Matthias Hornung

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.

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.

DX5+NKT cells induce the death of colitis-associated cells: involvement of programmed death ligand-1

NKT cells are activated by CD1d and show an immune regulating function. Here, we investigated whether DX5+NKT cells could be used to reduce colitis in a chronic colitis mouse model and studied the potential immunological mechanisms involved. Chronic colitis was induced either by transfer of enriched CD62L+CD4+ T cells to severe‐combined‐immunodeficient mice or by feeding dextran sodium sulfate to immune competent mice. DX5+NKT cells were transferred to mice with chronic colitis. Co‐transfer of DX5+NKT cells, but not CD8+ control cells, prevented the onset of colitis, and the immune regulatory effect of DX5+NKT cells was completely abrogated by injecting CD1d blocking antibody. Moreover, DX5+NKT cells reduced established colitis in both chronic colitis models. In vitro, DX5+NKT cells induced cell death of colon‐infiltrating lymphocytes isolated from diseased mice. This effect was inhibited in the presence of either anti‐CD1d or anti‐programmed death ligand‐1 (PD‐L1) blocking antibodies. The specific potency of DX5+NKT cells in regulating chronic colitis in two mouse models is demonstrated. In vitro testing suggests that DX5+NKT cells activated by CD1d induce cell death of colitis‐inducing lymphocytes, which is mediated through PD‐L1. Therefore, DX5+NKT cells could be important in the regulation of immune responses associated with 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.

The CD40 TRAF family member interacting motif carries the information to rescue WEHI 231 cells from anti-IgM-induced growth arrest

Engagement of the antigen receptor on WEHI 231 murine B lymphoma cells leads to growth arrest and induction of apoptosis. Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis. At the molecular level, CD40 has been shown to activate nuclear factor κB (NF‐κB) and stress‐activated protein kinase (SAPK). The aim of our present study was to define the stretch of the CD40 cytoplasmic tail responsible for mediating these effects in WEHI 231 cells. Using recombinant retroviruses with the enhanced green fluorescent protein as selection marker we transduced WEHI 231 cells with chimeric molecules consisting of the extracellular and transmembrane region of human CD40 or rat CD4 and selected portions of the murine CD40 tail. Chimeric molecules with cytoplasmic fragments encompassing the “CD40 tumor necrosis factor‐associated factor family member interacting motif” (TIM) were able to sustain growth and to uphold NF‐κB activity as efficiently as the whole intracellular region of CD40. While the potential of the motif relative to the whole cytoplasmic tail was independent of the heterologous part of the chimeras it was strongly influenced by its distance to the membrane. Placing the 17‐aminoacid stretch of the motif too close to the membrane, i.  e. only two or four amino acids apart, destroyed its capacity to mitigate the anti‐IgM effect. Activation of SAPK through the chimeric molecules always correlated with their ability to activate NF‐κB activity and to rescue the cells from apoptosis induced by antigen receptor ligation. Our data indicate that CD40‐TIM carries most if not all of the information needed to deliver the signals responsible for sustaining growth in anti‐IgM‐stimulated WEHI 231 cells.

Single-Incision Retroperitoneoscopic Adrenalectomy and Single-Incision Laparoscopic Adrenalectomy

OBJECTIVE Single-incision surgery is by now practicable in many fields of surgery, including surgery of the adrenal gland. We report on first experience with laparoscopic transperitoneal and retroperitoneoscopic single-incision adrenalectomy. PATIENTS AND METHODS Between September 2009 and February 2010, eight patients underwent single-incision adrenalectomy. Four patients received single-incision retroperitoneoscopic adrenalectomy, and four patients transperitoneal single-incision laparoscopic adrenalectomy. Technical feasibility and perioperative data are presented. RESULTS All patients had benign adrenal tumors (Conns adenoma, n = 7; pheochromocytoma, n = 1). Tumor size ranged between 1.2 and 2.4 cm. Mean operation time was 76 minutes for single-incision retroperitoneoscopic adrenalectomy and 82 minutes for single-incision laparoscopic adrenalectomy. Blood loss was irrelevant in both groups. CONCLUSIONS Single-incision adrenalectomy is safe and feasible in appropriate operation time, both by the retroperitoneoscopic technique and by the laparoscopic technique. It is also associated with good cosmetic outcome.

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.

Contrast-enhanced ultrasonography for localization of pathologic glands in patients with primary hyperparathyroidism

BACKGROUND The localization of enlarged parathyroid glands might, depending on size, histology, and concomitant goiter, be difficult in some patients. In the presented study, contrast-enhanced ultrasonography (CEUS) was applied as a new diagnostic tool to detect the site of parathyroid lesions. METHODS Thirty patients underwent operation for primary hyperparathyroidism (pHPT) between 8/2009 and 6/2010. Contrast-enhanced ultrasonography (CEUS) using a linear probe (6-9 MHz, LOGIQE9/GE), fundamental B scan, and Doppler ultrasonography, (99m)Technetium-sestamibi scintigraphy and magnetic resonance imaging (MRI) were performed in all patients preoperatively. The diagnostic sensitivity of the procedures, time requirements, and overall costs were analyzed. RESULTS Using CEUS, all 31 pathologic glands could be detected, compared with 23 using conventional ultrasonography, 25 using (99m)Technetium-sestamibi scintigraphy and 22 using MRI (P = .015). Costs and time requirement were less using CEUS as compared with (99m)Technetium-scintigraphy and MRI examinations (P = .002). Minimally invasive, video-assisted parathyroidectomy could be performed successfully based on CEUS findings in all but 7 patients who required concomitant thyroid surgery or had underwent previous thyroid operations. All patients showed normal serum levels of calcium and parathyroid hormone serum levels 3 months after parathyroidectomy. CONCLUSION CEUS represents a highly sensitive and cost-efficient method for localization of pathologic parathyroid glands in patients with pHPT. Future studies should confirm these findings in order to establish CEUS as a standard diagnostic procedure.

DX5 + NKT cells display phenotypical and functional differences between spleen and liver as well as NK1.1 - Balb/c and NK1.1 + C57Bl/6 mice

BackgroundNatural killer T cells represent a linkage between innate and adaptive immunity. They are a heterogeneous population of specialized T lymphocytes composed of different subsets. DX5+NKT cells are characterized by expression of the NK cell marker DX5 in the context of CD3. However, little is known about the phenotype and functional capacity of this unique cell population. Therefore, we investigated the expression of several T cell and NK cell markers, as well as functional parameters in spleen and liver subsets of DX5+NKT cells in NK1.1- Balb/c mice and compared our findings to NK1.1+ C57Bl/6 mice.ResultsIn the spleen 34% of DX5+NKT cells expressed CD62L and they up-regulated the functional receptors CD154 as well as CD178 upon activation. In contrast, only a few liver DX5+NKT cells expressed CD62L, and they did not up-regulate CD154 upon activation. A further difference between spleen and liver subsets was observed in cytokine production. Spleen DX5+NKT cells produced more Th1 cytokines including IL-2, IFN-γ and TNF-α, while liver DX5+NKT cells secreted more Th2 cytokines (e.g. IL-4) and even the Th17 cytokine, IL-17a. Furthermore, we found inter-strain differences. In NK1.1+ C57Bl/6 mice DX5+NKT cells represented a distinct T cell population expressing less CD4 and more CD8. Accordingly, these cells showed a CD178 and Th2-type functional capacity upon activation.ConclusionThese results show that DX5+NKT cells are a heterogeneous population, depending on the dedicated organ and mouse strain, that has diverse functional capacity.