Crypt fusion as a homeostatic mechanism in the human colon.

OBJECTIVE: The crypt population in the human intestine is dynamic: crypts can divide to produce two new daughter crypts through a process termed crypt fission, but whether this is balanced by a second process to remove crypts, as recently shown in mouse models, is uncertain. We examined whether crypt fusion (the process of two neighbouring crypts fusing into a single daughter crypt) occurs in the human colon. DESIGN: We used somatic alterations in the gene cytochrome c oxidase (CCO) as lineage tracing markers to assess the clonality of bifurcating colon crypts (n=309 bifurcating crypts from 13 patients). Mathematical modelling was used to determine whether the existence of crypt fusion can explain the experimental data, and how the process of fusion influences the rate of crypt fission. RESULTS: In 55% (21/38) of bifurcating crypts in which clonality could be assessed, we observed perfect segregation of clonal lineages to the respective crypt arms. Mathematical modelling showed that this frequency of perfect segregation could not be explained by fission alone (p<10-20). With the rates of fission and fusion taken to be approximately equal, we then used the distribution of CCO-deficient patch size to estimate the rate of crypt fission, finding a value of around 0.011 divisions/crypt/year. CONCLUSIONS: We have provided the evidence that human colonic crypts undergo fusion, a potential homeostatic process to regulate total crypt number. The existence of crypt fusion in the human colon adds a new facet to our understanding of the highly dynamic and plastic phenotype of the colonic epithelium.wellcome trust royal societ

Clonal transitions and phenotypic evolution in Barrett esophagus

BACKGROUND & AIMS: Barrett's esophagus (BE) is a risk factor for esophageal adenocarcinoma but our understanding of how it evolves is poorly understood. We investigated BE gland phenotype distribution, the clonal nature of phenotypic change, and how phenotypic diversity plays a role in progression. METHODS: Using immunohistochemistry and histology, we analyzed the distribution and the diversity of gland phenotype between and within biopsy specimens from patients with nondysplastic BE and those who had progressed to dysplasia or had developed postesophagectomy BE. Clonal relationships were determined by the presence of shared mutations between distinct gland types using laser capture microdissection sequencing of the mitochondrial genome. RESULTS: We identified 5 different gland phenotypes in a cohort of 51 nondysplastic patients where biopsy specimens were taken at the same anatomic site (1.0-2.0 cm superior to the gastroesophageal junction. Here, we observed the same number of glands with 1 and 2 phenotypes, but 3 phenotypes were rare. We showed a common ancestor between parietal cell-containing, mature gastric (oxyntocardiac) and goblet cell-containing, intestinal (specialized) gland phenotypes. Similarly, we have shown a clonal relationship between cardiac-type glands and specialized and mature intestinal glands. Using the Shannon diversity index as a marker of gland diversity, we observed significantly increased phenotypic diversity in patients with BE adjacent to dysplasia and predysplasia compared to nondysplastic BE and postesophagectomy BE, suggesting that diversity develops over time. CONCLUSIONS: We showed that the range of BE phenotypes represents an evolutionary process and that changes in gland diversity may play a role in progression. Furthermore, we showed a common ancestry between gastric and intestinal-type glands in BE

TP53-binding protein variants and breast cancer risk: a case-control study

INTRODUCTION: The TP53-binding protein (53BP1) has been shown to influence TP53-mediated transcriptional activation, thus playing a pivotal role in DNA damage signalling. Genetic aberrations in TP53 and in ATM and CHEK2 predispose to cancer. We have therefore examined the effects of 53BP1 single nucleotide polymorphisms (D353E, G412S, and K1136Q) and the novel 53BP1 6bp deletion (1347_1352delTATCCC) on breast cancer risk. METHODS: Allelic discrimination was performed to investigate the frequencies of 53BP1 D353E, G412S, and K1136Q and of 1347_1352delTATCCC in 353 patients with breast cancer and 960 control individuals. RESULTS: No significant association of 53BP1 D353E, G412S, or K1136Q with breast cancer risk was detected. 53BP1 1347_1352delTATCCC, leading to the loss of an isoleucine and a proline residue, showed a nonsignificant inverse association with breast cancer risk (odds ratio = 0.61, 95% confidence interval = 0.22 to 1.68, P = 0.34). CONCLUSION: The lack of association casts doubt on the putative effects of D353E, G412S, and K1136Q on breast cancer risk. Investigating a larger study cohort might elucidate the influence of the 6bp deletion 1347_1352delTATCCC. Studying the functional effect and the impact of this variant on the risk of other cancers may be revealing

Chemotherapy with BCNU in recurrent glioma: Analysis of clinical outcome and side effects in chemotherapy-naïve patients

Background: To date, standardized strategies for the treatment of recurrent glioma are lacking. Chemotherapy with the alkylating agent BCNU (1,3-bis (2-chloroethyl)-1-nitroso-urea) is a therapeutic option even though its efficacy and safety, particularly the risk of pulmonary fibrosis, remains controversial. To address these issues, we performed a retrospective analysis on clinical outcome and side effects of BCNU-based chemotherapy in recurrent glioma. Methods: Survival data of 34 mostly chemotherapy-naïve glioblastoma patients treated with BCNU at 1st relapse were compared to 29 untreated control patients, employing a multiple Cox regression model which considered known prognostic factors including MGMT promoter hypermethylation. Additionally, medical records of 163 patients treated with BCNU for recurrent glioma WHO grade II to IV were retrospectively evaluated for BCNU-related side effects classified according to the National Cancer Institute Common Toxicity Criteria for Adverse Events (CTCAE) version 2.0. Results: In recurrent glioblastoma, multiple regression survival analysis revealed a significant benefit of BCNU-based chemotherapy on survival after relapse (p = 0.02; HR = 0.48; 95 % CI = 0.26–0.89) independent of known clinical and molecular prognostic factors. Exploratory analyses suggested that survival benefit was most pronounced in MGMT-hypermethylated, BCNU-treated patients. Moreover, BCNU was well tolerated by 46 % of the 163 patients analyzed for side effects; otherwise, predominantly mild side effects occurred (CTCAE I/II; 45 %). Severe side effects CTCAE III/IV were observed in 9 % of patients including severe hematotoxicity, thromboembolism, intracranial hemorrhage and injection site reaction requiring surgical intervention. One patient presented with a clinically apparent pulmonary fibrosis CTCAE IV requiring temporary mechanical ventilation. Conclusion: In this study, BCNU was rarely associated with severe side effects, particularly pulmonary toxicity, and, in case of recurrent glioblastoma, even conferred a favorable outcome. Therefore BCNU appears to be an appropriate alternative to other nitrosoureas although the efficacy against newer drugs needs further evaluation

Osteoprotegerin in Exosome-Like Vesicles from Human Cultured Tubular Cells and Urine

Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and receptors may be present in exosomes and are expressed by proximal tubular cells. We have now studied the expression of selected TNF superfamily proteins in exosome-like vesicles from cultured human proximal tubular cells and human urine and have identified additional proteins in these vesicles by LC-MS/MS proteomics. Human proximal tubular cells constitutively released exosome-like vesicles that did not contain the TNF superfamily cytokines TRAIL or TWEAK. However, exosome-like vesicles contained osteoprotegerin (OPG), a TNF receptor superfamily protein, as assessed by Western blot, ELISA or selected reaction monitoring by nLC-(QQQ)MS/MS. Twenty-one additional proteins were identified in tubular cell exosomelike vesicles, including one (vitamin D binding protein) that had not been previously reported in exosome-like vesicles. Twelve were extracellular matrix proteins, including the basement membrane proteins type IV collagen, nidogen-1, agrin and fibulin-1. Urine from chronic kidney disease patients contained a higher amount of exosomal protein and exosomal OPG than urine from healthy volunteers. Specifically OPG was increased in autosomal dominant polycystic kidney disease urinary exosome-like vesicles and expressed by cystic epithelium in vivo. In conclusion, OPG is present in exosome-like vesicles secreted by proximal tubular epithelial cells and isolated from Chronic Kidney Disease urine.This work was supported by grants from the Instituto de Salud Carlos III (ISCIIIRETIC REDINREN RD06/0016, RD12/0021, PI11/01854, PI10/00072 PI09/ 00641 and PS09/00447); Comunidad de Madrid (Fibroteam S2010/BMD-2321, S2010/BMD-2378); Sociedad Española de NefrologÍa; European Network (HEALTH F2-2008-200647); DIALOK European project LSHB-CT-2007-036644; Fundacion Lilly and IRSIN/FRIAT to JE; Programa Intensificación Actividad Investigadora (ISCIII/ Agencia Laín-Entralgo/CM) to AO; Instituto de Salud Carlos III (FIS PI11/01401, CP09/00229); and Fundación Conchita Rábago de Jiménez DÍaz to GAL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip