The hybrid CL-SP-D molecule has the potential to regulate xenogeneic rejection by human neutrophils more efficiently than CD47.

OBJECTIVE: Innate immunity plays a vital role in xenotransplantation. A CD47 molecule, binding to the SIRPα expressed on monocyte/macrophage cells, can suppress cytotoxicity. Particularly, the SIRPα contains ITIM, which delivers a negative signal. Our previous study demonstrated that the binding between CL-P1 and surfactant protein-D hybrid (CL-SP-D) with SIRPα regulates macrophages' phagocytic activity. In this study, we examined the effects of human CD47 and CL-SP-D expression on the inhibition of xenograft rejection by neutrophils in swine endothelial cells (SECs). METHODS: We first examined SIRPα expression on HL-60 cells, a neutrophil-like cell line, and neutrophils isolated from peripheral blood. CD47-expressing SECs or CL-SP-D-expressing SECs were generated through plasmid transfection. Subsequently, these SECs were co-cultured with HL-60 cells or neutrophils. After co-culture, the degree of cytotoxicity was calculated using the WST-8 assay. The suppressive function of CL-SP-D on neutrophils was subsequently examined, and the results were compared with those of CD47 using naïve SECs as controls. Additionally, we assessed ROS production and neutrophil NETosis. RESULTS: In initial experiments, the expression of SIRPα on HL-60 and neutrophils was confirmed. Exposure to CL-SP-D significantly suppressed the cytotoxicity in HL-60 (p = 0.0038) and neutrophils (p = 0.00003). Furthermore, engagement with CD47 showed a suppressive effect on neutrophils obtained from peripheral blood (p = 0.0236) but not on HL-60 (p = 0.4244). The results of the ROS assays also indicated a significant downregulation of SEC by CD47 (p = 0.0077) or CL-SP-D (p = 0.0018). Additionally, the suppression of NETosis was confirmed (p = 0.0125) in neutrophils co-cultured with S/CL-SP-D. CONCLUSION: These results indicate that CL-SP-D is highly effective on neutrophils in xenogeneic rejection. Furthermore, CL-SP-D was more effective than CD47 at inhibiting neutrophil-mediated xenograft rejection.

Suppression of macrophage-mediated xenogeneic rejection by the ectopic expression of human CD177.

Cellular xenogeneic rejection by the innate immune system is a major immunological obstruction that needs to be overcome for the successful clinical use of xenografts. Our focus has been on macrophage-mediated xenogeneic rejection, since suppressing macrophage function has considerable potential for practical applications in the area of xenotransplantation. We report herein on an investigation of the suppressive effect of human CD177 (hCD177) against macrophage-mediated xenogeneic rejection. Wild type swine aortic endothelial cell (SEC) and an SEC transfectant with hCD177 (SEC/hCD177) were co-cultured with macrophages, and the degree of cytotoxicity was evaluated by WST-8 assays, and phagocytosis was examined using Calcein-AM labeling methods. The expression of anti/pro-inflammatory cytokines was evaluated by RT-qPCR and the phosphorylation of SHP-1 on macrophages in co-culture was evaluated by Western blotting. The result of cytotoxicity assays indicated that hCD177 suppressed M1 macrophage-mediated xenogeneic rejection (vs. SEC, p < 0.0001). Similarly, the result of phagocytosis assays indicated that hCD177 suppressed it (vs. SEC, p < 0.05). In addition, hCD177 significantly suppressed the expression of IL-1ß, a pro-inflammatory cytokine, in M1 macrophages (vs. SEC, p < 0.01). Luciferase assays using THP1-Lucia NF-kB also showed a significant difference in NF-kB activation (vs. SEC, p < 0.001). In addition, hCD177 was found to induce the phosphorylation of SHP-1 in M1 macrophages (vs. SEC, p < 0.05). These findings indicate that hCD177 suppresses M1 macrophage-mediated xenogeneic rejection, at least in part via in the phosphorylation of SHP-1.

The Innate Cellular Immune Response in Xenotransplantation.

Xenotransplantation is very attractive strategy for addressing the shortage of donors. While hyper acute rejection (HAR) caused by natural antibodies and complement has been well defined, this is not the case for innate cellular xenogeneic rejection. An increasing body of evidence suggests that innate cellular immune responses contribute to xenogeneic rejection. Various molecular incompatibilities between receptors and their ligands across different species typically have an impact on graft outcome. NK cells are activated by direct interaction as well as by antigen dependent cellular cytotoxicity (ADCC) mechanisms. Macrophages are activated through various mechanisms in xenogeneic conditions. Macrophages recognize CD47 as a "marker of self" through binding to SIRPα. A number of studies have shown that incompatibility of porcine CD47 against human SIRPα contributes to the rejection of xenogeneic target cells by macrophages. Neutrophils are an early responder cell that infiltrates xenogeneic grafts. It has also been reported that neutrophil extracellular traps (NETs) activate macrophages as damage-associated pattern molecules (DAMPs). In this review, we summarize recent insights into innate cellular xenogeneic rejection.

Effect of a C5a receptor antagonist on macrophage function in an intestinal transplant rat model.

BACKGROUND: C5a promotes alloreactivity via the C5a receptor 1 (C5aR1) on immune cells, but this has not been confirmed in the case of small intestine transplantation immunity. In the present study, we examined the effect of C5aR1 antagonist (PMX53) on macrophage function in small intestinal transplantation. METHODS: The model was created by heterotopic intestinal transplantation using donor Dark Agouti and recipient Lewis rats. PMX53 was administered starting on the day of operation until postoperative day 7. The graft survivals were compared, and HE staining of grafts, lymphocyte mixed reaction test (MLR, mixed culture of T cells from lymph nodes and spleen cells from donors), and changes in macrophage and T cell accumulation in grafts on day 6 after transplantation were evaluated. In addition, the effect of PMX53 on macrophage differentiation and activation was assessed using macrophages derived from bone marrow (BMDM). RESULTS: Graft survival was significantly prolonged in the therapeutic group compared to the untreated group. Histological evaluation showed that PMX53 inhibited the shortening of the graft villus, and the stimulation index of MLR was significantly lower in the therapeutic group compared to the untreated group. In the therapeutic group, the accumulation of macrophages in intestinal graft and monocyte in blood were reduced, compared with the untreated group. PMX53 decreased the differentiation in BMDM and the mRNA expression of IL-1ß and TNF-α in activated BMDM. CONCLUSION: Inhibition of C5a/C5aR1 signaling appears to regulate macrophage differentiation and suppress rejection in small intestine transplantation immunity.

Laparoscopic repair of sliding inguinal hernia in female children.

PURPOSE: To evaluate the safety and feasibility of laparoscopic repair of sliding inguinal hernia in female children. METHODS: Laparoscopic percutaneous extraperitoneal closure (LPEC) was performed in 482 female inguinal hernia children between 2006 and 2015. Fourteen of these patients were associated with sliding inguinal hernia, and these 14 patients were enrolled and reviewed retrospectively. RESULTS: The mean age and the body weight at the operation was 9.6 months and 7.8 kg. Seven patients required the reduction of the ovary under general anesthesia. Laparoscopy, however, revealed that five patients had severe sliding of fallopian tube into the inguinal canal. One of these five patients received a simple LPEC, but developed the recurrence due to the low ligation of the hernia sac, and needed the second hernia repair under inguinal approach. Other four patients with fallopian tube sliding required the dissection of the fallopian tube and peritoneal repair, or the conversion to inguinal approach; therefore they had longer surgical time compared to those without fallopian tube sliding. CONCLUSION: LPEC is safe and feasible for the sliding inguinal hernia repair except the cases with fallopian tube sliding. Patients with fallopian tube sliding required additional procedure or conversion to inguinal approach.

[A Case of Collision Tumor of Gastric Malignant Lymphoma and Gastric Cancer].

A 71-year-old man with anemia, weight loss, and loss of appetite was admitted. Ultrasound examination found thickening of the wall of the stomach. A type 3 gastric tumor was detected in the greater curvature of the gastric corpus via upper gastrointestinal endoscopy. Total gastrectomy, transverse colon resection, and Roux-en-Y anastomosis reconstruction was performed. In the postoperative pathological results, adenocarcinoma, tub2, and diffuse large B cell lymphoma collision was found. The patient underwent chemotherapy for malignant lymphoma and although it was a relatively advanced neoplasia, he is alive without a recurrence.

Primary diffuse large B cell lymphoma of the common bile duct causing obstructive jaundice.

We report a rare case of a diffuse large B-cell lymphoma (DLBCL) arising from the common bile duct (CBD). A 77-year old man presented with general fatigue and obstructive jaundice. Abdominal computed tomography revealed a well-circumscribed enhancing mass in the midportion of the CBD with proximal bile duct dilatation. Endoscopic retrograde cholangiopancreatography (ERCP) also showed a midportion of the CBD stricture. Direct peroral cholangioscopy revealed smooth mass in the midportion of the CBD, and narrow-band imaging (NBI) showed irregular tortuous microvessels. The brushing cytology of the CBD was performed, and it was diagnosed as suspicious for poorly differentiated adenocarcinoma or malignant lymphoma. We performed extrahepatic bile duct resection for accurate diagnosis. Histological and immunohistochemical examination of the resected specimen revealed DLBCL. Although systemic chemotherapy is the mainstay of treatment for DLBCL, he refused scheduled subsequent chemotherapy, and died of multiple liver metastases 6 months after surgery.

Management of granulomatous mastitis: a series of 13 patients who were evaluated for treatment without corticosteroids.

Granulomatous mastitis (GM) is a rare chronic inflammatory breast condition with unknown etiology. There is still no generally accepted optimal treatment for GM. Corticosteroid treatment and/or wide excision is most commonly reported in the literature. Incision and drainage or limited excision alone has little benefit because of a strong tendency of recurrence. Corticosteroids also have a high failure rate and possible side effects. In the current series, we treated GM patients without corticosteroids, except for one patient. We also devised multidirectional deep drainage for advanced and complicated abscesses, which are characteristic of GM. This retrospective study included 13 women who met the required histologic criteria of GM. The mean age of the patients was 41 years. All of the patients were premenopausal. Six patients had breast-fed in the last 5 years. Five patients were under medication with antidepressants. A total of 11 patients developed abscesses during the clinical course, and the abscesses penetrated the retromammary space in 4 patients. We treated 2 of these 4 patients with multidirectional deep drainage and obtained complete remission in 5 and 6.5 months, respectively. These times were much shorter than those in the other 2 patients. The time to resolution in 11 patients was 4 to 28 months. This overall outcome was comparable with that of corticosteroid treatment reported in the literature. Because the natural history of GM is thought to be self-limiting, close observation and minimally required drainage of abscesses without corticosteroid administration remain the treatment modality of choice.