**2.1 Crypt apoptosis**

Crypt apoptosis is considered a unique feature of ACR in SBT. The crypt is an architectural element that is located at the base of the villous epithelium and serves as the source of mucosal cells. Paneth cells, stem cells, and reserve stem cells are included in the crypt. Enterocytes are differentiated from reserve stem cells in the crypt and migrate to the tips of villi through the transit amplifying zone [20]. The kinetics of differentiation and loss of enterocytes contribute to the maintenance of quick renewal for mucosal homeostasis. The supply of enterocytes becomes interrupted by apoptosis in the crypt, and the shortening of villi becomes unavoidable. When ulceration occurs, the lesion is susceptible to infectious enteritis such as CMV- and EBV-related enteritis, for a significant period of time [21, 22].

Pathologically, the diagnosis of small bowel transplant rejection is based on the appearance of 6 or more apoptotic lesions per ten crypts [3, 4] (**Table 1**). The detection of crypt apoptosis is commonly used because of its high reproducibility. Nevertheless, discussions about the number of lesions per crypt were held at the Banff Conference 2019. In the case we experienced, if more than six apoptotic cells were detected in the crypts, subsequent ulceration is inevitable, and infection from the ulcer site might occur. Therefore, we considered that immunosuppressants should be administered when apoptotic cells were observed in the crypt [18].

Previous apoptosis findings have shown that the cells are eosinophilic with an intensely stained nucleus [17, 18] (**Figure 1**). Cells with lobulated nuclei, such as neutrophils and apoptotic cells, can be confused morphologically; therefore, careful observation is necessary. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining is one method to avoid this confusion. This staining procedure involves an enzyme-mediated reaction. First, the fragmented DNA is labeled with biotin containing terminal deoxynucleotidyl transferase. The labeled DNA then reacts with streptavidin for staining. Both labels with 3,3′-diaminobenzidine (DAB) and fluorescein isothiocyanate (FITC) are available for visualization of the apoptotic body [18].

As apoptosis progresses, fragmented cell debris (apoptotic bodies) are observed in or around the crypt. Increasing the dose of the immunosuppressive drug suppresses the progression of apoptosis. Therefore, quick detection of apoptosis is critical for effective immunosuppression therapy [18, 19].

The factors that cause such apoptotic responses in the crypt and lamina propria are poorly understood. It is possible that cytotoxic T lymphocytes (CTLs) can directly

**91**

*Pathology of Intestinal Transplantation: Rejection and a Case of Tolerance*

Lymphocytic apoptosis in the lamina propria [18]

Isloated apoptotic bodies in the lamina

A few apoptotic body cluster in the lamina

aggregate in the lamina

Phagocytosis of apoptotic bodies by macrophages [18]

Aggregation of macrophages [18]

macrophages

Granuloma consisting of

None

propria

propria

propria

related findings

Up to 6 apoptotic bodies per 10 crypts

per 10 crypts Confluent apoptosis

inflammation, epithelial injury

Severe/exfoliative Mucosal ulceration Apoptotic bodies

*Histological criteria for ACR of the intestinal allograft [10]. The findings under the lymphocyte and* 

attack the crypt of the graft. However, it is not always histologically evident that CTLs directly infiltrate near the crypt and remain near this area. There is also a noteworthy research report suggesting that CD8-positive CTLs are not always involved in ACR [23]. At the basic research level, rejection of the apoptosis-inducing factors perforin and granzyme B released from CTLs has been reported [24]. Therefore, the destruction of the mucosal immune system by local increases in complement and

*Histology of ACR of the intestinal allograft. The onset of ACR. Eosinophil infiltrates are observed in the ulcerated mucosa (left, 100×). Apoptotic bodies are observed in the crypt (indicated by arrows, middle, 200×;* 

In addition to the crypt apoptosis, apoptotic lymphocytes are identified by systematic immunostaining of lymphocyte surface antigens: T cell surface antigens CD3, CD4, and CD8; B cell surface antigens CD20 and CD79a; natural killer cell surface antigen CD56; and activated lymphocytes Fas and its ligand (FasL) [25]. FasL, also known as CD95L, is a surface antigen of activated cytotoxic T cells and NK cells are observed at the onset of rejection [18] (**Figure 2**, upper panels).

Apoptotic bodies are also been observed in the lamina propria and Peyer's patch (PP) distant from the crypt, and the macrophages that phagocytose them often aggregate to present granuloma-like findings. Notably, these bodies are stained with

inflammatory cytokines is thought to be the cause of apoptosis.

**2.2 Immunohistochemical monitoring**

*right, 100×, TUNEL-stained with 3,3'-diaminobenzidine).*

*DOI: http://dx.doi.org/10.5772/intechopen.94361*

Indeterminate Crypt apoptosis and

Mild >6 apoptotic bodies

*macrophage categories refer to our previous study [18].*

Moderate Increased

**Histologic grade**

**Table 1.**

**Figure 1.**

*Pathology of Intestinal Transplantation: Rejection and a Case of Tolerance DOI: http://dx.doi.org/10.5772/intechopen.94361*


#### **Table 1.**

*Organ Donation and Transplantation*

CMV enteritis and EBV enteritis become inevitable.

other findings as indicators of ACR [17–19].

**2. Diagnostic criteria for ACR**

**2.1 Crypt apoptosis**

time [21, 22].

the apoptotic body [18].

surgery may occur during the early phase after transplantation. Cytomegalovirus (CMV) enteritis and Epstein–Barr virus -related enteritis are severe side effects and post-transplantation lymphoproliferative disorders/diseases [13–15]. It is often difficult to make a differential diagnosis of the ACR findings. However, histologic diagnosis is critical for the selection of immunosuppressants and their respective doses. Tacrolimus, cyclosporin, and steroids are commonly prescribed in the early stages of rejection [16]. If an excessive dose is administered, the occurrences of

Among the various histological features, crypt epithelial cell apoptosis has been evaluated as a highly reproducible finding. However, other histological findings have been proposed at different institutions. We have also previously suggested

Crypt apoptosis is considered a unique feature of ACR in SBT. The crypt is an architectural element that is located at the base of the villous epithelium and serves as the source of mucosal cells. Paneth cells, stem cells, and reserve stem cells are included in the crypt. Enterocytes are differentiated from reserve stem cells in the crypt and migrate to the tips of villi through the transit amplifying zone [20]. The kinetics of differentiation and loss of enterocytes contribute to the maintenance of quick renewal for mucosal homeostasis. The supply of enterocytes becomes interrupted by apoptosis in the crypt, and the shortening of villi becomes unavoidable. When ulceration occurs, the lesion is susceptible to infectious enteritis such as CMV- and EBV-related enteritis, for a significant period of

Pathologically, the diagnosis of small bowel transplant rejection is based on the appearance of 6 or more apoptotic lesions per ten crypts [3, 4] (**Table 1**). The detection of crypt apoptosis is commonly used because of its high reproducibility. Nevertheless, discussions about the number of lesions per crypt were held at the Banff Conference 2019. In the case we experienced, if more than six apoptotic cells were detected in the crypts, subsequent ulceration is inevitable, and infection from the ulcer site might occur. Therefore, we considered that immunosuppressants should be administered when apoptotic cells were observed in the crypt [18].

Previous apoptosis findings have shown that the cells are eosinophilic with an intensely stained nucleus [17, 18] (**Figure 1**). Cells with lobulated nuclei, such as neutrophils and apoptotic cells, can be confused morphologically; therefore, careful observation is necessary. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining is one method to avoid this confusion. This staining procedure involves an enzyme-mediated reaction. First, the fragmented DNA is labeled with biotin containing terminal deoxynucleotidyl transferase. The labeled DNA then reacts with streptavidin for staining. Both labels with 3,3′-diaminobenzidine (DAB) and fluorescein isothiocyanate (FITC) are available for visualization of

As apoptosis progresses, fragmented cell debris (apoptotic bodies) are observed in or around the crypt. Increasing the dose of the immunosuppressive drug suppresses the progression of apoptosis. Therefore, quick detection of apoptosis is

The factors that cause such apoptotic responses in the crypt and lamina propria are poorly understood. It is possible that cytotoxic T lymphocytes (CTLs) can directly

critical for effective immunosuppression therapy [18, 19].

**90**

*Histological criteria for ACR of the intestinal allograft [10]. The findings under the lymphocyte and macrophage categories refer to our previous study [18].*

#### **Figure 1.**

*Histology of ACR of the intestinal allograft. The onset of ACR. Eosinophil infiltrates are observed in the ulcerated mucosa (left, 100×). Apoptotic bodies are observed in the crypt (indicated by arrows, middle, 200×; right, 100×, TUNEL-stained with 3,3'-diaminobenzidine).*

attack the crypt of the graft. However, it is not always histologically evident that CTLs directly infiltrate near the crypt and remain near this area. There is also a noteworthy research report suggesting that CD8-positive CTLs are not always involved in ACR [23]. At the basic research level, rejection of the apoptosis-inducing factors perforin and granzyme B released from CTLs has been reported [24]. Therefore, the destruction of the mucosal immune system by local increases in complement and inflammatory cytokines is thought to be the cause of apoptosis.
