Effects of tumour necrosis factor-alpha on BrdU incorporation in cultured human enterocytes

Bromodeoxyuridine incorporation is a useful method for studying the pattern of DNA synthesis in proliferating cells. The distribution pattern of incorporated BrdU in villus enterocytes of duodenal explants was analysed after exposure to TNFalpha in organ culture. TNFalpha caused a consistent, low level uptake of BrdU in the portion of the nucleus close to the nuclear membrane, this pattern was absent from the control cultures. As these epithelial cells are terminally arrested in G(0), the BrdU incorporation was thought not to be due to S phase DNA synthesis, but rather a response to the cytotoxic influence of TNFalpha. Microtitre plate proliferation assays of cell density and DNA synthesis were devised to study the effects of TNFalpha on confluent monolayers of the human foetal jejunal cell line I407 and the mouse fibrosarcoma cell line L929. Both cell lines showed a similar response to TNFalpha. Exposure to TNFalpha alone did not reduce cell numbers but did cause a significant increase in DNA synthesis (p < 0.05). When cycloheximtde was added in tandem with TNFalpha there was a significant reduction in cell number (p < 0.001) and level of DNA synthesis (p < 0.01) indicative of cell death. The DNA of cells exposed to TNFalpha and cycloheximide was fragmented when viewed on an electrophoresis gel. The results show that BrdU incorporation might be a good indicator of damage to the DNA of cells after cytotoxic insult. TNFalpha may be responsible for villus enterocyte damage in enteropathies such as coeliac disease and GVHR of the small bowel.     

Cell-mediated cytotoxicity against HLA-D-region products expressed in monocytes and B lymphocytes. I. Blocking by Unlabelled cells and inhibition by antisera

Cytotoxic effector cells against HLA-D-region produts were generated in cultures with HLA-A- and B-matched, HLA-D-mismatched stimulating cells. Monocytes from unrelated donors sharing HLA-D/DR antigens with the primary stimulator were used as target cells. Lysis of target cells was inhibited by addition of unlabelled monocytes having the same HLA-D/DR antigens. Inhibition of cytotoxicity was also observed with unlabelled B cells, but T lymphocytes had little effect. The distribution of the target antigens, therefore, fits the known distribution of the products of HLA-D. In other experiments, a human alloantiserum specific for HLA-DRw3 was found to inhibit cellular cytotoxicity specific for HLA-D/DRw3. Lysis by HLA-D/DRw3-specific effector cells was not inhibited by sera against HLA-DRw2 or DRw7 or by antibodies against HLA-B8 using HLA-B8 positive, DRw3 positive targer cells. A xenogeneic serum against human Ia antigens, produced in rabbits, blocked cytotoxicity directed at DRw2, DRw3 and DRw4. These results suggest that cell-mediated cyctotoxicity was directed against HLA-D/DR or very closely associated determinants.     

Distinct and overlapping effector functions of expanded human CD4+, CD8α+ and CD4-CD8α- invariant natural killer T cells

CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+), CD8α(+) and CD4(-)CD8α(-) double-negative (DN) subsets. CD4(+) iNKT cells expanded more readily than CD8α(+) and DN iNKT cells upon mitogen stimulation. CD8α(+) and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+) cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4(+) and CD8α(+) fractions, respectively. Only CD4(+) iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α(+), DN or CD4(+) iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.     

The Effect of Substrate Stiffness,Thickness, and Cross-Linking Density on Osteogenic Cell Behavior

Osteogenic cells respond to mechanical changes in their environment by altering their spread area, morphology, and gene expression profile. In particular, the bulk modulus of the substrate, as well as its microstructure and thickness, can substantially alter the local stiffness experienced by the cell. Although bone tissue regeneration strategies involve culture of bone cells on various biomaterial scaffolds, which are often cross-linked to enhance their physical integrity, it is difficult to ascertain and compare the local stiffness experienced by cells cultured on different biomaterials. In this study, we seek to characterize the local stiffness at the cellular level for MC3T3-E1 cells plated on biomaterial substrates of varying modulus, thickness, and cross-linking concentration. Cells were cultured on flat and wedge-shaped gels made from polyacrylamide or cross-linked collagen. The cross-linking density of the collagen gels was varied to investigate the effect of fiber cross-linking in conjunction with substrate thickness. Cell spread area was used as a measure of osteogenic differentiation. Finite element simulations were used to examine the effects of fiber cross-linking and substrate thickness on the resistance of the gel to cellular forces, corresponding to the equivalent shear stiffness for the gel structure in the region directly surrounding the cell. The results of this study show that MC3T3 cells cultured on a soft fibrous substrate attain the same spread cell area as those cultured on a much higher modulus, but nonfibrous substrate. Finite element simulations predict that a dramatic increase in the equivalent shear stiffness of fibrous collagen gels occurs as cross-linking density is increased, with equivalent stiffness also increasing as gel thickness is decreased. These results provide an insight into the response of osteogenic cells to individual substrate parameters and have the potential to inform future bone tissue regeneration strategies that can optimize the equivalent stiffness experienced by a cell.     

Deoxycholic acid promotes development of gastroesophageal reflux disease and Barrett's oesophagus by modulating integrin‐αv trafficking

The fundamental mechanisms underlying erosive oesophagitis and subsequent development of Barrett's oesophagus (BO) are poorly understood. Here, we investigated the contribution of specific components of the gastric refluxate on adhesion molecules involved in epithelial barrier maintenance. Cell line models of squamous epithelium (HET‐1A) and BO (QH) were used to examine the effects of bile acids on cell adhesion to extracellular matrix proteins (Collagen, laminin, vitronectin, fibronectin) and expression of integrin ligands (α₃, α_4, α₅, α₆ and α_ν). Experimental findings were validated in human explant oesophageal biopsies, a rat model of gastroesophageal reflux disease (GORD) and in patient tissue microarrays. The bile acid deoxycholic acid (DCA) specifically reduced adhesion of HET‐1A cells to vitronectin and reduced cell‐surface expression of integrin‐α_ν via effects on endocytic recycling processes. Increased expression of integrin‐α_v was observed in ulcerated tissue in a rat model of GORD and in oesophagitis and Barrett's intestinal metaplasia patient tissue compared to normal squamous epithelium. Increased expression of integrin‐α_ν was observed in QH BO cells compared to HET‐1A cells. QH cells were resistant to DCA‐mediated loss of adhesion and reduction in cell‐surface expression of integrin‐α_ν. We demonstrated that a specific component of the gastric refluxate, DCA, affects the epithelial barrier through modulation of integrin α_ν expression, providing a novel mechanism for bile acid‐mediated erosion of oesophageal squamous epithelium and promotion of BO. Strategies aimed at preventing bile acid‐mediated erosion should be considered in the clinical management of patients with GORD.