receive nab-paclitaxel 100 mg/m2 (arm A) or 125 mg/m2 (arm B) on days 1, 8, and 15 on a 28-day cycle, as first-line treatment for advanced disease. The primary endpoint was event-free survival (EFS), wherein an event was defined as disease progression (PD), functional decline (FD), or death. In each arm, the null hypothesis that the median EFS would be 64 7 months was tested against a one-sided alternative according to the Brookmeyer Crowley test. Secondary endpoints included objective response rate (ORR), clinical benefit rate (CBR), progression-free survival (PFS), overall survival (OS), and safety. Results: After a median follow-up of 32.6 months, 140 events were observed in 158 evaluable patients. Median EFS was 8.2 months (90% CI, 5.9-8.9; p = 0.188) in arm A vs 8.3 months (90% CI, 6.2-9.7, p = 0.078) in arm B. Progression-free survival, overall survival, and response rates were similar in both groups. A higher percentage of dose reductions and discontinuations due to adverse events (AEs) was noted in arm B. The most frequently reported non-haematological AEs were fatigue (grade [G] 2-3 toxicity occurrence in arm A vs B, 43% and 51%, respectively) and peripheral neuropathy (G2-3 arm A vs B, 19% and 38%, respectively). Conclusion: Pre-specified outcomes were similar in both treatment arms. However, 100 mg/m2 was significantly better tolerated with fewer neurotoxicity-related events, representing a more feasible dose to be recommended for older patients with advanced disease. Trial registration: EudraCT, 2012-002707-18. Registered on June 4, 2012. NIH ClinicalTrials.gov, NCT02783222. Retrospectively registered on May 26, 2016

The taste of D- and L-amino acids: In vitro binding assays with cloned human bitter (TAS2Rs) and sweet (TAS1R2/TAS1R3) receptors

The taste of different enantiomeric forms of amino acids has been deeply investigated because it represents the most impressive case of correlation between stereochemistry and flavour. Herein, we aimed to elucidate the molecular activity of d- and l-amino acids using an in vitro system based on a cellular model overexpressing sweet and bitter receptors, and to analyse the correlation between in vitro and sensory studies. With our work we demonstrated specific enantiomeric activities for several amino acids on TAS1R2-TAS1R3 sweet receptor. Moreover, we proved interaction of tryptophan and phenylalanine with a specific group of TAS2Rs bitter receptors, confirming and improving the results recently obtained in the tasting of amino acids. In addition, we provide the first systematic analysis of l- and d-amino acid actions on the sweet heterodimeric receptor

Microbiota-Dependent Upregulation of Bitter Taste Receptor Subtypes in the Mouse Large Intestine in High-Fat Diet-Induced Obesity.

Bitter taste receptors (Tas2rs in mice) detect bitterness, a warning signal for toxins and poisons, and are expressed in enteroendocrine cells. We tested the hypothesis that Tas2r138 and Tas2r116 mRNAs are modulated by microbiota alterations induced by a long-term high-fat diet (HFD) and antibiotics (ABX) (ampicillin and neomycin) administered in drinking water. Cecum and colon specimens and luminal contents were collected from C57BL/6 female and male mice for qRT-PCR and microbial luminal 16S sequencing. HFD with/without ABX significantly increased body weight and fat mass at 4, 6, and 8 weeks. Tas2r138 and Tas2r116 mRNAs were significantly increased in mice fed HFD for 8 weeks vs. normal diet, and this increase was prevented by ABX. There was a distinct microbiota separation in each experimental group and significant changes in the composition and diversity of microbiome in mice fed a HFD with/without ABX. Tas2r mRNA expression in HFD was associated with several genera, particularly with Akkermansia, a Gram-negative mucus-resident bacterium. These studies indicate that luminal bacterial composition is affected by sex, diet, and ABX and support a microbial dependent upregulation of Tas2rs in HFD-induced obesity, suggesting an adaptive host response to specific diet-induced dysbiosis

Fresh and frozen Lipogems-derived micro-fractured human adipose tissue generates mesenchymal stem cells with higher differentiation potential and in vivo repair efficacy

The differentiation potential and the ease of their isolation have made the multipotent mesenchymal stem cells very important for the development of a vast range of clinical applications in regenerative medicine. Here we report our findings on human adipose tissue-derived stem cells (hADSCs) obtained through the Lipogems device from micro-fractured lipoaspirate. The use of such a device allows the successful establishment of hADSCs colonies even without liberase H1 treatment. Cells maintained in mesenchymal classic medium are large and flat with short outgrowing processes that occasionally grow for several mm. These cells can also be obtained from lipogems preparation after preservation at 4\ub0C for up to 72 hours and , even, cryopreservation at -80\ub0C for over 1 month. Differently it is almost impossible to obtain hADSCs from cryopreserved lipoaspirate. The cell cycle analysis showed that 75% of cells are in G0/G1phase and 21% in S+G2/M, and only a marginal 0.2% apoptosis. No Chromosomal abnormalities. These hADSCs from either fresh or frozen lipogems preparations are bearer of typical mesenchymal markers at values above 90%, and express embryonic markers such as NANOG and OCT4 and neural markers such as nestin, neurod1, pax 6 and musashi. The superficial epitopes are maintained even when hADSCs were grown in culture after storage at -80\ub0C. Their driven osteogenic and adipogenic differentiation in vitro yields hADSCs with finer intracellular micro-organelles and fat deposits are more numerous and smaller in size. We shall also present how these lipogems-derived hADSCs can transdifferentiate in vivo in specific lineages determined by the site of transplantation and by the condition

Distribution, quantification, and characterization of substance P enteric neurons in the submucosal and myenteric plexuses of the porcine colon.

The pig is an important translational model for studying intestinal physiology and disorders for its many homologies with humans, including the organization of the enteric nervous system (ENS), the major regulator of gastrointestinal functions. This study focused on the quantification and neurochemical characterization of substance P (SP) neurons in the pig ascending (AC) and descending colon (DC) in wholemount preparations of the inner submucosal plexus (ISP), outer submucosal plexus (OSP), and myenteric plexus (MP). We used antibodies for the pan-neuronal marker HuCD, and choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS), markers for excitatory and inhibitory transmitters, for multiple labeling immunofluorescence and high-resolution confocal microscopy. The highest density of SP immunoreactive (IR) neurons was in the ISP (222/mm2 in the AC, 166/mm2 in the DC), where they make up about a third of HuCD-IR neurons, compared to the OSP and MP (19-22% and 13-17%, respectively, P < 0.001-0.0001). HuCD/SP/ChAT-IR neurons (up to 23%) were overall more abundant than HuCD/SP/nNOS-IR neurons (< 10%). Most SP-IR neurons contained ChAT-IR (62-85%), whereas 18-38% contained nNOS-IR with the highest peak in the OSP. A subpopulation of SP-IR neurons contains both ChAT- and nNOS-IR with the highest peak in the OSP and ISP of DC (33-36%) and the lowest in the ISP of AC (< 10%, P < 0.001). SP-IR varicose fibers were abundant in the ganglia. This study shows that SP-IR neurons are functionally distinct with variable proportions in different plexuses in the AC and DC reflecting diverse functions of specific colonic regions

Distribution, quantification, and characterization of substance P enteric neurons in the submucosal and myenteric plexuses of the porcine colon

The pig is an important translational model for studying intestinal physiology and disorders for its many homologies with humans, including the organization of the enteric nervous system (ENS), the major regulator of gastrointestinal functions. This study focused on the quantification and neurochemical characterization of substance P (SP) neurons in the pig ascending (AC) and descending colon (DC) in wholemount preparations of the inner submucosal plexus (ISP), outer submucosal plexus (OSP), and myenteric plexus (MP). We used antibodies for the pan-neuronal marker HuCD, and choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS), markers for excitatory and inhibitory transmitters, for multiple labeling immunofluorescence and high-resolution confocal microscopy. The highest density of SP immunoreactive (IR) neurons was in the ISP (222/mm 2 in the AC, 166/mm 2 in the DC), where they make up about a third of HuCD-IR neurons, compared to the OSP and MP (19–22% and 13–17%, respectively, P < 0.001–0.0001). HuCD/SP/ChAT-IR neurons (up to 23%) were overall more abundant than HuCD/SP/nNOS-IR neurons (< 10%). Most SP-IR neurons contained ChAT-IR (62–85%), whereas 18–38% contained nNOS-IR with the highest peak in the OSP. A subpopulation of SP-IR neurons contains both ChAT- and nNOS-IR with the highest peak in the OSP and ISP of DC (33–36%) and the lowest in the ISP of AC (< 10%, P < 0.001). SP-IR varicose fibers were abundant in the ganglia. This study shows that SP-IR neurons are functionally distinct with variable proportions in different plexuses in the AC and DC reflecting diverse functions of specific colonic regions