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The intestine is composed of an epithelial layer containing rapidly proliferating cells that mature into two regions, the small and the large intestine. Although previous studies have identified stem cells as the cell-of-origin for intestinal epithelial cells, no studies have directly compared stem cells derived from these anatomically distinct regions. Here, we examine intrinsic differences between primary epithelial cells isolated from human fetal small and large intestine, after in vitro expansion, using the Wnt agonist R-spondin 2. We utilized flow cytometry, fluorescence-activated cell sorting, gene expression analysis and a three-dimensional in vitro differentiation assay to characterize their stem cell properties. We identified stem cell markers that separate subpopulations of colony-forming cells in the small and large intestine and revealed important differences in differentiation, proliferation and disease pathways using gene expression analysis. Single cells from small and large intestine cultures formed organoids that reflect the distinct cellular hierarchy found in vivo and respond differently to identical exogenous cues. Our characterization identified numerous differences between small and large intestine epithelial stem cells suggesting possible connections to intestinal disease.

Exposure to high levels of ionizing radiation (IR) leads to debilitating and dose-limiting gastrointestinal (GI) toxicity. Using three-dimensional mouse crypt culture, we demonstrated that p53 target PUMA mediates radiation-induced apoptosis via a cell-intrinsic mechanism and identified the GSK-3 inhibitor CHIR99021 as a potent radioprotector. CHIR99021 treatment improved Lgr5+ cell survival and crypt regeneration after radiation in culture and mice. CHIR99021 treatment specifically blocked apoptosis and PUMA induction and K120 acetylation of p53 mediated by acetyl-transferase Tip60, while it had no effect on p53 stabilization, phosphorylation or p21 induction. CHIR99021 also protected human intestinal cultures from radiation by PUMA but not p21 suppression. These results demonstrate that p53 posttranslational modifications play a key role in the pathological and apoptotic response of the intestinal stem cells to radiation and can be targeted pharmacologically.

A patient with progressive metastatic pancreatic cancer was treated with a single infusion of 16.2×10 9 autologous T cells that had been genetically engineered to clonally express two allogeneic HLA-C*08:02–restricted T-cell receptors (TCRs) targeting mutant KRAS G12D expressed by the tumors. The patient had regression of visceral metastases (overall partial response of 72% according to the Response Evaluation Criteria in Solid Tumors, version 1.1); the response was ongoing at 6 months. The engineered T cells constituted more than 2% of all the circulating peripheral-blood T cells 6 months after the cell transfer. In this patient, TCR gene therapy targeting the KRAS G12D driver mutation mediated the objective regression of metastatic pancreatic cancer. (Funded by the Providence Portland Medical Foundation.) A 71-year-old woman with progressive pancreatic adenocarcinoma containing a mutated KRAS oncogene was given adoptive cellular therapy of her own T cells that had been altered to express two different T-cell receptors specific for her HLA type and the mutated KRAS in the tumor. Six months after treatment, the tumor burden had decreased by 72%, and the response was ongoing.

TGF-β is an immunosuppressive cytokine that is upregulated in colorectal cancer. TGF-β blockade improved response to chemoradiotherapy in preclinical models of colorectal adenocarcinoma. We aimed to test the hypothesis that adding the TGF-β type I receptor kinase inhibitor galunisertib to neoadjuvant chemoradiotherapy would improve pathological complete response rates in patients with locally advanced rectal cancer. This was an investigator-initiated, single-arm, phase 2 study done in two medical centres in Portland (OR, USA). Eligible patients had previously untreated, locally advanced, rectal adenocarcinoma, stage IIA–IIIC or IV as per the American Joint Committee on Cancer; Eastern Cooperative Oncology Group status 0–2; and were aged 18 years or older. Participants completed two 14-day courses of oral galunisertib 150 mg twice daily, before and during fluorouracil-based chemoradiotherapy (intravenous fluorouracil 225 mg/m2 over 24 h daily 7 days per week during radiotherapy or oral capecitabine 825 mg/m2 twice per day 5 days per week during radiotherapy; radiotherapy consisted of 50·4–54·0 Gy in 28–30 fractions). 5–9 weeks later, patients underwent response assessment. Patients with a complete response could opt for non-operative management and proceed to modified FOLFOX6 (intravenous leucovorin 400 mg/m2 on day 1, intravenous fluorouracil 400 mg/m2 on day 1 then 2400 mg/m2 over 46 h, and intravenous oxaliplatin 85 mg/m2 on day 1 delivered every 2 weeks for eight cycles) or CAPEOX (intravenous oxaliplatin 130 mg/m2 on day 1 and oral capecitabine 1000 mg/m2 twice daily for 14 days every 3 weeks for four cycles). Patients with less than complete response underwent surgical resection. The primary endpoint was complete response rate, which was a composite of pathological complete response in patients who proceeded to surgery, or clinical complete response maintained at 1 year after last therapy in patients with non-operative management. Safety was a coprimary endpoint. Both endpoints were assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, NCT02688712, and is active but not recruiting. Between Oct 19, 2016, and Aug 31, 2020, 38 participants were enrolled. 25 (71%) of the 35 patients who completed chemoradiotherapy proceeded to total mesorectal excision surgery, five (20%) of whom had pathological complete responses. Ten (29%) patients had non-operative management, three (30%) of whom ultimately chose to have total mesorectal excision. Two (67%) of those three patients had pathological complete responses. Of the remaining seven patients in the non-operative management group, five (71%) had clinical complete responses at 1 year after their last modified FOLFOX6 infusion. In total, 12 (32% [one-sided 95% CI ≥19%]) of 38 patients had a complete response. Common grade 3 adverse events during treatment included diarrhoea in six (16%) of 38 patients, and haematological toxicity in seven (18%) patients. Two (5%) patients had grade 4 adverse events, one related to chemoradiotherapy-induced diarrhoea and dehydration, and the other an intraoperative ischaemic event. No treatment-related deaths occurred. The addition of galunisertib to neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer improved the complete response rate to 32%, was well tolerated, and warrants further assessment in randomised trials. Eli Lilly via ExIST program, The Providence Foundation.

Alpha-tocopheryloxyacetic acid (α-TEA) is a pro-apoptotic agent that demonstrates in vivo anti-tumor activity in pre-clinical models in a T cell-dependent fashion. In IND-enabling studies, orally administered α-TEA lysine salt demonstrated a safety profile in mice and dogs providing rationale for initiating a Phase I clinical trial of α-TEA in patients with advanced cancer (NCT02192346). α-TEA lysine salt was administered to patients in gelatin capsules daily for 28 days. The main clinical objectives of the trial are to characterize α-TEA related toxicity, and determine the maximum tolerated dose, and pharmacokinetics of a-TEA in humans. Secondary objectives are to characterize the phenotype of T-cell subsets in the peripheral blood by flow cytometry. The diagnoses of patients in the ongoing trial include renal cancer, esophageal cancer, thyroid cancer, duodenal cancer, squamous cell oropharyngeal cancer and advanced squamous cell carcinoma of the head and neck. Six patients have been treated at the 2.4mg/kg dose level, and five patients at the 4.8mg/kg dose level. Two patients had atrial fibrillation possibly associated with α-TEA at the 4.8 mg/kg dose level. Grade 2/3 toxicities (24 hour diarrhea) were observed in Subject 1 that in retrospect was not α-TEA related. Subject 3 had grade 4 elevations of hepatocellular enzymes on day 28 from disease progression and grade 3 transient lymphopenia, possibly related to α-TEA. Three patients have stable disease, one lasting 5 months and another lasting 2+ months, which is ongoing. Of interest is that the two patients with stable disease demonstrated an expansion of the CD8+ T cell subset and another patient demonstrated increased CD8 effector memory and substantial increase in expression of the activation markers, CD38 and HLA-DR at day 8 post-treatment. The trial is still accruing patients and the pharmacokinetics, pharmacodynamics, and identification and characterization of α-TEA metabolites in human plasma are ongoing.Trial registrationClinicalTrials.gov identifier NCT02192346.

Parent involvement in public education has changed over time in the United States. Recently it has taken on a more radical dimension aimed at shifting the role of parents. These efforts are identified by some as parent empowerment and arguably may be part of a larger policy movement to secure parent voice in equity-focused education reform. The policy innovation allows parents with students in persistently underperforming schools to force a change in school governance. Since the passage of the first parent trigger law in California in 2010, three parent petition campaigns have forced a turnaround in school governance. The purpose of this study was to provide a baseline of understanding for the ways in which parent trigger legislation intersected educational policy and to investigate the extent to which the law supported the needs, values, and interests of local parent stakeholders. This qualitative study consisted of three ways in which to examine the legislative influence on parent empowerment: 1) a state-level document analysis of proposed and enacted parent trigger legislation; 2) interviews with the legislator and the education reform advocate responsible for authoring the first parent trigger law; and, 3) eleven interviews with key stakeholders involved in the first two successful efforts to use the parent trigger at Desert Trails Elementary in Adelanto, California and 24th Street Elementary in Los Angeles, California. A cross case comparison of the two school sites revealed that the needs and core beliefs of parent leaders aligned with the intent of the parent trigger law. However, an intermediary organization was required to help the parent stakeholders attain the resources, socio-political learning, and community building strategies necessary to effectively exercise their parental legal right. Moreover, factors within the local context affected the parent leaders' implementation of the law. Levels of relational trust either mitigated or exacerbated the process. Finally, the use of the law was experienced by parent leaders as both personally and collectively empowering in shifting their role to decision maker. This study has implications for researchers, policy makers, and practitioners considering parent trigger legislation and parent empowerment as a solution for failing schools.

The epithelial layers of the small intestine (SI) and large intestine (LI) are generated via self-renewal and differentiation of tissue-specific stem cells (SCs). Failure of intestinal SCs to respond properly to proliferation and differentiation signals can lead to the formation of cancer, almost exclusively found in the LI in humans. We hypothesize that there are distinct resident SCs in the SI versus LI leading to an increased frequency of human LI (colon) cancer. While fleeting observations of differences between SI and LI have been made in the past, a robust study of the origin of their differences has yet to be done. Here we show dramatic intrinsic differences between normal human SI and LI SCs that may have important implications for the disparity in their susceptibilities to cancer. Primary human fetal SI and LI cells were isolated and expanded in vitro using conditions that selected for SCs. Flow cytometry and limiting dilution analysis indicated differential SC marker expression as well as disparate populations of colony-forming cells. Gene array analysis showed separate hierarchical clustering and differential expression of transcripts involved in differentiation, proliferation and disease pathways of the SI and LI. Using a three-dimensional in vitro differentiation assay, SI and LI SCs formed organoids with architecture and cellular hierarchy similar to that found in vivo. Immunostaining and real-time PCR indicated that both SI and LI SCs retain the ability to differentiate into mature cells of the intestine. We also found that well-known proliferation and differentiation pathways in SI- and LI-derived organoids responded differently when exposed to the same exogenous stimuli. Notably, upon exposure to differentiation cues, an expected decrease in SC marker LGR5 in SI SCs was met with an unexpected increase in LI SCs. We also demonstrate similarities between human LI SCs and colon cancer SCs not present in SI SCs, supporting the notion that normal intestinal SCs are the cell of origin of cancer. Our characterization of human fetal SI and LI SCs revealed critical intrinsic differences that may affect their susceptibility to diseases such as cancer. Further elucidation of pathway differences may allow the exploitation of protective mechanisms to prevent or treat human colon cancer.

BackgroundSince it is the site of initial immune activation and priming of antigen-specific T cells, as well as the first location to which tumor cells metastasize, our research focuses on understanding the immune status and tumor-induced suppression within breast cancer (BrCa)-draining sentinel lymph nodes (SLN).MethodsSuppressive immune subsets were profiled by multi-color flow cytometry in two different BrCa SLN cohorts. In the first cohort, collected at the Providence Cancer Center (08/11-07/13), we looked at frequencies of HLA-DR- CD14+ myeloid cells and their expression of co-stimulatory and inhibitory receptors. Four metastasis+ SLN and 11 metastasis- SLN were assessed in this cohort. SLN in the second cohort were collected at the VUmc in Amsterdam and Kennemer Gasthuis in Haarlem (10/13-06/14) and contained 2 metastasis+ and 7 metastasis- SLN. In this cohort the frequencies of HLA-DR- CD14+ cells as well as CD25_hi FoxP3+ T regulatory cells (Treg) were analyzed. Since all tumors corresponding to the metastasis+ SLN turned out to be HER2 negative, only metastasis- SLN from HER2- tumors were included. Apart from 2 tumors in the Providence cohort, all tumors did express progesterone and/or estrogen receptors.ResultsElevated frequencies of HLA-DR- CD14+ immature myeloid cells could be detected in the metastasis+ SLN in both cohorts. This difference was statistically significant for the Providence cohort (p < 0.0001) (3.8 fold increase). No differences were observed for expression levels of the co-stimulatory molecules CD80 and CD86 or the inhibitory molecules PD-L1 and B7H4 on HLA-DR- CD14+ cells between positive or negative SLN and expression was low for all these markers. Due to the small number of metastasis+ SLN in the Dutch cohort statistics could not yet be performed, but a 2.3 fold increase in HLA-DR- CD14+ cells was seen. In this cohort, frequencies of Treg were found to be 4-fold higher in the metastasis+ SLN compared to metastasis- SLN (0.31 ± 0.22 vs. 1.22 ± 0.24 Treg of CD4 T cells). Moreover, a significant correlation was observed between the frequencies of HLA-DR- CD14+ immature myeloid cells and the frequencies of Treg in these BrCa SLN (r = 0.718, p < 0.01).ConclusionOur data suggest that tumor-derived factors negatively influence both the myeloid and the lymphoid compartments within SLN draining HER2 negative breast cancers.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

In 1880 neurologist George Miller Beard identified the diagnosis of neurasthenia. Popularly referred to as Americanitis, Beard treated an increasing number of people for symptoms of anxiety, malaise and gastric discomfort. Per Beard, the illness resulted from the rapidly changing mechanical landscape of modernity. Similarly, contemporary Media Ecology literature suggests that Americanitis continues amidst our current digital moment. Manifest as narcissistically anxious nervous exhaustion, digital Americanitis results due to technological encouragement of existential and communicative closure, thereby negatively implicating the human condition, human communication and communication ethics practices. As such, this project considers Marshall McLuhan’s Media Ecology to examine the communicative phenomena of Americanitis. Based on affable grounding assumptions as well as calls in recent literature, McLuhan’s work is read through the presently underrepresented Media Ecology scholarly approach of existential phenomenology. In particular, this Merleau-Pontean existential phenomenological reading enhances understanding of the implicit theory of human communication and communication ethics informing McLuhan’s Media Ecology criticism. Once elucidated, McLuhan’s theoretical assumptions, aims and ends comport with theoretical dimensions of Merleau-Ponty’s existential phenomenology to reveal how and why technology encourages our digital Americanitis . When placed into conversation, the two thinkers also offer possible responses to our ills—approaches to “taming” Americanitis.

The ability of S. polygaloides to accumulate nickel (Ni) was tested when grown in nutrient solution and serpentine and calcareous soil. Additionally, studies were conducted to determine the effect of nitrogen (N) form on Ni uptake (ammonium, NH$\\sb4\\sp+$ vs. nitrate, NO$\\sb3\\sp-$) and to characterize the Ni chemistry of the serpentine soil. S. polygaloides was exposed to chronic Ni concentrations of 0, 0.25, 0.50, and 1.0 mM Ni when grown in the nutrient solution. Plants grown in solutions containing 0.25 mM Ni had the greatest biomass (4.72 g), removed 40.03% of the Ni added to the nutrient solution into its shoots, and accumulated 8,420 mg kg$\\sp{-1}$ Ni in shoots (dry weight basis). S. polygaloides grown in solutions with Ni concentrations exceeding 0.25 mM had lower dry weights and reduced transpiration. Even though stressed, plants in the 0.50 mM treatment accumulated a maximum Ni concentration of 13,020 mg kg$\\sp{-1}.$ There was no statistical difference for dry weight or Ni uptake between N treatments for S. polygaloides on either soil. S. polygaloides accumulated the greatest biomass (10.68 g) and concentrated the most Ni (7,460 mg kg$\\sp{-1}$) when grown on the Millville soil mixed with 3,000 mg kg$\\sp{-1}$ Ni. Each plant grown on the Millville soil mixed with Ni removed 74 mg Ni versus 18 mg Ni when grown on the serpentine soil.