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Bartonella species are emerging human pathogens. Bats are known to carry diverse Bartonella species, some of which are capable of infecting humans. However, as the second largest mammalian group by a number of species, the role of bats as the reservoirs of Bartonella species is not fully explored, in term of their species diversity and worldwide distribution. China, especially Northern China, harbors a number of endemic insectivorous bat species; however, to our knowledge, there are not yet studies about Bartonella in bats in China. The aim of the study was to investigate the prevalence and genetic diversity of Bartonella species in bats in Northern China. Bartonella species were detected by PCR amplification of gltA gene in 25.2% (27/107) bats in Mengyin County, Shandong Province of China, including 1/3 Rhinolophus ferrumequinum, 2/10 Rhinolophus pusillus, 9/16 Myotis fimbriatus, 1/5 Myotis ricketti, 14/58 Myotis pequinius. Phylogenetic analysis showed that Bartonella species detected in bats in this study clustered into ten groups, and some might be novel Bartonella species. An association between Bartonella species and bat species was demonstrated and co-infection with different Bartonella species in a single bat was also observed. Our findings expanded our knowledge on the genetic diversity of Bartonella in bats, and shed light on the ecology of bat-borne Bartonella species.

BACKGROUND & AIMS:Intestinal microfold (M) cells are specialized epithelial cells that act as gatekeepers of luminal antigens in the intestinal tract. They play a critical role in the intestinal mucosal immune response through transport of viruses, bacteria and other particles and antigens across the epithelium to immune cells within Peyer's patch regions and other mucosal sites. Recent studies in mice have demonstrated that M cells are generated from Lgr5+ intestinal stem cells (ISCs), and that infection with Salmonella enterica serovar Typhimurium increases M cell formation. However, it is not known whether and how these findings apply to primary human small intestinal epithelium propagated in an in vitro setting. METHODS:Human intestinal crypts were grown as monolayers with growth factors and treated with recombinant RANKL, and assessed for mRNA transcripts, immunofluorescence and uptake of microparticles and S. Typhimurium. RESULTS:Functional M cells were generated by short-term culture of freshly isolated human intestinal crypts in a dose- and time-dependent fashion. RANKL stimulation of the monolayer cultures caused dramatic induction of the M cell-specific markers, SPIB, and Glycoprotein-2 (GP2) in a process primed by canonical WNT signaling. Confocal microscopy demonstrated a pseudopod phenotype of GP2-positive M cells that preferentially take up microparticles. Furthermore, infection of the M cell-enriched cultures with the M cell-tropic enteric pathogen, S. Typhimurium, led to preferential association of the bacteria with M cells, particularly at lower inoculum sizes. Larger inocula caused rapid induction of M cells. CONCLUSIONS:Human intestinal crypts containing ISCs can be cultured and differentiate into an epithelial layer with functional M cells with characteristic morphological and functional properties. This study is the first to demonstrate that M cells can be induced to form from primary human intestinal epithelium, and that S. Typhimurium preferentially infect these cells in an in vitro setting. We anticipate that this model can be used to generate large numbers of M cells for further functional studies of these key cells of intestinal immune induction and their impact on controlling enteric pathogens and the intestinal microbiome.

The myofibroblast is an important stromal cell of the gastrointestinal tract. Current in vitro and in vivo models either do not accurately recreate stromal-epithelial interactions or are not specific to myofibroblasts. We sought to create an animal model that would allow the study of myofibroblast-epithelial interactions. We isolated and cultured colonic myofibroblasts from FVB mice. Cells were α-SMA and vimentin positive but desmin negative on immunoblot analysis. We injected the myofibroblasts into the colonic submucosa of syngeneic adult mice (n = 8) via a miniendoscopic system. We then isolated green fluorescent protein (GFP) positive colonic myofibroblasts from C57BL/6-Tg(CAG-EGFP)1Osb/J mice and injected them into the colonic lamina propria of C57BL/6J mice at 1x10(5) (n = 14), 1x10(6) (n = 9), or 5x10(6) cells/mL (n = 4). A subset of mice were injected with serum-free media and ink without cells (n = 3). Mice underwent repeat endoscopy and euthanasia one or 7 days after injection. Colons were isolated and either fixed in 10% formalin or the inked sites were individually excised and lysed for DNA. We assessed the injection sites via histology and immunohistochemical stains for α-SMA and GFP. We used qPCR to quantify GFP DNA transcripts at the lamina propria injection sites. Submucosal injection of myofibroblasts resulted in the formation of a subepithelial wheal on endoscopy, which persisted to day 7. Myofibroblasts injected either into the submucosa or lamina propria maintained viability on post-injection day 7 as evidenced by positive α-SMA staining. qPCR of lamina propria injections showed a dose-dependent increase in GFP DNA transcripts on post-injection day 1, whereas the number of transcripts on day 7 was equivalent for the concentrations injected. We demonstrate short-term survival of primary cultured colonic myofibroblasts in syngeneic mice. This may prove to be a valuable model for studying the role of myofibroblasts in states of health and disease.

The microRNA miR-101 is downregulated in several cancers, including bladder cancer. However, miR-101's role in the invasion, metastasis, and chemosensitivity of bladder cancer cells remains unclear. This study was conducted to determine miR-101's role on the lymphangiogenic molecule vascular endothelial growth factor C (VEGF-C) and their effects upon bladder cancer cell migration, invasion, and chemosensitivity to cisplatin.Two bladder cancer cell lines (T24 and 5637) and the tool cell line 293T were employed here. Bladder cancer cells were transfected with either a miR-101 overexpression vector or a scrambled-sequence lentivirus, both of which exhibited a high transfection efficiency. Non-transfection was used as a mock negative control. Wound healing and Transwell assays were performed to measure cell migration and invasiveness. A luciferase reporter assay was performed to validate miR-101's interaction with VEGF-C's 3' untranslated region followed by RT-PCR and Western blot confirmation. An MTS assay was used to evaluate the cisplatin sensitivity of the cell lines.miR-101 overexpression significantly inhibited the migration and invasiveness while significantly enhancing cisplatin sensitivity. miR-101 negatively regulated VEGF-C protein expression, and VEGF-C overexpression rescued the effects of miR-101 overexpression, indicating that miR-101 negatively regulates VEGF-C protein expression post-transcriptionally. miR-101 and VEGF-C interference independently enhanced cisplatin cytotoxicity in bladder cancer cells.miR-101 suppresses VEGF-C expression, inhibits cell migration and invasion, and increases cisplatin sensitivity in bladder cancer cells. This study provides new insight into miR-101's role in bladder cancer and shows miR-101's promise as a potential molecular target for bladder cancer.

Intestinal epithelial stem cells (ISCs) are the focus of recent intense study. Current in vitro models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs) are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs in vitro and allows for their successful in vivo implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful in vivo engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation.

BACKGROUND:We previously reported in vitro maintenance and proliferation of human small intestinal epithelium using Matrigel, a proprietary basement membrane product. There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells. METHODS:Human small intestine was procured from fresh surgical pathology specimens. Small intestinal crypts were isolated using EDTA chelation. Intestinal subepithelial myofibroblasts were isolated from a pediatric sample and expanded in vitro. After suspension in Matrigel or type I collagen gel, crypts were co-cultured above a confluent layer of myofibroblasts. Crypts were also grown in monoculture with exposure to myofibroblast conditioned media; these were subsequently sub-cultured in vitro and expanded with a 1∶2 split ratio. Cultures were assessed with light microscopy, RT-PCR, histology, and immunohistochemistry. RESULTS:Collagen supported viable human epithelium in vitro for at least one month in primary culture. Sub-cultured epithelium expanded through 12 passages over 60 days. Histologic sections revealed polarized columnar cells, with apical brush borders and basolaterally located nuclei. Collagen-based cultures gave rise to monolayer epithelial sheets at the gel-liquid interface, which were not observed with Matrigel. Immunohistochemical staining identified markers of differentiated intestinal epithelium and myofibroblasts. RT-PCR demonstrated expression of α-smooth muscle actin and vimentin in myofibroblasts and E-Cadherin, CDX2, villin 1, intestinal alkaline phosphatase, chromogranin A, lysozyme, and Lgr5 in epithelial cells. These markers were maintained through several passages. CONCLUSION:Type I collagen gel supports long-term in vitro maintenance and expansion of fully elaborated human intestinal epithelium. Collagen-based methods yield familiar enteroid structures as well as a new pattern of sheet-like growth, and they eliminate the need for Matrigel for in vitro human intestinal epithelial growth. Future research is required to further develop this cell culture system for tissue engineering applications.

The intestinal crypt-niche interaction is thought to be essential to the function, maintenance, and proliferation of progenitor stem cells found at the bases of intestinal crypts. These stem cells are constantly renewing the intestinal epithelium by sending differentiated cells from the base of the crypts of Lieberkühn to the villus tips where they slough off into the intestinal lumen. The intestinal niche consists of various cell types, extracellular matrix, and growth factors and surrounds the intestinal progenitor cells. There have recently been advances in the understanding of the interactions that regulate the behavior of the intestinal epithelium and there is great interest in methods for isolating and expanding viable intestinal epithelium. However, there is no method to maintain primary human small intestinal epithelium in culture over a prolonged period of time. Similarly no method has been published that describes isolation and support of human intestinal epithelium in an in vivo model. We describe a technique to isolate and maintain human small intestinal epithelium in vitro from surgical specimens. We also describe a novel method to maintain human intestinal epithelium subcutaneously in a mouse model for a prolonged period of time. Our methods require various growth factors and the intimate interaction between intestinal sub-epithelial myofibroblasts (ISEMFs) and the intestinal epithelial cells to support the epithelial in vitro and in vivo growth. Absence of these myofibroblasts precluded successful maintenance of epithelial cell formation and proliferation beyond just a few days, even in the presence of supportive growth factors. We believe that the methods described here can be used to explore the molecular basis of human intestinal stem cell support, maintenance, and growth.

Somatic cell nuclear transfer (SCNT) is an important method of breeding quality varieties, expanding groups, and preserving endangered species. However, the viability of SCNT embryos is poor, and the cloned rate of animal production is low in pig. This study aims to investigate the gene function and establish a disease model of Banna miniature inbred pig. SCNT with donor cells derived from fetal, newborn, and adult fibroblasts was performed, and the cloning efficiencies among the donor cells were compared. The results showed that the cleavage and blastocyst formation rates did not significantly differ between the reconstructed embryos derived from the fetal (74.3% and 27.4%) and newborn (76.4% and 21.8%) fibroblasts of the Banna miniature inbred pig (P>0.05). However, both fetal and newborn fibroblast groups showed significantly higher rates than the adult fibroblast group (61.9% and 13.0%; P<0.05). The pregnancy rates of the recipients in the fetal and newborn fibroblast groups (60% and 80%, respectively) were higher than those in the adult fibroblast group. Eight, three, and one cloned piglet were obtained from reconstructed embryos of the fetal, newborn, and adult fibroblasts, respectively. Microsatellite analyses results indicated that the genotypes of all cloning piglets were identical to their donor cells and that the genetic homozygosity of the Banna miniature inbred pig was higher than those of the recipients. Therefore, the offspring was successfully cloned using the fetal, newborn, and adult fibroblasts of Banna miniature inbred pig as donor cells. [ABSTRACT FROM AUTHOR]

The intestinal crypt-niche interaction is thought to be essential to the function, maintenance, and proliferation of progenitor stem cells found at the bases of intestinal crypts. These stem cells are constantly renewing the intestinal epithelium by sending differentiated cells from the base of the crypts of Lieberkühn to the villus tips where they slough off into the intestinal lumen. The intestinal niche consists of various cell types, extracellular matrix, and growth factors and surrounds the intestinal progenitor cells. There have recently been advances in the understanding of the interactions that regulate the behavior of the intestinal epithelium and there is great interest in methods for isolating and expanding viable intestinal epithelium. However, there is no method to maintain primary human small intestinal epithelium in culture over a prolonged period of time. Similarly no method has been published that describes isolation and support of human intestinal epithelium in an in vivo model. We describe a technique to isolate and maintain human small intestinal epithelium in vitro from surgical specimens. We also describe a novel method to maintain human intestinal epithelium subcutaneously in a mouse model for a prolonged period of time. Our methods require various growth factors and the intimate interaction between intestinal sub-epithelial myofibroblasts (ISEMFs) and the intestinal epithelial cells to support the epithelial in vitro and in vivo growth. Absence of these myofibroblasts precluded successful maintenance of epithelial cell formation and proliferation beyond just a few days, even in the presence of supportive growth factors. We believe that the methods described here can be used to explore the molecular basis of human intestinal stem cell support, maintenance, and growth. [ABSTRACT FROM AUTHOR]