Thomas Pech

Perioperative Infliximab Application Ameliorates Acute Rejection Associated Inflammation After Intestinal Transplantation

As we have shown in the past, acute rejection‐related TNF‐α upregulation in resident macrophages in the tunica muscularis after small bowel transplantation (SBTx) results in local amplification of inflammation, decisively contributing to graft dysmotility. Therefore, the aim of this study is to investigate the effectiveness of the chimeric‐monoclonal‐anti‐TNF‐α antibody infliximab as perioperative single shot treatment addressing inflammatory processes during acute rejection early after transplantation. Orthotopic, isogenic and allogenic SBTx was performed in rats (BN‐Lewis/BN‐BN) with infliximab treatment. Vehicle and IV‐immunoglobulin‐treated animals served as controls. Animals were sacrificed after 24 and 168 h. Leukocyte infiltration was investigated in muscularis whole mounts by immunohistochemistry, mediator mRNA expression by Real‐Time‐RT‐PCR, apoptosis by TUNEL and smooth muscle contractility in a standard organ bath. Both, infliximab and Sandoglobulin® revealed antiinflammatory effects. Infliximab resulted in significantly less leukocyte infiltration compared to allogenic controls and IV‐immunoglobulin, which was accompanied by lower gene expression of MCP‐1 (24 h), IFN‐γ (168 h) and infiltration of CD8‐positive cells. Smooth muscle contractility improved significantly after 24 h compared to all controls in infliximab treated animals accompanied by lower iNOS expression. Perioperative treatment with infliximab is a possible pharmaceutical approach to overcome graft dysmotility early after SBTx.

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.

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.

Influence of Immunosuppression on Alloresponse, Inflammation and Contractile Function of Graft After Intestinal Transplantation

In small bowel transplantation (SBTx), graft manipulation, ischemia/reperfusion injury and acute rejection initiate a severe cellular and molecular inflammatory response in the muscularis propria leading to impaired motility of the graft. This study examined and compared the effect of tacrolimus and sirolimus on inflammation in graft muscularis. After allogeneic orthotopic SBTx, recipient rats were treated with tacrolimus or sirolimus. Tacrolimus and sirolimus attenuated neutrophilic, macrophage and T‐cell infiltration in graft muscularis, which was associated with reduced apoptotic cell death. Nonspecific inflammatory mediators (IL‐6, MCP‐1) and T‐cell activation markers (IL‐2, IFN‐γ) were highly upregulated in allogeneic control graft muscularis 24 h and 7 days after SBTx, and tacrolimus and sirolimus significantly suppressed upregulation of these mediators. In vitro organ bath method demonstrated a severe decrease in graft smooth muscle contractility in allogeneic control (22% of normal control). Correlating with attenuated upregulation of iNOS, tacrolimus and sirolimus treatment significantly improved contractility (64% and 72%, respectively). Although sirolimus reduced cellular and molecular inflammatory response more efficiently after 24 h, contrary tacrolimus prevented acute rejection more efficiently. In conclusion, tacrolimus and sirolimus attenuate cellular and molecular inflammatory response in graft muscularis and subsequent dysmotility of the graft after allogeneic SBTx.

Intestinal Regeneration, Residual Function and Immunological Priming Following Rescue Therapy After Rat Small Bowel Transplantation

Clinical evidence suggests that recurrent acute cellular rejection (ACR) may trigger chronic rejection and impair outcome after intestinal transplantation. To test this hypothesis and clarify underlying molecular mechanisms, orthotopic/allogenic intestinal transplantation was performed in rats. ACR was allowed to occur in a MHC‐disparate combination (BN‐LEW) and two rescue strategies (FK506monotherapy vs. FK506+infliximab) were tested against continuous immunosuppression without ACR, with observation for 7/14 and 21 days after transplantation. Both, FK506 and FK506+infliximab rescue therapy reversed ACR and resulted in improved histology and less cellular infiltration. Proinflammatory cytokines and chemotactic mediators in the muscle layer were significantly reduced in FK506 treated groups. Increased levels of CD4, FOXP3 and IL‐17 (mRNA) were observed with infliximab. Contractile function improved significantly after FK506 rescue therapy, with a slight benefit from additional infliximab, but did not reach nontransplanted controls. Fibrosis onset was detected in both rescue groups by Sirius‐Red staining with concomitant increase of the fibrogenic mediator VEGF. Recovery from ACR could be attained by both rescue therapy regimens, progressing steadily after initiation of immunosuppression. Reversal of ACR, however, resulted in early stage graft fibrosis. Additional infliximab treatment may enhance physiological recovery of the muscle layer and enteric nervous system independent of inflammatory reactions.

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.

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.

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life (1,3). Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known(6,7). Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory (8-11). The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.