Elsie Oppermann

Inactivating SOCS1 mutations are caused by aberrant somatic hypermutation and restricted to a subset of B cell lymphoma entities

STATs are constitutively activated in several malignancies. In primary mediastinal large B-cell lymphoma and Hodgkin lymphoma (HL), inactivating mutations in SOCS1, an inhibitor of JAK/STAT signaling, contribute to deregulated STAT activity. Based on indications that the SOCS1 mutations are caused by the B cell-specific somatic hypermutation (SHM) process, we analyzed B-cell non-HL and normal B cells for mutations in SOCS1. One-fourth of diffuse large B-cell lymphoma and follicular lymphomas carried SOCS1 mutations, which were preferentially targeted to SHM hotspot motifs and frequently obviously inactivating. Rare mutations were observed in Burkitt lymphoma, plasmacytoma, and mantle cell lymphoma but not in tumors of a non-B-cell origin. Mutations in single-sorted germinal center B cells were infrequent relative to other genes mutated as byproducts of normal SHM, indicating that SOCS1 inactivation in primary mediastinal large B-cell lymphoma, HL, diffuse large B-cell lymphoma, and follicular lymphoma is frequently the result of aberrant SHM.

Inhibition of endothelial receptor expression and of T-cell ligand activity by mycophenolate mofetil

The novel immunosuppressive drug mycophenolate mofetil (CellCept, MMF) blocks DNA-synthesis by the inhibition of the enzyme inosine monophosphate dehydrogenase (IMDH). IMDH is also involved in the synthesis of adhesion receptors which are known to play an important role in the regulation of cell-cell contacts. Therefore, application of MMF might lead to a reduction of cellular infiltrates in the course of transplant rejection. To evaluate the therapeutic value of MMF, we investigated to what extent MMF blocks T-lymphocyte infiltration in vitro with regard to (a) adhesion to endothelial cells, (b) horizontal migration along these cells and (c) penetration through the endothelial cells. The results demonstrated a strong inhibition of both CD4+ and CD8+ T-cell adhesion and penetration by MMF. The ID50 value for CD4+ T-cell adhesion was calculated to be 0.03 microM and the ID50 value for CD4+ T-cell penetration 1.21 microM. MMF did not significantly influence the horizontal migration of T-lymphocytes along the human vascular endothelial cell (HUVEC) borders. FACS-analysis revealed a diminished E-selectin and P-selectin expression on endothelial cell membranes in the presence of MMF. Although MMF did not interfere with the synthesis of T-cell adhesion ligands, the binding activity of lymphocytic leucocyte function associated antigen 1 (LFA-1), very late antigen 4 (VLA-4) and PSGL-1 (P-selectin glycoprotein ligand 1) to immobilized intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin was impaired. Moreover, MMF prevented VLA-4 and PSGL-1 receptor accumulation on the membranes of T-cell pseudopodia. It can be concluded that MMF possesses potent infiltration blocking properties. MMF evoked down-regulation of specific endothelial membrane molecules and the loss of protein localization in the lymphocyte protrusions might be predominantly responsible for the observed blockade of cell adhesion and penetration.

Transarterial chemoembolization alone and in combination with other therapies: a comparative study in an animal HCC model

The purpose of this study is to compare transarterial chemoembolization (TACE) alone and in combination with other therapies in an animal model. Subcapsular implantation of a solid Morris hepatoma 3924A in the liver was carried out in 50 male ACI rats (day 0). Tumor volume (V1) was measured by MRI (day 13). After laparotomy and retrograde placement of a catheter into the gastroduodenal artery (day 14), the following protocols of the interventional procedure were applied: TACE (mitomycin C + lipiodol) + immunotherapy (group A: TNFα + IL-2, group B: OK-432 + IL-2); TACE + antiangiogenesis therapy (group C: TNP-470, group D: endostatin); TACE alone in group E (control group). Tumor volume (V2) was assessed by MRI and the mean ratio of x (V2/V1) was calculated. Data were analyzed using Dunnett’s t test (comparing therapeutic groups with the control group) and the Student-Newman-Keuls test (comparing significant therapeutic groups). Multivariate analysis showed a significant reduction in the tumor growth rate (P<0.05) in groups B (x=6.53) and C (x=4.01) compared to the mean ratio of the control group E (x=9.14). Significant results were observed in group C (P<0.05) in comparison with the other therapeutic groups. TACE combined with immunotherapy (OK-432) and antiangiogenesis therapy (TNP-470) retards tumor growth compared with TACE alone in an HCC animal model.

Preservation of the Synthetic and Metabolic Capacity of Isolated Human Hepatocytes by Coculture With Human Biliary Epithelial Cells

Bioartificial liver support systems have demonstrated limited efficacy in compensation of liver detoxification and substitution of liver‐derived factors. However, in these devices, the biological substitution of the complex liver function has been restricted to xenogeneic or transformed hepatocytes. Therefore, we have examined the long‐term effect of coculturing normal human hepatocytes (HCs) with allogeneic biliary epithelial cells (BECs). We applied functional in vitro assays to examine their metabolic potential by ammonia detoxification to urea, cytochrome P450‐dependent lignocaine conversion to mono‐ethyl‐glycine‐xylidide (MEGX), and protein expression and secretion. As the liver has a pivotal role in the synthesis of coagulation factors, we measured antithrombin III (AT III), factor VII, and albumin, comparing HCs plated on collagen or inside 3‐dimensional collagen gels. Over 30 days, expression and secretion of albumin and clotting factors by human HCs were augmented by culture inside collagen gel, but were also enhanced and better maintained by coculture with BECs. Higher proportions of BECs cocultured with HCs substantially increased the protein synthesis and urea production. Remarkably, the almost absent cytochrome P450 activity of HC alone after 1 week could be reversed and maintained over 3 weeks by coculture with BECs. The pattern of these effects differed from the extent of interleukin‐6 (IL‐6) production and HC viability under the compared conditions. In conclusion, coculture of human HCs with BECs impressively restores the synthetic and metabolic liver function in vitro. These results suggest mechanisms of improved liver epithelial differentiation supported by coculture conditions. This technique offers new perspectives in bioartificial liver support, hepatocyte transplantation, and stem cell differentiation. (Liver Transpl 2005;11:410–419.)

Clinical relevance of maspin expression in bladder cancer

Transitional cell carcinoma (TCC) of the bladder is a solid tumor that induces angiogenesis to maintain nutrition and oxygenation of tumor cells. Maspin, a serpin with tumor suppressing activity, has recently been identified as an inhibitor of angiogenesis. This study examined the impact of maspin expression in the growth pattern of TCC of the bladder. Maspin was identified in a panel of normal tissues, in several bladder carcinoma cell lines, and 51 patient samples of TCC of the bladder. Expression was detected by RT-PCR and immunohistochemistry. Furthermore, the level of maspin was correlated to the growth rate of bladder tumor cell lines in vitro and in vivo. Maspin expression was found in high quantities in normal urothelium. Maspin expression was preserved in superficial bladder cancers but was significantly diminished in invasive carcinomas. Within the group of invasive TCCs, maspin expression was inversely correlated to the patient prognosis. Furthermore, low maspin expression level was coupled to an increased tumor cell growth in vivo. Down-regulation of maspin expression seems to be a specific event in the progression of invasive bladder carcinoma. Maspin might be a useful marker to determine the prognosis of invasive TCC. Furthermore, maspin re-expression might become a therapeutic option in the treatment of invasive, metastatic TCC.

In vitro analysis of verapamil-induced immunosuppression: Potent inhibition of T cell motility and lymphocytic transmigration through allogeneic endothelial cells

BACKGROUND Cyclosporine A (CsA) and tacrolimus prevent proliferation but not transendothelial migration of alloreactive lymphocytes into donor organs. As a result, serious adverse effects, such as nephrotoxicity and neurotoxicity, have been observed under CsA/tacrolimus therapy. The incorporation of new drugs with infiltration blocking properties might enhance the efficacy of the current immunosuppressive protocol, allowing lower CsA/tacrolimus dosage. Because Ca2+ plays a critical role in cell-cell interaction, the Ca2+-channel blocker verapamil might be a good cany. didate for supporting CsA/tacrolimus-based therapy. METHODS A T-cell endothelial cell coculture model or immobilized immunoglobulin G globulin chimeras were employed to investigate how S- and R- verapamil interfere with the lymphocytic infiltration process. The expression and arrangement of membranous adhesion receptors and cytoskeletal F-actin filaments were analyzed by fluorometric method in the presence of. verapamil. RESULTS Both verapamil enantiomers strongly inhibited lymphocyte infiltration. CD4+ and CD8+ T-cells were influenced to a similar extent with regard to horizontal locomotion (CD4+=CD8+), but to a different extent with regard to adhesion and penetration (CD4+ > CD8+). Moreover, penetration was blocked to a higher extent than was adhesion. ID50-values were 31 microM (CD4+-adhesion) and 11 microM (CD4+-penetration). Verapamil reduced P-selectin expression on endothelial cells and effectively down-regulated binding of T-cells to immobilized P-selectin immunoglobulin G globulins (ID50=4.4 microM; CD4+). A verapamil-induced reduction of intracellular F-actin in T-lymphocytes was proven to be mainly responsible for diminished cell locomotion. CONCLUSIONS The prevention of CD4+ T-cell penetration by verapamil might argue for its use as an adjunct to CsA/tacrolimus-based immunosuppressive therapy.

Chronic Ethanol Feeding Modulates Inflammatory Mediators, Activation of Nuclear Factor-κB, and Responsiveness to Endotoxin in Murine Kupffer Cells and Circulating Leukocytes

Chronic ethanol abuse is known to increase susceptibility to infections after injury, in part, by modification of macrophage function. Several intracellular signalling mechanisms are involved in the initiation of inflammatory responses, including the nuclear factor-κB (NF-κB) pathway. In this study, we investigated the systemic and hepatic effect of chronic ethanol feeding on in vivo activation of NF-κB in NF-κBEGFP reporter gene mice. Specifically, the study focused on Kupffer cell proinflammatory cytokines IL-6 and TNF-α and activation of NF-κB after chronic ethanol feeding followed by in vitro stimulation with lipopolysaccharide (LPS). We found that chronic ethanol upregulated NF-κB activation and increased hepatic and systemic proinflammatory cytokine levels. Similarly, LPS-stimulated IL-1β release from whole blood was significantly enhanced in ethanol-fed mice. However, LPS significantly increased IL-6 and TNF-α levels. These results demonstrate that chronic ethanol feeding can improve the responsiveness of macrophage LPS-stimulated IL-6 and TNF-α production and indicate that this effect may result from ethanol-induced alterations in intracellular signalling through NF-κB. Furthermore, LPS and TNF-α stimulated the gene expression of different inflammatory mediators, in part, in a NF-κB-dependent manner.

CCL2 Chemokine as a Potential Biomarker for Prostate Cancer: A Pilot Study

Purpose Prostate specific antigen is not reliable in diagnosing prostate cancer (PCa), making the identification of novel, precise diagnostic biomarkers important. Since chemokines are associated with more aggressive disease and poor prognosis in diverse malignancies, we aimed to investigate the diagnostic relevance of chemokines in PCa. Materials and Methods Preoperative and early postoperative serum samples were obtained from 39 consecutive PCa patients undergoing radical prostatectomy. Serum from 15 healthy volunteers served as controls. Concentrations of CXCL12, CXCL13, CX3CL1, CCL2, CCL5, and CCL20 were measured in serum by Luminex. The expression activity of CXCR3, CXCR4, CXCR5, CXCR7, CXCL12, CXCL13, CX3CR1, CXCL1, CCR2, CCR5, CCR6, CCR7, CCL2, and CCL5 mRNA was assessed in tumor and adjacent normal tissue of prostatectomy specimens by quantitative real-time polymerase chain reaction. The associations of these chemokines with clinical and histological parameters were tested. Results The gene expression activity of CCL2 and CCR6 was significantly higher in tumor tissue compared to adjacent normal tissue. CCL2 was also significantly higher in the blood samples of PCa patients, compared to controls. CCL5, CCL20, and CX3CL1 were lower in patient serum, compared to controls. CCR2 tissue mRNA was negatively correlated with the Gleason score and grading. Conclusion Chemokines are significantly modified during tumorigenesis of PCa, and CCL2 is a promising diagnostic biomarker.

Characterization of Bone Marrow Mononuclear Cells on Biomaterials for Bone Tissue Engineering In Vitro

Bone marrow mononuclear cells (BMCs) are suitable for bone tissue engineering. Comparative data regarding the needs of BMC for the adhesion on biomaterials and biocompatibility to various biomaterials are lacking to a large extent. Therefore, we evaluated whether a surface coating would enhance BMC adhesion and analyze the biocompatibility of three different kinds of biomaterials. BMCs were purified from human bone marrow aspirate samples. Beta tricalcium phosphate (β-TCP, without coating or coated with fibronectin or human plasma), demineralized bone matrix (DBM), and bovine cancellous bone (BS) were assessed. Seeding efficacy on β-TCP was 95% regardless of the surface coating. BMC demonstrated a significantly increased initial adhesion on DBM and β-TCP compared to BS. On day 14, metabolic activity was significantly increased in BMC seeded on DBM in comparison to BMC seeded on BS. Likewise increased VEGF-synthesis was observed on day 2 in BMC seeded on DBM when compared to BMC seeded on BS. The seeding efficacy of BMC on uncoated biomaterials is generally high although there are differences between these biomaterials. Beta-TCP and DBM were similar and both superior to BS, suggesting either as suitable materials for spatial restriction of BMC used for regenerative medicine purposes in vivo.

Mycophenolate mofetil increases adhesion capacity of tumor cells in vitro.

Background. The immunosuppressive drug mycophenolate mofetil (MMF) reduces expression of the heterophilic binding elements intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and thereby prevents attachment of alloactivated leukocytes to donor endothelium. The authors speculated that MMF might further diminish receptors of the immunoglobulin superfamily which, however, act as homophilic binding elements. Because decrease of homophilic adhesion receptors correlates with tumor dissemination and metastasis, MMF could trigger development or recurrence of neoplastic tumors. Methods. The authors analyzed the influence of MMF on homotypic adhesion receptors and its consequence for tumor cell attachment to an endothelial cell monolayer. Neuroblastoma (NB) cells, which self-aggregate by means of the homophilic-binding element neural cell adhesion molecule (NCAM), were used. Effects of MMF on the 140- and 180-kDa NCAM isoforms were investigated quantitatively by flow cytometry, Western blot, and reverse-transcriptase (RT) polymerase chain reaction (PCR). The relevance of NCAM for tumor cell binding was proven by treating NB with NCAM antisense oligonucleotides. Results. MMF profoundly increased the number of adherent NB cells, with a maximum effect at 0.1 &mgr;M, compared with controls. Decrease of NCAM on the cell surface was detected by flow cytometry. Western blot and RT-PCR demonstrated reduced protein and RNA levels of the 140- and 180-kDa isoforms. Treatment of NB cells with NCAM antisense oligonucleotides showed that reduced NCAM expression leads to enhanced tumor cell adhesion. Conclusions. MMF decreases NCAM receptors, which is associated with enhanced tumor cell invasiveness. The authors conclude that an MMF-based immunosuppressive regimen might increase the risk of tumor metastasis if this process is predominantly conveyed by means of homophilic adhesion proteins.