M. Anthuber

Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor

Conventional immunosuppressive drugs have been used effectively to prevent immunologic rejection in organ transplantation. Individuals taking these drugs are at risk, however, for the development and recurrence of cancer. In the present study we show that the new immunosuppressive drug rapamycin (RAPA) may reduce the risk of cancer development while simultaneously providing effective immunosuppression. Experimentally, RAPA inhibited metastatic tumor growth and angiogenesis in in vivo mouse models. In addition, normal immunosuppressive doses of RAPA effectively controlled the growth of established tumors. In contrast, the most widely recognized immunosuppressive drug, cyclosporine, promoted tumor growth. From a mechanistic perspective, RAPA showed antiangiogenic activities linked to a decrease in production of vascular endothelial growth factor (VEGF) and to a markedly inhibited response of vascular endothelial cells to stimulation by VEGF. Thus, the use of RAPA, instead of cyclosporine, may reduce the chance of recurrent or de novo cancer in high-risk transplant patients.

Overexpression of melanoma inhibitory activity (MIA) enhances extravasation and metastasis of A-mel 3 melanoma cells in vivo

The secreted MIA protein is strongly expressed by advanced primary and metastatic melanomas but not in normal melanocytes. Previous studies have shown that MIA serum levels correlate with clinical tumour progression in melanoma patients. To provide direct evidence that MIA plays a role in metastasis of malignant melanomas, A-mel 3 hamster melanoma cells were transfected with sense- and antisense rhMIA cDNA and analysed subsequently for changes in their tumorigenic and metastatic potential. Enforced expression of MIA in A-mel 3 cells significantly increased their metastatic potential without affecting primary tumour growth, cell proliferation or apoptosis rate in hamsters, compared with control or antisense transfected cells. Additionally, MIA overexpressing transfectants showed a higher rate of both tumour cell invasion and extravasation. Cells transfected with MIA antisense generally exerted an opposite response. The above changes in function attributed to the expression of MIA may underlie the contribution of MIA to the malignant phenotype.

Factors Associated With Disease Progression in Patients With Gastrointestinal Stromal Tumors in the Pre-Imatinib Era

The aim of this study was to determine the predictors of survival in 38 patients with curatively resected gastrointestinal stromal tumors (GISTs). The tumor was located in the stomach in 23 cases, the small bowel in 13, and the colon in 2. In 23 patients (61%), a mutation in exon 11 of the kit gene was detected. In 7 cases, all small gastric tumors, a mutation in the platelet-derived growth factor receptor a (PDGFRA) gene was detected. The overall 5-year survival rate was 70%. In 9 patients, GISTs relapsed, leading to an actuarial 5-year disease-free survival of 78%. By multivariate analysis, the presence of distant metastases, the proliferative (MIB-1) index, and deletional mutation in codons 557 and/or 558 of kit exon 11 correlated significantly with poor outcome. None of the PDGFRA mutant GISTs relapsed. These findings suggest a strong relationship between various tyrosine kinase receptor mutations and survival outcome in patients with GISTs.

Blocking MAdCAM-1 in vivo reduces leukocyte extravasation and reverses chronic inflammation in experimental colitis

BackgroundLeukocyte recruitment to sites of intestinal inflammation is a crucial multi-step process, leading ultimately to the accumulation of cells in the inflamed tissue. These interactions in the gut are critically dependent on the mucosal addressin cell adhesion molecule-1 (MAdCAM-1), which is expressed on endothelial cells within the mesenteric lymph nodes and the lamina propria of the intestine. Here, we investigate the pathophysiologic role of MAdCAM-1 in the intestinal microcirculation in vivo.MethodsUsing a standard mouse model, chronic colitis was established after four cycles of dextran sodium sulfate (DSS) application. MAdCAM-1 expression was investigated by immunohistochemistry and Western blotting, as well as real-time polymerase chain reaction (PCR). Intravital microscopy was used to study the role of MAdCAM-1 on leukocyte-endothelium interactions and leukocyte extravasation.ResultsSignificant changes in MAdCAM-1 were observed in mice with chronic DSS-induced colitis. Upregulation of MAdCAM-1 expression in chronic colitis was demonstrated on a protein and messenger ribonucleic acid (mRNA) level. Anti-MAdCAM-1 treatment lead to a marked reduction (>60%) of leukocyte sticking and extravasation in vivo, compared to the controls. This was parallelled by a significant reduction (45%) of intestinal inflammation, as measured by the histologic grading score.ConclusionThese in vivo results demonstrate a distinct role of MAdCAM-1 in inflammatory intestinal diseases, and suggest that therapeutic strategies targeting this adhesion molecule could be useful in the treatment of chronic colitis.

Rapamycin decreases leukocyte migration in vivo and effectively reduces experimentally induced chronic colitis

BackgroundImmunosuppressive calcineurin inhibitors, like cyclosporine (CsA), can be used for the clinical management of severe ulcerative colitis. However, patients treated with CsA are at a risk for developing kidney failure and may be more susceptible to colon cancer. Furthermore, severe neurotoxicity and hypertension are common problems. To avoid the side effects of CsA, new immunosuppressive drugs to treat colitis are needed. The aim of the present study was to test the immunosuppressive mammalian target of rapamycin inhibitor rapamycin in an experimental model of chronic colitis and to compare its effectiveness with CsA.MethodsChronic colitis was established in Balb/c mice after four feeding cycles of dextran sodium sulfate. Because leukocyte recruitment to sites of intestinal inflammation is crucial for the development of chronic colitis, intravital microscopy was used to study the effect of rapamycin and CsA on leukocyte–endothelium interactions and leukocyte extravasation. To assess the degree of colitis, histological sections were evaluated.ResultsBoth rapamycin and cyclosporine effectively reduced leukocyte sticking (>60%) in submucosal venules, as compared to controls. Furthermore, rapamycin, but not CsA, reduced (>35%) leukocyte extravasation in the mucosa. Both rapamycin and CsA treatments significantly improved the histologic inflammation score.ConclusionOur in vivo results demonstrate that rapamycin reduces leukocyte sticking and extravasation during chronic colitis induction and proves to be as effective as CsA at reducing experimental chronic colitis. These results support the use of rapamycin in clinical trials to avoid serious side effects of CsA therapy in chronic colitis patients.

Preferential Migration of CD62L+ Cells into the Appendix in Mice with Experimental Chronic Colitis

Background: Clinical and experimental studies suggest that appendectomy can protect against development of ulcerative colitis and Crohn’s disease. However, how T cells in the appendix affect the development of colitis has not been clarified. Aim: To investigate the in vivo migration and activation of colitis-inducing CD62L⁺ cells during development of chronic colitis. Methods: CD62L⁺CD4⁺ cells were fluorescently labeled and transferred to severe combined immunodeficient (SCID) mice to induce colitis. In vivo migration of T cells into the mucosa of the appendix and colon was quantified by in vivo microscopy after 7 weeks. In a second experiment, unlabeled CD62L⁺CD4⁺ cells were transferred, reisolated after 7 weeks, and adhesion molecule (integrin α₄β₇) and costimulatory molecule (CD154) expression was analyzed. Results: Six to eight weeks after CD62L⁺CD4⁺ cell transfer, SCID mice developed chronic colitis. In vivo microscopic analysis demonstrated a preferential migration of fluorescence-labeled CD62L⁺CD4⁺cells into the mucosa of the appendix versus the colon. Re-isolation of lamina propria cells from mice with colitis confirmed that CD62L⁺CD4⁺ cell migration was significantly enhanced in the appendix, compared to the colon (3.5-fold). Furthermore, a higher proportion of CD62L⁺CD4⁺ cells re-isolated from the appendix expressed integrin α₄β₇ and CD154 than from the colon. Conclusion: This study demonstrates the preferential migration of CD62L⁺CD4⁺cells into the appendix as compared to the colon. This migration pattern correlated with upregulation of integrin α₄β₇and CD154 (CD40 ligand) on T cells. Our results suggest an important role of the appendix in the pathogenesis of colitis.

Short-term treatment with anti-CD44v7 antibody, but not CD44v4, restores the gut mucosa in established chronic dextran sulphate sodium (DSS)-induced colitis in mice

Increased expression of CD44 variant isoforms have been shown on the inflammatory infiltrates in human and mouse colitis and blockade or deletion of CD44 isoforms inhibit experimental colitis. The objective of this study was to find out if short‐term treatment of CD44 antibodies specific to CD44v7, but not to other variant isoforms, suppresses leucocyte–endothelial interaction in chronic dextran sodium sulphate (DSS)‐induced colitis in mice. Chronic colitis was induced by oral administration of four cycles of 5% DSS in BALB/c mice. Expression of CD44 was investigated on isolated mononuclear cells of the gut immune system. In established colitis, mice were treated with antibodies against CD44v7 or CD44v4 three times in 7 days. Intravital microscopy was used to study leucocyte–endothelial interactions and leucocyte extravasation. As a marker of inflammatory infiltrates myeloperoxidase was quantified in gut tissue. CD44‐induced apoptosis was determined by fluorescence staining of hypodiploidic cell nuclei. In chronic DSS‐induced colitis both CD44 variant isoforms, v4 and v7 were significantly up‐regulated on mononuclear cells. However, whereas anti‐CD44v7 antibody treatment induced a marked restoration of the gut mucosa and significantly reduced endothelial sticking and extravasation of circulating leucocyte in vivo (P < 0·01), application of anti‐CD44v4 or an isotype control antibody had no anti‐inflammatory effect. A significant reduction of myeloperoxidase activity was detected after blockade of CD44v7, but not v4. Short‐term treatment with anti‐CD44v7 antibody blocks T cell extravasation and recruitment to the intestinal mucosa and cures established experimental colitis.

Rapamycin treatment at immunosuppressive doses affects tumor blood vessel circulation

RECENTLY WE reported that the immunosuppressive drug rapamycin also has potent anti-angiogenic effects that can control tumor growth in mice. The antiangiogenic effect was shown to be mediated, at least in part, by inhibition of vascular endothelial growth factor (VEGF) production and VEGF-induced endothelial cell signaling. This combined action reduced new blood vessel formation in established tumors, causing tumor growth to be limited by the lack of angiogenesis. Interestingly, we also noted by intravital microscopy that blood tended to form pools in the development phase of implanted tumors. In the present follow-up study, we more closely examined the flow dynamics within the blood vessels of control tumors and tumors in mice treated with usual immunosuppressive doses of rapamycin. The system we used was a dorsal skin-fold chamber, where blood vessel formation and flow dynamics could be directly observed by real-time intravital microscopy.

Einfluß von Immunsuppressiva auf die Mikrozirkulation und Leukozyten Endothelinteraktion nach Ischämie Reperfusion

As ischemia/reperfusion injury (I/R) and rejection are closely interrelated phenomena, we aimed to examine the impact of immunosuppressive drugs on microvascular I/R injury in vivo and in vitro. By means of intravital microscopy leukocyte-endothelial interaction, functional capillary density (FCD), and microvascular permeability were investigated after application of immunosuppressive doses of Cyclosporin A(CyA, 20 mg/kg), Tacrolimus (FK 506, 0.4 mg/kg) and Mycophenolat Mofetil (MMF, 20 mg/kg) (n = 6) in a model of 3 h pressure induced ischemia in the dorsal skinfold chamber of BalbC mice. The effect of CyA, FK 506 and MMF on in vitro leukocyte adherence and adhesion molecule expression (ICAM-1, VCAM-1, E-selectin determined by FACS) on IL-1s stimulated HUVEC cells were assessed. CyA and FK 506 significantly reduced microvascular I/R injury, indicated by reduced leukocyte adhesion, improved macromolecular permeability and FCD in vivo. In vitro, CyA and FK 506 attenuated PMN adherence, ICAM-1, VCAM-1 and E-selectin expression. Although MMF inhibited endothelial adhesion molecule expression as well, it was less effective in reducing PMN adhesion in vitro as well as attenuating microvascular I/R injury in vivo, indicating an endothelial specificity of MMF. The efficacy of CyA and FK506 on I/R injury raises the question of a earlier treatment of donors and may explain the inefficiency of new drugs attenuating I/R in clinical trials

Kolitis induzierende T-Zellen migrieren verstärkt in den Appendix

Background: Previous studies showed a distinct role of the cecum and appendix in the development of colitis [3] [2]. Transferred CD45RBhigh T cells targeting the colon induce colitis in scid mice [4]. However, the proportional distribution of migrating cells into colon, cecum and appendix has not been clarified. Purpose of the study was to investigate the proportion of migrated T cells into appendix and cecum vs. colon in vivo. Methods: CD4+ cells were isolated from the spleen of Balb/c mice using magnetic activated cell sorting (MACS). Then CD45Rbhigh cells were purified and labeled by a fluorescence kit. After control of purity and cell viability by FACS, Balb/c SCID mice were reconstituted with 500,000 CD45RBhigh cells i.p. (Tcell transfer). After onset of colitis (8 weeks) in vivo microscopy was performed. In inhalation anesthesia the colon was mobilized and exteriorized for investigation [1]. T cell migration into mucosa of appendix, cecum and colon and additionally into the spleen was quantified in vivo (T cells/mm2 mucosa). Lamina propria CD4+ cells were reisolated and adhesion molecule expression was analysed by flow cytometry. To reveal onset of colitis histologic specimens and clinical signs were scored. Results: Eight weeks after CD45RBhigh T cell transfer SCID mice showed severe signs of colitis clinically and by histology. Quantification by in vivo microscopy showed an increased predominant migration of fluorescence labelled T cells into the mucosa of cecum and appendix compared to the rest of the colon. Transferred T cells were not found in the spleen. Reisolation of lamina propria CD4+ cells from the different compartments showed that Tcell migration was significantly enhanced in the appendix and cecum compared to the colon (38,6 ± 2,8 appendix tissue vs. 10,8±0,7/μg colon tissue). Reisolated T cells from both compartments showed high expression of LFA-1 and ICAM-1 and low expression of a4β7 and costimulatory receptor CD154. Conclusion: The present study shows for the first time migration of colitis inducing T cells in vivo over a longer time period. Predominant migration of T cells into the appendix compared to the rest of the colon were found in vivo and in vitro. Our results support the pivotal role of the appendix in the pathogenesis of colitis. The specific interaction of appendix tissue and T cells must be the content of further studies.