Adrian Robins

Smooth and rough lipopolysaccharide phenotypes of Brucella induce different intracellular trafficking and cytokine/chemokine release in human monocytes.

Virulence of the intracellular pathogen Brucella for humans is mainly associated with its lipopolysaccharide (LPS) phenotype, with smooth LPS phenotypes generally being virulent and rough ones not. The reason for this association is not quite understood. We now demonstrate by flow cytometry, electron microscopy, and ELISA that human peripheral blood monocytes interact both quantitatively and qualitatively different with smooth and rough Brucella organisms in vitro. We confirm that considerably higher numbers of rough than smooth brucellae attach to and enter the monocytes in nonopsonic conditions; but only smooth brucellae replicate in the host cells. We show for the first time that rough brucellae induce higher amounts than smooth brucellae of several CXC (GRO‐α, IL‐8) and CC (MIP‐1α, MIP‐1β, MCP‐1, RANTES) chemokines, as well as pro‐ (IL‐6, TNF‐α) and anti‐inflammatory (IL‐10) cytokines released by challenged monocytes. Upon uptake, phagosomes containing rough brucellae develop selective fusion competence to form spacious communal compartments, whereas phagosomes containing smooth brucellae are nonfusiogenic. Collectively, our data suggest that rough brucellae attract and infect monocytes more effectively than smooth brucellae, but only smooth LPS phenotypes establish a specific host cell compartment permitting successful parasitism. These novel findings link the LPS phenotype of Brucella and its virulence for humans at the level of the infected host cells. Whether this is due to a direct effect of the LPS molecules or to upstream bacterial mechanisms remains to be established.

Dendritic cells are dysfunctional in patients with operable breast cancer

Background: Dendritic cells (DCs) play a crucial role in presenting antigens to T lymphocytes and inducing cytotoxic T cells. DCs have been studied in patients with breast cancer to define the factors leading to failure of an effective systemic and locoregional anticancer host response. Methods: Purified DCs were obtained from peripheral blood (PB) and lymph nodes (LNs) of women with operable breast cancer, using immunomagnetic bead selection. The stimulatory capacity of DCs in the allogeneic mixed leukocyte reaction (MLR) and autologous T cell proliferation test (purified protein derivative (PPD) as stimulator), the expression of surface markers on DCs and the production of cytokines in vitro by DCs from patients with operable breast cancer and from healthy donors (controls) were studied. Results: 70–75% purified DCs were isolated from PB and LNs. PBDCs and LNDCs from patients with operable breast cancer demonstrated a reduced capacity to stimulate in an MLR, compared with PBDCs from normal donors (p<0.01). Autologous T cell proliferation in patients had a decreased ability to respond to PPD, when compared with controls (p<0.01). However, T cells from patients responded as well as control T lymphocytes in the presence of control DCs. PBDCs and LNDCs from patients expressed low levels of HLA-DR and CD86, and induced decreased interleukin-12 (IL-12) secretion in vitro, compared with DCs from normal donors (p<0.01). Conclusion: These data suggest a defective DC function in patients with operable breast cancer. Switched-off DCs in patients with early breast cancer and decreased IL-12 production may be important factors for progressive tumour growth.

The Stem Cell Marker CD133 Associates with Enhanced Colony Formation and Cell Motility in Colorectal Cancer

CD133 is a membrane molecule that has been, controversially, reported as a CSC marker in colorectal cancer (CRC). In this study, we sought to clarify the expression and role of CD133 in CRC. Initially the size of the CD133−expressing (CD133+) population in eight well-described CRC cell lines was measured by flow cytometry and was found to range from 0% to >95%. The cell line HT29 has a CD133+ population of >95% and was chosen for functional evaluation of CD133 after gene knockdown by RNA interference. A time course assay showed that CD133 inhibition had no significant effect on cell proliferation or apoptosis. However, CD133 knockdown did result in greater susceptibility to staurosporine-induced apoptosis (p = 0.01) and reduction in cell motility (p<0.04). Since gene knockdown may cause “off-target” effects, the cell line SW480 (which has a CD133+ population of 40%) was sorted into pure CD133+ and CD133− populations to allow functional comparison of isogenic populations separated only by CD133 expression. In concordance with the knockdown experiments, a time course assay showed no significant proliferative differences between the CD133+/CD133− populations. Also greater resistance to staurosporine-induced apoptosis (p = 0.008), greater cell motility (p = 0.03) and greater colony forming efficiency was seen in the CD133+ population than the CD133− population in both 2D and 3D culture (p<0.0001 and p<0.003 respectively). Finally, the plasticity of CD133 expression in tumour cells was tested. Quantitative PCR analysis showed there was transcriptional repression in the CD133− population of SW480. Prolonged culture of a pure CD133− population resulted in re-emergence of CD133+ cells. We conclude that CD133 expression in CRCs is associated with some features attributable to stemness and that there is plasticity of CD133 expression. Further studies are necessary to delineate the mechanistic basis of these features.

Human T cell subset commitment determined by the intrinsic property of antigen: the proteolytic activity of the major mite allergen Der p 1 conditions T cells to produce more IL‐4 and less IFN‐γ

The house dust mite Dermatophagoides pteronyssinus allergen Der p 1 elicits IgE antibody responses in a significant proportion of patients suffering from dust mite allergy. We have recently shown that Der p 1 proteolytically cleaves a cell surface molecule involved in the homeostatic control of human IgE synthesis, namely the IL‐2 receptor (CD25) on T cells. As a result, these Tcells show markedly diminished proliferation and IFN‐γ secretion in response to stimulation by anti‐CD3 antibody. However, these observations still leave open the important issue of whether CD25 cleavage, and the consequent suppression of IFN‐γ secretion, leads to enhanced IL‐4 secretion, and whether such cytokine changes would be exhibited by both CD4 and CD8 T cells. Here we demonstrate for the first time that the proteolytic activity of Der p 1 biases human CD4 and CD8 T cells towards a type 2 cytokine profile. Our data provide compelling evidence for the role of the proteolytic activity of Der p 1 in creating a microenvironment conducive for IgE synthesis.

Immunity in the tumor‐bearing host and its modification by serum factors

Tumor‐bearing individuals initiate immune responses to tumor‐associated neoantigens. These responses are demonstrable by in vitro analyses of cell‐mediated and humoral immune reactions, although it is emphasized that in most human studies, their correlation with positive tumor rejection reactions has not as yet been fully substantiated. Detection of cell‐mediated immunity in tumor‐bearing hosts has led to the concept that its effectiveness in controlling tumor growth may be modified or impaired by antagonistic humoral factors. This proposal is discussed with regard to the role of blocking reactions operative at the level of the target tumor cell, and with respect to direct inhibition of lymphocyte cytotoxicity by interaction with humoral factors. Available evidence indicates that blocking reactions are probably mediated by tumor‐specific immune complexes, and although antibody‐mediated blocking has been detected, its relevance in abrogating cell‐mediated immunity in the tumor‐bearing host is questionable. Alternatively, inhibition of the reactivity of sensitized lymphocytes, by tumor antigen or immune complexes, may prove to be of more importance in the immunologic control of tumor growth.

Inflammatory bowel disease: dysfunction of GALT and gut bacterial flora (I)

The precise cause(s) of Crohns disease and ulcerative colitis are unknown. From animal models and human studies it is well established that gut bacterial flora are essential for inducing the bowel inflammation. Animal models, when kept in a germ-free environment, do not develop colitis until the gut flora is reconstituted. It is not clear whether the bacterial antigens (Ags) from the normal flora or some other pathogenic bacterial Ags induce/propagate the inflammatory process in inflammatory bowel disease (IBD). Despite extensive research it has not been possible to identify any specific bacteria or virus as a credible cause of IBD. Recent understanding of quorum sensing molecules (QSMs) secreted by bacteria helps to explain the community behaviour in bacterial species. When QSMs reach a defined concentration, they activate bacterial proliferation and a number of virulence genes. Also, these molecules have been found to modulate the immune system to the advantage of the gut bacteria. They have not been well studied, however, in the gut. Inappropriate secretion of QSMs may alter the gut-associated lymphoid tissue (GALT) and, thereby, deregulate the immune tolerance normally present. Usefulness of probiotics and their immune modulating effects are being increasingly reported. Probiotics are also being used in the treatment of IBD. The interaction between the epithelial cells and the gut flora is very important as this is the first line of contact; this interaction may determine the induction of tolerance and mucosal integrity or immune activity, tissue inflammation and abnormal permeability. The latter is documented in patients with IBD and their healthy relatives. This may be an important factor in disruption of mucosal integrity and GALT dysfunction.

Evolutionary trends of the first hypervariable region of the hepatitis C virus E2 protein in individuals with differing liver disease severity.

Hepatitis C virus (HCV) exists as a complex swarm of genetically related viruses known as a quasispecies. Recent work has shown that quasispecies complexity and evolutionary rates are associated with the outcome of acute infection. Knowledge of how the virus population evolves at different stages of chronic infection is less clear. We have studied rates of evolution of the first hypervariable region (HVR1) of the E2 envelope protein in six individuals with disparate liver disease severity. These data show that virus populations present in individuals with mild non-progressive liver disease evolve in a typical Darwinian fashion, with a consistent accumulation of non-synonymous (amino acid-changing) substitutions. By contrast, the virus population remains relatively static in individuals with severe progressive liver disease. Possible mechanisms for this disparity are discussed.

Characterization of putative stem cells in isolated human colonic crypt epithelial cells and their interactions with myofibroblasts

Colonic epithelial stem cells are believed to be located at the crypt base where they have previously been shown to express musashi-1. The colonic stem cell niche, which includes extracellular matrix and myofibroblasts (together with other cell types), is likely to be important in maintaining the function of the progenitor cells. The aims of our studies were to characterize stem cells in isolated and disaggregated human colonic crypt epithelial cells and investigate their interactions with monolayers of primary human colonic myofibroblasts. In unfractionated preparations of disaggregated colonic crypts, musashi-1 positive cells preferentially adhered to colonic myofibroblasts, despite the presence of excess blocking anti-beta(1)-integrin antibody. These adherent epithelial cells remained viable for a number of days and developed slender processes. Cells with side population characteristics (as demonstrated by ability to expel the dye Hoechst 33342) were consistently seen in the isolated colonic crypt epithelial cells. These side population cells expressed musashi-1, beta(1)-integrin, BerEP4, and CD133. Sorted side population crypt epithelial cells also rapidly adhered to primary colonic myofibroblasts. In conclusion, in preparation of isolated and disaggregated human colonic crypts, cells with stem cell characteristics preferentially adhere to primary human colonic myofibroblasts in a beta(1)-integrin-independent fashion.

Assessment of Be1 and Be2 cells in systemic lupus erythematosus indicates elevated interleukin-10 producing CD5+ B cells

Recent studies indicate that normal B cells can be primed to differentiate into two distinct cytokine- secreting effector subsets, Be1 and Be2. The aim of this study was to analyse, for the first time, Be1 and Be2 cells at the single cell level in SLE patients using the recently developed technique of flow cytometry for intracellular cytokines. Peripheral blood mononuclear cells (PBMC) from SLE patients and age- and sex-matched normal controls were cultured for 24 h in the presence or absence of phorbal myristate acetate and ionomycin (PMA=I) or lipopolysaccharide (LPS). The production of type 1 (IFN-g, IL-2) and type 2 (IL-4, IL-5, IL-6, IL-10, IL-13) cytokines by B cells (and IL-10 production by fractionated CD5+ and CD57 B cells) was investigated using an intracellular cytokine staining technique and flow cytometry. In the absence of PMA=I stimulation, the percentage of B cells from SLE patients was significantly lower than those of normal subjects and significantly more SLE B cells spontaneously produced IL-10 than controls. Moreover, CD5+ B cells from SLE patients were enriched for cells with signs of previous in vivo activation and for high levels of IL-10 production. A significant positive correlation was observed between the percentage of IL-10- and IL-6-producing PMA=I-stimulated B cells in SLE patients, but not in controls. There were no significant differences in the production of other cytokines by B cells of SLE patients and normal subjects. In conclusion, a general alteration of type 1 and type 2 cytokine production by B cells is not observed in SLE patients. The role of B cell cytokines in the pathogenesis of SLE appears to be exerted by elevated secretion of in vivo IL-10, which may play an important role in the immune dysregulation observed in SLE patients. Moreover, the cross regulation of IL-10 and IL-6 is disrupted in SLE patients.

Characterization of Humoral and Cellular Immune Responses Elicited by Meningococcal Carriage

ABSTRACT In order to study the immune response elicited by asymptomatic carriage of Neisseria meningitidis, samples of serum, peripheral blood mononuclear cells (PBMCs), and saliva were collected from a cohort of more than 200 undergraduate students in Nottingham, United Kingdom, who were subject to high rates of acquisition and carriage of meningococci. Serum immunoglobulin G levels were elevated following increases in the rate of carriage, and these responses were specific for the colonizing strains. In order to investigate T-cell responses, PBMCs from 15 individuals were stimulated with a whole-cell lysate of the H44/76 meningococcal strain (B:15:P1.7,16), stained to detect cell surface markers and intracellular cytokines, and examined by flow cytometry. The cells were analyzed for expression of CD69 (to indicate activation), gamma interferon (IFN-γ) (a representative T-helper 1 subset [Th1]-associated cytokine), and interleukin-5 (IL-5) (a Th2-associated cytokine). Following a brief meningococcal stimulation, the numbers of CD69+ IFN-γ+ CD56/16+ NK cells were much higher than cytokine-positive CD4+ events. Both IFN-γ+ and IL-5+ events were detected among the CD69+ CD4+ population, leading to the conclusion that an unbiased T-helper subset response was elicited by meningococcal carriage.