2Center for Anatomical, Pathological, and Forensic Medical Research, Graduate School of Medicine, Kyoto University, Yoshida-konoe-cho, Kyoto 606-8501, Japan
Despite improvements in the outcomes, acute cellular rejection (ACR) remains the major cause of intestinal graft failures following SBT [9-12]. For most patients that experience severe ACR, adequate recovery of mucosal function is difficult; bacterial and viral opportunistic infections are inevitable because of defensive mucosal barriers. Therefore, diagnosing ACR in the early phase is essential during postoperative care .
The production of IL-5 increases significantly relative to other cytokines in the allograft tissue during ACR . In parallel with this, eosinophil infiltrates have frequently been observed , as well as mixed cellular inflammation . C - reactive protein (CRP) is another indicator of ACR (Figure 1A); this protein is known to rise in inflammation following IL-6 secretion by macrophages. A CRP test has been shown to measure 1.0-3.0 mg/10-1L in patients without administration of an immunosuppressive reagent however, this value elevates to over 3.0 mg/10-1L at the onset of ACR. Following immunosuppressive therapy, this value promptly decreased to 1.0 mg/10-1L [22,23]. Nonetheless, low copy numbers of CMV infection in the transplants did not significantly elevate the CRP value. Therefore, CRP value is a differential diagnostic maker of ACR from CMV enteritis.
Among histologic criteria, crypt apoptosis in the mucosa is one of the most reliable observations; severe ulceration follows this and sufficient recovery of the mucosa becomes difficult when graft damage reaches the submucosal area . Repeat occurrences of this severe damage leads to chronic graft rejection in which fibrosis proceeds leading to irreversible inefficiency of absorption in the intestine. Apoptosis in the crypt is detectable using the TUNEL method or by the caspasecleaved keratin fragment marker.
In addition, Tsuruyama, et al.  reported that apoptotic bodies cluster in the Lamina Propria (LP) of villi at the onset of ACR (Figure 2C). Apoptotic crypts in grafts undergoing ACR are shown as intensely stained. In addition, macrophages phagocytosing apoptotic T cells are frequently observed with clustering in the LP (Figure 2D). These apoptotic cell clusters are significantly decreased following steroid pulse administration . Therefore, this apoptotic response in LP is one of the immunological reactions associated with ACR. Scoring of the degree of apoptosis in the LP is available for evaluation (score 0, no signals; score 1, scant and isolated signals; score 2, a few signal aggregations; score 3, signal aggregates surrounding
Crypt apoptosis and related findings
Lymphocytic apoptosis in LP
up to 6 apoptotic bodies per 10 crypts
>6 apoptotic bodies per 10 crypts
Isolated apoptotic bodies
A few apoptotic body cluster
Apoptotic bodies aggregate
A) Lymphocyte infiltrates including eosinophils and neutrophils (× 200).
B) Crypt apoptosis (×200). An Arrow indicates the eosinophilic apoptotic bodies in the crypt.
C) Double staining of NKT cells with TCRValpha24 (red, PE) and TUNEL (green, FITC) (× 200).
D) Apoptotic clusters stained with TUNEL. DAB was used for visualization. An arrow represents the cluster.
Asaoka, et al.  reported the activation of cytotoxic T cells (CTLs) in granzyme B/ perforin-mediated graft injury. Unlike apoptosis in graft versus host disease that may be associated with elevation in TNF-alpha production , Fas ligand (FasL) is frequently stained in cases of ACR . Therefore, the ACR of intestinal graft includes various activations of apoptosis-related molecules, by expression and release. After T cells undergo apoptosis , apoptotic bodies are then phagocytosed by macrophages. Similar findings in a liver allograft have been reported . Thus, these phagocytic findings may be common to multiple allografts in ACR.
The entry of cytotoxic T cells CTLs into PPs via HEVs is observed within 7–10 days after transplantation and ACR occurs in cases. When ACR persists, PPs disintegrate in severe ACR and mucosal recovery is not sufficient when fibrosis develops. This is one of the poor prognoses of intestinal transplants, because once the mucosal defensive mechanism is lost in the erosive site, the graft becomes susceptible to bacterial and viral infection. Therefore, the immunosuppressive therapy before PP disintegration is essential for the control of post-transplantation success rate .
Follicular B cells in PPs are stimulated by antigens in the lumen and differentiate from Immunoglobulin M (IgM+) to IgA+ B cells by class switching, which is mediated by Activation-Induced Cytidine Deaminase (AID). IgA+ B cells in PPs circulate throughout the body via the thoracic duct and differentiate into IgA+ B cells by the effect of IL-6 produced by intestinal epithelial cells. The primary antibodies secreted into the intestinal tract mucus are of the IgA class, and are transported to the gut luminal side by binding to multimeric antibody receptors that are retained on intestinal epithelial cells. Notably, host lymphocytes rapidly repopulate allograft PPs/ILFs within two years in the absence of ACR . Allograft ILFs revealed a higher maturation state than control samples, and IgA+ plasma cells were increased in a number in allograft mucosa . AID gene expression in allograft PPs/ILFs that the immunological burden may promote the maturation of B cells . Histological examination showed hyperplastic changes of PPs with an increase in expression of CD20, a mature B cell marker at the onset of ACR .
FoxP3+ Treg are another immunological modulator of intestinal allograft, and the graft-protective mechanism has been extensively investigated . Treg are recruited to the liver allograft at the onset of ACR and are maintained in tolerated liver allograft . Introduction of bone marrow mesenchymal cells into the intestinal allograft increased Treg in parallel with IL-10 and TGF-β . In clinical cases, the roles of Treg are not understood with respect to tolerance or induction of ACR of intestinal allografts. In experimental studies linked with human biopsy samples, IL-17 plays a critical role in ACR of intestinal transplantation [37,42], and may be a target for inhibiting ACR.
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