Martin von Websky

Role of resident macrophages in the immunologic response and smooth muscle dysfunction during acute allograft rejection after intestinal transplantation.

Resident muscularis macrophages initiate an inflammatory cascade during ischemia/reperfusion that is associated with dysmotility and the activation of immunologic processes. We hypothesized that these muscularis macrophages may also play a potential immunologic role for acute allograft rejection in intestinal transplantation. Orthotopic SBTx (BN‐Lew) was performed without immunosuppression. Animals were sacrificed 7 days after SBTx. The role of resident macrophages was evaluated by transplantation of macrophage‐depleted and gadolinium chloride‐treated gut. Leukocyte infiltration was investigated in muscularis whole mounts by immunohistochemistry. Mediator mRNA expression was determined by Real‐Time‐RT‐PCR. Apoptosis was evaluated by TUNEL. Smooth muscle contractility was assessed in a standard organ bath. In comparison to vehicle‐treated grafts, macrophage‐depleted grafts exhibited significantly lower mediator mRNA peak expression (IL‐6, IL‐2, IL‐10, MCP‐1, iNOS, TNFα, IFNγ, FasL), leukocyte infiltrates (ED1‐ and ED2 positive monocytes and macrophages, neutrophils, CD4+ and CD8+ lymphocytes), apoptosis rates and an improved histologic rejection grading. Vehicle‐treated grafts showed a 77% decrease in smooth muscle contractility compared to naïve controls, while macrophage‐depleted gut exhibited only a 51% decrease in contractile activity. Transplantation of macrophage‐depleted gut attenuates the functionally relevant molecular and cellular immunologic response within the graft muscularis in acute allograft rejection. Resident macrophages participate in initiating these processes.

Acute rejection and the muscularis propria after intestinal transplantation: the alloresponse, inflammation, and smooth muscle function.

Background. It has been shown that in transplantation the intestinal muscularis may act as an immunologically active layer via the activation of resident macrophages and the recruitment of leukocytes. Thus we hypothesized that inflammation within the intestinal muscularis is involved in the promotion of acute rejection in intestinal allografts and that this causes smooth muscle dysfunction. Methods. Orthotopic allogenic and small bowel transplantation (Brown-Norway rats–Lewis rats) was performed without immunosuppression. Animals were sacrificed 1, 4, and 7 days after small bowel transplantation. Isogenic transplanted grafts (Brown-Norway rats–Brown-Norway rats) as well as nontransplanted bowel served as controls. Mediator mRNA expression was determined by real-time reverse-transcriptase polymerase chain reaction. Leukocyte infiltration was evaluated in muscularis whole mounts by immunohistochemistry. Apoptosis was evaluated by TdT-mediated dUTP-X nick end labeling assay. Contractility was assessed in a standard organ bath under bethanechol stimulation. Statistical analysis was performed using a Student’s t test and one-way analysis of variance. Results. Transplanted animals showed a significant early inflammatory response within the graft muscularis because of reperfusion injury. Only allogenic transplanted animals exhibited a significant second molecular inflammatory peak in the muscularis during rejection (mRNA induction for interleukin (IL)-6, intercellular adhesion molecule-1, monocyte chemoattractant protein (MCP)-1, interferon-&ggr;, IL-2, tumor necrosis factor-&agr;, IL-10, inducible nitric oxide synthase). These findings were associated with significant leukocyte infiltration within the muscularis, increasing apoptotic cells and massive impairment of smooth muscle contractile activity by 78%. Conclusions. The data shows that transplantation results in an early and temporary inflammatory response within the intestinal graft muscularis, that is reactivated and intensified during acute allograft rejection. The immunoreaction within the intestinal muscularis leads to intestinal allograft smooth muscle dysfunction.

Perioperative Glycine Treatment Attenuates Ischemia/reperfusion Injury and Ameliorates Smooth Muscle Dysfunction in Intestinal Transplantation

Background. Ischemia/reperfusion evokes a functionally relevant inflammatory response within the muscularis propria of small bowel grafts by activation of resident macrophages and leukocyte recruitment. We hypothesized that immunomodulatory perioperative treatment with glycine attenuates the proinflammatory cascade and improves smooth muscle dysfunction of small bowel grafts. Methods. Orthotopic SBTx was performed in Lewis rats. Glycine (1 mg/g body weight) was infused (0.1 mL/g/hr) for 2 hr before harvest as preconditioning in the donor, and for 2 hr from the onset of reperfusion in the recipient. Transplanted vehicle (isotonic saline)-treated animals and naive animals served as controls. Rats were sacrificed after 3 hr and 24 hr. Leukocyte infiltration was investigated in muscularis whole mounts by immunohistochemistry. Mediator mRNA expression was determined by real-time-PCR. Jejunal circular smooth muscle contractility was assessed in a standard organ bath. Results. Compared with vehicle controls, glycine-treated graft muscularis expressed a significant alleviation in mRNA peak expression for IL-6, IL-1&bgr;, ICAM-1, MCP-1, TNF&agr;, COX-2, and iNOS. Also glycine-treated grafts exhibited significantly less infiltration with ED-1-positive macrophages and MPO-positive neutrophils as well as reduced apoptosis. Concurrent to these results, vehicle controls showed an 80% decrease in smooth muscle contractility, whereas glycine-treated animals exhibited only a 40% decrease in contractile activity compared with controls. Conclusions. The data indicate that perioperative glycine treatment reduces the molecular and cellular inflammatory response within the grafts and improves smooth muscle dysfunction after transplantation. Therefore, the glycine-activated chloride channel on resident and infiltrating leukocytes could be a promising pharmacologic target to attenuate ischemia/reperfusion injury after ITx.

Inducible nitric oxide synthase expression in the intestinal muscularis mediates severe smooth muscle dysfunction during acute rejection in allogenic rodent small bowel transplantation.

BACKGROUND Acute rejection in small bowel transplantation is associated with dysmotility. Therefore, host and organ not only face the threat of destructive immunological processes but also the risk of bacterial translocation, endotoxemia, and systemic inflammatory response syndrome. We hypothesized that dysmotility during acute rejection is based on an alloreactive leukocyte infiltrate and coexpression of the kinetically active mediator inducible nitric oxide synthase (iNOS) in the muscularis propria. MATERIALS AND METHODS Allogenic and isogenic rat small bowel transplantation (SBTx; Brown Norway [BN] to Lewis and BN to BN) was performed without immunosuppression. Animals were sacrificed 4 and 7 d after SBTx. Leukocyte infiltration and iNOS protein was investigated by immunohistochemistry and immunohistology. Real-time reverse transcription polymer chain reaction was used to detect iNOS expression. Griess reaction was used to evaluate NO production. Spontaneous, bethanechol-stimulated, and L-N(6)-(1-iminoethyl)-L-Lysin-blocked jejunal circular muscle contractions were measured in a standard organ bath in vitro. RESULTS On d 7 after SBTx, allogenic transplanted animals showed significant infiltration with ED-1- and ED-2-positive monocytes and macrophages within the muscularis parallel to the manifestation of acute rejection. Additionally, immunohistochemistry localized iNOS protein in leukocytes within the muscularis. Reverse transcription polymer chain reaction showed a significant increase in iNOS mRNA expression (460-fold) in allogenic transplanted muscularis compared to isogenic transplanted muscularis (2.5-fold). Compared to controls, allogenic grafts showed a 73% decrease in smooth muscle contractility, while isogenic grafts showed only an 8% decrease of contractility on d 7. L-N(6)-(1-iminoethyl)-L-Lysin application in vitro significantly improved muscle contractility and decreased NO production. CONCLUSION The data show that inflammation associated iNOS expression in the intestinal graft muscularis is involved in motoric graft dysfunction during acute rejection.

Combination therapy of tacrolimus and infliximab reduces inflammatory response and dysmotility in experimental small bowel transplantation in rats.

Background. Intestinal transplantation initiates a functionally relevant inflammatory response by activation of resident macrophages within the muscularis associated with dysmotility. Infliximab is used successfully as a potent anti-inflammatory agent for the treatment of chronic inflammatory bowel diseases and as rescue therapy in acute steroid-resistant rejection in selected settings in clinical small bowel transplantation. We hypothesize that additional perioperative treatment with infliximab diminishes initiation of the inflammatory cascade and improves motility in small bowel grafts using a standard tacrolimus immunosuppressive protocol. Methods. Orthotopic intestinal transplantation was performed in rats. In two treatment groups (24/168 hr), infliximab was administered intravenously directly after reperfusion and tacrolimus was injected intramuscularly after transplantation and once a day. Two other treatment groups (24/168 hr) received standard immunosuppressive therapy with tacrolimus. Isogenic and allogenic transplanted vehicle-treated animals (24/168 hr) and native gut served as control. Results. Infliximab-treated grafts exhibited significantly less leukocyte infiltration at 24/168 hr after transplantation and at 168 hr significantly less apoptosis in the tunica muscularis compared with tacrolimus monotherapy. Additional infliximab treatment resulted in increased smooth muscle contractility (30%) after 24 hr compared with tacrolimus control. Conclusions. Dysmotility of transplanted small bowel results from reperfusion injury and acute rejection. Additional perioperative treatment with infliximab reduces early unspecific inflammatory responses and complements immunosuppressive therapy with tacrolimus.

Functional assessment of intestinal motility and gut wall inflammation in rodents: analyses in a standardized model of intestinal manipulation.

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.(1,2) These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.(3) The inflammation is characterized by liberation of proinflammatory mediators such as IL-6(4) or IL-1β or inhibitory neurotransmitters like nitric oxide (NO).(5) Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.(2) Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.(6) In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Conception, Pregnancy, and Lactation Despite Chronic Intestinal Failure Requiring Home Parenteral Nutrition.

BACKGROUND Short-term parenteral nutrition is commonly accepted to be safe in pregnancy, but knowledge about the management of pregnancy during long-term home parenteral nutrition (HPN) is sparse. METHODS AND RESULTS A systematic literature review revealed that the published experience is limited to 15 pregnancies with parenteral nutrition from preconception to delivery and beyond. Maternal morbidity was surprisingly low, and fetal outcome was good; however, micronutrient deficiencies may have contributed to fetal anomalies. Herein, we additionally report the case of a 26-year-old Caucasian woman with long-term HPN dependence secondary to short bowel syndrome caused by recurrent thromboembolic mesenteric infarctions who delivered a healthy fetus at 37 weeks of gestation. Individual macronutrient support and adequate micronutrient supplementation ensured normal maternal weight gain and fetal development. Based on the individual maternal risk of recurrent thrombosis, anticoagulant treatment was carefully titrated throughout pregnancy. Furthermore, loss of abdominal domain with a rigid maternal abdominal wall secondary to short bowel syndrome and multiple laparotomies resulted in food intolerance during the third trimester. Still, with multidisciplinary efforts, both mother and the breast-fed infant were in good health at 12 months after delivery. CONCLUSIONS Taking the reported literature into consideration, we conclude that under the premise of optimal medical care, the risk:benefit ratio for pregnancy of HPN-dependent women seems to be justifiable. To minimize the risks, we recommend preconception counseling and early referral to a tertiary center offering both a high-risk pregnancy unit and a nutrition service. In particular, maternal micronutrient levels should be monitored.

A natural tetrahydropyrimidine, ectoine, ameliorates ischemia reperfusion injury after intestinal transplantation in rats.

Background/Aims: Ischemia reperfusion (I/R) injury after small bowel transplantation leads to inflammatory reactions and loss of structural integrity with subsequent graft contractile dysfunction in the early postoperative phase. The natural tetrahydropyrimidine ectoine (1-,4-,5-,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid; THP) protects the ileal mucosa and muscularis against effects of I/R injury in an experimental model of isolated graft reperfusion. The effects of THP treatment were evaluated in an established experimental intestinal transplant model. Methods: Isogenic, orthotopic small bowel transplantation was performed in Lewis rats (6 h cold ischemia time). Perioperative THP treatment (intraluminal/intravascular) groups were compared to vehicle-treated animals (after 3 and 24 h) and non-transplanted controls (n = 5/group). Park’s score defined the effects of I/R injury. The infiltration of neutrophils, monocytes and macrophages, mRNA expression of IL-6 and TNF-α, serum levels of IL-6 and NO and smooth muscle contractility were evaluated. Results: Improved graft outcome after intraluminal and intravascular THP treatment was defined by considerably ameliorated neutrophil infiltration and less histological signs of I/R injury (p ≤ 0.05). In the presence of THP, mRNA expression of IL-6 and TNF-α and IL-6 and NO serum levels were reduced and smooth muscle function was improved. Conclusion: THP treatment offers protection against the effects of I/R injury in intestinal transplantation in vivo, however, only as supplementary treatment option.

Effects of immunosuppressive therapy after experimental small bowel transplantation in rats

Significant improvements of graft and patient survival have been achieved over the past 20 years in the field of intestinal transplantation. Tacrolimus monotherapy with corticosteroids, or in combination with sirolimus is the most commonly used immunosuppressive regimen. Early (24h) after experimental allogenic small bowel transplantation in rats, sirolimus reduces the cellular and molecular inflammatory response with subsequent graft dysmotility more efficiently than tacrolimus, with contrary effects at 7 days after transplantation. This study evaluates three immunosuppressive strategies in the post-acute phase after intestinal transplantation - tacrolimus or sirolimus monotherapy and the combination therapy of tacrolimus with infliximab. After orthotopic intestinal transplantation between Brown Norway and Lewis rats, animals received 14 days of immunosuppressive treatment. Histology, infiltration of neutrophils, monocytes and macrophages, cytokine and mediator mRNA expression (real time RT-PCR) and smooth muscle function in a standard organ bath were assessed at 14 days after transplantation in all treatment groups and isogenic controls. Allogenic transplanted rats without immunosuppressive therapy and non-transplanted animals served as further control. Tacrolimus prevented acute rejection and graft dysmotility more effectively (p ≥ 0.05) than sirolimus. Reduced mRNA expression levels of CD4, IFN-γ, IL-6, IL-10, iNOS, NFκB, TNF-α and MCP-1 (p ≤ 0.05) were observed in tacrolimus treated animals compared to sirolimus. Additional infliximab application did not influence the cellular and molecular inflammatory response in the post-acute phase after transplantation. In conclusion, the severe cellular and molecular inflammatory response in allogenic transplanted grafts without immunosuppressive therapy is ameliorated by all three immunosuppressive regimens, but tacrolimus was found to be more efficient than sirolimus at 14 days after transplantation. Our findings do not rule out the usage of sirolimus as single immunosuppressive therapy, but indicate and confirm the potency and effectivity of tacrolimus as basis immunosuppressant in the field of intestinal transplantation.

The novel guanylhydrazone CPSI-2364 ameliorates ischemia reperfusion injury after experimental small bowel transplantation.

Background Resident macrophages within the tunica muscularis are known to play a crucial role in initiating severe inflammation in response to ischemia reperfusion injury after intestinal transplantation contributing to graft dysmotility, bacterial translocation, and possibly, acute rejection. The p38 mitogen-activated protein kinase is a key player in the signaling of proinflammatory cytokine synthesis in macrophages. Therefore, we investigated the effects of CPSI-2364, an apparent macrophage-specific inhibitor of the p38 mitogen-activated protein kinase pathway in an isogenic intestinal rat transplantation model. Methods Recipient and donor animals were treated perioperatively with CPSI-2364 (1 mg/kg, intravenously) or vehicle solution. Nontransplanted animals served as control. Animals were killed 30 min, 3 hr, and 18 hr after reperfusion. Results CPSI-2364 treatment resulted in significantly less leukocyte infiltration and significantly improved graft motor function (18 hr). Messenger RNA expression of proinflammatory cytokines (interleukin 6) and kinetic active mediators (NO) was reduced by CPSI-2364 in the early phase after transplantation. Histologic evaluation revealed the protective effects of CPSI-2364 treatment by a significantly less destruction of mucosal integrity at all time points. Perioperative treatment with CPSI-2364 improves graft motor function through impaired inflammatory responses to ischemia reperfusion injury by inhibition of proinflammatory cytokines and suppression of nitric oxide production in macrophages. Conclusions CPSI-2364 presents as a promising complementary pharmacological approach preventing postoperative dysmotility for clinical intestinal transplantation.