Cornelia Doebis

Heme oxygenase-1 inhibits rat and human breast cancer cell proliferation: mutual cross inhibition with indoleamine 2,3-dioxygenase

Heme oxygenase‐1 (HO‐1) is the rate limiting enzyme of heme catabolism whereas indoleamine 2,3 dioxygenase (IDO) catabolizes tryptophan through the kynurenine pathway. We analyzed the expression and biological effects of these enzymes in rat and human breast cancer cell lines. We show that rat (NMU and 13762) but not human cells (MCF‐7 and T47D) express HO‐1. When overexpressed, we found this enzyme to have anti‐proliferative and proapoptotic effects by antioxidant mechanisms in these four cell lines. We show that IDO is expressed by rat and human breast cancer cells. IDO inhibition with 1‐MT and siRNA leads to diminished proliferation in rat cells. In contrast, HO‐1 negative human cell lines increase proliferation upon IDO inhibition. Since we also demonstrate that IDO inhibits the anti‐proliferative HO‐1, we propose that IDO has opposite effects on proliferation depending on the coexpression or not of HO‐1. We also describe that HO‐1 inhibits IDO at the post‐translational level through heme starvation. In vivo, we show that rat normal breast expresses HO‐1 and IDO. In contrast, N‐nitrosomethylurea‐induced breast adenocarcinomas only express IDO. In conclusion, we show that HO‐1/IDO cross‐regulation modulates apoptosis and proliferation in rat and human breast cancer cells.—Hill, M., Pereira, V., Chauveau, C., Zagani, R., Remy, S., Tesson, L., Mazal, D., Ubillos, L., Brion, R., Ashgar, K., Mashreghi, M. F., Kotsch, K., Moffett, J., Doebis, C., Seifert, M., Boczkowski, J., Osinaga, E., Anegon, I. Heme oxygenase‐1 inhibits rat and human breast cancer cells proliferation: mutual cross inhibition with indoleamine 2,3‐dioxygenase. FASEB J. 19, 1957–1968 (2005)

Autoregulation of Th1-mediated inflammation by twist1

The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor κB (NF-κB)–dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-κB, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohns disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lymphocytes limited the expression of the cytokines interferon-γ, IL-2, and tumor necrosis factor-α, and ameliorated Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis.

Deficient EBV-Specific B- and T-Cell Response in Patients with Chronic Fatigue Syndrome

Epstein-Barr virus (EBV) has long been discussed as a possible cause or trigger of Chronic Fatigue Syndrome (CFS). In a subset of patients the disease starts with infectious mononucleosis and both enhanced and diminished EBV-specific antibody titers have been reported. In this study, we comprehensively analyzed the EBV-specific memory B- and T-cell response in patients with CFS. While we observed no difference in viral capsid antigen (VCA)-IgG antibodies, EBV nuclear antigen (EBNA)-IgG titers were low or absent in 10% of CFS patients. Remarkably, when analyzing the EBV-specific memory B-cell reservoir in vitro a diminished or absent number of EBNA-1- and VCA-antibody secreting cells was found in up to 76% of patients. Moreover, the ex vivo EBV-induced secretion of TNF-α and IFN-γ was significantly lower in patients. Multicolor flow cytometry revealed that the frequencies of EBNA-1-specific triple TNF-α/IFN-γ/IL-2 producing CD4+ and CD8+ T-cell subsets were significantly diminished whereas no difference could be detected for HCMV-specific T-cell responses. When comparing EBV load in blood immune cells, we found more frequently EBER-DNA but not BZLF-1 RNA in CFS patients compared to healthy controls suggesting more frequent latent replication. Taken together, our findings give evidence for a deficient EBV-specific B- and T-cell memory response in CFS patients and suggest an impaired ability to control early steps of EBV reactivation. In addition the diminished EBV response might be suitable to develop diagnostic marker in CFS.

Intrabody-mediated phenotypic knockout of major histocompatibility complex class I expression in human and monkey cell lines and in primary human keratinocytes

Cultured keratinocyte allografts from unrelated donors can be readily grown as sheets in large-scale cell culture and have been used as an immediate skin cover for severely burned patients. Despite the absence of passenger leukocytes and the unlimited amount of material that can be obtained for permanent skin coverage, the allografts are susceptible to rejection. Since MHC class I (MHCI) antigens serve as targets for allograft rejection, we investigated whether ‘phenotypic knockout’ of human MHCI could be achieved through expression of an ER-directed anti-human MHCI single-chain intrabody (sFvhMHCI) that is directed against a monomorphic, conformational epitope, expressed across species lines, on the MHCI heavy chain. Co-immunoprecipitation of both MHCI heavy chain and β2-microglobulin occurred in transfected monkey COS-1 cells, while Jurkat T cells stably expressing the ER-directed sFvhMHCI intrabody showed that complete phenotypic knockout of MHCI cell surface expression could be achieved. Infection of several human cell lines of divergent tissue sources and different HLA haplotypes resulted in marked down-regulation of MHCI expression, even under conditions where inflammatory cytokines (eg γ-IFN) which up-regulate MHCI expression were used. Finally, when adenovirus encoding the anti-human MHCI intrabody was used to transduce primary human keratinocytes, a marked reduction of surface MHCI expression was observed. These in vitro studies set the groundwork for in vivo studies to determine if intrabody-mediated knockout of MHCI can impair alloantigen expression and prolong the survival of keratinocyte allografts.

Decline of surface MHC I by adenoviral gene transfer of anti-MHC I intrabodies in human endothelial cells—new perspectives for the generation of universal donor cells for tissue transplantation

The seeding of small‐calibre vascular polytetrafluoroethylene (PTFE) grafts with endothelial cells provides an increase in biocompatibility of the graft surface. The harvest and ex vivo culture of autologous endothelial cells is highly delicate. Allogeneic human umbilical vein endothelial cells (HUVEC) could be a potential cell source—however, rejection might occur due to major histocompatibility complex (MHC) I mismatches. Lowering cell surface MHC I expression on endothelial cells by gene transfer of an anti‐MHC I intrabody might reduce graft failure. The intrabody consists of a single‐chain variable fragment (sFv) of an anti‐MHC I antibody, carrying a terminal KDEL sequence to retain the molecule together with the MHC I inside the endoplasmic reticulum.

The Lymphocyte Transformation Test for Borrelia Detects Active Lyme Borreliosis and Verifies Effective Antibiotic Treatment

Borrelia-specific antibodies are not detectable until several weeks after infection and even if they are present, they are no proof of an active infection. Since the sensitivity of culture and PCR for the diagnosis or exclusion of borreliosis is too low, a method is required that detects an active Borrelia infection as early as possible. For this purpose, a lymphocyte transformation test (LTT) using lysate antigens of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii and recombinant OspC was developed and validated through investigations of seronegative and seropositive healthy individuals as well as of seropositive patients with clinically manifested borreliosis. The sensitivity of the LTT in clinical borreliosis before antibiotic treatment was determined as 89,4% while the specificity was 98,7%. In 1480 patients with clinically suspected borreliosis, results from serology and LTT were comparable in 79.8% of cases. 18% were serologically positive and LTT-negative. These were mainly patients with borreliosis after antibiotic therapy. 2.2% showed a negative serology and a positive LTT result. Half of them had an early erythema migrans. Following antibiotic treatment, the LTT became negative or borderline in patients with early manifestations of borreliosis, whereas in patients with late symptoms, it showed a regression while still remaining positive. Therefore, we propose the follow-up monitoring of dis-seminated Borrelia infections as the main indication for the Borrelia-LTT.

Cellular Players and Role of Selectin Ligands in Leukocyte Recruitment in a T-Cell-Initiated Delayed-Type Hypersensitivity Reaction

Delayed-type hypersensitivity (DTH) reactions are characterized by a strong cellular infiltrate, including neutrophils, macrophages, and T lymphocytes. In all these cell types, both E- and P-selectin-dependent adhesion pathways play a significant role in recruitment into the inflamed skin. Accordingly, inhibition of selectin-mediated interactions (eg, by antibodies) results in impairment of acute DTH reactions. However, whether inhibition of a specific cell type is responsible for the anti-inflammatory effect or whether all leukocytes are affected remains unclear. To address this question, we used fucosyltransferase-VII knockout mice that lack functional selectin ligands as either donors or recipients in a DTH model elicited by Th1 cell and antigen transfer. We found that selectin-mediated adhesion is required by Th1 effector cells to enter the DTH reaction site and, additionally, to elicit the DTH reaction. On the other hand, elimination of selectin binding in the recipients neutrophils and macrophages by use of fucosyltransferase-deficient mice receiving wild-type Th1 effector cells resulted in a strongly reduced infiltration of neutrophils and macrophages but unimpaired footpad swelling. These findings demonstrate a major role for both E- and P-selectin in the recruitment of different leukocyte cell types. However, only the presence of selectin ligands on T cells was critical for the inflammatory reaction. These findings reveal T cells as the predominant targets for selectin blockade that aim to suppress skin inflammation.

T cells as pioneers: antigen-specific T cells condition inflamed sites for high-rate antigen-non-specific effector cell recruitment

Cellular infiltration is a classic hallmark of inflammation. Whereas the role of T cells in many types of inflammation is well established, the specific impact of antigen recognition on their migration into the site and on the accumulation of other effector cells are still matters of debate. Using a model of an inflammatory effector phase driven by T‐cell receptor (TCR) transgenic T cells, we found (i) that antigen‐specific T cells play a crucial role as ‘pioneer cells’ that condition the tissue for enhanced recruitment of further T effector cells and other leucocytes, and (ii) that the infiltration of T cells is not dependent on antigen specificity. We demonstrate that a small number of antigen‐specific T cells suffice to initiate a cascade of cellular immigration into the antigen‐loaded site. Although antigen drives this process, accumulation of T cells in the first few days of inflammation was not dependent on T‐cell reactivity to the antigen. Both transgenic and wild‐type T effector cells showed enhanced immigration into the site of antigen challenge after the initial arrival and activation of antigen‐specific pioneer cells. This suggests that bystander accumulation of non‐specific effector/memory T cells is a general feature in inflammation. Furthermore, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ were identified as mediators that contribute to conditioning of the inflammatory site for high‐rate accumulation of T effector cells in this T‐cell‐driven model.

Efficient in vitro transduction of epithelial cells and keratinocytes with improved adenoviral gene transfer for the application in skin tissue engineering.

The adenovirus-mediated transfer of therapeutic genes into keratinocytes may be a useful approach to treat several skin diseases or to improve the graft take of in vitro generated skin equivalents used for wound coverage. However, in contrast to many other tissues, keratinocytes are relatively difficult to transduce by adenoviral vectors. To achieve high efficiency of adenoviral transduction into epithelial cells we investigated the effects of the polycation polybrene on the infection process. The human (HaCaT, A549) and rat (NBT II, MHICI) epithelial cell lines, as well as human and rat primary keratinocytes, were transduced with recombinant Ad(beta)-gal adenovirus, encoding for the reporter gene E. coli beta-galactosidase, in the presence of various polybrene concentrations. We determined the amount of beta-gal positive cells by X-gal staining and the beta-gal expression by ONPG-assay after 24 h. In all tested human and rat epithelial cell lines, as well as in human and rat primary keratinocytes, the addition of polybrene during adenoviral transduction of Ad(beta)-gal resulted in a marked increase of beta-gal positive cells and beta-gal protein expression. The efficacy of polybrene showed a clear dose dependency. The improvement of adenoviral gene transfer into various types of human and rat epithelial cells by polybrene allows us to reduce the amount of recombinant virus particles resulting in a decreased inflammation induced by this therapeutic agent. In addition, the efficient transduction and expression with enhanced adenoviral transfer of therapeutic genes into primary keratinocytes provides a powerful tool for analysing the functions and the regulation of a gene of interest in vitro.

Modulation of Graft Arteriosclerosis in a Rat Carotid Transplantation Model

BACKGROUND Venous autografts used in cardiovascular surgery tend to deteriorate over time due to arteriosclerotic complications. Cadaveric vascular allografts represent a possible alternative for this application, but donor endothelial cells (ECs) and antigen presenting cells of the graft trigger alloresponses mediated by MHC class I (MHC I) antigen, leading to graft failure. Vascular allograft rejection might be prevented by reducing cell surface expression of MHC I and thereby lowering the immunogenicity of the grafts. MATERIAL AND METHODS An Intrabody approach was used to reduce MHC I expression in vascular allografts. The efficacy of an adenovirus (Ad) carrying an anti-MHC I Intrabody gene (Ad-Intrabody) was first tested in vitro using rat aortic ECs. The effect of the Ad-Intrabody was then studied in vivo by a model of rat carotid artery transplantation. Grafts were analyzed 7 and 28 days after transplantation by immunohistochemistry and real time reverse transcriptase-polymerase chain reaction. RESULTS Ad-Intrabody gene transfer reduced MHC I surface expression of rat ECs and inhibited in vivo alloimmune responses to carotid allografts. Decreased T cell and macrophage infiltration was observed within Ad-Intrabody transduced arterial allografts at day 28. This was associated with an inhibition of intimal thickening formation. Analysis of mRNA showed diminished levels of T cell markers and Interferon-gamma expression in the Ad-Intrabody-treated group compared with control groups. CONCLUSIONS Ex vivo adenoviral gene transfer of an Intrabody against MHC I into rat carotid arteries prior to transplantation reduced both graft arteriosclerosis and inflammation in the absence of any systemic immunosuppression.