Urokinase and the intestinal mucosa: evidence for a role in epithelial cell turnover

Background—The functions of urokinase inintestinal epithelia are unknown.
Aims—To determine the relation of urokinaseexpressed by intestinal epithelial cells to their position in thecrypt-villus/surface axis and of mucosal urokinase activity toepithelial proliferative kinetics in the distal colon.
Methods—Urokinase expression was examinedimmunohistochemically in human intestinal mucosa. Urokinase activitywas measured colorimetrically in epithelial cells isolated sequentiallyfrom the crypt-villus axis of the rat small intestine. In separate experiments, urokinase activity and epithelial kinetics (measured stathmokinetically) were measured in homogenates of distal colonic mucosa of 14 groups of eight rats fed diets known to alter epithelial turnover.
Results—From the crypt base, an ascendinggradient of expression and activity of urokinase was associated withthe epithelial cells. Median mucosal urokinase activities in each ofthe dietary groups of rats correlated positively with autologous mediannumber of metaphase arrests per crypt (r=0.68; p<0.005)and per 100 crypt cells (r=0.75; p<0.001), but not withcrypt column height.
Conclusions—Localisation of an enzyme capable ofleading to digestion of cell substratum in the region where cells areloosely attached to their basement membrane, and the association of its activity with indexes of cell turnover, suggest a role for urokinase infacilitating epithelial cell loss in the intestine.

Keywords:urokinase; intestinal epithelium; colon; epithelialproliferation

     

Individual histories and selection in heterogeneous populations

The strength of selection in populations has traditionally been inferred by measuring changes in bulk population parameters, such as mean reproductive rates. Untangling the effect of selection from other factors, such as specific responses to environmental fluctuations, poses a significant problem both in microbiology and in other fields, including cancer biology and immunology, where selection occurs within phenotypically heterogeneous populations of cells. Using “individual histories”—temporal sequences of all reproduction events and phenotypic changes of individuals and their ancestors—we present an alternative approach to quantifying selection in diverse experimental settings. Selection is viewed as a process that acts on histories, and a measure of selection that employs the distribution of histories is introduced. We apply this measure to phenotypically structured populations in fluctuating environments across different evolutionary regimes. Additionally, we show that reproduction events alone, recorded in the population’s tree of cell divisions, may be sufficient to accurately measure selection. The measure is thus applicable in a wide range of biological systems, from microorganisms—including species for which genetic tools do not yet exist—to cellular populations, such as tumors and stem cells, where detailed temporal data are becoming available.     

Guidelines for the Bandgap Combinations and Absorption Windows for Organic Tandem and Triple-Junction Solar Cells

Organic solar cells have narrow absorption windows, compared to the absorption band of inorganic semiconductors. A possible way to capture a wider band of the solar spectrum—and thus increasing the power conversion efficiency—is using more solar cells with different bandgaps in a row, i.e., a multi-junction solar cell. We calculate the ideal material characteristics (bandgap combinations and absorption windows) for an organic tandem and triple-junction solar cell, as well as their acceptable range. In this way, we give guidelines to organic material designers.     

In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

The human adenovirus phylogenetic tree is split across seven species (A–G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D—such as their cellular tropism, receptor usage, and in vivo biodistribution profile—remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)—a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.     

Inhibitory effect of oestradiol on activation of rat hepatic stellate cells in vivo and in vitro

Background—Hepatic stellate cellsplay a key role in the pathogenesis of hepatic fibrosis.
Aims—To examine the inhibitoryeffect of oestradiol on stellate cell activation.
Methods—In vivo, hepatic fibrosiswas induced in rats by dimethylnitrosamine or pig serum. In vitro, ratstellate cells were activated by contact with plastic dishes resultingin their transformation into myofibroblast-like cells.
Results—In the dimethylnitrosamineand pig serum models, treatment with oestradiol at gestation relateddoses resulted in a dose dependent suppression of hepatic fibrosis withrestored content of hepatic retinyl palmitate, reduced collagencontent, lower areas of stellate cells which express α smooth muscleactin (α-SMA) and desmin, and lower procollagen type I and III mRNA levels in the liver. In cultured stellate cells, oestradiol inhibited type I collagen production, α-SMA expression, and cell proliferation. These findings suggest that oestradiol is a potent inhibitor of stellate cell transformation.
Conclusion—The antifibrogenic roleof oestradiol in the liver may contribute to the sex associateddifferences in the progression from hepatic fibrosis to cirrhosis.

Keywords:hepatic stellate cells; hepatic fibrosis; oestradiol; α smooth muscle actin; retinyl palmitate

     

Heterogeneity in responses by primary adult human colonic epithelial cells to purified enterotoxin of Bacteroides fragilis

Background—Enterotoxigenic strains ofBacteroides fragilis (ETBF) have been implicated indiarrhoeal illness in livestock and children, but their role in adulthuman colonic disease is unknown.
Aims—To investigate responses by primary adulthuman colonic epithelial cells to purified B fragilistoxin (BFT).
Methods—BFT was purified from culture supernatantof a highly toxigenic strain of ETBF. Morphological changes to primarycolonic epithelial cells, in response to purified BFT, were studied in organ culture of colonic biopsy specimens from 15adults.
Results—BFT induced epithelial cell cytotoxicityin colonic biopsy specimens from 12/15 subjects. The BFT inducedmorphological changes were characterised by epithelial cell rounding,separation from adjacent cells, and detachment from the basementmembrane. In severely affected specimens, almost all the epithelialcells were affected. There was heterogeneity between subjects in the rate at which BFT induced epithelial cell cytotoxicity occurred. Furthermore, in colonic biopsy specimens from three subjects, exposureto BFT did not induce any significant morphological changes toepithelial cells.
Conclusion—BFT is capable of inducingcytotoxicity in primary adult human colonic epithelial cells. Such aneffect of ETBF derived BFT on epithelial cells in the colon in vivowould be expected to lead to mucosal inflammation and diarrhoea.Heterogeneity in responses by primary colonocytes probably reflects theoutcome of host-BFT interactions. Such interactions in vivo coulddetermine the occurrence of colonic disease in some individuals but not others.

Keywords:Bacteroides fragilis; enterotoxin; epithelial cells; apoptosis

     

Ceramide, a mediator of interleukin 1, tumour necrosis factor α, as well as Fas receptor signalling, induces apoptosis of rheumatoid arthritis synovial cells

OBJECTIVES—To examine the effects of ceramide, which is a lipid second messenger of cell surface receptors, including tumour necrosis factor α (TNFα), interleukin 1 (IL1), and Fas receptors, on rheumatoid arthritis (RA) synovial cells.
METHODS—Synovial cells from RA patients and normal skin fibroblasts were cultured with cell permeable ceramide (C2-ceramide). Apoptosis was assessed by microscopic observation of morphological changes, nuclear staining, and DNA electrophoresis. DNA synthesis was examined by thymidine incorporation.
RESULTS—C2-ceramide induced reversible morphological changes of synovial cells such as cell rounding within four hours. Subsequently, irreversible nuclear changes characteristic to apoptosis were observed at 48 hours. DNA synthesis was not promoted. The addition of ceramide exerted similar effects on cultured dermal fibroblasts.
CONCLUSION—Ceramide induced apoptosis in RA synovial cells. Ceramide could be a second messenger specific for apoptosis of RA synovial cells.

Keywords: ceramide; apoptosis; rheumatoid arthritis     

Expression of a marker for colonic crypt base cells is correlated with poor prognosis in human colorectal cancer

Background—There is a need for markers in colorectal cancer which will allow subclassification of stage groups into subgroups with high versus low risk of recurrent disease.
Aims—To develop monoclonal antibodies that recognise antigens on immature crypt base cells, on the assumption that in a neoplasm undifferentiated but not the terminally differentiated cells will be responsible for tumour progression.
Methods—Colon crypt cells which were isolated from human colonic mucosa by EDTA/EGTA incubation were studied. By stepwise harvesting, crypt base cell enriched fractions were obtained, and after incubation with antibodies against dominant antigens, used as immunogens.
Results—Of one crypt base cell specific antibody (5E9), the reactive epitope appeared to be a non-terminal carbohydrate in the mucin O-glycans of the colon. The epitope did not seem to be colon specific, but was expressed in a variety of other tissues. In colorectal carcinomas, 5E9 immunoreactivity identified a subgroup of patients with a tendency for worse prognosis.
Conclusion—A mucin associated maturation epitope was identified in colonic crypt base cells, the expression of which in Dukes'' stage B3 colorectal carcinoma may be associated with poor prognosis.

     

Increased state of activation of CD4 positive T cells and elevated interferon γ production in pouchitis

Background—Immunoregulatory abnormalities of Tcells might be of importance in the pathogenesis of pouchitis afterileoanal pouch anastomosis (IAP).
Aims—To characterise T cell subsets, their stateof activation, and production of cytokines in inflamed and non-inflamedpouches in patients with ulcerative colitis (UC) and familialadenomatous polyposis (FAP). The influence of T cell activation onmucosal transformation was also studied.
Patients—Mucosal biopsy specimens were taken from42 patients with IAP (33 with UC and nine with FAP).
Methods—Mononuclear cells were isolated bystandard techniques and characterised by three colour flow cytometry.Interferon γ (IFN-γ) production was studied using the ELISPOT technique.
Results—In patients with UC with pouchitis therewas a significant increase in the CD4:CD8 ratio, expression ofactivation markers on CD3+ cells, and number of IFNγ producingmononuclear cells compared with patients with UC without pouchitis(CD4:CD8 ratio 1.3 (range 0.7-2.7) versus 0.6 (0.1-1.0), p=0.012). Inaddition, a positive correlation between increased crypt depth and thenumber of CD4+ cells (r=0.57) was shown.
Conclusion—The observed increase in activatedmucosal CD4+ T cells and IFN-γ production might lead to mucosaldestruction and crypt hyperplasia as seen in pouchitis.

Keywords:pouchitis; T cell activation; mucosaltransformation

     

Detection of anti-colon antibodies in inflammatory bowel disease using human cultured colonic cells

Background—Investigationof anti-colon antibodies may be simplified if a sensitive method andhomogeneous source of antigen were available.
Aims—To examine theanti-colon antibody response using human colonic carcinoma cell linesas antigen.
Subjects—Patients withinflammatory bowel disease and other gastrointestinal disorders andhealthy controls were studied.
Methods—Comparativeenzyme linked immunosorbent assays (ELISAs) were performed to assessthe value of whole Caco-2, HT-29, and LS-180 cells as antigen. Theantigenic determinants of the immune response were characterised bywestern blot analysis.
Results—Serademonstrated immunoreactivity against each of the cell lines, butdifferent epitopes were recognised. Applying whole Caco-2 cells asantigen in an ELISA, the prevalence of anti-colon antibodies wassignificantly greater in patients with ulcerative colitis (36%) thanCrohn's disease (13%), other gastrointestinal disorders (13%) andhealthy controls (0) (p<0.05). The immune response was not associatedwith one predominant antigen.
Conclusions—Fixedwhole cell ELISA is a simple and feasible method for studying theanti-colon antibody response. This response is non-specific, beingdirected against multiple antigens, and is likely to be anepiphenomenon of inflammatory bowel disease, more so for ulcerativecolitis than Crohn's disease.

Keywords:anti-colon antibodies; inflammatory boweldisease

     

Novel Textile Scaffolds Generated by Flock Technology for Tissue Engineering of Bone and Cartilage

Textile scaffolds can be found in a variety of application areas in regenerative medicine and tissue engineering. In the present study we used electrostatic flocking—a well-known textile technology—to produce scaffolds for tissue engineering of bone. Flock scaffolds stand out due to their unique structure: parallel arranged fibers that are aligned perpendicularly to a substrate, resulting in mechanically stable structures with a high porosity. In compression tests we demonstrated good mechanical properties of such scaffolds and in cell culture experiments we showed that flock scaffolds allow attachment and proliferation of human mesenchymal stem cells and support their osteogenic differentiation. These matrices represent promising scaffolds for tissue engineering.     

Colonic epithelial cell proliferation in hereditary non-polyposis colorectal cancer

Background—Despite the recentdiscovery of four genes responsible for up to 90% of all cases ofhereditary non-polyposis colorectal cancer (HNPCC), there will still befamilies in whom predictive testing is not possible. A phenotypicbiomarker would therefore be useful. An upwards shift of theproliferative compartment in colonic crypts is reported to be one ofthe earliest changes in premalignant mucosa.
Aims—To assess the role of cryptcell proliferation as a phenotypic biomarker in HNPCC.
Patients—Thirty five patients at50% risk of carrying the HNPCC gene (21 of whom subsequently underwentpredictive testing and hence gene carrier status was known) and 18controls.
Methods—Crypt cell proliferationwas measured at five sites in the colon using two different techniques.Labelling index was determined using the monoclonal antibody MIB1 andwhole crypt mitotic index was measured using the microdissection andcrypt squash technique. The distribution of proliferating cells within the crypts was also assessed.
Results—There were no significantdifferences in the total labelling index or mean number of mitoses percrypt, nor in the distribution of proliferating cells within the crypt,between the study and control groups at any site. When the 21 patients in whom gene carrier status was known were analysed separately therewere no significant differences in the measured indices ofproliferation between the HNPCC gene carriers and non-gene carriers.
Conclusion—Crypt cell proliferationis not a discriminative marker of gene carriage in HNPCC.

Keywords:cell proliferation; hereditary non-polyposiscolorectal cancer

     

Role of endothelial cell denudation and smooth muscle cell dedifferentiation in neointimal formation of human vein grafts after coronary artery bypass grafting: therapeutic implications

OBJECTIVE—To provide better insights into the genesis of neointimal thickening in human vein grafts early after surgery.
DESIGN—Retrospective study.
SETTING—Tertiary referral centre.
SUBJECTS—18 distal anastomotic sites of patent grafts, obtained at necropsy from eight patients who died over differing periods (ranging from two days to nine months) after the procedure.
MAIN OUTCOME MEASURES—Immunohistochemical evaluation of smooth muscle cell phenotype modulation in relation to proliferative activity.
RESULTS—The earliest changes are characterised by loss of surface lining endothelial cells and insudation of blood corpuscular elements admixed with fibrin-platelet thrombus. At sites of injury vimentin positive and actin negative spindle shaped cells appear in the intima, while the related pre-existent media shows focal absence of actin positive smooth muscle cells. Proliferative activity colocalises at these sites. With time distinct neointimal thickening occurs, associated with disappearance of proliferative activity and a phenotypic shift of the smooth muscle cells.
CONCLUSIONS—The observation that luminal endothelial cell denudation, with insudation of the intima with blood elements, occurs in the very early stages suggests that these phenomena are responsible for the observed dedifferentiation of pre-existent smooth muscle cells, known to be a prerequisite for cell proliferation and the evolution of intimal thickening. It is likely, therefore, that platelet released growth factors play a pivotal role, which thus may provide a target for preventive pharmacological intervention.


Keywords: smooth muscle cell proliferation; vein graft stenosis; platelet derived growth factor; platelet receptor inhibitors     

The same systemic autoimmune disease provokes arthritis and endocarditis via distinct mechanisms

The immune mechanisms that provoke concomitant inflammation of synovial joints and cardiac valves in disorders such as rheumatic fever and systemic lupus erythematosus remain poorly defined. Here, we report the discovery of spontaneous endocarditis—in addition to their well-studied autoimmune arthritis—in K/BxN T cell receptor (TCR) transgenic mice. The same adaptive immune system elements were required for initiation of arthritis and endocarditis, and both diseases were dependent on autoantibodies. In contrast, the participation of key innate immune system molecules and perhaps T cells as effectors of inflammation differed between the 2 target tissues. Arthritis in K/BxN TCR transgenic mice depended primarily on complement C5 and not FcRγ-using receptors; conversely, endocarditis depended essentially on FcRγ receptors and not C5. Elucidating how a single systemic autoimmune disease engages distinct immune effector pathways to damage different target tissues is essential for optimizing the treatment of such disorders.     

Induction of intestinal lesions in nu/nu mice induced by transfer of lymphocytes from syngeneic mice infected with murine retrovirus

T Mizuochi,^dH Asakura,^bM Fujiwara^a

Background—Murine leukemia virus, LP-BM5, inducessevere immunodeficiency with abnormal lymphoproliferation insusceptible C57BL/6 mice. In a previous study, it was shown that aSjögren's syndrome-like systemic exocrinopathy is induced in thevirus infected mice.
Aims—To examine lymphocyte functions of the virusinfected mice.
Methods—Four-week old mice were inoculated withthe virus and their spleen cells were transferred into syngeneic nu/numice. Their organs were examined by light and electron microscopy.Phenotypes of the colon infiltrating cells were examined by flow cytometry.
Results—All nu/nu recipients had died by six weeksafter cell transfer, showing runting disease like cachexia withdiarrhoea and anal bleeding. Histopathological examination revealedthat systemic exocrinopathy was adoptively transferable and that the colon became thickened due to mononuclear cell infiltration into themucosal and submucosal layer with hyperplasia of intestinal epithelialcells. No virus particles were found in the colon. Flow cytometricanalyses revealed that most of the infiltrating CD4+ T cells showedCD45RB^low. No intestinal lesions were observed in the virusinfected mice nor in nu/nu mice inoculated with normal lymphocytes.
Conclusion—Lymphocytes of the virus infected miceinduced colitis and hyperplasia of intestinal epithelial cells as wellas systemic exocrinopathy in nu/nu mice. Our experimental system maygive some insight into intestinal lesions associated with virus infection.

Keywords:murine leukemia virus; nude mice; enterocolitis; colitogenic cells

     

PDGF-D−PDGFRβ signaling enhances IL-15–mediated human natural killer cell survival

The axis of platelet-derived growth factor (PDGF) and PDGF receptor-beta (PDGFRβ) plays prominent roles in cell growth and motility. In addition, PDGF-D enhances human natural killer (NK) cell effector functions when binding to the NKp44 receptor. Here, we report an additional but previously unknown role of PDGF-D, whereby it mediates interleukin-15 (IL-15)–induced human NK cell survival but not effector functions via its binding to PDGFRβ but independent of its binding to NKp44. Resting NK cells express no PDGFRβ and only a low level of PDGF-D, but both are significantly up-regulated by IL-15, via the nuclear factor κB signaling pathway, to promote cell survival in an autocrine manner. Both ectopic and IL-15–induced expression of PDGFRβ improves NK cell survival in response to treatment with PDGF-D. Our results suggest that the PDGF-D−PDGFRβ signaling pathway is a mechanism by which IL-15 selectively regulates the survival of human NK cells without modulating their effector functions.

Natural killer (NK) cells—a distinct lymphocyte subset in the circulation—play critical roles in antiviral and antitumor immunity (1). One advantage of NK cells is that they recognize “nonself” cells without being activated by specific antigens, allowing a more rapid response than with T cells. This broad cytotoxicity and rapid killing make NK cells ideal for cancer immunotherapy (2). Of note, chimeric-antigen-receptor (CAR)-NK cells have several therapeutic advantages over CAR-T cells (2–4). However, NK cells’ shorter lifespan may limit their clinical efficacy. A better understanding of the mechanisms that regulate NK cell survival might therefore improve their clinical application for cancer immunotherapy.Platelet-derived growth factor (PDGF) is one of the main growth factors that regulate cell growth and division (5). The PDGF family consists of PDGF-A, PDGF-B, PDGF-C, and PDGF-D (5). These ligands bind to two tyrosine kinase receptors, PDGFRα and PDGFRβ (5). Upon activation by PDGFs, PDGF receptors dimerize and undergo autophosphorylation on tyrosine residues in the intracellular domain, which mediates the binding of cofactors and subsequently activates signal transduction, including Ras/Raf/MEK/Erk mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase/protein kinase B (P13K/AKT) (5). PDGFs play prominent roles in cell differentiation, cell growth, cell transformation, and cell migration (5). A recent study showed that PDGF-D is a ligand of NKp44, one of the natural cytotoxicity receptors expressed by activated human NK cells (6). PDGF-D binding to NKp44 prompted NK cells to secrete interferon (IFN)-γ and tumor necrosis factor (TNF)-α, arresting the growth of tumor cells (6). However, little is known about the role of PDGFR signaling in NK cell immunity.In this study, we report a previously unknown role of PDGF-D: regulation of interleukin 15 (IL-15)–mediated cell survival—not effector functions in human NK cells—that is dependent on PDGFRβ but independent of NKp44. Our findings suggest that the PDGF-D−PDGFRβ signaling pathway is a mechanism by which IL-15 selectively regulates the survival of human NK cells but not their effector functions.     

Heterodimeric integrin complexes containing β1-integrin promote internalization and lethality of anthrax toxin

To kill macrophages, the lethal factor component of Bacillus anthracis toxin binds to a carrier protein (PA), which then interacts with the CMG2 receptor protein on the cell surface and is endocytosed into the cytoplasm. CMG2, as well as TEM8, a second PA receptor not present on macrophages, contain a von Willebrand A domain that is crucial for toxin binding. Here we report that integrin β1, another cell surface von Willebrand A domain protein, can mediate and potentiate anthrax toxin endocytosis. By using microarray-based analysis to globally correlate gene expression profiles with toxin sensitivity, we associated toxin effects with the integrin-activating proteins osteopontin and CD44. Further study showed that PA binds to α4β1– and α5β1–integrin complexes, leading to their conjoint endocytosis, and also interacts—weakly relative to CMG2 but comparably to TEM8—with purified α5β1 complex in vitro. Monoclonal antibody directed against β1-integrin or its α integrin partners reduced PA/integrin endocytosis and anthrax toxin lethality, and hyaluronic acid—which interferes with CD44-mediated integrin activation—had similar effects. Remarkably, whereas deficiency of CMG2 protected macrophages from rapid killing by large toxin doses (>50 ng/mL), by 24 h the toxin-treated cells were dead. Such late killing of CMG2-deficient cells by high dose toxin as well as the late death observed during exposure of CMG2-producing macrophages to low-dose toxin (<1 ng/mL), was dependent on integrin function. Effects of inactivating both CMG2 and integrin were synergistic. Collectively, our findings argue strongly that β1-integrin can both potentiate CMG2-mediated endocytosis and serve independently as a low-affinity PA receptor.     

Interferon-γ impacts at multiple points during the progression of autoimmune diabetes

The role of interferon-γ in autoimmune diabetes was assessed by breeding a null mutation of the interferon-γ receptor α chain into the nonobese diabetic mouse strain, as well as into a simplified T cell receptor transgenic model of diabetes. In contrast to a previous report on abrogation of the interferon-γ gene, mutation of the gene encoding its receptor led to drastic effects on disease in both mouse lines. Nonobese diabetic mice showed a marked inhibition of insulitis—both the kinetics and penetrance—and no signs of diabetes; the transgenic model exhibited near-normal insulitis, but this never evolved into diabetes, either spontaneously or after experimental provocation. This failure could not be explained by perturbations in the ratio of T helper cell phenotypes; rather, it reflected a defect in antigen-presenting cells or in the islet β cell targets.